定量检测人超敏C-反应蛋白双抗体夹心ELISA方法的建立及初步临床应用

目的：建立定量检测人超敏C-反应蛋白（CRP）双抗体夹心EUSA方法。方法：采用Protein A亲和层析法纯化本室制备的抗CRP单克隆抗体（mAbs），并行SDS—PAGE和Western blotting对纯化抗体特性进行鉴定；利用简易过碘酸钠法对抗CRPmAbs进行标记HRP后行抗体配对实验；通过方阵滴定法确定包被抗体和酶标抗体的最适工作浓度；以纯化的CRP抗原为标准品建立标准曲线；以重复性、灵敏性和回收率实验评价ELISA方法。初步对人血浆中的超敏CRP水平进行检测。结果：最佳配对组合为抗CRP mAb 1C10和HRP标记的抗CRP mAb2 C11，最适工作浓度分别为10μg／mL和l：2000，该方法的批内、批间变异系数分别为3．1％～9．7％和3．6％～13．6％，灵敏度达8．3ng／mL，回收率为90％～109％。用ELISA法测定左右冠无明显狭窄者68例，狭窄小于50％者59例和狭窄大于50％患者67例的血浆hs．CRP水平，冠脉狭窄小于50％组（3．7±1．2）mg／L与冠脉无狭窄组（1．8±0．7）mg／L比较明显升高（P〈0．05）；狭窄大于50％组（8．9±3．3）mg／L与狭窄小于50％组比较明显升高（P〈0．05）。结论：建立了一种可用于检测人超敏CRP的双抗体夹心ELISA方法。

Establishment of a sandwich ELISA for quantitative measurement of human supersensitivity C-reactive protein and primary clinical applica-tion study

AIM: To establish a sandwich ELISA for quantitative measurement of supersensitivity C-reac-tive protein(hs-CRP). METHODS: Anti-CRP monoclonal antibodies (mAbs) prepared by our laboratory were purified by Protein A affinity chromatography and analyzed by SDS-PAGE and Western-blotting to test their characteristics. All the mAbs were labeled with horseradish peroxidase by sodium oxidation method and antibody mating test were performed using anti-CRP mAb as coating antibody and HRP labeled anti-CRP mAb as labeled antibody, in which optimal concentrations were defined by square mateix titration. Standard curve was performed using purified CRP and the sensitivity, reproducibility and recovery rate test of ELISA was evaluated. The CRP levels in plasma were measured in this assay. RESULTS: The optimal paired antibodies were anti-CRP mAb 1C10 and HRP labeled anti-CRP mAb 2C11, and the optimal concentrations were 10 μg/mL and 1: 2000,respectively. The coefficient of variation were 3.1% to 9.7 % within assay and 3.6% to 13.6% between assay. The sensitivity in this assay was 8.3 ng/mL. The recovery rate was 90%to 109%. According to the result,68 normal persons' hs-CRP level in plasma of coronary arteriography were detected by ELISA, 59 with stenosis < 50% and 67 with stenosis ≥50%. The results showed that the plasma hs-CRP level in patients with stenosis < 50% (3.65 ± 1.15) mg/L was significantly higher ( P < 0.05)than hs-CRP in normol persons(1.75 ± 0.74) mg/L, the plasma hs-CRP level in stenosis ≥ 50% patients ( 8.93 ± 3.29) mg/L was significantly higher (P < 0.05) than stenosis < 50% patients. CONCLUSION: A sandwich ELISA for detecting hs-CRP was established.