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目的制备抗大肠杆菌精制内毒素的兔多克隆抗体并建立检测内毒素的间接竞争性酶联免疫吸附试验(ELISA)方法。方法大肠杆菌精制内毒素作为免疫原,采用逐渐加强免疫剂量的方法免疫家兔,制备抗体,将抗体用于ELISA检测精制内毒素;并与复合型内毒素进行反应。结果随着免疫次数增加,抗血清效价逐渐提高,最后一次免疫后血清效价达到1∶6 400;在包被抗原浓度100μg/mL和兔多抗稀释比1:500最佳使用条件下建立间接竞争性ELISA,内毒素浓度与吸光度(A)值成反比例关系,线性回归方程为:y=-0.1 ln(x)+0.765,检测最低内毒素浓度为50 ng/mL;兔血清与复合型内毒素反应效价为1∶16 000。结论成功制备出抗精制内毒素兔多克隆抗体,且能与复合型内毒素反应。
Objective To prepare rabbit polyclonal antibodies against E. coli and to establish an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of endotoxin. Methods Escherichia coli endotoxin was used as immunogen to immunize rabbits by gradually boosting the immunizing dose. Antibodies were prepared and the antibodies were detected by ELISA for endotoxin purification. The recombinant endotoxin was used to react with complex endotoxin. Results With the increase of immunization times, the titer of antisera gradually increased, and the serum titer reached 1: 400 after the last immunization; established under the optimal conditions of 100μg / mL antigen and 1: 500 dilution ratio of rabbit polyclonal antibody Indirect competitive ELISA, endotoxin concentration and absorbance (A) value is inversely proportional to the linear regression equation: y = -0.1 ln (x) +0.765, detection of the lowest endotoxin concentration of 50 ng / mL; rabbit serum and complex Endotoxin response titer 1:16 000. Conclusion The polyclonal antibody against rabbit anti-endotoxin was successfully prepared and could react with complex endotoxin.