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 ha ve been developed for the detection of type II pyrethroids, including deltamethrin, cypermethrin, and lambda-cyhalothrin. Two approaches wer e employed in hapten synthesis to raise antibodies with different cros s-reactions: (1) use of three spacer attachment points to offset diffe rent parts of molecules from the points of attachment and (2) use of l inkers 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; Hapte ns based on the derivatives of the pyrethroid metabolites were also pr epared. The second approach involved the use of a linker with a bulky (cyclohexane ring) functionality for preparation of an enzyme conjugat e. While most combinations of antibody and conjugate could be used in immunoassays for detection of deltamethrin in the 10-100 mu g/L range, in most cases the limits of detection of the assays (for total isomer s of a particular target pyrethroid) were lowered 10-50 fold by treatm ent of the pyrethroid standards with dilute alkali to produce a differ ent isomer mix. Fifteen antisera prepared using 8 haptens were each sc reened with 14 peroxidase conjugates, and 26 antibody/conjugate combin ations were selected for further study on the basis of the assay sensi tivity, dynamic behavior, and specificity far deltamethrin, cypermethr in, and cyhalothrin. These immunoassays provided 50% inhibition of ant ibody binding (IC50) values between 1.5 and 4.2 mu g/L of isomerized t otal deltamethrin and limits of detection of 0.2-0.7 mu g/L. The most sensitive immunoassay for total deltamethrin was obtained using cyperm ethric acid-KLH as the immunogen and a conjugate based on a derivative of cypermethrin coupled through the middle of the molecule to peroxid ase. These provided an IC50 of 2 mu g/L and a limit of detection of 0. 2 mu g/L of isomerized total deltamethrin. However, no particular hapt en design produced antisera of clearly superior sensitivity or specifi city for deltamethrin. Differing cross-reactions with the closely rela ted pyrethroids, deltamethrin, cypermethrin, and cyhalothrin, were obt ained, and for several antibodies the cross-reaction as well as the li mits of detection could be altered by varying the conjugate combinatio ns. Each of the 12 antibody/enzyme conjugate combinations that sensiti vely detected deltamethrin were very stereospecific, detecting the alp ha S, 1R cis, (DM1), and alpha R, 1R cis (DM2) isomers only; the assay sensitivity was greater for the latter isomer.