Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples.

The subject invention is related generally to monoclonal antibodies and 
more specifically to monoclonal antibodies reactive with synthetic 
pyrethroids found in foods and environmental samples. 
IDENTIFICATION OF TERMS 
Abbreviations or definitions used in the disclosure herein are as follows: 
cELISA, competition enzyme-linked immunosorbent assay; 
BSA, bovine serum albumin; 
KLH, keyhole limpet hemocyanin; 
GC/EC, gas chromatography/electron capture; 
hapten, a small molecule which carries an antigenic determinant, but is not 
immunogenic 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; 
Soxhlet apparatus, a solvent extraction still in which condensed solvent 
vapors repeatedly pass through a sample to extract organic materials; 
synthetic pyrethroids, a large group of non-naturally occuring compounds, 
related to pyrethrum, a mixture of esters of chrysanthemic and pyrethric 
acids, which are used as insecticides, including the widely used 
compounds, permethrin, cypermethrin and deltamethrin, and less commonly 
used compounds, phenothrin, fenpropathrin, flucythranate, fenvalerate and 
tetramethrin; 
cypermethrin, alpha-cyano-3-phenoxybenzyl cis, 
trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate; 
fenpropathrin, 
alpha-cyano-3-phenoxybenzyl-2,2,3,3-tetramethylcyclopropanecarboxylate; 
deltamethrin, 
-/+alpha-cyano-3-phenoxybenzyl-(+/-)-cis,trans-3-(2,2-dibromovinyl)-2,2-di 
methylcyclopropanecarboxylate; 
fenvalerate, cyano(3-phenoxyphenyl)methyl 
4-chloroalpha-(1-methylethyl)benzeneacetate; 
flucythranate, 
cyano(3-phenoxyphenyl)methyl(S)-4-(difluoromethoxy)-alpha-(1-methylethyl) 
benzeneacetate; 
permethrin, 3-phenoxybenzyl 
(lRS),cis,trans-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate; 
phenothrin, 3-phenoxybenzyl(lRS)cis,transchrysanthemate; 
tetramethrin, N-(3,4,5,6-tetrahydrophthalimino) methyl 
(dl)-cis,trans-chrysanthemate 
BACKGROUND OF THE INVENTION 
Monoclonal antibodies have considerable usefulness as diagnostic and 
therapeutic agents in clinical, commercial and research applications. 
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 
known and repeatable, fabrication of monoclonal antibodies reactive to 
small organic chemicals, such as carcinogens, pesticides, toxic chemicals 
and DNA adducts is less straight-forward. Production of antibodies to 
small organic molecules may sometimes be achieved by first linking a small 
molecule, which is termed a hapten, to a carrier protein, and the immune 
reactive cells may respond to an antigenic determinant site of this 
complex. Initial chemical treatment of the small molecule hapten, may be 
required in order for it to be conjugated to an immunoreactive carrier 
protein. The immunoreactive cells are induced to form antibodies which 
have recognition sites to various reactive sites located (1) on the hapten 
molecule, (2) the carrier protein, (3) the hapten carrier-protein complex, 
or (4) any combination of the hapten, the linkage chemistry and the 
carrier protein. The specific reactive site is not known and is 
unpredictable. The site and chemistry of the hapten conjugation to the 
carrier protein may influence the specificity of the antibodies produced. 
Antibodies with specific binding to reactive sites on small organic 
molecules are sensitive indicators, which may be used to 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. This work with synthetic 
pyrethroids demonstrates that conjugation of the hapten antigenic site as 
far as possible from the 3-phenoxybenzyl group favors the production of 
antibodies which preferentially recognize the phenoxybenzyl group. The 
phenoxybenzyl group is shared by many synthetic pyrethroids, and thus 
antibodies induced against one of the reactive groups of synthetic 
pyrethroids may recognize several members of the class of synthetic 
pyrethroids which carry that reactive group. 
Synthetic pyrethroids are a large group of insecticides which are widely 
used in the United States and other countries. The synthetic pyrethroids 
most commonly used in the United States are permethrin, cypermethrin and 
deltamethrin; other synthetic pyrethroids include phenothrin, 
fenpropathrin, flucythranate, fenvalerate, and tetramethrin, among other 
compounds. The three most commonly used compounds contain both a 
phenoxybenzyl and a cyclopropane moiety. The other less used synthetic 
pyrethroids contain at least one of these groups, or a phenoxyphenyl 
moiety in place of the phenoxybenzyl moiety. In 1982, synthetic 
pyrethroids represented as much as 30% of the world insecticide market. 
The synthetic pyrethroids in use differ widely in their chemical structure, 
toxicity and photostability. Historically, the agricultural use of 
synthetic pyrethroids, such as allethrin and bioallethrin, was limited due 
to the unstable character of those compounds in the air and water. 
However, recently developed synthetic pyrethroids such as permethrin, 
cypermethrin and deltamethrin are more stable and thus have greater 
agricultural utility. These compounds are widely used for insect control 
in food processing plants because they display low mammalian toxicity. 
Widespread use of these more stable compounds, however, has led to concern 
about the possiblity that pesticide residues might remain in foodstuffs 
and the environment. Both cypermethrin and permethrin have been listed as 
potentially oncogenic pesticides by the U.S. Environmental Protection 
Agency. (See Regulating Pesticides in Food: The Delaney Paradox, National 
Research Council Board on Agriculture, National Academy Press, Washington, 
D.C., 1987.) 
Residue limits in meats and fats have been established in the United States 
for permethrin, cypermethrin and deltamethrin. Residue limits for many 
pyrethroids have been set by the Food and Agricultural Organization and 
the World Health Organization. However, the lack of convenient, rapid 
detection systems has hampered environmental identification and 
quantification of pyrethroids. Conventional analysis involves multi-step 
sample clean-up procedures followed by gas chromatography, and due to 
thermal instabilities, detection is limited to electron capture (GC/EC). 
