DE20023799U1 - Detecting infections by acid-resistant microorganisms, particularly for diagnosing Helicobacter pylori, comprises immunochromatographic detection of antigen in feces - Google Patents

Abstract

Detecting infection by an acid-resistant microorganism (A), in a mammal, comprises using immunochromatography. Detecting infection by an acid-resistant microorganism (A), in a mammal, comprises (a) preparing an immunochromatographic test strip having a sample application zone (I); (b) applying a fecal sample, containing an antigen (Ag) of (A) to (I); (c) incubating the sample with: (i) a first receptor (R1) to form a complex with Ag; or (ii) at least two different R1 to form a three-part complex with Ag, where R1 are specific for an Ag which, after passage through the intestines, at least in some mammals, retains a native (or corresponding) structure against which the mammal produces antibodies (when immunized or infected with (A), or its extracts, lysates or derived proteins (or fragments) or synthetic peptides); (d) immobilizing a second receptor (R2), able to bind to the complex formed between Ag and R1 to an analytical region; (e) transporting the first complex; (f) forming a second complex with R2 in the analytical region; and (g) detecting the complex. Independent claims are also included for the following: (1) an immunochromatography test device, especially for the new process, that comprises (I), an incubation system, an analytical region and a system for transporting the Ag-R1 complex; (2) monoclonal antibodies (MAb), their fragments or derivatives, that have a variable region comprising at least one of 24 specified CDRs (complementarity-determining regions), given in the specification; (3) an aptamer (i) that binds specifically to the same epitope as MAb; (4) an epitope (ii) that binds specifically to MAb or (i); (5) an antibody, or its fragments or derivatives, that bind specifically to (ii); and (6) a kit containing at least one test device of (1).

Description

In The description of this invention is a number of published Called documents. The subject matter of these documents is by reference incorporated into the description.

The The invention relates to a rapid immunochromatographic test, in particular a test strip for detecting infection of a mammal an acid resistance Microorganism. There is (a) a stool sample of the mammal under Use (aa) of a receptor incubated under conditions that a complex formation of an analyte or an antigen from the acid-resistant Allow microorganism with the receptor; or (ab) at least two different receptors incubated under conditions the complex formation of an analyte or antigen from the acid-resistant Allow microorganism with at least two receptors and wherein the receptor according to (aa) or the receptors according to (ab) an analyte or specifically binds / bind an antigen, the at least one Part of the mammals after the intestinal passage has a structure that is the native structure or corresponds to the structure against which a mammal after infection or immunization with the acid-resistant Microorganism or an extract or lysate thereof or a protein from or a fragment thereof or a synthetic peptide Antibody produced; and (b) the formation of at least one analyte or antigen receptor complex according to (a) proves. Preferably, the acid resistant Microorganism a bacterium, in particular Helicobacter pylori, Helicobacter hepaticus, Campylobacter jejuni or Mycobacterium tuberculosis. Further preferred is that the receptor / Receptors to an epitope / epitope of a catalase, urease or metalloproteinase binds. Furthermore, the invention relates to diagnostic compositions and test devices containing the aforementioned components and packaging containing them.

Of the Detection of the infection of a mammalian organism with a microbial pathogen or parasite today can affect different, invasive, semi-invasive or non-invasive species. All invasive methods require endoscopy and biopsy. Using these techniques will increase the physical integrity of the person being examined injured, e.g. when taking a biopsy. A withdrawal from Biopsy is laborious, causes high costs and usually means a big Burden for the patient. Because the infection with certain microorganisms, for example, with H. pylori not over the entire gastric mucosa must be distributed is by biopsy removal at a non-infected site false-negative result possible. Another disadvantage of all invasive methods is the influence results of previous treatment with proton pump inhibitors, Bismuth or antibiotics.

Semi-invasive or non-invasive diagnostic techniques are changing in parameters fixed without an intervention in the organism can be. For this purpose, body fluids are preferred and excretions such as serum, breath, urine, saliva, sweat or stool, sampled and analyzed.

Diagnostic techniques can be determined by the parameters proved in direct and indirect Divide methods. In direct methods, the presence the pathogen or parasite, its constituents or their degradation products by electron microscopy, optical characterization, mass spectrometry, Measurement of radioactive decay products or specific enzymatic Reactions detected. Often, however, these methods are high associated with equipment expenditure (for example breath test). Indirect procedures On the other hand, they rely on the detection of reactions of the host organism to the pathogen or parasite, e.g. the presence of antibodies against antigens of the pathogen in the serum or saliva of the host.

After this the intervention in the organism in invasive techniques for the organism in most cases stressful and frequent also with high expenditure on equipment as well as a health Risk associated with non-invasive techniques Comparatively simple detection of samples of the above body fluids and excretions are the method of choice Host in the same way to a particular pathogen or a particular The parasite reacts, delaying the host's reaction and also after removal of the pathogen or parasite can persist in the organism, direct procedures are always preferable. Ideally, therefore, a diagnosis is made by the non-invasive, direct detection of the pathogen or parasite in body fluids or eliminations created. This allows in contrast to indirect Procedure determining the current infection status.

In addition, a diagnostic procedure should be optimized for other aspects: high reproducibility, sensitivity and specificity, guaranteed availability of the materials to be used in constant quality, low production and implementation costs, and ease of use Dependent on complex equipment are the parameters to be considered here.

Out the reasons above take procedures based on the high selectivity and binding affinity of certain Substance classes (e.g., antibodies, Receptors, lectins, aptamers) for molecular structures which can be chosen to suit each to be determined substance are highly specific, in the medical Diagnostics an increasingly wide space. In particular the possibility the immobilization of these substances on solid surfaces and the coupling to radioactive nuclides, enzymes with suitable substrates color reactions trigger or to colored particles having the highly specific binding affinity (e.g. in the ELISA = enzyme-linked immunosorbent assay) led to the development of cost-effective, simple and less time-consuming detection method for the body's own like foreign substances.

In found the initial stages of development of these detection methods exclusively polyclonal antibodies Use. However, these have some, well known to those skilled in the art Disadvantages, especially limited availability and often cross-reactivity. The Development of Methods for the Production of Monoclonal Antibodies (Köhler & Milstein (1975)), the progress in the isolation of receptors and their directed Expression in cellular Host systems, the development of lectins with high affinity for certain carbohydrates and the discovery that single-stranded nucleic acid molecules (aptamers) could specifically bind molecular structures, these disadvantages largely remove. Today can be done with comparatively simple methods the specificity and sensitivity of detection methods.

by virtue of high specificity Such methods are particularly suitable for detecting individual, defined substances such as haptens, peptides or proteins, provided that the recognized structural element is constant within the one to be examined Sample population and specific for the substance to be detected. They are also for a measurement in body fluids well suited, and thus provide an obvious option for the direct Detection of pathogens in this sample matrix For example, in the prior art methods of diagnosis e.g. from Entamoeba histolytica (Haque (1993), J. Infect. Dis. 167: 247-9), enterohemorrhagic Escherichia coli (EHEC, Park (1996) J. Clin Microbiol 34: 988-990), Vibrio cholerae (Hasan (1994), FEMS Microbiol. Lett. 120: 143-148), torovirus-like Particles (Koopmans (1993), J. Clin. Microbiol. 31: 2738-2744) or Taenia saginata (Machnicka (1996) Appl. Parasitol 37: 106-110) Chair described.

The The pathogens described above have in common that they are present in the gut of their host, in all cases humans, are capable of life and reproduction. So you own Mechanisms that help you survive and the multiplication in the presence of active in the intestine degradation and Allow digestive systems. Thus it is probable that in the Excretion with the chair a high number of intact or almost intact Go away pathogens or parasites. With detection reagents, for example antibodies which recognize the intact pathogens or parasites, they can usually easily detected in stool or in prepared stool specimens become.

It However, there are a number of pathogens and parasites, on the one hand due to the relationships of the tissues affected by them (e.g., lung, Stomach, pancreas, duodenum, liver) to the gastrointestinal tract in the stool may occur, On the other hand, however, in the intestine itself are not viable and / or capable of reproduction.

To These pathogens and parasites include, for example, Helicobacter pylori (H. pylori) and Helicobacter hepatis, Mycobacterium tuberculosis and other mycobacteria, Chlamydia pneumoniae, Legionella pneumophilae, Pneumocystis carinii, and others. Other pathogens such as Legionella pneumophilae can be identified by antigens that are transmitted via the Kidney into the urine specifically detect. However, this succeeds only if the amount in the urine is sufficient for the proof. Of the Proof in the stool would be this is a welcome Alternative. However, intestinal transit is involved in these organisms a strong attack by the digestive and degradative mechanisms connected to the intestinal flora. For The molecular pathways specific to the pathogens considered can be destroyed or greatly reduced in their concentration.

Degradation of the pathogens in the gut has also proved to be a problem for other acid-resistant bacteria for safe detection in stool samples. The number of germs in the stomach of an infected person is low in comparison to other bacteria that colonize the intestine. In addition, after leaving the stomach, germs and germ fragments must travel a long way through the intestine, which is rich in proteases. These circumstances have the consequence that only small amounts of intact proteins can be found in the stool, whereby one can not assume that always the same fragments of certain proteins go through the intestinal tract unscathed. This also requires that for an ELISA or immunochromatic Quick combination of two epitopes on an antigen is no longer necessarily given as in the native protein, and closely spaced epitopes have the greatest likelihood of showing a positive result in a detection that requires two epitopes on the same molecule. Ideally, only one epitope on the same molecule is required for detection, and this epitope must be present twice in the case of a monomer. For example, in the case of a dimer, a single presence on each subunit would be sufficient. The individually different distribution of antigens detected in the stool of infected persons additionally indicates individual characteristics in the processing of the antigens during intestinal transit. A first approach to reducing this problem has been provided by the disclosure of EP-A-0 806 667. In this application it was demonstrated that polyclonal antibodies could be induced with the lysate of a particular H. pylori strain recognizing a broader variability of strains from different geographic regions. However, this application does not indicate which antigens are recognized by the serum. Given that immunity sera may vary despite all standardization efforts, the method developed in the above-mentioned application must be considered as sub-optimal for wide application. In addition, for the provision of the polyclonal sera always new animals must be immunized. The corresponding methods are both time-consuming and cost-intensive.

Ideally would be a safe detection of infection of an acid-resistant pathogenic as mentioned above Organism / parasites with a single or a limited number For this pathogenic organism / parasite specific reagent / reagents possible.

The EP 291 194 describes an analytical test device containing a porous support with a wet mobile specific binding reagent for an analyte and a permanently immobilized unlabelled specific binding reagent for the same analyte.

task Thus, the present invention is a corresponding simple one and inexpensive test provide.

These The object is achieved by the embodiments characterized in the claims solved.

The The invention relates to a method for detecting an infection of a mammal with an acid-resistant Microorganism, wherein (a) a stool sample of the mammal under Use (aa) of a receptor incubated under conditions that a complex formation of an antigen from the acid-resistant microorganism allow with the receptor; or (ab) at least two different ones Receptors incubated under conditions that cause complex formation an antigen from the acid-resistant Allow microorganism with at least two receptors and wherein the receptor according to (aa) or the receptors according to (ab) Specifically binds / binds antigen, at least in part of the mammal after the intestinal passage has a structure that is the native structure or corresponds to the structure against which a mammal after infection or immunization with the acid-resistant Microorganism or an extract or lysate thereof or a protein from or a fragment thereof or a synthetic peptide antibody produced; and (b) the formation of at least one antigen receptor complex according to (a) proves.

Of the Invention is also the idea underlying an immunochromatographic rapid test, such as to provide a test strip suitable for detection to deliver a named infection.

Of the Chair Quick Test Included a plurality of layers or regions, preferably from possibly different ones porous Materials exist. The test strip has a sample application area, the actual test carrier (Test or analysis area), and an absorber layer (absorption area) on. In a preferred embodiment the multiple layers or areas are fixed on a polyester carrier arranged. In the sample application area are in a preferred embodiment in the dried state the for the detection required specific immunological receptors or more preferably specific antibodies to the antigen. These are preferably with visible colored particles, e.g. colloidal gold or polystyrene (Latex) etc., marked.

