NZ230675A - Atrazine and atrazine derivative detection using a monoclonal antibody immunoassay - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £30675 2306 75 Priority Psts): I..v.O.. ■ r- Q Corn"'-*** PoodJicat-on FIleo: '7.'... 1 Class: (j;...C?.C.i.fv.1.??.::■/.:y...?✓.y.. 'icatiwj C^ts: l-.O. Journal, No: ZiAW'M' NO DRAWINGS ?rr°r,ts Form :;o. 3 1 5 SEP 1989 NEW ZEALAND patents act 1953 ^ complete specification IMMUNOLOGICAL DETECTION OF ATRAZINE AND ATRAZINE DERIVATIVES )f/Ws, CIBA-GEIGY AG/ A Swiss Corporation, of Klybeckstrasse 141, 4002 Basle, SWITZERLAND hereby declare the invention, for which ty'-'e pray that a patent may be granted to rte/us, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page la) 230675 p! - !q- -17227/= Tmmunological detection of atrazine and atrazine derivatives The present invention relates to monoclonal antibodies which are distinguished by high selectivity and affinity for atrazine and/or atrazine derivatives and/or for inactive atrazine metabolites and which are therefore suitable in an outstanding manner for use in an immunoassay for the rapid and efficient detection of atrazine and/or atrazine derivatives as well as of inactive atrazine metabolites.

Another aspect of the present invention relates to hvbridoma cell lines which produce said monoclonal antibodies as well as immunological methods for the detection of atrazine and/or atrazine derivatives as well as of inactive atrazine metabolites by use of said monoclonal antibodies, and to the test kits which can be used within the scope of these detection methods.

The use of synthetic herbicides for the purpose of crop protection and the environmental pollution associated therewith have become the centre of public discussion increasingly frequently in recent times. s-Triazines and s-triazine derivatives such as atrazine, simazine or propazine have, since the discovery of their herbicidal potency in the 1950s, been employed on a large scale for the purpose of crop protection, especially for controlling annual broad-leaved weeds as well as various wild grasses in maize crops.

Conservative estimates suggest that of the order of 45 million and up to 100 million hectares of maize fields are currently treated with s-triazines or s-triazine derivatives. (followed by page 2) 2306 75 It has long been known that s-triazines such as, for example, atrazine, simazine or propazine are broken down relatively slowly in soil and other biotopes.

This persistence of the s-triazines mi the soil is also of great importance for the agriculturalist. When atrazine is used in the amounts which need to be applied, for example, in maize crops it is possible even within one crop rotation for problems to arise with alrazine-sensitive following crops so that the agriculturalist is forced in some circumstances to have an elaborate laboratory soil analysis carried out. The analytical methods used therein are extremely time-consuming and cost-intensive because they depend on highly specialised and elaborate apparatus.

For the stated reasons it must therefore be regarded as an extremely pressing task to improve the existing detection methods for synthetic herbicides, especially for s-triazines and s-triazine derivatives, as well as the breakdown products thereof, too, that is to say to develop methods which are less costly, more efficient and easier to operate and which can also be used outside the laboratory under field conditions and which, for example, provide the agriculturalist quickly and reliably with information on whether and in what concentrations a particular active ingredient or metabolite is present in, for example, soil or water.

Of particular importance in this connection is the possibility of differentiating between the active ingredient and its inactive breakdown products, because only in this way is it possible to determine quantitatively the actual amount of active ingredient still present in the soil.

Atrazine and its main breakdown product hydroxyatrazine were formerly detected in soil and water samples primarily by means of TLC (thin-layer chromatography) as well as with the aid of spectroscopic methods. By contrast, nowadays mainly HPLC (high pressure liquid chromatography) (Ramsteiner and Hormann, 1979) and GC (gas chromatography) (Ramsteiner K, 1985) methods are used. 230675 All the said methods for the detection of synthetic herbicides are, however, associated with numerous disadvantages. Thus, for example, for the determination of atrazine or hydroxyatrnzine in soil samples by means of HPLC it is necessary to interpolate elaborate purification and concentration sreps before the actual ch'otn.itographic analysis is carried ou t.

Another disadvantage of the HPLC methods derives from the fact that photometric detectors, which are relatively non-specific, are used therein. Besides mass-spectroscopic detection, the chromatographic analyses are based on the determination of retention times in the particular system. However, these values are relative and thus not structure-specific.

Analysis of hydroxyatrazine by gas chromatography in conjunction with determination by mass spectroscopy can be carried out only with considerable elaboration because of the low volatility of this compound.

In order to avoid these disadvantages, which have been described above, of the established analytical methods, there have been recent attempts to develop immunological methods - as are already employed routinely in clinical diagnostics for detecting a wide variety of antigens - in the agricultural sector too, especially for the quantitative and qualitative determination of agricultural chemicals in soil, water or air samples.

Thus, for example, the development of immunological methods for detecting certain herbicides such as 2,4-dichlorophenoxyacetic acid (Fleeker, 1986) or chlorsulfuron (Kelley et al. , 1985) as well as various pesticides such as diflubenzuron (Wie and Hammock, 1982), metalaxyl (Newsome, 1985) or parathion (Ercegovich et al■, 1981) has now started.

A method has even been disclosed already for the immunological detection of atrazine (US-P 4,530,786), but this is based, as are the above-mentioned methods too, on the use of polyclonal antisera obtained from animals previously immunized with an appropriate antigen. 230675 - u - Polyclonal antiser.i have a very heterogeneous composition, that is to say they contain a large number of different antibodies which react with different epitopes of the particular antigen. This heterogeneous composition of polyclonal antisera comes about because when an experimental anrmal is immunize! with a particular antigen there is always simultaneous stimulation of several antibody-producing cell clones, each of which recognizes a different epitope on the antigen molecule, and therefore different antibodies are produced by the stimulated cell clones.

This is why immune sera from immunized animals are always polyclonal and thus heterogeneous with regard both to their specificity and to their membership of the individual classes of immunog1obu1ins.

The result of this heterogeneity in the composition of polyclonal antisera may therefore be that structurally closely related compounds, such as, for example, atrazine and its main breakdown product hydroxy-atrazine, cannot be differentiated to a sufficient extent when polyclonal antibodies are used in an immunoassay.

By contrast, the object which this invention was intended to achieve related primarily to the provision of a highly selective immunoassay which is simple to operate and efficient for rapid and reliable detection of atrazine and/or atrazine derivatives as well as of inactive atrazine breakdown products, but especially of hydroxyatrazine and/or hydroxy-atrazine derivatives, which make it possible to differentiate atrazine and/or atrazine derivatives from their breakdown products.

It has now been possible, surprisingly, to achieve this object within the scope of the present invention, specifically by providing monoclonal antibodies with high specificity and affinity for atrazine and/or atrazine derivatives as well as by providing monoclonal antibodies with high specificity and affinity for the breakdown products of atrazine and/or atrazine derivatives, but especially for the hydroxyl analogues of atrazine and/or atrazine derivatives such as, for example hydroxyatrazine, hydroxysimazine, hydroxypropazine etc., by using the hybridoma/monoclonal antibody technology known per se. 230675 The use of hybrid somatic cell linos (hybr i doina s) as source of antibodies against very particular antigens derives from the work of Kohler and Milstein (Nature 256: 495-97, 1975).

The antibodies which can be obtained by the methods described therein differ very greatly from those obtained from the antisera of conventionally immunized animals.

The principle of the hybridoma/monoclonal antibody technology is based on the observation that when two somatic cells are fused 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) which has been taken from a previously immunized donor animal, whereas the ability for continuous cell division in culture is contributed by the other fusion partner, a tumour cell line (often a myeloma).

Each of these hybrid cell lines synthesizes a homogeneous immunoglobulin which represents only a single representative from the large number of possible antibodies which can be synthesized in vivo by an animal in response to an antigen.

Since every immunoglobulin-producing clone is characterized by a single type of antibody, the name monoclonal antibodies has become established.

There are many advantages of monoclonal over polyclonal antibodies: a) monoclonal antibodies can be obtained in large numbers and in high purity, b) the preparation of monoclonal antibodies is homogeneous with regard to the antigen reactivity and does not change over the course of time either, t c) hybridomas producing monoclonal antibodies can be stored for years or decades without losing their specific properties, i.e. the production of specific monoclonal antibodies, d) monoclonal antibodies are more suitable than polyclonal antisera for use as standard reagents because the latter are adversely affected by a large range of variation, for example with regard to a) the taking of blood from immunized animals for obtaining the antiserum, B) continuous availability of material for additional immunizations, y) the limited life span of the donor animals.

Monoclonal antibodies, which have now been prepared against a large number of antigens, are well established primarily in medical diagnostics, and the latter is now inconcoiveable without them.

By contrast, the use of monoclonal antibodies in the agricultural sector has hitherto been primarily confined to screening for plant diseases and development of vaccines for productive livestock.

In connection with plant diseases, for example, Hsu H.T., et a 1. , (ASM News, 50(3): 91-101, 1984) have listed a total of 18 species of plant viruses 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.

The present invention has succeeded for the first time, by use of the hybridoma/monoclonal antibody technology which is known per se and has been briefly described previously, in making available both monoclonal antibodies with high specificity and affinity for atrazine and/or atrazine derivatives and monoclonal antibodies with high specificity and affinity for inactive breakdown products of atrazine and/or atrazine derivatives, which are suitable, by reason of their specificity and the consequent low mutual cross-reactivity, in an outstanding manner for use in an immunoassay for the rapid and reliable detection of atrazine and/or 23 0 6 7 5 atrazine derivatives on the one hand and the inactive breakdown products thereof on the other hand, and thus can also be used for differentiating atrazine and/or atrazine derivatives from their inactive metabolites.

The •jresent invention thus primarily relates to monoclonal antibodies, and the derivatives thereof, which have a high specificity and affinity for atrazine and/or atrazine derivatives and which show essentially no cross-reactivity with metabolites of atrazine and/or atrazine derivatives, but especially no cross-reactivity with the inactive hydroxyl analogues of atrazine and/or atrazine derivatives.

This application furthermore relates to monoclonal antibodies, and the derivatives thereof, which have a high specificity and affinity for the inactive breakdown products of atrazine and/or atrazine derivatives, especially for the hydroxyl analogues of atrazine and/or atrazine derivatives, and which show essentially no cross-reactivity with atrazine and/or atrazine derivatives.

Particularly preferred within the scope of this invention are monoclonal antibodies, and the derivatives thereof, which have a high specificity and affinity for atrazine and which show essentially no cross-reactivity with hydroxyatrazine or hydroxyl analogues of atrazine derivatives such as hydroxysimazine, hydroxypropazine etc.

Likewise preferred within the scope of this invention are monoclonal antibodies and the derivatives thereof, which have a high specificity for hydroxyatrazine and which show essentially no cross-reactivity with atrazine or atrazine derivatives such as, for example, propazine, simazine, ametryn, atratone, etc.

Likewise embraced by the present invention are monoclonal antibodies, and the derivatives thereof, which have a high specificity and affinity for atrazine and which show a pronounced cross-reactivity, but in particular a cross—reactivity of more than 50 %, preferably of more than 80 %, with certain atrazine derivatives of similar structure, especially with those O 7 £ J selected from tlie group comprising propazine, promctryn and promoton, but which show essentially no cross-reactivity witli other atrazine derivatives or with hydroxyl analogues of atrazine and/or atrazine derivatives.

