A panel of antisera was prepared using analogues and derivatives of metabolites of the organochlorine insecticide, p,p′-DDT as haptens. The assays developed exhibited differing cross-reactions for different DDT analogues and metabolites, and the choice of hapten for the detecting enzyme conjugate had almost as much effect on assay specificity and sensitivity as the structure of the hapten used for antibody production. Those assays developed using hapten I, based on esters of bis(pchlorophenyl)acetic acid (DDA), typically detected DDA with greater sensitivity than p,p′-DDT or p,p′-DDE. The most sensitive assay for p,p′-DDT (lower limit of detection of 0.3 µg/L) was obtained using an immunogen based on bis(p-chlorophenyl)ethanol (hapten IV), although a significant crossreaction with dichlorodiphenyltrichloroethane (DDD) and DDE was obtained. The most specific assay for p,p′-DDT was obtained using an immunogen (hapten VI) that includes all elements of the DDT structure, except that one of the p-chloro groups was replaced by β-alanine carboxamide for coupling to carrier proteins. Antibodies based on a similar DDE hapten (V) exhibited specificity for p,p′-DDE over p,p′-DDT. Greater specificity and sensitivity for dicofol were obtained by using an immunogen derived from ester hydrolysis of chlorbenzilate (hapten II). The assays provided methods for detection of p,p′-DDT plus p,p′-DDE either by using the antibody raised to hapten IV with conjugate based on hapten Ib or by using the assay based on hapten V, with treatment of samples with warm alcoholic KOH, which converted DDT to DDE. Some of the immunoassays were applied to the detection of DDT and DDE in water, soil, and selected foods.

The leaves of the perennial pasture grass Phalaris aquatica L. (phalaris) contain two groups of known toxins, indole alkaloids, primarily dimethyltryptamines and N-methyltyramines, which cause illnesses in grazing animals, especially sheep. Using amino-reactive and phenolic hydroxyl-reactive homobifunctional reagents, simple methods were devised for coupling toxins representative of those in phalaris to carrier proteins and enzymes for ELISA development. ELISAs were produced for both groups of toxins. Dimethyltryptamines were most sensitively detected [lower limit of detection (LLD) of 1 microg/L for bufotenine] using rabbit anti-bufotenine antibodies, coupled to ovalbumin using divinyl sulfone, with detection using a peroxidase conjugate prepared using the same hapten coupled with 1, 4-butanediol diglycidyl ether. The assay cross-reacted with other toxins of the same class (N,N-dimethyltryptamine and N, N-dimethyl-5-methoxytryptamine) but not with the structurally related amino acids histidine and tryptophan. The most sensitive N-methyltyramine assay (LLD of 1 microg/mL for N-methyltyramine) utilized antisera to tyramine with N-methyltyramine coupled to peroxidase. Significant cross-reaction was seen with the low-grade toxin hordenine, but detection of tyramine was poorer, whereas the amino acid tyrosine was not detected. These assays could be applied to the analysis of simple extracts of Phalaris leaves with minimal interference. A good correspondence was observed between toxin levels by ELISA and estimates from a more tedious thin-layer chromatography method. The method has now been incorporated in a Phalaris breeding program.

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