[go: up one dir, main page]

WO1992014154A1 - Serodiagnostic d'agglutination destine a des antigenes mycobacteriens dans des echantillons biologiques - Google Patents

Serodiagnostic d'agglutination destine a des antigenes mycobacteriens dans des echantillons biologiques Download PDF

Info

Publication number
WO1992014154A1
WO1992014154A1 PCT/US1992/001131 US9201131W WO9214154A1 WO 1992014154 A1 WO1992014154 A1 WO 1992014154A1 US 9201131 W US9201131 W US 9201131W WO 9214154 A1 WO9214154 A1 WO 9214154A1
Authority
WO
WIPO (PCT)
Prior art keywords
particles
agglutination
enhancer
monoclonal antibody
naked eye
Prior art date
Application number
PCT/US1992/001131
Other languages
English (en)
Inventor
Nicolae Istrate
Original Assignee
Dynagen, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dynagen, Inc. filed Critical Dynagen, Inc.
Publication of WO1992014154A1 publication Critical patent/WO1992014154A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form

Definitions

  • This invention relates to agglutination methods useful for detecting analytes in biological samples.
  • the invention is particularly useful for detecting mycobacteria in a sample from an individual suspected to have or being tested for tuberculosis.
  • the mycobacteria are a diverse assemblage of acid-fast, Gram-positive bacteria, some of which are important disease-causing agents in humans and animals, Bloom et al. , Rev. Infect. Pis. , 5:765-780 (1983); Chaparas, CRC Rev. Microbiol. , :139-197 (1982).
  • the two most common diseases caused by mycobacteria are tuberculosis and leprosy, the causative agents being Mycobacterium tuberculosis and Mycobacterium leprae, respectively.
  • mycobacterial species are capable of causing tuberculosis or tuberculosis-like disease. Wallace, R.J. , et al. , Review of Infectious Diseases, 5 ⁇ :657-679 (1984).
  • Mycobacterium avium for example, causes tuberculosis in fowl and in other birds. Members of the M. avium-intracellulare
  • MAI immuno-deficiency syndrome
  • AIDS immuno-deficiency syndrome
  • the members of the MAI-complex are resistant to standard anti-tuberculosis drugs. Pitchenik, A.E., et al. , Annals of Internal Medicine, 101:641-645 (1984).
  • tuberculosis results from respiratory infection with M. tuberculosis. Infection may often be asymptomatic, but could result in disease, producing pulmonary or other lesions which lead to severe debilitation or death.
  • tuberculosis remains a significant health problem, especially in developing countries.
  • an estimated 11 million people are affected by the disease and about 3.5 million new cases occur each year.
  • U.S. Congress, OTA "Status of Biomedical Research and Related Technology for Tropical Diseases", OTA-H-258, Washington, D.C. 1985.
  • certain groups of individuals such as those who are HIV-positive have a markedly increased incidence of tuberculosis.
  • Early diagnosis of TB is particularly important because the disease is preventable, treatable and curable.
  • immunoassays Due to the disadvantages of using a diagnostic method reliant on microscopic observations, immunoassays have been studied for their applicability in diagnosing TB.
  • Several immunological methods for detecting mycobacterial antigens such as enzyme linked im unosorbent assay (ELISA) and radioimmunoassay (RIA) have been mentioned as possible alternatives to microscopy (Daniel, Reviews of Infectious Diseases Vol II Supplement 2, March-April, 1989, pp.S471-S478) .
  • Methods 129 (1990), pp. 9-14) describe an immunoassay for detecting mycobacterial antigens using a latex agglutination assay.
  • the latex particles are coated with a polyclonal antiserum reactive with mycobacterial antigens. Agglutination was detected with an optical counter, and apparently was not detectable by visual observation with the naked eye.
  • polyclonal antisera is typical in latex agglutination assays, but has drawbacks.
  • the principal drawback is that the use of polyclonal antiserum does not permit standardization. Different batches of polyclonal antisera will have different properties.
  • Monoclonal antibodies have been reported in connection with latex agglutination assays in only a few instances. Monoclonal antibodies are reactive with a single antigen, and it is problematic to obtain a monoclonal antibody to a bacillus with sufficient specificity and titer so as to permit agglutinaton in such an assay.
  • microscopy which is considered to have low sensitivity, can detect 10,000 intact bacteria/ml of sputum. Although microscopy detects bacteria in only 40-60% of diseased individuals when uncultured sputum is used as the sample, it nevertheless is the best method currently available and is an order of magnitude better than the agglutination assay of the '192 patent. Moreover, the '192 patent did not use sputum from diseased individuals. Rather, cultured mycobacteria were mixed with the sputum of non-diseased donors. Thus, the ability of the '192 agglutination assay to detect mycobacteria in the sputum (which includes non-intact bacteria) of diseased individuals remains suspect.
  • a rapid, reliable method for detecting mycobacteria including the causative agent responsible for tuberculosis.
  • This method is capable of detecting mycobacterial antigen in biological samples, including sputum, at levels that are clinically acceptable.
  • This method may be conducted and the results evaluated by a person having limited expertise. The person only has to be capable of identifying the presence or absence of agglutination.
  • the method includes contacting the biological sample with particles coated with antibody directed towards a mycobacterial antigen.
  • the antibody is a monoclonal antibody.
  • the biological sample is contacted under conditions which permit agglutination to occur. After a short period of time, the agglutination of the particles is detected as an indication of the presence of mycobacteria in the biological sample.
  • conditions are
  • the method permits detection at clinically acceptable levels, that is, 10,000 bacteria/ml of biological fluid, and more preferably permits detection in samples obtained from fluids having levels of 1000 bacteria/ml.
