In order to establish double antibodies sandwich ELISA(DAS-ELISA)for detection of chicken HMGB1 protein (chHMGB1),the soluble form recombiant chHMGB1 protein (rchHMGB1) was obtained with IPTG induction firstly.Then to obtain the mouse monoclonal antibody and rabbit polyclonal antibody against rchHMGB1 protein respectively,the BABL/c mice and New Zealand white rabbits were immunized with purified rchHMGB1 protein.And the rabbit polyclonal antibody was subjected to HRP labeling.A DAS-ELISA method for quantitative determination of rchHMGB1 fusion protein was established with the monoclonal antibodies as the coated antibody,and HRP labeled rabbit polyclonal antibody as detection antibody.The specificity,detection limit and stability of the method were evaluated.The optimal working concentration of coated antibody and detection antibody both were 1∶500.The antigen concentration in the range of 72.5 ng/mL-75 μg/mL showed a good linear relationship and the correlation coefficient R2＞0.99.The detection limit of the method was 36.25 ng/mL for rchHMGB1.The standard curves oltained by continuous detection the antiges for 6 days were stable.The DAS-ELISA method of rchHMGB1 had high sensitivity and repeatability,and would lay a foundation for further study of chiHMGB1 protein in the process of disease pathogenesis.%为建立鸡高迁移率族蛋白1 (chHMGB1)蛋白酶联免疫定量分析方法,本研究原核表达重组chHMGB1蛋白(rchHMGB1).以纯化的rchHMGB1作为免疫原分别免疫BABl/c小鼠和新西兰大白兔以分别制备针对rchHMGB1的鼠单克隆抗体(MAb)和兔多克隆抗体(PAb),并对兔PAb进行辣根过氧化物酶(HRP)标记.以鼠抗rchHMGB1 MAb做为包被抗体,HRP标记兔PAb为检测抗体,建立了定量测定rchHMGB1的双抗体夹心ELISA方法.对该方法进行了特异性、检测限和稳定性等试验.结果显示,包被抗体和检测抗体的最佳工作浓度均为1∶500稀释,抗原浓度在72.5 ng/mL～75 μg/mL范围内呈良好线性关系,相关系数R2＞0.99.该方法可以特异性检测rchHMGB1,最低检测限为36.25 ng/mL,连续6d检测抗原绘制的标准曲线稳定性良好.本实验建立的rchHMGB1双抗体夹心ELISA方法,灵敏度较高,重复性好,为进一步研究chHMGB1在疾病感染过程中发挥的病理学机制奠定基础.
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