内毒素多克隆抗体制备及其在ELISA中应用  

Preparation of polyclonal antibody against E.coli endotoxin and application of icELISA

作　　者：周卓晟[1,2] 罗中捷[1,3] 郝春慧[1] 郭寅生[1] 刘红艳[1] 黄正[1] 

机构地区：[1]华中科技大学同济医学院公共卫生学院教育部环境与健康重点实验室,湖北武汉430030 [2]深圳市龙岗区龙岗卫生监督分所 [3]广州市卫生监督所

出　　处：《中国公共卫生》2012年第11期1467-1469,共3页Chinese Journal of Public Health

基　　金：国家自然科学基金(30771774)

摘　　要：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 antibody against E.coli endotoxin and to establish indirect competitive enzyme-linked immunosorbent assay（icELISA） for the detection of endotoxin.Methods Purified E.coli endotoxin was used as the immunogen to inject rabbit with gradually increased doses.Then the antibody was used for ELISA detection of endotoxin and reacted with the cell-bound E.coli endotoxin.Results The endotoxin antiserum titer gradually increased with successive increased number of immunization times.The serum titer was 1∶ 6 400 after the last immunization.At the fully optionized antigen concentration of 100μg/mL and rabbit polyclonal antibody dilution of 1∶ 500,the icELISA results showed that concentration of endotoxin was in inverse proportion to the value of A,with a linear regression equation of y=-0.1ln（x） ＋ 0.765,correlation coefficient R2 of 0.982,and the detection limit of 50 ng/mL.The titer of reaction with cell-bound E.coli O157∶ H7 endotoxin was 1∶ 16 000.Conclusion The rabbit polyclonal antibody against endotoxin was prepared successfully and could react with cell-bound E.coli endotoxin.

关 键 词：内毒素 多克隆抗体 酶联免疫吸附试验(ELISA) 

分 类 号：R115[医药卫生—公共卫生与预防医学]