抗体夹心酶联免疫吸附法测定重组溶葡萄球菌酶研究

Detection of Recombinant Lysostaphin Using Antibody Sandwish Enzyme-linked Immunoadsorbent Assay

摘要:

用重组溶葡萄球菌酶免疫家兔获得抗血清，经亲和层析纯化后用HRP标记，以双向免疫扩散法确定抗血清效价，以Western blot鉴定抗体的特异性，建立双抗夹心法标准曲线，鉴定其最小检出限、精确度、回收率。实验显示多克隆抗体能与溶葡萄球菌酶特异性结合，双抗夹心ELISA法检测抗原的最小检出限为0.98ng/mL，标准曲线在0.98～500ng/mL范围内线性良好。3份同批样本分别重复6次测定，平均批内变异系数为6.4%；3份不同批样本分别重复6次测定，平均批间变异系数为6.5%。血清中加入已知量的标准抗原，测得平均回收率为98.6%。此法检测重组溶葡萄球菌酶的可测范围广，灵敏度和精密度高，变异系数较小。结果证实建立的检测血清中重组溶葡萄球菌酶含量的双抗夹心酶联免疫吸附测定法(Enzyme-linked immunosorbent assay，ELISA) 灵敏、准确、可靠。

Abstract:

The double-antibody-sandwich enzyme-linked immunoadsorbent assay (ELISA) for detection of rLysostaphin in humans had been developed and established through this study. rLysostaphin of high purity (>95%) produced in Shanghai Hi-Tech United Bio-Technological Research & Development Co., Ltd (SHUBRD) was used to produce a rabbit anti-rLysostaphin polyclonal antibody. The standard curve of rLysostaphin polyclonal antibody that was constructed showed that the lowest range of detection was found at 0.98 ng of rLysostaphin/mL, and the curve exhibited linearity preferably from 0.98 to 500 ng of rLysostaphin/mL. When three serum samples of the same batch were assayed for 6 replicates, and more 3 samples from different batches for 6 replicates, the average intra-assay and inter-assay coefficient variances (CV) were 6.4% and 6.5%, respectively. The relative recovery rate was 98.6% when quantitative standard antigens were added to the serum. The present method for detection of rLysostaphin in serum is specific, highly sensitive, highly precise, and exhibited a low CV and will be helpful in the further study of rLysostaphin pharmacokinetics and holds promise in clinical applications.