This application furthermore relate;- to monoclonal antibodies, and the derivatives thereof, which have a high specificity and affinity for hydroxyatrazine and which show a cross-reactivity, but in particular a cross-reactivity of at least 40 %, with various hydroxyl analogues of atrazine derivatives, but which show essentially no cross-reactivity with atrazine and/or atrazine derivatives.

Very particularly preferred within the scope of this invention are monoclonal antibodies, and the derivatives thereof, which have a high specificity for hydroxyatrazine and which show a pronounced cross-reactivity, but in particular a cross-reactivity of up to 100 %, with the structurally very similar hydroxypropazine but which show essentially no cross-reactivity with atrazine or atrazine derivatives such as, for example, propazine, simazine, ametryn, atratone, etc. as well as with hydroxyl analogues of atrazine derivatives such as hydroxysimazine, hydroxydesmetryn, hvdroxyterbuthylazine etc.

By derivatives of monoclonal antibodies are meant within the scope of the present invention, for example, antibody fragments which still have the high specificity and affinity for the antigenic determinants of atrazine and/or atrazine derivatives or hydroxyatrazine and/or hydroxyl analogues of atrazine derivatives, furthermore radioactively labelled monoclonal antibodies which are labelled, for example, with radioactive iodine (12*1, i3ij)t carbon (1 ■* C), sulfur (35S), tritium (3H) or the like, conjugates of monoclonal antibodies with biotin or avidin, with enzymes such as horseradish peroxidase, alkaline phosphatase, B-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malic dehydrogenase or glucose-6-phosphate dehydrogenase, furthermore conjugates of monoclonal antibodies with bioluminescent (for example luciferase) , chemoluminescent (for example acridinium esters) or fluorescent (for example phycobiliproteins) agents. The present application likewise embraces bispecific as well as so-called cross-linked 230675 antibodies. This list of examples of possible antibody derivatives serves merely for illustration of the present invention and is not intended to limit the subject-matter of the invention in any way.

«****"\ The term "essentially no c^oss-reactivity" is intended to mean within the scope of this invention that the reactivity of the atrazine- or hydroxy-atrazine-speci f ic monoclonal antibodies with non-specific epitopes of other compounds, in particular structurally related compounds, is less ■""" than 20 %, but is preferably less than 5% and very particularly prefer ably less than 1 %.

The definition of the percentage cross-reactivity within the scope of this invention is given by the following relation: (Atrazine/hydroxyatrazine concentration for 50 % inhibition/concentration of the s-triazine/hydroxytriazine analogues for 50 % inhibition) x 100.

A 50 % inhibition can be determined, for example, with the aid of a competitive ELISA assay (cf. Example 8). The latter then corresponds, for example, to that antigen concentration which results in a 50 % inhibition of the antibody binding to the carrier-bound antigen.

The present invention further relates to hybridoma cell lines which synthesize, and preferably secrete into the surrounding medium, the monoclonal antibodies characterized in detail previously.

The present invention particularly relates to a hybridoma cell line which produces a monoclonal antibody which has a high specificity and affinity for atrazine and/or atrazine derivatives and which shows essentially no cross-reactivity with the inactive hydroxyl analogues of atrazine and/or atrazine derivatives.

Likewise embraced is a hybridoma cell line which produces a monoclonal antibody which has a high specificity and affinity for the hydroxyl analogues of atrazine and/or atrazine derivatives and which shows essentially no cross-reactivity with atrazine and atrazine derivatives. 2306 75 This invention further relates to a hybridoma cell lino which produces a monoclonal antibody which has a high specificity and affinity for atrazine and which shows essentially no cross-reactivity with hydroxyatrazine or other hydroxyl analogues of atrazine derivatives.

A particularly preferred hybridoma cell line synthesizes, and secretes into the surrounding medium, a monoclonal antibody which has a high specificity and affinity for atrazine and which shows a very pronounced cross-reactivity, but in particular a cross-reactivity of more than 50 %, preferably of more than 80 %, with atrazine derivatives of similar structure, in particular with those selected from the group comprising propazine, prometryn and prometon, but which shows essentially no cross-reactivity with other atrazine derivatives as well as with hydroxyatrazine or hydroxyl analogues of atrazine derivatives.

A hybridoma cell line which has the identifying characteristics of ECACC 8808/2501, as well as the clones and subclones thereof, is very particularly preferred.

The present invention further relates to a hybridoma cell line which produces a monoclonal antibody which has a high specificity and affinity for hydroxyatrazine and which shows essentially no cross-reactivity with atrazine and/or atrazine derivatives.

Likewise particularly preferrred within the scope of this invention is a hybridoma cell line which synthesizes a monoclonal antibody which has a high specificity and affinity for hydroxyatrazine and which shows a cross-reactivity, but in particular a cross-reactivity of more than AO %, with compounds of similar structure, such as hydroxysimazine, hydroxy-propazine and hydroxydesmetryn, but which shows essentially no cross-reactivity with atrazine and/or atrazine derivatives.

A hyridoma cell line which has the identifying characteristics of ECAC 8808/2502, as well as the clones and subclones thereof, is very particularly preferred. 230675 - n - Likewise preferred within the scope of this invention is a hybridoma cell line which synthesizes, and secretes into the surrounding medium, a monoclonal antibody which has a high specificity and affinity for hydroxyatrazine and which shows a pronounced cross-reactivity, but in particular a c ros s-1 e.'C t i <.-i t y ot up t>-> 100 %, with the structurally very similar hvdroxypropazine but which shuws essentially no cross-reactivity with other hydroxyl analogues of atrazine derivatives such as, for example, hydroxysimazine, hydroxydesmetryn or hydroxyterbuthylazines , as well as with atrazine and/or atrazine derivatives.

A hybridoma cell line which has the identifying characteristics of ECACC 8808/2503, as well as the clones and subclones thereof, is very particularly preferred.

Likewise embraced by the present invention are variants and mutants of the hybridoma cell lines characterized in detail previously, which arise spontaneously or else can be produced artificially with the aid of known methods and which still have the characteristic properties of the starting material, that is to say are still able to produce, and secrete into the surrounding medium, the antibodies according to the invention, or derivatives thereof.

Likewise embraced by the present invention are methods for the preparation of said hybridoma cell lines as well as methods for the preparation of said monoclonal antibodies.

By clones and subclones of hybridoma cell lines are meant hybridomas which originate from the starting clone by repeated cloning and which still have the features of the starting clone which are essential to the invention.

This invention further relates to a method for the immunological detection of atrazine and/or atrazine derivatives, for example in soil, water or air samples as well as in biological material such as, for example, in plant or animal extracts, by use of the monoclonal antibodies according to the invention. 230675 The present invention furthermore relates to a method for the immunological detection of li yd roxya t raz i ne and/or hydroxyl analogues of atrazine derivatives, for example in soil, water or air samples as well as in biological material such as, for example, in plant or animal extracts, by use of the monoclonal antibodies according to i lie invention.

A method for the detection of hydroxyatrazine is very particularly preferred.

Likewise part of the present invention are compositions for the qualitative and quantitative determination of atrazine and/or atrazine derivatives or of breakdown products of atrazine and/or atrazine derivatives, but in particular of hydroxyl analogues of atrazine and/or atrazine derivatives, such as, for example, hydroxyatrazine, hvdroxypropazine, hydroxysimazine etc., in the form of ready-to-use test kits which contain at least one of the monoclonal antibodies according to the invention as reagent and which are suitable for use under field conditions for a rapid and reliable detection of atrazine and/or atrazine derivatives as well as of breakdown products of atrazine and/or atrazine derivatives, but in particular of hydroxyatrazine and/or hydroxyl analogues of atrazine derivatives.

The monoclonal antibodies according to the invention are prepared using methods which are known per se and which are essentially based on the methods developed by Kohler and Milstein (Nature, 256: 495-497, 1 975).

Since the target substances atrazine and hydroxyatrazine which are to be analysed and for which the specific monoclonal antibodies are to be developed are relatively small and simple molecules which after administration to an experimental animal are not by themselves able to induce an appropriate immune response there, it is necessary before the actual immunization first to apply preparatory measures.

Compounds of this type, which by reason of their size and simple structure are not able to induce an immune response, are called haptens or incomplete antigens and are thus contrasted with complete antigens (= immunogens) which both act as antigen and are able to induce an immune 230675 response. Hapten molecules of this type can be conjugated to high molecular weight compounds (carrier molecules), by which means they become comparable in their properties to complete antigens, that is to say they now have the ability to induce an immune response.

Some of the antibodies formed curing the course of the immunization reaction are able to react wit.lt specific epitopes on the hapten molecule, irrespective of whether the hapten molecule is present by itself or else remains coupled to the carrier molecule.

The term hapten which is frequently used hereinafter is intended to mean within the scope of this invention primarily the atrazine and/or hydroxyatrazine molecules used for the immunization.

Hence, within the scope of this invention, the atrazine and hydroxyatrazine molecules acting as haptens are coupled, before the immunization of experimental animals, to a high molecular weight carrier which is suitable for conferring complete antigenic activity on said atrazine and hydroxyatrazine molecules.

By suitable carrier molecules are meant within the scope of this invention primarily macromolecular compounds which have freely accessible reactive groups for the coupling reaction with the hapten and which are able to confer on the hapten, by coupling to it, an immunogenic potency or else to enhance the immunogenicity thereof which is already present.

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

Very particularly preferred for the use according to the invention as carrier molecule are lysine-rich proteins with a molecular weight between 10,000 and 1,500,000 such as, for example, bovine serum albumin (BSA: MW 66,200), human serum albumin (HSA: MW 58,000) or keyhole limpet protein (KLH; MW 1,000,000), which can be obtained commercially and thus are available in any desired amount. 230675 Of course, it is also possible within the scope of the present invention to use other macromolecular compounds as carrier molecules as long as they meet the abovementioned requirements, such as, for example, porcine thyroglobulin, B2 microglobulin, hemoevnnin, immunoglobulins, toxins (cholera, tetanus, diphtheria toxin -;tc.), polysaccharides, lipopoly-saccharides, natural or synthetic po1yndeny 1 ic and polyuridvlic acids, polyalanyl and polylysine polypeptides or cell membrane components such as, for example, formaline or glutaraldehyde-treated erythrocyte cell membranes.

Likewise suitable for use as carrier molecule in the method according to the invention is, for example, the purified IgG fraction against mouse IgG (H+L) from rabbits according to the method described by 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 else, preferably, via a spacer which is, where appropriate, first attached to the hapten molecule.

In this connection, the coupling of the substance which is to be analysed 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 induce a specific immune response, that is to say induce the formation of specific antibodies.

Primarily suitable as a spacer for the conjugation of the hapten (atrazine and/or atrazine derivatives as well as the hydroxyl analogues of atrazine and/or atrazine derivatives) to the carrier molecule are compounds which contain at least one or else several reactive groups which are able to enter into an interaction with the freely accessible reactive groups of the carrier molecule.

Particularly preferred within the scope of this invention is the use of a spacer molecule which comprises between 3 and 10 bridge carbon atoms and which have as reactive group(s) one or more reactive groups such as, for example, amino, carboxyl or SH group(s). 230675 These reactive groups can be induced to react by methods known per se with the reactive groups of the hapten and carrier molecule with the formation of a hapten-carrier conjugate.