  • an agglutination reaction is enhanced with an enhancer.
  • the enhancer is a reagent capable of promoting the agglutination reactions preferably in a manner which assures that agglutination will be visually detectable by the naked eye if the appropriate antigens are present in the biological sample.
  • This invention also pertains to particles coated with monoclonal antibodies which may be used in the above-described methods and kits containing the reagents necessary to conduct the methods of the invention.
  • Another aspect of the invention pertains to the use of a negative control latex which allows one to differentiate if latex agglutination is due to antigen-antibody interaction (specific agglutination) or whether something else in the sample causes non-specific agglutination with the antibody coated on the latex beads.
  • Figure 1 is a graph used in selecting monoclonal antibodies for the method(s) of this invention.
  • Figure 2 is a table illustrating the relationship between the concentration of enhancer and autoagglutination
  • Figure 3 illustrates a preferred kit according to the invention.
  • Figure 4 shows a slide of the kit of Fig. 3.
  • the agglutination method of the invention involves contacting a biological sample with a particle coated with an antibody.
  • the antibody is specific towards an analyte being tested for in the sample and may include as equivalents any agent capable of binding to the analyte. Agglutination is detected as an indication of the presence of the analyte in the sample.
  • a monoclonal antibody may be used, and both the contacting and detection steps of the invention preferably are conducted within a relatively short period of time, e.g. preferably within about fifteen minutes.
  • the antibody is directed towards an analyte indicative of a mycobacterium.
  • the antibody is directed towards an analyte indicative of a mycobacterium.
  • the antibody is
  • a monoclonal antibody directed toward an antigen from a mycobacterium is contacted with particles coated with a monoclonal antibody directed towards a mycobacterial antigen under conditions which permit agglutination to occur.
  • the agglutination of the particles is detected as an indication of the presence of mycobacteria in the sample. Examples of mycobacteria which may be detected include Mycobacterium tuberculosis and Mycobacterium leprae.
  • the mycobacterial antigen detected may be lipoarabinomannan (LAM) antigen.
  • LAM is a highly immunogenic lipopolysaccharide. LAM is a prominent component of the cell walls of both M. leprae and M. tuberculosis and has been implicated as a major B cell stimulant in tuberculosis and leprosy. Portions of LAM are exposed on the surface of mycobacteria.
  • the particles used in this invention may be any particles capable of agglutinating in a detectable manner.
  • the prior art has disclosed particles typically in a size range of from about 0.l ⁇ to about 15 ⁇ ; however, larger and smaller particles may be used.
  • a preferred size particle for this invention is 0.15 ⁇ - l.O ⁇ , more preferably about 0.2 ⁇ .
  • red blood cells include red blood cells, glass beads, liposomes, pollen spores, metal oxide particles, latex, and carbohydrates, e.g. dextran, agarose or cellulose.
  • the preferred particles are latex particles.
  • latex is art recognized and typically refers to particles made of natural or synthetic rubber or plastic. Latex particles are commercially available and are prepared using addition polymerization processes in aqueous media. Monomers used in preparing latex include acrolein, acrylate, methyl acrylate, methacrylate, methyl methacrylate, glycidyl methacrylate, styrene, vinyl toluene, t-butyl styrene and copolymers containing mixtures of these monomers. The polymers and copolymers optionally may contain cross-linking agents such as divinyl benzene and butadiene.
  • the particles also may be colored, thereby enhancing the ease of visual detection of agglutination.
  • the color may be selected to provide a contrast between the particles and the background color of a slide, for example.
  • the preferred particles of this invention are red latex beads which can be obtained from Rhone Poulenc, France.
  • the red beads are polystyrene having an average diameter of about 0.2 ⁇ . Such beads provide a desirable contrast against an opaque slide that is white. - 11 -
  • coated particle is intended to encompass a particle having antibody on its surface.
  • the surface of a particle may be coated using methods capable of directly or indirectly attaching antibodies.
  • the antibodies may be absorbed directly on the surface of the particle or attached to the particle through a spacer molecule, e.g. a molecule capable of bonding to both the surface of the particle and to the antibody.
  • the antibodies are directly attached or absorbed to the particle using passive coating techniques well known to those of ordinary skill in the art. Such coating techniques tend to preserve the specificity and activity of the immunological reagent.
  • the antibodies of this invention are preferably those directed towards mycobacterial antigen. Directed towards is intended to encompass antibodies capable of binding with an antigenic portion of a mycobacterium.
  • the term antibody is intended to include whole antibodies, antibody fragments, chimeric antibodies containing portions from two different species, and synthetic peptides identical to or functionally nalogous to the antibody.
  • Antibody fragments such as F(ab') 2 , Fab and F v may be produced by standard techniques of enzyme digestion.
  • synthetic peptides such as F(ab') 2 , Fab and F v may be produced by standard techniques of enzyme digestion.
  • Fab and F analogues can be produced by genetic engineering techniques. See e.g., Better, M. et al. (1988) Science 240:1041; Huston, J.S. et al. (1988) Proc. Natl. Ac d. Sci. USA 85:5879-5883.
  • the preferred form of antibody is whole, monoclonal antibody. It should be understood that more than one type of monoclonal antibody may be attached to a particle.
