Development of Immunoassays for Type II Synthetic Pyrethroids. 1. Hapten Design and Application to Heterologous and Homologous Assays.

Immunoassays differing in selectivities for pyrethroid insecticides have been developed for the detection of type II pyrethroids, including deltamethrin, cypermethrin, and lambda-cyhalothrin. Two approaches were employed in hapten synthesis to raise antibodies with different cross-reactions: (1) use of three spacer attachment points to offset different parts of molecules from the points of attachment and (2) use of linkers with and without bulky groups in the enzyme conjugate to reduce antibody affinities for the spacer arm in the immunoassay. The first approach resulted in the preparation of three series of haptens with a spacer attached (1) at the aromatic moiety of pyrethroid, (2) through the middle of the molecule, and (3) at the cyclopropane moiety. Haptens based on the derivatives of the pyrethroid metabolites were also prepared. The second approach involved the use of a linker with a bulky (cyclohexane ring) functionality for preparation of an enzyme conjugate. While most combinations of antibody and conjugate could be used in immunoassays for detection of deltamethrin in the 10-100 µg/L range, in most cases the limits of detection of the assays (for total isomers of a particular target pyrethroid) were lowered 10-50 fold by treatment of the pyrethroid standards with dilute alkali to produce a different isomer mix. Fifteen antisera prepared using 8 haptens were each screened with 14 peroxidase conjugates, and 26 antibody/conjugate combinations were selected for further study on the basis of the assay sensitivity, dynamic behavior, and specificity for deltamethrin, cypermethrin, and cyhalothrin. These immunoassays provided 50% inhibition of antibody binding (IC(50)) values between 1.5 and 4.2 µg/L of isomerized total deltamethrin and limits of detection of 0.2-0.7 µg/L. The most sensitive immunoassay for total deltamethrin was obtained using cypermethric acid-KLH as the immunogen and a conjugate based on a derivative of cypermethrin coupled through the middle of the molecule to peroxidase. These provided an IC(50) of 2 µg/L and a limit of detection of 0.2 µg/L of isomerized total deltamethrin. However, no particular hapten design produced antisera of clearly superior sensitivity or specificity for deltamethrin. Differing cross-reactions with the closely related pyrethroids, deltamethrin, cypermethrin, and cyhalothrin, were obtained, and for several antibodies the cross-reaction as well as the limits of detection could be altered by varying the conjugate combinations. Each of the 12 antibody/enzyme conjugate combinations that sensitively detected deltamethrin were very stereospecific, detecting the alphaS, 1R cis, (DM1), and alphaR, 1R cis (DM2) isomers only; the assay sensitivity was greater for the latter isomer.