WO1992012427A1

WO1992012427A1 - Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

Info

Publication number: WO1992012427A1
Application number: PCT/US1991/000151
Authority: WO
Inventors: Larry H. Stanker; Martin Vanderlaan; Bruce E. Watkins
Original assignee: United States Department Of Energy
Priority date: 1991-01-08
Filing date: 1991-01-08
Publication date: 1992-07-23
Also published as: EP0553077A1; EP0553077A4

Abstract

Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at low concentrations in samples.

Description

MONOCLONAL ANTIBODIES TO CYCLODIENE INSECTICIDES AND METHOD FOR DETECTING THE SAME

The United States Government has rights in this invention pursuant to Contract No. W-7405-ENG-48 between the U.S. Department of Energy and the University of California for the operation of the Lawrence Livermore National Laboratory.

TECHNICAL FIELD The subject invention is related generally to assay of insecticides found in foods and environmental samples and more particularly to production and use of monoclonal antibodies for specific detection of cyclodiene insecticides.

BACKGROUND ART Identification of Terms

Abbreviations or definitions used in the disclo- sure herein are as follows: cELISA: competition enzyme-linked immunosorbent assay. BSA: bovine serum albumin.

KLH: keyhole limpet hemocyanin.

GC/EC: gas chromatography/electron capture.

GABA: gamma-amino butyric acid, a neurotransmitter which increases the permeability of the post- synaptic membrane to Cl^~ ion.

hapten: a small molecule which carries an antigenie determinant, but is not i munogenic until it is chemically coupled to a larger protein carrier. The hapten-carrier complex then stimulates an immune competent cell to form antibodies to the hapten and the complex;

HPLC: high pressure liquid chromatography.

cyclodiene insecticides: chlorinated hydrocarbon insecticides, containing an endo ethylene bridge which include heptachlor, chlordane, aldrin, dieldrin, endrin, p

Strobane , endosulfan. toxaphene and BHC (mixed isomers) benzene hydrochloride.

heptachlor: 1 ,-exo-,4,5,6,7,8,8,-Heptachloro-3a,4, 7, 7a-tetrahydro-4,7-methanoindene.

heptachlor epoxide: the product of heptachlor oxidation, which occurs in soil, in animals, and in or on crops when treated with heptachlor.

chlordane: 2,3,4,5,6,7,8,8-Octachloro-2,3,3a,4, 7,7a-hexahydro-4,7-methanoindene.

aldrin: 1 ,2,3,4,10,10-Hexachloro-l ,4,4a,5,8,8a hexahydro-1 ,4-exo, endo-5,8,-dimethano- naphthalene (HHDN).

dieldrin: (1R, 4S, 5S, 8R)-1 ,2,3,4,10,10 -hexachloro-1 ,4,4a,5,8,8a -hexahydro-1 ,4:5,8-dimethano-naphthalene

(principal constituent, known as HEOD).

endosulfan: 6,7,8,9,10,10-Hexachloro-l ,5,5a,6, 9,9a-hexahydro-6,9, -methano-2,4,3-benzoCe3- dioxathiepin-3-oxide. endrin: (1R, 4S, 5R, 8S) 1 ,2,3,4, 10, 10-hexachloro

-6 , 7-epoxy-l ,4, 4a, 5 , 6, 7, 8 , 8a-octahydro-exo-

5,8,dimethano-naphthalene .

D

Strobane : polychlorinates of camphene, pinene and related terpenes. (Production discontinued by Tenneco Chemicals, Inc.).

norbornene: isomer resulting from the maximal interaction between the unsaturated systems of the reaction between cyclopentadiene and maleic anhydride. The acid anhydride ring system is fused to the newly formed methylene-bridged cyclohexene nucleus (bicycloC2.2.13hept-2-ene; norbornene) through its endo positions.

camphene: C-_Q H-_Q.

toxaphene: (camphechlor) isomeric reaction mixture of chlorinated camphenes containing 67-69% chlorine.

BHC: 1 ,2,3,4,5,6-Hexachlorocyclohexane; also known as benzene hexachloride. There are five isomers, of which one, gamma BHC, has insecticidal activity and is known as the pesticide lindane. Kepone : Decachloro-octahydro-1 ,3,4,-metheno-2H- cyclobuta(cd) pentalen-2-one (Discontinued by Allied Chemical Co.).

Cyclodiene insecticides are a large group of polychlorinated cyclic hydrocarbons with endomethylene bridged structures. A variety of chlorinated hydrocarbon insecticides were prepared by the Diels-Alder reaction following the discovery of chlordane in 1945, and various derivatives were widely used in the United States and other countries. Until recent discontinuance of production, or limitations on domestic usage, the cyclodiene insecticides most commonly used in the United States were

D heptachlor, chlordane, aldrin, dieldrin, endrin, Strobane , endosulfan, toxaphene and BHC.

Cyclodiene insecticides may have one or more 5 or 6 carbon rings which have been heavily halogenated. Cyclodienes may have a single cyclopentadiene ring or a dicyclopentadiene ring structure. The structure of bicyclo[2.2.13hept-2-ene has a joined pair of five carbon rings, which is formed by the joint sharing of three carbons, and is termed the norbornene functionality. The norbornene structure is a common structure of many cyclodiene insecticides. Cyclodienes may have a dicyclopentadiene (C_-Q H, ~ ) ring structure in which 3 carbons are shared commonly between two 5 carbon rings and also 2 carbons may be shared commonly between one of those rings and another 5 carbon or substituted five-carbon ring. Each of the ring structures is extensively halogenated. with the ring hydrogens most commonly being replaced with chlorine. Cyclodiene insecticides act upon the central nervous system by inhibition of ATPase activity which affects ion transport and interferes with nerve cell receptors.

The cyclodiene insecticides in use differ widely in their chemical structure, toxicity and photostabiiity. Heptachlor was heavily used in agriculture as an insecticide and termiticide until these applications were phased out in the mid-1970's. Currently, heptachlor use is restricted, however, previous widespread use of these compounds has led to concern about the possibility that pesticide residues might remain in foodstuffs and the environment.

