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首页> 外文会议>2010 4th International Conference on Bioinformatics and Biomedical Engineering >A Novel Protocol for Making of Monoclonal Antibody against AFP
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A Novel Protocol for Making of Monoclonal Antibody against AFP

机译：制备抗AFP单克隆抗体的新方案

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Abstract-Alpha-fetoprotein (AFP) is a major serum protein (Mr, =70,000) synthesized during fetal life. Reappearance of AFP in adult serum often signals pathological conditions, particularly hepatocarcinomas and teratocarcinomas. To obtain monoclonal antibodies against AFP for further study of the structure and biological function of AFP, a novel method for making hybridoma cell lines producing monoclonal antibodies for AFP by immunization of animals with synthetic peptides were proposed. One epitope peptide was synthesized based on the bioinformatics analysis of the AFP and coupled with keyhole limpet hemocyanin ( KLH ). BALB/c mice were immunized with the coupled AFP polypeptide to make monoclonal antibody. Hybridomas were generated by the fusion of the spleenocytes from these mice with Sp2/0 myeloma cells. Hybridomas producing anti-AFP antibodies were screened by enzyme-linked immunosorbent assay ( ELISA), and the monoclonal antibodie specificities were determined by and indirect-ELISA.The favorite epitope of AFP was seleceted by bioinformatics methods and synthesized successfully, which coupled with carrier KLH as immunogen to immunize BALB/c mice, two hybridoma cell lines (2F12 and 3G8) stable in secreting anti-AFP specificity monoclonal antibodies (mAbs) were successfully generated, Titers of tybridoma cell culture supernatant and asicites are both above 1:32 and 100,000 respectively.Creatively preparing mAb against AFP by ways of immunizing mice with the coupled epitope to KLH.The mAb should be suitable for studies of the cellular localization of AFP's biosynthesis and its processing after interaction with receptor. It will also be useful in the immunoaffinity purification of the AFP in sreum and as a useful reagent for the analysis of biochemical, structural, and functional properties of AFP
机译：摘要甲胎蛋白（AFP）是胎儿生命期间合成的一种主要血清蛋白（Mr，= 70,000）。成人血清中AFP的重新出现通常预示着病理状况，尤其是肝癌和畸胎癌。为了获得针对AFP的单克隆抗体以进一步研究AFP的结构和生物学功能，提出了一种通过用合成肽免疫动物来制备产生针对AFP的单克隆抗体的杂交瘤细胞系的新方法。根据AFP的生物信息学分析，合成了一种表位肽，并与匙孔血蓝蛋白（KLH）偶联。用偶联的AFP多肽免疫BALB / c小鼠以制备单克隆抗体。杂交瘤是通过将这些小鼠的脾细胞与Sp2 / 0骨髓瘤细胞融合而产生的。通过酶联免疫吸附试验（ELISA）筛选产生抗AFP抗体的杂交瘤，并通过间接ELISA确定单克隆抗体的特异性。通过生物信息学方法选择并成功合成了AFP的最爱抗原决定簇，并与载体KLH偶联作为免疫BALB / c小鼠的免疫原，成功产生了两种稳定分泌抗AFP特异性单克隆抗体（mAbs）的杂交瘤细胞系（2F12和3G8），tybridoma细胞培养上清液和腹水的滴度均在1:32和100,000以上通过用偶联表位的小鼠免疫KLH的方法创造性地制备抗AFP的mAb.mAb应该适合于研究AFP的生物合成的细胞定位及其与受体相互作用后的加工过程。它也可用于皮肤中AFP的免疫亲和纯化，并可用作分析AFP的生化，结构和功能特性的有用试剂

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