  • a particularly preferred monoclonal antibody for one embodiment of the invention is designated ML9D3, produced by the ML9D3 cell line, ATCC Accession No. HB 10684, Rockville, Md. This monoclonal antibody is directed toward the LAM antigen.
  • monoclonal antibody-producing cell lines examples include hybridoma cell lines, myeloma cell lines, or viral or oncogenically transformed lymphoid cells.
  • Hybridoma cells which can produce the specific antibodies for use with this invention may be made by the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975) or similar procedures employing different fusing agents. Briefly, the procedure is as follows: the hybridoma which secretes the monoclonal antibodies are produced by immunizing an animal with an antigen. Lymphoid cells (e.g. splenic lymphocytes) are then obtained
  • hybrid cells from the immunized animal and fused with immortalizing cells (e.g. myeloma or heteromyeloma) to produce hybrid cells.
  • immortalizing cells e.g. myeloma or heteromyeloma
  • the hybrid cells are screened to identify those which produce the desired antibody and then are cloned and tested to prove that the cells produce only monoclonal antibodies.
  • the hybridoma cells producing the desired antibody can be subsequently expanded.
  • the hybridomas are expanded by injecting them intraperitoneally into mice under conditions which allow ascites fluid to develop.
  • the ascites fluid is collected from the mice, pooled together and centrifuged.
  • the supernatant from this process is termed neat ascites.
  • the conditions which permit agglutination to occur are those which lead to the agglutination of the particles in a manner which is detectable, preferably visually with the naked eye. For this, approximately 100 clumps must be seen to determine agglutination. Clumps must be about 50 ⁇ in size to be visible with the naked eye. Approximately 10 bonds are required per particle to cause clumping.
  • the conditions should be such that agglutinaton is detectable at levels of 10,000 bacteria/ml of biological fluid, and preferably at levels of 1000 bacteria/ml of sputum. This will ensure detection of greater than 50% of those individuals having a mycobacterial disease. Examples of such conditions
  • a selection process such as an ELISA assay may be used to determine whether a particular lot or batch of monoclonal antibodies would be useful in the method of this invention if coated on the particles.
  • 1 ⁇ g/ l of LAM antigen can be coated on a solid phase, e.g. multiwell plate.
  • LAM may be prepared as described below in Example 6 and in copending U.S. application serial no. 07/654,321, filed February 12, 1991, and entitled "Purified LAM and Synthetic Analogs Thereof", the entire disclosure of which is incorporated herein by reference.
  • the coated solid phase then is incubated with various dilutions of monoclonal antibodies at 37°C for approximately one hour.
  • the unbound monoclonal antibodies are separated from the solid phase.
  • the solid phase with attached antibodies is subsequently incubated with a second antibody directed towards the monoclonal antibody and conjugated to an enzyme (incubation with secondary antibody is for 1 hr at RT) .
  • the solid phase-antibody-secondary antibody conjugate is incubated for an additional hour at 37°C in the presence of the substrate for the enzyme.
  • enzymatic reaction is measured using spectrophotometric means, e.g. absorbance, as an indication of the amount of monoclonal antibody bound to the antigen, A curve may be established by plotting the absorbance vs. dilution and the "titer" of the monoclonal antibodies may be determined ( Figure 1) .
  • spectrophotometric means e.g. absorbance
  • the quantitation of specific activity or titer is expressed as the reciprocal of the dilution of monoclonal antibody which exhibits 50% of maximum absorbance. This can be determined from a curve established by plotting the % of maximum absorbance of particular dilutions of monoclonal antibodies versus the dilution of the monoclonal antibodies (MAb) as shown in Figure 1.
  • the % of maximum absorbance of each dilution can be calculated by using the following formula:
  • the readout absorbance (B) is 0.783(B), and the nonspecific absorbance (NSB) is 0.110, and the maximum absorbance (B0) is always considered 1.000 (100%
  • the solution has a titer of antibodies against LAM of at least 70K [approximately 3 mg antibody per ml], and more preferably 100K [approximately 5 mg antibody per ml], at 50% absorbance, then the lot or batch used in preparing the dilutions may be used to coat particles for use in this invention. It should be understood that the foregoing titers are merely cut-off limits useful for deciding whether a lot or batch of antibodies is suitable for coating particles according to this invention.
  • the solution still may be useful in the agglutination assays of the invention after various manipulations, e.g. purification and/or concentration processing steps. Problems may be encountered if attempting solely concentration of the antibodies because the other components of the ascites, enzymes etc., also become concentrated. Concentration alone thus may lead to nonspecific binding and a decrease in stability. However, concentration coupled with purification may be desirable not only to attain adequate titer, but also to increase the stability of the monoclonal antibodies.
  • the amount of monoclonal antibody coated on the particles is dependent on a number of variables.
  • 100 ⁇ l of neat ascites having a titer of at least 10OK [approximately 5 mg antibody per ml] of antibodies against LAM at 50% absorbance, i.e. a monoclonal antibody selected using the cut-off limits described above, is combined with 4 ml of a 0.5% suspension of latex beads (0.2 ⁇ polystyrene). It should be understood that these parameters are interrelated. For example, if the volume of neat ascites is increased, then a larger volume of solution containing latex
  • beads or a solution of latex beads having an increased concentration of beads should be used.
