模拟表位结合夹心化学发光法检测抗A和抗B效价  

Development of Mimotopes Combined with Sandwich Chemiluminescence Immunoassay for the Determination of Anti-A and Anti-B Titers

作　　者：周水梅 沈长新[1,2] 宋晶晶 王娇 林晨瑶 黄爽 贺学宇[3] ZHOU Shui-mei;SHEN Chang-xin;SONG Jing-jing;WANG Jiao;LIN Chen-yao;HUANG Shuang;HE Xue-yu(Genetic Diagnosis Center, Zhongnan Hospital of Wuhan University, Wuhan,Hubei,430071,China;Department of Blood Transfusion, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, 430071,China;Institute of Hepatobiliary, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, 430071,China)

机构地区：[1]武汉大学中南医院基因诊断中心,湖北武汉430071 [2]武汉大学中南医院输血科,湖北武汉430071 [3]武汉大学中南医院肝胆研究院,湖北武汉430071

出　　处：《现代生物医学进展》2019年第15期2822-2828,共7页Progress in Modern Biomedicine

基　　金：湖北省卫生计生委采供血专项基金项目(WJ2015CB005)

摘　　要：目的:获得A/B血型抗原的模拟多肽,并将其与夹心化学发光免疫分析(chemiluminescence immunoassay, CLIA)相结合,以开发更有效的方法来测量A/B抗体的效价。方法:分别用NaM87-1F6和HEB-29对噬菌体12肽库进行三轮淘选。对经酶联免疫吸附实验(ELISA)确定的阳性菌斑进行测序分析,根据其氨基酸组成合成模拟多肽,并通过竞争性ELISA、凝集抑制等方法检测其抗原性。随后将获得的模拟多肽与CLIA结合,检测A、B抗体效价,制作标准曲线以及与现有的微柱凝胶法(CAT)进行相关性对比。结果:经过三轮淘选和相关鉴定后得到13个与抗A抗体和19个与抗B抗体特异性相互作用的噬菌体克隆,其中分别有9个(MTRIQLRMMRLH)和12个(PQMSRHRMRMLP)展示相同的氨基酸序列,据此合成的多肽分别命名为MTR和PQM。竞争性ELISA结果表明,MTR能够特异性拮抗A抗体和A抗原的替代多肽的免疫结合;PQM能够特异性拮抗B抗体和B抗原的替代多肽的免疫结合。凝集抑制实验表明MTR/PQM可以特异性抑制A/B型红细胞与抗A/B之间的相互作用。结合模拟肽的夹心CLIA具有较宽的线性范围(2-2048)和良好的线性相关系数(对于A抗体R2=0.9566,对于B抗体R2=0.9762)。基于CLIA方法和CAT测得的抗体滴度结果高度相关(P<0.001)。结论:MTR和PQM有良好的A或B抗原性,具有成为其人工替代品的潜能。模拟多肽与夹心CLIA相结合能够实现对A、B抗体滴度的定量检测,且其结果准确可靠。Objective: The aim of this study was to acquire the mimic peptides of A/B antigen and combined it with sandwich chemiluminescence immunoassay(CLIA)to develop more effective way to measure the titer of A/B antibody. Methods: Three-round of biopanning was performed on the phage 12 peptide library with NaM87-1 F6 and HEB-29 as targets,respectively. Positive plaques determined by enzyme-linked immunosorbent assay(ELISA) were then sequenced, and the mimetic peptides were synthesized according to their amino acid sequences. Competitive ELISA and agglutination inhibition were carried out to confirm the interaction between anti-A/B and the synthesized peptides. The mimetic peptides was then combined with CLIA to detect A and B antibody titers. Standard curves were prepared and the results obtained by the method based on CLIA and the micro-column agglutination technique(CAT) method were compared and the correlation between them was analyzed. Results: After three rounds of panning and related identification, 13 and 19 phage clones that specifically interacted with anti-A and anti-B antibodies were obtained, of which 9(MTRIQLRMMRLH) and 12(PQMSRHRMRMLP) showed the same amino acid sequences, respectively. The synthesized peptides were named MTR and PQM, respectively.The results of competitive ELISA indicated that MTR can specifically antagonize the binding of anti-A and the replacement polypeptide of the A antigen;PQM can specifically antagonize the immunological binding of anti-B and the replacement polypeptide of the B antigen. Agglutination inhibition experiments showed that MTR/PQM could specifically inhibit the interaction between group A/B red blood cells and anti-A/B. The synthesized peptides combined with CLIA method proposed here had a broad linear range(2-2048) and a good linear correlation coefficient(for A antibody R^2=0.9566, for B antibody R^2=0.9762). The results obtained by the method based on CLIA were highly correlated with the results obtained by the CAT method(P<0.001). Conclusion: MTR and PQM have good A

关 键 词：噬菌体展示 A、B抗体 化学发光 血型A B抗原 

分 类 号：R-33[医药卫生] R446.6