Gardner J S, Chiu A L, Maki N E, Harris J F
J Immunol Methods. 1985 Dec 27;85(2):335-46. doi: 10.1016/0022-1759(85)90142-5.
We describe a quantitative method of analysis for assessing stability of human monoclonal antibody production by hybridomas. Clones derived from fusion between the SHM-D33 heteromyeloma line and EBV-stimulated human lymphocytes were studied for antibody presence using a fluorescent labelling technique. Frequencies of antibody-negative variants in clonal populations were measured, and measurements on parallel clonal populations were subjected to Luria-Delbrück fluctuation analysis to compute rates of generations of antibody-negative cells. Independent hybridoma clones exhibited a range of stabilities and the corresponding rates varied between 5 X 10(-4) and 6 X 10(-2) cell-1 generation-1. Rates of generation of antibody-negative variants for the more stable heteromyeloma hybridomas compared well with those of 2 established mouse hybridoma lines tested (less than 10(-3) cell-1 generation-1). There was a positive correlation between frequency of antibody-negative variants measured in clonal populations grown to large numbers of cells (greater than 10(7) per culture) and their rate of loss of antibody production. Large variations in frequency of antibody-negative variants were observed in parallel clonal populations, suggesting that loss of ability to produce antibody is due to random, mutation-like events including chromosome loss (Luria and Delbrück, 1943). High frequencies of antibody-negative variants may indicate imminent loss of antibody-producing capacity by a clone growing in suspension culture.
我们描述了一种用于评估杂交瘤产生人单克隆抗体稳定性的定量分析方法。利用荧光标记技术，研究了源自SHM-D33异骨髓瘤细胞系与EB病毒刺激的人淋巴细胞融合产生的克隆，以检测抗体的存在情况。测定了克隆群体中抗体阴性变体的频率，并对平行克隆群体的测定结果进行了Luria-Delbrück波动分析，以计算抗体阴性细胞的产生率。独立的杂交瘤克隆表现出一系列稳定性，相应的产生率在5×10⁻⁴至6×10⁻²细胞⁻¹代⁻¹之间变化。与所测试的2个已建立的小鼠杂交瘤细胞系（小于10⁻³细胞⁻¹代⁻¹）相比，更稳定的异骨髓瘤杂交瘤产生抗体阴性变体的速率与之相当。在生长至大量细胞（每培养物大于10⁷）的克隆群体中测得的抗体阴性变体频率与其抗体产生丧失率之间存在正相关。在平行克隆群体中观察到抗体阴性变体频率的巨大差异，这表明产生抗体能力的丧失是由于包括染色体丢失在内的随机的、类似突变的事件（Luria和Delbrück，1943）。抗体阴性变体的高频率可能表明在悬浮培养中生长的克隆即将丧失产生抗体的能力。