  • the monoclonal antibody preferably is present on the surface of the particle at a density sufficient such that the particles are capable of autoagglutinating in a manner detectable with a naked eye in the presence of an enhancer (discussed in greater detail below) at a preselected concentration.
  • an enhancer discussed in greater detail below
  • a preselected concentration of enhancer is used as a standard , and only coated beads capable of autoagglutinating as seen by the naked eye in the presence of this preselected concentration of enhancer are used. It should be understood that this concentration of enhancer is used for selection purposes only. It is not the same concentration of enhancer actually used in the agglutination method of this invention.
  • the concentration of enhancer used in the agglutination method of the invention is selected as described below.
  • Enhancers are additives which promote nonspecific agglutination generally. Thus, an enhancer at sufficient concentration may be added to a solution containing antibody-coated beads and thereby cause agglutination of the coated beads without the addition of antigen. This ability to promote agglutination can be exploited to turn a
  • enhancers include polyethylene glycol (PE , ol. wt. 8000), polyvinylpyrrolidone (PVP, mol. wt. 40,000, K value or intrinsic viscosity of 29-32), dextran (mol. wt. 70,000) and combinations thereof.
  • the concentration of enhancer used in the agglutination assays may be selected as follows. Various concentrations of the enhancer are prepared. At the higher concentrations, the enhancer typically will cause autoagglutination of the coated particles visible to the naked eye in the absence of antigen. Lower concentrations are tested until no visually detectable autoagglutination occurs. The concentration of enhancer immediately below the minimum concentration causing visually detectable autoagglutination preferably is selected for use in the method of this invention. Enhancers selected in this manner are capable of providing autoagglutination visible to the naked eye when a -
  • biological sample containing the appropriate antigen is part of the agglutination reaction mixture.
  • the agglutination of the particles may be detected by using any means capable of measuring the agglutination of the particles in a quantitative or qualitative manner.
  • the preferred means of detection is visual detection with the naked eye. Detection may also be carried out using the aid of a microscope or other optical instruments, i.e. optical counter. However, when appropriate concentrations and conditions as set forth above are selected, detection aids are unnecessary and the test thus is greatly simplified and economized.
  • the biological samples used within the method of this invention may be any biological sample in which agglutination is detectable. It may include, for example, viable bacteria, killed or fragments of bacteria and/or soluble antigen derived from bacteria, depending upon the preferred processing method employed. For safety considerations, it may be desirable to inactivate the bacteria by processing procedures such as bioling in NaOH, which particular procedure would result in substantial solubilization of the bacteria.
  • the type of biological sample selected may depend on the type of disease suspected or the condition of the individual being tested. For example, the type of disease suspected or the condition of the individual being tested.
  • genital specimens e.g. uterocervico-vaginal secretions
  • urine may be obtained from individuals suspected of having urogenital tuberculosis.
  • Cerebrospinal fluid may be obtained if tuberculous meningitis is suspected.
  • biological samples include serum, whole blood, urine, feces, tissue specimens, (e.g. pus, exudates and biopsy specimens), cold abscess drainage, peritoneal ascitic fluid, uterocervicovaginal secretions, cerebro-spinal fluid, pulmonary secretions, (e.g. bronchoalveolar and gastric lavage), pleural fluid and sputum.
  • Mycobacteria may infect almost any tissue of the body.
  • the successful detection of the bacteria may depend on the techniques used in collecting and processing the biological samples.
  • the processing techniques typically are either decontamination and/or concentration procedures, which kill or reduce to negligible levels all bacteria other than mycobacteria and/or which concentrate the number of mycobacteria per volume of sample.
  • types of decontaminaton and/or concentration processes which can be used include the N-acetyl-L-cysteine-(NALC) sodium hydroxide, benzalkonium chloride (zep ⁇ iran) trisodium phosphate, and sputolysin-oxalic acid methods. Versions of these methods are art recognized. 0.
  • Gastric lavage should be processed immediately or neutralized with a basic solution (e.g. 10% sodium bicarbonate) and refrigerated until processed as with sputum. If more than ten ml of watery-appearing aspirate is obtained, cent if gation may be appropriate (e.g. 3600 x g for 30 minutes) and only the sediment saved for processing in the decontamination and/or concentration steps.
  • a basic solution e.g. 10% sodium bicarbonate
  • cent if gation may be appropriate (e.g. 3600 x g for 30 minutes) and only the sediment saved for processing in the decontamination and/or concentration steps.
  • Urine samples can be divided into about four volumetric aliquots, e.g. 50 ml, and centrifuged to form a sediment or pellet (e.g. 3600 x g for 30 minutes). The supernatant fluid may be decanted. The combined sediment then may be collected, brought up to 10 ml and used in the decontamination and/or concentration steps (preferably as in Example 5) .
  • one or two grams of formed stool or 5 ml of liquid stool can be transferred to a 50 ml centrifuge tube and distilled water added to bring the volume up to 10 ml.
  • the suspension is vortexed - 23
  • Pus and wound aspirates can be transferred to a 50 ml centrifuge tube with 10 ml distilled water. The specimen is vortexed vigorously and allowed to stand for 20 minutes; then the suspension is processed as with sputum.