Residues of heptachlor and heptachlor epoxide in meats, fats and milk have been monitored in the United States by the

U.S.D.A. Heptachlor epoxide is a major metabolite in body tissue and milk of cows which have grazed on heptachlor-treated pasture. However, the lack of convenient, rapid detection systems has hampered environmental identification and quantification of cyclodienes. Conventional analysis for these compounds includes total organic chlorine content or multi-step sample clean-up using organic solvent extraction procedures followed by gas chromatography and electron capture (GC/EC).

Refinements of the general technique for hybridoma production developed by Kohler and Milstein in 1975 (Nature 256:

495-497) make it possible to produce large quantities of monoclonal antibodies which are able to recognize specific antigenic determinants. While development of antibodies reactive to protein antigenic sites is repeatable, fabrication of monoclonal antibodies reactive to small organic chemicals, such as carcinogens, pesticides, toxic chemicals and DNA adducts sequences linking of a small molecule, termed a hapten, to a carrier protein prior to immunization of an animal. The specificity of the antibodies produced may be influenced by the site and chemistry of the hapten conjugation to the carrier protein.

Antibodies with specific binding to reactive sites on small organic molecules are sensitive indicators, which may distinguish chemical isomers (Stanker et al , Toxicology 45: 229-243 1987). With small haptens, the greatest antibody specificity for a reactive group appears to occur when that reactive group is most distant from the site of the linkage binding to the carrier protein. Previous attempts at immunization have produced polyclonal antibodies which have poor specificity for heptachlor.

DISCLOSURE OF THE INVENTION Accordingly, it is an object of the present invention to provide monoclonal antibodies and the hybridomas that produce such monoclonal antibodies which will react specifically and sensitively with cyclodienes, particularly those antibodies with a high degree of specificity for a class of halogenated hydrocarbons which possess the cyclopentadiene or dicyclopentadiene ring, and more particularly those antibodies with a high degree of specificity for a class of cyclodienes which possess the norbornene structure. Another object is to provide a method for production of monoclonal antibodies which identify cyclodiene insecticides, particularly those with the cyclopentadiene or dicyclopentadiene functionality, more preferably those with the norbornene structure, and including production of a monoester of heptachlor hapten which will elicit formation of these antibodies.

A further object is to provide a method for the specific and sensitive detection and separation of cyclodiene insecticides from samples, particularly a class of cyclodiene insecticides which possess the cyclopentadiene or dicyclopentadiene functionality, and more particularly a class of cyclodiene insecticides which possess the norborene structure, by binding to specific monoclonal antibodies.

Another object is to provide a method for separation of halogenated cyclic organic compounds from tissue or environmental samples and solubilizing them in a vehicle appropriate for assay of halogenated cyclic organic compounds by specific monoclonal antibodies.

Another object is to provide a method for the detection and isolation of cyclodiene insecticides, particularly those with cyclopentadiene or dicyclopentadiene functionalities, and more particularly those with the norbornene structure, in environmental and food samples.

Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following and the accompanying drawings and their descriptions which form part of the disclosure, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

To achieve the foregoing and other objects and in accordance with the purpose of the present invention as embodied and broadly described herein, the subject invention is directed to the monoclonal antibody, and the hybridoma which produces it, which is specifically reactive to the cyclodienes which have a cyclopentadiene or dicyclopentadiene functionality, and more specifically, to cyclodiene insecticides which have the norbornene structure. Such compounds include, but are not limited to those shown in Table 1 (appearing on page 33), and derivatives thereof. The present invention also provides a method for the production and use of monoclonal antibodies reactive with the antigenic determinants on compounds selected from the group consisting of the cyclodiene insecticides which have a cyclopentadiene or dicyclopentadiene functionality, or more particularly to those with the norbornene structure.

The method for the production of monoclonal antibodies to cyclodiene insecticides in accordance with the subject invention is adapted from general methods for production of monoclonal antibodies, such as that described by Kohler and Milstein (1975), which is incorporated by reference. Cyclodiene-specific antibodies are produced by immunizing a suitable mammal with an immunogenic carrier protein conjugate of the desired antigen compound and obtaining im unosensitized cells from the mammal, which are capable of producing antibodies to the antigen. The immunosensitized cells are fused with immortally reproducing cells of the same species, or of another mammal species. The hybrid cells produced are cultured in a suitable host, or in a culture medium, and clones of hybrid cells, referred to as hybridomas, are isolated. The hybridomas continuously produce specific antibodies which react with the sensitizing antigen and from them cells are selected which produce monoclonal antibodies of desired reactivity. These antibodies are grown in culture medium or in a host, and harvested and purified, if desired.

The monoclonal antibodies produced are capable of recognizing cyclodiene insecticides, particularly those with the cyclopentadiene or dicyclopentadiene functionalities, and more particularly those with norbornene structure. The cell lines developed in accordance with the instant invention are capable of producing highly specific monoclonal antibodies which may be used to distinguish the presence of cyclodiene insecticides in foods and environmental samples. These antibodies are contemplated to be useful for the sensitive detection of cyclodiene insecticides in tissue and surface samples.

The disclosed cyclodiene insecticide specific antibodies produced according to the present method may be used as attachment agents in an affinity column for the concentration and purification of cyclic pesticide structures which may contain substitutions on the ring structure. The subject method of use provides for preparation of samples containing polar soluble compounds so that they may be rapidly and automatically screened for polar compounds, by a nonpolar immunoassay with monoclonal antibodies.

The procedure for isolation of assay samples is suitable for materials found in environmental samples and in common foods. The extraction procedure developed for assay of food or environmental samples by specific monoclonal antibodies may also be used when samples are assayed by GC/EC. A kit format of the diagnostic method may be used for field testing of environmental surface wipe samples.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 presents a representative synthetic pathway for the production of the hapten-carrier protein linkage of the immunogen used to immunize mice to produce monoclonal antibodies to cyclodiene insecticides.

