多肽抗体检测原发性肝癌血清标志物ELISA方法的建立及应用

血清多肽是癌症诊断信息的重要来源,建立、优化了检测多肽标志物的直接ELISA法,并应用于肝癌血清中的多肽标志物的检测。制备及纯化针对多肽标志物Pep5的单克隆抗体并进行辣根过氧化物酶标记,用其建立检测相应抗原的直接ELISA法。方法线性范围为1.5-20 ng/mL,检测限为1.24 ng/mL;标准品批内及批间CV分别小于3.66%及4.89%,血清样本批内及批间CV分别小于11.69%及18.18%;线性范围内(9、12和15 ng/mL)的回收率分别为98.98%,99.61%和101.58%。应用该方法共检测160例正常血清、104例肝硬化及156例肝癌患者血清,正常组与肝硬化组及肝癌组间差异显著(P<0.001),Pep5诊断肝癌的敏感性和特异性分别为80.8%和96.2%。同时检测94例HCC血清中的AFP和Pep5,AFP检出率为63.8%,Pep5检出率为90.4%,AFP联合Pep5检测时,能将HCC的检出率提高至94.7%。

Detection and Application of Serum Markers of Primary Hepatocellular Carcinoma by ELISA Using Synthetic Polypeptide Antibody

Serum peptides are the rich sources for cancer-specific diagnostic information.To develop an ELISA method for detection of the serum markers of primary hepatocellular carcinoma(HCC),the anti-peptide monoclonal antibody was prepared,purified and labeled with horseradish peroxidase(HRP)for detection of the corresponding antigens by direct ELISA.The linear range of the method was 1.5-20 ng/mL with the detection limit of 1.24 ng/mL.The intra-and inter-assay coefficients of variation(CV)of synthetic polypeptide were less than 3.66% and 4.89%.The intra-and inter-assay CV of sera were 11.69% and 18.18% respectively,or less.The recovery test(9,12,15 ng/mL)showed variation between 98.98% and 101.58%.Sera from 160 healthy blood donors,104 patients with cirrhosis and 156 HCC,were analyzed by this method.There was statistically significant difference in values between healthy subjects and the patients with cirrhosis and HCC patients(P< 0.001),the sensitivity and specificity of peptide in the diagnosis of HCC were 80.8%and 96.2%,respectively.Meanwhile,the positive rate of AFP was 63.8% and the positive rate of peptide was 90.4% in 94 HCC serum samples.The detection rate of HCC increased to 94.7% by combined detection of AFP with peptide.