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

Abstract

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.

Effect of hapten structures on specific and sensitive enzyme-linked immunosorbent assays for N-methylcarbamate insecticide metolcarb

“…With the increasing of detection request for chemical residue, immunoassay of total and multi-residue was focused on gradually. So, with higher CRs, some immunoassay for a type of compounds is also expected and some class specific antibodies organophosphorus [20][21][22] or pyrethoid insecticides have been reported [23][24][25]. Therefore, it is of great use to study the effect of coating hapten structure on immunoassay specificity.…”

Class-specific immunoaffinity monolith for efficient on-line clean-up of pyrethroids followed by high-performance liquid chromatography analysis

“…Immunoassays have been developed for monitoring of these compounds with the merits of rapidity, sensitivity and relatively low cost [8][9][10][11][12][13]. They are especially useful to check if the ଝ This paper is part of the special issue "Immunoaffinity Techniques in Analysis", T.M.…”

Application of computer-assisted molecular modeling for immunoassay of low molecular weight food contaminants: A review

“…Small molecules with masses less than about 1000-10,000 Da are usually not immunogenic [6]; therefore, they are required to be conjugated to a carrier protein (capable of generating an immune response) by using a linker or spacer arm. Research on hapten design and application to heterologous and homologous assays in synthetic pyrethroids was conducted by Lee et al [7], and a review of approaches used to synthesize haptens used in immunoassay of organophosphate and synthetic pyrethroid insecticides was conducted by Skerritt and Lee [8]. Basic issues concerning structural aspects of hapten-antibody specificity in steroids were reviewed by Fránek [9].…”