鼠抗人B7-H4单克隆抗体的制备及可溶性B7-H4酶联检测试剂盒的研制

发布时间：2018-06-18 13:00

本文选题：B7-H4 + 单克隆抗体　；参考：《苏州大学》2012年硕士论文

【摘要】：B7-H4(B7x, B7S1)是B7家族的新成员，可以通过抑制T细胞增殖、细胞因子的产生及阻遏细胞周期进程发挥负性调控作用。人B7-H4位于1号染色体上，cDNA长约1.8kb，在基因组上跨越66kb，含有6个外显子和5个内含子，编码282个氨基酸，属于免疫球蛋白超家族。人B7-H4分子的mRNA在淋巴和非淋巴组织中都有表达，但免疫组化显示，B7-H4蛋白仅在部分正常组织中微弱表达。近年来许多研究陆续发现B7-H4蛋白在多种肿瘤组织中异常表达，如卵巢癌、乳腺癌、肺癌等，，这提示了B7-H4分子可能在肿瘤细胞逃避机体免疫系统攻击中发挥一定的作用，此外B7-H4在维持机体免疫耐受过程中也起到重要的作用。因此，B7-H4分子的作用为肿瘤、自身免疫性疾病和移植排斥反应等疾病提供了新的治疗思路。阻断B7-H4分子与其受体的相互作用，有望成为干预自身免疫性疾病和肿瘤生物治疗的潜在靶点。制备特异性鼠抗人B7-H4单克隆抗体及研制特异、灵敏、稳定的人可溶性B7-H4酶联检测试剂盒，为进一步研究B7-H4在肿瘤发生发展中的作用提供了有力的工具，同时也为临床相关疾病的诊断、治疗提供了新的手段。一、鼠抗人B7-H4单克隆抗体的制备及其生物学特性的初步研究【目的】制备鼠抗人B7-H4的单克隆抗体（mAb）并对其生物学特性进行鉴定。【方法】以本室构建的稳定高表达B7-H4分子的L929/B7-H4基因转染细胞为免疫原，免疫6~8周龄的BALB/c小鼠，取小鼠脾脏细胞与小鼠骨髓瘤细胞(SP2/0)融合后，采用流式细胞术（FCM）筛选阳性克隆，制备稳定分泌抗B7-H4mAb的细胞株，用快速试纸法鉴定mAb的Ig亚类，并采用Western blot、Dot blot、位点竞争抑制实验和T细胞增殖抑制阻断实验对所获的mAb进行鉴定。【结果】成功获得3株稳定分泌抗人B7-H4mAb的阳性克隆，分别命名为5C8、5G3、8F10。快速试纸法鉴定5C8为IgG2a类、5G3和8F10均为IgG1类。Western blot和Dot blot结果表明：5C8、5G3、8F10均能与人B7-H4蛋白特异性结合；位点竞争实验显示（1）：3株单抗与商品化单抗H74结合在不同位点；（2）：与本室已有的2株单抗1F10和2B2位点竞争结果为：3株单抗与1F10结合在不同位点；5C8和5G3与2B2识别位点可能相近，8F10与单抗2B2结合在不同位点；（3）：这3株单抗相互位点竞争结果为：5C8与5G3结合在不同位点；8F10与5C8和5G3识别位点可能相近。T细胞增殖抑制阻断实验显示，这3株单抗均能阻断B7-H4分子对T细胞增殖抑制作用。【结论】成功获得3株鼠抗人B7-H4单克隆抗体，为研究B7-H4分子在肿瘤中的作用提供了手段。二、可溶性B7-H4酶联检测试剂盒的研制【目的】建立人可溶性B7-H4（sB7-H4）酶联检测试剂盒。【方法】以本研究已获得的3株单抗（5C8、5G3、8F10）和本室已获得的另外2株鼠抗人B7-H4单抗（1F10、2B2）为基础，采用单抗1F10作为包被抗体，以标记生物素(biotin)的单抗8F10作为检测抗体，建立双单抗夹心的人可溶性B7-H4（sB7-H4）酶标检测方法，并对正常人外周血血清中的sB7-H4含量进行了检测。【结果】成功研制了人sB7-H4酶联检测试剂盒，其灵敏度为3.13ng/mL。该试剂盒4℃放置1个月，离散度(CV%)±2.80，回收率为92.98%~107.4%，提示该检测试剂盒具有良好的灵敏度、稳定性和准确性。用该试剂盒测得正常人血清中sB7-H4的正常值为40.00±11.93ng/mL。【结论】成功研制了检测人sB7-H4酶联检测试剂盒。综上所述，成功获得3株能稳定分泌鼠抗人B7-H4单克隆抗体的杂交瘤细胞株，并研制了检测人sB7-H4酶联检测试剂盒。为进一步研究B7-H4分子生物学功能奠定了物质基础。
[Abstract]:B7 - H4 ( B7 - H4 ) is a new member of B7 family . B7 - H4 molecule plays an important role in inhibiting T cell proliferation , cytokine production and cell cycle progression .