Thus, for example, i'. is possible to bond a spacer molecule via a reactive amino group with the aid of dialdehydes (for example glutardi-aldehyde) to one of the free amino groups of the carrier molecule.

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

Particularly preferred within the scope of this invention is the use of a spacer molecule with a carboxyl group which can be linked with the aid of water-binding agents such as, for example, a carbodiimide, preferably N,N1-dicyclohexylcarbodiimide, to a free amino group of the carrier molecule.

A specific embodiment of the present invention starts from atrazine, hydroxyatrazine or one of their derivatives, preferably from 2,6-di-chloro-4-(isopropylamino)-s-triazine, which is reacted with 6-amino-valeric acid with the formation of 2-chloro-4-(isopropylamino)-6-(6-aminovaleric acid)-s-triazine. This compound can then be converted very straightforwardly by known methods, such as, for example by addition of aqueous mineral acids, in another reaction step into the corresponding hydroxyl analogue.

The actual coupling reaction is preferably carried out by the active ester method. This entails the derivatives of atrazine or hydroxyatrazine first being solubilized in a suitable solvent. Particularly suitable solvents are aprotic solvents which have a low rate of evaporation, such as, for example, N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO).

The carboxyl groups are subsequently derivatized to an active ester in such a way that the derivatives of atrazine and/or hydroxyatrazine which have previously been solubilized are reacted, for example, with N-hydroxysuccinimide, N-hydroxysulfosuccinimide, N,N'-dicyclohexy 1 -carbodiimide or N', N'-carbonyldiimidazole or with derivatives of these compounds.

The active e-ter is then removed from the reaction mixture and added to BSA or KLH. An incubation time of 0.1 to 12 hours, preferably of 4 to 5 hours, is followed by removal of the precipitate. The supernatant can then be used, where appropriate after interpolation of further purification steps, for the actual immunization reaction.

Besides the active ester method which is preferred within the scope of this invention, it is also possible to use alternative methods for the coupling of the hapten to the carrier molecule, such as, for example, the mixed anhydride method. This entails the carboxyl group of the bridge member being linked to the carrier molecule by use of acetic anhydride or of the carbodiimide derivative 1-ethy1 — 3—(3'-dimethylaminopropyl)carbodiimide .

The donor animals are immunized by administration, one or more times, of the haptens which are coupled to a high molecular weight carrier molecule. Administration 2 or 3 times, which takes place at intervals of 7 to 30 days, but in particular 13 to 15 days, is particularly preferred.

The administration form preferred within the scope of the present invention is injection, which can take place both intravenously, intra-peritoneally or subcutaneously.

A combination of subcutaneous and intraperitoneal injection is preferred.

A rest period of 0.5 to A months is followed by another single administration of the hapten conjugate in a dosage of 100 pg to 1000 pg.

In a period of 1 to 6 days after the last dosage the donor animals are sacrificed, and a spleen cell suspension is prepared. o 230675 This entails the isolated spleen cells being suspended in a suitable buffer (for example a BSS buffer) and stored in the form of a cell suspension until they are fused with suitable myeloma cells.

These fusions were initially complicated by the fact that the myeloma cell lines also synthesized monoclonal antibodies so that the hybrid produced two types of monoclonal antibodies, one having its origin in the myeloma cell and a second defined by the genetic information in the immunocompetent cell.

Hence, preferably used within the scope of the present invention are those tumour cells which are themselves unable to produce any monoclonal antibodies, such as, for example SP2/0-Agl4 (Shulman et al ■ , 1978) or X63-Ag8.653, which makes the analysis of the resulting fusion products very much more straightforward. It is advantageous for the success of fusion if the spleen cells are present in a 2 to 20-fold excess in relation to the myeloma cells.

The fusion of spleen and myeloma colls is carried out in a special fusion medium which has a composition which provides optimal conditions for the intended cell fusion.

Said fusion medium is preferably a buffer solution which contains one of the fusion promoters customarily used for the fusion of cells, such as, for example, sendai viruses or other paramyxoviruses, where appropriate in UV-inactivated form, calcium ions, surface-active lipids such as, for example, lysolecithin or polyethylene glycol. Particularly preferred within the scope of this invention is the use of polyethylene glycol (PEG), in particular of polyethylene glycol (PF.G) with a mean MW of 600 to 6000, and in a concentration of 30 % to 60 %. A PEG concentration of 40 % - 50 % is particularly preferred. The optimal fusion temperature is between 18°C and 39°C. A temperature of 37°C is particularly preferred.

After the fusion of the immunocompetent spleen cells with the myeloma cells has taken place the fused antibody-producing hybrid cells are selected with the aid of methods known per se. a 230675 18 Various possibilities exist for the selection of successful fusion events (hybrid cells) from the 2 types of parent cells. Routinely, one million and more cells of each parent type is used in the fusion protocol. Since the fusion does not take place with a 100 % frequency, it may become a difficult enterprise to separate the fusion products from the large excess of unfused or self-fused parent cells.

As already mentioned previously, the hybridoma cells are produced by fusion of short-lived antibody-producing (spleen) B cells as well as long-lived myeloma cells.

The desired result is a long-lived cell line which produces antibodies. Since the spleen cells have only a limited life span in culture, it is therefore possible in principle simply to wait until all the unfused as well as all the self-fused spleen cells have died. However, there then still remains the task of separating the long-lived antibody-producing cells from the long-lived cells producing no antibodies.

A conventional selection system is based on the availability or nonavailability of the enzyme hypoxanthine-guanine phosphoribosyl-trans-ferase (HGPRT). This enzyme is a constituent of the purine salvage pathway in mammalian cells.

These cells are additionally able to carry out de novo synthesis of pu rines.

It is probable that under normal conditions these two synthetic pathways operate to a certain extent in parallel.

However, if a cell has no HGPRT, the salvage pathway is blocked and the purines must be prepared from non-purine material.

Used for the selection of HGPRT-negative myeloma cells are, as a rule, so-called purine antimetabolites such as, for example, 8-azaguanine, which has a very similar structure to that of the purine guanine and is therefore able to replace the latter in some of its normal reactions. 2306 75 Azaguanine is incorporated in the DN'A, which leads to an impairment of normal growth behaviour and finally to the death of cells. Since azaguanine must be replaced by the salvage pathway, none of the cells which have no HGPRT activity are able to utilize azaguanine and thus grow in the presence thereof.

A selective system which operates with the same enzyme but by reversing the sign, in that HGPRT-positive cells are selected in this case, is described by J.W. Littlefield (1964).

This selection system is based on the use of the so-called HAT medium which contains, inter alia, hypoxanthine, aminopterin and thymidine (HAT medium) as constituents. Aminopterin is an antimetabolite which inhibits de novo purine synthesis as well as the methylation of deoxyuridylate to thymidyla t e.

Hypoxanthine can act as accessory purine in the case where aminopterin blocks de novo purine synthesis, while thymidine makes the necessity for methylation of deoxyuridylate superfluous.

This is why all HGPRT-positive cells proliferate in the presence of aminopterin, while the HGPRT-negative cells die.

In the hybrid system which is used for selection within the scope of this invention, the myeloma cells are preferably resistant to azoguanine and sensitive to aminopterin that is to say they are HGPRT negative. The antibody-producing cells are, on the other hand, HGPRT positive.

It is possible by fusing the cells and cultivating in an HAT medium to select the cells which have successfully fused together because the myeloma cells which are responsible for the proliferation are able to grow only in the presence of an HGPRT activity and this activity must be provided by the HGPRT-positive cell line.

The HGPRT-positive antibody-producing cell lines are not in fact killed in this medium. However, they survive for only a certain time and are not able to proliferate. 230675 n Thus, fusion of the cells in an HAT medium results in a system in which, although myeloma cells and the antibody-producing cells are able to grow for a period which is sufficient for the production of hybrid cells, only the hybrid cells are able to survwe and proliferate.

In a preferred embodiment of the present invention, the fused hybrid cells are cultivated in the presence of so-called feeder cells which are macrophages isolated previously from the peritoneum of untreated non-immunized experimental animals. For the cultivation and the selection of the fused hybrid cells, the cell suspension is divided into several aliquots, and the individual aliquots are continually examined for the development of hybrid coll cultures as well as for the formation of antibodies.

Cultivation of the fused hybrid cells on microtiter plates is particularly preferred within the scope of this invention.

This entails the cell suspension obtained after the fusion being distributed over the individual wells in a microtiter plate and cultivated for a period of 7 to 30 days under suitable conditions promoting the growth of the fused hybrid cells (for example HAT/HT media).

The supernatants from grown hybrid cultures are continually examined for the formation of antibodies.

Positive hybrid cell cultures are then singled out using known methods, but preferably using the limiting dilution method, and subsequently cloned in suitable cultivation media.

The supernatants from the grown cell clones are likewise examined for the formation of antibodies.

The screening of the hybridoma cell clones according to the invention, which have been prepared as described hereinbefore, for the formation of suitable monoclonal antibodies is preferably carried out using one of the 230675 immunoassays customarily used for this purpose, such as, for example, an enzyme-coupled immunoassay or a radioimmunoassay.

In the enzyme-coupled immunoassay the hapten conjugates which have been characterized in detail previously are first adsorbed onto a solid carrier. The remaining free binding sites are subsequently saturated, and thus blocked, by addition of carrier molecules.

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

The present invention further relates to the preparation of monoclonal antibodies by use of methods which are known per se and which are characterized in that the hybridoma cell lines according to the invention, which have been characterized in detail previously, or else clones or subclones thereof, which synthesize, and secrete into the surrounding medium, the antibodies according to the invention, are cultivated in vitro or in vivo by known methods.

The in vitro cultivation of the hybridoma cells according to the invention is carried out in suitable cultivation media, in particular in the standardized culture media customarily used, such as, for example, Dulbecco's modified Eagle medium (DMEM) or RPMI 16A0 medium, which can be supplemented, where appropriate, by addition of mammalian sera such as, for example, fetal calf serum, or by growth-promoting additives and trace element s.

The isolation of the monoclonal antibodies preferably starts with a precipitation of the immunoglobulin fraction from the relevant supernatants from the hybridoma cultures, for example using ammonium sulfate. This is followed by further working up and purification steps which are known to those skilled in the art and include, for example, the use of chromatographic methods such as, for example, gel filtration, ion exchange chromatography, DEAE-cellulose chromatography, protein A or immunoaffinity chromatography. p> 2 3 0 6 7 5 Large amounts of the monoclonal antibodies according to the invention can also be obtained, however, using in vivo methods.

Thus, for example, it is possible to inject antibody-producing hybridoma cell clones into suitable >na initials, which induce the development of antibody-producing tumours in the treated animals. It is possible after a period of 1 to 3 weeks to isolate the antibodies from the body fluids of the animals treated in this way.

In a particular embodiment of the present invention, female Balb/c mice which have, where appropriate, been pretreated with a hydrocarbon such as, for example, pristane receive intraperitoneal injection of a hybridoma cell clone according to the invention.

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

The monoclonal antibodies are isolated in a manner exactly analogous to the isolation described previously from the supernatants from hybridomas cultivated in vitro.

The present invention further relates to the use of the antibodies according to the invention in one of the conventional immunoassays for the detection of atrazine and/or atrazine derivatives and/or of inactive breakdown products of atrazine and/or atrazine derivatives as well as for differentiating atrazine and/or atrazine derivatives from their inactive breakdown products in soil, air and water samples as well as, where appropriate, in extracts from plants or other biological material.

