Lee N, McAdam DP, Skerritt JH
CSIRO Plant Industry, North Ryde, NSW 2113, and Canberra, ACT 2601, Australia.
J Agric Food Chem. 1998 Feb 16;46(2):520-534. doi: 10.1021/jf970438r.
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.
已开发出对拟除虫菊酯类杀虫剂具有不同选择性的免疫测定法，用于检测II型拟除虫菊酯，包括溴氰菊酯、氯氰菊酯和高效氯氟氰菊酯。在半抗原合成中采用了两种方法来产生具有不同交叉反应的抗体：（1）使用三个间隔连接点，使分子的不同部分与连接点偏移；（2）在酶结合物中使用带有和不带有庞大基团的连接子，以降低免疫测定中抗体对间隔臂的亲和力。第一种方法导致制备了三组带有连接间隔基的半抗原：（1）连接在拟除虫菊酯的芳族部分；（2）穿过分子中部；（3）连接在环丙烷部分。还制备了基于拟除虫菊酯代谢物衍生物的半抗原。第二种方法涉及使用具有庞大（环己烷环）官能团的连接子来制备酶结合物。虽然大多数抗体和结合物组合可用于免疫测定以检测10 - 100 μg/L范围内的溴氰菊酯，但在大多数情况下，通过用稀碱处理拟除虫菊酯标准品以产生不同的异构体混合物，可将测定的检测限（针对特定目标拟除虫菊酯的总异构体）降低10 - 50倍。使用8种半抗原制备的15种抗血清分别用14种过氧化物酶结合物进行筛选，并根据测定灵敏度、动态行为以及对溴氰菊酯、氯氰菊酯和氯氟氰菊酯的特异性，选择了26种抗体/结合物组合进行进一步研究。这些免疫测定法提供的抗体结合抑制率（IC(50)）值在1.5至4.2 μg/L的异构化总溴氰菊酯之间，检测限为0.2 - 0.7 μg/L。使用氯氰菊酸 - KLH作为免疫原以及基于通过分子中部与过氧化物酶偶联的氯氰菊酯衍生物的结合物，获得了对总溴氰菊酯最灵敏的免疫测定法。这些方法提供的IC(50)为2 μg/L，异构化总溴氰菊酯的检测限为0.2 μg/L。然而，没有特定的半抗原设计能产生对溴氰菊酯具有明显更高灵敏度或特异性的抗血清。获得了与密切相关的拟除虫菊酯溴氰菊酯、氯氰菊酯和氯氟氰菊酯不同的交叉反应，并且对于几种抗体，交叉反应以及检测限可通过改变结合物组合来改变。灵敏检测溴氰菊酯的12种抗体/酶结合物组合中的每一种都具有非常高的立体特异性，仅检测αS, 1R顺式（DM1）和αR, 1R顺式（DM2）异构体；对后一种异构体的测定灵敏度更高。