  • Pieces of tissue thought to be contaminated are finely minced using a tissue grinder tub and pestle. Ten ml of distilled water is added, vortexed vigorously, and then allowed to stand for 20 to 30 minutes. The material then is transferred to a 50 ml centrifuge tube. An equal volume of NALC-NaOH is added and then mixed vigorously and allowed to stand for 20 minutes. Twenty-five ml of phosphate buffer is added. The tube is mixed vigorously and centrifuged at 3,600 X g for 20 minutes. The supernatant fluid is decanted and the pellet is suspended in 100 to 200 ⁇ l Tris buffered saline and used in the assays of the invention. - 24 -
  • Cerebrospinal fluid may be centrifuged to concentrate the bacteria and the supernatant fluid discarded.
  • the pellet containing the bacteria may be resuspended in distilled water or in an aqueous solution with thorough mixing and the mixed solution may be used in the agglutination methods of the invention.
  • Pleural fluid may be collected in sterile anticoagulant (e.g. in the presence of ethylene diamine tetraacetic acid or heparin), centrifuged to form a pellet of concentrated bacteria and the supernatant fluid discarded.
  • the pellet may be resuspended in distilled water or an aqueous solution and the mixed solution may be assayed according to the invention.
  • the pleural fluid becomes clotted, it may be liquified using sputolysin and/or vigorous mixing.
  • a pellet from 20 ml of pleural fluid is resuspended in 50 to 100 microliters of buffer and then assayed according to the invention.
  • Blood may be collected in sterile anticoagulant.
  • the blood may be allowed to stand at room temperature until separation of the plasma and other blood components has occurred.
  • the leukocyte-rich plasma may be removed and centrifuged at 400 x g until a pellet is formed (e.g. 15 minutes) .
  • the pellet may be resuspended under
  • the lysed cells may then be centrifuged (3600 x g) until a pellet is formed (e.g. 30 minutes).
  • the pellet may be resuspended in a buffer solution (preferably 100 - 200 ⁇ l) and used in the agglutination methods of the invention.
  • Sputum may be processed by mixing with a basic solution, often a NaOH-NALC solution.
  • the combination of the basic solution and the sputum may be mixed thoroughly, kept for 15 in., and subsequently centrifuged.
  • the supernatant fluid may be decanted and a buffer having a pH of about 6.5 may be added to the pellet.
  • the pH of the sputum may be tested with pH paper to determine whether the sputum is substantially neutral.
  • the details of processing sputum are set forth in detail in Examples 5, below.
  • kits useful for testing biological samples of individuals for the presence of mycobacteria include a plastic container 12 containing particles coated with a monoclonal antibody directed towards mycobacterial antigen.
  • the monoclonal antibodies preferably are the same as those described above.
  • the kit 10 further may have an enhancer as described above and preferably in a
  • the kit further may contain slides having a designated region for performing the assays of the invention.
  • Fig. 4 shows an opaque, white slide 16 having a region 18 designated by a circle drawn on the slide.
  • the region 22 also may be designated by other means such as a recessed region.
  • the kits also may include at least one reagent container 20 including reagents for processing a biological sample.
  • the kits further may include other containers for positive or negative control reagents, and preferably includes a container for negative control latex.
  • the kit may include instructions related to the use of the kit in aiding in the diagnosis of an individual suspected of having or being tested for tuberculosis.
  • a 15 ml polystyrene centrifuge tube was blocked by delivering six ml of a blocking reagent [3% milk caseine, 5% fetal calf serum in coating buffer (20 mM Tris-HCl, 0.15 M NaCl, pH 7.5)] to the tube and - 27 -
  • the supernatant fluid was discarded using vacuum aspiration and the pellet formed during centrifugation was resuspended in two ml of coating buffer.
  • the suspension was vortexed vigorously (and/or pipetted back and forth) to disperse the latex particles. An additional four ml of coating buffer was added and the suspension was centrifuged in the cold again at 27,000xg for fifteen minutes. The supernatant fluid was discarded and the pellet was washed once more as described above.
  • the washed pellet was resuspended in two ml of coating buffer and the latex beads were dispersed in the coating buffer using a pansonicator. The homogeneity of the latex beads was confirmed by microscopic examination. The suspension was delivered to the previously blocked polystyrene tube and an additional 2 ml of coating buffer was added. 1
  • the latex beads are ready to be examined for the presence of antibody on their surface and for the specific activity of these antibodies.
  • the neat ascites may be purified prior to sensitizing the beads. This may be desirable because it increases the stability of the antibody coated beads in relatively high temperature conditions (e.g. - 29
  • Example 3 Selection of Concentration of Enhancer An enhancer (PEG) is dissolved in saline (0.8% NaCl in deionized water) at concentrations ranging from 7% to 0.5%. These various concentrations of enhancer are run in a standardized agglutination assay to determine the minimum concentration of enhancer which causes agglutination. Then, the concentration below that which results in agglutination visible to the naked eye is selected as that for use in the assay of Example 5.
  • PEG enhancer
  • saline 0.8% NaCl in deionized water
  • the enhancer concentration selected for use in the agglutination assay of the invention is chosen as that concentration below, but close to, the - 31 -
  • enhancer concentration that shows autoagglutination. In this example, that concentration either is 4.0% enhancer or 3.5% enhancer. Four % enhancer would provide a slightly more sensitive assay than one using an enhancer concentration of 3.5%.
  • M. tuberculosis for use as a positive control in the latex agglutination test was prepared.
  • Mycobacterium tuberculosis strain TMC 107 (Erdman) is grown for eight weeks in a glycerol-alanine-salts medium as a shaker culture.