The test carrier further preferably consists of a special test membrane such as nitrocellulose. On this test membrane, particularly preferred are further specific receptors which are directed against the antigen, immobilized as a test line. As a functional control, a further capture line, for example receptors or antibodies directed against the labeled receptor or antibody, can be immobilized on the test membrane. An absorber layer at the end of the test strip advantageously ensures that the sample flow is based on the capillary action of the porous materials in contact with one another is right.

In the method according to the invention In one embodiment, the sandwich immunoassay is in the sample application area of the test strip is a labeled first specific receptor for the analyte or the antigen, for example a labeled antibody (antibody conjugate) deposited or dried. As a test line is a second specific Receptor for immobilized the analyte or the antigen. During the test will be the first specific receptor such as an antibody conjugate through the sample fluid solved and over transports the test membrane. Is the specific analyte or the Antigen present in the sample, complexes form during the test run from labeled first receptor or antibody conjugate and antigen or Analyte off. At the capture line, this complex binds to the second specific receptor and forms there the so-called sandwich complex out. By the associated accumulation of the labeled receptors or antibody conjugates A visible test signal is generated at the test line. Is not an analyte or Antigen present in the sample, no sandwich complex is formed out, and there is no signal generation.

In a preferred embodiment The sandwich process is at the test line rather than a specific one Receptor streptavidin immobilized. The simple sandwich method as a catcher on the test line used specific receptor for the antigen is conjugated to biotin and together with a labeled specific The receptor is deposited in the sample application area of the test strip. In a particularly preferred embodiment is the label colloidal gold. During the test execution forms a sandwich complex, consisting of a labeled receptor, the antigen and the biotin-labeled Receptor already while the migration through the sample application area and the test membrane out and over at the test line the biotin-labeled specific receptor by the immobilized there Bound streptavidin.

In a further preferred embodiment is the gold-labeled specific receptor in a first conjugate region Sample area of the test strip and biotin-labeled specific receptor deposited in a second conjugate area of the sample application area.

In a further preferred embodiment The labeled first specific receptor can not be replaced by one labeled first specific receptor, for example antibodies, replaced become. This first specific receptor is then replaced by a another labeled receptor, this first specific receptor binds, detects, with the other labeled receptor the second specific receptor, which is immobilized as a test line, not binds.

In a further preferred embodiment may be the specific second receptor immobilized on the test line by a non-immobilized second specific receptor, For example, antibodies replaced become. The analyte-receptor complex is then replaced by one at the Test line immobilized receptor, this non-immobilized second specific receptor binds, the other being bound immobilized receptor the first labeled specific receptor does not bind.

In a particularly preferred embodiment the non-immobilized second specific receptor is bound at the receptor immobilized on the test line.

at Use of antibodies This can be done, for example, that the first and second specific antibodies come from different species.

For example The first specific non-labeled receptor is a mouse antibody that second specific receptor one rabbit antibody and the other labeled Receptor an anti-mouse antibody.

In a particularly preferred embodiment is the specific unlabeled receptor in a first range of conjugate the sample application area of the test strip and the labeled receptor, which binds the specific unlabeled receptor, in one second conjugate area of the sample application area of the test strip landfilled.

In In a further particularly preferred embodiment, the first conjugate region is in the sample application region in the flow direction before or above the second conjugate region.

Particularly suitable for the realization of the present invention is a rapid immunochromatographic test. This test is a dry reagent test, with all the specific ones required for analysis Reagents are contained in a test strip composed of a plurality of porous materials, preferably in the dried state. Such a test is based on the principle that the analysis is started by adding a liquid sample and the sample liquid migrates due to capillary forces through a test strip composed of several porous materials. During the migration of the sample liquid, specific binding reagents are dissolved and complex formation occurs between analyte contained in the sample and specific binding reagents. The complexes of analyte and specific binding reagents are trapped at a defined zone, which is preferably designed as a test line, by specific binding reagent immobilized at this test site. These trapped complexes are formed by the accumulation of visible particles coupled to the binding reagents, e.g. For example, colored polystyrene (latex), colloidal gold, etc. are visualized. Thus, the test according to the invention is also feasible for non-professionals. In addition, it allows easy and hygienic handling. In the implementation, only one step is required in the particularly preferred embodiments, namely the application of a sample. The visually evaluable result is then available very quickly within a few minutes (2-30 minutes).

In another embodiment is the immunochromatographic according to the invention Quick test in the test device described in WO98 / 58587 used. This combination allows fast and easy sample taking, preparation and analysis.

In addition avoids the rapid test according to the invention in the analysis of stool samples the previously existing disadvantage, that the dissolved in the buffer Sample has a high solids content, which is the sample flow through the fine porous Prevent or complicate materials of the test strip.

Furthermore can the disadvantages of the prior art are avoided stool samples heavily diluted Need to become. Also need when performing the method according to the invention stool samples diluted in the range of 1: 5 to 1:20 before the test not centrifuged and from larger solid Components are released. In addition, the present invention enables to provide a highly sensitive test for laboratory-independent use.

Especially by shifting the preferred biotinylated receptor from The test line in the application area will both have a greatly extended incubation period for the Binding of the capture antibody (second Receptor) to the antigen (analyte) as well as a higher affinity of the binding Streptavidin / biotin in the ratio for binding antibody / antigen and thus achieve a better binding kinetics at the test line, although the test strips remain nearly unchanged in their dimensions can.

Of the Term "acid-resistant Microorganism "im According to this invention every microorganism, due to its properties / adaptation mechanisms to the host the physical and chemical influences of the Digestive tract resists, so that it by a preferably immunological detection or detectable using aptamers is. examples for such acid-resistant Microorganisms are Helicobacter pylori, Helicobacter hepaticum, Mycobacterium fuberculosis, Mycobacterium pseudotuberculosis and Mycobacterium cansassii.

For the purposes of this invention, the term "stool sample of the mammal" means any stool sample that can be used for the detection method according to the invention. In particular, stool specimens which have been prepared according to methods known per se for diagnostic tests are included. The treatment is carried out for example in accordance RIDASCREEN ^® Entamoeba enzyme immunoassay (R-Biopharm GmbH, Darmstadt).

"Conditions that allow a complex formation "are easily adjustable by the skilled person; s. also Harlow and Lane, a.a.O., and are physiological conditions, for example.

The term "after the intestinal passage has a structure which corresponds to the native structure" in the sense of this invention means that the epitope of an antigen after intestinal passage is recognized by a receptor, for example a monoclonal antibody, derivative or fragment thereof or the aptamer, the / which was obtained from or binds to the same antigen / epitope that did not pass through the intestinal passage. In other words, the epitope / antigen specifically bound by the above-mentioned receptor has survived the intestinal transit in terms of its structure unscathed or substantially undamaged and has not been degraded. As a source for the native structure of the epitope / antigen, for example, a bacterial extract digested with a French press, which can be prepared by conventional methods (cf., eg, Sambrook et al., "Molecular Cloning, A Laboratory Manual", 2nd edition 1989, CSH Press , Cold Spring Harbor, USA) or a bacterial lysate which has been further purified by standard methods (eg Sambrook et al., supra).

Of the Term "after the Intestinal passage has a structure that corresponds to the structure, against a mammal after infection or immunization with the acid-resistant microorganism or an extract or lysate thereof or a protein thereof or a fragment thereof or a synthetic peptide produces antibodies "means according to the invention, that of the Receptor recognized epitope corresponds to an epitope, that of the immune system of a mammal, preferably of a human, presented becomes. The mechanisms of antigen presentation as well as mechanisms that lead to the processing of antigens and the resulting antibody diversity are known in the art and, for example, in Janeway and Travers, Immunology, 2nd edition 1997, Spektrum Akademischer Verlag GmbH, Heidelberg. These epitopes can become differ from the native epitopes. The contact of the mammal with the microorganisms or the proteins or fragments or the Synthetic peptides may be due to natural infection (except at the synthetic peptides) or by immunization. For immunization can also extracts, lysates, synthetic peptides etc. of the microorganism / protein be used. Suitable immunization schemes are available known in the art and described for example in Harlow and Lane, a.a.O. Suitable antibodies can for example by immunization and / or screening for surrogates such as synthetic peptides, recombinantly produced proteins, extracts, Lysates or partially digested proteins are obtained.

"Synthetic peptides" include those Peptides containing at least one epitope of the native or through the intestine passaged antigen. The peptides can have the same primary structure as the antigen or fragments thereof. You can, however also another primary structure (primary Amino acid sequence e.g. conservative exchanges).

Of the Term "specific binds "means according to the invention, that the receptor no or substantially no reactivity with other epitopes in samples uninfected mammal having. Usually the receptor binds only to an epitope of an antigen that is in the Stool sample occurs.

Of the Term "immune complex" as used in the sense of the invention complexes comprising monoclonal and / or polyclonal Antibody.

So can in this embodiment The invention, a processed stool sample, for example via a Scavenger receptor be bound to a solid phase and the infecting agent with be detected in the labeled form receptor. Provided that the antigen present after the intestinal passage is (still) in (homo) is present in di- or multimeric form, the same receptor can both as a catcher as well as used as a detector.

From Meaning is for the inventive method Furthermore, that for a successful detection of only one epitope of an antigenic protein in the must be substantially consistent after intestinal transit detectable. This Epitope can also occur several times on a homo dimer or multimer. The probability that this Epitope is found in detectable form, but is essential higher, as if a proof test on more than one epitope to be detected must build up.

Finally brings the method according to the invention, that only needs one receptor, Cost and standardization benefits with it.

Based on the surprising invention Findings that certain Antigens from the mentioned microorganisms after intestinal transit have a substantially consistently detectable epitope structure, must also a second embodiment as essential for the invention applies. This embodiment is based on the fact that different Bind receptors to different epitopes of the same antigen. Of the The term "essentially" means that this Epitope / epitopes and thus a corresponding infection with the Microorganism at more than 60 to 70%, preferably at least 75%, stronger preferably more than 85%, more preferably more than 90%, even more preferably more than 95%, and most preferably more than 98% of those affected can / can. Ideally, infections are detected in 100% of those affected.

Surprisingly, it has been found according to the invention that with a single receptor which specifically binds an epitope of an antigen of an acid-resistant microorganism, or two receptors which specifically bind two epitopes of the same antigen, a relatively reliable diagnosis of the infection with these bacteria / pathogens can be carried out , The invention includes embodiments in which other epitopes having the aforementioned properties, recognized by other receptors, such as monoclonal antibodies or fragments or derivatives thereof or aptamers. The latter embodiments are suitable for further increasing the safety in the position of the diagnosis. These other receptors may advantageously be antibodies, fragments or derivatives which specifically recognize urease, preferably β-urease, the 26 kD protein or Hsp 60, all preferably from H. pylori. The detection of one or more of these proteins / protein fragments can be performed in the same test and in an independent test with another part of the same sample.

The results according to the invention are especially surprising, therefore since the prior art had taught away from this. So for example found in H. pylori that main antigens not the desired one in ELISA tests specificity and sensitivity exhibit; see. Newell et al., Serodiag. Immunother. Infect. Dis. 3 (1989), 1-6. About that In addition, EP-A 0 806 667 teaches that reliable detection of H. pylori infections due to receptors such as monoclonal antibodies genetic variability H. pylori strains not possible be.

The inventive method is opposite the mentioned State of the art particularly advantageous because with only a receptor a relatively safe diagnosis is possible. Preferably for the Detection, for example in ELISA or rapid test pairs of receptors, like antibodies, fragments, Derivatives thereof or aptamers used, the two receptors of the same or different epitopes on the same pair Bind antigen. For example, H. pylori catalase forms multimers Structures of several equal subunits. In the ELISA, quick test or other assays thus the same receptors as catcher receptors as well as Detection receptors are used.

Preferably may be due to a combination of different mAbs that are different Epitopes of catalase are directed or by a combination two detection systems for various antigens (e.g., catalase / urease) increase sensitivity and specificity be achieved.