Analysis by high-pressure liquid chromatography (HPLC) has also been 
described, but this separation technique cannot adequately resolve the 
various synthetic pyrethroids. Detection is also a problem with HPLC 
analysis. The complexity of standard chemical extraction and purification 
of compounds of such low incidence has engendered a need for other means 
by which to identify, rapidly and specifically, and to quantify these 
materials. Specific characterization of the presence and concentration of 
these compounds by assay with anti-pyrethroid monoclonal antibodies would 
permit, rapid, automatable analysis of these materials in foods and 
environmental samples. 
SUMMARY 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 synthetic pyrethroids, 
particularly those antibodies with a high degree of specificity for a 
class of synthetic pyrethroids which possess the phenoxybenzyl or 
cyclopropane functionalities, and more particularly those antibodies with 
a high degree of specificity for a class of synthetic pyrethroids which 
possess the phenoxybenzyl and cyclopropane functionalities. 
Another object is to provide a method for production of monoclonal 
antibodies which identify synthetic pyrethroids, preferably those with the 
phenoxybenzyl or cyclopropane moities, more preferably those with the 
phenoxybenzyl and cyclopropane moieties, and including production of a 
monoester of cyclopropanedicarboxylic acid hapten of phenothrin which will 
elicit formation of these antibodies. 
A further object is to provide a method for the specific and sensitive 
detection and separation of synthetic pyrethroids from samples, 
particularly a class of synthetic pyrethroids which possess the 
phenoxybenzyl or cyclopropane functionalities, and more particularly a 
class of synthetic pyrethroids which possess the phenoxybenzyl and 
cyclopropane functionalities, by binding to specific monoclonal 
antibodies. 
Another object is to provide a method for the detection of synthetic 
pyrethroids, particularly those with phenoxybenzyl or cyclopropane 
functionalities, and more particularly those with the phenoxybenzyl and 
cyclopropane functionalities, in environmental and food samples. A kit 
format of the detection system may be used for field analysis of surface 
wiped or extracted 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 forgoing 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 antibodies and the 
hybridomas which produce them which are specifically reactive to the 
synthetic pyrethroids which have a phenoxybenzyl or a cyclopropane group, 
and more specifically, to synthetic pyrethroids which have both a 
phenoxybenzyl group and a cyclopropane ring. Such compounds include, but 
are not limited to those shown on Table I, 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 synthetic pyrethroids 
which have a phenoxybenzyl or a cyclopropane moiety, or more preferably to 
a phenoxybenzyl and a cyclopropane moiety, and the hybrid cell lines which 
are capable of continuously producing these antibodies. 
The method for the production of monoclonal antibodies to synthetic 
pyrethroids in accordance with the subject invention is adapted from the 
general method of Kohler and Milstein (1975) which is herein incorporated 
by reference. The production of pyrethroid-specific antibodies comprises 
immunizing a suitable animal with an antigen selected from the group 
consisting of synthetic pyrethroids, a monoester of 
cyclopropanedicarboxylic acid hapten of phenothrin, and immunogenic 
carrier protein conjugates of these compounds; obtaining from the animal 
immunosensitized cells, capable of producing antibodies to the antigen of 
choice; fusing the immunosensitized cells with immortally reproducing 
cells of the same species or of another animal species; culturing the 
hybrid cells in a suitable host or in a culture medium; isolating clones 
of hybrid cells, referred to as hybridomas, which continuously produce 
specific antibodies which react with the sensitizing antigen; selecting 
hybridomas which produce monoclonal antibodies of desired reactivity; 
producing these antibodies in culture medium or in a host; harvesting the 
antibodies from the culture medium or from the host used for growing the 
cells; and purifying the monoclonal antibodies, if desired. 
According to a further aspect of the present invention, 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 synthetic pyrethroids in foods and 
environmental samples. These antibodies are contemplated to be useful for 
the sensitive detection of synthetic pyrethroids in tissue and surface 
samples. More specifically, the subject method of use provides for rapid, 
automatable screening of samples for synthetic pyrethroid compounds, with 
the use of synthetic pyrethroid specific monoclonal antibodies. The 
monoclonal antibodies produced are capable of recognizing synthetic 
pyrethroids, especially those with the functional groups, phenoxybenzyl or 
cyclopropane, and more especially, those with phenoxybenzyl and 
cyclopropane functional groups. The disclosed method may be used for 
identification and quantification of synthetic pyrethroid materials found 
in environmental samples and common foods. A kit format of the diagnostic 
method may be used for field testing of environmental surface wipe 
samples. 
The synthetic pyrethroid-specific antibodies produced according to the 
present method may be used as attachment agents in an affinity column for 
the concentration and purification of synthetic pyrethroids. The 
antibodies produced also have application as detection agents for the 
indentification and quantification of synthetic pyrethroids following 
extraction. The extraction procedure for food or environmental samples 
assayed by specific monoclonal antibody assay may also be used when 
samples are assayed by GC/EC.

DETAILED DESCRIPTION OF THE INVENTION 
The subject invention is directed to a group of monoclonal antibodies, and 
the hybridomas which continuously produce them, which react specifically 
to the described group of synthetic pyrethroids, which contain the 
phenoxybenzyl or the cyclopropane functionalities, and more preferably to 
those which react with synthetic pyrethroids which contain both 
phenoxybenzyl and cyclopropane groups. The present invention also provides 
a method for the production of monoclonal antibodies reactive to synthetic 
pyrethroids with phenoxybenzyl or cyclopropane groups, more preferably to 
those reacting with synthetic pyrethroids with both a phenoxybenzyl and 
cyclopropane groups. 
Techniques for the immunization of laboratory animals with synthetic 
peptides, which are analogs of protein fragments, and identification of 
specific protein epitopes, which are immunologically recognized features 
of a protein, as represented by antibody-inducing synthetic peptides, are 
known to those skilled in the art. However, when the antigen compounds are 
short peptide fragments or small nonproteinaceous molecules, injection of 
these compounds may fail to produce an adequate immune reaction in a test 
subject. Modified small molecules, termed haptens, may be conjugated to a 
known immunogen or to a carrier protein which is a known immunogen. By 
linkage into a small molecule-protein conjugate, the hapten may be 
rendered immunogenic. Antibodies produced in response to this immunogenic 
conjugate will sometimes 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. 