  • the Erdman strain was obtained from the Trudeau Mycobacterium Culture Collection, Trudeau Institute, Sarenac Lake, NY, culture number TMC 107 and is available at the ATCC, No. 35801, Rockville, Md., U.S.A.
  • Other strains may be employed, including the rapid growing, attenuated strain of M. tuberculosis H37Ra, obtained from K. Takayama, Madison, WI, described by Takayama, K. , et al. (1975), J. Lipid Res. , 16, 308-317 and available from the ATCC, No. .
  • the cultures are autocleved at 80°C for 1 h, cooled and filtered.
  • the harvested cells are washed several times with distilled water and stored frozen (-20 C) until ready for breakageN Harvested cells (-130 g wet weight) were resuspended in PBS
  • Triton X100 containing 0.5% Triton X100 and 0.02% NaN., (200 ml) .
  • a thick suspension is desirable in order to achieve complete breakage of cells.
  • LAM, lipomannan (LM) and phosphoinositol mannoside (PIM) have a great affinity for detergent. Use of Triton X100 when breaking the cells helped to keep most of these amphipathic molecules in solution thereby giving maximum yield during acetone precipitation.
  • the suspension was sonicated while cooling in an ice bath for 10 min with a W-385 Sonicator Ultrasonic Liquid Processor (Heat
  • the sonicate was passed four times through a French pressure cell (Model SA073; American Instruments Co., Urbana, IL) at 20,000 lb sq. in.
  • the sonicate pressate was centrifuged twice at 27,000 x g for 45 min.
  • the pellet was washed twice with the above buffer (50 ml each time) and recentrifuged.
  • the supernatant fluids were combined and recentrifuged (at 27,000x g) in order to remove most of the cell wall. (The supernatant fluid appeared translucent after centrifugation.)
  • the precipitate was collected by centrifugation at 10,000 x g and air dried.
  • N-acetyl-L-cysteine (NALC powder) is added to an equal volume of a sputum sample in a 50 ml centrifuge tube and vortexed thoroughly. Preferable at least 1 ml of sputum is present. The mixture is allowed to stand for fifteen minutes. The tube is filled up to the 45 ml mark with distilled water and centrifuged at 2800 x g to 3000 x g for fifteen minutes. Subsequently, the supernatant fluid is carefully decanted and 100-200 ⁇ l of P0 ⁇ ⁇ buffer (pH 6.8) is added to the pellet.
  • P0 ⁇ ⁇ buffer pH 6.8
  • the processed sputum is neutralized, if necessary, with neutralizing reagent (I HC1) .
  • the pH of the sputum to be used in the agglutination assay must be approximately pH 7 to allow appropriate physiological conditions for antibody-antigen binding to occur.
  • agglutination assay 20 ⁇ l enhancer, 40 ⁇ l processed sputum sample, and 20 ⁇ l sensitized beads is delivered to a slide.
  • the various reagents are mixed with a mixing stick and the slide is placed on a mechanical rotator for five minutes. The presence of agglutination is then detected.
  • a negative control latex is comprised of 20 ⁇ l enhancer, 40 ⁇ l of the same processed sputum, - 35 -
  • sensitized negative latex beads mixed with sputum and enhancer
  • the negative latex beads are prepared as set forth in Example I, using neat ascites to coat the beads with the exception that the sensitized beads are then stored for at least 7 days at 37°C prior to use. This results in beads sensitized with monoclonal antibody lacking specific activity i.e. the beads will agglutinate with anti-mouse antibody, but not with a sample of positive control antigen or a sample containing mycobacteria.
  • the negative control latex may also be obtained by sensitization of the latex beads with purified monoclonal antibody, but without the use of BSA as a coadsorbent. In this case also the beads bind to anti-mouse antibody, but lack specific activity.
  • Mycobacterium tuberculosis strain TMC 107 (Erdman) is grown for eight weeks (or strain H37Ra for a lesser time) in a glycerol-alanine-salts medium as a shaken culture.
  • the cultures were autoclaved at 80°C for 1 h, cooled and filtered using sterile 0.22 micron
  • the suspension was sonicated while cooling in an ice bath for 10 min with a W-385 Sonicator Ultrasonic Liquid Processor (Heat
  • the sonicate was passed four times through a French pressure cell (Model SA073; American Instruments Co., Urbana, IL) at 20,000 lb per sq. in.
  • the sonicate pressate was centrifuged at 27,000 x g for 45 min, two times. The pellet was washed twice with the above buffer (50 ml each time) and recentrifuged. The supernatant fluids were combined and recentrifuged (at 27,000 x g) in order to remove most of the cell wall. (The supernatant fluid appeared translucent after centrifugation. )
  • distilled acetone was added (to a final concentration of 90% acetone) to precipitate mainly polysaccharides. Some proteins were also precipitated during this procedure. (Considering that very large volumes of solvents were used, it was more efficient when the supernatants were divided into two 1000 ml Erlenmeyer flasks.) The acetone precipitate was stored at 4°C for 48 h.
  • the precipitate was collected by centrifugation at 10,000 x g and air dried. Dry precipitate (1 g) was suspended in 6 ml of 6 M guanidine HC1 in 10mM Tris HC1, pH 7.4 by pansonication. (The insoluble material remaining is removed by low speed centrifugation (2000xg) prior to application to the column.) The soluble material is applied to a sephacryl S-400 column (1.5 x 150 cm) in the same buffer. Fractions (2 ml) are collected and monitored by PAGE. Fractions are pooled according to enrichment with LAM, LM and PIM and dialyzed extensively against water (5 to 6 changes of water) . There is no resolution between LAM, LM or PIM after this preliminary column fractionation. The dialyzed fractions were freeze dried to yield approximately 500 mg of impure material.
  • LAM has many LPS-like biological activities. To ensure that LPS contamination was not present in preparations, lyophilized LAM was redissolved in pyrogen-free water, filtered through 0.45 ⁇ m PTFE filtration unit and passed through 2.0 ml of Detoxi-Gel column (Pierce Chemical, Rockford, IL), refiltered through a second 0.20 ⁇ m sterile filter - 39 -