One Another advantage of the method according to the invention is its design as a direct and non-invasive procedure, what is stated in the introduction Amenities for the patient as well as the reliability increased in determining the stage of the disease.

preferred embodiments The invention will now be described by way of example with reference to the figures. Show it:

1 Cloned DNA sequence encoding the heavy chain V region of a catalase-specific monoclonal antibody (HP25.2m / 2H10).

2 Cloned DNA sequence encoding the light chain V region of a catalase-specific monoclonal antibody (HP25.2m / 2H10).

3 Cloned DNA sequence encoding the heavy chain V region of a catalase-specific monoclonal antibody (HP25.6m / 1B5).

4 Cloned DNA sequence encoding the light chain V region of a catalase-specific monoclonal antibody (HP25.6m / 1B5).

5 Cloned DNA sequence encoding the light chain V region of a β-urease specific monoclonal antibody.

6 Cloned DNA sequence encoding the heavy chain V region of a β-urease specific monoclonal antibody.

7 Cloned DNA sequence encoding the light chain V region of another β-urease specific monoclonal antibody.

8th Cloned DNA sequence encoding the heavy chain V region of another β-urease specific monoclonal antibody.

9 Structure of a preferred test strip according to the invention.

10 Construction of a preferred test strip according to the invention with control line.

The chair rapid test according to the invention 10 consists of a sample order area ( 1 . 2 ), a test area (test membrane) ( 3 ) and an absorption area ( 4 ).

In a preferred embodiment, the sample application area ( 1 ) of two superimposed conjugate areas. The upper conjugate region or the conjugate nonwoven preferably contains the specific receptor labeled, for example, with gold. The conjugate region or conjugate web (not shown) underneath contains, for example, the biotin-labeled specific receptor.

One good sample flow and even distribution of immunoreagents in the sample suspension during of the test procedure are for the sensitivity of an immunochromatographic rapid test of great importance. Both parameters are mainly used by the properties of the porous Materials, as well as their dimensions and mutual arrangement affected.

at Quick tests for the examination of stool specimens are also the Requirement in the sample solution contained solids before the transfer to filter efficiently on the test membrane.

The sample application area of the test is made of a conjugate fleece ( 1 ) and a subsequent filter ( 2 ). Through the filter, the solids content of the stool suspension, which would prevent a sample flow through the test membrane, separated. The test membranes, with which a high sensitivity is to reach, have a distribution of pore sizes in the range between 2 and 20 .mu.m, preferably 5 .mu.m. The filter has an exclusion size of 1-2 μm. For this purpose, materials made of glass fiber or polyester-glass fiber mixtures are particularly suitable. Also suitable are mixtures of natural and synthetic fibers that have been developed for blood separation.

The following Filters are suitable for example: Whatman GF / A, GF / B, GF / C, GF / D, F145, F147, F487, GF / DVA; Ahlstrom 111, 141, 142, 151, 164; Pall A / B, A / C, A / D, A / E, A / F. For the conjugate fleece is made of open-pore fiberglass material, Polyester or polypropylene such as Whatman F 075-14; Schleicher and Schuell GF 10, GF33, GF 53, Accuflow P, Accuflow G; Ahlstrom 8980, 2033, 2040, 8975; Millipore QR 01, QR 35, QR 51, QR61.

The Konjugatvlies serves in this structure in addition to its function as a conjugate carrier layer as a pre-filter, the coarse solids of the suspended stool sample holds back before they hit the filter. It is also advantageous in this structure, that sample liquid and solved Conjugate together through the downstream filter and flow so that the incubation time of analyte or antigen and conjugate is prolonged.

The Dimensions of conjugate fleece, filter and test membrane and as well the transitions between The materials of the sample application area are designed so that a sufficient Filtering the stool suspension is achieved.

During the Sample application is allowed the sample liquid only in contact with the conjugate fleece, but not with the filter reach.

Of the inventive structure guaranteed that one sufficient filter surface in contact with the sample fluid flowing through the conjugate web can come. Furthermore possible the structure of the invention a minimum distance between conjugate fleece and test membrane to a trespassing of insufficiently filtered sample fluid on the test membrane excluded.

The dimensions of the individual areas or materials correspond to the following information:
The preferred width of the test strip is between 3 mm and 10 mm, particularly preferably 5 mm. The preferred length of the test strip is 50-100 mm, more preferably 75 mm. The preferred length of the conjugate fleece is in the range of 5-30 mm, more preferably 25 mm. The overlap of conjugate fleece and filter is in this case preferably between 5 and 15 mm, particularly preferably 10 mm. Preferred are filters of length between 10 and 20 mm, more preferably 15 mm. The overlap between filter and test membrane is preferably between 1 and 3 mm, more preferably 1 mm.

The absorber layer at the end of the test strip ( 4 ) according to the invention has both a sufficient capacity for receiving the sample liquid migrated through the test strip, as well as a relative fine-pored structure to maintain the capillary action. Cellulose glass fiber materials such as Whatman 17 CHR, 3MM, 31 ET, WF1.5, D28, CD 427-05, CD 427-07, CD 427-08; Schleicher & Schuell 2992, GB 003, GB 004; Pall 111, 113, 133, 165, 197; Ahlstrom 320, 222, 238, 237. Particularly preferred for test strips with a width of 5 mm, a dimension of 10-30 mm in length for the analysis area. The overlap of the test membrane on the absorber layer is preferably at least 1 mm.

Especially Preferably, the rapid test of the invention finds use in one Apparatus for receiving and analyzing samples, as in WO 98/58587 (PCT / EP98 / 03764). The object This document is incorporated by reference into the specification.

In a preferred embodiment is the acid-resistant An acid-resistant microorganism Bacterium.

in the The prior art is known as a series of acid-resistant bacteria. In a particularly preferred embodiment, this is acid-resistant Bacterium a bacterium of the genus Helicobacter, Campylobacter or the genus Mycobacterium.

In Another particularly preferred embodiment is the bacterium a bacterium of the species Helicobacter pylori, Helicobacter hepaticum, Campylobacter jejuni or a bacterium of the species Mycobacterium tuberculosis.

In a further preferred embodiment is the receptor / are the receptor (s) antibodies, (a) fragments) or (a) derivatives thereof or aptamer (s).

Of the Term "receptor" as used in the sense However, the present invention also other binding partners such as for example, avidin, streptavidin or polystreptavidin and biotin.

"Fragments" or "derivatives" of monoclonal antibodies for the purposes of this invention have the same binding specificity as the monoclonal antibodies. Such fragments or derivatives can be prepared by conventional methods; see. eg Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, USA, 1988. Examples of fragments are Fab, F (ab ') ₂ or Fv fragments. Examples of derivatives are scFv fragments. Derivatives may also be chemically synthesized substances that have the same or improved binding properties as the antibodies. Such substances can be prepared, for example, by peptidomimetics or by various rounds of phage display and subsequent selection for improved binding properties. According to the invention, aptamers are understood as meaning nucleic acids such as RNA, ssDNA (ss = single-stranded), modified RNA or modified ssDNA, which bind a large number of target sequences with high specificity and affinity. The term "aptamer" is known in the art and defines, for example, in Osborne et al., Curr. Opin. Chem. Biol. 1 (1997), 5-9, or in Stull and Szoka, Pharm. Res. 12 (1995), 465-483.

Of the Term "antigen-antibody complex" in the sense of this Invention not just complexes that consume the antigen with the native antibody, but also those who have it with its fragments or derivatives received.

Covered by the Invention are embodiments, where only monoclonal antibodies or fragments or derivatives thereof or only aptamers used be, as well as embodiments, in which in a test different types of detection reagents be used. So it is possible the existence first monoclonal antibody with a second antibody derivative or a first aptamer with a second antibody fragment is used, to name just two examples. In this respect, the terms "first" and "second" refer to the first and second Detection reagent. It does not mean that there are always two antibodies, derivatives or fragments thereof or always two aptamers are used.

The Use of monoclonal antibodies, fragments or derivatives granted or by aptamers an easy-to-maintain standard of reliability of the diagnostic procedure, which is a big advantage compared to previously known and for introduced this purpose Diagnostic procedure is. Furthermore, this is omitted, for example, in the process EP-A 0 806 667 always necessary new immunizing and subsequent Testing of experimental animals.

In a further preferred embodiment, the antigen is the antigen of a catalase. The kata Lase has the particular advantage that it could be detected in all previously known acid-resistant bacteria. According to the invention, it could be determined as a further advantage that the catalase is very resistant to digestion in the intestinal tract, which simplifies the detection of significant amounts. Finally, the catalase or fragments thereof, even after the passage of the intestine, often has a more ordered structure, for example in tetrameric form, which facilitates detection with only one receptor type.

Surprisingly was found according to the invention, that in a population of mammals, in particular of human patients whose stool is infected with acid-resistant Bacteria was tested, essentially all members of this Consistently exhibited recurring catalase epitopes in stool, so that with high probability with only one corresponding receptor, preferably monoclonal antibodies, Fragments or derivatives thereof or aptamers a relatively safe Diagnosis can be made. In particular, since the catalase a Having tetrameric antigenic structure, this diagnosis may advantageously For example, in ELISA, rapid immunochromatographic test or in similar arranged fixed system.

Especially it is preferred that the Catalase is the catalase of H. pylori.

In a further preferred embodiment the antigen is a metalloproteinase, more preferably the Metalloproteinase from H. pylori.

In another preferred embodiment the antigen is a urease, preferably from H. pylori.

In a further preferred embodiment is for the proof in addition used a mixture of receptors, the mixture of receptors as a catcher the antigen acts when the receptor acts as a detector of the antigen is used and the mixture acts as a detector of the antigen, if the receptor as a catcher of the antigen is used. For the proof can both different mixtures of receptors as catcher and Detector of the antigen as well as the same mixture as catcher and Detector of the antigen can be used. When using the same Mixture as a catcher and detector of the antigen, the capture is preferably in labeled Form and the detector immobilizes on the test line.

These embodiment The invention allows a particularly reliable diagnosis, in particular if the antigen is not in dimeric or multimeric conformation the intestinal passage is present. This embodiment allows only one the two in the most standardized immunological detection methods used receptor types is a monoclonal antibody, while for example, the second type of receptor may be a polyclonal serum can.

In a particularly preferred embodiment the mixture of receptors is a polyclonal antiserum.

In Another particularly preferred embodiment will be for the evidence Mixtures of receptors used, being a mixture of receptors as a catcher the antigen acts and a mixture of receptors as a detector of the antigen, and preferably at least one mixture polyclonal serum is.

In Another particularly preferred embodiment is a mixture of receptors both as catchers as well as a detector of the antigen, preferably this mixture is a polyclonal antiserum.

In one additional particularly preferred embodiment was the polyclonal antiserum against a lysate of the microorganism, preferably H. pylori.

In In another particularly preferred embodiment, the lysate is a Lysate with enriched antigen.

In another preferred embodiment the lysate is a lysate with depleted immunodominant antigen.

The aforementioned two embodiments also include that the lysate is a lysate of enriched antigen, preferably enriched catalase, as well as depleted immunodominant antigen, preferably major antigenic urease. In particular, the combination mentioned leaves a good and for the method according to the invention particularly suitable immunization yield. One way of carrying out corresponding enrichment or depletion processes is described in more detail in the examples.

According to one Another particularly preferred embodiment was the polyclonal Antiserum against a purified or a (semi) synthetically produced Antigen, preferably a catalase, metalloproteinase or urease enzyme, preferably from H. pylori.

The Receptors, preferably the monoclonal antibodies, fragments or derivatives of it or the aptamers can according to the invention linear or recognize conformational epitopes and bind specifically. In a another preferred embodiment At least one of the receptors binds to a conformational epitope.

In a particularly preferred embodiment tie all Receptors conformational epitopes.