Synthetic pyrethroids are small organic molecules, and in an effort to 
render such a molecule immunogenic, it was necessary to conjugate such 
molecules to carrier protein. Various modifications of synthetic 
pyrethroids can be made to achieve this purpose. For example, permethrin 
does not have functional groups through which it could be conjugated to a 
carrier protein, so it was necessary to synthesize an analog hapten, the 
monoester of cyclopropanedicarboxylic acid of phenothrin, for this 
purpose. Antibodies selected for their binding to the analog hapten of 
permethrin were also similarly responsive to the related pyrethroid 
compounds, permethrin and phenothrin, as well as the synthesized hapten, 
but were not responsive to the carrier protein alone. 
The method for the production of monoclonal antibodies which are capable of 
distinguishing the presence of synthetic pyrethroids with the 
phenoxybenzyl or cyclopropane functionality, and more preferably of 
distinguishing presence of synthetic pyrethroids with the phenoxybenzyl 
and cyclopropane functionalities in accordance with the subject invention, 
comprises immunizing a suitable animal, preferably mice, rats, hamsters, 
rabbits, goats, sheep, cows and horses, still more preferably mice, with 
an antigen selected from the group consisting of the described synthetic 
pyrethroids with the phenoxybenzyl or cyclopropane functionality and a 
monoester of cyclopropanedicarboxylic acid hapten of phenothrin, which has 
been conjugated to an immunogenic carrier protein. Repeated administration 
of the antigen is made in a suitable amount, preferably between about 10 
.mu.g to about 200 .mu.g, with about 100 .mu.g (i.p.) per animal, per 
administration of pyrethroid carrier protein conjugate being especially 
preferred. The antigen may be mixed with complete Freund's adjuvant, 
preferably in a 1:1 mixture, 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 or of another animal 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 synthetic pyrethroids 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 synthetic 
pyrethroids. The hybridomas may be propagated in a suitable host animal or 
grown in a suitable culture or carrier medium. Host animals include but 
are not limited to mice, rats, hamsters, guinea pigs, rabbits, goats, 
sheep, cows and horses, and the like. Suitable culture media include, but 
are not limited to, ascites fluid and hybridoma supernatant, other than 
the one of the culture media specified above. 
According to a further aspect of the present invention, in accordance with 
its objects and purposes, the hybridoma cell lines, designated as Py-1, 
Py-3, and Py-4, were developed. The clones of said cell lines are capable 
of producing monoclonal antibodies of high specificity to distinguish the 
presence of the described synthetic pyrethroids with the phenoxybenzyl or 
cyclopropane functionalities, more preferably to distinguish the presence 
of the described synthetic pyrethroids with the phenoxybenzyl or 
cyclopropane functionalities. The antibodies are given the same name as 
the cell line from which they are derived. These cell lines are deposited 
with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, 
Rockville, Md. 20852 USA, and have been accorded Accession Numbers as 
follows: Py-1, ATCC No. HB 9996; Py-3, ATCC No. HB 9997 and Py-4, ATCC No. 
HB 9998. 
These deposits were made pursuant to a contract between the ATCC and the 
assignee of this patent application. The contract with the ATCC provides 
for permanent availability of said strains and progeny thereof to the 
public upon issuance of a U.S. patent related to this application 
describing and identifying the deposit or upon the publication or laying 
open to the public of any U.S. or foreign patent application, whichever 
comes first and for the availability of these strains and the progeny 
thereof to one determined by the U.S. Commissioner of Patents and 
Trademarks to be entitled thereto according to 35 USC 122 and the 
Commissioner's rules pursuant thereto (including 37 CFR 1.14 with 
particular reference to 886 OG 638). The assignee of the present 
application has agreed that if the strains on deposit should die or be 
lost or destroyed when cultivated under suitable conditions, it will be 
promptly replaced upon notification with a viable culture of the same 
strain. 
The depository, under the terms of the Budapest Treaty assures that said 
cultures deposited will be maintained in a viable and uncontaminated 
condition for a period of at least five years after the most recent 
request for the furnishing of a sample of a deposited hybridoma cell line 
was received by the ATCC and, in any case, for a period of at least 30 
years after the date of the deposit. 
Availability of the deposited strains is not to be construed as a license 
to practice the invention in contravention of the rights granted under the 
authority of any government in accordance with it's patent laws. 
Also, the present invention is not to be considered limited in scope by the 
strains 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. Further, various modifications 
of the invention in addition to those shown and described herein apparent 
to those skilled in the art from the preceding description are considered 
to fall within the scope of the appended claims. 
Specificity of the antibodies is determined by an assay modified from the 
direct binding ELISA assay method of Stanker et al. (1986) (J. Immunol. 
136: 4171-4180). Briefly, 96-well microtiter plates (Dynatech "Immulon-II. 
Alexandria, Va.) were coated with either Hb or synthetic peptide 
conjugated to BSA by incubating 100 .mu.l/well of a 100 .mu.g/ml solution 
of antigen for 18 hrs. at 4.degree. C. The wells were then washed three 
times with phosphate-buffered saline (PBS) and nonspecific protein binding 
sites blocked by incubatin the plates for 1 hr. at room temperature in a 
1% solution of ovalbumin (400 .mu.l/well). After additional washes in PBS, 
test culture fluid or ascites fluid was added to the wells (50 
.mu.l/well), and the plates were incubated for 1 hr. at 37.degree. C. They 
were then washed extensively with a detergent solution (0.05% Tween-20). 
Peroxidase-conjugated anti-mouse serum (U.S. Biochemicals) was then added 
(50 .mu.l/well of a 1/800 dilution), and the plates were incubated for an 
additional 1 hr. at 37.degree. C. Finally, 2.2 
azino-di-(3-ethylbenzthiazoline sulfonic acid) substrate was added, and 
the enzyme activity was determined by multiple scannings by using a 
Titertek Multiskan 96-well microtiter plate reader (Flow Laboratories, 
McLean Va.). Absorbance measurements at 405 nm were recorded as a function 
of time and were analyzed by using computer analyses in which slopes of OD 
405/hr. are calculated by using linear regression. 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 assay is dependent upon the type of binding antigen 
used to coat the microtiter plate. Any of the several described synthetic 
pyrethroid antigens may be used to coat the microtiter plate, including a 
protein conjugate of a monoester of cyclopropanedicarboxylic acid hapten 
of phenothrin. The compound 3-phenoxybenzoic acid (3-pba) which has been 
complexed with bovine serum albumin (BSA), is a preferred binding antigen. 