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

On décrit un sérodiagnostic servant à détecter une mycobactérie dans un échantillon prélevé d'un fluide biologique. Le test consiste à mettre l'échantillon en contact avec des particules enrobées d'un anticorps monoclonal agissant contre une mycobactérie dans des conditions qui permettent une agglutination visible à l'oeil nu lorsque moins de 10,000 bactéries sont présentes par ml de fluide biologique. De préférence, l'agglutination est amplifiée par un agent amplificateur présent en une concentration tout juste inférieure à la concentration minimale en agent amplificateur qui permet une auto-agglutination de particules détectables à l'oeil nu en l'absence d'un antigène. On décrit aussi des kits et des éléments du kit servant à effectuer le sérodiagnostic d'agglutination.
PCT/US1992/001131 1991-02-12 1992-02-12 Serodiagnostic d'agglutination destine a des antigenes mycobacteriens dans des echantillons biologiques WO1992014154A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US65425691A 1991-02-12 1991-02-12
US654,256 1991-02-12

Publications (1)

Publication Number Publication Date
WO1992014154A1 true WO1992014154A1 (fr) 1992-08-20

Family

ID=24624110

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/001131 WO1992014154A1 (fr) 1991-02-12 1992-02-12 Serodiagnostic d'agglutination destine a des antigenes mycobacteriens dans des echantillons biologiques

Country Status (2)

Country Link
AU (1) AU1564592A (fr)
WO (1) WO1992014154A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012190A3 (fr) * 1994-10-13 1996-06-27 Brigham & Womens Hospital Presentation d'antigenes hydrophobes a des lymphocytes t par des molecules cd1
WO1997034149A1 (fr) * 1996-03-12 1997-09-18 Stefan Svenson Methode diagnostique de mycobacteriose et trousse d'essai immunologique
US5679347A (en) * 1992-12-10 1997-10-21 Brigham And Women's Hospital Methods of isolating CD1-presented antigens, vaccines comprising CD1-presented antigens, and cell lines for use in said methods
US5853737A (en) * 1992-12-10 1998-12-29 Brigham And Women's Hospital Method for inducing a CD1-restricted immune response
US6238676B1 (en) 1992-12-10 2001-05-29 Brigham And Women's Hospital Presentation of hydrophobic antigens to T-cells by CD1 molecules
RU2188428C2 (ru) * 2000-06-02 2002-08-27 Научно-исследовательский ветеринарный институт Нечерноземной зоны РФ Способ выявления микобактериальных антигенов
US6465633B1 (en) 1998-12-24 2002-10-15 Corixa Corporation Compositions and methods of their use in the treatment, prevention and diagnosis of tuberculosis
ES2199058A1 (es) * 2002-06-06 2004-02-01 Univ Malaga Reactivo de latex para la deteccion de anticuerpos frente a micoplasma neumoniae.
US7063844B2 (en) 1992-12-10 2006-06-20 The Brigham And Women's Hospital, Inc. Presentation of hydrophobic antigens to T-cells by CD1 molecules
EA009817B1 (ru) * 2004-06-07 2008-04-28 Лысуха, Николай Николаевич Способ выявления туберкулезной инфекции
US20220205989A1 (en) * 2016-09-19 2022-06-30 Massachusetts Institute Of Technology Systems including janus droplets
US12274993B2 (en) 2019-09-18 2025-04-15 Massachusetts Institute Of Technology Systems and methods for affecting interactions of electromagnetic radiation with janus droplets for sensitive detection of species

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4362531A (en) * 1980-04-15 1982-12-07 Technicon Instruments Corporation Agglutination immunoassays carried out with agent to reduce non-specific interferences
US4777130A (en) * 1984-12-05 1988-10-11 Andra Biologicals Isolation of mycobacterial a 60 antigen for diagnostic purposes
US4812414A (en) * 1987-09-18 1989-03-14 Eastman Kodak Company Immunoreactive reagent particles having tracer, receptor molecules and protein of pI less than 6
US4828981A (en) * 1983-08-24 1989-05-09 Synbiotics Corporation Immunoassays for determining Dirofilaria immitis infection using antiidiotype monoclonal antibody reagents
US4851333A (en) * 1984-02-24 1989-07-25 Genetic Systems Corporation Method and composition for the detection and diagnosis of Legionella pneumophila

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4362531A (en) * 1980-04-15 1982-12-07 Technicon Instruments Corporation Agglutination immunoassays carried out with agent to reduce non-specific interferences
US4828981A (en) * 1983-08-24 1989-05-09 Synbiotics Corporation Immunoassays for determining Dirofilaria immitis infection using antiidiotype monoclonal antibody reagents
US4851333A (en) * 1984-02-24 1989-07-25 Genetic Systems Corporation Method and composition for the detection and diagnosis of Legionella pneumophila
US4777130A (en) * 1984-12-05 1988-10-11 Andra Biologicals Isolation of mycobacterial a 60 antigen for diagnostic purposes
US4812414A (en) * 1987-09-18 1989-03-14 Eastman Kodak Company Immunoreactive reagent particles having tracer, receptor molecules and protein of pI less than 6

