抗人红细胞膜血型糖蛋白A单克隆抗体的制备及鉴定

Monoclonal antibodies against glycophorin A

目的：制备抗人红细胞膜血型糖蛋白Ａ（ＧＰＡ）的特异单克隆抗体，对研究ＧＰＡ基因突变和ＧＰＡ蛋白糖基化变异有重要意义。方法：分别用ＯＭ和纯化Ｍ－ＧＰＡ蛋白以及ＯＮ型红细胞和纯化Ｎ－ＧＰＡ蛋白免疫ＢＡＬＢ／ｃ小鼠，取脾细胞与小鼠骨髓瘤细胞Ｓｐ２／０融合，经正常和酶处理人细胞凝集筛选，间接免疫荧光，酶联免疫分析及蛋白质印迹鉴定，制备了鼠抗人ＧＰＡ的单克隆抗体。结果：获得抗ＧＰＡ蛋白１～３９位肽段及相关糖链的５株单抗，除２Ｅ９以外均为糖链依赖型。其中抗Ｍ，Ｎ－ＧＰＡ单抗２株：２Ｅ９Ｉｇ（Ｇ１，λ）和９Ｅ９Ｉｇ（Ｇ３，κ）；２Ｅ９抗Ｍ，Ｎ－ＧＰＡ６－３９位同源肽段，９Ｅ９抗Ｍ，Ｎ－ＧＰＡ１～２６位肽段。Ｎ－ＧＰＡ单抗３株：３Ｃ３Ｉｇ（Ｇ１，κ），５Ｆ９Ｉｇ（Ｇ３，κ）和６Ａ８Ｉｇ（Ｇ３，λ），均为抗Ｎ－ＧＰＡ１～６位肽段抗体。４株单抗与胰蛋白酶处理的红细胞不能反应，６Ａ８使胰蛋白酶处理的Ｍ型红细胞凝集而不能凝集胰蛋白酶处理的Ｎ型红细胞，推测Ｍ和Ｎ型红细胞的ＧＰＢ蛋白存在某种差异。

Objective: To prepare monoclonal antibodies (McAb) against human GPA for studying human erythrocyte glycophorin A(GPA) gene mutation and glycosylation variation. Methods: Hybridizing Sp2/0 cells with the spleen cells from BALB/c mice immunized with O,MM type human erythrocytes and purified M GPA, O,NN type human erythrocytes and purified N GPA respectively, follolvel by selecting by hemagglutination of untreated and enzyme treated human erythrocytes and identifing by indirect immuno fluorescence microscopy, ELISA and Western blotting, 5 mouse McAbs have been obtained. Results: The 5 McAbs are directed to GPA NH 2 terminal 1-39 peptide, and are sialic acids dependent except 2E9. Both 2E9 Ig(G1,λ) and 9E9 Ig(G3,κ) are antibodies to M,N type GPA, the epitope of 2E9 is located at M,N GPA homologous 6-39 residues and that of 9E9 is on M,N GPA 1-26 residues; The determinant of the other 3, i.e, 3C3 Ig(G1,κ),5F9 Ig (G3,κ)and 6A8 Ig(G3,λ), are located on N GPA1-6 peptide. 4 of the 5 McAbs don′t react to trypsin treated erythrocytes, 6A8 can agglutinate trypsin treated MM but not NN erythrocytes, suggesting that GPBs on the MM and NN cells may be different in some extent. Conclusion: These McAbs are valuable for studying GPA variation resulting from gene mutation and glycosylation variation.