层粘连蛋白酶联免疫分析方法的建立及初步应用

A quantitative enzyme-linked immunoabsorbent assay for human laminin

目的 建立酶联免疫吸附试验 ,对层粘连蛋白进行定量检测。方法 将鼠抗人层粘连蛋白单克隆抗体包被于聚苯乙烯微孔板 ,兔抗人层粘连蛋白抗体作第一抗体 ,酶标羊抗兔抗体作为第二抗体 ,在对有关试验条件进行优化后 ,检测 73份献血员标本。结果 本法的标准曲线为 2 5～ 16 0 0ng/ml,但在 5 0～ 80 0ng/ml范围内线性理想 ,其线性回归系数大于 0 .95。批内和批间变异系数分别为 8.6 %和 9.3% ,灵敏度为 2 5ng/ml,平均回收率为 98.1%。 17份样品用本方法与放射免疫法同时检测 ,其相关系数为 0 92 96。 73例献血员血清层粘连蛋白水平为 (115 .7± 17.3)ng/ml。结论 本方法可替代放射免疫法 。

Objective To develop a quantitative ELISA for detection of laminin.Methods A sandwich enzyme-linked immunosorbent assay for human laminin,which consists of a monoclonal antibody of mouse anti-human laminin as the fixing antibody coated on microtitre plates, another polyclonal antibody of rabbit anti-human laminin was used as connecting antibody, and HRP-conjugated goat anti-rabbit IgG as displaying antibody,was developed.Results The working range of standard curve was within 25～1600 ng/ml of laminin. The coefficients of variation were 8.6% for inter-assay and 9.3% for intra- assay,respectively.The minimum detectable limit was 25ng/ml,and the mean recovery rate of laminin was 98.1%. The correlation coefficient between the ELISA and RIA was 0.93. The concentration of serum laminin from healthy donors was( 115.7 ±17.3)ng/ml.Conclusion The sandwich ELISA can be used for quantitative determination of laminin in serum and tissue fluid.