一种定量检测人血清高敏C反应蛋白的化学发光免疫方法

Chemiluminescent immunoassay for high-sensitivity C-reactive protein

旨在建立一种可定量检测人血清高敏CRP的化学发光检测方法（High—sensitivity C—reactive protein quantifiable chemiluminescent immunoassay，hs—CRPCLIA）。首先利用亲和层析和离子交换层析技术从肝硬化病人腹水中纯化出高纯度的天然CRP作为免疫原制备了22株CRP单克隆抗体（单抗1，其中13株单抗在磷酸胆碱配体捕获ELISA中呈阳性，然后利用方正滴定法筛选出单抗10C5和10C11建立了hs-CRP CLIA。试剂盒评估结果显示：该方法对血清中干扰物质IgG、血红蛋白、甘油三酯等无非特异性反应；该方法检测灵敏度高，在0．04～20．38mg／L范围内定量检测人血清CRP标准品呈良好线性关系限0〉0．993）；该方法准确性高、可重复性好，平均回收率为99％，批内差为4．2％～5．8％，批间差为9．0％～11．5％；该方法与进口商品化高敏CRPELISA试剂盒平行比较检测90份血清标本，结果显示两者有良好的可比性（r=0．968）。综上，建立的hs—CRPCLIA是一种准确、可靠、可定量的高灵敏C反应蛋白检测方法，该方法的临床应用，有利于改善我国心脏病风险评估及肠炎性疾病预后判断。

We developed a high-sensitivity C-reactive protein quantifiable chemiluminescent immunoassay （hs-CRP CLIA）. The high-purity native CRP was purified from hepatic cirrhosis patient ascetic fluid by affinity and ion exchange chromatography and used as an immunogen to develop the monoclonal antibodies （mAbs） against CRP. Twenty-two mAbs were identified reactive with CRP in ELISA and 13 of them were reactive in the phosphorycholine ligand capture ELISA. The mAbs 10C5 and 10C11 were selected to develop the hs-CRP CLIA. The linearity and performance of the hs-CRP CLIA was characterized. It was showed not reactive when testing against other serum materials （IgG, hemoglobin and triglyceride）. The reliable correlation （R^2 〉 0.993） was obtained between testing value （RLU/S） and the concentration of human serum CRP calibrator. The linearity fell in the range of 0.04-20.38 mg/L. The assay has good accuracy and reproducibility, the mean recovery was 99% and the precision of the intra- and inter assay was CVs （4.2%-5.8%） and （9.0%-11.5%）, respectively. In testing of 90 human sera, this assay performed well and correlated comparably with a commercial hs-CRP ELISA kit. Thus, hs-CRP CLIA is an accurate, reliable, quantifiable assay for detection of high-sensitive C-reactive protein in serum, it may be useful to improve the risk assessment of cardiovascular disease and the prognosis of inflammatory bowel disease.