In a particularly preferred embodiment, the heavy chain of the catalase epitope-binding antibody [HP25.2m / 2H10] has at least one of the following CDRs, preferably the CDR3 and more preferably all three of the following CDRs:
CDR1: NYWIH
CDR2: YINPATGSTSYNQDFQD
CDR3: EGYDGFDS

In a further particularly preferred embodiment, the DNA sequence encoding the heavy chain of the catalase epitope-binding antibody [HP25.2m / 2H10] has at least one of the following CDRs, preferably the CDR3 and more preferably all three of the following CDRs:
CDR1: AACTACTGGATTCAC
CDR2: TACATTAATC CTGCCACTGG TTCCACTTCT TACAATCAGG ACTTTCAGGA C
CDR3: GAGGGGTACG ACGGGTTTGA CTCC

In another particularly preferred embodiment, the light chain of the catalase epitope-binding antibody [HP25.2m / 2H10] comprises at least one of the following CDRs, preferably the CDR3, and more preferably all three of the following CDRs:
CDR1: SASSSVNYMY
CDR2: DTS CLASS
CDR3. QQWSSNPYT

Furthermore, in a particularly preferred embodiment, the DNA sequence encoding the light chain of the catalase epitope-binding antibody (HP25.2m / 2H10) comprises at least one of the following CDRs, preferably the CDR3 and more preferably all three of the following CDRs:
CDR1: AGTGCCAGCT CAAGTGTAAA TTACATGTAC
CDR2: GACACATCCA AATTGGCTTC T
CDR3: CAGCAGTGGA GTAGTAATCC GTACACG

In a particularly preferred embodiment, the heavy chain of the catalase epitope-binding antibody [HP25.6m / 1B5] has at least one of the following CDRs, preferably the CDR3 and more preferably all three of the following CDRs:
CDR1: DTYVH
CDR2: KIDPANGKTKYDPIFQA
CDR3: PIYYASSWFAY

In a further particularly preferred embodiment, the DNA sequence encoding the heavy chain of the catalase epitope-binding antibody [HP25.6m / 1B5] comprises at least one of the following CDRs, preferably the CDR3 and more preferably all three of the following CDRs:
CDR1: GACACCTATGTGCAC
CDR2: AAGATTGATCCTGCGAATGGTAAAACTAAATATGACCCGATATTC CAGGCC
CDR3: CCCATTTATTACGCTAGTTCCTGGTTTGCTTAC

In another particularly preferred embodiment, the light chain of the catalase epitope-binding antibody [HP25.6m / 1B5] comprises at least one of the following CDRs, preferably the CDR3, and more preferably all three of the following CDRs:
CDR1: KASQDVGTSVA
CDR2: WTSTRHT
CDR3: QQYSSSPT

Furthermore, in a particularly preferred embodiment, the DNA sequence encoding the light chain of the catalase epitope-binding antibody [HP25.6m / 1B5] comprises at least one of the following CDRs, preferably the CDR3 and more preferably all three of the following CDRs:
CDR1: AAGGCCAGTCAGGATGTGGGTACTTCTGTTGCC
CDR2: TGGACATCCACCCGGCACACT
CDR3: CAGCAATATAGCAGCTCTCCCACG

In another preferred embodiment is the β-urease specific antibody that of one of the German collection of microorganisms and cell cultures (DSMZ) on June 23, 1998 according to the statutes of the Budapest Under the accession numbers DSM ACC2360 or DSM ACC2362 Hybridomas HP8m / 4H5-D4-C9 or HP9.1m / 3C2-F8-E2 produced antibodies. Of the β-urease-specific in the figures antibody HP8m / 1H5-G2-B4 is produced by a daughter clone of the deposited hybridoma HP8m / 4N5-D4-C9 produced. The two by mother and daughter clone produced antibodies are encoded by identical DNA sequences and have the same Properties on.

In this other particularly preferred embodiment of the method according to the invention, the heavy chain of the antibody binding an epitope of the β-urease has at least one of the following CDRs, preferably the CDR3 and more preferably all three of the following CDRs:
CDR1: GFTFSSHFMS
CDR2: SISSGGDSFYPDSLKG
CDR3: DYSWYALDY
or:
CDR1: GYAFSTSWMN
CDR2: RIYPGDGDTNYNGKFKG
CDR3: EDAYYSNPYSLDY

In a further particularly preferred embodiment, the DNA sequence coding for the heavy chain of the antibody binding an epitope of the β-urease comprises at least one of the following CDRs, preferably the CDR3 and more preferably all three following CDRs:
CDR1: GG CTACGCATTC AGTACCTCCT GGATGAAC
CDR2: CGGATTTATC CTGGAGATGG AGATACTAAC TACAATGGGA AGTTCAAGGG C
CDR3: GAG GATGCCTATT ATAGTAACCC CTATAGTTTG GACTAC
or:
CDR1: GG ATTCACTTTC AGTAGCCATT TCATGTCT
CDR2: TCCATTAGTA GTGGTGGTGA CAGTTTCTAT CCAGACAGTC TGAAGGGC
CDR3: GACTAC TCTTGGTATG CTTTGGACTA C

In another particularly preferred embodiment of the method according to the invention, the light chain of the antibody binding to an epitope of the β-urease comprises at least one of the following CDRs, preferably the CDR3 and more preferably all three of the following CDRs:
CDR1: RASQSIGTRIH
CDR2: YGSESIS
CDR3: QQSNTWPLT
or:
CDR1: HASQNINVWLS
CDR2: KASNLHT
CDR3: QQGRSYPLT

Furthermore, the DNA sequence encoding the light chain of this antibody preferably has the following CDRs:
CDR1: A GGGCCAGTCA GAGCATTGGC ACAAGAATAC AC
CDR2: TAT GGTTCTGAGT CTATCTCT
CDR3: CAACAA AGTAATACCT GGCCGCTCAC G
or:
CDR1: C ATGCCAGTCA GAACATTAAT GTTTCGTTAA GC
CDR2: AAG GCTTCCAACT TGCACACA
CDR3: CAACAG GGTCGAAGTT ATCCTCTCAC G

It is further particularly preferred that the heavy and light chains having the above CDRs occur together in an antibody, fragment or derivative thereof, the catalase or the β-urease or a fragment thereof, preferably from H. pylori specifically binds. However, the invention also includes embodiments in which these heavy or light chains are combined with other light or heavy chains, wherein the bonding properties can be substantially maintained or improved. Corresponding methods are known in the art. Particularly preferred antibodies have in the variable regions of the light and heavy chains in the 1 and 2 . 3 and 4 . 5 and 6 respectively. 7 and 8th shown on or the regions are encoded by the DNA sequences shown there. The CDRs can be integrated into various FRs ("framework regions") by methods known in the art.

In a preferred embodiment Following with the stool sample before incubation with the antibodies Steps performed: The stool sample is 1: 3 to 1:25, preferably about 1:15 in one Sample buffer suspended. Such Probenpufter are in the state of Technique known. An exemplary sample buffer contains 150 mM PBS, 0.1% SDS.

In a preferred embodiment the sample buffer consists of 150 mM PBS, 0.5% serum and 2% detergent. Here, the serum from bovine, mouse, rat, pig or human and the detergent is selected from the group of ionic (preferably Tween 20) or nonionic detergents (preferably SDS).

In another embodiment, the detection according to the invention can also be used for the detection of H. pylori in gastric gases, respiratory condensate, saliva, dental plaque, mucosal smears, biopsies, whole blood or serum. The recovery of respiratory gases may be accomplished by administering to the patient cold CO ₂ -containing drinks which cause a release of gastric gases in the form of "burps". These gases can be collected in suitable containers or obtained in accordance with respiratory condensate in the manner known to the skilled person, for example by means of a Vorrichtung according to DE 19718925 or a device according to DE 19505504 , The condensates thus obtained can then be introduced into the test according to the invention in liquid form, all steps of the method according to the invention being described above, with the exception that a sample as described here is used instead of a stool sample. Dental plaque and mucosal swabs are obtained according to the methods known in the art and can be used as saliva, whole blood and serum in a suitable concentration and modification of the resuspension buffer in the detection according to the invention.

In a further preferred embodiment the detection of the formation of the at least one antigen receptor complex / antigen receptor / receptor mixture complex in step (b) by means of an immunochromatographic method.

In a particularly preferred embodiment the method according to the invention becomes the same receptor for binding in the immunological procedure used for the solid phase as well as for the detection of the epitope. During the scavenger receptor in unmodified form to a solid phase, for example nitrocellulose, can be bound is the receptor used for detection optionally provided with a marker.

In a further embodiment may be the capture receptor in biotinylated form and immobilized on the solid phase Streptavidin can be bound to these.

on the other hand may be the capture receptor not biotin-labeled and the epitope of the microorganism, preferably the bacterial epitope, over a third biotin-labeled receptor can be detected, wherein this receptor is preferably an antibody, fragment or derivative of which or is an aptamer, or a species-specific or Ig-class specific antibody or a corresponding aptamer can be. This third biotin-labeled Receptor specifically binds the capture receptor, and the analyte-receptor complex will be on the test line used in this embodiment consists of immobilized streptavidin, via the third biotin-labeled Bound receptor.

As a marker for the receptor used for detection, colloidal gold, selenium, colored polystyrene or latex particles, carbon particles and (known in the art from the prior art) Disperse colors are used.

on the other hand can, as mentioned above, the receptor used for detection also not marked and thus the epitope of the microorganism also over one third labeled receptor to be detected against this unmarked receptor, this receptor being preferred an antibody, Fragment or derivative thereof or an aptamer is a species-specific or Ig-class specific antibody or a corresponding one May be aptamer.

In a particularly preferred embodiment is the label colloidal gold.

such Markers are known in the art; see. for example Harlow and Lane a.a.O. The same applies to aptamers. The above described embodiment is particularly favorable for the detection of catalase, possibly even after the intestinal transit yet is present as a tetramer. Of course, also in this embodiment Combinations of antibodies, Fragments, derivatives and aptamers, e.g. combinations of antibodies etc. that bind to different epitopes of the same antigen.

In another preferred embodiment the method according to the invention is the monoclonal antibody a mouse antibody.

Of Further, in a preferred embodiment, the receptors are on a carrier fixed.

The Fixation of the receptors, preferably the antibodies, fragments or derivatives thereof or the aptamer to a carrier is particularly advantageous for the execution from routine checks. The combination antibody carrier / aptamer carrier let yourself Furthermore, pack well as a test kit or in kit form.

In a particularly preferred embodiment is the carrier material a porous one Support material.

In Another particularly preferred embodiment is the carrier material a test strip.

In addition exists in a preferred embodiment the carrier material cellulose or a cellulose derivative.

Of the Mammal, its stool, stomach gas, breath condensate, saliva, dental plaque, mucosal swab, biopsy, Whole blood or serum can be examined by the method according to the invention can, can an animal, for example, a pet like a cat or a dog, a farm animal, e.g. a pig or other animal like a mouse, a tiger, a gerbil or a ferret.

In a preferred embodiment is the mammal a human.

In a further preferred embodiment is the inventive method an automated process. An automated procedure can be performed for example by means of a robot, wherein the robot a part or all Performs method steps. Corresponding robots are known in the art.

Furthermore The invention relates to a monoclonal antibody, fragment or derivative of which has a V-region which is a combination of the having CDRs shown above or one of the above produced hybridomas is produced.

Preference is given to monoclonal antibodies, fragments or derivatives thereof, at least one of the in the 1 and 2 . 3 and 4 . 5 and 6 respectively. 7 and 8th have shown V regions. Preferably, these antibodies have two of the 1 and 2 . 3 and 4 . 5 and 6 respectively. 7 and 8th illustrated V regions. It is also preferred that these V-regions of the in the 1 and 2 . 3 and 4 . 5 and 6 respectively. 7 and 8th Coded DNA sequences are encoded.

In a particularly preferred embodiment of the invention, the monoclonal antibody, the fragment or derivative thereof is a mouse antibody or a fragment or derivative thereof or a chimeric, preferably a humanized antibody or a fragment or derivative thereof. The derivative may also be a fusion protein. It is further preferred that the antibody is labeled, for example with a colloid, with a mark consisting of gold, selenium, latex, colored polystyrene, carbon particles known to those skilled disperse colors.

The Production of chimaerized, humanized and human antibodies and the other derivatives are well known in the art (e.g. Vaughan et al., 1998; Orlandi et al., 1989, Harlow and Lane, supra).

The The invention also relates to an aptamer having the same epitope as the monoclonal antibodies, specifically binds the fragment or derivative thereof. The production Such aptamers can be prepared by methods known in the art respectively.

Farther For example, the invention relates to an epitope derived from any of the above described monoclonal antibody, Fragment or derivative thereof or aptamer is specifically bound.

Furthermore For example, the invention relates to other antibodies, derivatives or fragments of which, the epitope according to the invention bind specifically. These antibodies can for example monoclonal antibodies, those using the epitope as hapten / component of an antigen according to usual Process can be produced.