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, and 
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 proteins of the animal 
species which was the source of the pyrethroid-specific antibodies. 
Substrate and buffer are provided for the reaction of the enzyme-tagged 
indicator molecule. The optical signal from the enzyme substrate reaction 
indicates the amount of pyrethroid-specific antibody which remains bound 
to the immobilized antigen on the reaction plate. 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 coupling the enzyme reaction endpoint to a biotin-avidin 
complex. Optimal enzyme sensitivity occurs when a minimal amount of 
coating antigen (3-pba-BSA) is used. The total binding capacity of the 
plate is large so that extra unconjugated carrier protein does not degrade 
binding. 
The present invention also provides an improved method for identifying 
synthetic pyrethroids in the presence of other compounds present in food 
or environmental samples. The method is contemplated to be useful for the 
sensitive detection of synthetic pyrethroids and other pyrethroid 
metabolites. The specific affinity for synthetic pyrethroids of the 
instant monoclonal antibodies, when fixed to a binding surface, is 
suitable for isolation of pyrethroid metabolites from samples. 
Evaluation of synthetic pyrethroid contamination of foods requires 
preliminary sample preparation to remove other organic material which may 
interfere with the sensitive antibody detection assay. Pyrethroids tend to 
concentrate in fat. They can be removed by rendering the fat by heating to 
100.degree. C. and extracting the pyrethroids with an organic/aqueous 
solvent mixture. Assay samples of food or tissue are prepared by 
homogenization and a preliminary extraction with a organic aqueous mixture 
of acetonitrile and water in the proportions of about 50-95 parts to 50-5 
parts, with 85:15 being especially preferred. The fat layer is removed by 
chilling, and then the upper phase is extracted with hexane in a 
separatory funnel. Salt solution is added to improve separation of the 
aqueous wash layers, followed by additional distilled water washes. The 
hexane fraction is recovered by draining through and drying over anhydrous 
sodium sulfate. The pyrethroid components are removed from the hexane 
extracts by binding to a pretreated alumina oxide column. The pyrethroid 
components are eluted from the alumina column with benzene, the benzene 
solvent evaporated and the sample resolublized in acetonitrile. Pyrethroid 
content of the sample is then assayed in aqueous buffer by competition 
ELISA procedures. 
The binding specificity for the described synthetic pyrethroids of the 
monoclonal antibodies of this invention may be utilized in a binding 
column to selectively discriminate the described synthetic pyrethroids or 
pyrethroid metabolites. Compounds with the 3-phenoxybenzyl and 
cyclopropane functionalities may reversibly bind specificly to monoclonal 
antibodies, produced by this invention, which are fixed in a column. 
Differential binding of pyrethroid compounds to a column of fixed 
monoclonal antibody may 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. These examples are not to be 
construed as limiting the invention to the precise forms or modes 
disclosed. In fact, several improvements and modifications are possible. 
It is intended that such improvements and modifications are encompassed by 
the appended claims. 
EXAMPLES 
1. Synthesis of A Pyrethroid Hapten 
The synthetic pyrethroid compound, permethrin, is a small molecule, which 
by itself is not immunogenic. A related hapten is formed by addition of 
reactive groups, which will render it immunogenic by enabling it to couple 
to an immunogenic carrier protein. Conjugation of carrier protein to the 
binding site most distant from the preferential recognition site, the 
3-phenoxybenzyl group, forms a hapten which elicits an antibody which will 
recognize several different synthetic pyrethroids. 
The hapten synthesis outlined in FIG. 2 is achieved by suspending 
chrysanthemum monocarboxylic acid (compound 4) (1.28 g, 7.6 mM, mixed 
isomers) in 20 ml of methylene chloride. Oxalyl chloride (740 .mu.l, 8.4 
mM) was added followed by one drop of dimethylformamide. The mixture was 
stirred for 2 hours at room temperature. Then 3-phenoxybenzyl alcohol 
(2.00 g, 10 mM) and pyridine (2 mL) were added and the mixture stirred for 
an additional 12 hours. Ether was added, and the mixture was washed with 
saturated aqueous sodium bicarbonate, followed by water. The organic layer 
was dried over anhydrous sodium sulfate, evaporated, and purified by 
column chromatography on neutral alumina (eluted with benzene) to yield 
2.2 g of phenothrin (compound 5). 
A mixture of ozone and oxygen gas (3:97, w/w) (Welsbach Ozone Systems, 
Sunnyvale, Calif. #T-408) was passed through a solution containing 1.40 g 
of phenothrin (compound 5) in 200 ml of ethyl acetate (4 mM) and 10 ml of 
formic acid at 0.degree. C. for 20 min at a rate of 1.2 l/min. A solution 
of 30% aqueous hydrogen peroxide (3.0 ml) was then added and the mixture 
kept at 4.degree. C. for 12 hours. The solution was extracted twice with 
water and then 2M NaOH. The aqueous layer was acidified with 12 M HCl and 
then was extracted with methylene chloride. The organic layer was dried 
over anhydrous sodium sulfate and evaporated. The resulting oil was 
purified by column chromatogaphy on silica gel (methanol/chloroform, 3:97) 
to yield 360 mg (26%) of (compound 6) as an oil. The composition of the 
phenothrin-hapten was verified by physical analysis. FIG. 2 shows the 
pathway for the synthesis of the phenothrin hapten for construction of the 
protein-hapten conjugate used for immunization. 
2. Immunization of Animals with Antigen 
The hapten-protein conjugates used for immunization were produced by 
conjugation of the phenothrin hapten (compound 6 of FIG. 2) to keyhole 
limpet hemocyanin (KLH) to form (py-KLH), and to bovine serum albumin 
(BSA) to form (py-BSA) using the mixed anhydride method of Erlanger et al. 
