According to references, some generic immunizing haptens of each type of pyrethroids have been described [7,31,32,33]. insecticides can be prepared for multi-residue assays. Keywords: hapten, class-specific antibody, sensitivity, immunoassay, pyrethoid insecticides Abbreviations THFtetrahydrofuranDMFdimethyl formamideNHSN-hydroxysuccinimideDCCN,N-dicyclohexylcarbodiimideBSAbovine serum albuminOVAovalbuminCRcross-reactivityELISAenzyme-linked immunosorbent assayTMBTetramethylbenzidineGAM-HRPperoxidase-labeled goat anti-mouse immunoglobulinsI50concentration giving 50% inhibition of maximum responseLODlimit of detectionPBS150 mM phosphate buffer, pH 7.4PBSTPBS containing 0.05% (v/v) Tween 20 Introduction Pyrethroid insecticides have been being used widely from agricultural uses [1,2,3,4] to home pest control [5,6] and are effective against a broad range of pests. The synthetic pyrethroids and natural pyrethrins can be divided into two groups of compounds on the basis of their chemical structure and mechanism of action at insect target sites (Physique 1): The type I compounds are simple cyclic alcohol esters of 2,2-dimethyl-3-(2-methyl-1-propenyl)-cyclopropanecarboxylic acid, and the type II compounds are esters of an aryl cyanohydrin [7]. Synthetic OF-1 pyrethroid residues have been seen in agricultural products [8,9] and food [10]. Lower and lower and lower amounts are being allowed worldwide by regulatory agencies [11]. Many pyrethroids act Rabbit polyclonal to TGFB2 as a neurotoxin, they are also highly toxic to aquatic life, particularly fish [12,13], so it is very important to develop a rapid, sensitive, specific methods to monitor pyrethroid residues in food products. Open in a separate window Physique 1 Structures of some synthetic pyrethroids. Current analytical methods for pyrethroid insecticides involve multistep sample cleanup procedures followed OF-1 by gas chromatography on devices equipped with an electron capture detector (GC-EC) [14,15], gas chromatography-mass spectrometry (GC-MS) [16,17], liquid chromatography combined with postcolumn fluorimetry derivatization and fluorescence detection (HPLC-FD) [18,19], or high performance liquid chromatography-mass spectrometry (HPLC-MS) [20]. These methods meet the requirements for sensitivity and accuracy of pyrethroids measurements. On the other hand, they are relatively intensive, time-consuming, expensive, and not particularly suitable for large numbers of samples. Immunoassays have been considered as a valuable supplement to existing, and rapidly developing, chromatographic techniques, because they have attractive features including high sensitivity and selectivity, rapid detection, and the possibility of analysis of difficult matrices without extensive pre-treatment [21]. When an immunoassay for a single analyte was developed in the past years, people all expected CRs as lower as you possibly can [22,23,24,25,26,27]. With the increase in requests for detection of chemical residues, there has been a gradual focus on total and multi-residues immunoassays. So, with higher CRs, some immunoassays for a type of compounds are also expected and some class specific antibodies against organophosphorus [28,29,30], type I or type II pyrethoid insecticides have been reported [7,31,32]. Here we aimed to develop a combination of coating conjugates and antibodies with equal high sensitivity to both type I and type II pyrethoids. Open in a separate window Scheme 1 Synthetic schemes for the preparation of generic haptens 1, 2, 4, OF-1 and 5 and the structure of hapten XQ. Results and Discussion Hapten design and conjugate verification Obtaining haptens with proper chemical structures is the key to development of novel antibodies and immunoassays. According to the characteristics of the two types of synthetic pyrethroid pesticides [32], here we designed three types of haptens (Scheme 1): the first (hapten 1) is for type I pyrethroids without a cyano group, the second (hapten 2 and XQ) for type II pyrethroids with a cyano group, and the third (hapten 4 and 5) for both types of pyrethroids with loss of the ester group. Most of these haptens were synthesized simply with just one or two actions and without any rigorous condition. Through scanning the UV-Vis spectrum, hapten/protein ratios were calculated by measuring the absorbance of the hapten, the protein, and the hapten-protein conjugate at the same wavelength, and the ratios of BSA-hapten 1, BSA-hapten 2, OVA-hapten 1, 2, 4, 5, XQ were 14, 13, 9, 6, 8 and 4. Here, hapten 1 and hapten 2 were used as immunizing haptens. To attempt to produce specific antibodies against both types of pyrethroid insecticides, a mixture of conjugates of hapten 1 and hapten 2 was also regarded.