The monoclonal antibodies according to the invention can thus be used in all known immunoassays based on the specific binding between antigen and the corresponding monoclonal antibody, such as, for example, in an radioimmunoassay (RIA), an enzyme-coupled immunoassay (ELISA), an immunofluorescence test etc. 230675 In the case of the RIA test, the monoclonal antibody according to the invention can bo used as such or else in the form of a radioactively labelled derivative. It is possible in this connection for all modifications disclosed to date of the RIA test to be used for detecting the target substances relevant within the scopi of this invention, such as, for example, an RIA test in homogeneous or solid phase, a heterogeneous RIA test as well as a single or double (sandwich) RIA test with direct or indirect (competitive) detection of the antigen. The same also applies to the use of an enzyme-coupled immunoassay.

Preferred within the scope of this invention is 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 hydroxyl analogues.

The principle of the competitive immunoassay is based on a competition between a free antigen and a labelled antigen or one which is bound to a solid carrier for the relevant binding sites on the antibody molecule.

A distinction is made in principle between two possible ways of carrying out this competitive immunoassay. a) The first method is based on the competition between free antigen and the antigen which is bound to a solid carrier for the free binding sites on the antibody which is provided with a marker. In this connection, the binding of the antigen to a solid carrier can take place either directly or else via a carrier molecule.

The concentration of free antigen is determined in this case via the decrease in the labelled antibody which is bound to the antigen immobilized on the carrier.

This decrease is proportional to the amount of free antigen contained in the sample. 23 0 6 75 - 2h - b) An alternative method is based on competition between free and labelled antigen for the relevant binding sites on the antibody, which in this case is bound to a solid carrier.

The concentration of free antigen is determined via th'- decrease in labelled antigen, which decrease varies as a junction of the concentration of free antigen.

Examples of suitable solid carrier material for binding the antigen or the antibody are the plastic surface of a microtitre plate or of a test tube, the surface of beads made of polystyrene, polypropylene, polyvinyl chloride, glass or plastic or else the surface of strips of filter paper, dextran-cellulose or nitrocellulose, or similar materials. The latter are coated with one of the monoclonal antibodies according to the invention or with an antigen, it being possible for the binding to the carrier material to be brought about by simple adsorption or else where appropriate after previous activation of the carrier material with, for example, glutaraldehyde or cyanogen bromide.

Particularly preferred vitliin the scope of this invention is the use of the monoclonal antibody according to the invention in an enzyme-coupled immunoassay [ELISA (enzyme linked immuno sorbent assay)].

In this connection it is possible for the monoclonal antibody according to the invention to 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-coupled derivative of the antibody according to the invention or else of enzyme-coupled antibodies which are known per se and which recognize an epitope of an antibody according to the invention and hind thereto.

Particularly preferred within the scope of this invention is the use of an ELISA assay in which one of the carrier materials described previously is first coated with an antigen. The carrier-bound antigen is subsequently incubated with a test solution which contains the antigen to be 230675 detected as well ns one of the antibodies according to the invention. In this connection, the antigen to be detected can be present either in free form or else as constituent of a water or soil sample.

After an incubation time of 10 mirutes to 2 hours the complete mixture is incubated with an enzyme-labe1 led antibody which recognizes the monoclonal antibody according to the invention and binds to the latter. One example of an enzyme-labelled antibody of this type is a phosphatase-labelled goat anti-sheep immunoglobulin, or a corresponding goat anti-mouse antibody, both of which can be obtained commercially.

The amount of bound antibody protein can be determined by means of an enzyme-substrate reaction, for example using spectroscopic methods.

Likewise preferred within the scope of this invention is an ELISA assay which is based on the competition of labelled and of free antigen for the antibody which is bound to one of the carrier materials mentioned previously.

The quantity of free antigen present in a particular sample is in this case determined via the decrease in labelled antigen, the precision of this decrease becoming greater with the amount of free antigen contained in the sample.

Another possible way of carrying out the ELISA assay comprises the following methodological measures: One of the carriers described previously, 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. After this the entire mixture is incubated with a polyclonal immune serum against said antigen, for example a sheep immune serum, and the bound antibodies in the polyclonal serum are revealed using enzvme-labelled antibodies which recognize and bind to the latter. The amount of bound protein is determined by means of an enzyme-substrate reaction. 230675 One example of an enzyme-labelled antibody of this type is a phosphatase-labelled goat anti-sheep immunoglobulin which is commercially available.

Another possible way of carrying out the ELISA assay comprises one of the carrier materials which has been described previously arid is coated with a monoclonal antibody according to the invention being incubated with a test solution and with a second solution which contains an enzyme-conjugated monoclonal antibody.

This entails the free monoclonal antibody recognizing a different epitope on the antigen than the enzvme-coupled antibody. The amount of bound enzyme can be determined by moans of an enzyme-substrate reaction, for example in the form of a colour change which can be detected visually.

This colour change is proportional to the amount of antigen in the test solution.

Moreover, the antibody according to the invention can also be used in an ELISA test in which the latter is labelled with an enzyme, and the carrier is coated with a monoclonal anti-antigen antibody which recognizes a different epitope than the monoclonal antibody according to the invention.

The present invention furthermore relates to compositions for the qualitative and quantitative determination of atrazine and/or atrazine derivatives and/or of breakdown products of atrazine and/or atrazine derivatives, but in particular of hydroxyl analogues of atrazine and/or atrazine derivatives, such as, for example, hydroxyatrazine, hydroxy-propazine, hydroxysimazine etc., in the form of a test kit, which composition can, besides the monoclonal antibodies according to the invention and/or the derivatives thereof, optionally also contain other monoclonal or polyclonal antibodies, but in particular labelled monoclonal or polyclonal antibodies, as well as other additives. n 230675 Particularly preferred within the scopc of this invention are test kits which are based on one of the customarily used immunoassays selected from the group comprising radioimmunoassay, enzyme-coupled immunoassay and chemiluminescence assay.

Very particularly preferred test kits are those iri which the detection of atrazine and/or atrazine derivatives and/or of breakdown products of atrazine and/or atrazine derivatives, but in particular of hydroxyl analogues of atrazine and/or atrazine derivatives, such as, for example, hydroxyatrazine, hydroxypropazine, hydroxysimazine etc., is based on a competitive immunoassay, but in particular on an enzyme-coupled immunoassay (ELISA).

Test kits for a radioimmunological detection of atrazine and/or atrazine derivatives and/or of breakdown products of atrazine and/or atrazine derivatives, but in particular of hydroxyl analogues of atrazine and/or atrazine derivatives, such as, for example, hydroxyatrazine, hydroxy-propazine, hydroxysimazine etc., can contain, for example, the following constituents: (a) a suitable carrier material which can be uncoated or else coated with one of the antibodies according to the invention or an antigen conj ugate; (b) where appropriate freeze-dried or conccntrated solutions of one of the antibodies according to the invention and/or of a radioactively labelled derivative thereof or radioactively labelled antigen or standardized solutions of the antigen; (c) buffer solutions and (d) where appropriate polypeptides, detergents and other additives which, for example, prevent nonspecific adsorption and aggregate formation, as well as (e) pipettes, reaction vessels, calibration plots, pack inserts etc.

Tests kits for immunological detection of atrazine and/or atrazine derivatives and/or of breakdown products of atrazine and/or atrazine derivatives, but in particular of hydroxyl analogues of atrazine and/or 23 0 6 75 atrazine derivatives, such as, for example, hydroxyatrazine, hydroxy-propazine, hydroxysimazine etc., which are based on an enzyme-coupled immunoassay (ELISA) can contain, for example, the following constituents: (a) a suitable carrier material which can be uncoaled or else coated with one of the antibodies according to tlu- invention or with an antigen conjugate; (b) where appropriate freeze-dried or concentrated solutions of one of the antibodies according to the invention and/or of a second enzyme-labelled monoclonal or polyclonal antibody which is directed against the antigen to be determined or against an antibody recognizing the antigen; (c) enzyme substrates in solid or dissolved form; (e) the antigen or standardized solutions of the antigen; (f) buffer solutions; (g) where appropriate polypeptides, detergents and other additives which, for example, prevent non-specific adsorption and aggregate formation, as well as (h) pipettes, reaction vessels, calibration plots, colour tables, pack inserts etc.

A test kit for the detection of atrazine and/or atrazine derivatives and/or of breakdown products of atrazine and/or atrazine derivatives, but in particular of hydroxyl analogues of atrazine and/or atrazine derivatives, such as, for example, hydroxyatrazine, hydroxvpropazine, hydroxysimazine etc., which is based on the chemiluminescence test, can contain, for example, the following constituents: (a) a suitable carrier material which can be uncoated or else coated with one of the antibodies according to the invention or with an antigen conj ugate; (b) where appropriate freeze-dried or concentrated solutions of one of the antibodies according to the invention and of a second polyclonal antibody which is able to recognize the first antibody according to the invention and is linked to a chemiluminescent marker; (c) solutions containing a component which induces the emission of light, such as, for example, H2O2 and NaOH; (d) buffer solutions; o 230675 (e) where appropriate polypeptides, detergents and other additives which prevent non-specific adsorption and aggregate formation, as well as (f) pipettes, reaction vessels, pack inserts etc.

Carrier materials which --.an be used within the scpe of the present invention primarily comprise insoluble polymeric materials, selected from the group comprising polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, polyvinyl chloride, polyacrylamide, nitrocellulose, crosslinked dextran, fluorinated resins, agarose, crosslinked agarose, polysaccharides etc. However, besides these, other materials are also conceiveable, such as, for example, glass, metal, nylon-based mesh, etc.

The carrier materials specifically mentioned previously can have very different shapes and, depending on the specific purpose of use intended in each case, have a wide variety of forms. These comprise, for example, dishes, beads, plates, rods, cells, small bottles, tubes, filaments, meshes etc.

Often used for the production of test kits are, for example, microtitre plates made of transparent plastic materials such as, for example, polyvinyl chloride or polystyrene, which can be uncoated or else coated with one of the antibodies according to the invention, with free antigen or with an antigen conjugate. Likewise used are beads, tubes or rods made of polystyrene as well as polystyrene latex, in which case the surrounding latex material can be removed from the polystyrene particles by centrifugation.

Another constituent of the test kit according to the invention comprises markers or indicators which can be used to detect the presence of a complex-forming reaction, but in particular of an immunological reaction which preferably results in an antigen-antibody complex or else in a ligand-receptor complex, it being possible to draw not only qualitative but also, where appropriate, quantitative conclusions about the antigen to be detected. Suitable markers or indicators are both single atoms and molecules which can be involved either directly or else indirectly in the generation of a detectable signal. These markers or indicators can be linked either directly to the antigen to be detected or to one of the 230675 monoclonal antibodies according to the invention or else be incorporated in the latter. However, they can also be present as single substances or as constituent of a separate compound which is neither the antigen to be detected itself nor one of the monoclonal antibodies according to the invention hut which in turn is able to react with the receptor molecule, for example in the form of a complex formation.

These compounds which are present separately are preferably a second antibody molecule which can 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, for example, the antigen to bo detected or one of the monoclonal antibodies according to the invention, but preferably to a receptor molecule which is present in the form of a complex.

Other additional reagents are necessary in many cases and then result in a detectable signal only in cooperation with the marker. This particularly applies when enzymes are involved.