The The present invention further relates to a diagnostic Composition containing at least one receptor, preferably at least one monoclonal antibody, fragments or derivatives thereof or aptamers as defined above, fixed to a support material.

Of Furthermore, the present invention relates to a test device to detect at least one epitope as defined above, comprising (a) at least one receptor, preferably a monoclonal Antibody, Fragments or derivatives thereof or an aptamer is as above defined, fixed to a substrate; (b) a device for the preparation and analysis of stool samples; and optionally (c) a mixture of receptors as above Are defined.

As mentioned above, the invention further relates to a device for processing and analysis of stool samples as described in WO 98/58587. These Device includes sample collection, preparation and test unit (Test strips) in one device.

The The invention further relates to the subject matter containing a test device (a) at least one receptor, preferably a monoclonal antibody, fragments or derivatives thereof or an aptamer as defined above, wherein the receptor is conjugated with colloidal gold, polystyrene (Latex) or other coloring particles whose size is typical in the range between 5nm and 100nm, preferably between 40nm and 60 nm (particularly preferred is the particle size of 40 nm to 60nm for Gold and a particle size of 200nm and 500nm for Latex); (b) a device for the preparation and analysis of stool samples, as described for example in WO 98/58587; and optionally (c) a mixture of receptors as defined above.

alternative for devices for the preparation and analysis of stool samples, the Compositions and kits also reprocessing devices (as far as required) and analysis of gastric gases, respiratory condensate, saliva, Dental plaque, mucosal smears, biopsies, whole blood or serum exhibit.

Furthermore The present invention relates to a kit containing (a) at least a receptor, preferably a monoclonal antibody, fragments or derivatives thereof or an aptamer as defined above, fixed to a support material; optionally also (b) an apparatus for conditioning and Analysis of stool samples as described for example in WO 98/58587; and optionally (c) a mixture of receptors as above Are defined.

The Figures show:

1 : Cloned DNA sequence encoding the heavy chain V region of a catalase-specific monoclonal antibody [HP25.2m / 2H10]. The encoded amino acid sequence is shown in single letter code. The Kabat et al. certain CDR regions 1-3 are underlined.

2 : Cloned DNA sequence specific for the V region of a catalase specific light chain monoclonal antibody [HP25.2m / 2H10). The encoded amino acid sequence is shown in single letter code. The Kabat et al. certain CDR regions 1-3 are underlined.

3 : Cloned DNA sequence encoding the heavy chain V region of a catalase-specific monoclonal antibody [HP25.6m / 1B5]. The encoded amino acid sequence is shown in single letter code. The Kabat et al. certain CDR regions 1-3 are underlined.

4 : Cloned DNA sequence encoding the light chain V region of a catalase-specific monoclonal antibody [HP25.6m / 1B5]. The encoded amino acid sequence is shown in single letter code. The Kabat et al. certain CDR regions 1-3 are underlined.

5 : DNA sequence encoding a light chain of a first urease-specific monoclonal antibody (DMS ACC2360). The encoded amino acid sequence is also shown in single letter code. The Kabat et al. certain CDR regions 1-3 are highlighted by framing.

6 : DNA sequence coding for a heavy chain of a first urease-specific monoclonal antibody (DMS ACC2360). The encoded amino acid sequence is also shown in single letter code. The Kabat et al. certain CDR regions 1-3 are highlighted by framing.

7 : DNA sequence encoding a light chain of a second urease specific monoclonal (DMS ACC2362). The encoded amino acid sequence is also shown in single letter code. The Kabat et al. certain CDR regions 1-3 are highlighted by framing.

8th : DNA sequence coding for a heavy chain of a second urease-specific monoclonal antibody (DMS ACC2362). The encoded amino acid sequence is also shown in single letter code. The Kabat et al. certain CDR regions 1-3 are highlighted by framing.

9 : Quick Test Strip General Construction: Sample Order Area ( 1 ); Test or analysis area ( 3 ) and absorption area ( 4 ). In the sample application area, the receptors required for detection, such as specific antibodies for the analyte or the antigen labeled with visible colored particles, eg colloidal gold or polystyrene (latex), or other binding partners, are in the dried state. The test carrier usually consists of a special test membrane such as nitrocellulose. On this test membrane further specific receptors, which are directed against the analyte or the antigen, immobilized as a test line. Between sample application area and test area there is a filter ( 2 ).

10 : Setup of Quick Test Strip with Check Line: Sample Order Area ( 1 ); Test or analysis area ( 3 ) and absorption area ( 4 ). In the sample application area, in the dried state, there are the receptors for the antigen required for the detection, which are marked with visible colored particles, eg colloidal gold or polystyrene. In a preferred embodiment, the sample application region can consist of two conjugate regions lying above one another, which contain, for example, a gold-labeled receptor in one region and a biotin-labeled receptor in the other. The test carrier usually consists of a special test membrane such as nitrocellulose. On this test membrane are other specific receptors that are directed against the analyte (antigen), as a test line ( 6 ) immobilized. In a preferred embodiment, streptavidin may be immobilized as a test line. As a functional check, a further control or interception line ( 7 ), for example a receptor directed against the labeled receptor. Between sample application area and test carrier there is a filter ( 2 ).

The examples illustrate the Invention.

Example 1: Isolation of H. pylori antigens

1.1 Cultivation of H. pylori

H. pylori (strain NCTC 11637) was streaked in Petri dishes on Wilkins-chalkern agar with the addition of 10% horse blood and amphotericin B, vancomycin and cefsoludin (Sigma Chemicals) and 1-2 Days under a microaerophilic atmosphere (Anaerocult GasPAk, Merck) at 37 ° C incubated. The contents of 2 dishes were suspended in 350 ml BHIB medium supplemented with antibiotics as above in a 1 L bottle (Schott), the medium for 4-8 min with a gas mixture of 10% CO ₂ , 5% O ₂ , 85 % N ₂ fumigated and the bottle closed. The culture was shaken for 2 days at 37 ° C on a rotary shaker. The contents of the bottle were then transferred to a sterile 10 L bottle and made up with 4.7 L BHIB medium. The bottle was then incubated for a further 2 days at 37 ° C on a rotary shaker. The entire volume was then centrifuged at 5000 g for 15 min, supernatant decanted and the bacterial pellet weighed. For storage, the pellet was resuspended in a physiological saline solution with the addition of 15% glycerol in the ratio 2: 1 (w / v) and frozen at -80 ° C. To verify the identity of the cultured bacteria, microscopic inspection of the bacteria and tests for urease, oxidase and catalase activity were performed.

Example 2: Preparation of H. pylori antigens

Preparation of H. pylori lysate

H. pylori bacterial pellet (Example 1) was treated 1:10 with PBS, pH 7.5 and resuspended on ice. The bacterial cells were on ice with the small probe of an ultrasound device (Sonifier, Branson), at 25-30% intensity 10 × 60 s with each 60 s pause sonicated. The open-minded bacterial cells were 2 × 20 min, at 4 ° C and centrifuged at 10,000 rpm (Sorvall, SS-34). The supernatant was used as antigen preparation for the production used by polyclonal antisera.

Preparation of H. pylori catalase

frozen Bacteria pellet was in proportion 1: 2 (w / v) with digestion buffer (20mM Tris / HCl pH 7.0, 1mM EDTA, 1mM phenyl methyl sulfonyl fluoride (PMSF), 0.05% sodium azide and 10% (v / v) isobutanol) and at room temperature (RT) on an overhead mixer until completely thawed and additionally further shaken for about 15 minutes. After centrifugation at 20,000 RPM (Sorvall SS34), 4 ° C for 20 min, became the supernatant decanted and over a 0.45 μm Filter filtered.

Of the clear supernatant was in proportion 1: 3 with Buffer A (20 mM Tris HCl pH 7.0, 1 mM EDTA, 1 mM PMSF, 0.05% Sodium azide) and equilibrated to buffer A SourceQ column (16/10) (Pharmacia). Of the Pass from the SourceQ column the enzyme contained catalase and was free of major H. pylori antigens such as urease, HSP60 and alkyl hydroperoxide reductase.

to Isolation of catalase became the run of the SourceQ column one Subjected to molecular sieve chromatography (Superdex 200) (16/60). The catalase was together with another approximately 150 kDa protein (Neutrophil Activating Protein, NAP) in approximately equal proportions isolated.

In higher Purity was obtained with catalase when passing from the SourceQ column a 2 M sodium acetate solution, pH 4.9 was brought to 40 mM sodium acetate and transferred to a SourceS column (8/28). After a wash with Buffer A to remove unbound Proteins were catalysed with buffer B (40 mM sodium acetate, 1 M NaCl, pH 4.9) using a linear NaCl gradient (buffer A plus 0% to 100% buffer B) elutes. Catalase elutes at about 370 mM NaCl.

Example 3: Characterization the catalase:

The purified protein showed in reducing conditions on SDS-PAGE a molecular weight of about 58 kDa and a purity of ≥ 90%.

To identify the isolated protein, microsequencing was performed. The protein was digested in the SDS-PAGE gel with LysC protease. The extracted protein mixture was separated by RP-HPLC. Sequence analysis of the LysC peptide revealed the following amino acid sequence:
ERLHDTIGESLAHVTHK

These Sequence is identical to the corresponding LysC peptide from H. pylori catalase (Manos J. et al. (1998) Helicobacter 3 (1), 28-38; Genbank Accession No AAC16068.1)

Example 4: Preparation polyclonal and monoclonal antibody (pAk; mAb)

Production of polyclonal antisera:

polyclonal Antisera to H. pylori lysate, H. pylori lysate depleted Main antigens such as urease, HSP60 and alkyl hydroperoxide reductase (see Example 2: isolation and purification), H. pylori lysate with enriched Catalase (for example, by adding catalase to the lysate) as well as polyclonal antisera against purified catalase can by Immunization of a selected Mammalian (e.g. Mouse, rabbit, goat, etc.) with the corresponding catalase epitopes containing immunogenic preparations to be obtained.

The antibody can by protein A affinity chromatography purified from sera and as a capture antibody in the sandwich ELISA (see Example 8) for assessing the suitability of monoclonal antibodies for antigen detection be used in patient chair.

polyclonal Rabbit antisera were obtained from pab Productions (Herbertshausen) made from H. pylori lysate. From these antisera were by means of Protein A affinity chromatography polyclonal antibodies purified and as catching antibodies in the Sandwich ELISA (see Example 8) to assess the suitability of monoclonal antibody for the Detection of H. pylori antigens used in patient chair.

Production of monoclonal Antibody:

The Preparation of monoclonal antibodies was carried out by methods known to those skilled in the art (Harlow & Lane, 1988, Peters & Baumgarten, 1990).

immunization

Out H. pylori lysate prepared antigen preparations (see example 2) were used to immunize mice (BALB / c × C57 / Black, F1 generation, 8-12 Weeks old). The primary immunization was 50 μg of antigen Emulsified 1: 1 with Freund's complete adjuvant (Difco) and intraperitoneally injected (200 μl / mouse). With 4 monthly boosts, the mice each received 25 μg of antigen with incomplete Freund's adjuvant. From retroorbital removed Blood became antiserum as positive control in ELISA (see fusion screening) won.

fusion

Two days after the last immunization, the spleens were removed from the mice and the spleen cells were fused with poly (ethylene glycol) 4000 with the myeloma cells P3 × 63Ag8.653 (ATCC CRL-1580, Kearney et al., 1979) in a ratio of 5: 1. The fused cells were suspended in HAT medium (cloning medium (= RPMI 1640 medium, 20% FCS, 200 U / ml rhIL-6) with nypoxanthine-aminopterin-thymidine supplement (Sigma)) and at a cell density of 2-6x 10 ⁴ cells / well in 96-well microtiter plates plated. The cultivation of the hybridomas was carried out at 37 ° C, 5% CO ₂ and 95% relative humidity.

