高迁移率族蛋白B1单克隆抗体制备与竞争ELISA测定法的建立

目的制备高迁移率族蛋白B1(HMGB1)单克隆抗体和建立HMGB1的直接竞争ELISA测定方法。方法采用PEG细胞融合技术、有限稀释法和间接ELISA法制备和筛选杂交瘤细胞。直接竞争ELISA法测定大鼠血清HMGB1水平。结果共获得7株HMGB1特异性杂交瘤细胞株。建立的直接竞争ELISA方法的测定范围为1~320 ng/ml,回收率为97%~106%,变异系数为2.6%~7.3%。用该方法测定大鼠血清HMGB1水平发现,糖尿病大鼠模型HMGB1水平高于正常大鼠(P<0.001)。结论直接竞争ELISA可用于HMGB1的测定,该方法较双抗体夹心ELISA法更简便、快速。

Prepartion and Application of High Mobility Group Box1 Monoclonal Antibody

Objective To prepare monoclonal antibody specific for HMGB1 and establish a direct competitive ELISA for assaying serum HMGB1.Methods Hybridoma technique,limited dilution,and indirect ELISA were used in the preparation of HMGB1 monoclonal antibody.The direct competitive ELISA was used in the detection of rat serum HMGB1.Results Seven clones of hybridoma secreting HMGB1 monoclonal antibody were obtained.A competitive ELISA based on HMGB1-HRP conjugate was established.And the competitive ELISA was found to detect HMGB1 ranging from 1 to 320 ng/ml and to have a recovery ranging form 97% to 106% and a CV from 2.6% to 7.3%.By using the competitive ELISA to detect the serum level of HMGB1 of rats,it was found that the HMGB1 level in the rats with diabetes mellitus was much higher than that in normal ones(P<0.001).Conclusion The competitive ELISA based on HMGB1-HRP conjugate is a practical method for detection of HMGB1,and it is easier and more rapid than sandwich ELISA based on antibodies.