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目的比较两种不同活化方法制备的酪胺化Hib荚膜多糖(PRP-Ty)的特性,分别作为包被抗原建立测定人血清中Hib荚膜多糖特异抗体含量的ELISA方法。比较并确定包被抗原,对建立的ELISA方法进行初步验证。方法分别用CNBr和CDAP作为活化剂制备PRP-Ty,经Sepharose CL-4B层析分析相对分子质量分布范围,并确定适宜PRP-Ty抗原包被浓度的间接ELISA方法,以人Hib荚膜多糖IgG抗体定量标准品作为阳性标准,通过四参数非线性拟合计算人血清Hib荚膜多糖IgG抗体含量。结果 CNBr活化法制备的酪胺化Hib荚膜多糖(PRP-TyCNBr)较天然Hib荚膜多糖相对分子质量向小相对分子质量方向偏移,而CDAP活化法制备的酪胺化Hib荚膜多糖(PRP-TyC DAP)较天然Hib荚膜多糖相对分子质量分布无显著变化;两种PRP-Ty在0.65～2.00μg/mL的包被浓度范围内均有良好的包被活性,方法的灵敏度均达到0.02μg/mL IgG抗体检测水平。结论 PRP-TyC DAP作为包被物的ELISA测定方法可以更加真实可靠地反映人血清IgG抗体水平。
Objective To compare the characteristics of tyramized Hib capsular polysaccharide (PRP-Ty) prepared by two different activation methods and establish the ELISA method for determining the content of Hib capsular polysaccharide specific antibody in human serum as coating antigen respectively. The antigens were compared and identified, and the established ELISA method was preliminarily verified. Methods PRP-Ty was prepared by using CNBr and CDAP as activators respectively. The relative molecular weight distribution was analyzed by Sepharose CL-4B chromatography and the indirect ELISA method was established to determine the optimal PRP-Ty antigen coating concentration. Human Hib capsular polysaccharide IgG Antibodies quantitative standards as a positive standard, by four-parameter non-linear fitting human serum Hib capsular polysaccharide IgG antibody content. Results The tyramine-Hib capsular polysaccharide (PRP-TyCNBr) prepared by the CNBr activation method showed a shift from the relative molecular mass of native Hib capsular polysaccharide toward the small relative molecular mass, whereas the tyramized Hib capsular polysaccharide prepared by CDAP activation PRP-TyC DAP) had no significant change compared with the native Hib capsular polysaccharide. Both PRP-Ty had good coating activity in the range of 0.65 ~ 2.00μg / mL, the sensitivity of the method reached 0.02 μg / mL IgG antibody detection level. Conclusion ELISA assay of PRP-TyC DAP as a coating can reflect human serum IgG antibody more accurately and reliably.