Markers or indicators which can be used within the scope of the present invention are well known to those skilled in the art of immunology and immunochemistrv. They comprise, for example, radioactively labelled elements or substances, enzymes or chemiluminescent substances. The following list of possible markers or indicators is intended merely to illustrate by way of example the wide variety of substances and reagents which can be used without, however, restricting the subject-matter of the invention in any way by this.

Examples of suitable markers or indicators are to be found among the group of radioactive elements. In this connection, particularly preferred elements of this type are those which either themselves emit y rays, such as, for example, 12 * I , 125I, 12SI, 132I, 51 Cr or else induce emission of these rays, such as, for example, MC, 1SE, 13N'. Likewise suitable are so-called B emitters such as 111In, 1^C and 3H. 230675 Other suitable markers comprise chemiluminescent substances, but in particular fluorescent substances, which can be bonded very simply by chemical means to the antigen or to an antibody without denaturing the latter. The resulting fluorochrome can be detected very straightforwardly using fluurometric methods. Fluorochromos which may be mentioned specifically at this point aie those selected from the group comprising fluorescein isocyanato, fluorescein isothiocvanate, 5-dimethylamine-1-naphthalenesulfonyl chloride, tetramethylrhodamine isothiocyanate, lissamine, rhodamine 8200 sulfonyl chloride etc.

Further fluorescent agents as well as a description of analytical techniques are to be found in DeLuca "Immunofluorescence Analysis" in: Antibody As a Tool, Harchalonis et al., John Wiley & Sons, Ltd., pp 189-231 (1982).

Particularly preferred within the scope of this invention is the use of enzymes as marker or indicator substances, such as, for example, horseradish peroxidase, alkaline phosphatase, B-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malic dehydrogenase, glucose-6-phosphate dehydrogenase etc.

When enzymes are used as marker substances it is necessary to add additional reagents which allow the formation of an immune complex to be followed via the enzyme activity, as well as, where appropriate, a stop reagent with which it is possible to stop the enzyme reaction.

Particularly preferred in this connection are reagents which result in a colour reaction. In the case of horseradish peroxidase, mention may be made at this point of, for example, hydrogen peroxide which, in combination with an additional oxidized dyestuff precursor such as, for example, diaminobenzidine or o-phenylenediamine, results in a brown or yellow colour. When glucose oxidase is used as marker substance it is possible to employ, for example, 2,2'-azino-di( 3-ethyl-benzothiazoline-6-sulfonic acid) [ABTS] as substrate.

Thus the present invention further relates to the use of test kits which contain at least one of the monoclonal antibodies according to the invention as reagent for the rapid and efficient, qualitative and/or 2 3 0 6 ft quantitative detection of atrazine and/or atrazine derivatives and/or of breakdown products of atrazine and/or atrazine derivatives, but in particular of hydroxyl analogues of atrazine and/or atrazine derivatives, such as, for example, hydroxyatrazine, hydroxypropazine, hydroxysimazine etc., as well as for differentiating atrazine and/or atrazine derivatives from their breakdown products.

I. Non-1imiting working examples Example 1: Synthesis of atrazine/hvdroxvatrazine conjugates 1.1: Triazinvl-valeric acid coupling components a) 2-Chloro-4-isopropylamino-6-(1-carboxybutyl-A-amino)-s-triazine .66 ml of diazobicyclo[5.A.0]undec-5-en are added to a mixture of 10.35 g of 2,6-dichloro-A-isopropylamino-s-triazine and 6.15 g of 5-aminovaleric acid in 150 ml of dry chloroform, and the reaction solution is stirred at the reflux temperature for 1 hour and then evaporated in vacuo (room temperature). The oilv residue is stirred with 60 ml of 2N hydrochloric acid at 0°C and, after standing for two hours, the precipitate is filtered, washed with water and diethyl ether and dried. 4.3 g of the desired product, 2-chloro-4-isopropylamino-6-( 1-carboxybutyl-4-amino)-s-triazine (melting point 191-192°C) are obtained. b) 2-Hydroxy-4-isopropylamino-6-(l-carboxybutyl-4-amino)-s-triazine • hydrochloride 1 g of 2-chloro-A-isopropylamino-6-(1-carboxybutyl-A-amino)-s-triazine is stirred in 7 ml of 6N hydrochloric acid at room temperature for A hours and concentrated in vacuo at 45°C, and the crystals are filtered and dried. 0.98 g of the desired product, 2-hydroxy-4-isopropylamino-6-(1-carboxybutyl-4-amino)-s-triazine • hydrochloride (melting point 1A9-151°C) is obtained. 23 0 6 1.2: Triazinc-protein conjugates The atrazine and hydroxyatrazine derivatives are conjugated either to bovine serum albumins (BSA; Fluka) or to keyhole limpet haemocyanin (KLH; Calbiochem) using the activated ester method (Kulkarni et a 1., 1981).

This entails specifically the carboxyl group of the derivative being solubilized in N,N-dimethylformamide (DMF) (6 mg/200 pi) at room temperature, and subsequently a A molar excess of ct-hydroxvsuccinimide (9.1 mg/200 pi) as well as N,N1-dicyclohexylcarbodiimide (16 mg/200 pi) being added.

The precipitate formed in the reaction is removed by centrifugation at 12,000 g and room temperature for 3 minutes, and the activated ester is subsequently added to BSA or KLH which has previously been solubilized in a phosphate-buffered saline solution (PBS buffer). The molar ratio of [derivat ive]/[BSA ] is about 55/1 (24 mg of derivative/5.A ml of BSA) in this case.

After incubation at a temperature of 4°C for 4 hours, the precipitate which has formed is removed by centrifugation at 2,000 g and A°C for 10 minutes, and the remaining supernatant is extensively dialysed against PBS before it is then used for the immunization experiments.

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

Example 2: Immunization Groups each of 5 female BALB/c mice (Sisseln animal farm, Switzerland), which are between A and 6 weeks old, 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 which is mixed in a ratio of 1:1 with 0.1 ml of complete Freund's adjuvant. 50 pi of this injection solution are injected intraperitoneally, and the remaining 150 pi subcutaneously. 2 3 0 6 7 5 In the second and third injection series, which takes place 14 and 30 days, respectively, after the first administration, the complete Freund's adjuvant is replaced by incomplete. 1 week after the last injection, blood serum is taken from the experimental animals, and the blood titres are determined using an ELISA test where the microtitre plates have previously been coated with BSA-conju-gated hapten (see Section 6).

After a rest period of 2 months, another single intraperitoneal injection of the KLH conjugate in a dosage of 500 pg/200 pi of PBS is carried out.

Example 3: Fusion protocol 3.1. Obtaining feeder cells (peritoneal macrophages) Untreated Balb/c mice about 6 to 8 weeks old are sacrificed one day before the planned fusion and sterilized by immersion in 70 % strength alcohol.

Subsequently a sterile incision is made in the fur and the upper abdominal skin without injuring the peritoneum. A sterile 5 ml plastic syringe and a sterile 18 gauge injection needle are used to inject 4 ml 2 + 2 + of BSS (without Ca" and Mg ) and 1 ml of air into the abdominal cavity.

The abdomen is gently massaged (leaving the syringe and needle in the abdominal cavity) and then the previously injected BSS buffer is withdrawn again from the peritoneum and placed in a sterile Falcon tube. This procedure is repeated twice more. The macrophages obtained in this way are cooled in ice and subsequently washed 2 x with 20 ml of BSS each time.

This entails the macrophages being centrifuged at 300 g and a temperature of 5°C for 10 min each time. The pellet is then resuspended in 50 ml of HAT medium, and the cell suspension is distributed on 4 Costar plates with a total of 24 wells (0.5 ml/well). 2306 75 The macrophages prepared in this way are subsequently stored in an incubator at a temperature of 37°C and a CO2 concentration of 6 %.

About 4 x 106 macrophages are required for each fusion process. 3.2. Culturinp, the myeloma cell line Sp 2/0-Ar 14 The said myeloma cell line Sp 2/0-Ag 14 is a myeloma cell line which does not itself secrete any antibodies and which is described by M. Shulman et al■ (1978). This myeloma cell line can be obtained from the American Type Culture Collection in Rockville, Maryland. 50 ml of a well-grown culture which contains at least 10 million cells are required for each fusion. The myeloma cells are preferably cultivated in T 175 Falcon flasks (from Beckton & Dickenson).

One day before the fusion, the cultivation medium (RPMI 1640) is replaced by fresh RPMI 1640 medium. On the day of fusion the Sp 2/0-Agl4 cells are harvested, placed in a sterile 50 ml plastic tube and centrifuged at 300 g and a temperature of 5°C for 10 min. (MSE centrifuge, model Chilspin, UK).

After the centrifugation, the supernatant is drawn off and discarded. The 2+ 2 + cells are washed 2 x with about 30 ml of BSS buffer (Ca and Mg" free) each time (10 min at 300 g, 5°C) and subsequently resuspended in 5 ml of BSS.

An aliquot of the cell suspension is taken for determination of the cell count and is stained with fluorescein diacetate (FDA). The myeloma cells are stored in ice until used further. 3.3. Preparation of a spleen cell suspension The spleen is removed from a Balb/c mouse, which has previously been immunized as in Example 2, under sterile conditions and while cooling with ice.

The previously immunized Balb/c mouse is sacrificed by cervical dislocation, and the spleen is removed under sterile conditions. For this, the mouse is briefly immersed in 70 % strength elhanol and dissected with sterile instruments. The spleen is carefully removed and placed on a fine nylon mesh. There it is cut up finely with scispors and then carefully, using the plunger from a 5 ml syringe, pushed through the mesh without thereby damaging too many cells. The mesh is rinsed with BSS throughout the procedure.

The cell suspension obtained in this way is placed in 50 ml plastic tubes and centrifuged at 300 g and a temperature of 5°C for 10 min (MSE centrifuge, model Chilspin; UK). The cells are subsequently washed 2 x with 20 ml of BSS each time (10 min; 300 g; 5°C; MES Chilspin) and, after the centrifugation, the cell pellet is resuspended in 10 ml of BSS.

The spleen cells are left on ice until fused with Sp 2/0-Ag 14 myeloma cells. 3.4. Fusion: Spleen cells and Sp 2/0-Ag 14 mveloma cells The ratio of myeloma cells to spleen cells for the fusion should be 1:10.

Spleen cells (in BSS buffer) and Sp 2/0-Ag 14 myeloma cells (in BSS buffer) are mixed in the stated ratio and centrifuged at 300 g and a temperature of 5°C for 10 min (MSE centrifuge, model Chilspin). The pellet is once again resuspended in BSS buffer, and the suspension is subsequently centrifuged again.

The pellet is cautiously stirred up and placed in a water bath at 37°C.

Then 1 ml of prewarmed and sterile PEG 4000 (MERCK) is added dropwise, over a period of 60 sec, to the cells, during which the entire mixture is constantly but gently agitated. The cells are subsequently shaken for a 2+ 2 + further 30 sec before 5 ml of a prewarmed BSS buffer (without Ca , Mg" ) are likewise added dropwise over a period of about 5 min while stirring cont inuou sly. 230675 The cells fused in the manner described are then spun down (10 min; 300 g; 20°C, MSE centrifuge, model Chilspin), and the supernatant is drawn off and discarded. The cell pellet is resuspended in 50 ml of HAT medium, and the cell suspension obtained in this way is distributed over the 4 prepared Costar plates (microtitre plates with 'A wells, diameter of each well 24 mm; total area for cell growth 2.0 cm ) (0.5 ml/well).