1959 (J. Biol. Chem. 234:1090-4). By this procedure, 
iso-butylchlorocarbonate is reacted with compound (6) to form an acid 
anhydride intermediate which readily couples to the free amine groups of 
the protein. 
An equally useful method of conjugation of the hapten directly to the 
carrier protein was performed as follows: 50 mg of carrier protein was 
dissolved in 5 ml of double distilled water and added to 50 mg of hapten 
dissolved in 5 ml of water. The pH was adjusted to 7.0 by dropwise 
addition of dilute NaOH. Fifty mg of EDC 
(1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide hydrochloride) was added, 
the mixture stirred overnight at room temperature and dialyzed for 48 
hours against 4 changes of phosphate buffered saline (0.01 M phosphate, 
0.155 M NaCl, pH 7.2). KLH conjugates were used for immunization and BSA 
conjugates were used for ELISA screening of hybridoma clones. Antibodies 
to pyrethroids could be raised by repeated injection of a test animal with 
synthetic pyrethroid-keyhole limpet hemocyanin conjugate (py-KLH). In the 
preferred method, the synthetic pyrethroid-keyhole limpet hemocyanin 
conjugate (py-KLH) was used to immunize 6-month old BALB/cBkl mice by 
intraperitoneal injections of 100 .mu.g py-KLH conjugate, mixed 1:1 with 
complete Freund's adjuvant. 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 .mu.g synthetic 
pyrethroid-bovine serum albumin conjugate (py-BSA) in sterile saline. 
Hybridoma fusions, of lymphocytes to SP2/0 myeloma cells were made by 
standard methods(See Bigbee, W.L. et al. Molecular Immunology 20: 
1353-1362 (1983)), and grown under the conditions described by Stanker et 
al. (1986) (J. Immunol. 136: 4174-4180) The fusion protocol was a 
variation of that described by Oi and Herzenberg (1981). Briefly, 4.75 ml 
of polyethylene glycol (PEG 1540) (Polysciences, Warrington, Pa.) was 
mixed with 5 ml of serum-free SDMEM, 0.75 ml of dimethylsulfoxide (DMSO) 
and 50 .mu.l of 1 M NaOH. NaOH was used to bring the mixture to about pH 
7.8 as judged by the phenol red in the SEMEM. The spleen lymphocytes 
(approximately 10.sup.8 cells) and approximately 10.sup.7 log phase 
myeloma cells were co-centrifuged and pelleted in a 50-ml conical 
centrifuge tube (Corning, Corning, N.Y.). The pellet was re-suspended in 1 
ml of the 50% PEG solution over a period of 1 min. The slurry of cells was 
stirred for an additional minute and 2 ml of serum-free SDMEM was added 
over the next 2 min. followed by an additional 7 ml of serum-free SDMEM 
over the next 2 min. The resulting 10-ml suspension was diluted to 50 ml 
in SDMEM containing 2.5% RS and l.mu.M aminopterin. All the solutions were 
pre-warmed to 37.degree. C. and maintained near that temp. during fusion 
with the use of a 37.degree. C. Temp-Blok (American Scientific Products. 
Evanston, Ill.). The fused cells were then spread over 30 96-well 
microculture plates and were allowed to grow in media consisting of equal 
parts of M3 medium and Hanna serum-free medium (Hanna Biologicals, 
Berkeley, Calif.), containing 40 .mu.M aminoptrin and 2% fetal calf serum 
at 37.degree. C. in a humid 5% CO.sub.2 atmosphere for 10 to 14 days 
before screening for antibody-producing hybridomas. Fusion is not limited 
to the use of SP2/0 myeloma cells and the use of other immortally 
reproducing 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. Characterization of Antibodies by ELISA Assays 
A direct binding ELISA was used to screen for antibodies to synthetic 
pyrethroids in the culture fluids of growing hybridomas. The direct 
binding ELISA method of Stanker et al. (1986)(J. Immunol. 136: 4174-4180) 
was modified as follows: 
The 96-well Immulon-II microliter plates (Dynatech Laboratories, 
Alexandria, VA) were coated with a pyrethroid hapten-protein complex, 
preferably 3-phenoxy benzoic acid-bovine serum albumin complex (3-pba-BSA) 
in the amount of about 0.002-0.5 .mu.g per well, preferably about 0.2 
.mu.g per well, in carbonate-bicarbonate buffer (pH 9) for 18 hours at 
4.degree. C., blocked for 1 hour at room temperature with a 1% solution of 
ovalbumin, and then incubated for 1 hour at 37.degree. C. with the 
hybridoma supernatants. The plates were carefully washed with a solution 
of surfactant compounds, especially preferred was 0.05% Tween-20 .TM. 
(Polyoxethylenesorbitan Monolaurate) in water. For visualization of the 
binding of the pyrethroid-specific antibody from mouse, peroxidase, 
conjugated with goat anti-mouse antiserum, (U.S. Biochemicals, Cleveland, 
Ohio) diluted 1:500 in conjugate dilution buffer (0.005 M, 0.075 N NaCl, 
0.001% Tween-20, pH 7.2) was added to each well. Following a second one 
hour incubation at 37.degree. C., the plates were washed again and the 
substrate, 2,2 amino-di-3-ethylbenzthiazoline sulfonic acid (ABTS), added. 
Absorbance measurements at 405 nm were taken as a function of time and the 
resulting data were transferred to a Macintosh computer and subsequently 
analyzed using the "Cyberdoma" ELISA software described by Slezak et al. 
(1983)(J. Immunol. Methods 65: 83-95). Enzyme which participated in the 
color reaction was indicative of the presence of mouse antibody bound in 
the wells. Hybridoma cells from wells showing a positive response in the 
ELISA screen were expanded and subcloned twice by limiting dilution to 
insure 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 
hydroxylapatite chromatography (Stanker et al. (1985) J.Immunol. Methods 
76: 157-169). Isotype determination was done by ELISA using mouse heavy- 
and light- chain specific antisera (Southern Biotechnology Assoc., 
Birmingham, Ala.). 
