基于单克隆抗体的双酚A半抗原直接包被的酶联免疫吸附法的建立

Establishment of Hapten Coated Enzyme-Linked Immunosorbent Assay for Bisphenol A Based on Monoclonal Antibody

传统的人工抗原包被酶联免疫吸附法(ELISA)依赖疏水相互作用将人工抗原与酶标板结合,会影响半抗原的呈现与识别,且多采用多克隆抗体为检测抗体,这些均会导致检测灵敏度下降和标准化难度增大。该研究选择双酚酸(BVA)作为双酚A(BPA)的半抗原与蛋白质偶联制备人工抗原免疫BALB/c小鼠,获得一株可分泌BPA单克隆抗体(MAb)的杂交瘤细胞株。利用3-氨丙基三乙氧基硅烷硅化处理酶标板,将BVA直接包被在酶标板上,建立了一种基于MAb的半抗原直接包被的BPA间接竞争ELISA法。该检测方法最低检测限IC10为0.29 ng/mL,半数抑制率IC50为5.4 ng/mL,水样中加标回收率为94.04%~102.31%。与人工抗原包被BPA ELISA检测方法(IC10:0.5 ng/mL,IC50:16.65 ng/mL,水样中加标回收率为89.72%~105.25%)相比,检测灵敏度显著性提高。

Traditional artificial antigen coated ELISA relies on hydrophobic interaction to bind the artificial antigen to the enzyme plate,and uses polyclonal antibodies as the detect antibodies,these can affect the presentation and recognition of hapten,resulting in decreased detection sensitivity and increased difficulty in standardization.As the hapten of bisphenol A(BPA),4,4-bis(4-hydroxyphenyl) valeric acid(BVA) was conjugated with protein to prepare artificial antigen,BALB/c mice were immunized.A hybrid tumour cell line which can secrete monoclonal antibody(MAb) of BPA was prepared.Using 3-aminopropyl triethoxyl silane to treat the enzyme label plate,BVA was directly coated on the enzyme label plate,and a BPA indirect competitive ELISA(icELISA) based on MAb was established.The minimum detection limit IC10of this method was 0.29 ng/mL,the half inhibition rate IC50 was 5.4 ng/mL,and the standard recovery rate was 94.04%~102.31%.Compared with the artificial antigen coated icELISA for BPA(IC10 was 0.5 ng/mL,IC50 was 16.65 ng/mL,standard recovery rate were 89.72%~105.25%),the sensitivity of hapten coated icELISA was improved significantly.