CAJ | 학술논문

制备了抗拟除虫菊酯类农药( Pyrethroids)的广谱性单克隆抗体,并鉴定其免疫学特性；以间苯氧基苯甲酸( PBA)为半抗原,用活性酯法将其与牛血清蛋白( BSA)偶联制得人工抗原PBA-BSA免疫Balb/c小鼠,5次免疫后选择效价最高、对PBA识别能力最强的小鼠取脾细胞与SP2/0骨髓瘤细胞在PEG1500作用下融合,经间接ELISA筛选及有限稀释法进行亚克隆,分离阳性细胞株,腹水诱导法大量制备单克隆抗体,并用Protein G亲和柱纯化,间接ELISA法测定抗体效价、亚型、亲和力常数及对拟除虫菊酯类农药的作用；UV结果显示,PBA-BSA成功偶联,获得1株稳定分泌抗拟除虫菊酯类农药单克隆抗体的杂交瘤细胞株4H11,其培养腹水抗体效价为1∶6.5×106,其抗体亚类为IgG1,对PBA的亲和力常数为2.5×10 L/mol,对PBA的IC50为208.9μg/L,检出限为21μg/L,对高效氯氰菊酯、氟氰戊菊酯、氰戊菊酯和氯氰菊酯的 IC50分别为1.01,2.15,3.16和3.67μg/L。
제비료항의제충국지류농약( Pyrethroids)적엄보성단극륭항체,병감정기면역학특성；이간분양기분갑산( PBA)위반항원,용활성지법장기여우혈청단백( BSA)우련제득인공항원PBA-BSA면역Balb/c소서,5차면역후선택효개최고、대PBA식별능력최강적소서취비세포여SP2/0골수류세포재PEG1500작용하융합,경간접ELISA사선급유한희석법진행아극륭,분리양성세포주,복수유도법대량제비단극륭항체,병용Protein G친화주순화,간접ELISA법측정항체효개、아형、친화력상수급대의제충국지류농약적작용；UV결과현시,PBA-BSA성공우련,획득1주은정분비항의제충국지류농약단극륭항체적잡교류세포주4H11,기배양복수항체효개위1∶6.5×106,기항체아류위IgG1,대PBA적친화력상수위2.5×10 L/mol,대PBA적IC50위208.9μg/L,검출한위21μg/L,대고효록청국지、불청무국지、청무국지화록청국지적 IC50분별위1.01,2.15,3.16화3.67μg/L。
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.