Figure 2 shows representative competition ELISA data for the monoclonal antibody from hybridoma Hept-2, (closed circles) when heptachlor is used as a competitor. Bars represent +/- one standard deviation.

Figure 3 shows competition ELISA data for monoclonal antibody from Hept-2 when reacted with competitors: heptachlor, (closed circle); heptachlor epoxide, (open circle); aldrin, (open square); chlordane, (open diamond^: and endrin, (open triangle). BEST MODE FOR CARRYING OUT THE INVENTION

The subject invention is directed to a group of monoclonal antibodies, and the hybridomas which continuously produce them, which react specifically with the described group of cyclodiene insecticides, which contain the cyclopentadiene or dicyclopentadiene ring, and more particularly to those which contain the norbornene structure. The present invention also provides a method for making monoclonal antibodies reactive to chlorinated cyclodiene insecticides with the cyclopentadiene or dicyclopentadiene rings, more particularly to those which contain the norbornene structure. The subject invention provides a method for preparation of samples containing fat-soluble halogenated cyclic organic compounds so that they may be assayed by aqueous-based immunoassay.

Immunogen

Cyclodiene insecticides are small organic molecules, and in order to render them immunogenic, it was necessary to conjugate such molecules to carrier protein by a method such as that described in

Stanker, et al in Toxicol. 45- 229-243 (1987), which is incorporated by reference. Various modifications of cyclodienes may be made in an effort to achieve this purpose. In particular, a functional group was introduced into chlordene by which it could be conjugated to a carrier protein. In the preferred example, hexachlorocyclo- pentadiene and cyclopentadiene are allowed to react together and the resulting intermediate is oxidized to form 1-hydrochlordene. This intermediate was connected with a succinate linker to a carrier protein to form the immunogen. The carrier protein was selected from any of several immunogenic proteins such as keyhole limpet hemocyanin, serum albumins and thyroglobulin. In the preferred mode, the analog hapten of heptachlor was linked through succinate to BSA as the protein conjugate.

Immunization to Raise Antibodies Techniques for the immunization of laboratory mammals with proteins are known to those skilled in the art. However, when the antigenic compounds are short peptide fragments or small nonproteinaceous molecules, immunization with these compounds may fail to produce an adequate immune reaction. Modified small molecules, termed haptens, may be conjugated to a known immunogen or to a carrier protein which is a known immunogen and by such a linkage be rendered immunogenic. Mammalian antibodies raised in response to an immunogenic conjugate may recognize the small molecule apart from the carrier protein. Carrier proteins may be selected from any of a group of proteins which are immunogenic. Suitable carrier proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumins, including bovine serum albumin (BSA), globulins including thyroglobulins and the like. Keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) are conveniently employed in the subject invention.

The method for the production of monoclonal antibodies which are capable of distinguishing the presence of cyclodiene insecticides with the cyclopentadiene or dicyclopentadiene ring structure and more particularly of distinguishing the presence of cyclodienes with the norbornene structure in accordance with the subject invention, comprises immunizing a suitable mammal, preferably mice, rats, hamsters, rabbits, goats, sheep, cows and horses, still more preferably mice, with an antigen hapten conjugate; preferably with an antigen of heptachlor succinate hapten, which has been conjugated to an immunogenic carrier protein. Repeated intraperitoneal (i.p.) administration of the antigen is made to a first species of mammal in a suitable amount, preferably between about 10 ug to about 200 ug, with about 50 ug

(i.p.) per animal, per administration of heptachlor carrier protein conjugate being especially preferred. The antigen may be mixed with adjuvant, preferably Ribi adjuvant (Ribi Immunoche ical Research

Corp., Hamilton MT), and administered at intervals of about two weeks for several, preferably three immunizations.

Immunosensitized spleen cells or lymphocytes, preferably sensitized spleen cells, which are now capable of producing antibodies to the antigen of choice, are removed from the animal. The sensitized spleen cells or lymphocytes are fused with immortally reproducing cells, preferably myeloma cells of the first species of mammal or of another species, to produce hybrid cells. The hybrid cells are cultured in a suitable host or in a culture medium. The clones of hybrid cells, known as hybridomas, which continuously produce or secrete specific antibodies to an antigen of the aforenamed group, are isolated. Hybridomas, which produce monoclonal antibodies that distinguish the presence of cyclodienes, are selected, quantities of these monoclonal antibodies are generated, antibodies from the culture medium or from the host used for growing the cells are harvested, the monoclonal antibodies isolated and purified, if preferred, and monoclonal antibodies so produced are used to assay samples for the presence of cyclodienes.

The hybridomas may be propagated in a suitable host animal or grown in a suitable culture or carrier medium. Host animals are mammals which include, but are not limited to, those described previously. Suitable culture media include, but are not limited to, ascites fluid, hybridoma supernatant or synthetic media, such as D-MEM, RPMI or Iscocos, as well as one of the culture media specified above. Screening for Antibodies Antibodies were screened for their ability to bind to the analog hapten of heptachlor-BSA and were also selected for those similarly responsive to the related cyclodiene compounds, heptachlor epoxide, chlordane, aldrin, as well as the synthesized antigen, heptachlor-KLH, but were not responsive to the carrier proteins alone. The selection procedure also included screening for antibody which would be active in the solvent system of the immunoassay. This screening eliminates clones that recognize the linkage chemistry of the conjugate protein. Those clones which test positively with respect to the hapten regardless of carrier protein and negative with respect to the two carrier proteins in the presence of immunoassay solvent were subcloned. Those clones which could recognize free heptachlor, heptachlor epoxide and/or chlordane were evaluated by c-ELISA.

According to a further aspect of the present invention, in accordance with its objects and purposes, the hybridoma cell line. designated as Hept-2, was developed. The clone of said cell line is capable of producing monoclonal antibody of high specificity with which to distinguish the presence of the described cyclodienes with the cyclopentadiene or dicyclopentadiene functionality, more preferably to distinguish the presence of the described cyclodienes with the norbornene structure. This cell line was deposited on December 11, 1990, with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 USA, and has been accorded Accession Number ATCC No. HB 10623. This hybridoma deposit was made pursuant to a contract under the terms of the Budapest

Treaty, between the ATCC and the assignee of this patent application. Also, the present invention is not to be considered limited in scope by the strain deposited, since the deposited embodiments are intended only to be illustrative of particular aspects of the invention. Any hybridoma cell lines which are functionally equivalent to these deposited are considered to be within the scope of this Invention.

