鸡抗人B7同源物4(B7-H4)IgY多克隆抗体的制备及鉴定  

Preparation and characterization of chicken anti-human B7-H4 IgY polyclonal antibody

作　　者：张丽 范振军 张文婷 黄焕绍 王家俊 黑爱莲 丛延广 徐军发 ZHANG Li;FAN Zhenjun;ZHANG Wenting;HUANG Huanshao;WANG Jiajun;HEI Ailian;CONG Yanguang;XU Junfa(Institute of Laboratory Medicine,College of Medical Technology,Guangdong Medical University,Dongguan 523808;Laboratory medicine center,The first Dongguan Affiliated Hospital,Guangdong Medical University,Dongguan 523710;Sino-Swed TongKang Bio-Tech Limited,Shenzhen 518000,China)

机构地区：[1]广东医科大学医学技术学院检验医学研究所,广东东莞523808 [2]广东医科大学附属东莞第一医院检验医学中心,广东东莞523710 [3]华瑞同康生物技术(深圳)有限公司,广东深圳518000

出　　处：《细胞与分子免疫学杂志》2024年第3期257-266,共10页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(82270013);广东省教育厅创新团队项目(2022KCXTD010)。

摘　　要：目的制备抗人B7同源物4(B7-H4)IgY多克隆抗体,建立合适的双抗体夹心ELISA检测人血清中可溶性B7-H4(sB7-H4)蛋白。方法筛选人源B7-H4蛋白合适的B细胞表位肽免疫新海兰灰蛋鸡,收集鸡蛋纯化鸡抗人B7-H4 IgY抗体;检测抗体的纯度、浓度及效价,并用ELISA、Western blot、流式细胞术验证抗体的特异性和功能;建立双抗体夹心ELISA法并检测临床样本;同时采用商品化的ELISA试剂盒对相同样本进行对比检测。结果制备了鸡抗人B7-H4 IgY抗体并证明该抗体对人B7-H4蛋白有高特异性;采用该抗体检测临床样本发现卵巢癌和良性卵巢肿瘤患者血清中sB7-H4含量明显高于健康人,这与使用商品试剂盒获得的检测结果一致;采用IgY抗体建立的ELISA灵敏度高于商品试剂盒。结论成功制备了鸡抗人B7-H4 IgY多克隆抗体,并用其建立了一种适合检测人血清中sB7-H4蛋白的双抗体夹心ELISA。Objective To prepare anti-human B7 homolog 4(B7-H4)egg yolk immunoglobulins(IgY)polyclonal antibody and establish a double-antibody sandwich ELISA for the detection of soluble B7-H4(sB7-H4)protein in human serum.Methods Bioinformatics was used to screen specific B cell epitope peptides of human sB7-H4.New Hyland Grey laying hens were immunized with these peptides,and the eggs from the immunized hens were collected to purify chicken anti-human B7-H4 IgY antibody.The purity,concentration and titer of the antibody were detected,and its specificity and function of the antibodies were verified by using ELISA,Western blot analysis and flow cytometry,respectively.A double-antibody sandwich ELISA was established to detect sB7-H4 in clinical samples by using the IgY antibody.Comparative detection was performed using a commercialized ELISA kit on the same set of clinical samples.Results The chicken anti-human B7-H4 IgY antibodies were successfully prepared and proven to be highly specific for the human B7-H4 protein.The ELISA established with the IgY polyclonal antibody detected significantly higher levels of soluble B7-H4 in the serum of patients with ovarian cancer and benign ovarian tumors compared to healthy controls.These results were consistent with the detection results obtained using a commercialized ELISA kit.However,the ELISA with IgY antibody exhibited higher sensitivity than the commercialized kit.Conclusion The chicken polyclonal antibody against human B7-H4 IgY is successfully prepared,and a double-antibody sandwich ELISA suitable for detecting sB7-H4 protein in human serum is established.

关 键 词：可溶性B7同源物4(sB7-H4) IgY多克隆抗体 双抗体夹心ELISA 

分 类 号：R446.6[医药卫生—诊断学] R392-33[医药卫生—临床医学] R392.11R730.43