DD289559A5 - PROCESS FOR THE PREPARATION OF MONOCLONAL ANTIBODY AGAINST ATRAZINE AND / OR DERIVATIVES, THEIR USE IN IMMUNASSAYS AND TEST KITS - Google Patents

Abstract

The invention relates to a method for the production of monoclonal antibodies and their use in immunoassays for the immunological detection of atrazine and / or atrazine derivatives of hydroxyatrazine and / or hydroxyatrazine derivatives in the form of ready-to-use test kits. The process according to the invention is characterized in that a) atrazine and / or an atrazine derivative or hydroxyatrazine and / or hydroxy analogues of atrazine derivatives are conjugated with a carrier molecule, b) a donor animal is immunized with said conjugate, c) an immune component B cell from the isolated immunized donor animal, d) said immune component B-cell fused with a continuous cell division capable tumor cell line, e) the resulting fusion product isolated, cultured in a suitable culture medium and then cloned, f) the cloned hybridoma (hybridoma) on the formation of monoclonal G. Examining the antibody bodies, and g) culturing the hybridoma cell lines or clones or subclones thereof produced according to method steps a) to e) in vivo or in vitro by means of known methods and isolating the producing monoclonal antibodies {antibody, monoclonal; Proof; atrazine; Atrazinderivate; hydroxyatrazine; Hydroxyatrazine derivatives, immunological}

Description

Field of application of the invention

The present invention relates to processes for the preparation of monoclonal antibodies characterized by a high selectivity and affinity towards atrazine and / or atrazine derivatives or to inactive atrazine metabolites. and are therefore excellently suited for use in an immunoassay for the rapid and effective detection of atrazine and / or atrazine derivatives as well as inactive atrazine metabolites.

Characteristic of the known state of the art

The use of synthetic herbicides for the purposes of crop protection and the associated environmental pollution have recently become the focus of public discussion.

s-triazines and s-triazine derivatives such as atrazine, simazine or propazine have been used extensively since the discovery of their herbicidal potency in the 1950's for the purposes of crop protection, particularly for control of annual broadleaf weeds and various wild grasses in maize crops.

Cautious estimates suggest that corn fields currently range in size from 45 million to

100 million hectares are treated with s-triazines or s-triazine derivatives.

It has long been known that s-triazines, such as. As atrazine, simazine or propazine in the soil and other biotopes only relatively

be slowly degraded.

This persistence of s-triazines in the soil is also of great importance to the farmer. When using atrazine in Application rates as z. B. are necessary in maize crops, already within a crop rotation problems in

Atrazinempfindlichen subsequent crops occur, so that the farmer may be forced to elaborate

Soil analysis in the laboratory. The analysis methods used there are extremely time and kooteuintensiv,

because they are tied to highly specialized and expensive equipment.

For the reasons given, it must therefore be regarded as an urgent task, the existing one Detection method for synthetic herbicides, in particular for s-triazines and s-triazine derivatives and for their Degradation products to be improved, d. H. To develop more cost effective, more efficient and more manageable procedures, too

can be applied outside the laboratory under field conditions and which, for example, quickly and reliably provide the farmer with information on whether and at what concentrations a particular active substance or metabolite, eg. In the

Soil or water is present. Of particular importance in this context is the ability to differentiate the active agent

its inactive decomposition products, as this is the only way to quantify what is actually left in the soil

Active ingredient is possible. Atrazine and its major degradation product hydroxyatrazine were previously detected in soil and water samples

primarily by means of TLC (thin-layer chromatography) and by means of spectroscopic methods. Today, by contrast, HPLC (High Pressure Liquid Chromatography) (Ramsteiner and Hörmann, 1979) and GC (Gas

Chromatography) (Ramsteiner K, 1985). However, all of these methods for the detection of synthetic herbicides are associated with numerous disadvantages. So

For example, for the determination of atrazine or hydroxyatrazine in soil samples by HPLC prior to performing the actual chromatographic analysis, elaborate purification and concentration steps must be interposed.

Another disadvantage of HPLC methods is the fact that photometric detectors are used there

become relatively unspecific. In addition to the mass spectroscopic detection based chromatographic

Analyzes on the determination of retention times of the respective system. These values are relative and not so

structure-specific.

The gas chromatographic analysis of hydroxyatrazine in conjunction with a mass spectroscopic determination is

due to the low volatility of this compound feasible only with considerable effort.

In order to avoid these disadvantages of the established analytical methods described above, it has recently been attempted

also to develop immunological methods for the agricultural sector, as already routinely used in clinical diagnostics for the detection of the most diverse antigens, in particular for the quantitative and qualitative ones

Determination of agrochemicals in soil, water or air samples. For example, immunological procedures for the detection of certain herbicides, such as

2,4-dichlorophenoxyacetic acid (Fleeker, 1986) or chlorsulfuron (Kelley et al., 1985) and various pesticides, such as

Diflubenzuron (Wie and Hammock, 1982), metalaxyl (Newsome, 1985) or parathion (Ercegovich et al., 1982). A process has also been described for the immunological detection of atrazine (US Pat. No. 4,530,786), which however, like US Pat

also the aforementioned methods based on the use of polyclonal antisera obtained from animals previously immunized with a corresponding antigen.

Polygonal antisera are very heterogeneous in their composition, i. H. they contain a variety of different antibodies,

which react with different epitopes of the respective antigen. This heterogeneous composition is polyclonal

Antisera is due to the fact that in the immunization of a test animal with a particular antigen always

several antibody-producing cell clones are stimulated simultaneously, each of which has a different epitope on the

Recognizes antigen molecule and therefore produced by the stimulated cell clones different antibodies. For this reason, immune sera from immunized animals are always polygonal and therefore heterogeneous, both in terms of their specificity and

as well as their affiliation to the individual classes of immunoglobulins.

This heterogeneity in the composition of polyclonal antisera may therefore lead to structurally closely related Compounds, such as the atrazine and its main degradation product hydroxyatrazine when using polyclonal Antibodies can not be sufficiently differentiated in the context of an immunoassay. Object of the invention

The invention provides a method for producing monoclonal antibodies which have high specificity and affinity for atrazine and / or atrazine derivatives and their use in easily manageable, effective and highly selective immunoassays rapid and reliable detection of atrazine and / or atrazine derivatives as well as of inactive atrazine degradation products but in particular of hydroxyatrazine and / or hydroxatrazine derivatives.

-6- 289 Explanation of the essence of the invention

The object to be solved in the context of this invention, however, was primarily to provide a method for producing monoclonal antibodies having a high specificity and affinity for atrazine and / or atrazine derivatives, and to provide an easily manageable, effective and highly selective immunoassays for a rapid and reliable detection of atrazine and / or atrazine derivatives and of inactive atrazine derivatives, but in particular of hydroxyatrazine and / or hydroxyatrazine derivatives, which enable a differentiation of atrazine and / or atrazine derivatives against their degradation products.

This object has now been surprisingly achieved in the context of the present invention by providing methods for producing monoclonal antibodies with high specificity and affinity for atrazine and / or

Atrazinder'ivate and by providing methods for producing monoclonal antibodies with high specificity and affinity for the degradation products of atrazine and / or Atrazinderivaten, but in particular for the hydroxy analogues of atrazine and / or Atrazinderivaten such. Hydroxyatrazine, hydroxysimazine, hydroxypropazine, etc., using the conventional hybridoma / monoclonal antibody technology.

The use of hybrid somatic cell lines (hybridomas) as a source of antibodies against very specific antigens is based on the work of Köhler and Milstein (Nature 256: 495-497, 1975).

The antibodies which can be obtained by the method described there are very different from those obtained from antisera of conventionally immunized animals.

The principle of hybridoma / monoclonal antibody technology is based on the observation that when two somatic cells merge, the resulting hybrid cell has characteristic features of both parent types.

In the case of monoclonal antibody production, the ability to synthesize the specific antibody derives from an immunocompetent B cell (usually a spleen cell) taken from a previously immunized Donur animal, while the ability to continuously divide in culture through the other fusion partner, a tumor cell line (often a myeloma) is contributed.

Each of these hybrid cell lines synthesizes a homogeneous immunoglobulin representing only a single member of the large number of potential antibodies that can be synthesized by an animal in response to an antigen in vivo.

Since each immunoglobulin-producing clone is characterized by a single type of antibody, the term monoclonal antibody has come to be used.

The advantages of monoclonal versus polyclonal antibodies are numerous:

a) monoclonal antibodies can be obtained in large numbers and in a high degree of purity,

b) the production of monoclonal antibodies is homogeneous with respect to antigen reactivity and does not change with time,

c) monoclonal antibody-producing hybridomas can be stored for years and decades without losing their specific properties, i. to lose production of specific monoclonal antibodies,

d) monoclonal antibodies are more suitable for use as standard reagents than polygonal antisera, as the latter are adversely affected by a wide variation, for example with respect to α) the collection of immunized animals to obtain the antiserum, β) a constant availability of material for additional immunizations , γ) the limited lifespan of the donor animals.

Monoclonal antibodies, which have since been produced against a variety of antigens, are well established in medical diagnostics and are indispensable.

In contrast, the use of monoclonal antibodies in the agricultural sector has so far been limited primarily to the screening of plant diseases and the devel opment of vaccines for livestock.

In the context of plant diseases, for example, Hsu, H.T., et al. (ASM News, 50 (3): 91-101, 1984) lists a total of 18 species of plant hosts against which monoclonal antibodies have been developed, including "Carnation Etched Ring Virus", "Potato Leaf Roll Virus", "Southern Bean Mosaic Virus". , Tobacco Mosaic Virus, Tomato Ring Spot Virus and Tulip Breaking Virus.

In the context of the present invention, it has now been possible for the first time using monoclonal antibodies with high specificity and affinity for atrazine and / or atrazine derivatives as well as monoclonal antibodies with high specificity and affinity for the hybridoma / monoclonal antibody technology described above inactive decomposition products of atrazine and / or atrazine derivatives which, because of their high specificity and consequent low mutual cross-reactivity, are excellently suited for use in an immunoassay for the rapid and reliable detection of atrazine and / or atrazine derivatives on their and their inactive On the other hand, degradation products are suitable and can thus also be used for differentiating atrazine and / or atrazine derivatives from their inactive metabolites.

The present invention thus relates primarily to processes for the preparation of monoclonal antibodies and their derivatives, which have a high specificity and affinity towards atrazine and / or atrazine derivatives and which substantially no cross-reactivity with metabolites of atrazine and / or atrazine derivatives, but in particular no cross-reactivity with the inactive hydroxy analogues of atrazine and / octane atrazine derivatives.

A further subject of this application relates to processes for the preparation of monoclonal antibodies and their derivatives, which have a high specificity and affinity for the inactive degradation products of atrazine and / or atrazine derivatives, in particular towards the hydroxy analogues of atrazine and / or atrazine derivatives, and which substantially no cross-reactivity with atrazine and / or atrazine derivatives.

Particularly preferred in the context of this invention are methods for producing monoclonal antibodies and their derivatives which have a high specificity and affinity for atrazine and which show substantially no cross-reactivity with hydroxyatrazine or hydroxy analogs of atrazine derivatives, such as hydroxysimazine, hydroxypropazine, etc. Also preferred in the context of this invention are processes for the preparation of monoclonal antibodies and their derivatives which have a high specificity towards hydroxyatrazine and which show substantially no cross-reactivity with atrazine or atrazine derivatives, such as, for example, For example, propazine, simazine, ametryn, atraton, and the like.