The Costar plates are incubated at a temperature of 37°C and a CO2 concentration of 6 %.

Example 4: Cultivation of the hybrid cells On the 1st day after the cell fusion 1 ml of HAT medium is added to each well on the culture plates. 3 to 4 days after the cell fusion the fused cells are inspected under the microscope. At the same time the used medium is aspirated off and replaced by 1 ml of fresh HAT medium. After a further 3 days (6-7 days after cell fusion) the culture medium is again changed. Each well is scanned under the microscope for hybrids from day 7 to 10 after cell fusion, and the medium is renewed 2 to 3 x a week.

As soon as hybrids have grown in a well the HAT medium therein can be replaced by HT medium. The supernatant from washed hybrid cultures (at least 10 % of the well) is drawn off with a sterile Pasteur pipette and tested for the presence of antibodies.

As soon as the well is grown completely over with positive hybride colonies, the latter can be transfered to new Costar plates in RPMI 1640 medium, with the contents of a well which is completely grown over being distributed over 2 to 3 new wells.

Example 5: Cloning of the positive hvbrid cells The cells in a positive well are detached using a pipette and transferred in 1 ml of medium into a tube. An aliquot is subsequently taken for determining the cell count and is stained with FDA (dilution 1:2 with FDA: 50 yl of cells + 50 pi of dyestuff). The preferred cell count is 105 to 10s cells/ml. 230675 The hybrid cells are subsequently diluted in a ratio of 1:100 with HT medium (for example 100 pi of cells + 9.9 ml of HT medium). ml of HT medium are placed in each of two 50 ml Falcon tubes, and 5 ml of a macrophage suspension are used to make up each tube to a total of 30 ml. The macrophages are previously isolated from a mouse and resuspended in 10 ml of HT medium (cf. Section 3.1).

The hybrid cells are diluted in these macrophage-containing Falcon tubes until a cell density of (i) 270 cells/30 ml or (ii) 90 cells/30 ml is reached. These mixtures are subsequently distributed over Costar plates (microtitre plates with 96 wells), with 200 pi being added to each well. This corresponds to a cell count of (i) 1.8 cells/well or (ii) 0.6 cells/well. 1.5 microtitre plates are thus required for each dilution.

After 7 days the individual wells are checked under the microscope, and the wells which contain cell clones are identified. The dilution at which about 50 % of the wells contain cell clones is used for the ELISA test. This ought as a rule to be the dilution with 0.6 cells/well.

After about 7-10 days, the supernatants from the positive wells (with clones) are tested in an ELISA test for the presence of monoclonal antibodies, and the positive clones are grown on Costar plates (with 24 wells) in RPMI 1640 medium. Aliquots of these positive clones are stored in liquid nitrogen.

Example 6: Hvbridoma screening (ELISA test) Firstly, 100 pi of a solution of BSA-conjugated hapten in sodium carbonate buffer (50 mM, pH 9.6) are placed in the individual wells of a microtitre plate, and this mixture is incubated in a humidity chamber at 4°C overnight. The wells are subsequently washed 5 x each with 0.1 % PBS-Tween buffer. 230675 To block the unoccupied binding sites on the microtitre plate, 200 pi of PBS-BSA solution (1 %) are placed in each well. This mixture is incubated at room temperature for 1-2 hours and subsequently washed with a 0.1 % strength PBS-Tween buffer. 200 pi of the hybridoma supernatant which is diluted in the ratio 1:2 with PBS-Tween (0.1 %) are then added to each well, and the complete mixture is incubated at room temperature for 2 hours. The wells are subsequently again washed 5 x with a 0.1 % strength PBS-Tween buffer.

This is followed by incubation with phosphatase-conjugated anti-mouse antibody (Kirkegaard & Perry Lab.). Firstly, 100 pi of a goat antibody against mouse IgG, which has been purified by an affinity chromatography and is in the form of a 1:1500 dilution in PBS-Tween (0.1 %) (Kirkegaard & Perry Laboratories) and is labelled with alkaline phosphatase, are added to each well.

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

Then 150 pi of a substrate-containing solution (1 mg/ml p-nitrophenyl phosphate) are added to each well. An incubation time of 2 hours in the dark is followed by spectroscopic determination at 405 nm. Positive hybridoma cells which secrete a specific antibody give a strongly positive signal at the selected wavelength.

Example 7: Expansion of the hybridoma cells in the mouse To stimulate ascites production, female Balb/c mice (20-25 mg) (Sisseln animal farm, CM) are pretreated with 0.3 ml of pristane oil (Aldrich Chemical) which is injected intraperitoneally. 1 to 3 weeks after the pristane administration the mice receive a second injection (0.2 ml of pristane oil, i.p.). At the same time as this second injection the animals receive 2 x 106 hybridoma cells in 0.2 ml PBS. 2 3 0 6 7 5 - AO - The ascites fluid resulting from this treatment is collected, centrifuged at 800 g and stored at a temperature of -20"C. After the ascites fluid has been thawed it is centrifuged at 30,000 g for 1 hour. The top layer, which contains mainly lipids, is removed. The protein concentration is subsequently determined ind adjusted to a level 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 subsequently dissolved in 20 mM Tris-HCl buffer, pH 7.9, which contains 50 mM NaCl, and dialysed against the same buffer at 4°C overnight.

The IgG fraction is further worked up by means of anionic 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, until a final NaCl concentration of 25 mM is reached, and 10 mg of protein/ml of gel are loaded onto the column. Elution is achieved by increasing the NaCl concentration from 25 mM to 200 mM (linear gradient). Monoclonal antibodies are generally eluted in the region of 80 mM NaCl.

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

The purity in the present case is >90 %.

Example 8: Atrazine and hydroxyatrazine detection Atrazine and hydroxyatrazine are detected using a two-stage competitive ELISA test.

BSA-conjugated hapten is firstly adsorbed in a sodium carbonate buffer (pH 9.6) (0.2 pg/well) onto microtitre plates and incubated at a temperature of 4°C overnight. The remaining free binding sites on the solid carrier material are then blocked by addition of BSA in the form of a 1 % solution. 2306 75 The plates are subsequently washed with PBS which is enriched with 0.1 % (v/v) polysorbate 20 (PBS-Tween). 50 pi of the ronoclonal antibodies purified previously (2 pg/ml) or else the supernatant from the cell clones (in a dilution of 1:5 to 1:22.5) are incubated a) with 950 pi of a standard solution which contains an increasing quantity of triazine or triazine analogue, b) with triazine-containing water samples or c) triazine-containing soil extracts. (All dilutions are carried out in PBS-Tween).

After an incubation time of 1 hour at room temperature, 200 pi of the mixture is added to each well in the microtitre plate, and the complete mixture is incubated for a further hour.

The wells are subsequently washed 5 x and charged with 100 pi/well of goat anti-mouse IgG antibody which is conjugated to alkaline phosphatase (dilution 1:1500) and incubated for a period of 1.5 hours.

After renewed washing, 150 pl/well of the substrate p-nitrophenyl phosphate dissolved in 1 mg/ml diethanolamine buffer (1 mM, pH 9.8) are added to the wells.

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

To measure the amount of atrazine/hydroxyatrazine contained in a sample, first a calibration plot is constructed (Fig. 1), with B/Bo x 100 being plotted against the concentration of inhibitor. (Bo represents absorbance measured without addition of a s-triazine inhibitor to the antibody, and B, absorbance measured with various concentrations of s-triazine inhibitors). The Iso indicates the concentration of antigen which inhibits 50 % 230675 - 1*2 - of the binding of the antibody to the solid phase. The I50 is calculated using an adaptation of the curve fitting program ENZFITTER (Leather-barrow, Elsevier-Biosoft) based on a four-parameter logistic curve (Raab GM, 1983). The quantitative determination of atrazine and hydroxyatrazine in soil or water samples within the scope of the ELISA is also calculated using the ENZFITTER programme, with curve fitting being based on standards included on each microtitre plate.

Example 8.1: Analysis of soil samples Aliquots (2 g) of standardized soil samples of various origins (see Table II) are extracted with 20 ml of a methanol/water [80/20 (v/v)] mixture in an extractor for 4 hours. For the competitive ELISA the soil extracts are usually diluted in a ratio of 1:40 in PBS-Tween (0.1 %) in order to prevent possible denaturation of the monoclonal antibodies by the methanol. The diluted soil samples (9 50 pi) are subsequently incubated with the specific antibodies (50 pi) in the manner described previously.

For the HPLC determination of hydroxyatrazine the methanol extracts are purified further by cation exchange chromatography (for example on a Dowex 50W-X4 column, followed by adsorption on an Amberlite XAD-2 polystyrene-divinylbenzene exchanger resin). Subsequently a gel filtration, for example on a Bio-Gel P-2 column, is carried out. The samples are then applied to the HPLC, and hydroxyatrazine is determined at a wavelength of 240 nm.

The comparison atrazine determination is carried out by means of gas chromatography using a thermo-ionic detector and/or subsequent mass spectroscopy.

Example 8.2: Analysis of water samples For the competitive ELISA 100 pi of a 10-fold concentrated PBS-Tween buffer are added to 850 pi of a water sample. This mixture is finally incubated with 50 pi of the anti-atrazine antibody MAb 4063-21-1. 230675 For the HPLC determination, the s-triazines contained in the water sample (20 ml) are first concentrated on a RP-18 precoluinn and subsequently further fractionated on an analytical RP-18 column. The atrazine determination is carried out at 230 nm. 11. Resu11 s a) Preparation of monoclonal antibodies Only a few days after cell fusion has taken place between myeloma cells and the spleen cells of a mouse which had previously been immunized with the hydroxyl conjugate described in Section 1 it is possible to detect proliferating cell colonies in 93 of the total of 96 wells in the microtitre plates. This corresponds to a fusion efficiency of 97 %. of these cell colonies produce monoclonal antibodies which show a very strong reaction in the ELISA test. The latter are cloned by means of the limited dilution method (compare Section 5). 9 of the synthesized monoclonal antibodies belong to the IgGl subclass, and one to the IgG2b subclass.

The monoclonal antibodies obtained in the manner described previously can be divided into 2 groups on the basis of their cross-reactivity determined in an ELISA test.

In the first group (represented by ^LAb 4009-65-3) the cross-reactivity is mainly confined to hydroxypropazine while, by contrast, in the second group (represented by MAb 4009-77-20) cross-reactivities occur also with other s-triazines such as, for example, hydroxysimazine, hydroxydesmetryn etc. (compare Table 1).

On the other hand, no cross-reactivity with active triazines such as, for example, atrazine occurs in either group. Thus the binding of the monoclonal antibodies is confined to compounds which have a hydroxyl group in position 2 of the triazine ring. 23 0 6 75 The lower detection limit for hydroxyatrazine is in the region of 0.1 ng/ml of buffer for MAb 4009-85-3 and of 0.05 ng/ml of buffer for MAb 4009-77-20. The corresponding 150 values are 0.95 ng/ml and 0.5 ng/ml.

When atrazine conjugates (represented by MAb 4063-21 -1 ) are used there i'. obtained a total of 5 monoclonal antibodies, all of which show an approximately comparable extent of cross-reactivity. A cross-reactivity with various active s-triazines is found in this case.