Assay for Cyclodiene Insecticides

Specificity of the antibody is evaluated by a method such as that modified from the direct binding ELISA assay method of Stanker et al . (1986) (J. Immunol. 136: 4171-4180), which is incorporated by reference.

Antibodies of this invention may be used in the form of hybridoma supernatant, or as ascites fluid, or as the isolated and purified monoclonal antibodies. The sensitivity of the assey is dependent upon the type of binding antigen used to coat the microtiter plate. Any of the several described cyclodiene antigens may be used to coat the microtiter plate, the preferred antigen being a BSA protein conjugate of heptachlor.

The binding specificities of antibodies to standards and assay samples were evaluated by competition ELISA assays. In a competition assay system, an antigen, which is a large molecule with protein or carbohydrate moieties which can precisely bind with steric interaction with the conformation of the antibodies, is fixed to the reaction surface of a test plate. Antigen-specific antibody is added along with an aliquot of sample extract or standard test solution. The free-floating antibody partitions between the fixed antigen bound to the test reaction surface and the antigen of the added sample or standard, which is free-floating in the solution.

After a reaction period, the free-floating antigen-antibody complex is washed away. The plate is rewashed and incubated with an enzyme-tagged indicator molecule which will immunospecifically bind to the proteins of the animal species which was the source of the cyclodiene-specific antibodies. Substrate and buffer are provided for the reaction of the enzyme-tagged indicator molecule. The optical signal generated from the enzyme substrate reaction indicates the amount of cyclodiene-specific antibody which remains bound to the immobilized antigen on the reaction plate. Limited color in the microtiter reaction well indicates a high concentration of antigen in the sample. The utility of such a reaction system is dependent on the surface binding of the antigenic hapten-protein complex. The sensitivity of such a binding system can be amplified by any of several means such as by coupling the enzyme reaction endpoint to a biotin-avidin complex. Optimal enzyme detection sensitivity occurs when a minimal amount of coating antigen (heptachlor-BSA) is used. The total binding capacity of the plate is large so that extra unconjugated carrier protein does not degrade binding.

Immunoassay of Organic Compounds in Food or Environmental Samples

The present invention also provides an improved method for identifying organic compounds and pesticides in the presence of other compounds found in food or environmental samples. The method is contemplated to be useful for the sensitive detection of organic compounds, pesticides and their metabolites in samples by their specific binding affinity to monoclonal antibodies which are fixed to a surface.

It is necessary to modify the isolation conditions when nonpolar compounds, non-electrolytic compounds which generally consist of carbon or nitrogen, with or without other elements, are to be assayed by a specific monoclonal antibody in a polar or electrolytic reaction medium of the immunoassay.

Evaluation of organic compounds or pesticide contamination of samples requires preliminary sample preparation to remove other organic material which may interfere with the specific antibody immunoassay. Pesticides tend to concentrate in fat and this fat may interfere with the detection immunoassay. Fat can be removed by rendering of the tissue with heating to 110°C for 30 minutes and extraction of the rendered fat with activated resin, such as silica gel, alumina, C-18 reverse phase resin or florisil, preferably florisil, followed by a resuspension of the sample in a polar-non-polar solvent mixture for immunoassay.

Food or tissue samples are prepared for assay by heating and dissolving the rendered fat in organic solvent. Assay samples from soft samples, such as soils, grains and muscle tissue are ground in organic solvent, such as hexane and subjected to column fractionation. Muscle tissue of fish and other lean animals is homogenized in organic solvent, and milk is simply extracted with an organic solvent, preferably hexane. The hexane-extracted fat fraction is applied to an activated glass column in which the resin is used in a ratio of 50-100 times the weight of the fat sample. The fat soluble components are eluted from an activated glass column, preferably a florisil column, with organic solvent such as hexane and the solvent evaporated under a stream of inert gas (N₂ @ 30°C). The residues are then resuspended in alcohol, preferably 100% methanol and subsequently diluted to 25% methanol prior to assay with cyclodiene-specific monoclonal antibodies of the invention. Other sample preparation techniques, when used for HPLC or GC analysis, use a smaller amount of florisil and may leave residues which may interfere with the immunoassay.

This sample preparation method is suitable for a variety of organic compounds, including those with one or more 5 or 6 carbon ring structures. It is especially suitable for heterocyclic pesticides, their production by-products and metabolites. A kit format of the detection system may be used for field analysis of surface wiped or extracted samples. The specific antibodies developed here can be used in a diagnostic kit for office or field testing of samples which are suspected of containing cyclodiene insecticides or metabolites of such. A card or a cup test format may be used in which the antibody is affixed to an absorbent layer, or is mixed in a permeable layer which is bound to an absorbent layer.

Isolation of Cyclodienes

The binding specificity of the described monoclonal antibodies of this invention for cyclodienes may be utilized in a binding column to selectively discriminate cyclodienes. Compounds with the norbornene structure may reversibly bind specifically to monoclonal antibodies, produced by this invention, which are fixed in a column. Differential binding of cyclodiene compounds to a column of fixed monoclonal antibody may also be used to remove, purify or concentrate these compounds. An exemplary separation method is binding and release of a compound by alteration of the ionic concentration of the column.

The following examples, presented by way of illustration, serve to explain the present invention in more detail.

EXAMPLES:

1. Synthesis of Cyclodiene Hapten and Immunogen

The cyclodiene compound, heptachlor, is a small molecule. which by itself has limited immunogenicity. A hapten is formed b condensation of hexachloropentadiene and cyclopentadiene to develop an intermediate, chlordene, and oxidation of the intermediate to form 1-hydroxychlordene by the method of Buchel et al . Chem. Ber.