Also included in the present invention are methods of making monoclonal antibodies and their derivatives,

which have a high specificity and affinity towards atrazine and which have a pronounced cross-reactivity, in particular a cross-reactivity of more than 50%, preferably more than 80%, with certain structurally similar

Atrazlnderivaten show, especially those selected from the group consisting of propazine, and Prometryn Prometon, but essentially no cross-reactivity with other Atrazinderivaten or with hydroxy analogues of atrazine

and / or atrazine derivatives.

A further subject of this application relates to processes for the preparation of monoclonal antibodies and their derivatives which

have a high specificity and affinity for hydroxyatrazine and have a cross-reactivity, but especially one

Cross reactivity of at least 40%, with different hydroxy analogues of Atrazinderivaten show, but in the

essentially show no cross-reactivity with atrazine and / or atrazine derivatives.

Very particularly preferred within the scope of this invention are processes for the preparation of monoclonal antibodies and their Derivatives which have a high specificity towards hydroxyatrazine and which have a pronounced cross-reactivity,

but especially a cross-reactivity of up to 100%, with the structurally very similar HyHroxypropazin show, but essentially have no cross-reactivity with atrazine or Atrazinderivaten such. For example, propazine, simazine, ametryn,

Atraton et al. and hydroxy analogues of atrazine derivatives such as hydroxysimazine, hydroxydesetrmine, hydroxyterbuthylazine

etc. have.

Among derivatives of monoclonal antibodies are within the scope of the present invention, for. B. to understand antibody fragments,

which still has the high specificity and affinity for the antigenic determinants of atrazine and / or atrazine derivatives or

Hydroxyatrazine and / or hydroxy analogues of Atrazinderivaten own, further radiolabeled monoclonal Antibodies containing, for example, radioactive iodine ( ¹²⁶ I, ³¹ I) carbon ( ¹⁴ C), sulfur ( ³⁶ S), tritium ( ³ H) or the like

are labeled, conjugates of monoclonal antibodies with biotin or avidin, with enzymes such as horseradish peroxidase, alkaline

Phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic acid anhydrase, acetylcholinesterase, lysozyme, Malate dehydrogenase or glucose-6-phosphate dehydrogenase, and conjugates of monoclonal antibodies

bioluminescent (eg luciferase), chemiluminescent (eg acridinium ester) or fluorescent (eg.

Phycobiliproteins) agents. Also included in the present application are bispecific and so-called "cross-linked" Antibody. This exemplary listing of possible antibody derivatives is merely illustrative of the present invention Invention and is not intended to limit the subject invention in any way. The term "substantially no cross-reactivity" is to be understood in the context of this invention that the Reactivity of the specific for atrazine or hydroxyatrazine monoclonal antibody with non-specific epitopes of others Compounds, in particular structurally related compounds less than 20%, but preferably less than 5% and

most preferably less than 1%.

The percentage of cross-reactivity, in the context of this invention, is defined by the following relationship

be reproduced:

(Atrazine / Hydroxyatrazine Concentration for 60% Inhibition / Concentration of s-Triazine / Hydroxytriazine Analogs for 50% Inhibition) χ 100.

A 50% inhibition can be determined, for example, by means of a competitive ELISA assay (see Example 8). These

then corresponds for example to that antigen concentration which leads to a 50% inhibition of antibody binding to the carrier-bound antigen.

Hybridoma cell lines which synthesize the previously characterized monoclonal antibodies and preferably in the

secrete surrounding medium are described.

In particular, a hybridoma cell line is described which produces a monoclonal antibody of high specificity

and affinity for atrazine and / or atrazine derivatives, and which exhibits substantially no cross-reactivity with the inactive hydroxy analogues of atrazine and / or atrazine derivatives.

Also described is a hybridoma cell line that produces a monoclonal antibody that has high specificity and specificity Affinity to the hydroxy analogues of atrazine and / or Atrazinderivaten and has essentially no Cross reactivity with atrazine and / or Atrazinderivaten shows. Furthermore, a hybridoma cell line is described which produces a monoclonal antibody having a high specificity

and having affinity for atrazine and having substantially no cross-reactivity with hydroxyatrazine or others

Hydroxy analogues of Atrazinderivaten shows. Particularly preferred is a hybridoma cell line that synthesizes a monoclonal antibody and converts it into the surrounding Secreted medium which has high specificity and affinity for atrazine and which has a very strong cross-reactivity,

but in particular a cross-reactivity of more than 50%, preferably of more than 80%, with structurally similar

Atrazindeiivaten shows, especially those selected from the group consisting of propazine, and Prometryn Prometon, but essentially no cross-reactivity with other Atrazinderivaten and hydroxyatrazine or Hydroxy analogs of atrazine derivatives shows. Most preferred is a hybridoma cell line which has the distinguishing characteristics of ECACC 8808/2501

owns, as well as their clones and subclono.

Furthermore, a hybridoma cell line is described which produces a monoclonal antibody having a high specificity

and having affinity for hydroxyatrazine and having substantially no cross-reactivity with atrazine and / or

Atrazine derivatives shows. Also particularly preferred within the scope of this invention is a hybridoma cell line which is a monoclonal antibody

which has a high specificity and affinity for hydroxyatrazine and which has a cross-reactivity, in particular a cross-reactivity of more than 40%, with structurally similar compounds such as hydroxysimazine, hydroxypropazine and

Hydroxydesmetryn, but having substantially no cross-reactivity with atrazine and / or atrazine derivatives. Most preferred is a hybridoma cell line which has the distinguishing characteristics of ECACC 8808/2502

owns, as well as their clones and subclones.

Also preferred in the context of this invention is a hybridoma cell line that synthesizes a monoclonal antibody

and secreted into the surrounding medium, which has a high specificity and affinity for hydroxyatrazine and which has a highly pronounced cross-reactivity but in particular a cross-reactivity of up to 100%, with structurally very similar

similar hydroxypropazine, but having substantially no cross-reactivity with other hydroxy analogues of Atrazinderivaten, such as hydroxysimazine, Hydroxydesmetryn or Hydroxyterbuthylazin, and with atrazine and / or Atrazinderivaten.

Most preferred is a hybridoma cell line having the distinguishing characteristics of ECACC 8808/2503, as well as their clones and subclones.

Likewise encompassed by the present invention are variants and mutants of the hybridoma cell lines previously characterized in more detail, which arise spontaneously or else can be prepared artificially by known processes and which still have the characteristic properties of the starting material, ie. H. which are still able to produce antibodies of the invention or derivatives thereof and to secrete into the surrounding medium.

Also within the scope of the present invention are methods of making said hybridoma cell lines and methods of making said monoclonal antibodies.

Cloning and subcloning of hybridoma cell lines is to be understood as meaning hybridomas which emerge from the parent clone by repeated clonorone and which still have the features of the starting clone essential to the invention.

Another object of this invention relates to a method for the immunological detection of atrazine and / or Atrazinderivaten, z. B. in soil, water or air samples and in biological material such. In plant or animal extracts, using the monoclonal antibodies of the invention.

Furthermore, the present invention relates to a method for the immunological detection of hydroxyatrazine and / or hydroxy analogues of Atrazinderivaten, z. B. in soil, water or air samples and in biological material such. In plant or animal extracts, using the monoclonal antibodies of the invention.

Very particularly preferred is a method for the detection of hydroxyatrazine.

Also part of the present invention are means for the qualitative and quantitative determination of atrazine and / or Atrazinderivaten or degradation products of atrazine and / or Atrazinderivaten, but especially of hydroxy analogues of atrazine and / or Atrazinderivaten, such as. Hydroxyatrazine, hydroxypropazine, hydroxysimazine, etc. in the form of ready-to-use test kits containing at least one of the monoclonal antibodies of the invention as a reagent and for use under field conditions for rapid and reliable detection of atrazine and / or Atrazinderivaten, as well as degradation products of atrazine and / or atrazine derivatives, but especially of hydroxyatrazine and / or hydroxy analogues of atrazine derivatives are suitable.

The monoclonal antibodies according to the invention are prepared by methods known per se which are based essentially on the methods developed by Köhler and Milstein (Nature, 256: 495-497, 1975).

Since the target substances to be analyzed, atrazine and hydroxyatrazine, for which specific monoclonal antibodies are to be developed, are relatively small and simple molecules which, after application to an experimental animal alone, are not able to trigger a corresponding immune response there preparatory measures are taken before the actual immunization.

One refers to those compounds which are due to their size and simple structuring not to induce an immune reaction in length, as Heptene or incomplete antigens and thus it faces the complete antigens (= immunogens), which act both as an antigen and an immune response induce assets. Such hapten molecules can be conjugated to high molecular weight compounds (carrier molecules), making them comparable in their properties to complete antigens, i. H. they now have the ability to trigger an immune response.

Some of the antibodies formed in the course of the immunization reaction are then able to react with specific epitope on the hapten molecule, regardless of whether the hapten molecule is alone or still coupled to the carrier molecule.

In the context of this invention, the term hapten, which is frequently used in the following, should primarily be understood to mean the atrazine and / or hydroxyatrazine molecules used for the immunization.

In the context of this invention, therefore, the atrazine and hydroxyatrazine molecules acting as haptens are coupled before the immunization of experimental animals to a high molecular carrier ('carrier') which is suitable for imparting to said atrazine and hydroxyatrazine molecules complete antigenic activity.

Suitable carrier molecules in the context of this invention are primarily macromolecular compounds which have freely accessible reactive groups for the coupling reaction with the hapten and which, by coupling to the hapten, are able to impart an immunogenic potency to the hapten or to increase their already existing immunogenicity.

Particularly preferred in the context of this invention are macromolecular compounds which contain freely accessible reactive amino groups.

Very particularly preferred for use according to the invention as a carrier molecule are lysine-rich proteins having a molecular weight of between 10,000 and 1,500,000, such as e.g. "Bovine serum albumin" (BSA: Mü 66200), human serum albumin (HSA: mw 58000) or "keyhole limpet protein" (KLH: mw 1,000,000), which are commercially available and are therefore available in any amount.

Of course, in the context of the present invention, other macromolecular compounds can be used as carrier molecules, provided that they meet the above requirements, such. B. "porcine thyroglobulin", B2 microglobulin, hemocyanin, immunoglobulins, toxins (cholera, tetanus, diphtheria toxin, etc.), polysaccharides, lipopolysaccharides, natural or synthetic polyadenyl and Polyuridylsäuren, polyalanyl and polylysine polypeptides or cell membrane components, such as For example, formalin or glutaraldehyde-treated erythrocyte cell membranes.

Also suitable for use as a carrier molecule in the process according to the invention is, for example, the purified IgG fraction against mouse IgG (H + L) from rabbit according to the method described in H. Kawamura and J.A. Berzofsky (J. Immunol., 136: 58,

1986).

The conjugation of the hapten to the carrier molecule can be carried out either directly or preferably via a bridge member, which is optionally initially attached to the hapten molecule.

The coupling of the substance to be analyzed to the carrier molecule must be carried out in such a way that the relevant structural elements of the target substances remain freely accessible and are thus able to trigger a specific immune response, ie. H. to induce the formation of specific antibodies.

As bridge members for a conjugation of the hapten (atrazine and / or atrazine derivatives and the hydroxy analogues of atrazine and / or atrazine derivatives) to the Csrriermolekül are primarily compounds which contain at least one or more reactive groups which are capable of to interact with the freely accessible reactive groups of the carrier molecule.

Particularly preferred in the context of this invention is the use of bridge members which comprise between 3 and 10 bridging carbon atoms and which as reactive group (s) one or more reactive groups, such as. B. amino, carboxyl or SH group (s) possess.

