抗犬VEGF单克隆抗体及夹心ELISA试剂盒的制备及应用

Preparation and Application of Canine VEGF Monoclonal Antibody and Sandwich ELISA Kit

本研究旨在制备犬血管内皮生长因子(VEGF)单克隆抗体和研制犬VEGF双抗夹心的ELISA检测试剂盒。研究利用原核表达的方法制备出具有良好反应原性的重组融合蛋白犬VEGF-His,并在此基础上进行了犬VEGF单克隆抗体的制备及鉴定,结果表明,所获得的犬VEGF单克隆抗体对犬VEGF蛋白具有良好的反应原性和较高的特异性。利用此单克隆抗体,与VEGF的兔多克隆抗体血清分别作为夹心ELISA检测试剂盒的上下层抗体使用,建立了以犬VEGF为抗原的夹心ELISA试剂盒的检测方法,并对正常健康犬及乳腺肿瘤患犬血清进行了检测,得出了此试剂盒的阳性和阴性临界值。实验结果表明,此试剂盒具有良好的敏感性、特异性和重复性,且利用本试剂盒对临床样本进行验证时,阳性符合率为87. 5%,阴性符合率为97. 56%,具有较高的临床应用价值。

The purpose of this study was to prepare monoclonal antibodies against canine VEGF and develop a double antibodies sandwich ELISA kit.The recombinant fusion protein canine VEGF-His was prepared by prokaryotic expression method.Based on this,the preparation and identification of canine VEGF monoclonal antibody were carried out.The identification results showed that the antibody can react with natural canine VEGF.The protein has good reactogenicity and high specificity.Using this monoclonal antibody,together with the VEGF rabbit polyclonal antibody serum as the upper and lower antibodies of the sandwich ELISA kit,the detection method of the sandwich ELISA kit using canine VEGF as antigen was established,The serum of healthy and breast tumors in canines was tested and the positive and negative thresholds of the kit were obtained.The kit has good sensitivity,specificity and reproducibility.When using this kit to verify clinical samples,the positive coincidence rate is 87.5%,and the negative coincidence rate is 97.56%,which has high clinical application value.It can be used for early diagnosis of clinical canine breast tumors.