双抗体夹心ELISA定量检测血清Cap43蛋白方法的建立与评价

Development and initial application of double antibodies sandwich ELISA method for quantitatively detecting Cap43 protein in serum

目的建立定量检测血清Cap43蛋白表达双抗体夹心ELISA法,并初步探讨血清Cap43蛋白表达水平在肺癌患者与正常人群间有无差异,以及该蛋白表达水平的高低与肺癌组织学分型间的关系。方法建立双抗体夹心ELISA方法对肺癌患者与正常人群血清中Cap43蛋白的含量进行定量检测。结果双抗夹心ELISA包被Ⅰ抗羊抗人NDRG1多克隆抗体(PcAb)、Ⅰ抗兔抗人Cap43PcAb以及辣根酶(HRP)标记的羊抗兔Ⅱ抗的最佳工作浓度分别为1∶750,1∶1200,1∶350,标准蛋白曲线的回归方程为:y=0.53566+0.00046x,R2=0.9939,P<0.0001。应用该方法初步检测结果显示:肺癌组血清标本Cap43蛋白含量明显高于正常对照组(P<0.01),且肺腺癌组病人血清Cap43含量高于肺鳞癌组(P<0.01)。该方法的敏感度(SE)为84.51%,特异度(SP)为35.00%。结论建立了一种敏感度高,重复性好的定量检测血清中Cap43蛋白的双抗体夹心ELISA方法;Cap43蛋白在肺癌患者血清中呈现高表达,且在肺腺癌组病人血清中表达水平高于肺鳞癌组。

[Objective] To develop a double antibody sandwich ELISA for quantitation of Cap43 protein in serum and initially study correlation between contents of Cap43 protein expression in serum from lung cancer patients and healthy adults and tumor histological pattern in lung cancer. [Methods] Develop a double antibody sandwich ELISA for quantitatively detecting Cap43 protein in serum from lung cancer patients and healthy adults. [Results] The optimal concentration of the first coating antibody： goat anti-human Cap43 PcAb, the first antibody： rabbit antihuman Cap43 and HRP-second antibody： goat anti-rabbit Cap43 PcAb were 1：750, 1：1200, 1：350. The standard protein curve regression equation was： y=0.53566＋0.00046x; its linear detection was 10 to 3000 ng/mL （R^2=0.9939, P 〈0.0001）. The intial quantitatibve detection showed the level of Cap43 protein expression in serum in lung cancer group was higher than healthy adults group （P 〈0.05）. The contents of Cap43 protein expression in serum in lung adenocarcinoma group were more than lung squamous carcinoma （P 〈0.05）. This detection＇s sensivity and speciticity are 84.51%, 35.00% respectively. [Conclusion] A sensitive, repeatable double antibody sandwich ELISA is estab： lished for quantitation of Cap43 protein in serum. The intial detecting results reveal Cap43 protein is high expressed in serum from lung cancer patients and the level of the protein expression in serum in lung adenocaroinoma is higher than lung squamous carcinoma.