These reactive groups can be reacted with the reactive groups of the hapten and carrier molecule using known methods to form a hapten-carrier conjugate. Thus, for example, it is possible to bind a bridge member via a reactive amino group with the aid of dialdehydes (eg glutardialdehyde) to one of the free amino groups of the carrier molecule.

If the bridge member has a reactive SH group, the conjugation of the hapten to the carrier molecule can be carried out by oxidation with free SH groups of the carrier.

Particularly preferred in the context of this invention is the use of bridge members having a carboxyl group which can be linked to a free amino group of the carrier molecule by means of water-binding agents, such as, for example, a carbodiimide, preferably N, N'-dicyclohexylcarbodiimide.

In a specific embodiment of the present invention, atrazine, hydroxyatrazine or one of its derivatives, preferably 2,6-dichloro-4- (isopropylamino) -s-triazine, are used and this is reacted with δ-aminovaleric acid to give 2-chloro -4- (isopropylamino) -6- (5-aminovaloriansäure) -s-triazine. This compound can then by known methods such. B. by the addition of aqueous mineral acids, in a further reaction step are very easily converted into the corresponding hydroxy analogues.

The actual coupling reaction is preferably carried out by means of the active ester method. The atrazine or hydroxyatrazine derivatives are first solubilized in a suitable solvent. Suitable solvents are in particular aprotic solvents which have a low evaporation rate, such as. B. Ν, Ν-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) into consideration.

Subsequently, the carboxyl groups are derivatized to an active ester by the previously solubilized atrazine and / or Hydroxyatrazinderivate z. B. with N-hydroxysuccinimide, N-hydroxysulfosuccinimide, N.N'-dicyclohexylcarbodiimide or Ν, Ν'-carbonyldiimidazole or with derivatives of these compounds are reacted. The active ester is then separated from the reaction mixture and added to BSA or KLH. After an incubation period of 0.1 to 12 hours, preferably 4 to 5 hours, the precipitate is removed. The supernatant can then optionally be used after the interposition of further purification steps for the actual immunization reaction. In addition to the preferred in the context of this invention active ester method, alternative methods for coupling the hapten to the carrier molecule can be used, such. B. the mixed anhydride method. In this case, the carboxyl group of the bridge member is attached to the carrier molecule using acetic anhydride or the carbodiimide derivative 1-ethyl-3- (3'-dimethylaminopropyldicarbodiimide.

The immunization of the donor animals is carried out by one or more applications of coupled to a high molecular weight carrier Heptene. Particularly preferred is a 2 to 3 times application, which takes place at intervals of 7 to 30 days but especially from 13 to 15 days.

The preferred form of administration in the context of the present invention is the injection, which can be carried out both intravenously, intraperitoneally or subcutaneously.

Preferred is a combination of subcutaneous and intraperitoneal injection.

After a rest period of 0.5 to 4 months, a further single application of the hapten conjugate takes place in a dosage of 100 pg to 1000 mg.

In a period of 1 to 6 days after the last dose, the donor animals are sacrificed and a spleen cell suspension is prepared.

The isolated spleen cells are thereby suspended in a suitable buffer (eg a BSS buffer) and stored in the form of a cell suspension until their fusion with suitable myeloma cells.

Initially, these fusions were complicated by the fact that the myeloma cell lines also monoclonalally synthesized antibodies, so that the hybrid produced two types of monoclonal antibodies, one that had its origin in the myeloma cell and a second one, by the genetic information of the immunocompetent cell was determined. In the context of the present invention, therefore, it is preferable to use those tumor cells which themselves can not produce monoclonal antibodies, e.g. SP2 / O-Ag 14 (Shulman et al., 1978) or X 63-Ag £ .653, greatly simplifying the analysis of the resulting fusion products. For fusion success, it is advantageous if the spleen cells are present in a 2 to 20-fold excess relative to the myeloma cells.

Fusion of spleen and myeloma cells is performed in a special fusion medium that has a composition that provides optimal conditions for the intended cell fusion.

Preferably, said fusion medium is a buffer solution containing one of the fusion promoters commonly used for fusion of cells, such as e.g. B. Sendai viruses or changes. Paramyxoviruses, optionally in UV-inactivated form, calcium ions, surface-active lipids, such as lysolecithin or polyethylene glycol. Particularly preferred in the context of this invention is the use of polyethylene glycol (PEG), in particular of polyethylene glycol (PEG) with an average MW of 600 to 6000, and in a concentration of 30% to 60%. Particularly preferred is a PEG concentration of 40% -50%. The optimum fusion temperature is between 18 ⁰ C and 39 ° C. Particularly preferred is a temperature of 37 ° C.

After fusion of the immunocompetent spleen cells with the myeloma cells, the fused antibody-producing hybrid cells are selected by methods known per se.

There are various possibilities for reading out successful fusion events (hybrid cells) from the 2 types of parent cells. Routinely, one million and more cells of each parent type are used in the fusion protocol. Because fusion does not occur at a 100% frequency, it can be a difficult task to separate the fusion products from the vast majority of unfused or self-fused parent cells. As mentioned previously, the hybridoma cells are formed by the fusion of short-lived antibody-producing (spleen) B cells and long-lived myeloma cells.

The desired result is a long-lasting cell line that produces antibodies. In principle, since the spleen cells only have a limited lifespan in culture, it is easy to wait until all non-fusion spleen cells and all self-fused spleen cells have died. However, there remains the task of separating the long-lived antibody-producing cells from the long-lived non-antibody-producing cells. A common selection system is based on the availability or unavailability of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). This enzyme is part of the purine "salvage pathway" in mammalian cells.

These cells are also able to synthesize purines de novo.

Under normal circumstances, both synthetic routes are likely to operate to some extent in parallel. However, if a cell does not have HGPRT, the salvage pathway is blocked and the purines must be made from non-purine material.

For the selection of HGPRT-negative myeloma cells, so-called purine antimetabolites, such as, for example, are generally used. For example, the 8-azaguanine is structurally very similar to purine guanine and can therefore replace it in some of its normal reactions.

Azaguanine is incorporated into the DNA, resulting in impaired normal growth behavior and ultimately cell death. Since azaguanine must be replaced via the salvage pathway, all those non-HGPRT-active users can not use azaguanine and thus grow in its presence.

A selective system which works with the same enzyme but with opposite signs, in which case HGPRT positive cells are selected, is described in J.W. Littlefield (1964).

This selection system is based on the use of the so-called HAT medium, which i.a. Hypoxanthine, aminopterin and thymidine (HAT medium) as constituents. Aminopterin is an antimetabolite that inhibits de novo purine synthesis and methylation of deoxyuridylate to thymidylate.

Hypoxanthine can act as an auxiliary trace in the event that aminopterin blocks da novo purine synthesis while thymidine obviates the need for methylation of deoxyuridylate.

Therefore, in the presence of aminopterin, all HGPRT positive cells will proliferate while the HGPRT negative cells die off.

In the hybrid system used for selection in this invention, the myeloma cells are preferably resistant to azoguanine and sensitive to aminopterin, i. H. they are HGPRT negative. In contrast, the antibody-producing cells are HGPRT positive.

By fusing the cells and cultivating in a HAT medium, the cells successfully fused together can be selected, since the myeloma cells responsible for proliferation can only grow in the presence of HGPRT activity and reject this activity from the HGPRT. positive cell line must be delivered.

Although the HGPRT-positive antibody-producing cell lines are not killed in this medium. But they survive only for a certain time, and are unable to proliferate.

Thus, by fusing the cells into a HAT medium, a system is created in which the myeloma cells and the antibody-producing cells can grow for a time sufficient for the production of hybrid cells, but in which only the hybrid cells survive and can proliferate.

In a particular embodiment of the present invention, the fused hybrid cells are cultured in the presence of macrophages previously isolated from the peritoneum of untreated, unimmunized experimental animals, so-called "feeder cells." For the cultivation and selection of the fused hybrid cells, the cell suspension is divided into several aliquots and the individual aliquots are constantly being investigated for the formation of hybrid cell cultures and for the formation of antibodies.

Particularly preferred in the context of this invention is the cultivation of the fused hybrid cells on microtiter plates. The cell suspension obtained after the fusion is distributed to the individual wells of a microtiter plate and cultivated for a period of 7 to 30 days under suitable conditions promoting the growth of the fused hybrid cells (eg HAT / HT media).

The supernatants of grown hybrid cultures are constantly examined for the formation of antibodies. Positive hybrid cell cultures are then isolated using known methods, but preferably by means of the limiting dilution method and then cloned in suitable culture media. The supernatants of the grown cell clones are also checked for the formation of antibodies. The screening of the hybridoma cell clones according to the invention produced according to the above description for the formation of suitable monoclonal antibodies is preferably carried out with the aid of one of the immunoassays commonly used for this purpose, such. As an enzyme-linked immunoassay or a radioimmunoassay. In the enzyme-coupled immunoassay, the previously characterized hapten conjugates are first adsorbed to a solid support. The remaining free binding sites are then saturated by the addition of carrier molecules and thus blocked.

For the detection of monoclonal antibodies, aliquots of the supernatants of said hybridoma cell clones are incubated with the carrier-bound hapten conjugates.

Another object of the present invention relates to the production of monoclonal antibodies using methods known per se, which are characterized in that the above-characterized hybridoma cell lines according to the invention or clones or subclones thereof, which synthesize the antibodies according to the invention and secrete into the surrounding medium, Cultivated in vitro or in vivo using known methods.

The in vitro cultivation of the hybridoma cells according to the invention is carried out in suitable culture media, in particular in the commonly used, standardized culture media such. Dulbacco's Modified Eagle Medium (DMEM) or RPM11640 medium, optionally supplemented by the addition of mammalian sera, e.g. ß. Fetal calf serum, or may be supplemented by growth-promoting additives and trace elements.

The isolation of the monoclonal antibodies preferably begins with a precipitation of the immunoglobulin fraction from the respective supernatants of the hybridoma cultures, eg. B. with the aid of ammonium sulfate. This is followed by further work-up and purification steps which are known to the person skilled in the art and which include, for example, the wounding of ultrasound chromatographic methods, such as e.g. G., Gel filtration, ion exchange chromatography, DEAE-Celluloso chromatography, protein A, or immunoaffinity chromatography.

However, large amounts of the monoclonal antibodies according to the invention can also be obtained with the aid of in vivo methods.

For example, it is possible to inject antibody-producing hybridoma cell clones into suitable mammals which induce the development of antibody-producing tumors in the treated animals. After a period of 1 to 3 weeks, the antibodies can be isolated from the body fluids of the animals thus treated. In a particular embodiment of the present invention, female Balb / c mice optionally supplemented with a hydrocarbon such as e.g. Pristane, an inventive hybridoma cell clone Injected intraperitoneally.

One to three weeks after injection of the hybridoma cell clone, the ascites fluid is collected and stored until further processing.

The isolation of the monoclonal antibody is carried out in exactly analogous manner to the previously described isolation from the supernatants of in vitro cultured hybridomas.

Another object of the present invention relates to the use of the antibodies according to the invention in one of the conventional immunoassays for the detection of atrazine and / or Atrazinderivaten or of inactive degradation products of atrazine and / or Atrazinderivaten and for the differentiation of atrazine and / or Atrazinderivaten against their inactive degradation products Soil, air and water samples and, where appropriate, in extracts of plants or other biological material. The monoclonal antibodies of the invention can thus be used in all known immunoassays which rely on the specific binding between antigen and the corresponding monoclonal antibody, e.g. in a radioimmunoassay (RIA), an enzyme-linked immunoassay (ELISA), an immunofluorescence test, etc.