Fusion screening by direct ELISA

The culture supernatants from overgrown wells were screened (about 10 days after the fusion) in a direct ELISA on 96-well microtiter plates (MaxiSorb, Nunc):
The ELISA plates were coated with 2 μg / ml immunization antigen in carbonate buffer, pH 9.6 (100 μl / well, overnight, 5 ° C). The coating solution was filtered off with suction and still blocked binding sites with 2% skimmed milk powder in PBS (w / v) blocked (200 .mu.l / well 1 h, room temperature). After washing the plate twice with PBS pH 7.3 with 0.025% Tween-20 (v / v), the culture supernatants of the primary clones were pipetted undiluted into the wells (100 μl / well) and the plates were incubated for 1-2 h at room temperature. As a positive control, antiserum was used as the negative control medium. After washing again, the bound antibodies were detected with a peroxidase-labeled secondary antibody (rabbit anti-mouse Ig-POD (DAKO) in PBS with 0.1% bovine serum albumin, 20 min, room temperature). After washing the plate four times, substrate solution (K-Blue, Neogen or citric acid buffer, pH 4.5 with TMB + H ₂ O ₂ ) was added. The peroxidase converts the colorless substrate tetramethylbenzidine (TMB, Sigma) to a colored complex. After 10 minutes, the reaction was stopped by adding 1N sulfuric acid. Culture supernatants of clones producing antigen-specific antibodies showed a clear staining against the colorless negative culture supernatants.

establishing and culturing the hybridomas

positive Clones were recloned twice according to the principle of limiting dilution, to get monoclones (Coller & Coller, 1983). The first recloning was carried out in cloning medium Hypoxanthine-thymidine supplement (Sigma), the second in cloning medium. Reclones were again screened for their antigen specificity by direct ELISA. Of the Clone finally reconnected in flat bottles of production medium (RPMI 1640 medium with 5% IgG reduced FCS) adapted. The cells were cryopreserved and culture supernatant for the Antibody Purification produced.

Example 5: Characterization the antibody from culture supernatant

Out a repertoire of 30 specific (against the immunization antigen Antibody-producing) Clones were H. pylori -infected by good reactivity on stool samples Patients selected in sandwich ELISA (see Example 6)

isotype

at the established clones were determined for isotype in the culture supernatant of the monoclonal antibody performed with the isotyping kit IsoStrip (Roche Diagnostics). This revealed 8 clones of type IgG1 and one clone of type IgG2a (see Table 2).

Western

The Culture supernatants were checked in the Westernblot for the ability to do that Specifically recognize immunization antigen. 15 μg purified per gel Boiled antigen in reducing sample buffer (Laemmli, 1970) and to a 12% SDS-picolylacrylamide minigel (8.6 cm x 7.7 cm x 0.1 cm, Biometra). After electrophoretic separation at 25-30 mA were the proteins (antigen) by means of semidry blot method on a nitrocellulose membrane immobilized.

The Membrane was blocked with 2% skimmed milk powder in PBS (30 min, room temperature) and washed three times with TBS / Tween-20 (0.2%) for 5 min. For the following Incubation step was the membrane in an Accutran cross-blot screening unit (Schleicher and Schuell) clamped, using a grid plate with 34 cross channels. In each of the resulting tracks 250 ul TBS / Tween-20 were submitted and 250 μl each added to the hybridoma culture supernatants to be tested. The incubation was carried out at room temperature for 2 h with shaking.

To washing three times TBS / Tween-20, the membrane was 1 h with the POD-conjugated Secondary antibody (rabbit anti-mouse Ig-POD, DAKO). The membrane was washed three times and the immune complex by adding the 3,3-diaminobenzidine substrate solution (DAB, Sigma) visualized. The antibody-binding Protein bands were subsequently by an insoluble one Peroxidase substrate visualized.

6 Hybridoma culture supernatants showed one band corresponding to catalase (58 kDa), 3 supernatants were negative in the Western blot, but showed a positive reaction with native antigen in the ELISA. They probably recognize a conformational epitope. The results are summarized in Table 2.

Example 6: Screening of mAb culture supernatants on Patient samples (mixed polyclonal / monoclonal system)

Those monoclonal antibody, the fusion detection using direct ELISA positive antigen detection were shown as culture supernatants in the sandwich ELISA in terms of their patient recognition and antigen detection limit examined.

Stool samples were available as internal development samples, whose infection status (groups 0 and 4) was assessed by histological examination and / or ¹³ C-urea breath test. In patients of group 0, an infection with H. pylori could be safely ruled out, in patients of group 4 an infection can be reliably detected.

The coating of the ELISA plates (MaxiSorb, Nunc) was carried out overnight at 5 ° C. with 100 μl of a solution of a polyclonal rabbit anti-catalase antibody or polyclonal rabbit anti-H. At pylori body (pAc, about 20 μg IgG / ml 0.1 M carbonate buffer, pH 9.5). To saturate the remaining binding sites, 200 μl of 150 mM PBS pH 7.2 with 0.2% fish gelatin (w / v) per well were added and incubated for 30 minutes at room temperature. Thereafter, the ELISA plate was washed twice with 250 μl of PBS with the addition of 0.025% Tween-20 (wash buffer 1). Human chair was suspended 1:10 (w / v) with 150mM PBS with the addition of 2% skimmed milk powder and 1mM EDTA.

to Determination of the antigen detection limit was a H. pylori negative Stool suspension with 50 ng / ml catalase (see Example 2) and diluted in 1: 2 steps with H. pylori negative stool suspension. ever 100 μl of the Stool suspensions were incubated per well for one hour (duplicate in patient samples). The plate was washed 4x with washing buffer 2 (PBS with 0.2% Tween-20). Subsequently, 100 μl of culture supernatant of hybridomas (diluted 1: 5 in PBS) admitted and for Incubated at RT for 60 min. The detection of the bound antibodies took place by adding a conjugated secondary antibody (rabbit anti-mouse IgG-POD, DAKO, Copenhagen, Denmark). The peroxidase (POD) sets the colorless substrate tetramethylbenzidine (TMB, Sigma) into a blue product. After 5 to 10 minutes, or as soon as even the negative control showed a slight blue coloration, the reaction became by adding 1N sulfuric acid (100 μl / well) stopped. The strenght the color reaction was measured in the ELISA reader (MWG spectral). The measurement was made at 450 nm against the reference wavelength 620 nm. Before adding the detection antibody or the substrate solution was the ELISA plate 3-4 times each with washing buffer 1 washed.

The lowest concentration, at which an extinction is still greater or equal to twice the control (H. pylori negative stool sample without antigen admixture) was determined as the detection limit.

Table 1: HP25.2m / 2H10: Sensitivity and specificity in sandwich ELISA with patient samples

• AK: antibodies; HP: H. pylori; pAK polyclonal antibody

Of the monoclonal antibodies HP25.2m / 2H10 demonstrated a sensitivity of 68% in the sandwich ELISA with patient samples (out of 25 positive samples 17 were correctly recognized) and a specificity of 82% (out of 17 samples, 14 were correctly recognized).

Table 2: Characterization of the monoclonal antibodies against catalase

• Ag: antigen; WB: Western blot; NWG: detection limit

Results

table 2 holds the results of the isotype determination, the Western blot analysis, the detection limit determination and the patient recognition for the monoclonal antibody in culture supernatant against catalase together. In the mixed polyclonal-monoclonal sandwich ELISA system the mAb HP25.2m / 2H10 shows a sensitivity of 68% and a specificity of 82%. An improvement of sensitivity and specificity showed by the use of purified mAbs (instead of culture supernatant) in a purely monoclonal ELISA system.

there can either a monoclonal antibody, which is directed against the same epitope of the antigen, or two different monoclonal antibodies which are directed against different epitopes of the same antigen (see Example 8), as catcher and detector antibodies be used.

Example 7: Cleaning of monoclonal antibodies from hybridoma culture supernatants

The Purification of mAb from serum-free hybridoma culture supernatants by means of a modified protein-G affinity chromatography (Pharmacia Biotech, 1994).

The filtered (0.45 μm) Culture supernatants were directly above passed a protein G matrix. The protein detection in the run or eluate was carried over the measurement of the optical density at 280 nm. After a washing step with 150 mM PBS, pH 7.2 until reaching the background detector value was eluted with 0.1 M glycine / HCl, pH 3.3. The regeneration of Protein G matrix was 0.1 M glycine / HCl, pH 2.7.

Example 8: Characterization the purified monoclonal antibody and antibody selection for the test

Those Antibody, which are the best detection properties in stool sample detection in the measurement from the culture supernatant were further characterized in the purified state. To the one were by surface plasmon resonance the affinity constants certainly. Furthermore, the binding regions of the antibodies were mapped (Epitope mapping). After all a selection of suitable antibody pairs was made using stool samples in the sandwich chair ELISA and in the quick test.

characterization of Antibody-antigen interactions using surface plasmon resonance spectroscopy (SPR) spectroscopy

With of SPR spectroscopy the affinity constants the monoclonal antibody be determined. This allows suitable antibodies for development from ELISA and quick test.

Carrying out the Surface plasmon resonance spectroscopy at Pharmacia BIAcore

All Steps were taken on a Pharmacia BIAcore Processing Unit CA 186 according to the manufacturer's instructions (BIAcore Methods Manual).

catalase was over Amino coupling immobilized on the dextran matrix of the BIAcore CM5 sensor chip. To activate the dextran matrix, 45 μl of a 1: 1 mixture of 0.05 M N-hydroxysuccinimide (NHS) and 0.2 M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) solution at a flow rate of 5 μl / min over the Sensor chip passed. Subsequently catalase (35 μl; 50 μg / ml in 10 mM sodium acetate pH 5.0) to the dextran matrix. remaining NHS esters were with 1 M ethanolamine (35 μl) disabled. Non-covalently bound to the dextran matrix catalase was removed by regeneration of the sensor chip with HCl (10 mM, 15 μl).

By Addition of the catalase-specific monoclonal antibodies were these reacted with immobilized catalase and the Mass accumulation measured at the detector. There were different antibody solutions Concentration in the range between 20 and 670 nM used. These were at a flow rate of 25 μl / min each over the injected on the sensor chip CM5 immobilized catalase.

KD = koff:kon From the time course of the resonance signal, the values for the rate constants of the adsorption (k _on ) and desorption (k _off ) of the antibody can be calculated (BIAevaluation software 3.0). Of the 6 monoclonal antibodies tested, 4 showed very good affinities K _D > 5E - 10 (Table 3). Table 3: Results of the affinity determination of the monoclonal antibodies against catalase

K D = k off k on

Epitope mapping of monoclonal antibody against catalase

The Epitope mapping was done by Pepscan Systems (Netherlands) carried out. It was a peptide library (30-mer with an overlap of 27 amino acids) of Catalase prepared on plastic cards and incubated with the antibodies. The identified epitopes (peptides to which antibodies have bound) are in Tab. 4 listed. HP25.2m / 2H10 showed nonspecific peptide recognition, i.e. that this Antibodies very much probably binds to a discontinuous structural epitope. HP25.6m / 1B5 also showed the main recognition region (see Tab. 4) other unspecific peptide bonds that involve the participation of a Expect structural component. If one transmits the determined epitopes on the structure of E. coli catalase (Bravo et al., 1999), it is striking that the antibody Bind HP25.6m / 1B5, 1A5 4E3, 1G4 and 1H4 in the enzyme center (amino acid 190-360), an area that is nearby the catalytic domain lies.

Table 4: Results of epitope mapping of catalase mAbs

overlapping Detection regions of the antibodies are underlined.

Determination of suitable Antibody pairs on hand of patient chair

The antibody against Catalase were first titrated against each other. Then with the so optimized ELISA systems Patient chair samples tested, as well as the detection limits of catalase determined in a human neutral chair (Table 5).

Structure of the sandwich ELISA:

The Coating of the ELISA plates (MaxiSorb; Nunc) was carried out for 1 h 37 ° C with 100 μl of a mAb solution in Carbonate buffer, 0.1M, pH 9.5. To block the still free binding sites were 200μl 150mM PBS pipetted with 0.2% fish gelatin (w / v) per well and at 30 min Room temperature incubated. Anschießend followed by a two-time Wash with 250μl Wash Buffer 1 (PBS with 0.025% Tween). Humane chair was in the ratio 1:10 (w / v) with 150mM PBS with the addition of 2% skimmed milk powder and 1mM EDTA suspended. Purified H. pylori catalase was used to determine the antigen detection limit in known concentrations of the stool suspension of H. pylori negative patient (neutral chair) was added. The stool sample suspensions were centrifuged for 5 min at 7000g. Per 100μl of the supernatant was per well for 1h incubated. The samples were plotted as double values. The plate was subsequently 4 × with Wash Buffer 2 (250μl PBS with the addition of 0.2% Tween). Then, 100 μl of a biotin-coupled solution was added PBS; 0.1% BSA was added and for Incubated at RT for 60 min. Detection of bound antigen / antibody complexes was done by adding a conjugate of streptavidin with POD (Dianova). The POD then sets in the next Step the colorless substrate TMB (Sigma) into a blue product around. After 5 to 10 minutes, or as soon as the negative control a light blue color showed that the reaction was stopped by adding 1N sulfuric acid (100 μl / well). The strenght the color reaction was measured in the ELISA reader (MWG spectral). The measurement takes place at 455 nm against the reference wavelength 620 nm.