2 : 405 (1966).

The 1-hydroxychlordene hapten is rendered immunogenic by coupling it to an immunogenic carrier protein through a succinate moiety. The conjugated hapten-protein elicits formation of an antibody which will recognize several different cyclodienes.

In particular, the condensation reaction is a Diels-Alder addition of hexachlorocyclopentadiene and cyclopentadiene to yield 4,5,6,7,8,8,-hexachloro-3a,4,

7,7a-tetrahydro-4,7-methanoindene which is oxidized with selenium dioxide to l-exo-hydroxy-4,5,6,7,8,8-hexachloro- 3a,4,7,7,a-tetrahydro-4,7-methanoindene. The hapten synthesis is outlined in Figure 1 which shows the formation of the heptachlor hapten and construction of the protein-hapten conjugate used for immunization. The composition of the heptachlor-hapten was verified by physical analysis.

The hapten was rendered immunogenic by coupling to an immunogenic carrier protein through a succinate moiety. In particular, l-exo-hydroxy-4,5,6,7,8,8- hexachloro-3a,

4,7,7a-tetrahydro-4,7-methanoindene was treated with succinic anhydride in pyridine. The resulting solution was evaporated in vacuo, dissolved in 5% aqueous sodium bicarbonate, washed with chloroform, and the aqueous solution was acidified with concentrated hydrochloric acid to give the crude he isuccinate. The crude material was conjugated to bovine serum albumin and keyhole limpet hemocyanin by a N-hydroxysuccinimide procedure such as that described by Lauer et al . Experientia 30: 558 (1974), which is incorporated by reference.

The hapten-protein conjugates used for immunization were produced by conjugation of the 1-hydroxychlordene hemisuccinate to keyhole limpet hemocyanin (KLH) to form (heptachlor-KLH), and to bovine serum albumin (BSA) to form (heptachlor-BSA) using acid anhydride intermediate which readily couples to the free amine groups of the protein. KLH conjugates were used for immunization of animals and BSA conjugates were used for ELISA screening of hybridoma clones.

2. Immunization of Animals with Antigen

Antibodies to cyclodienes were raised by repeated injection of a mammal, in the preferred mode, mice, with a heptachlor-keyhole limpet hemocyanin conjugate (heptachlor-KLH). In the preferred method, the heptachlor-keyhole limpet hemocyanin conjugate, preferably 1-hydroxychlordene-KLH, made as described above, was used to immunize 6-month old BALB/cBkl mice by repeated intraperitoneal injections, preferably three, of 50 ug heptachlor-KLH conjugate, mixed with Ribi adjuvant. In the preferred mode, the mice received a single injection every other week for three injections.

3. Production of Hybridomas

Four days prior to lymphocyte fusion, mouse hapten-specific serum titer was boosted with an intrasplenic injection of 100 ug cyclodiene-bovine serum albumin conjugate, preferably

1-hydroxychlordene-BSA conjugate in sterile saline. Hybridoma fusions, of lymphocytes to SP2/0 myeloma cells were made by standard methods such as those described by Bigbee, W.L. et al . Molecular

Immunology 20: 1353-1362 (1983)), and grown under suitable conditions such as those described by Stanker et al . ⁽1986^{) (}J_^ Immunol. 136: 4174-4180). The papers describing the methods of

Bigbee et al . and Stanker et al . are incorporated by reference.

Fusion is not limited to the use of SP2/0 myeloma cells and the use of other immortally reproducing mammalian cells is contemplated to be within the scope of this invention. Following fusion, hybridomas were screened in a direct binding ELISA for the ability of the antibodies which they produced to recognize the appropriate

BSA-hapten conjugates.

4. Screening for Cyclodiene Specific Antibodies by Direct

Binding ELISA Assays

A direct binding ELISA assay, a modification of the method of Stanker et al . (1986) (0. Immunol. 136: 4174-4180), was used to screen against a variety of heptachlor and heptachlor-related antigens for antibodies to cyclodienes in the culture fluids of growing hybridomas.

Microliter plates were coated with a cyclodiene hapten-protein complex, preferably 1-hydroxychlordene-bovine serum albumin complex in the amount of about 0.002-0.5 ug per well, preferably about 0.2 ug per well, in carbonate-bicarbonate buffer

(pH 9). They were incubated for 1 hour at 37°C with the hybridoma supernatants. The plates were carefully washed with a solution of

TM surfactant compounds, especially preferred was 0.05% Tween-20 (Polyoxethylenesorbitan Monol aurate) i n a concentration of

0.0001-0.1%, i n water.

For visualization of the binding of the cyclodiene-specific antibody, a fluorometric endpoint of an enzymatic reaction such as, mouse peroxidase, conjugated with goat anti-mouse antiserum (United

States Biochemicals, Cleveland, OH) was used.

Absorbance measurements at 405 nm were taken and subsequently analyzed by a procedure such as that using the

"Cyberdoma" ELISA software described by Slezak et al . (1983) (J. Immunol. Methods 65: 83-95).

Hybridoma cells from wells showing a positive response in the ELISA screen were expanded and subcloned twice by limiting dilution to ensure their monoclonal origin. Ascites fluid was prepared in irradiated mice according to Stanker et al . (1986) (J.Immunol. Methods 136: 4174-4180), and the monoclonal antibodies purified from the ascites by a method such as hydroxylapatite chromatography as described by (Stanker et al . (1985) J. Immunol .

Methods 76: 157-169), or by protein G affinity purification reagent as described by Pharmacia, the manufacturer of the reagent. Isotype determination was done by ELISA using mouse heavy- and light-chain specific antisera (Southern Biotechnology Assoc, Birmingham, AL).

Hybridomas from the fusions were cultured and antibodies that recognized both hapten conjugates, but not either carrier protein, was observed and evaluated for their ability to recognize unconjugated heptachlor and heptachlor epoxide in a competition

ELISA. Antibodies from only two of the hybridomas recognized the unconjugated compounds. The hybridoma which produced antibody with the highest relative affinity was named Hept-2.