In the RIA test, the monoclonal antibody according to the invention can be used as such or else in the form of a radiolabelled derivative. In this case, all known to date modifications of the RIA test for the detection of relevant in the context of this invention target substances can be used, such. B. a homogeneous or solid phase RIA test, a heterogeneous RIA test and a single or double ("sandwich") RIA test with direct od-v indirect (competitive) detection of the antigen.The same is true for the use The use of a monoclonal antibody according to the invention in a competitive immunoassay for the detection of atrazine and / or atrazine derivatives or their hydroxy analogs is preferred in this invention. In principle, one differentiates between two possibilities for carrying out this competitive immunoassay: an antigen bound to a solid carrier and a free antigen around the relevant binding sites on the antibody molecule.

a) The first method is based on the competition between the antigen bound to a solid support and free antigen around the free binding sites on the antibody which are labeled with a marker. The binding of the antigen to a solid support may be either direct or via a carrier molecule take place.

The determination of the concentration of free antigen in this case takes place via the decrease in the labeled antibody bound to the carrier-fixed antigen.

This decrease is proportional to the amount of free antigen contained in the sample.

b) An alternative method is based on free and labeled antigens competing with each other for the relevant binding sites of the antibody, which in this case is bound to a solid support.

The determination of the concentration of free antigen is carried out via the decrease in labeled antigen, which varies depending on the concentration of free antigen.

For example, the plastic surface of a microtiter plate or of a test tube, the surface of spheres of polystyrene, polypropylene, polyvinyl chloride, glass or plastic or else the surface of filter paper, dextran, can be used as the solid carrier material which is suitable for the binding of the antigen or the antibody. Cellulosic or nitrocellulose strips or similar materials. These are coated with one of the monoclonal antibodies of the invention or an antigen, wherein the binding to the carrier material by simple adsorption or else optionally after previous activation of the carrier material with z. As glutaraldehyde or cyanogen bromide can be mediated. Particularly preferred in the context of this invention is the use of the monoclonal antibody according to the invention in an enzyme-linked immunoassay (ELiSA ("Enzyme Linked Immuno Sorbent Assay")).

In this case, the monoclonal antibody according to the invention can be used as such or in the form of an enzyme-coupled derivative.

The ELISA assay is based either on the use of an enzyme-linked derivative of the antibody according to the invention or of per se known enzyme-coupled antibodies which recognize and bind to an epitope of an antibody according to the invention.

Particularly preferred in the context of this invention is the use of an ELISA assay in which one of the carrier materials described above is first coated with an antigen. The carrier-bound antigen is then incubated with a test solution containing the antigen to be detected and one of the antibodies according to the invention. The antigen to be detected can be present either in free form or as part of a water or soil sample.

After an incubation period of 10 minutes to 2 hours, the entire batch is incubated with an enzyme-labeled antibody which recognizes and binds to the monoclonal antibody according to the invention. An example of such an enzyme-labeled antibody is a phosphatase-labeled goat anti-sheep immunoglobulin, or a corresponding goat anti-mouse antibody, which can be obtained commercially.

The amount of bound antibody protein can be determined by an enzyme-substrate reaction, e.g. B. using spectroscopic shear method determine.

Also preferred in the context of this invention is an ELISA test based on the competition of labeled and free antigen with the antibody bound to one of the abovementioned carrier materials.

The proportion of free antigen present in a particular sample is in this case determined by the decrease in the labeled antigen, the more precise the more free antigen the sample contains.

A further possibility for carrying out the ELISA assay comprises the following method measures: One of the previously described carriers, which is coated with one of the antibodies according to the invention, is incubated with a test solution which contains the antigen to be detected. Thereafter, the entire batch is incubated with a polyclonal immune serum against said antigen, for example a sheep immune serum, and the bound antibodies of the polyclonal serum are developed using enzyme-labeled antibodies which recognize and bind them. The amount of bound protein is determined by an enzyme-substrate reaction.

An example of such an enzyme-labeled antibody is a phosphatase-labeled goat anti-sheep immunoglobulin, which is commercially available.

Another way to perform the ELISA assay is to incubate one of the previously described support materials coated with one of the monoclonal antibodies of the invention with a test solution and a second solution containing an enzyme-conjugated monoclonal antibody.

The free monoclonal antibody recognizes a different epitope on the antigen than the enzyme-linked antibody. The amount of bound enzyme can be determined by a Jr enzyme-substrate reaction, e.g. in the form of a color change that can be visually perceived.

The color change is proportional to the amount of antigen in the test solution.

In addition, the antibody according to the invention can also be used in an ELISA test in which it is labeled with an enzyme and the carrier is coated with an anti-antigen monoclonal antibody which recognizes an epitope other than the monoclonal antibody according to the invention.

The present invention further relates to agents for the qualitative and quantitative determination of atrazine and / or atrezine derivatives or degradation products of atrazine and / or Atrazinderivaten, but in particular of hydroxy analogues of atrazine and / or Atrazinderivaten such. As hydroxyatrazine, hydroxypropazine, hydroxysimazine, etc., in the form of a ^r est kit, in addition to the monoclonal antibodies of the invention and / or derivatives thereof, optionally also ai.'lere monoclonal or polygonal antibodies, but in particular labeled monoclonal or polygonal antibodies, as well as other additives.

Particularly preferred in the context of this invention are test kits based on one of the commonly used immunoassays selected from the group consisting of radioimmunoassay, enzyme-linked immunoassay and chemiluminescent assay.

Very particular preference is given to test kits in which the detection of atrazine and / or atrazine derivatives or degradation products of atrazine and / or atrazine derivatives, but especially of hydroxyanogens of atrazine and / or atrazine derivatives, such as. Hydroxyatrazin, hydroxypropazine, hydroxysimazine, etc., ε 'a competitive immunoassay, but in particular based on an enzyme-conjugated immunoassay (ELISA).

Test kits for a radioimmunological Nachwels of atrazine and / or Atrazinderivaten or degradation products of atrazine and / or Atrazinderivaten, but especially of hydroxy analogues of atrazine and / or Atrazinderivaten such. As hydroxyatrazine, hydroxypropazine, hydroxysimazine, etc., may contain, for example, the following ingredients:

(a) a suitable carrier material which may be uncoated or coated with one of the antibodies of the invention or an antigen conjugate;

(b) optionally freeze-dried or concentrated solutions of one of the antibodies of the invention and / or a radiolabeled derivative thereof or radiolabeled antigen or standardized solutions of the antigen;

(c) buffer solutions and

(d) optionally polypeptides, detergents and other additives which prevent, for example, non-specific adsorption and aggregate formation and

(e) pipettes, reaction vessels, calibration curves, leaflets etc.

Test kits for the immunological detection of atrazine and / or Atrazinderivaten or degradation products of atrazine and / or Atrazinderivaten, but especially of hydroxy analogues of atrazine and / or Atrazinderivaten such. Hydroxyatrazine, hydroxypropazine, hydroxysimazine, etc. based on an enzyme-linked immunoassay (ELISA), may for example contain the following components:

(a) a suitable carrier material which may be uncoated or coated with one of the antibodies of the invention or an antigenic conjugate;

(b) optionally freeze-dried or concentrated solutions of one of the antibodies of the invention and / or a second enzyme-labeled monoclonal or polyclonal antibody directed against the antigen or antibody detecting the antigen;

(c) enzyme substrates in solid or dissolved form;

(e) the antigen or standardized solutions of the antigen;

(f) buffer solutions;

(g) optionally polypeptides, detergents and other additives which prevent, for example, non-specific adsorption and aggregate formation and

(h) pipettes, reaction vessels, calibration curves, color plates, leaflets etc.

A test kit for the detection of atrazine and / or Atrazinderivaten or degradation products of atrazine and / or Atrazinderivaten, but especially of hydroxy analogues of atrazine and / or Atrazinderivaten such. As hydroxyatrazine, hydroxypropazine, hydroxysimazine, etc., which is based on a Chemllumlzenzzenztest, may for example contain the following components:

(a) a suitable carrier material which may be uncoated or coated with one of the antibodies of the invention or an antigenic conjugate;

(b) optionally freeze-dried or concentrated solutions of one of the antibodies of the invention and a second polyclonal antibody capable of recognizing the first antibody of the invention and linked to a chemiluminescent label;

(c) solutions containing a component that triggers the emission of light, such as. H ₂ O ₂ and NaOH;

(d) buffer solutions;

(e) optionally polypeptides, detergents and other additives which prevent nonspecific adsorption and aggregate formation and

(f) pipettes, reaction vessels, leaflets etc.

Support materials that can be used in the present invention include primarily insoluble polymeric materials selected from the group consisting of polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, polyvinyl chloride, polyacrylamide, nitrocellulose, cross-linked dextran, fluorinated resins, agarose , cross-linked agarose, polysaccharides, etc. In addition, however, other materials are conceivable, such. Glass, metal, nylon mesh, etc.

The support materials mentioned above in detail may be configured very differently and have very different shapes depending on the particular intended purpose. These include, for example, trays, spheres, plates, sticks, cells, vials, tubes, fibers, nets, etc. Frequent use in the manufacture of test kits, for example, are microtiter plates made of transparent plastic materials, e.g. As polyvinyl chloride or polystyrene, which may be uncoated or coated with one of the antibodies of the invention, with free antigen or an antigen conjugate. Also used are beads, tubes or rods of polystyrene and polystyrene latex, wherein the surrounding latex material can be separated by centrifugation of the polystyrene particles.

Another component of the test kit according to the invention are markers or indicators which make it possible to detect the presence of a complex formation reaction, but in particular of an immune reaction, which preferably leads to an antigen-antibody complex or else to a ligand-receptor complex qualitative as well as quantitative statements about the antigen to be detected are possible. As markers or indicators, both single atoms and molecules come into consideration, which may be involved either directly or indirectly in the generation of a detectable signal. These markers or indicators can either be linked directly to the antigen to be detected or linked to one of the monoclonal antibodies according to the invention or else be present in them. However, they may also be present as individual substances or as part of a separate compound which is neither the antigen to be detected nor one of the monoclonal antibodies according to the invention but which in turn is capable of reacting with the receptor molecule, e.g. in the form of a complex formation.

These separately present compounds are preferably a second antibody molecule which may be of both monoclonal and polyclonal origin, a complement protein or fragments thereof, S. aureus protein A, etc. These separate compounds recognize and bind specifically to a receptor molecule. such as As the antigen to be detected or one of the monoclonal antibodies of the invention, but preferably to a receptor molecule, which is in the form of a complex.

In many cases, additional, additional reagents are necessary, which then lead to a detectable signal only in conjunction with the marker. This is especially true when enzymes are involved.

Markers or indicators that can be used in the present invention are well known to those skilled in the immunology and immunochemistry arts. They include, for example, radioactively labeled elements or substances, enzymes or chemiluminescent substances. The following list of possible markers or indicators is merely intended to exemplify the great variety of useful substances and reagents without, however, in any way restricting the subject matter of the invention.