When suitable detector antibody for the Combination with all other antibodies tested was found to be HP25.2m / 2H10 (Table 5). Due to the affinity data were used as catcher antibodies in the Quick test HP25.6m / 1B5, 1G4 and 1H4 tested. It turned out HP25.6m / 1B5 as best capture antibody out.

Table 5: Results of mating the monoclonal antibodies against catalase

Patient recognition (detection of 8 critical G4 and 4 G0 patient samples)
N = limit of detection [ng / ml] of the catalase in neutral
critically positive = samples that proved to be particularly problematic in detection

Example 9: Production of conjugates for use in immunochromatographic rapid tests

Coupling of monoclonal antibodies to biotin

The monoclonal antibodies were coupled to biotin following purification. The coupling was carried out by known methods (Harlow & Lane, 1988). The monoclonal antibodies were conjugated at a concentration of about 1-2 mg / ml. Before coupling, the antibodies were rebuffered by dialysis in 0.1 M sodium acetate buffer, pH 8.3 and 0.1 M sodium bicarbonate buffer, pH 8.3, respectively. For each 1 mg of antibody, 50 μg of N-hydroxysuccinimidobiotin (NHS-d-biotin; Sigma) in DMSO was added and mixed. The mixture was incubated for 1 h at room temperature. Thereafter, the biotinylated antibodies were released from uncoupled NHS-d-biotin by extensive dialysis against 0.15 M PBS, 0.05 NaN ₃ , pH 7.5.

Coupling of monoclonal antibodies of colloidal gold

The coupling of monoclonal antibodies to colloidal gold was done by known standard methods (Frens, 1973, Geoghegan and Ackerman, 1977; Slot et al., 1985). Gold colloid with a particle size of 40 nm, OD (520 nm) = 1 (British BioCell, Cardiff, England) was adjusted to pH 9 with 0.1 MK ₂ CO ₃ . Purified mAb was dialyzed against 2 mM borate buffer, pH 9.2, and diluted to a concentration of 0.1 mg / ml. For coupling, 2 ml of the mAb solution was added dropwise to 20 ml of the gold solution with rapid Stir and incubate for 30 min at room temperature. The optimal IgG concentration and the appropriate coupling pH were determined individually for each mAb To stabilize the gold-IgG conjugate, 2 ml bovine serum albumin was added at a concentration of 10% and incubated for a further 5 min. Gold colloid not coupled with IgG and free IgG were then separated by centrifugation. For this purpose, the coupling batch was centrifuged for 30 min at 15,000 rpm (Sorvall, SS-34) and the clear supernatant was suctioned off by means of a vacuum. The gold-IgG conjugate, deposited as a loose dark red stained sediment at the bottom of the centirifuge tube, was taken up in 2 ml of 20 mM Tris, pH 8.2 with the addition of 1% bovine serum albumin and 0.05% NaN ₃ .

Example 10: Immunochromatographic Quick test

With the antibody pair HP25.2m / 2H10 and HP25.6m / 1B5 an immunochromatographic test was set up in the sandwich-principle. This test is as in 9 and 10 shown schematically, from a sample application area ( 1 ), a filter ( 2 ), a test or analysis area ( 3 ) and an absorption area ( 4 ).

When Signal-forming immunoreagent was purified mAb HP25.2m / 2H10 coupled to gold (British BioCell, Cardiff, England). The mAb-gold conjugate was in deionized water with the addition of 5% sucrose (Sigma, Deisenhofen) to an OD (520 nm) of 3 and onto a conjugate fleece made of fiberglass (Pall, Dreieich) applied. Subsequently was the conjugate fleece vacuum dried.

As a test area ( 10 . 3 ) nitrocellulose was coated at 95-175 sec / 4 cm flow rate (Millipore, Redford, MA, USA) with the immunoreagents forming the test and control line. For this purpose, a special applicator for test strips (Imagene, Hanover, NH, USA) was used. As a test line ( 10 . 6 ) was purified mAb HP25.6m / 1B5, applied in phosphate buffer pH 7.4 in the concentration 1-2 ug / cm. As a control line ( 10 . 7 ) a polyclonal anti-mouse antibody (Dianova, Hamburg) was applied at a concentration of 0.1-0.3 μg / cm.

Subsequently, the coated nitrocellulose and the coated conjugate fleece were adhered together with the other components of the test strip to polyester support (G & L, Santa Clara, Calif., USA) and cut into 5 mm wide individual strips. As a filter ( 10 . 2 Glass fiber materials (Ahlstrom, Mt., Holly Springs, PA, USA, Pall, Dreieich, Whatman, Maidstone, England) were used in a 1-2 cm width. For the absorption area ( 10 . 4 Absorbent cellulosic or cellulosic glass fiber materials in a width of 2-3 cm were used (Pall, Dreieich, Schleicher & Schuell, Dassel, Whatman, Maidstone, England).

Example 11: Immunochromatographic Quick test using streptavidin as a test line

deviant from the rapid test described in Example 10 was there as Test line mAb HP25.6m / 1B5 coupled to biotin and on a second conjugate fleece of the sample application area dried up.

When Test line was recombinant streptavidin (Roche, Mannheim) in a concentration of 10-20 mg / ml coated in phosphate buffer, pH 7.4.

In this sandwich construction are during the test execution both antibody conjugates mobile and wander during the test execution over the Test strips. In the presence of antigen already forms during the Hike the entire sandwich complex off to the test line over the Binding of biotin to streptavidin is trapped.

Example 12: Detection of H. pylori in human stool by immunochromatographic Quick test

patient samples

For the evaluation of the immunochromatographic rapid test, 200 stool samples of patients from ten different clinics or gastroenterological practices were available in Germany. Samples were taken from both patients who did not have gastrointestinal disorders and gastrointestinal disorders, as well as those who underwent examination for complaints or diseases of the gastrointestinal tract. The infection status of the patients was assessed by ¹³ C urea breath test or by histological examination of a gastric biopsy specimen patients wi The results obtained in these two gold standard accepted methods were not included in the evaluation. The laboratory staff were not aware of the infection status of the tested patients.

Test procedure

to execution In the rapid test, the stool samples were dissolved 1:15 in sample buffer and 500 μl of the sample liquid applied to the application zone (sample application area) of the test strip. After 15 minutes the test was visually evaluated. The test signal at the test line was available as (test result positive) or not available (negative test result). Reading the Test result was each independently of three persons, who had no qualification as laboratory staff. additionally the tests were half-quantitative by the laboratory staff with 0 (negative), 1 (weak positive) and 2 (strong positive).

table Figure 6 shows the results compared by the chair rapid test on the two reference methods in an overall evaluation 200 patient samples were obtained. Of the 100 H. pylori-positive Samples were tested as 95 positive, with 5 samples occurring a false negative result. Of the 100 H. pylori-negative Samples were tested as correctly negative for 94 samples, for 6 samples a false positive result occurred. Sensitivity and specificity of the rapid test 95% and 94% compared to the reference methods.

Table 6: Test results of the gold standard reference methods and the rapid test (semi-quantitative) in the study of a total of 200 stool samples

• n.d .: not determined; 0: negative; 1: weak positive; 2: strong positive

• Sensitivity: 95%
• specificity: 94%

Example 13: Cloning and sequence determination of the functional variable regions of immunoglobulins Hybridoma cell lines

Total RNA became from antibody-producing Isolated hybridoma cell lines according to Chomczynski (Chomczynski, 1987). The corresponding cDNA was synthesized by standard methods (Sambrook et al., 1989)

The DNA regions containing the kappa light chain as well as the heavy chain Fd segment (VH or CH1) the respective antibody were amplified by PCR. It came in table 7 listed Oligunucleotide primer set for use. Those from the individual hybridoma cell lines isolated cDNA served as a template.

The used primer set leads to a 5'-Xho each I and a 3'-Spe I interface in the heavy chain Fd fragments as well as one each 5'-Sac I and one 3'-Xba I interface in the kappa light chains. For PCR amplification of the heavy Chain Fd-encoding DNA fragments were 11 different 5 'VH primers (MVH 1-8 and MULH1-3) each combined with the 3'-VH primer MIgG2a (HP25.2m / 2H10) or with the 3 'VH primer MIgG1 (HP25.6m / 1B5). To amplify the DNA fragments, which are useful for the kappa light chains 11 different 5 'VK primers (MUVK 1-7 and MULK1-4) were used, respectively with the 3 'VK primer 3'MUCK combined.

The The following temperature program was used in all PCR amplifications Application: Denaturation at 94 ° C for 30 s, primer annealing at 52 ° C for 60 s, polymerization at 72 ° C for 90 s. This program was for Maintained 40 cycles, followed by a 10 min completion of the fragments at 72 ° C.

The Results of the PCR amplifications were separated by agarose gel electrophoresis and DNA bands of the expected molecular weight isolated.

For the antibody 25.2m / 2H10 the isolated bands were subsequently subjected to restriction digestion Use of the enzymes Xho I and Spe I (heavy chains) or Sac I and Xba I (light chains) and the fragments obtained cloned into the plasmid vector Bluescript KS (Stratagene) after this first with the restriction enzymes Xho I and Spe I or Sac I and Xba I had been split.

Plasmid preparations of the cloned heavy and light chain fragments were then sequence analyzed. Sequences encoding immunoglobulin heavy and light chain functional variable regions (VH and VL, respectively) were selected. In this way, exactly one functional VH and one functional VL region could be identified for this hybridoma cell line. The functional VH and VL sequences are in 1 / 2 played. The first four amino acids of the VH region were complemented by recloning. Cloning and sequencing were performed by standard methods (Sambrook et al., 1989).

For the antibody 25.6m / 1B5, the isolated bands were then directly sequenced and a functional light and a functional heavy chain identified. The heavy chain Fd fragment and the light chain were then subjected to a restriction digest using the enzymes Xhol and Spel (heavy chain) or Sacl and XbaI (light chain) and the fragments obtained in the plasmid vector pBSIIIHisEx (Connex) cloned after this with the restriction enzymes Xho I and Spe I or Sac I and Xba I had been cleaved and resequenced.

In this way, exactly one functional VH and one functional VL region could be identified for this hybridoma cell line. The functional VH and VL sequences are in 3 / 4 played. The VH and VL sequences show the mature N termini as determined by leader primer sequencing. Cloning and sequencing were performed by standard methods (Sarnbrook et al., 1989).

