Borrebaeck C A
Mol Immunol. 1984 Oct;21(10):841-5. doi: 10.1016/0161-5890(84)90137-8.
Antigen-specific hybridomas can be produced using spleen cells that have been immunized in culture as parental cells in the hybridization step. The in vitro immunization of non-immune mouse spleen cells was supported by a lymphokine preparation that contained T-cell-replacing factor (TRF). The influence of TRF, produced by a mixed-thymocyte reaction, on immunization in culture has been investigated using bacterial cells as the immunogen. The cell vols of stimulated splenocytes were monitored and it was found that the induction of antigen-specific blast cells, which could subsequently be immortalized by fusing them with myeloma cells, was completely abolished in immunizations which were not supported by TRF. If serum-free conditions were used during the in vitro immunization step, the frequency of antigen-specific blast cells increased, which resulted in a higher yield of specific hybridomas. This was due to the reduced background stimulation achieved by omitting serum proteins. The relationship between immunogenic dose and response, measured as the specific efficiency obtained in hybridization experiments with in vitro immunized cells, was recorded using different amounts of sperm whale myoglobin as antigen. An antigen-specific response was recorded with as low as 1 ng antigen/ml.
可使用在培养中已免疫的脾细胞作为杂交步骤中的亲本细胞来制备抗原特异性杂交瘤。一种含有T细胞替代因子（TRF）的淋巴细胞因子制剂支持对非免疫小鼠脾细胞进行体外免疫。已使用细菌细胞作为免疫原研究了由混合胸腺细胞反应产生的TRF对培养中免疫的影响。监测了受刺激脾细胞的细胞体积，发现抗原特异性母细胞的诱导在没有TRF支持的免疫中完全被消除，这些抗原特异性母细胞随后可通过与骨髓瘤细胞融合而永生化。如果在体外免疫步骤中使用无血清条件，抗原特异性母细胞的频率会增加，这导致特异性杂交瘤的产量更高。这是由于省略血清蛋白实现了背景刺激的降低。使用不同量的抹香鲸肌红蛋白作为抗原，记录了免疫原剂量与反应之间的关系，反应以体外免疫细胞杂交实验中获得的特异性效率来衡量。低至1 ng抗原/ml时记录到了抗原特异性反应。
