Production and Identification of Broad Spectrum Monoclonal Antibody against a Group of Pyrethroid Insecticides
10.11895/j.issn.0253-3820.140081
- VernacularTitle:抗拟除虫菊酯类农药广谱性单克隆抗体的研制及鉴定
- Author:
Mengtang WEN
;
Yuan LIU
;
Shuai YAN
;
Xiao ZHANG
;
Heng WANG
;
Xianjin LIU
- Publication Type:Journal Article
- Keywords:
Pyrethroid pesticides;
Broad specificity;
Monoclonal antibody;
Enzyme-linked immunosorbent assay
- From:
Chinese Journal of Analytical Chemistry
2014;(9):1245-1251
- CountryChina
- Language:Chinese
-
Abstract:
The objective of this study is to generate broad spectrum monoclonal antibody ( mAb ) against a group of pyrethroid insecticides and to identify its immunological characteristics. The generic hapten 3-phenoxy-benzoic acid ( PBA) was conjugated to carrier protein BSA by activated ester method. Balb/c mice were immunized with PBA-BSA. The titer of polyclonal antibody ( pAb ) was detected by indirect enzyme-linked immunosorbent assay ( ELISA) after five times immunization. The mouse with high titer and sensitivity was selected for cell fusing. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by indirect non-competitive ELISA based on the coating antigen PBA-ovoalbumin ( PBA-OVA ) . High-sensitivity and high-specificity mAb was prepared after subcloning using limiting dilution method. Purified mAb was obtained after purified by saturated ammonia sulfate precipitation and protein G affinity column. The immunological characteristics of mAb such as titer, antibody subtypes, affinity constant and the sensitivity to pyrethroid insecticides were characterized by indirect ELISA; The results of UV spectroscopy and SDS-PAGE showed that PBA-BSA artificial antigen was synthesized successfully. A hybridoma cell line (4H11) secreting anti-pyrethroid mAb was established. The titre of ascites was up to 1:6. 5×106, and the mAb was IgG1 subtype. The affinity constant of the mAb to PBA was about 2. 5×107 L/mol, with a IC50 value of 208. 83 μg/L and a detection limit of 21. 23 μg/L to PBA. Simultaneously, beta-cypermethrin, flucythrinate, cypermethrin and fenvalerate were sensitively recognized by the mAb with the IC50 of 1. 01, 2. 15, 3. 16 and 3. 67μg/L, respectively.