The cross-reactivities occurring with hydroxylated metabolites are, by contrast, negligibly small, (compare Table 1) For MAb 4063-21-1 the detection limit for atrazine is 0.05 ng/ml of buffer with an Iso (50 % inhibition) of 0.45 ng/ml. b) Recovery studies These recovery studies are carried out by adding a known amount of hydroxyatrazine or atrazine to a methanol/water soil extract. They serve mainly to investigate any interfering effects from organic material during the immunoassay.

Tab. II shows that there is virtually complete recovery irrespective of the composition of the soil samples used. The recovery rate is 97 % even when the humus content is nearly 10 %. c) Detection of atrazine in water samples 21 different water samples were analysed by means of HPLC and ELISA using MAb 4063-21-1.

A concentration step must be carried out for the HPLC determination but is dispensed with when the monoclonal antibody according to the invention is used in an ELISA assay.

Tab. Ill shows that the detection limit for atrazine when the immmuno-assay is used is about 0.05 ppb. The correlation between ELISA and HPLC determination for the atrazine-containing samples (> 0.05 ppb) is 23 0 6 75 determined by regression analysis. This reveals a good agreement between the two methods (r = 0.91, p < 0.0005), the slightly elevated values in the ELISA determination not being significant.

The water samples were additionally investigated for traces of .hydroxyatrazine. This metabolite was undetectable with either of the two methods (HPLC and ELISA).

Deposi t ion: The hybridoma cell lines prepared and used within the scope of the present invention have been deposited at the European Collection of Animal Cell Cultures (ECACC) in Salisbury, UK, which is recognized as an international depository, in accordance with the requirements of the Budapest treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure. The said international depository provides a statement on the viability of the deposited samples.

Cell line Date of Depository Date of viability deposit number* certificate Hybridoma 25.08.1988 8808/2501 25.08.1988 Clone AO63— 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 * Depository number assigned by the above-identified international depository. 230675 - UG - Media and buffers used A) RPMI 1640 medium RPMI 1640 (Soromed) wit.li the following additions: Calf serum "/ fa L-Glutamine 4 mM Gentamicin 0. 01 % Sodium pyruvate 1 mM 2-Mercaptoethanol 50 MM Insulin pM T ransferrin pM Selenium (ITS) pM B) HAT medium 1 litre of RPMI 1640 medium with 20 ml addition of HAT conc. 50 x from Boehringer (hypoxanthine 680.5 mg/1; aminopterin 8.8 mg/1; thymidine 193.8 mg/1) C) HT medium 1 litre of RPMI 1640 medium with 20 ml addition of HT conc. 50 x from Boehringer (hypoxanthine 680.5 mg/1; thymidine 193.8 mg/1).

D) BSS buffer (Earle's saline without Ca and Mg, pH 7.4) KCl 7.3 mM NaCl 116 mM NaHCOj 26 mM NaH2P0u*2H20 1 mM Glucose 5.5 mM Phenol red 48 pM 1 % addition (v/v) of a penicillin/streptomycin solution (Seromed) (10,000 U of penicillin, 10 mg/ml streptomycin). 230675 - t*i - E) Sodium carbonatc buffer (pH 9.6) NazCOj A 7 7 mg NallCOj 879 mg NaN3 (0.5M) ].8 ml ad 300 ml H2O F) PBS buffer (pll 7.0) NaCl 8.5 g NazHP0^-2H20 1.28 g Nail2 POu • 2H2 0 0 . A 36 g ad 1000 ml H20 G) PBS-Tween 20 (0.1 %) 1 ml of Tween 20 (Serva) + 1000 ml of PBS H) PBS-BSA (1 %) BSA 5 g NaN3 (0.5M) 3 ml ad 500 ml PBS I) Substrate buffer (Diethanolamine buffer, pH 9.8) Diethanolamine 97 ml NaN3 (0.5M) 6 ml MgClz'6H20 100 mg ad 1000 ml H2O, pH adjusted to pli 9.8 with conc. HCL.

Preparation of the substrate: Immediately before use, one substrate tablet (= 5 mg) of the p-nitrophenyl phosphate substrate (Sigma 104) is dissolved in 5 ml of substrate buffer. t I \ ) - 4 8 Table 1: Cross-reactivity of various s-triazine and hydroxytriazine analogues with an anti-atrazin MAb (MAb 4063-21-1) and two anti-hydroxyatrazine MAbs (MAb 4009-85-3 and MAb 4009-77-20) Inhibitor MAb 4063-21-1 (ECACC 8808/2501) MAb 4009-85-3 (ECACC 8808/2503) Mab 4009-77-20 (ECACC 8808/2 502) I 50 % cross- I 50 h cross- I 50 % cross- ng/ml reactivity ng/ml reactivity ng/ml reactivity (a) (b) (a) (c) (a) (c) Atrazine 0.45 100.0 >1000 <0. 1 >1000 <0. 05 Propaz ine 0.5 90.0 >1000 <0. 1 >1000 <0. 05 Sima z ine 18 2.5 Desmetryn 90 0.5 Terbuthylazine 9 .0 Trietazine 4 11.3 Ametryn 9 .0 >1000 <0. 1 >1000 <0. 05 Atraton 7 6.4 >1000 <0. 1 >1000 <0. 05 Prometryn 0.8 56. 3 S ime t ryn 27 1 . 7 Prometon 0.6 75.0 OH-At razine 21 2.1 0.95 100. 0 0.5 100. 0 OH-Propazine .5 8.2 0.95 100. 0 0.6 83. 3 OH-Simazine 180 0.3 65 1. 1.2 41 . 7 OH-Desmetryn 360 0.1 8 11. 9 0.7 71. h OH-Terbuthylazine 8 .6 19 . 0 3.3 .2 a) Inhibitorconcentration for 50 % inhibition in the competitive ELISA-test b) cross-reactivity, defined as: (atrazine concentration for 50 % inhibition)/(concentration of the s-triazine analogues for 50 % inhibition) x 100 c) cross-reactivity, defined as: (hydroxyatrazine concentration for 50 % inhibition)/(concentration of the s-triazine analogues for 50 % inhibition) x 100 ro CaI CD CJl o (J : ) 49 - Table II. Recovery of hydroxyatrazine f rom soil extracts* previously inoculated with hydroxyatrazine Soil sample % Soil Humus composition % Sand % Silt % Loam pH Hydroxyatrazine added (ppb) Hydroxyatrazine recovery rate (%) MAb 4009-85-3 (ECACC 8808/2503) Risilon (Israel) 1 . .8 80.8 3. 8 13.6 7.8 60 200 600 91 92 90 93 Vetroz (Switzerland) 9, .3 18.1 60. , 4 21 . 5 7.3 100 97 Stein (Switzerland) 43 17. .4 34.6 7.1 100 110 Collombey (Switzerland) 1 . ,4 83.9 13. .6 2.5 7.4 100 107 Les Evouettes (Switzerland) 2, .6 .7 64 . 3 6.2 100 94 *enriched soil extracts (methanol extracts) are diluted 1:40 in PBS-Tween (0.1 %) for the ELISA determination ro oi o CJl i ) o 50 - Table III. Determination of atrazine in water samples Comparison between HPLC and ELISA (MAb 4063-21-1; ECACC 8808/2501) Water sample ELISA/HPLC HPLC ELISA ratio Atrazine Desethylatrazine s- •T riazine Pg/1 Mg/1 yg/i 1 0. .09 0. ,05 0. 1.1 2 0. , 10 0. .15 0. 14 1.4 3 0. ,41 0. , 59 0. 38 0.9 4 0. . 10 0. .11 0. 1.5 0. .08 0. .06 0. 11 1.4 6 0. ,06 0. ,05 0. 09 1. 5 7 <0. ,05 0. , 11 0. 07 >1.0 8 <0. ,05 0.05 0. 06 >1.0 9 0. 06 <0. 05 0. 07 1.2 <0. 05 <0. 05 0. 07 >1.0 11 <0. ,05 <0. 05 0. 08 >1.0 12 0. ,14 0. .29 0. 0.7 13 0. ,22 0. .14 0. 19 0.8 14 0. .23 0. ,13 0. 34 1.5 0. .23 0. .13 0. 24 1.0 16 0, . 36 0. .24 0. 49 1.4 17 0, . 35 0. .23 0. 43 1.2 18 0.

.*G 0. .24 0. 33 0.8 19 0, .40 0. .27 0. 41 1.0 0. .50 0. .32 0. 50 1.0 21 0. .45 0. .29 0. 54 1.2 control 0. 01 H20 (HPLC) ro CM o> ^4 CJ1

Claims (63)

230675 51 Literature Ercegovich CD ct al, J. Agric. Food Chem., 29: 559-563, 1981 Fleeker 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 Kohler G, Milstein, Nature, 256: 495-497, 1975 Kulkarni NP et al, Cancer Res■, 41: 2700-2706, 1981 Littlefield JW, Science, 14 5: 709, 1964 Nevsome WH, J. Agric. Food Chem., 33: 528-530, 1985 Raab GM, Clin. Chem., 29: 1757-1761, 1983 Ramsteiner KA, Horman WD, J. Agric. Food Chem., 27: 934-938, 1 979 Ramsteiner K., "Atrazin". In Methodensammlung zur Ruckstandsanalytik von Pflanzenschutzmitteln (Collection of Methods for Analysis of Residues of Crop Protection Agent); Deutsche Forschungsgemeinschaft Ed.; Verlag Chemie, 1985; Method 6-D Shulman M et al, Nature, 2 76: 269-270, 1978. Wie SI, Hammock BD, J. Agric. Food Chem., 30: 949-957. 1982 Patent literature US-P 4,530,786 230675 - 52 - WHAT ifWE CLAIM IS:- Pa ton6 c 1 .i i nio—

1. A hybridoma cell line which produces a monoclonal antibody which has a high specificity and affinity for atrazine and/or atrazine derivatives and which shows essentially no cross-reactivity with the inactive hydroxyl analogues of atrazine and/or atrazine derivatives.

2. A hybridoma cell line which produces a monoclonal antibody which has a high specificity and affinity for the hydroxyl analogues of atrazine and/or atrazine derivatives and which shows essentially no cross-reactivity with atrazine and/or atrazine derivatives.

3. A hybridoma cell line according to claim 1, which produces a monoclonal antibody which has a high specificity and affinity for atrazine and which shows essentially no cross-reactivity with hydroxyatrazine or other hydroxyl 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 shows a pronounced cross-reactivity with structurally similar atrazine derivatives selected from the group comprising propazine, promotryn and prometon but which has essentially no cross-reactivity with other atrazine derivatives or hydroxyl analogues of atrazine and/or atrazine derivatives.

5. A hybridoma cell line according to claim 4, which has the identifying characteristics of ECACC 8808/2501.

6. A hybridoma cell line according to claim 2, which produces a monoclonal antibody which has a high specificity and affinity for hydroxyatrazine and which shows essentially no cross-reactivity with atrazine and/or atrazine derivatives.

7. A hybridoma cell line according to claim 6, which produces a monoclonal antibody which has a high specificity and affinity for hydroxyatrazine and which shows a very pronounced cross-reactivity with hydroxy- r< <■ i or,. • o .;'; ; . > - 53 - propazine hue which shows essentially no cross-reactivity with hydroxyl analogues of other atrazine derivatives or with atrazine and/or atrazine derivatives.