Suitable markers or indicators can be found, for example, within the group of radioactive elements. In particular, those elements are preferred which either emit themselves γ-rays, such as. B. ^'24 J,' ²⁶ J, ¹²⁸ J, ¹³² J, ⁶¹ Cr or induce an emission of these rays, such. B. ¹¹ C, ¹⁸ F, ' ³ N. Also suitable are so-called. Ss-emitters such as' ¹¹ In, ¹⁴ C and ³ H. Other suitable markers include chemiluminoszierende substances, but especially fluorescent substances, which are very easy to chemically with can be linked to the antigen or an antibody without denaturing it. The resulting fluorochrome can be detected very easily by means of fluorometric methods. Specifically to call at this point are fluorochromes selected from the group consisting of fluorescein isocyanate, Fluoresceinisothiocyanatjö-dimethylamino-i-naphthalinsulfonylchlorid.Tetraniethylrhodaminisothiocyanat, lissamine, rhodamine 8200 sulfuryl chloride, etc.

Additional fluorescent agents as well as a description of analysis techniques can be found in DeLuca, Immunofluorescence Analysis, in: Antibody As a Tool, Marchalonis et al., John Wiley & Sons, Ltd., pp. 189-231 (1982).

Particularly preferred in the frame. Of this invention is the use of enzymes as marker or indicator substances, such as. Horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, etc. When enzymes are used as marker substances, it is necessary to add additional reagents containing it allow to follow the formation of an immune complex on the enzyme activity, and optionally a stop reagent, with which the enzyme reaction can be terminated.

Particular preference is given to reagents which lead to a color reaction. In the case of horseradish peroxidase may be mentioned at this point, for example, hydrogen peroxide, which in combination with an additional, oxidized Farbstoffprecursor such. As diaminobenzidine or o-phenylenediamine leads to a brown or yellowing. When using the glucose oxidase as a marker, for example, 2,2'-azino-di- (3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) can be used as a substrate. Another object of the present invention thus relates to the use of test kits containing at least one of the monoclonal antibodies of the invention as a reagent for the rapid and effective, qualitative and / or quantitative detection of atrazine and / or Atrazinderivaten or degradation products of atrazine and / or atrazine derivatives, but especially of hydroxy analogues of atrazine and / or Atrazinderivaten such. As hydroxyatrazine, hydroxypropazine, hydroxysimazine, etc. and for the differentiation of atrazine and / or atrazine derivatives against their degradation products.

I. Nlchtllmltlerende embodiments

Example 1: Synthesis of Atrazine / Hydroxyate Conjugates 1.1: Triazinyl-Valeric Acid Coupling Components

a) 2-Chloro-4-isopropylamino-6- (1-carboxybutyl-4-amino) -s-triazine

To a mixture of 10.35 g of 2,6-dichloro-4-isopropylamino-s-triazine and 6.15 g of 5-aminovaleric acid in 150 ml of dry chloroform are added 15.66 ml of diazobicyclo [5.4.0] undec-5-ene, 1 hour stirred at reflux temperature and the reaction solution was evaporated in vacuo (room temperature). The oily residue is stirred at ⁰ ° C. with 60 ml of 2N hydrochloric acid, after standing for 2 hours, the precipitate is filtered, washed with water and diethyl ether and dried. This gives 4.3 g of the desired product, 2-chloro-4-isopropylamino-6- (1-carboxybutyl-4-amino) -s-triazine (mp 191-192 ⁰ C).

b) 2-hydroxy-4-isopropylamino-6- (1-carboxybutyl-4-amino) -s-triazine hydrochloride

1 g of 2-chloro-4-isopropylamino-6- (1-carboxybutyl-4-amino) -s-triazine is stirred for 4 hours at room temperature in 7 ml of 6 N hydrochloric acid, concentrated in vacuo at 45 ⁰ C, the Crystals filtered and dried. This gives 0.98 g of the desired product, 2-hydroxy-4-isopropylamino-6- (1-carboxybutyl-4-amino) -s-triazine hydrochloride (mp 149-151 ⁰ C). 1.2: Triazine-Proteln conjugates

The atrazine and hydroxyatrazine derivatives are conjugated to either bovine serum albumin (BSA; Fluka) or keyhole limpet hemocyanin (KLH; Calbiochem) using the activated ester method (Kulkarni et al., 1981). In particular, the carboxyl group of the derivative in Ν, Ν-dimethylformamide (DMF) (6ιτ ^ / 200μΙ) is solubilized at room temperature and then with a 4molaren excess of a-hydroxysuccinimide (9.1 mg / 200μΙ) and N.N'- Dicyclohexylcarbodiimid (16ιτ ^ / 200μΙ).

The precipitate formed in the reaction is removed by centrifugation at 12000g for 3 minutes at room temperature and the activated ester subsequently added to BSA or KLH previously solubilized in phosphate buffered saline (PBS buffer). The molar ratio of (derivative) / (BSA) is about 55/1 (24 mg derivative / 5.4 ml BSA). After 4 hours of incubation at a temperature of 4 ° C, the precipitate formed is removed by centrifugation at 2000 g for 10 minutes at 4 ° C and the remaining supernatant is dialyzed extensively against PBS before being used for the immunization experiments.

The extent of coupling reaction is determined by SDS gel electrophoresis and by absorption spectrophotometry. The molar ratio of hydroxyatrazine or atrazine to BSA is about 11: 1.

Example 2: Immunization

Groups of 5 female BALB / c mice (Tierfarm Sisseln, Switzerland) aged 4 to 6 weeks receive 3 series of intraperitoneal or subcutaneous injections with KLH-conjugated atrazine or hydroxyatrazine (50 pg / injection).

The first injection contains 0.1 ml of the conjugate in PBS mixed in a ratio of 1: 1 with 0.1 ml of complete Freund's adjuvant.

50μΙ of this solution for injection are injected intraperitoneally, the remaining 150μΙ subcutaneously.

In the second and third series of injections, 14 and 30 days after the first application, the complete Freund adjuvant will be replaced by incomplete ones.

One week after the laston injection, blood serum is drawn from the experimental animals and the blood titer is determined by an ELISA test, the microtiter plates being previously coated with BSA-conjugated hapten (see section 6).

After a rest period of 2 months, another single intraperitoneal injection of the KLH conjugate is carried out in a dosage of Ε · 00μς / 200μΙ PBS.

Example 3: Fusion Protocol

3.1. Obtaining Feeder cells (Peritoneal Macrophages).

Untreated Balb / c mice, approximately 6 to 8 weeks old, are sacrificed one day prior to the intended fusion and sterilized by immersion in 70% alcohol.

Subsequently, the coat and the upper abdominal skin are trimmed sterile without injuring the peritoneum. Using a sterile 5 ml plastic syringe and a sterile 18-liter injection needle, inject 4 ml of BSS (without Ca ²⁺ and Mg ²⁺ ) and 1 ml of air into the abdominal cavity.

After lightly massaging the abdomen (leaving the syringe and needle in the abdominal cavity), the previously injected BSS buffer is withdrawn from the peritoneum and placed in a sterile falcon tube. This procedure is repeated 2 more times. The macrophages thus obtained are cooled with ice and then washed twice with 20 ml BSS each time.

The macrophages are centrifuged for 10 min. At 300 g and a temperature of 5 ⁰ C centrifuged. The pellet is then resuspended in 50 ml of HAT medium and the cell suspension is spread on 4 Costar plates with a total of 24 wells (0.5 ml / well).

Subsequently, the thus prepared macrophages are stored at a temperature of 37 ⁰ C and a CO ₂ concentration of 6% in an incubator.

Approximately 4 χ 10 ⁶ macrophages are required per fusion process.

3.2. Cultivation of the myeloma cell inle Sp 2/0-Ag 14

The mentioned myeloma cell line Sp 2/0-Ag 14 is a myeloma cell line which itself does not secrete antibodies and which is described in M. Shulman et al. (1978). This myeloma cell line can be obtained from the American Type Culture Collection of Rockville, Maryland.

Each fusion requires 50ml of a well grown culture containing at least 10 million cells. Cultivation of the myeloma cells is preferably carried out in T175 "Falcon" bottles (Beckton & Dickenson).

One day prior to fusion, the culture medium (RPM 11640) is replaced with fresh RPMI 1640 medium. On the day of fusion, the SP 2/0-Ag 14 cells are harvested, placed in a sterile 50 ml plastic tubes and 10 min. At 300 g and a temperature of 5 ⁰ C centrifuged (MSE centrifuge, model Chilspln, UK).

After centrifugation, the supernatant is withdrawn and discarded. The cells are washed twice each with about 3Ox each with about 30 ml BSS buffer (Ca ³⁺ and Mg ²⁺ free) (10 min. At 300g, 5 ⁰ C) and then resuspended in 5 ml BSS.

An aliquot of the cell suspension is taken to determine cell number and stained with fluorescein diacetate (FDA). Until further processing, the myeloma cells are kept on ice.

3.3. Production of a spleen cell suspension

The removal of the spleen from a previously immunized according to Example 2 Balb / c mouse under sterile conditions and under cooling with ice.

The previously immunized Balb / c mouse is sacrificed by cervical dislocation and the spleen is removed under sterile conditions. The mouse is briefly immersed in 70% ethanol and prepared with sterile cutlery. The spleen is carefully removed and placed on a fine nylon net. There it is finely cut with scissors and then carefully pushed through the net with the aid of a 5 ml syringe punch, without destroying too many cells. During the whole process, the net is flushed with BSS. The cell suspension thus obtained is placed in 50 ml plastic tubes and centrifuged for 10 min at 300 g and a temperature of 5 ° C (MSE centrifuge, model Chilspin, UK). The cells are then 2 times with 20 ml each 8SS washed (10min .; 300g; 5 ⁰ C; M.ES Chilspin) and the cell pellet is resuspended after centrifugation in 10 ml BSS. Until fusion with Sp 2/0-Ag 14 myeloma cells, the spleen cells are left on ice.

3.4. Fusion: spleen cells and Sp2 / 0-Ag 14 myeloma cells

The ratio of myeloma cells to spleen cells should be 1:10 for the fusion.

Spleen cells (in BSS buffer) and Sp 2/0-Ag 14 myeloma cells (in BSS buffer) are combined in the indicated ratio and centrifuged for 10 min. At 300 g and a temperature of 5 ⁰ C (MSE centrifuge model Chilspin). The pellet is resuspended in BSS buffer and the suspension is then centrifuged again.

The pellet is stirred gently and placed in 37 ⁰ C warm water bath. It is then added 1 ml of prewarmed and sterile PEG-4000 (MERCK) over a period of 60sec. added dropwise to the cells, the entire batch is constantly moved. The cells are then left for 30 seconds. The mixture is then shaken for a further 5 ml, before 5 ml of a previously heated BSS buffer (without Ca ²⁺ , Mg ²⁺ ) are likewise added dropwise over a period of about 5 minutes with continued stirring.

The cells fused in the manner described are then centrifuged off (10 min, 300 g, 2O ⁰ C, MSE centrifuge, model Chilspin), the supernatant is withdrawn and discarded. The cell pellet is resuspended in 50 ml of HAT medium and the resulting cell suspension is distributed on the prepared 4 Costar plates (24-well microtiter plates, 24 mm diameter per well, 2.0 cm ² total cell area) (0.5 ml / well).

The incubation of the Costar plates is carried out at a temperature of 37 ° C and at a CO ₂ concentration of 6%.

Example 4: Cultivation of hybrid cells

On the 1st day after cell fusion, 1 ml of HAT medium per well is added to the culture plates. 3 to 4 days after cell fusion, microscopic control of the fused cells is performed. At the same time, the spent medium is aspirated and replaced with 1 ml of fresh HAT medium. After a further 3 days (6-7 days after cell fusion), the culture medium is changed again. From the 7th to the 10th day after the cell fusion, each well is screened microscopically for hybrids and the medium renewed 2 times to 3 times a week. Once hybrids have grown in a well, the HT medium can be replaced there by HT medium. The supernatant from washed hybrid cultures (at least 10% of well) is lifted off with a sterile Pasteur pipette and tested for the presence of antibodies.

