We have isolated three clones of hybrid cells which synthesize antibodies specific for determinants on simian virus 40 (SV40) T antigens. Mouse myeloma NS1 cells were fused with spleen cells from mice that had been immunized with SV40-transformed mouse cells. Hybrid cells were selected in HAT medium and cloned in soft agar. We used an enzyme-linked immunosorbent assay for detection and quantification of mouse antibodies against SV40 T antigens. Monoclonal antibodies from 3 of the 24 clones that scored as positive in the enzyme-linked immunosorbent assay were verified by immunoprecipitation to be specific for SV40 T antigens. Two clones (7 and 412) produced antibodies that recognized denaturation-sensitive antigenic determinants unique to large T antigen. Antibodies from clone 7 appeared to have a low affinity for large T antigen. Antibodies from clone 412 had a higher affinity for large T antigen but did not recognize a subclass of large T antigen that was recognized by tumor serum. Antibodies of the third clone, clone 122, recognized a denaturation-stable antigenic determinant of the 53,000-dalton mouse nonviral T antigen in SV40-transformed cells. Antibodies from clone 122 also recognized similar (51,000to 56,000-dalton) nonviral T antigens in SV40-transformed or lytically infected cells from five mammalian species and in four uninfected mouse lines. From these observations, we have concluded that (i) the 94,000-dalton SV40 large T antigen may exist as immunologically distinguishable subclasses, and (ii) the nonviral T antigens of five mammalian species share at least one antigenic determinant. Cells infected or transformed by simian virus 40 (SV40) synthesize new proteins, the SV40 tumor or T antigens, which are not virion components. The T antigens have been immunoprecipitated from cell extracts by using tumor serum, that is, antisera from hamsters or mice bearing tumors induced by SV40-transformed cells. The T antigens that have been thus characterized are large T antigen, small t antigen, and the host-coded nonviral T antigens. Large T antigen is a phosphoprotein of 85,000 daltons (85K) which migrates in denaturing gels at an apparent mass of 94K. Large T antigen is specified by the early region of the SV40 genome and is located predominantly in the nuclei of infected and transformed cells (36, 38, 45, 46). Some large

Two families of carboxylic acid derivatives of the herbicides atrazine and simazine were synthesized for use as haptens in the development of immunoassays. One family was made by using monosubstituted (alky1amino)cyanuric chlorides and a variety of w-amino acids, giving spacers of varying lengths attached to one secondary amino group. The second family resulted from the replacement of the 2-chloro group of atrazine or simazine with 3-mercaptopropanoic acid. These haptens were conjugated to carrier proteins via N-hydroxysuccinimide active esters to make immunogens and ELISA antigens. Mono(alky1amino)cyanuric chlorides were also conjugated directly to bovine serum albumin and thyroglobulin under physiological conditions. ELISA and competitive inhibition ELISA results demonstrated that antibodies from immunized rabbits bound all of the conjugated and free s-triazines tested, including the parent compounds atrazine and simazine. The degree of binding was dependent on the N-alkyl groups present and on the length and position of attachment of the spacer arm. These results illustrate the usefulness of this synthetic approach in easily producing immunogenic haptens for a class of compounds that are difficult to work with because of their low solubility.The s-triazine herbicides are among the most commonly detected pesticides in water (Muir et al., 1978; Glotfelty e t al., 1984;Ervin and Kittleson, 1988;LeMasters and Doyle, 1989). This observation is due to a combination of factors, including widespread and heavy use, chemical and biological stability, and mobility in water. From 1983 through 1987 over 3 000 000 pounds of simazine and atrazine (Figure 1; 3a and 3b) were used in California alone (California Department of Food and Agriculture, [1983][1984][1985][1986][1987]. For these reasons, the State of California considers them as prime indicator compounds of groundwater contamination. Of additional concern is the poorly understood potential of the s-triazines and their nitroso and other derivatives for teratogenic, mutagenic, and carcinogenic effects (Janzowski et al., 1980; Waters et al., 1981). Thus, a rapid and inexpensive assay is needed to allow rapid screening of numerous surface and subsurface water samples, as well as for a variety of other analytical applications. The value of immunoassays for the analysis of pesticide residues has been well established (Hammock and Mumma, 1980; Newsome, 1986; Harrison e t al., 1988; Van Emon et al., 1989; Jung e t al., 1989). Recent literature includes descriptions of immunoassays for three specific s-triazine herbicides, atrazine (Huber, 1985; Bushway et al., 1988;Dunbar et al., 1985;Schlaeppi et al., 1989), cyanazine (Robotti et al., 1986), and terbutryne (Huber and Hock, 1985).Since the commercially important s-triazine herbicides constitute a large class of structurally related compounds that should be amenable to immunoassay as a group, we chose a synthetic approach that allows for the development of a wider range of antibody specificities and ultimate assay designs tha...

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Immunochemical assays for small molecules such as pesticides are rapidly gaining acceptance among analytical chemists. These techniques are rapid, sensitive, cost effective and can easily cope with large sample loads. This review lists the advantages and disadvantages of the technique and describes the steps in assay development using examples from this laboratory, particularly the thiocarbamate herbicide molinate and the triazine herbicides. The focus is primarily on hapten synthesis strategies, assay format considerations, sample preparation and assay validation.

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A direct ELISA for the thiocarbamate herbicide molinate was used to study distribution and dissipation of the compound in a treated rice field. No sample preparation other than buffering and dilution was required for the analysis of field water samples. Analyses were performed in 96-well microplates and required less than 0-5 man-hour per sample (three dilutions per sample, four replicate wells per dilution). Spiked samples and selected field samples were split for analysis by ELISA and gas chromatography. Two control samples of 92 and 93 ppb (after dilution) had between run coefficients of variation of 13.8 and 13.9% for 37 ELISA runs. A nested ANOVA analysis revealed that the largest source of error for the ELISA was due to within replicate variability, partly attributable to interwell variability of the 96-well plates. Practical aspects of reducing assay error and handling ELISA data are discussed. Quality control data showed that reliability of the direct ELISA is comparable to the gas chromatography method for molinate. ELISA data from field samples showed concentration differences among sites in the same check which coincided with differences in water flow. The half-life of molinate in the field, as determined by ELISA, was comparable to the value determined by chromatography.

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