Isotype of the monoclonal antibody produced by Hept-2 was determined to be IgG_{2a kappa} -._{ght chain} by direct-binding ELISA with isotype-specific antisera (Southern Biotech, Mobile, AL). 5. Assessment of Cyclodienes by Competition Enzyme-linked Immunoabsorbent Assays

A competition enzyme-linked im unosorbent assay (c-ELISA) was developed to quantify heptachlor standards in solution and to assess the specificity of the antibodies for various cyclodienes and derivatives. Any of several coating antigens were used, however, preliminary work to optimize the sensitivity of the assay showed greatly improved sensitivity if heptachlor-bovine serum albumin was used. Microtiter plates were coated with 0.025 ug/well heptachlor-BSA and blocked with 300 ul of assay buffer per well for 1 hour at room temperature. In the competition ELISA, the competitors of antibody binding were dissolved in methanol and were added to assay buffer. Competitor was added so that each well contained 100 ul of competitor in a 50% methanol-assay buffer solution. An equal volume of assay buffer containing monoclonal antibody was added such that there was a final concentration of 100-200 ng antibody/well in a 25% methanol solution in assay buffer comprising antibody and competitor. These antibodies can tolerate up to 40% methanol with only minimal loss of activity. Peroxidase-conjugated goat anti-mouse IgG antibody was added. The plates were incubated for one hour at room temperature and the endpoint assessed by observation of the color change of

2,2,azino-di-3-ethylbenzthiozoline sulfonic acid (ABTS) substrate actuated by alkaline phosphatase conjugated to goat anti-mouse antibody. Because the sensitivity of c-ELISA's can be influenced by both the amount of specific anti-hapten antibody used and the amount of immobilized binding antigen, both of these parameters were optimized. In the preferred method, the amount of antigen used to coat the microtiter plates was varied from 0.005 to 10 ug/well, with 0.025 ug/well being more preferred. Antigen was absorbed onto the wells overnight by evaporation from the antigen dissolved in distilled water at 37°C. Maximal sensitivity of the assay system was observed when the binding antigen was allowed to evaporate onto the wells. To detect a limited quantity of coating antigen, however, the signal must be additionally amplified. Any of several enzyme-directed signal amplification systems were suitable. A preferred method of amplification of the enzyme-catalyzed optically-detected signal, when run with low background levels of plated antigen, is with an avidin-peroxidase/biotin-anti-mouse immunoglobulin system. The avidin-peroxidase/biotin-anti-mouse immunoglobulin system was used as the biotin can be easily coupled to antibodies or enzymes without loss of activity. Because of the exceptionally high binding affinity, avidin (10 /M) forms bridging complexes between biotinylated molecules. Both the specific-binding monoclonal antibody and the peroxidase-enzyme indicator system are bound to biotin. Linkage of the biotin molecules with avidin complexes the indicator molecules and increases detection signal. Cyclodiene specific antibody was conjugated to biotin by standard methods, such as that described by P. Tijssen in Chapter 3 of "Practice and Theory of Enzyme Immunoassays" (R. H. Burdon and P. H. Van Knippenberg, Eds., Elsevier, Amsterdam (1985)). Biotinylated-N-hydroxy succinimide (BNHS) ester was reacted with the cyclodiene-specific antibodies to make the biotinylated i munoreactants. In a preferred method, cyclodiene- specific antibody was identified by the binding of a biotinylated anti-mouse IgG immunoglobulin to the antibody, and using biotinylated peroxidase, Vectastain (Vector Laboratories, Burlinga e, CA) for bridging to the avidin-biotin amplification complex.

The antibody of the hybridoma Hept-2 was titrated against immobilized antigen (0.025 ug antibody/well) in a direct binding ELISA. When the antibody of the hybridoma Hept-2 was used at a concentration of 0.02 ug/well, the level used in subsequent cELISA's, approximately 50% of the plateau activity was reached. In

TM the preferred method, Tween-20 , in the concentration range of about 0.0001-0.1% , preferably a concentration of 0.01%, was routinely used 0.2 ml of assay buffer.

The competition ELISA data was normalized by using the optical density of wells in which antibody was bound to the solid phase antigen (heptachlor-BSA) in the absence of any competitor as the defined value of 100% activity. The test wells, each containing different amounts of competitor, were normalized to the 100% activity wells. Percent inhibition was calculated by subtracting the normalized percent activity from 100. Figure 2 shows competition ELISA data for the monoclonal antibody from hybridoma

Hept-2 when reacted with heptachlor. The I for heptachlor is

2-4 ng/well .

Figure 3 shows competition ELISA data for monoclonal antibody from hybridoma Hept-2 when reacted respectively with heptachlor, heptachlor epoxide, aldrin, chlordane, and endrin, as competitors. The I₅₀ values observed for these competitors were all in the range of 1-10 ng/well. Replicate assays generally had less than 10% variation. A smaller I₅₀ value indicates a greater relative affinity of the antibody for the compound. I₅₀ values were estimated graphically from competition ELISA data. Table 1 represents the cross-reactivity of the monoclonal antibody from hybridoma Hept-2 with various cyclic organic compounds including

(with 100% reactivity defined as the activity to heptachlor): heptachlor (100); heptachlor epoxide (100); chlordane (75); aldrin (100): endrin (150); dieldrin (300); endosulfan(mix) (173); alpha-endosulfan (43); beta-endosulfan (250); endosulfan sulfate

(182); toxaphene (100); Lindane (BHC)(mixed isomers) (7.3); and

D

Kepone (5). The values listed in Table 1 represent the averages from at least two independent assays. 6. Preparation of Food and Environmental Samples for

Immunoassay

Samples from food or environmental sources are prepared for specific immunoassay by isolation and treatment of the lipid fraction which may contain organic or pesticide materials. Assay material from soft samples such as soils, grains and muscle tissue is ground in organic solvent, such as hexane, and subjected to column fractionation. The lipid portion of a milk sample is in the low density cream which is assayed for presence of cyclodiene contaminants.