Table 7: List of primers used for PCR amplification of functional variable regions of heavy and light immunoglobulin chains (orientation 5'-3 ')

Literature:

• Coller & Coller, 1983: Coller, H.A., Coller, B.S., Meth. Enzymol. 121: 412-417
• Harlow & Lane, 1988: Harlow, E., Lane, D., Antibodies: A laboratory manual, Cold Spring Harbor Laboratory, New York
• Kearney et al., 1979: Kearney, J. Immunol. 123: 1548-1550
• Laemmli, 1970: Laemmli, E.K., Nature 227: 680-685
• Peters & Baumgarten, 1990: Peters, J.H., Baumgarten, H. (ed.), Monoclonal Antibodies, Springer Publisher, Berlin
• Fägerstam et al., 1990: Fägerstam, L. G. et al., J. Mol. Recognit. 3: 208-214
• Malmqvist, 1996: Methods 9: 525-532
• Eschweiler et al., 1993: Eschweiler, B., et al., Zentralbl. F. Bakt. 280: 73-85
• Pharmacia Biotech, 1994: Monoclonal Antibody Purification Handbook
• Chomczynski, 1987: Anal. Biochem. 162: 156-159
• Sambrook et al., 1989: Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, second edition
• Vaughan et al., 1998: Nature Biotechnology 16: 535-539
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• Janeway & Travers, 1997: Immunology, 2nd edition, Spektrum Akademischer Verlag GmbH, Heidelberg
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Claims (45)

Kit for detecting infection of a mammal with an acid-resistant Microorganism of the genus Heliobacter, containing (a) at least a monoclonal antibody, Fragment or derivative thereof or an aptamer that is an epitope an antigen of an acid-resistant Microorganism specifically binds, (b) another monoclonal Antibody, which specifically binds an antigen or epitope as defined in (a) and / or a polyclonal antiserum, wherein the polyclonal antiserum opposes is an antigen or epitope as defined in (a) and / or was won against (a) a lysate of an acid-resistant microorganism; (B) a lysate containing antigen from an acid-resistant microorganism enriched; (c) a lysate containing immunodominant antigen from an acid resistance Depleted microorganism; and or (d) a purified, Semisynthetic or synthetically produced antigen from a Acid-resistant microorganism; and (c) a device for preparing and analyzing the stool sample; preferably the microorganism, the lysate and / or the antigen from H. pylori; and where either the one defined under (a) monoclonal antibodies, the fragment thereof, the derivative thereof, the aptamer and / or the in (b) defined further monoclonal antibodies or the polyclonal antiserum to a carrier material are bound. Kit according to claim 1, characterized in that either the monoclonal antibody defined under (a), the Fragment thereof, the derivative thereof, the aptamer and / or the in (b) defined further monoclonal antibodies or the polyclonal antiserum to a carrier material are fixed. Kit according to claim 1 or 2, characterized that the antigen is a catalase, urease, Hsp 60 or metalloproteinase antigen is. Kit according to one of claims 1 to 3, characterized that the acid resistant Microorganism a bacterium of the species Heliobacter pylori or Heliobacter hepaticum is. Kit according to one of claims 1 to 4, characterized that the kit is either a monoclonal antibody as defined in (a) antibody and a monoclonal antibody as defined in (b), or a monoclonal antibody as defined in (a) and as defined in (b) polyclonal antiserum. Kit according to one of claims 1 to 5, characterized that the antibody or the polyclonal antiserum as defined in (a) as a catcher antigen and the antibody or antibody as defined in (b). the polyclonal antiserum as a detector for detection of the antigen serves. Kit according to one of claims 1 to 6, characterized that the polyclonal antiserum as a catcher and the antibody as Detector of the antigen serves. Kit according to one of claims 1 to 6, characterized that the polyclonal antiserum as detector and the antibody as the catcher of the antigen serves. Kit according to one of claims 1 to 8, characterized that at least one antibody or the polyclonal antiserum is labeled, wherein the label preferably selected is made of colloidal gold, selenium, colored polystyrene particles, colored Latex particles, biotin, carbon particles and dispersed colors. Kit according to one of claims 1 to 9, characterized that the carrier material is a test strip, preferably a test strip of porous carrier material. Kit according to one of claims 1 to 10, characterized in that the kit further comprises a resuspension buffer for diluting the Stool sample before incubation with the antibody and / or with the antiserum contains. Kit for the determination of H. pylori in a stool sample, full (a) a carrier material, to the a genus specific monoclonal antibody for H. pylori Antigen is bound, (b) a protein-based sample solvent; and (c) a variety of labeled antibodies specific for H. pylori antigen are. Kit according to claim 12, characterized in that the protein-based sample solvent Bovine serum albumin is. Kit according to claim 12 or 13, characterized that the plurality of labeled antibodies is a labeled polyclonal Antiserum is. Test device for detecting infection of a mammal with an acid resistance Microorganism of the genus Heliobacter, containing (a) at least a monoclonal antibody, Fragment or derivative thereof or an aptamer that is an epitope an antigen of an acid-resistant Microorganism specifically binds, with the antibody, the fragment or derivative thereof or the aptamer is labeled, wherein the label is preferably selected of colloidal gold, selenium, colored polystyrene particles, colored Latex particles, biotin, carbon particles and disperse colors; and (B) another monoclonal antibody which has one as defined in (a) Antigen or epitope specifically binds and / or a polyclonal Antiserum, wherein the polyclonal antiserum is against a protein as in (a) is defined antigen and / or epitope and / or won was against (a) a lysate of an acid-resistant microorganism; (B) a lysate containing antigen from an acid-resistant microorganism enriched; (c) a lysate containing immunodominant antigen from an acid resistance Depleted microorganism; and or (d) a purified, Semisynthetic or synthetically produced antigen from a Acid-resistant microorganism; and (c) a device for preparing and analyzing the stool sample; preferably the microorganism, the lysate and / or the antigen from H. pylori; and where either the one defined under (a) monoclonal antibodies, the fragment thereof, the derivative thereof, the aptamer and / or the in (b) defined further monoclonal antibodies or the polyclonal antiserum to a carrier material are bound. Test device according to claim 15, characterized in that either the monoclonal antibody defined under (a), the Fragment thereof, the derivative thereof, the aptamer and / or the in (b) defined further monoclonal antibodies or the polyclonal antiserum to a carrier material are fixed. Test device according to claim 15 or 16, characterized characterized in that the antigen is a catalase, urease, hsp 60 or metalloproteinase antigen. Test device according to one of claims 15 to 17, characterized in that the acid-resistant microorganism a bacterium of the species Heliobacter pylori or Heliobacter hepaticum is. Test device according to one of claims 15 to 18, characterized in that the test device either a as defined in (a) monoclonal antibody and one as defined in (b) monoclonal antibody contains or a monoclonal antibody as defined in (a) and a polyclonal antiserum as defined in (b). Test device according to one of claims 15 to 19, characterized in that as defined in (a) antibody or the polyclonal antiserum as catcher antigen and the antibody or antibody as defined in (b). the polyclonal antiserum as a detector for detection of the antigen serves. Test device according to claim 20, characterized in that that the polyclonal antiserum as a catcher and the antibody as Detector of the antigen serves. Test device according to claim 20, characterized in that that the polyclonal antiserum as detector and the antibody as the catcher of the antigen serves. Test device according to one of claims 15 to 22, characterized in that at least one antibody labeled wherein the label is preferably selected from colloidal gold, Selenium, colored polystyrene particles, colored latex particles, biotin, Carbon particles and disperse colors. Test device according to one of claims 15 to 23, characterized in that the carrier material is a test strip is, preferably a test strip of porous carrier material. Test device according to one of claims 15 to 24, characterized in that the test device further comprises a Resuspension buffer for dilution the stool sample before incubation with the antibody and / or with the antiserum contains A package containing a kit according to any one of claims 1 to 14 or a test device according to one of claims 15 to 25th Use of a monoclonal antibody, fragment or derivative thereof or an aptamer that is an epitope of a Antigens of an acid-resistant Microorganism in a stool sample specifically binds to the diagnosis an infection of a mammal with an acid-resistant Microorganism of the genus Heliobacter, wherein the microorganism preferably H. pylori. Use of a monoclonal antibody, fragment or derivative thereof or an aptamer that is an epitope of a Antigens of an acid-resistant Microorganism specifically binds in a stool sample, in combination With (i) another monoclonal antibody which has the epitope of a Antigens of an acid-resistant Microorganism specifically binds and / or (ii) a polyclonal Antiserum, wherein the polyclonal antiserum against the antigen or Epitope is directed and / or won against a. a lysate an acid-resistant Microorganism; b. a lysate made with an acid-resistant antigen Enriched microorganism; c. a lysate that is immunodominant Antigen from an acid-resistant Depleted microorganism; and / or d. a purified, Semisynthetic or synthetically produced antigen from a Acid-resistant Microorganism, being the acid-resistant Microorganism is preferably H. pylori; for the diagnosis of a Infection of a mammal with an acid-resistant Microorganism of the genus Heliobacter, wherein the microorganism preferably H. pylori. A test strip for detecting infection of a mammal with an acid-resistant Heliobacter genus microorganism in a stool sample containing; (a) at least one monoclonal antibody, fragment or derivative thereof, or an aptamer which specifically binds an epitope of an antigen of an acid-resistant microorganism, (b) another monoclonal antibody comprising an antigen or epitope as defined in (a) specifically binds and / or a polyclonal antiserum, wherein the polyclonal antiserum to an antigen as defined in (a) or epitope and / or recovered against (a) a lysate of an acid-resistant microorganism; (b) a lysate enriched with antigen from an acid-resistant microorganism; (c) a lysate depleted of immunodominant antigen from an acid-resistant microorganism; and / or (d) a purified, semisynthetic or synthetically prepared antigen from an acid-resistant microorganism; preferably the microorganism, the lysate and / or the antigen is H. pylori; and wherein either the monoclonal antibody defined in (a), the fragment thereof, the derivative thereof, the aptamer and / or the further monoclonal antibodies or the polyclonal antiserum defined in (b) in the dried state in the sample application area ( 1 ) are located. Test strip according to claim 29, characterized in that the monoclonal antibody defined under (a), the fragment thereof, the derivative thereof or the aptamer in the dried state in the sample application area ( 1 ) and the further monoclonal antibody or polyclonal antiserum defined in (b) ( 6 ) is immobilized. Test strip according to claim 29, characterized in that the further monoclonal antibody or the polyclonal antiserum defined under (b) in the dried state in the sample application area ( 1 ) and the monoclonal antibody defined in (a), the fragment thereof, the derivative thereof or the aptamer as a test line ( 6 ) is immobilized. Test strip according to one of claims 29 to 31, characterized in that the test strip has a test or analysis area ( 3 ), wherein in the test or analysis area ( 3 ) the test line ( 6 ) and, where appropriate, another control or interception line ( 7 ) is located. Test strip according to one of Claims 29 to 32, characterized in that the test strip has an absorption region ( 4 ), wherein the test strip the absorption area ( 4 ) in the transport direction substantially at its end. Test strip according to one of claims 29 to 33, characterized in that the sample application area ( 1 ) a conjugate fleece and a downstream in the transport direction filter ( 2 ), which is suitable to filter substantially the solids content of the stool or the stool suspension. Test strip according to one of claims 29 to 34, characterized in that the antibody defined in (a) by the suspension is soluble and / or the further antibody as defined in (b) or the polyclonal antiserum insoluble by suspension immobilized or dried on the test strip is / are. Test strip according to one of claims 29 to 34, characterized in that the further as defined in (b) more antibodies or the polyclonal antiserum solvable by the suspension and / or the antibody defined in (a) insoluble by suspension immobilized or dried on the test strip is / are. Test strip according to one of claims 29 to 36, characterized in that the antigen is a catalase, urease, Hsp 60 or metalloproteinase antigen. Test strip according to one of claims 29 to 37, characterized in that the acid-resistant microorganism a bacterium of the species Heliobacter pylon or Heliobacter hepaticum is. Test strip according to one of claims 29 to 38, characterized in that the test strip either a as defined in (a) monoclonal antibody and one as defined in (b) monoclonal antibody contains or a monoclonal antibody as defined in (a) and a polyclonal antiserum as defined in (b). Test strip according to one of claims 29 to 39, characterized in that as defined in (a) antibody or the polyclonal antiserum as catcher antigen and the antibody or antibody as defined in (b). the polyclonal antiserum as a detector for detection of the antigen serves. Test strip according to claim 40, characterized that the polyclonal antiserum as a catcher and the antibody as Detector of the antigen serves. Test strip according to claim 40, characterized in that the polyclonal antiserum as a detector and the antibody serves as the scavenger of the antigen. Test strip according to one of claims 29 to 42, characterized in that at least one antibody or the polyclonal antiserum is labeled, the label preferably selected is made of colloidal gold, selenium, colored polystyrene particles, colored latex particles, biotin, carbon particles and dispersed Colours. Test strip according to one of claims 29 to 43, characterized in that either the one defined under (a) monoclonal antibodies, the fragment thereof, the derivative thereof, the aptamer and / or the in (b) defined further monoclonal antibodies or the polyclonal antiserum to a carrier material bound or fixed, wherein the carrier material is preferably made of Cellulose or a cellulose derivative. Dry reagent test characterized in that the test contains a test strip according to any one of claims 29 to 44 and that all the specific reagents necessary for analysis in the several porous Materials composite test strips preferably in dried Condition are included.