Objective: Currently, detection method for cancer mainly includes biopsy, ultrasound and blood-based biomarkers detection. Although all of these methods are being employed in the clinical practice, they are often characterized by low sensitivity or/and specificity. In this study, we took the liver cancer as an example and combined immuno-MS and ELISA to verify the potential biomarker role of pep5 for hepatocellular carcinoma detection. Methods: A polypeptide named pep5 was designed and its antibody was used to detect liver cancer by methods of immuno-MS and ELISA. Proteins were determined using immunoprecipitation and Immunohistochemistry. Result: The data showed that combination of immuno-MS and ELISA can largely improve the sensitivity and specificity of the cancer detection. While the sensitivity and specificity are 74.83% and 71.52% (immuno-MS alone), 67.72% and 97.35% (ELISA alone), the combination of immuno-MS and ELISA can lead to a sensitivity and specificity of 72.78% and 100%, respectively. Immuno-MS and ELISA assay showed that pep5 antibody binds with both pep5 and its precursor protein. Conclusion: The combination of immuno-MS and ELISA may be a new strategy of the detection of cancer, and the pep5 also can be a hopeful serum biomarker for liver cancer.

应用血清多肽SPGO1、SPG03、SPG04的多抗(PcAb)酶联免疫吸附方法(ELISA),用于鼻咽癌放疗敏感性的检测.以碳二亚胺偶联法制备多肽免疫抗原,免疫动物制备兔血清多抗,用Protein A亲和层析法纯化多抗,选择3种多抗的工作滴度,应用多抗ELISA法检测63份鼻咽癌血清样本.紫外(UV)和聚丙烯酰胺凝胶电泳(SDS-PAGE)联合监测表明多肽偶联到BSA(牛血清白蛋白)上;动物免疫获得3种兔血清多克隆抗体各40 mL;纯化后的多抗纯度在90％以上,效价均在1:100 000以上;3种多抗的检测滴度分别为1:20000,1:100000,1:10 000;间接ELISA最低检测限为0.03 μg/mL,相对标准偏差(RSD)为5.6％.t检验分析3种多肽区分鼻咽癌放疗敏感组与不敏感组,均呈显著差异(P ＜0.000 1).ROC曲线表明,3种多肽检测鼻咽癌放疗敏感性的特异性在90％以上,灵敏度为73％～93％.3种多肽标志物均能显著区分鼻咽癌放疗敏感组与不敏感组,所应用的多抗ELISA方法检测鼻咽癌放疗敏感性特异性好,灵敏度高,可用于鼻咽癌个体化治疗的放疗敏感性辅助诊断.

Objective To establish an enzyme-linked immunosorbent assay(ELISA)for polypeptide5(Pep5)in human serum and apply it to the serological diagnosis of the progression of liver injury.Methods Microplate was precoated with sample diluents,blocked with 5% milk,and samples were detected with anti Pep5 McAb labled with HRP.And 690clinical samples were detected by method metioned above.Results Sensitivity was 1.25ng/mL.Linear measurement range was 0-24ng/mL.Recovery rate was 99.09%-107.08%.Intra-and inter-assay coefficinet of variation were 7.32%and 6.45%respectively,or less.The cross-reaction rate with acetylcholinesterase,laminin,hyaluronic and procollagen were all less than 1.00%.Clinical detecting showed that levels of Pep5increased with the exacerbation of liver injury(P<0.01).Compared with acetylcholinesterase kit,the relative coefficient(r)was 0.68.Conclusion Pep5could be used for the quantitative serological tests for the progression of liver injury,which might be reliable,simple and convenient,and could provide scientific evidences for the further study of Pep5function and development of related diagnostic kits.

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.

Serum peptides are the rich sources for cancer-specific diagnostic information and because of their complexity, low abundance, degradation, a lot of new peptidomic technologies were developed in recent years. A method based on peptide antibodies and immuno-mass spectrometry for detection of the serum peptide biomarkers was developed for detecting peptide biomarker candidates in the clinic tumor sera. The linear range of the method was 1-40 pmol with a detection limit of 0.5 pmol. The relative standard deviation was 9.5% for 5 pmol standard of synthetic peptide 5(n = 3). Furthermore, this method was successfully applied to determine the serum peptide biaomarker of 35.2 pmol/mL. The method of immuno-mass spectrometry combining peptide antibodies was paticular suitable for the detection of low concentration biomarkers in clinical samples, and it will be significant for earlier diagnosis of cancers.