Meat samples which contain structured fat and epithelial tissue are heated to render any fat. In the preferred method, samples are heated to 80-150°C for 60 minutes, a temperature of

100°C being more preferred. A tissue sample is supported by silicanized glass wool during the rendering process. The heated samples are dissolved in organic solvent, preferably hexane at a ratio of 0.1 g of rendered fat in the sample to 1 ml of hexane.

The hexane solubilized fat of any meat, soft tissue or milk sample is applied to a previously washed 5-10 gm florisil (Baker or

Fisher Scientific, Co.) column and eluted with an excess of hexane.

In the preferred mode, the volume of the florisil column is at least ten times the volume of the hexane-fat mixture. In the preferred mode, at least 20 ml of hexane is used to elute the florisil column. The sample eluate is dried under a stream of inert gas at room temperature, preferably N„ at 30°C. The florisil column used for sample clean-up is prepared by activiation of the gel to remove water and deactivated by addition of a defined amount of water back to the silica gel. In the preferred mode, the florisil is activated by heating, at 650 C overnight, and then stored at 130°C in an open container. For optimal results, the resin is selectively deactivated by addition of water back to the dessicated resin by addition with an atomizer with shaking between water additions. The preferred deactivated resin contains 7% w/w water. The deactivated resin is allowed to sit overnight before use, and can be stored in a closed container at room temperature.

Hexane-extracted samples eluted from the florisil column are assayed by the cELISA assay are dried down and resuspended in

100% methanol, and then diluted to 25% methanol with the aqueous assay buffer.

7. Assay of Cyclodienes in Food Samples

Antibodies produced by the above method may be used in a testing screen to identify the presence of cyclodienes in foods or environmental samples when prepared by the above method. The antibodies described can be used to distinguish the presence of the most commonly known cyclodiene insecticides.

The data herein presented indicate that the antibodies developed by the described method are suitable for detection of cyclodienes in samples from beef fat, which have been spiked with

10,000 and 100 ppb of the cyclodiene, heptachlor. The expected level of cyclodiene contamination in samples correlates well with that observed in spiked samples, when a greater than 95% recovery was assumed.

Heptachlor was detected in heptachlor-spiked fish muscle and in heptachlor-spiked samples of cream which are extracted with hexane, bound to and extracted from a florisil column with hexane, when the samples were assayed with monoclonal antibody from hybridoma Hept-2.

The above-described examples show that monoclonal antibodies produced by the hybridomas made as described, as exemplified by the monoclonal antibody of hybridoma Hept-2, are highly specific to cyclodiene insecticides with the cyclopentadiene or dicyclopentadiene structure, and more particularly to those with the norbornene structure, and are able to distinguish compounds with similar binding activities. Selective binding of cyclodienes to monoclonal antibodies made in this way, indicate that such monoclonal antibodies, as exemplified by monoclonal antibody from Hept-2, is used to identify, simply and conveniently, low concentrations of cyclodienes present in food or environmental samples. The instant invention describes a method for preparation of samples which contain lipid-soluble cyclic materials, such as those commonly found in pesticides, for assay by specific monoclonal antibody in aqueous or non-polar immunoassay.

The above embodiments were chosen and described in order to best explain the principles and the practical applications of the subject invention thereby to enable those skilled in the art to utilize the invention in various other embodiments and various modifications as are suitable for the particular use contemplated.

It is intended that the scope of the invention is best defined by the appended claims.

INDUSTRIAL APPLICABILITY Severe environmental contamination has occurred due to cyclodiene insecticides because these highly lipophilic compounds, such as dieldrin and heptachlor epoxide, have half-lives in the soil of 2-10 years. Bioaccu ulation of these compounds has resulted in their widespread distribution in human fat and milk. Fish have been found in natural waters with a 100,000-300,000-fold accumulation of these materials. These compounds are extremely toxic to fish, birds and small mammals. Cyclodienes have been listed as potentially oncogenic pesticides by the U.S. Environ enal Protecion Agency.

The complex standard chemical extraction and purification methods required to detect cyclodiene compounds which appear in such low incidence has motivated the search for other means to rapidly and specifically quantify these materials. Specific characterization of the presence and concentration of cyclodienes with the instant cyclodiene-specific monoclonal antibodies permits rapid, automatable analysis of cyclodiene compounds in foods and environmental samples.

Monocloal antibodies have considerable usefulness as diagnostic agents and as binding agents for removal of stable cyclodiene insecticides. TABLE 1

Reactivity of Monoclonal Antibody of Hept-2

Compound % Crossreactivity

heptachlor 100 heptachlor epoxide 100 chlordane 75 aldrin 100 endrin 150 dieldrin 300

Endosulfan (mix) 173 a-Endosulfan 43 b-Endosulfan 250

Endosulfansulfate 182

Toxaphene 100 Lindane (BHC) 7.3

Kepone 5 hexa-Cl-cydopentadiene 1.6 hexa-Cl-butadiene 0.4

1,2,4-triCl-Bz 0 hexa-Cl-ethane 0

2-Cl-naphthalene 0

1,2-diCl-Bz 0

1,3-diCl-Bz 0

1,4-diCl-Bz 0

2,5-diCl-nitroBz 0

2,4,6-triCl-phenol 0

2-Cl-phenol 0

2,4-diCl-phenol 0

2,4-DiMe-phenol 0

2-nitro-phenol 0

4-nitro-phenol 0

2,4-dinitro-phenol 0.2

4,6-dinitro-cresol 0.14 penta-Cl-phenol 0 phenol 0

4-Cl-3-Me phenol 0

2,4,5-triCl-phenol 0

4,5-diCl-catecol 0

2,4-diCl-6-nitrophenol 0

2,2,2-tri-Cl-ethanol 0.4

DDT 0

Chlorobenzene 0

2,4,5-triCl-phenoxyacetic acid 0

2,4-D 0

A value of 0 indicates that the cross reactivity was less than 0.06%

Claims

CLAIMS 1. Monoclonal antibodies, produced by a cell which is the fusion product of an immortal mammalian cell and a immunosensitized cell from a mammal which has been immunized with a hapten of a halogenated cyclic organic compound, which have reactive sites and specific affinities for compounds of the class of cyclodienes which possess the cyclopentadiene functionality.