Once the wells are fully grown with positive hybrid colonies, they can be transferred to new Costar plates in RPMI 1640 medium, with the contents of a well grown well distributed to 2 to 3 new wells.

Example 5 Cloning of the Positive Hybrid Cells

The cells of a positive well are dissolved using a pipette and transferred into 1 ml of medium in a tube.

Subsequently, an aliquot is taken to determine the cell number and stained with FDA (dilution 1: 2 with FDA: 50ul cells + 50 μΙ dye). The preferred cell number is 10 ⁵ to 10 "cells / ml.

Subsequently, the hybrid cells are diluted in a ratio of 1: 100 with HT medium (e.g., 100μΙ cells + 9.9ml HT medium).

25 ml of HT medium are placed in each of two 50 ml Falcon tubes and filled with 5 ml of a macrophage suspension to a total of 30 ml per tube. The macrophages are previously isolated from a mouse and resuspended in 10 ml of HT medium (see Section 3.1).

In these falcon tubes containing the macrophages, the hybrid cells are diluted until a cell density of (i) 270 cells / 30 ml or (ii) 90 cells / 30 ml is reached. Subsequently, these batches are distributed on Costar plates (96-well microtiter plates), with 200 μΙ per well added. This corresponds to a cell count of (i) 1.8 cells / well or (ii) 0.6 cells / well. Each dilution requires 1.5 microtiter plates in this way.

After 7 days, the individual wells are microscopically controlled and the wells containing cell clones are detected. The dilution in which approximately 50% of the wells contain cell clones is used for the ELISA test. This should usually be the dilution at 0.6 cells / well.

After about 7-10 days, the supernatants of the positive wells (with clones) are tested for the presence of monoclonal antibodies in an ELISA assay and the positive clones propagated on Costar plates (with 24 holes) in RPM 11640 medium. Aliquots of these positive clones are stored in liquid nitrogen.

Examples: Hybridoma Screening (ELISA Test)

First, 100 μl of a solution of BSA-conjugated hapten In sodium carbonate buffer (50 mM, pH 9.6) are added to the individual wells of a microtiter plate and this batch is incubated at 4 ° C. in a moist chamber overnight.

Subsequently, the wells are each washed 5 times with a 0.1% PBS-Tween buffer.

To block the unoccupied binding sites on the microtiter plate, 200 μΙ of a PBS-BSA solution (1%) is added to each well. This approach is incubated for 1-2 hours at room temperature and then washed with a 0.1% PBS-Tween buffer.

200μΙ of the hybridoma supernatant, diluted 1: 2 with PBS-Tween (0.1%), are then added to each well, and the entire batch incubated for 2 hours at room temperature. Subsequently, the wells are again washed 5x with a 0.1% PBS-Tween buffer.

This is followed by incubation with phosphatase-conjugated anti-mouse antibody (Kirkegaard & Perry Lab.). First, ΙΟΟμΙ of a goat antibody purified by an affinity chromatography against mouse IgG diluted 1: 1500 in PBS-Tween (0.1%) (Kirkegaard & Perry Laboratories) and labeled with alkaline phosphatase is added to each well.

The incubation time is 1.5 hours at room temperature. Subsequently, the individual wells are washed again with PBS-Tween (0.1%) (5x).

Thereafter, 150μΙ of a substrate-containing solution (1 mg / ml p-nitrophenyl phosphate) is added to each well. After an incubation period of 2 hours in the dark, the spectroscopic determination is carried out at 405 nm. Positive hybridoma cells which secrete a specific antibody give a strong positive signal at the selected wavelength.

Example 7: Expansion of hybridoma cells in the mouse

For the stimulation of ascites production female Balb / c mice (20-25 mg) (animal farm Sissel, CH) are pretreated with 0.3 ml pristane oil (Aldrich Chemical), which is injected intraperitoneally.

1 to 3 weeks after prietan administration, the mice are given a second injection (0.2 ml pristane oil, ip). Simultaneously with this second injection, the animals receive 2x10 ⁶ hybridoma cells in 0.2 ml PBS.

The results from this treatment ascites fluid is collected, centrifuged at 800 g and stored at a temperature of -20 ⁰ C. After defrosting, the ascites fluid is centrifuged for 1 hour at 30,000 g. The topmost layer, which mainly contains lipids, is removed. Subsequently, the protein concentration is determined and adjusted to a value of 10 mg / ml by addition of PBS.

The immunoglobulin G fraction (IgG) is precipitated by dropwise addition of 0.9 parts by volume of a saturated ammonium sulfate solution at 0 ° C. After 1 hour, the IgG fraction is pelleted by centrifugation at 22,000 g for 1 hour. The pellet is then dissolved in 20 mM Tris-HCl-Puifer, pH 7.9, containing 50 mM NaCl and dialyzed against the same buffer overnight at 4 ° C.

The further work-up of the IgG fraction is carried out by means of an anion exchange chromatography on a DE-52 diethylaminoethylcellulose (Whatman) column. The sample is diluted 1: 2 (v / v) with 20 mM Tris-HCl, pH 7.9, to a final NaCl concentration of 25 mM and 10 mg protein / ml gel is applied to the column. Elution is achieved by increasing the NaCl concentration from 25 mM to 20 mM (linear gradient). In general, the elution of monoclonal antibodies in the range of 8OmM NaCl.

The fractions are dialyzed against PBS overnight at a temperature of 4 ° C and stored at -70 ° C. The degree of purity is determined by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as by isoelectric focusing.

In the present case, the degree of purity is> 90%.

Example 8: Atrazine and Hydroxyatrazine Nachwols

The detection of atrazine and hydroxyatrazine is carried out by means of a two-stage, competitive ELISA test.

BSA-conjugated hapten is first adsorbed in microtiter plates in a sodium carbonate buffer (pH 9.6) (0.2 g / well) and incubated overnight at a temperature of 4 ° C. The remaining free binding sites of the solid support are then blocked by addition of BSA in the form of a 1% solution.

The plates are then washed with PBS supplemented with 0.1% (v / v) polysorbate 20 (PBS-Tween).

50μΙ of the previously purified monoclonal antibody (2 μΙ) or the supernatant of the cell clones (in a dilution of 1: 5 to 1: 22.5) are a) with 950μΙ of a standard solution containing an increasing proportion of triazine or triazine analogues , b) incubated with triazine-containing water samples or c) triazine-containing soil extracts. (All dilutions are made in PBS-Tween).

After an incubation period of 1 hour at room temperature, 200μΙ of the mixture is added to each well of the microtiter plate and the entire batch is incubated for another 1 hour.

The wells are then washed 5 times and with ΙΟΟμΙ / well goat anti-mouse IgG antibody conjugated to alkaline phosphatase present (dilution 1: 1500) charged and incubated for a period of 1.5 hours.

After rinsing again, 150 μl / well of the substrate p-nitrophenyl phosphate dissolved in 1 mg / ml diethanolamine buffer (1 mM, pH 9.8) is added to the wells.

A color change proportional to the amount of antibody reacted with the antigen bound to the solid phase is recorded at a wavelength of 405nm. The dilutions of the individual samples are chosen so that absorption values in the range between 0.3 and 0.5 are obtained without addition of an inhibitor (Bo). For the controls (without antibodies and with undetectable amounts of antigen) values of R <0.005 are obtained. All samples are determined in triplicate.

To determine the amount of atrazine / hydroxyatrazine contained in a sample, a calibration curve is first prepared (FIG. 1), wherein B / Boχ100 is plotted against the concentration of inhibitor. (Bo means absorbency measured without addition of a triazine inhibitor to the antibody, and B absorbency on addition of various concentrated triazine inhibitors.) The εεo value indicates that concentration of the antigen at which the antibody binding to the solid phase is 50% inhibited. The I _M value is determined by means of an ENZFITTER specially adapted to the present conditions

(Leatherbarrow, Elsevier-Biosoft) Curve calculation program determined based on a 4-parameter logistic curve (Raab GM, 1983).

Also the quantitative atrazine ozw. Hydroxyatrazine Determination in Soil or Water Samples ELISA is performed using the ENZFITTER program, with curve fitting based on standards running on each microtiter plate.

Example 8.1: Analysis of soil samples Aliquots (2g) of standardized soil samples of different origin (see Table II) are allowed to stand in an extractor for 4 hours

extracted with 20ml of a methanol / water (80/20 v / v) mixture. For the competitive ELISA, the soil extracts are usually diluted 1:40 in PBS-Tween (0.1%) to avoid possible denaturation of the monoclonal

Prevent antibodies from the methanol. Subsequently, the diluted soil samples (950μΙ) on the previously

described manner with the specific antibodies (50μΙ) incubated.

For the HPLC determination of hydroxyatrazine, the methanol extracts are removed by cation exchange chromatography (eg.

on a Dowex BO W-X4 column, followed by adsorption to an Amberlite XAD-2 polystyrene divinyl benzene

Exchange resin) further purified. Finally, a gel filtration, z. B. on a Bio-gel P-2 column performed. The Samples are then placed on the HPLC and hydroxyatrazine is determined at a wavelength of 240nm. The comparative determination of atrazine is carried out by means of gas chromatography, using a thermo-ionic Detector, and / or subsequent mass spectrometry performed. Example 8.2: Analysis of water samples For the competitive ELISA, 100μΙ of a 10X concentrated PBS Tween buffer becomes 850μΙ of a water sample

added. This approach is finally incubated with SOpI of anti-atrazine antibody MAb 4063-21-1.

For the HPLC determination, the s-triazines contained in the water sample (20 ml) are first placed on an RP-18 feed column

concentrated and then further separated on an analytical RP-18 column. Atrazine determination is carried out at 230nm.

II. Results

a) Preparation of monoclonal antibodies

Just a few days after the cell fusion between myeloma cells and the spleen cells of a mouse, which was previously treated with the in Section 1 described immunoconjugate can be found in 93 of the total 96 wells of Detect microtiter plates proliferating cell colonies. This corresponds to a fusion efficiency of 97%.

Ten of these cell colonies produce monoclonal antibodies, which show a very strong response in the ELISA test. These are cloned using the limited dilution method (see Section 5).

9 of the synthesized monoclonal antibodies belong to the IgG 1 subclass, one into the IgG 2b subclass.

The monoclonal antibodies recovered in the manner described above can be detected by virtue of their cross-reactivity as described in r.inem ELISA test is determined to be divided into 2 groups. In the first group (represented by MAb 4009-85-3), cross-reactivity is mainly restricted to hydroxypropazine,

whereas in the second group (represented by MAb 4009-77-20), cross-reactivities also occur with other s-triazines, such as e.g. Hydroxysimazine, hydroxydesmetry, etc. (see Table 1).

In both groups, however, no cross-reactivity with active triazines, such as. As atrazine, on. Thus, the bond remains the

monoclonal antibodies to compounds having a hydroxy group in position 2 of the triazine ring.

The lower detection limit for hydroxyatrazine is in the range of 0.1 ng / ml buffer for MAb 4009-85-3 and 0.05 ng / ml, respectively Buffer for MAb 4009-77-20. The corresponding U values were 0.95ng / ml and 0.5ng / ml. Using atrazine conjugates (represented by MAb 4063-21-1), a total of 5 monoclonal antibodies are obtained

all show a comparable degree of cross-reactivity. In this case, one finds cross-reactivity with various active s-triazines.

