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目的:制备小鼠抗人c-erbB2mAb,并进行特异性鉴定。方法:应用计算机软件分析人源c-erbB2抗原表位,人工合成羧基端含优势表位的13肽,与钥孔戚血蓝蛋白(KLH)偶联后,免疫BALB/c小鼠。取免疫小鼠脾细胞与Sp2/0骨髓瘤细胞常规融合,依次经HAT选择培养、间接ELISA法、克隆化和免疫组化染色法筛选出稳定分泌抗天然人源c-erbB2mAb的杂交瘤细胞株。用交叉反应试验和阻断试验检测mAb的特异性。结果:获得1株可稳定分泌抗天然人源c-erbB2抗体的杂交瘤细胞株。该mAb与已知的c erbB2抗原阳性的乳腺癌标本起反应;与其他不表达c-erbB2分子的细胞不起反应。用合成的13肽阻断后,失去与c-erbB2抗原的反应性。结论:用合成的13肽作为免疫原成功地制备出1株抗c-erbB2的mAb。
OBJECTIVE: To prepare mouse anti-human c-erbB2 mAb and to identify it specifically. METHODS: Human c-erbB2 epitopes were analyzed by computer software. The 13-mer peptide with the dominant epitope was synthesized and conjugated to keyhole limpet hemocyanin (KLH). BALB / c mice were immunized. The spleen cells of immunized mice were routinely fused with Sp2 / 0 myeloma cells, and then the hybridoma cell lines stably secreting anti-human c-erbB2 mAb were screened by HAT selection, indirect ELISA, cloning and immunohistochemistry . The specificity of the mAb was tested using cross-reactivity and blocking assays. Results: One hybridoma cell line stably secreting anti-human c-erbB2 antibody was obtained. This mAb reacts with known c erbB2 antigen-positive breast cancer specimens; it does not react with other cells that do not express c-erbB2 molecules. After blocking with the synthetic 13 peptide, the reactivity with the c-erbB2 antigen was lost. Conclusion: One anti-c-erbB2 mAb was successfully prepared using the synthesized 13 peptide as an immunogen.