血清多肽SPG01、SPG03、SPG04检测鼻咽癌放疗敏感性的初步研究

Preliminary Study of Detection on Nasopharyngeal Carcinoma Radiosensitivity by Peptide Biomarker of SPG01,SPG03 and SPG04

应用血清多肽SPG01、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∶20 000,1∶100 000,1∶10 000;间接ELISA最低检测限为0.03μg/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。

Applying the PcAb-ELISA（ polyclonal antibody Enzyme-Linked Immunosorbent Assay） of serum peptides of SPG01,SPG03 and SPG04 for detection of nasopharyngeal carcinoma（ NPC） radiotherapy sensitivity. Prepareing three peptide immunogens by Carbodiimide coupling method and obtaining the serum polyclonal-antibody through immune rabbits. The Protein A affinity chromatography way for the purification of PcAb was used and the detection titer of three antibodies was selected by ELISA. The PcAbELISA for detecting of 63 nasopharyngeal carcinoma serum samples was utilined. The unite monitor by UV and SDS-PAGE demonstrated that peptide was coupled on BSA（ Bovine serum albumin） and collected 40 mL polyclonal antibody for each peptides. The purity of the PcAbs was assessed at 90% and the antibodies titer above at 1∶ 100 000 in all after they were purified by Protein A.Finally,the antibody test titer of SPG01,SPG03 and SPG04 as 1∶ 20 000,1∶ 100 000,1∶ 10 000 respectively was chosen. The lowest detectable limit of PcAb-ELISA is 0. 03 μg /mL and its relative standard deviation（ RSD） is 5. 6%. Three peptides can distinguish between radiotherapy sensitive group and insensitive group of nasopharyngeal carcinoma with P 0. 000 1 through t-test analysis.ROC（ Receiver operating characteristic） curve showed that the specificity was up to 90% and the sensitivity was between 73% and93%. Nasopharyngeal carcinoma radiotherapy related serum peptide biomarkers of SPG01,SPG03 and SPG04 can be made as a notable distinction between radiosensitive group and rdioresistant group. The PcAb-ELISA method has good specificity and high sensitivity and can be used for one auxiliary diagnosis of radiation sensitivity in individualized treatment of NPC.