The occurring cross-reactivities with hydroxylated metabolites, on the other hand, are negligible, (see Table 1). For MAb 4063-21-1, the detection limit for atrazine is 0.05 ng / ml buffer at 0.45 ng I ₆₀ -WeH (50% inhibition).

b) Recovery studies

These recovery studies are performed by adding a known amount of hydroxyatrazine or atrazine to a methanol / water bottom extract. They primarily serve to investigate the possibly interfering influences of organic material during the immunoassay.

Tab. II shows that almost complete recovery occurs regardless of the composition of the soil samples used. Even with a humus content of approximately 10%, the recovery rate is 97%.

c) Atrazine eluates in water samples

Twenty-one pieces of water were analyzed by HPLC and ELISA using MAb 4063-21-1.

For the HPLC determination, a concentration step has to be carried out which, when using the

Monoclonal antibody according to the invention is omitted in an ELISA assay.

Table III shows that the detection limit for atrazine using the immunoassay is about 0.05 ppb. For the atrazine-containing For samples (> 0.05ppb) the correlation between ELISA and HPLC determination is determined by regression analysis. there

a good agreement between the two methods becomes apparent (r = 0.91, ρ <0.0005), whereby the slightly increased values of the ELISA determination are not significant.

In addition, the water samples were also tracked down by hydroxyatrazine. With neither method

(HPLC and ELISA), this metabolite could be detected.

Deposit: The hybridoma cell lines produced and used in the present invention were used in the International Depository recognized the European Collection of Animal Cell Cultures (ECACC) in Salisbury, UK,

according to the requirements of the Budapest Treaty for the International Recognition of the Deposit of

Microorganisms for the purpose of patenting, deposited. A statement on the viability of the deposited samples will be made by the said International Depository.

cell line deposit date Deposit No. · Date of life capacity certificate hybridoma 25.08.1988 8808/2501 25.08.1988 Clone 4063-21-1 hybridoma 25.08.1988 8808/2502 25.08.1988 Clone 4009-77-20 hybridoma 25.08.1988 8808/2503 25.08.1988 Clone 4009-85-3

* Deposit number issued by the International Depository Office, as described above.

Used media and buffers A) RPMI1640 Medlum RPM11640 (Seromed) with the following additions:

calf serum 15% L-Glutamln 4 mM gentamicin 0.01% Sodium pyruvate 1 mm 2-mercaptoethanol 50 μΜ insulin 5μΜ transferrin 5μΜ Selenium (ITS) 5IN

B) HAT-Medlum

1 liter of RPMI 1640 medium with 20ml of HAT cone added. Boehringer 5Ox (hypoxanthine 680.5 mg / l, aminopterin 8.8 mg / l, thymidine 193.8 mg / l)

C) HT-medlum

1 liter of RPMI 1640 medium with 20ml supplement of HT cone. 5Ox of Boehringer hypoxanthine 680.5mg / l; Thymidine 193.8 mg / l)

O) BSS buffer (Earle's saline, without Ca and Mg, pH 7.4)

KCI 7.3 mM NaCl 116mm NaHCO ₃ 26mM NaH ₂ PO ₄ 2H ₂ O 1 mm glucose 5.5 mM phenol 48 μΜ

1% addition (v / v) of a penicillin / streptomycin solution (Sc romed) (10000E penicillin, 10mg / ml streptomycin).

E) Sodium carbonate buffer (pH 9.6) Na ₂ CO ₃ 477 mg NaHCO ₃ 879 mg NaN ₃ (0.5 M) 1.8 ml ad 300 ml H ₂ O

F) PBS buffer (pH 7.0)

NaCl 8.5 g

Na ₂ HPO ₄ -2H ₂ O 1.28g

NaH ₂ PO ₄ -2H ₂ O 0.436 g ad 1000 ml H ₂ O.

G) PBS-Tween-20 (0.1%)

1 ml Tween-20 (Serva) + 1000 ml PBS

H) PBS-BSA (1%) BSA 5 g

NaN ₃ (0.5 M) 3 ml

ad 500 ml PBS

I) substrate buffer (diethanolamine buffer, pH P, 8) diethanolamine 97 ml

NaN ₃ (0.5 M) 6 ml

MgCl ₂ -6H ₂ O 100 mg

ad 1000 ml H ₂ O, adjust the pH to pH 9.8 with HCI cone.

Preparation of the substrate: Immediately prior to use, a substrate tablet (= 5 mg) of the p-nitrophenyl phosphate Substrate (Sigma-104) dissolved in 5ml substrate buffer.

Table I Cross-reactivity of various s-triazine and hydroxytriazine analogues with an anti-atrazine MAb (MAb 4063-21-1) and two anti-hydroxyatrazine MAbs (MAb 4009-85-3 and MAb 4009-77-20)

inhibitor MAb 4063-21-1 % cross MAb 4009  85-3 % cross MAb 4009-77-20 % cross (ECACC 8808/2501) reactivity (ECACC 8808/2503) reactivity (ECACC 8808/2502) reactivity 150 (B) 150 (O 150 (C) ng / ml 100.0 ng / ml <0.1 ng / ml <0.05 (A) 90.0 (A) <0.1 (A) <0.05 atrazine 0.45 2.5 > 1000 > 1000 Propazine. 0.5 0.5 > 1000 > 1000 simazine 18 5.0 Desmetryn 90 11.3 terbutylazine 9 5.0 <0.1 <0.05 trietazine 4 6.4 <0.1 <0.05 ametryn 9 56.3 > 1000 > 1000 atraton 7 1.7 > 1000 > 1000 prometryne 0.8 75.0 simetryn 27 2.1 100.0 100.0 prometon 0.6 8.2 100.0 83.3 OH atrazine 21 0.3 0.95 1.5 0.5 41.7 OH propazine 5.5 0.1 0.95 11.9 0.6 71.4 OH simazine 180 5.6 65 5.0 1.2 15.2 OH Desmetryn 360 8th 0.7 OH terbuthylazine 8th 19 3.3

a) Inhibitor concentration for 50% inhibition in the competitive EUSA test

b) Cross-reactivity, defined as: (atrazine concentration for 60% inhibition) / (concentration of s-triazine analogues for 50% inhibition) χ

c) Cross-reactivity, defined as: (hydroxyatrazine concentration for S0% inhibition) / (concentration of s-triazine analogs for 60% β inhibition) χ 100

Table II Recovery of hydroxyatrazine from soil extracts previously inoculated with hydroxyatrazine *

soil sample % Humus soil composition % Silt % Clay pH hydroxyatrazine hydroxyatrazine %Sand Addition (ppb) Recovery rate (%) MAb 4009-85-3 1.8 3.8 13.6 (ECACC 8808/2503) Risilon 80.8 7.8 20 91 (Israel) 60 92 200 90 9.3 60.4 21.5 600 93 Vetroz 18.1 7.3 100 97 (Switzerland) 5 17.4 34.6 stone 43 7.1 100 110 (Switzerland) 1.4 13.6 2.5 Collombey 83.9 7.4 100 107 (Switzerland) 2.6 64 10.3 Les Evouettes 25.7 6.2 100 94 (Switzerland)

* Fortified soil extracts (Methannl extracts) are diluted 1:40 in PBS-Tween (0.1%) for ELISA determination.

TABLE III Determination of atrazine in water samples Comparison between HPLC and ELISA (MAb 4063-21-1, ECACC 8808/2501)

water sample atrazine HPLC ELISA Ver'iältnis 0.09 desethylatrazine s-triazine ELIIiA / HPLC 0.10 0.05 0.10 1 0.41 0.15 0.14 1.1 2 0.10 0.59 0.38 1.4 3 0.08 0.11 0.15 0.9 4 0.06 0.06 0.11 1.5 5 <O, œ5 0.05 0.09 1.4 6 <0.05 0.11 0.07 1.5 7 0.06 0.05 0.06 > 1.0 8th <0.05 <0.05 0.07 > 1.0 9 <0.05 0.07 1.2 10 > 1.0

water sample atrazine HPLC 'JLISA relationship M9 / I desethylatrazine s-triazine ELISA / HLPC <0.05 Mg / I M9 / I 0.14 <0.05 0.08 11 0.23 0.29 0.10 > 1.0 12 0.23 0.14 0.19 0.7 13 0.23 0.13 0.34 0.8 14 0.36 0.13 0.24 15 0.35 0.24 0.49 16 0.40 0.23 0.43 17 0.40 0.24 0.33 18 0.50 0.27 0.41 ( 19 0.45 0.32 0.50 20 0.29 0.54 21 0.01 control 1.5 H ₂ O (HPLC) 1.0 , 4 1.2 ),8th 1.0 , 0 1.2

literature

Ercegovich, CD., Et al., J. Agrlc. Food Cham., 29: 559-563, 1981 Fleeker, J., J. Assoc. Off. Anal. Chem., 70: 874-878, 1987 Hsu.HT, et al., ASM News, 50 (3): 91-101, 1984 Kawamura, H., Berzojsky, JA, J. Immunol., 136: 58,1986 Kelley, M., et al., J. Agric. Food Chem., 33: 962-965, 1985 Köhler, G., Milstein, Nature, 256: 495-497, 1975, Kulkarni, NP, et al .. Cancer Res., 41: 2700-2706, 1981 Littlefield, JW, Science, 145: 709, 1964 Newsome, WH, J. Agric. Food Chem., 33: 528-530, 1985 Raab, G.M., CIIn. Chem., 29: 1757-1761, 1983 Ramsteiner, K.A., Hörman, W.D., J. Agrlc. Food Chem., 27: 934-938, 1979. Ramsteiner, K., "Atrazine." In Methods Collection for Residue Analysis of Crop Protection Agents; Deutsche Forschungsgemeinschaft Ed., Verlag Chemie, 1985; Method 6-D Shulman, M., et al., Nature, 276 : 269-270, 1978, Like, SI, Hammock, BD, J. Agric. Food Chem., 30: 949-957, 1982

patent literature

U.S. Patent 4,530,786

Claims (63)