Hybridomas from the fusions were cultured in 30, 96-well microculture 
dishes. Approximately 500 wells were observed to be secreting antibody 
that recognized py-BSA conjugate but not the BSA itself. Those cells 
showing the strongest response and specificity (approximately 250) were 
expanded and tested against py-BSA, py-KLH, BSA and KLH. Antibody that 
recognized both hapten conjugates, but not either carrier protein was 
observed in 29 wells. Thirteen of these were subcloned and evaluated for 
their ability to recognize unconjugated permethrin and cypermethrin in a 
competition ELISA. Antibodies from only three of the 13 hybridomas 
recognized the unconjugated compounds. These antibodies are named Py-1, 
Py-3, and Py-4. 
Isotypes of the monoclonal antibodies were determined by direct-binding 
ELISA with isotype-specific antisera (Southern Biotech, Mobile, Ala.). All 
three antibodies were determined to be IgG2a antibodies with kappa light 
chains. 
5. Competition Enzyme-linked Immunoabsorbent Assays 
A competition enzyme-linked immunosorbent assay (c-ELISA) was developed to 
quantify permethrin standards in solution and to assess the specificity of 
the antibodies for various natural and synthetic pyrethroids. Any of 
several coating antigens could be used, however, preliminary work to 
optimize the sensitivity of the assay showed greatly improved sensitivity 
if the phenothrin-bovine serum albumin used as the synthetic pyrethroid 
coating antigen on the ELISA plate was replaced with 3-phenoxybenzoic acid 
(3-pba) conjugated to BSA. The 3-pba conjugate could be prepared by using 
EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) as a cross-linking 
agent. The 3-pba conjugate was used as the coating agent for all 
subsequent experiments. Microtiter plates were coated with 0.5 .mu.g/well 
3-pba-BSA and blocked with ovalbumin. 
In the competition ELISA, the competitors were dissolved in acetonitrile 
and were added to phosphate-buffered saline(PBS)-Tween buffer such that 
the resulting solution was 6% acetonitrile. Competitor was added so that 
each well contained 100 .mu.l of competitor in a 6% acetonitrile-PBS 
solution. An equal volume of PBS containing monoclonal antibody was added 
such that there was a final concentration assay buffer containing antibody 
and competitor. These antibodies can tolerate up to 5% acetonitrile 
without loss of activity. The plates were incubated for one hour at 
37.degree. C. and the endpoint assessed by observation of the color change 
of p-nitrophenyl phosphate 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 
antigen, both of these parameters were optimized. In the preferred method, 
the amount of antigen used to coat the microtiter plates was varied from 
10-0.5 .mu.g/well, with 0.5 .mu.g/well being more preferred. The method of 
application of the antigen to the plate was important to the assay. 
Antigen could be absorbed onto the wells overnight at 4.degree. C. or 
antigen could be allowed to evaporate from the antigen solution onto the 
wells at 37.degree. C. Using Py-1 antibody and permethrin as competitor, 
maximal sensitivity was observed when the antigen was allowed to evaporate 
onto the wells. In the preferred method microliter plates were prepared by 
evaporation of 100 .mu.l of a 5 .mu.g/mL solution of coating antigen 
(3-pba-BSA) at 37.degree. C.. 
To detect a limited quantity of coating antigen, however, the signal must 
be additionally amplified. A preferred method of amplification of the 
enzyme-catalyzed optically-detected signal is with an 
avidin-peroxidase/biotin-anti-mouse immunoglobulin system which improves 
the sensitivity of the immunoassay when run with low background levels of 
plated antigen. The avidin-peroxidase/biotin-anti-mouse immunoglobulin 
system may be used to improve the efficacy of enzyme-linked immunoassay as 
the biotin can be easily coupled to antibodies or enzymes without loss of 
acitivity. The exceptionally high binding affinity of avidin /M) for 
biotin results in the formation of 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 results in complexing of the 
indicatormolecules and increased sensitive detection of the spectral 
signal. 
Synthetic pyrethroid specific antibodies may be conjugated to biotin by 
methods, such as that described by P. Tijssen in Chapter 3 of "Practice 
and Theory of Enzyme Immunoassays" (R. H. Burdon and P. H. Vanknippenberg, 
Eds., Elsevier, Amsterdam (1985)). Biotinylated-N-hydroxy succinimide 
(BNHS) ester can be reacted with the synthetic pyrethroid-specific 
antibodies to make the biotinylated immunoreactants. In a preferred 
method, synthetic pyrethroid-specific antibody can be identified by the 
binding of a biotinylated anti-mouse IgG immunoglobulin to the antibody. 
Biotinylated peroxidase, Vectastain (Vector Laboratories, Burlingame, 
Calif.) can be used as the detection signal after bridging to the 
avidin-biotin amplification complex. 
The antibody Py-1 was titrated against immobilized antigen (0.5 .mu.g 
antibody/well) in a direct binding ELISA. At a Py-1 antibody concentration 
of 0.02 .mu.g/well, the level used in subsequent cELISA's, 40% of the 
plateau activity was reached. To achieve a similar "50%" of plateau 
activity, antibodies Py-3 and Py-4 were used, as unpurified culture 
fluids, in a dilution of 1/200. A detergent, in the preferred method, 
Tween-20.TM., in the concentration range of about 0.0001-0.01% , 
preferably a concentration of 0.001%, was routinely used. Detergent 
concentrations greater than 0.015% interfered with the antibody activity, 
but if the detergent was omitted entirely, the non-specific binding 
activity was increased. 
The competition ELISA data was normalized using 100% activity as the 
optical density in wells in which antibody bound to the solid phase 
antigen (3-pba-BSA) in the absence of any competitor. 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. 
FIG. 3 shows competiton ELISA data for three monoclonal antibodies, Py-1, 
Py-3 and Py-4 with permethrin as the competitor (error bars represent +/- 
one standard deviation). The concentration of permethrin which caused a 
50% inhibition (I.sub.50) of antibody activity in the assay is in the low 
nanogram range for all three antibodies. The average I.sub.50 for 49 
samples of Py-1 antibody by permethrin, in assays run during a 5-month 
period, is 1.55 ng +/-0.6 ng. 
