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为了研究抗独特型抗体对天花粉蛋白特异的IgE反应的调节作用,建立了分泌针对天花粉蛋白的IgE单抗的独特型决定簇的大鼠-小鼠异种杂交瘤细胞株(6 C_5)。以分泌抗天花粉蛋白IgE单抗的小鼠淋巴细胞杂交瘤细胞株(TE—1)诱生的腹水,通过免疫亲和层析,分离得到TE-1单抗免疫Wistar大鼠。将免疫大鼠脾细胞与小鼠骨髓细胞(NS-1)进行异种间细胞融合,通过严格的筛选和克隆化,最终得到3侏生长稳定、连续分泌抗体的异种杂交瘤细胞株(6 C_5、3 E_3和8 G_6),并能顺利地经受冰冻保种和复苏。用间接ELISA法对所获得的单抗进行鉴定,即比较三个单抗对下列包被抗原的反应性,其中包括TE-1(天花粉蛋白特异的IgE单抗)、3A 12(天花粉蛋白特异的IgA单抗)、ADNP和142(抗DNP IgE单抗,来自两个不同的杂交瘤株),以及M Ig(正常小鼠血清Ig)。结果表明6C_5单抗只与TE-1呈阳性反应,对其余各种包被抗原的反应均呈阴性,说明杂交瘤株6C_5具有抗TE-1单抗独特型决定簇的特异性,实验经多次重复,因此可以结论,成功地建立了一株能分泌抗独特型抗体的异种淋巴细胞杂交瘤株。此外,8G_6与所有包被抗原均呈强阳性,说明它具有抗各类小鼠Ig共同决定簇的特异性。3 E_3的特异性尚未最终确定。在方法学上的改进包括细胞融合时所用的HAT选择培液中的次黄嘌呤和胸腺嘧啶核苷的剂量较常量加倍,而氨基喋呤的剂量较常量减半。并且缩短在HAT培液中和HT培液中培养时间。这些可能为杂种淋巴细胞杂交瘤株的建立提供了有利条件。为了保证ELISA检测的特异性,作包被抗原的TE-1单抗与免疫大鼠用的TE-1单抗的来源不同,它来自无血清培养液培养的TE-1的上清液。同时,间接ELISA法所采用的酶联第二抗体(兔抗大鼠Ig),通过小鼠Ig的吸收等,证明其与小鼠Ig无交叉反应后才使用。
In order to investigate the regulatory effect of anti-idiotypic antibodies on trichosanthin-specific IgE responses, a rat-mouse xenogeneic hybridoma cell line (6 C_5) secreting a unique determinant of trichosanthin IgE monoclonal antibody was developed. The immunized Wistar rats were isolated from the ascites induced by TE-1, a mouse lymphocyte hybridoma cell line secreting anti-TCS IgE monoclonal antibody. The spleen cells of immunized mice were fused with the mouse bone marrow cells (NS-1) for different kinds of cells. Through strict screening and cloning, 3 kinds of hybridoma cell lines with stable growth and continuous secretion of antibodies (6 C 5, 3 E_3 and 8 G_6), and can successfully withstand the cryopreservation and recovery. The McAbs obtained were identified by indirect ELISA to compare the reactivity of the three monoclonal antibodies against the following coated antigens, including TE-1 (Trichosanthin-specific IgE mAb), 3A12 (Trichosanthin-specific IgA monoclonal antibody), ADNP and 142 (anti-DNP IgE monoclonal antibody from two different hybridoma strains), and M Ig (normal mouse serum Ig). The results showed that 6C_5 McAb only reacted positively with TE-1 and all other antigens were negative, indicating that the hybridoma strain 6C_5 has the specificity of anti-TE-1 mAb determinant. Therefore, it can be concluded that a heterologous lymphocyte hybridoma strain capable of secreting anti-idiotypic antibodies has been successfully established. In addition, 8G_6 was strongly positive with all coated antigens, indicating that it has the specificity of anti-mouse Ig determinant. The specificity of 3 E_3 has not yet been finalized. Dose of hypoxanthine and thymidine in the HAT selection culture solution used for cell fusion includes doubling the dose and the dose of aminopterin is halved from the constant amount, in a methodological improvement. And shortens the incubation time in HAT medium and HT medium. These may provide favorable conditions for the establishment of hybrid lymphocyte hybridoma strains. To ensure the specificity of the ELISA assay, the TE-1 mAb coated with the antigen was derived from the supernatant of TE-1 cultured in serum-free medium, unlike the TE-1 mAb used in immunized rats. Meanwhile, indirect enzyme-linked immunosorbent secondary antibody (rabbit anti-rat Ig) was used after it was confirmed that it had no cross-reaction with mouse Ig by the absorption of mouse Ig.