Preparation of monoclonal antibody against human B7 - H4 and its biological characteristics

Objective To prepare the monoclonal antibody ( mAb ) of murine B7 - H4 against human B7 - H4 and to identify its biological characteristics .

BALB / c mice immunized with B7 - H4 were transfected into BALB / c mice immunized with B7 - H4 molecules . After fusion with SP2 / 0 mouse myeloma cells ( SP2 / 0 ) , the positive clones were screened by flow cytometry ( FCM ) . The Ig subclass of mAb was identified by rapid test paper . Western blot , dot blot , site competition inhibition assay and T cell proliferation inhibition test were used to identify the mAb .

The positive clones were identified as 5C8 , 5G3 and 8F10 , respectively . The results showed that 5C8 , 5G3 and 8F10 were IgG1 . Western blot and Dot blot showed that both 5C8 , 5G3 and 8F10 could bind to human B7 - H4 protein .
The site competition experiment showed that : ( 1 ) Three monoclonal antibodies were combined with commercial monoclone antibody H74 at different sites ;
( 2 ) The results were as follows : 3 monoclonal antibodies and 1F10 were bound to different sites ;
5C8 and 5G3 may be similar to the 2B2 identification sites , and 8F10 and 2B2 bind to different sites ;
( 3 ) The competitive results of these three isolates were : 5C8 and 5G3 bound at different sites ;
8F10 was similar to 5C8 and 5G3 recognition sites . T cell proliferation inhibition block experiment showed that these three monoclonal antibodies could block the inhibition of B7 - H4 molecule on T cell proliferation .

Conclusion The monoclonal antibodies against B7 - H4 have been successfully obtained in three murine anti - human B7 - H4 molecules , which provide a means to study the role of B7 - H4 molecules in tumors .

Development of Soluble B7 - H4 ELISA Kit

Objective To establish a human soluble B7 - H4 ( sB7 - H4 ) ELISA kit .

Based on three monoclonal antibodies ( 5C8 , 5G3 , 8F10 ) and another two mouse anti - human B7 - H4 monoclonal antibodies ( 1F10 , 2B2 ) which have been obtained in this study , monoclonal antibody 1F10 was used as the antibody to detect the antibody , biotin - labeled monoclonal antibody 8F10 was used as the detection antibody , and the detection method of human soluble B7 - H4 ( sB7 - H4 ) was established , and the levels of sB7 - H4 in serum of human peripheral blood were detected .

The sensitivity of the kit was 3.13ng / mL . The sensitivity was 3.13ng / mL . The results showed that the detection kit had good sensitivity , stability and accuracy . The normal value of sB7 - H4 in human serum was 40.00 卤 11.93ng / mL .

Conclusion The ELISA kit for detecting human sB7 - H4 was successfully developed .

In conclusion , three hybridoma cell strains capable of stably secreting anti - human B7 - H4 monoclonal antibodies against human B7 - H4 were successfully obtained , and a kit for detecting human sB7 - H4 was developed .
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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