FIG. 4 shows competition ELISA data for antibody Py-1 when reacted 
respectively with phenothrin, permethrin, deltamethrin, cypermethrin, and 
fenvalerate, as competitors. The I.sub.50 values observed for these 
competitors were 1.5, 1.7, 15, 30 and 350 ng/well, respectively. The 
specificities of monoclonal antibodies Py-1, Py-3 and Py-4 are illustrated 
in Table 1 by the I.sub.50 values observed with the following competitors: 
the hapten (compound 6 of FIG. 2), permethrin (mixed isomers), permethrin 
(trans isomer), 3-phenoxybenzoic acid, 3-phenoxybenzaldehyde, phenothrin, 
deltamethrin, fenvalerate, cypermethrin, fenpropathrin, tetramethrin, 
flucythranate, and chrysanthemic acid. Standard deviations of replicate 
assays were generally less than 0.2. The three monoclonal antibodies 
showed similar 50% inhibition values for permethrin, with Py-1 at 1.5 ng, 
Py-3 at 1.7 ng and Py-4 at 12 ng of competitor. Antibodies Py-1 and Py-3 
appear to have similar reactivities, whereas antibody Py-4 is less 
sensitive for all competitors tested, except the hapten. A smaller 
I.sub.50 value indicates a higher relative affinity of the antibody for 
the compound. I.sub.50 values were estimated graphically from competition 
ELISA data. When 50% inhibition could not be attained, the value of the 
highest level of competitor tested is reported with a greater than (&gt;) 
symbol. The values listed in Table 1 represent the averages from at least 
12 independent assays. 
TABLE I 
______________________________________ 
Average.sup.a 50% inhibition values (I.sub.50) in nanograms 
of competitors listed for Py-1, Py-3 and Py-4. 
Monoclonal Antibodies 
Compound Py-1 Py-3 Py-4 
______________________________________ 
Phenothrin 1.5 0.5 6 
Permethrin (mixed isomers) 
1.5 1.7 12 
Permethrin (trans isomers) 
1.7 nd.sup.b 
nd 
Hapten.sup.c 2 0.3 0.6 
Deltamethrin 15 10 10 
Fenpropathrin 17 10 25 
3-Phenoxybenzaldehyde 
22 nd nd 
Cypermethrin 30 20 22 
Flucythranate 120 45 275 
3-Phenoxybenzoic acid 
200 140 330 
Fenvalerate 350 160 4000 
Tetramethrin &gt;600.sup.d 
nd nd 
Chrysanthemic acid 
&gt;600 nd nd 
______________________________________ 
.sup.a Averages calculated from at least 12 assays. 
.sup.b nd = not done 
.sup.c a monoester of cyclopropanedicarboxylic acid hapten of phenothrin 
.sup.d 50% inhibition was not observed at the highest concentration of 
competitor 
6. Specificity of Monoclonal Antibodies in An Immunoassay in Meat Samples 
To determine whether components of meats interfere with the antibody 
binding or immunoassay of permethrin, extracts of commercial ground beef 
samples were prepared by a modification of the method of Braun and Stanek, 
(1982) (Assoc. Off. Anal. Chem. 65: 685-689), and added to the assay 
system. 
Briefly, ground beef (5 g) was mixed with 50 ml of an acetonitrile: water 
(85:15) solution and homogenized with a Polytron (Brinkman Instruments, 
Westbury, N.Y.) at setting 8 for 2 min. The sediment was removed by 
centri-setting fuging for 2 min at 100.times.G and freezing for several 
hours to coalesce the fat. A portion of the upper acetonitrile fraction, 
8.5 ml, was mixed with 10 ml hexane in a separatory funnel (30 sec.). A 
solution of 2% NaCl (40 mls) was added, shaken for 1 min and allowed to 
stand for 2 min. The hexane fraction was extracted again with 5 ml of 
water (30 sec.). The hexane fraction was recovered, the funnel rinsed with 
an additional 5 ml of hexane and the hexane fractions pooled and dried 
over anhydrous sodium sulfate. The pooled hexane fraction was passed over 
a 1 cm pretreated alumina oxide column (AG4)(100-200 mesh) (Bio-Rad 
Laboratories, Richmond, Calif.), which was then rinsed with hexane. The 
column had previously been washed with methylene chloride for 24 hours in 
a Soxhlet apparatus, dried at 130.degree. C. for 24 hours and stored at 
110.degree. C. The bound pyrethroids were released from the column with a 
wash of 10 ml of benzene, and the fraction was dried under a gentle stream 
of nitrogen. The sample was resuspended in 60 .mu.l of acetonitrile, 
followed by an additional 964 ul of phosphate buffered saline (PBS) with a 
concentration of 0.001% Tween-20.TM.. The sample was then used in a cELISA 
as described above. 
For detection of permethrin in meats, a meat sample was extracted in an 
acetonitrile-water solution, partitioned against hexane, purified on an 
alumina column and analyzed with the cELISA method as described above. 
Samples were spiked with known quantities of permethrin, and cELISA curves 
compared with those of a standard competition curve run with permethrin in 
assay buffer. In FIG. 5 are depicted cELISA data of meat samples which 
were spiked with analytical standards of permethrin and analyzed with 
monoclonal antibody Py-1 . Concentrations of analytical standards, 500 ppb 
(closed square); and 100 ppb (open square) were added to meat samples, and 
then extracted for assay with monoclonal antibodies. Open triangles 
represent permethrin standards analyzed in sample buffer. There was no 
competition for the antibody by unknown components in the meat when crude 
extract or hexane-partitioned material, spiked with permethrin, was 
analyzed. Addition of permethrin to the benzene extracts yielded 
competition curves which were similar to those observed with permethrin 
standards. Permethrin standards were not lost to the separation procedures 
of the chromatography or alumina column partitioning. 