2. Monoclonal antibodies as described in Claim 1, further defined in that they have specific affinities for compounds of the group consisting of heptachlor, heptachlor epoxide, chlordane,

D endrin, aldrin, endosulfan, toxaphene, BHC and Kepone .

3. A monoclonal antibody as described in Claim 2, further defined as a product of hybridoma Hept-2, which is deposited at the ATCC with Accession No. HB 10623.

4. Hybridomas, produced by the fusion of immortally-reproducing mammalian cells with immunosensitized cells from a mammal which was repeatedly injected with a hapten of halogenated cyclic organic compounds, or progeny thereof, which produce monoclonal antibodies that have reactive sites which react specificly with compounds of the class of cyclodienes which possess the cyclopentadiene functionality.

5. Hybridomas as described in Claim 4, further defined in that they react specifically with compounds of the group consisting of heptachlor, heptachlor epoxide, chlordane, endrin, aldrin, endosulfan, toxaphene, BHC and Kepone .

6. An hybridoma as described in Claim 5, further defined as Hept-2, which is deposited at the ATCC with Accession Nos.

HB 10623.

7. A method for the production of monoclonal antibodies which bind specifically to compounds of the class of cyclodienes which possess the norbornene structure, comprising: immunizing an animal by repeated administrations to a mammal of an antigen, selected from a group consisting of dicyclopentadienes with a norbornene structure and heptachlor derivatives, that has been conjugated to an immunogenic carrier protein, to raise immunosensitized cells capable of producing antibodies to said antigen; fusing said immunosensitized cells to immortally reproducing cells to produce hybrid cells, referred to as hybridomas; isolating and harvesting from said hybridomas, quantities of monoclonal antibodies which specifically react to said compounds of the class of cyclodienes which possess the norbornene structure.

8. The method of Claim 7, wherein said antigen is monoester of 1-hydroxychlordene, which is bound to an immunogenic carrier protein.

9. A method for detection of organic ring compounds by immunoassay, comprising the steps of: rendering the fat of a sample by heating; extraction of fat of said sample with a polar solvent to yield an organic material fraction; attachment of said organic material fraction to an activated silica gel column; extraction of said activated silica gel column with polar solvent; evaporation of said polar solvent to yield a residual organic fraction; solubilization of said residual organic fraction into a polar-nonpolar solution; and assay of said polar-nonpolar solution for organic ring compounds with monoclonal antibodies. 10. A method for detection of organic ring compounds as in

Claim 9 further defined as the class of cyclodienes which possess the norbornene functionality comprising: solubilization of said sample into polar-nonpolar solution; and assay of said polar-nonpolar solution by a competition ELISA binding method, wherein said cyclodiene-specific monoclonal antibody is partitioned between bound 1-hydroxychlordene conjugated to bovine serum albumin antigen coated on a reaction plate and the antigen of said solubilized sample.

11. The method according to Claim 10 wherein said cyclodiene-specific monoclonal antibody is from hybridoma Hept-2.

12. A method for separation of cyclodienes from a sample by reversible attachment to immobilized cyclodiene-specific antibodies of Claim 1.

AMENDED CLAIMS

[received by the International Bureau on 19 August 1991 (19.08.91); original claim 8 cancelled; original claims 1, 4 and 7 amended; other claims unchanged (4 pages)]

1. Monoclonal antibodies, produced by a cell which is the fusion product of an immortal mammalian cell and a immunosensitized cell from a mammal which has been immunized with 1-hydroxychlordene hemisuccinate hapten reacted with a protein, which have reactive sites and specific affinities for compounds of the class of cyclodienes which possess the cyclopentadiene functionality.

D endrin, aldrin, endosulfan, toxaphene, BHC and Kepone .

4. Hybridomas, produced by the fusion of immortally-reproducing mammalian cells with immunosensitized cells from a mammal which was repeatedly injected with 1-hydroxychlordene hemisuccinate hapten reacted with a protein, or progeny of said hybridomas, which produce monoclonal antibodies that have reactive sites which react specificly with compounds of the class of cyclodienes which possess the cyclopentadiene functionality.

5. Hybridomas as described in Claim 4, further defined in that they react specifically with compounds of the group consisting of heptachlor, heptachlor epoxide, chlordane, endrin, aldrin, p endosulfan, toxaphene, BHC and Kepone .

HB 10623.

7. A method for the production of monoclonal antibodies which bind specifically to compounds of the class of cyclodienes which possess the norbornene structure, comprising: immunizing an animal by repeated administrations to a mammal of an antigen, a monoester of 1-hydroxychlordene hemi succinate that has been conjugated to an immunogenic carrier protein, to raise immunosensitized cells capable of producing antibodies to said antigen; fusing said immunosensitized cells to immortally reproducing cells to produce hybrid cells, referred to as hybridomas; isolating and harvesting from said hybridomas, quantities of monoclonal antibodies which specifically react to said compounds of the class of cyclodienes which possess the norbornene structure. 8. cancel led

9. A method for detection of organic ring compounds by immunoassay, comprising the steps of: rendering the fat of a sample by heating; extraction of fat of said sample with a polar solvent to yield an organic material fraction; attachment of said organic material fraction to an activated silica gel column; extraction of said activated silica gel column with polar solvent; evaporation of said polar solvent to yield a residual organic fraction; solubilization of said residual organic fraction into a polar-nonpolar solution; and assay of said polar-nonpolar solution for organic ring compounds with monoclonal antibodies.

10. A method for detection of organic ring compounds as in Claim 9 further defined as the class of cyclodienes which possess the norbornene functionality comprising: solubilization of said sample into polar-nonpolar solution; and assay of said polar-nonpolar solution by a competition ELISA binding method, wherein said cyclodiene-specific monoclonal antibody is partitioned between bound 1-hydroxychlordene conjugated to bovine serum albumin antigen coated on a reaction plate and the antigen of said solubilized sample.