抗血型M、N及抗血型糖蛋白A/B单克隆抗体的制备及其特性鉴定

目的:制备抗血型M、N及抗血型糖蛋白A/B(GPA/GPB)的单克隆抗体(mAb),并进行特性鉴定。方法:用人“O”型血红细胞作为免疫源,免疫BALB/c小鼠。采用淋巴细胞杂交瘤技术制备mAb,用谱红细胞筛选阳性克隆;采用直接、间接血凝试验检测杂交瘤细胞培养上清及腹水中mAb的效价。分别用快速定性试纸和酶处理红细胞检测mAb的Ig亚类及抗原表位。用Western blot鉴定抗GPA/GPB mAb的特异性。结果:获得4株分泌抗M、1株抗N及3株抗GPA/GPB mAb的杂交瘤细胞株。杂交瘤细胞培养上清mAb的效价介于1×2-4~1×2-8之间,腹水mAb的效价在1×2-7~1×2-12之间。除1株mAb 1C1C9C4为IgM外,其他7株mAb均为IgG。通过杂交瘤细胞培养上清与谱红细胞的反应格局,结合mAb抗原表位的检测,确定4株和1株mAb可分别特异性结合于GPA的M、N抗原表位;另3株mAb 6D7C9、7C9H4和7C9G11与“O”型血红细胞膜的Western blot结果显示,均可结合GPA、GPB蛋白。结论:成功地建立了4株分泌抗M、1株分泌抗N及3株分泌抗GPA/GPB mAb的杂交瘤细胞株,可用于MNSs血型系统的研究及鉴定,并为制备双功能抗体用于病毒、肿瘤疾病的诊断和治疗打下了坚实的基础。

Preparation and identification of monoclonal antibodies against Type M/N and Glycophorin A/B

AIM: To prepare monoclonal antibodies(mAbs) against Type M/N and monoclonal antibodies against Glycophorin A and Glycophorin B (GPA/GPB) and identify rare blood group MkMk. METHODS: 8 week-old female BALB/c mice were immunized with type "O" red blood cells, Splenocytes from the immunized mice were fused with Sp2/0 myeloma cells, and positive hybridoma clones were screened by panel erythrocytes. The titer of supernatant and ascitic fluid was tested by direct and indirect agglutination. The subclasses and isotypes were identified by monoclonal antibody isotyping kit. The complemental structures of the combining sites of their antibody antigen were determined by enzyme-treated red cells. The specificity of the GPA/GPB mAbs was identified by Western blot analysis. RESULTS: Eight hybridoma cell lines against Type M/N and GPA/GPB were obtained. The titer of supernatant was between 1 x 2(-4) - 1 x 2(-8), and the titer of ascitic fluid was between 1 x 2(-7) - 1 x 2(-12). The immunoglobulin subclasses of all the mAbs were IgG except 1C1C9C4, which was IgM. Four anti-M mAbs and one anti-N mAb were deterimined by agglutination test using panel erythrocytes and the test of the combining sites in antiody antigen. Western blot analysis proved that three mAbs recognized GPA/GPB protein. CONCLUSION: Eight hybridoma cell lines against Type M/N and GPA/GPB have been obtained successfully. Among them, four mAbs recognize M, one recognizes N and three recognize GPA/GPB. Our study may be useful to research into MN blood group and for serological diagnosis. The anti-GPA/GPB mAbs can be used to prepare diabodies for the treatment and diagnosis of some of diseases.