1. A hybridoma cell line that produces a monoclonal antibody that has high specificity and affinity for atrazine and / or atrazine derivatives and that exhibits substantially no cross-reactivity with the inactive hydroxy analogs of atrazine and / or atrazine derivatives. 2. A hybridoma cell line that produces a monoclonal antibody that has high specificity and affinity for the hydroxy analogs of atrazine and / or atrazine derivatives and that exhibits substantially no cross-reactivity with atrazine and / or atrazine derivatives. A hybridoma cell line according to claim 1 which produces a monoclonal antibody having high specificity and affinity for atrazine and which exhibits substantially no cross-reactivity with hydroxyatrazine or other hydroxy analogues of atrazine derivatives. 4. A hybridoma cell line according to claim 3, which produces a monoclonal antibody which has a high specificity and affinity for atrazine and which exhibits a marked cross-reactivity with structurally similar atrazine derivatives selected from the group consisting of propazine, prometryn and prometon, but which in the essentially has no cross-reactivity with other atrazine derivatives or hydroxy analogs of atrazine and / or atrazine derivatives. A hybridoma cell line according to claim 4, which has the characterizing characteristics of ECACC 8808/2501. A hybridoma cell line according to claim 2 which produces a monoclonal antibody which has high specificity and affinity for hydroxyatrazine and which exhibits substantially no cross-reactivity with atrazine and / or atrazine derivatives. 7. A hybridoma cell line according to claim 6, which prochruises a monoclonal antibody which has high specificity and affinity for atrazine and which exhibits pronounced cross-reactivity with hydroxypropazine, but which has substantially no cross-reactivity with hydroxy analogues of other atrazine derivatives or with atrazine and / or Atrazine derivatives shows. A hybridoma cell line according to claim 6 which produces a monoclonal antibody which has high specificity and affinity for hydroxyatrazine and which exhibits cross-reactivity with hydroxy analogues of atrazine derivatives but which exhibits substantially no cross-reactivity with atrazine and / or atrazine derivatives. 9. Hyjidoma cell line according to claim 7, which has the characterizing features of ECACC 8808/2503. A hybridoma cell line according to claim 8 which has the characterizing features of ECACC 8808/2502. 11. mutants and variants of a hybridoma cell line according to any one of claims 1 to 10, which still have the characteristic properties of the starting material; d. H. which are still able to synthesize the antibodies of the invention and to secrete into the surrounding medium. 12. Monoclonal antibodies and their derivatives which have a high specifiacy and affinity towards atrazine and / or atrazine derivatives and which show substantially no cross-reactivity with the inactive hydroxy analogs of atrazine and / or atrazine derivatives. 13. Monoclonal antibodies and their derivatives which have a high specificity and affinity for the inactive hydroxy analogues of atrazine and / or atrazine derivatives and which show substantially no cross-reactivity with atrazine and / or atrazine derivatives. 14. Monoclonal antibodies and their derivatives according to claim 12, which have a high specificity and affinity for atrazine and which show substantially no cross-reactivity with hydroxyatrazine or hydroxy analogs of atrazine derivatives. 15. monoclonal antibodies and their derivatives according to claim 12, which have a high specificity and affinity for atrazine and show a strong cross-reactivity with structurally similar Atrazinderivaten selected from the group consisting of propazine, Prometryn and Prometon, but essentially no cross-reactivity with other atrazine derivatives or with hydroxy analogues of atrazine and / or atrazine derivatives. 16. Monoclonal antibodies and their derivatives according to claim 15, characterized in that the cross-reactivity with propazine is more than 80%. 17. monoclonal antibodies and their derivatives according to claim 13, which have a high specificity and affinity for hydroxyatrazine and which show substantially no cross-reactivity with atrazine and / or atrazine derivatives. 18. monoclonal antibodies and their derivatives according to claim 17, which have a high specificity and affinity for hydroxyatrazine and show a cross-reactivity with Hyciroxyanalogen of Atrazinderivaten, but show substantially no cross-reactivity with atrazine and / or Atrazinderivaten. 19. Monoclonal antibodies and their derivatives according to claim 18, characterized in that the cross-reactivity with hydroxy analogues of atrazine derivatives is at least 40%. 20. monoclonal antibodies and their derivatives according to claim 17, which have a high specificity and affinity for hydroxyatrazine and show a strong cross-reactivity with hydroxypropazine, but show substantially no cross-reactivity with Hydroxyana'ogen other Atrazinderivate or with atrazine and / or Atrazinderivaten , 21. Monoclonal antibodies and their derivatives according to claim 20, characterized in that the cross-reactivity with hydropropazine is up to 100%. 22. Monoclonal antibody and its derivatives which are produced by a hybridoma cell line according to one of claims 1 to 10. A monoclonal antibody and its derivatives according to claim 15 which is produced by a hybridoma cell line having the characterizing characteristics of ECACC 8808/2501. A monoclonal antibody and its derivatives according to claim 18 which is produced by a hybridoma cell line having the characterizing characteristics of ECACC 8808/2502. A monoclonal antibody and its derivatives thereof according to claim 21 which is produced by a hybridoma cell line having the characterizing characteristics of ECACC 8808/2503. 26. A method for producing a hybridoma cell line according to any one of claims 1 or 2, characterized in that a) atrazine and / or an atrazine derivative or hydroxyatrazine and / or hydroxy analogues of atrazine derivatives conjugated with a carrier molecule b) immunizing a donor animal with said conjugate c) isolated an immunocompetent B cell from the immunized donor animal d) said immunocomponent B cell is fused with a tumor cell line capable of continuous cell division e) isolated the resulting fusion product, cultured in a suitable culture medium and then cloned and f) the cloned hybrid cells are examined for the formation of monoclonal antibodies. 27. The method according to claim 26, characterized in that one uses as the carrier molecule macromolecular compounds having freely accessible reactive groups for the coupling reaction with atrazine and / or atrazine derivatives and / or hydroxy analogues of Atrazinderivaten and which are capable of said compounds an immunogenic To give potency. 28. The method according to claim 27, characterized in that one uses a lysine-rich protein having a molecular weight between 10,000 and 1,500,000. 29. The method according to claim 27, characterized in that one selects a protein selected from the group consisting of "Bovine Serum Albumin" (BSA), "Keyhole Limpet Protein" (KLP), Human Serum Albumin (HSA), "Porcine Thyroglobulin", B2 Microglobulin, hemocyamine, immunoglobulins; toxins; polysaccharides; lipopolysaccharides; natural or synthetic polyadenylic and polyuridylic acids; polyalanyl and polylysine polypeptides; or cell membrane components. 30. The method according to claim 26, characterized in that the conjugation to the carrier molecule takes place directly. 31. The method according to claim 26, characterized in that the conjugation to the carrier molecule via a bridge member (spacer) takes place. 32. The method according to claim 31, characterized in that said bridge member has one or more reactive groups which are capable of interacting with the reactive groups of the carrier molecule. 33. The method according to claim 32, characterized in that said reactive groups are carboxyl, amino or SH groups. 34. The method according to claim 26, characterized in that the coupling reaction is carried out by means of the active ester method. 35. The method according to claim 26, characterized in that the immunization of the donor animals is carried out by one to several times application of carrier-bound atrazine and / or atrazine derivatives. 36. The method according to claim 26, characterized in that the immunization of the donor animals by single or repeated application of carrier-bound hydroxyatrazine and / or hydroxy analogues of atrazine derivatives. 37. The method according to any one of claims 35 or 36, characterized in that the application takes place in the form of an intravenous, intraperitoneal or subcutaneous injection. 38. The method according to claim 26, characterized in that used for the fusion of immunocompetent cells from the donor animal tumor cell lines that do not produce monoclonal antibodies themselves. 39. The method according to claim 38, characterized in that one uses myeloma cell lines having the characteristic characteristics of Sp2 / O-Ag 14 or X63-Ag8.653, 40. The method according to claim 26, characterized in that one uses as a fusion medium, a buffer solution, one of the commonly used for the fusion of cells fusion promoters selected from the group consisting of: Sendai viruses or other paramyxoviruses, calcium ions, surface-active lipids or polyethylene glycol. 41. The method according to claim 40, characterized in that said fusion promoters are polyethylene glycol having an average molecular weight of 600 to 6000. 42. The method according to claim 41, characterized in that the polyethylene glycol concentration in the fusion medium is 30% -60%. 43. The method according to claim 26, characterized in that one uses for the selection of fused hybrid cells, the HAT selection medium. 44. The method according to claim 26, characterized in that culturing the hybrid cells in the presence of isolated macrophages ("feeder cells"). 45. The method according to claim 26, characterized in that isolated positive monoclonal antibody hybrid cell cultures using the limiting dilution method and then cloned in suitable culture media. 46. Method according to claim 26, characterized in that the hybridoma cell clones cloned according to claim 45 are examined with the aid of a immunoassay for the formation of suitable monoclonal antibodies. 47. The method according to claim 46, characterized in that one uses an enzyme-linked immunoassay or a radioimmunoassay. 48. A process for the preparation of monoclonal antibodies, characterized in that one cultivates the hybridoma cell lines produced according to one of claims 26 to 42 in vivo or in vitro by means of known methods and isolated the monoclonal antibodies produced. 49. The method according to claim 48, characterized in that one carries out In Vltro cultivation in suitable culture media. 50. The method according to claim 49, characterized in that one uses standardized culture media selected from the group consisting of "Dulbecco's Modified Eagle Medium" (DMEM) or RPM11640, which are optionally supplemented by the addition of mammalian serums, by growth-promoting additives or by trace elements can. 51. The method according to claim 48, characterized in that said monoclonal antibodies are prepared by expansion of the hybridoma cell lines produced according to any one of claims 23 to 39 in a donor animal in vivo. 52. A method for the immunological detection of atrazine and / or Atrazinderivaten, characterized in that one uses a monoclonal antibody according to any one of claims 12 öder 14 to 16 in one of the known immunoassays. 53. A method for the immunological detection of atrazine and / or Atrazinderviaten according to claim 52, characterized in that a monoclonal antibody which is produced by a hybridoma cell line which has the distinguishing characteristics of ECACC 8808/2501 or clones or subclones thereof, in one of the known immunoassays used. 54. A method for the immunological detection of hydroxyatrazine and / or Hydroxyatrazinderivaten, characterized in that one uses a monoclonal antibody according to any one of claims 13 or 17 to 21 in one of the known immunoassays. 55. A method for the immunological detection of hydroxyatrazine and / or Hydroxyatrazinderivaten according to claim 54, characterized in that a monoclonal antibody which is produced by a hybridoma cell line having the distinguishing characteristics of ECACC 8808/2502 or clones or subclones thereof, in one of the known immunoassays used. 56. Method for the immunological detection of hydroxyatrazine and / or hydroxyatrazine derivatives according to claim 54, characterized in that a monoclonal antibody produced by a hybridoma cell line having the characteristic features of ECACC 8808/2503 or clones or subclones thereof, in one of the known immunoassays used. 57. The method according to any one of claims 52 to 56, characterized in that it is a competitive immunoassay. 58. The method according to any one of claims 52 to 57, characterized in that said immunoassay is a radioimmunoassay (RIA), an enzyme-linked assay (ELISA) or a chemiluminescent assay. 59. Means for the immunological detection of atrazine and / or Atrazinderivaten in the form of a ready-test kit, characterized in that said test kit in addition to the commonly used carrier materials, reagents and other additives at least one monoclonal antibody according to any one of claims 12 or 14 bis Contains 16 as a reagent. 60. Means for the immunological detection of Hydroxyatrazin and / or hydroxy analogues of Atrazinderivaten in the form of a ready-test kit, characterized in that said test kit in addition to the commonly used carrier materials, reagents and other additives, at least one monoclonal antibody according to any one of claims 13 or Contains 17 to 21 as a reagent. 61. Means for the immunological detection of atrazine and / or atrazine derivatives in the form of a ready-to-use test kit according to claim 59, characterized in that said test kit in addition to the commonly used carrier materials, reagents and other additives contains at least one monoclonal antibody from a Hybridoma cell line which has the distinguishing characteristics of ECACC 8808/2501 or is produced from clones or subclones thereof. 62. Means for the immunological detection of hydroxyatrazine and / or hydroxy analogues of Atrazinderivaten in the form of a ready-test kit according to claim 60, characterized in that said test kit in addition to the commonly used carrier materials, reagents and other additives contains at least one monoclonal antibody as a reagent produced by a hybridoma cell line which has the distinguishing characteristics of ECACC 8808/2502 or clones or subclones thereof. 63. Means for the immunological detection of Hydroxyatrazine and / or hydroxy analogues of Atrazinderivaten in the form of a ready-test kit according to claim 60, characterized in that said test kit contains in addition to the commonly used carrier materials, reagents and other additives at least one monoclonal antibody as a reagent which is produced by a hybridoma cell line having the distinguishing characteristics of ECACC 8808/2503 or clones or subclones thereof. '