8. A hybridoma cell line according to claim 6, which produces a monoclonal antibody which has a high specificity and affinity for hydroxyatrazine and which shows a cross-reactivity with hydroxyl analogues of atrazine derivatives but which shows essentially no cross-reactivity with atrazine and/or atrazine derivatives.

9. A hybridoma cell line according to claim 7, which has the identifying characteristics of ECACC 8808/2503.

10. A hybridoma cell line according to claim 8, which has the identifying characteristics of ECACC 8308/2502.

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; that is to say are still able to synthesize and to secrete into the surrounding medium the antibodies according to any one of claims 12-25.

12. Monoclonal antibodies and the derivatives thereof which have a high specificity and affinity for atrazine and/or atrazine derivatives and which show essentially no cross-reactivity with the inactive hydroxyl analogues of atrazine and/or atrazine derivatives.

13. Monoclonal antibodies and the derivatives thereof which have a high specificity and affinity for the inactive hydroxyl analogues of atrazine and/or atrazine derivatives and which show essentially no cross-reactivity with atrazine and/or atrazine derivatives.

14. Monoclonal antibodies and the derivatives thereof according to claim 12, which have a high specificity and affinity for atrazine and which show essentially no cross-reactivity with hydroxyatrazine or hydroxyl analogues of atrazine derivatives. 0»S 230 6 7 5 - 54 -

15. Monoclonal antibodies and t)io derivatives thereof according to claim 12, which have a high specificity and affinity for atrazine and which show a very pronounced cross-reactivity with structurally similar atrazine derivatives selected from the group comprising propazine, prometryn and prometon but which have essentially no cross-reactivity with other atrazine derivatives or with hydroxyl analogues of atrazine and/or atrazine derivatives.

16. Monoclonal antibodies and the derivatives thereof according to claim 15, characterized in that the cross-reactivity with propazine is more than 80 %.

17. Monoclonal antibodies and the derivatives thereof according to claim 13, which have a high specificity and affinity for hydroxyatrazine and which show essentially no cross-reactivity with atrazine and/or atrazine derivatives.

18. Monoclonal antibodies and the derivatives thereof according to claim 17, which have a high specifity and affinity for hydroxyatrazine and which show a cross-reactivity with hydroxyl analogues of atrazine derivatives, but which show essentially no cross-reactivity with atrazine and/or atrazine derivatives.

19. Monoclonal antibodies and the derivatives thereof according to claim 18, characterized in that the cross-reactivity with hydroxyl analogues of atrazine derivatives is at least 40 %.

20. Monoclonal antibodies and the derivatives thereof according to claim 17, which have a high specificity and affinity for hydroxyatrazine and which show a very pronounced cross-reactivity with hydroxypropazine but which show essentially no cross-reactivity with hydroxyl analogues of other atrazine derivatives or with atrazine and/or atrazine derivatives.

21. Monoclonal antibodies and the derivatives thereof according to claim 20, characterized in that the cross-reactivity with hydroxypropazine is up to 100 %. - 55 -

22. Monoclonal antibody and the derivatives thereof which are produced b a hybridoma cell line according to any one of claims 1 to 10.

23. Monoclonal antibody and derivatives thereof according to claim 15, which is produced by a hybridoma cell line which has the identifying characteristics of ECACC 8508/2501.

24. Monoclonal antibody ana derivatives thereof according to claim 18, which is produced by a hybridoma call line which has the identifying characteristics of ECACC 3808/2502.

25. Monoclonal antibody and derivatives thereof according to claim 21, which is produced by a hybridoma cell line which has the identifying characteristics of ECACC 8808/2503.

26. Method for the preparation of 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 hydroxyl analogues of atrazine derivatives are conjugated with a carrier molecule b) a rton-human donor animal is immunized with said conjugate c) an immunocompetent B cell is isolated from the immunized donor animal d) said immunocompetent B cell is fused with a tumour cell line which is capable of continuous cell division e) the resulting fusion product is isolated, cultivated in a suitable culture medium and subsequently cloned and f) the cloned hybrid cells are investigated for the formation of monoclonal antibodies.

27. Method according to claim 26, characterized in that used as carrier molecule are macromolecular compounds which have reactive groups freely accessible for the coupling reaction with atrazine and/or atrazine - 30 - derivatives or hydroxyatrazine and/or hydroxyl analogues of atrazine derivatives and which are able to confer an immunogenic potency on said compounds.

28. Method according to claim 27, characterized in that a lysine-rich protein wich a molecular veight between 10,000 and 1,500,000 is used.

29. Method according to claim 27, characterized in that the carrier is selected from the group comprising bovine serum albumin (BSA), keyhole limpet protein (KLP). human serum albumin (HSA), porcine thyroglobulin, B2 microglobulin, haemocyanin, immunoglobulins, toxins, polysaccharides, lipopolysaccharides, natural or synthetic polyadenylic and polyuridylic acids, polyalanyl and polylysine polypeptides, and cell membrane components.

30. Method according to claim 26, characterized in that the conjugation to the carrier molecule takes place directly.

31. Method according to claim 26, characterized in that the conjugation to the carrier molecule takes place via a spacer molecule.

32. Method according to claim 31, characterized in chat said spacer molecule has one or more reactive groups which are able to enter into interaction with the reactive groups of the carrier molecule.

33. Method according to claim 32, characterized in that said reactive groups take Che form of carboxyl, amino or SH groups.

34. MeChod according Co claim 26, characCerized in that Che coupling reacCion is carried out by means of the active ester method.

35. Method according to claim 26, characterized in chat the immunization of Che donor animals is carried out by administracion one or more times ' ' / of carrier-bound atrazine and/or aCrazine derivatives. < ) v > t, : k> - 57 -

36. Mechod according to claim 26, characterized in that the immunization of the donor animal is carried out by administration one or more times of carrier bound hydroxyatrazine and/or hydroxyl analogues of atrazine derivatives.

37. Method according to any one of claims 35 or 36, characterized in '.hat the administration takes place in the form of an intravenous, intraperitoneal or subcutaneous injection.

38. Method according to claim 26, characterized in that used for the fusion of immunocompetent cells from the donor animal are tumour cell lines which themselves produce no monoclonal antibodies.

39. Method according to claim 38, characterized in that myeloma cell lines which have the identifying characteristics of Sp2/0-Agl4 or X63-Ag8.653 are used.

40. Method according to claim 26, characterized in that used as fusion medium is a buffer solution which contains one of the fusion promoters which is customarily used for the fusion of cells and is selected from the group comprising: sendai viruses or other paramyxoviruses, calcium ions, surface-active lipids or polyethylene glycol.

41. Method according to claim 40, characterized in that said fusion promoters take the form of polyethylene glycol with a mean molecular veight of 600 to 6,000.

42. Method according to claim 41, characterized in that the the polyethylene glycol concentration in the fusion medium is 30 %-60 % bv volume.

43. Method according to claim 26, characterized in that a HAT selection medium is used for the selection of fused hybrid cells.

44. Method according to claim 26, characterized in that the hybrid cells are cultivated in the presence of isolated macrophages ("feeder cells"). - 53 -

45. Method according to claim 25, characterized in that positive hybrid cell cultures producing monoclonal antibodies are singled out using the limiting dilution method and subsequently cloned in suitable cultivation media.

46. Method according to claim 26, charicterized in that the hybridoma cell clones cloned according to claim 45 are investigated using an immunoassay for the formation of suitable monoclonal antibodies.

47. Method according to claim 46, characterized in that an enzyme-coupled immunoassay or a radioimmunoassay is used.

48. Method for the preparation of monoclonal antibodies, characterized in that the hybridoma cell lines of any one of claims 1 to 11 are cultivated in vivo or in vitro using known methods, and the produced monoclonal antibodies are isolated.

49. Method according to claim 48, characterized in that an in vitro cultivation in suitable cultivation media is carried out.

50. Process according to claim 49, characterized in that standardized culture media selected from the group comprising Dulbecco's modified Eagle medium (DMEM) or RPMI 1640, which can be supplemented where appropriate by addition of mammalian sera, by growth-promoting additives or by trace elements, is used.

51. Method according to claim ^8, characterized in that said monoclonal antibodies are prepared by expansion in vivo. in a non —human donor animal.

52. Method for the immunological detection of atrazine and/or atrazine derivatives, characterized in that a monoclonal antibody according to any one of claims 12 or 14 to 16 is used in one of the known immunoassays.

53. Method for the immunological detection of atrazine and/or atrazine derivatives according to claim 52, characterized in that a monoclonal antibody which is produced by a hybridoma cell line which has the identifying characteristics of ECACC 88108/2501, or of clones^o^rjxsVjfc-**' , clones thereof, is'usea in one of the known immunoassays. '-'/y V*? fEB 1992r «; i j f, i ;) - 59 -

54. Method for the immunological detection of hydroxyatrazine and/or hydroxyatrazine derivatives, characterized in chat a monoclonal antibody according to one of claims 13 or 17 to 21 is used in one of the known immunoassays.

55. Method for che immunological decection of hydroxyatrazine and/or hydroxyatrazine derivatives according to claim 54, characterized in that a monoclonal antibody which is produced by a hybridoma cell line which has the identifying characteristics of ECACC 8303/2502, or of clones or subclones thereof, is used in one of the known immunoassays.

56. Method for the immunological detection of hydroxyatrazine and/or hydroxyatrazine derivatives according to claim 5-, characterized in that a monoclonal antibody which is produced by a hybridoma cell line which has the identifying characteristics of ECACC 8808/2503, or of clones or subclones thereof, is used in one of the known immunoassays.

57. Method according to one of claims 52 Co 56, characcerized in thac it takes the form of a competitive immunoassay.

58. Method according to one of claims 52 to 57, characterized in that the said immunoassay takes the form of a radioimmunoassay (RIA), an enzvme-coupled assay (ELISA) or a chemoluminescence assay.

59. Composition for the immunological detection of atrazine and/or atrazine derivatives in the form of a ready-to-use test kit, charac-terized in that said test kit contains, besides the customarily used carrier materials, reagents and other additives, at least one monoclonal antibody according to one of claims 12 or 14 to 16 as reagent.

60. Composition for the immunological detection of hydroxyatrazine and/or hydroxyl analogues of atrazine derivatives in the form of a ready-to-use test kit, characterized in that said test kit contains, besides the customarily used carrier materials, reagents and other additives, at least one monoclonal antibody according to one of claims 13 or 17 to 21 as reagent. 60

61. Composition 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 contains, besides the customarily used carrier materials, reagents and other additives, at least one monoclonal antibody which is produced by a hybridoma cell line •-•hich has the identifying characteristics of ECACC 8808/2501, or of clones or subclones thereof.

62. Composition for the immunological detection of hydroxyatrazine and/or hydroxyl analogues of atrazine derivatives in the form of a ready-to-use test kit according to claim 60, characterized in that said test kit contains, besides the customarily used carrier materials, reagents and other additives, at least one monoclonal antibody as reagent which is produced by a hybridoma cell line which has the identifying characteristics of ECACC 8808/2502, or of clones or subclones thereof.

63. Composition for the immunological detection of hydroxyatrazine and/or hydroxyl analogues of atrazine derivatives in the form of a ready-to-use test kit according to claim 60, characterized in that said test kit contains, besides the customarily used carrier materials, reagents and other additives, at least one monoclonal antibody as reagent which is produced by a hybridoma cell line which has the identifying characteristics of ECACC 8808/2503, or of clones or subclones thereof. F0 7.5/WB/cv* CIBA-GEIGY AG