采用酶联免疫分析法对血清淀粉样蛋白P成分进行定量分析。

Quantitation of serum amyloid P component by an enzyme-linked immunoassay.

作者信息

Serban D, Rordorf-Adam C

出版信息

J Immunol Methods. 1986 Jun 24;90(2):159-64. doi: 10.1016/0022-1759(86)90071-2.

Abstract

A solid phase enzyme-linked immunoassay (ELISA) for the murine acute-phase reactant, serum amyloid P component (SAP), was developed. The assay is based on our finding of a calcium-dependent binding of SAP to trinitrophenyl-conjugated proteins. The wells of polystyrene microtiter plates are coated with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH), then incubated with SAP-containing samples. The amount of SAP is determined by indirect ELISA, wells being sequentially incubated with rabbit anti-SAP antiserum and horseradish peroxidase-linked donkey anti-rabbit IgG conjugate. The limit of sensitivity of the assay is 0.6 ng/ml SAP. Comparison with data on sera obtained by rocket immunoelectrophoresis assay yielded a correlation coefficient of 0.89. The binding of SAP to TNP-KLH was inhibited by calcium chelators and low concentrations of non-ionic detergents. Chromatography of serum on TNP-Sepharose provided a efficient and simple way of purifying SAP. The assay was also adapted for the quantitation of human SAP. The use of the assay in studying the binding specificities of SAP and its physiological role is discussed.

摘要

我们开发了一种用于检测小鼠急性期反应物血清淀粉样蛋白P成分（SAP）的固相酶联免疫吸附测定（ELISA）方法。该测定方法基于我们发现的SAP与三硝基苯基偶联蛋白的钙依赖性结合。将聚苯乙烯微量滴定板的孔用三硝基苯基化的匙孔血蓝蛋白（TNP-KLH）包被，然后与含SAP的样品孵育。通过间接ELISA测定SAP的量，孔依次与兔抗SAP抗血清和辣根过氧化物酶标记的驴抗兔IgG结合物孵育。该测定方法的灵敏度极限为0.6 ng/ml SAP。与火箭免疫电泳测定获得的血清数据进行比较，相关系数为0.89。钙螯合剂和低浓度的非离子去污剂可抑制SAP与TNP-KLH的结合。血清在TNP-琼脂糖上的色谱分析提供了一种高效、简单的纯化SAP的方法。该测定方法也适用于人SAP的定量分析。文中讨论了该测定方法在研究SAP的结合特异性及其生理作用方面的应用。