The movement of permethrin through the route of the extraction procedure in 
ground beef samples was further confirmed by analysis of the recovery of 
.sup.14 C-labeled permethrin. Analysis of spiked samples (500 ng/g of 
permethrin, marked with a trace amount of radiolabeled permethrin) 
revealed 72% +/-1.7 of the permethrin in the acetonitrile extract (after 
fat removal), 65.5% +/-3.5 in the hexane fraction and 62% +/-2.9 of the 
total radiolabel was recovered in the final benzene fraction. 
7. Assay of Pyrethroids in Food Samples 
Antibodies produced by the above method can be used in a testing screen to 
identify the presence of synthetic pyrethroids in foods in the diet. A 
panel of different antibodies can be used to distinguish the presence of 
the most commonly known pyrethroid insecticides. 
The data herein presented indicate that the antibodies developed by the 
described method are suitable for detection of pyrethroids in ground beef 
samples which have been spiked with 500 and 100 ppb of permethrin (FIG. 
5). Good correlation was found between the estimated and measured levels 
of permethrin in all spiked samples which contained more than 50 ppb. 
(FIG. 6) The antibodies did not react with other constituents of meat. The 
expected level of permethrin contamination correlates well with that 
observed in spiked samples, when a 62% recovery was assumed. 
The sensitivity of the assay of pyrethroids in food samples could be 
improved by combining the immunoassay with a preliminary HPLC separation 
of components of the sample. Certain retention times on HPLC could be 
determined and then antibodies could be used as highly selective detectors 
for compounds migrating at defined distances in the HPLC. The 
immunochemical cross-reactivity would reflect the structural similarity of 
synthetic pyrethroids. The combination of HPLC retention time and 
immunochemical detection would serve as a basis for quantifying synthetic 
pyrethroids. The combination of the two isolation techniques would also 
provide for the identification of new compounds or pyrethroid metabolites. 
8. Methods for Purification of Pyrethroids 
The antibodies herein developed may be covalently bound to a column and 
used to extract pyrethroids from samples of foods or environmental 
materials which are eluted through the column. Subsequent elution and 
repeated extraction may be used to concentrate pyrethroids and metabolites 
for further evaluation. 
9. Kit for Field Detection of Pyrethroids 
The monoclonal antibodies described herein when placed in a kit format 
could be used for a rapid, field portable assay for the detection of 
pyrethroid insecticide residues. A kit assay for field inspectors would 
use pyrethroid-specific antibodies including those described herein and 
not require use of sophisticated optical systems for detection of antibody 
binding reaction endpoints. 
The kit would contain pyrethroid-specific monoclonal antibodies which are 
immobilized on the surface of a reaction vessel or plate. The 
immobilization surface could be on a pre-coated disposable tube or on a 
porous surface card, where the antibodies contact or overlay an absorbant 
material. Pyrethroid-specific monoclonal antibodies may be immobilized 
with a protein A bridge or similar protein binding complex. This binding 
agent would be of selected sensitivity which does not interfere with the 
pyrethroid binding of the specific monoclonal antibody. 
The indicator enzyme-conjugated hapten, used as the reporter molecule, is 
selected for optimal binding to and release from the surface bound 
pyrethroid-specific antibody. Indicator enzyme bound to one of several 
antigen analogs may function to bind to and release from the surface-bound 
antibody, with the indicator enzyme 3-pba antigen complex, especially 
preferred. The enzyme-antigen conjugate may be preloaded on the antibody 
prior to the addition of test sample, or known standard samples, or it may 
be applied concurrently with the test sample. The indicator enzyme-antigen 
conjugate and the test sample and/or standard solutions are then allowed 
to compete for antibody-binding sites. In a preferred method, indicator 
enzyme antigen conjugate is pre-loaded. When test samples are added which 
contain pyrethroids, they compete with the indicator enzyme-conjugated 
hapten for a binding position on the immobilized antibody. Substrate is 
added to react with the indicator enzyme which remains bound to the 
immobilized antibody. Absorbance measurements are used to quantify the 
amount of indicator enzyme conjugate displaced by the test sample. 
The kit would also provide a prepackaged sample-collecting absorbant pad, 
which contains organic solvent for solublization of the test sample from 
exposed environmental surfaces. 
The above-described examples confirmed that monoclonal antibodies produced 
by the hybridomas designated as Py-1 , Py-3, and Py-4 are highly specific 
to synthetic pyrethroids with the phenoxybenzyl or cyclopropane 
functionalities, and more particularly to those with the phenoxybenzyl and 
cyclopropane functionalities, and are able to distinguish compounds with 
similar binding activities. Binding values suggest presence of a common 
epitope recognized by these antibodies. The low I.sub.50 values indicate 
similar degrees of recognition of permethrin, phenothrin and the hapten 
(compound 6 of FIG. 2) by the monoclonal antibody Py-1 . The substitution 
of a cyano group for a hydrogen on the alpha carbon of the benzyl moiety 
correlates with a reduced antibody binding which is one-tenth of that for 
deltamethrin and cypermethrin. Selective binding of pyrethroids to this 
group of monoclonal antibodies indicate that these antibodies can be used 
to identify, simply and conveniently, low concentrations of synthetic 
pyrethroids present in food or environmental samples. 
The subject invention thus provides monoclonal antibodies that are able to 
distinguish the presence of synthetic pyrethroids. The instant invention 
also provides cell lines which continuously secrete these monoclonal 
antibodies and methods for their production. The monoclonal antibodies of 
the subject invention bind specifically to synthetic pyrethroids and are 
able to distinguish compounds containing the phenoxybenzyl group and which 
may have the cyclopropane group. These monoclonal antibodies may be used 
in diagnostic applications including those where recognition of synthetic 
pyrethroids in the presence of other materials in foods or environmental 
samples is desirable. The described monoclonal antibodies are contemplated 
to be useful for monitoring foodstuff and environmental samples for 
contamination by pyrethroid insecticides. 
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. The foregoing description of some 
preferred embodiments of the invention, therefore, have been presented 
only for purposes of description and illustration of the subject 
invention. It is not intended to be exhaustive or to limit the invention 
to the precise forms disclosed, and many modifications and variations 
thereof would become obvious to those skilled in the art from the 
teachings and disclosure herein. It is intended that the scope of the 
invention is best defined by the appended claims.