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
INFECTION AND IMMUNITY, Volume 55, No. 11, issued November 1987, GAYLORD et al., "Most Mycobacterium leprae carbohydrate-reactive monoclonal antibodies are directed to lipoarabinomannan", see Abstract and page 2861. *
JOURNAL OF IMMUNOLOGICAL METHODS, Volume 129, No. 1, issued 08 May 1990, CAMBIASO et al., "Immunological detection of mycobacterial antigens in infected fluids, cells and tissues by latex agglutination", pages 9-14. *
LANCET, Volume 2, issued 01 December 1984, KRAMBOVITIS et al., "Rapid diagnosis of Tuberculosis meningitis by latex particle agglutination", pages 1229-1231. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7063844B2 (en) 1992-12-10 2006-06-20 The Brigham And Women's Hospital, Inc. Presentation of hydrophobic antigens to T-cells by CD1 molecules
US5679347A (en) * 1992-12-10 1997-10-21 Brigham And Women's Hospital Methods of isolating CD1-presented antigens, vaccines comprising CD1-presented antigens, and cell lines for use in said methods
US5853737A (en) * 1992-12-10 1998-12-29 Brigham And Women's Hospital Method for inducing a CD1-restricted immune response
US6238676B1 (en) 1992-12-10 2001-05-29 Brigham And Women's Hospital Presentation of hydrophobic antigens to T-cells by CD1 molecules
WO1996012190A3 (fr) * 1994-10-13 1996-06-27 Brigham & Womens Hospital Presentation d'antigenes hydrophobes a des lymphocytes t par des molecules cd1
WO1997034149A1 (fr) * 1996-03-12 1997-09-18 Stefan Svenson Methode diagnostique de mycobacteriose et trousse d'essai immunologique
US6465633B1 (en) 1998-12-24 2002-10-15 Corixa Corporation Compositions and methods of their use in the treatment, prevention and diagnosis of tuberculosis
RU2188428C2 (ru) * 2000-06-02 2002-08-27 Научно-исследовательский ветеринарный институт Нечерноземной зоны РФ Способ выявления микобактериальных антигенов
ES2199058B1 (es) * 2002-06-06 2005-02-16 Universidad De Malaga Reactivo de latex para la deteccion de anticuerpos frente a micoplasmaneumoniae.
ES2199058A1 (es) * 2002-06-06 2004-02-01 Univ Malaga Reactivo de latex para la deteccion de anticuerpos frente a micoplasma neumoniae.
EA009817B1 (ru) * 2004-06-07 2008-04-28 Лысуха, Николай Николаевич Способ выявления туберкулезной инфекции
US20220205989A1 (en) * 2016-09-19 2022-06-30 Massachusetts Institute Of Technology Systems including janus droplets
US12061194B2 (en) * 2016-09-19 2024-08-13 Massachusetts Institute Of Technology Systems including Janus droplets with binding moieties for a virus, a pathogen or a bacterium
US12274993B2 (en) 2019-09-18 2025-04-15 Massachusetts Institute Of Technology Systems and methods for affecting interactions of electromagnetic radiation with janus droplets for sensitive detection of species

Also Published As

Publication number Publication date
AU1564592A (en) 1992-09-07

Similar Documents

Publication Publication Date Title
AU740158B2 (en) Beta(1-3)-glucan diagnostic assays
US4847199A (en) Agglutination immunoassay and kit for determination of a multivalent immune species using a buffered salt wash solution
US4683196A (en) Method and materials for the identification of lipopolysaccharide producing microorganisms
EP0280559B1 (fr) Essai immunologique d'agglutination et trousse de réactifs pour la détermination d'une espèce immunollogique polyvalente utilisant une solution saline de nettoyage tamponnée
EP0209273B1 (fr) Méthode pour la détection, pour la séparation et pour la purification de la toxine A de clostridium difficile et de ses récepteurs
US6413715B2 (en) β(1-3)-glucan diagnostic assays
Ivanyi et al. Immunodiagnostic assays for tuberculosis and leprosy
WO1992014154A1 (fr) Serodiagnostic d'agglutination destine a des antigenes mycobacteriens dans des echantillons biologiques
KR880001809B1 (ko) 성인 t 세포 백혈병 연관항원의 제조방법
Haikala et al. Rapid detection of rotavirus in stool by latex agglutination: comparison with radioimmunoassay and electron microscopy and clinical evaluation of the test
EP0516529A2 (fr) Détection des anticorps spécifiques
EP0101166B1 (fr) Procédé pour le dosage d'anticorps d'une maladie infectieuse
JP3186831B2 (ja) 血液型試薬としての凝集複合体
WO1985002685A1 (fr) Procede et substances permettant l'identification de micro-organismes produisant du lipopolysaccharide
WO1992014155A1 (fr) Anticorps monoclonaux diriges contre des antigenes mycobacteriens
JPS6314911B2 (fr)
Yugi et al. Serologic diagnosis of bovine tuberculosis
JP3337575B2 (ja) 抗ストレプトリジンｏ抗体の決定方法
GB1592825A (en) Rubella virus immunoassay materials and methods for making and using same
WO1995002186A1 (fr) Nouveau dosage diagnostique destine a la detection de la syphilis
JPS60249058A (ja) Ａｔｌウイルス抗体の測定方法および試薬
White et al. Diagnostic and therapeutic significance of staphylococcal teichoic acid antibodies
CN111948396A (zh) 一种阿达木单抗抗药抗体的胶体金试剂盒及其制备方法
KR100254414B1 (ko) 항-결핵균 항체 및 나균 항원에 대한 항혈청을 포함하는 결핵균 항원 검출용 진단키트
Parija Recent trends in the serodiagnosis of hydatid disease

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA FI HU JP NO

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA