人血清中Hib荚膜多糖抗体定量的ELISA方法建立及初步验证

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。

Establishment and preliminary verification of ELISA for quantitating PRP IgG antibodies in human sera

Objective To establish and carry out preliminary verification of ELISA for quantitating PRP IgG antibodies in human sera, the coating antigens are PRP-TycNBr and PRP-TycDAp. To compare the characteristics of PRP-TycNsr and PRP- Tyc oAP prepared with two different activation methods. Methods Preparation of PRP-Ty as coating antigens with two dif- ferent activators （ CNBr and CDAP） , comparison of distrubution of molecular weight for PRP-Ty and PRP on Sepharose CL- 4B chromatography, determination of indirect ELISA with the optimum coating concentration, calculation of PRP IgG anti- body concentration in human sera with the human PRP IgG reference as standard and four-parameter non-linear fit method. Results The molecular weight of PRP-TycNBr is obviously less than PRP and that of PRP-Tyc DAP is not remarkably changed from PRP. Two kinds of PRP-Ty have good coating activity in the range of 0.65-2.00μg/mL. The sensitivity of the method is in determination human lgG to 0.02 μg/mL. Conclusion The established ELISA method with PRP-Tyc DAP as coating antigen is able to closely reflect antibody against native PRP in human sera.