Massino Y S, Dergunova N N, Kizim E A, Smirnova M B, Tereshkina E B, Kolyaskina G I, Dmitriev A D
Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences, Moscow.
J Immunol Methods. 1997 Feb 14;201(1):57-66. doi: 10.1016/s0022-1759(96)00212-8.
On the model of a hybrid hybridoma (quadroma) to alpha-endorphin (END) and horseradish peroxidase (HRP), we have elaborated a general approach to analyse H and L chain interactions in hybrid hybridomas and to evaluate their efficiency as producers of bispecific antibodies (bAbs). This strategy is based on quantitative analysis of quadroma produced Abs by affinity chromatography and radioimmunoassay. First, Abs produced by quadroma cells in culture media (IgG pools from three quadroma clones) were fractioned with respect to specificity. Second, Ab concentrations in each fraction (bispecific, anti-END, anti-HRP and inactive) were measured by specific radioimmunoassays, using rabbit antiserum against mouse IgG and 125I-labelled affinity purified quadroma Abs. Then the experimentally obtained Ab distributions were compared with the predicted Ab distributions for different models of IgG chain recombination in quadroma cells (random H/L pairing, preferential homologous H/L association). As follows from these models, in a random H/L recombination the yield of bAbs in quadroma produced IgG cannot exceed 12.5%, and the ratio of bAbs and inactive Abs cannot exceed 0.5. In the analysed clones the yield of bAbs amounted to about 30% of total IgG, and the ratio of bAbs and inactive Abs was about 5-8, giving strong evidence for preferential homologous H/L association in these cells. The ratio of anti-HRP and anti-END Abs was about 10:1, suggesting unequal production of parental IgG chains in quadroma cells. The result of quantitative analysis of quadroma IgG was further supported by two-dimensional gel analysis of affinity-purified fractions of quadroma IgG and of two parental mAbs.
在针对α-内啡肽（END）和辣根过氧化物酶（HRP）的杂交杂交瘤（四瘤）模型上，我们精心设计了一种通用方法，用于分析杂交杂交瘤中重链（H链）和轻链（L链）的相互作用，并评估它们作为双特异性抗体（bAbs）产生者的效率。该策略基于通过亲和色谱法和放射免疫测定对四瘤产生的抗体进行定量分析。首先，将培养培养基中四瘤细胞产生的抗体（来自三个四瘤克隆的IgG池）按特异性进行分级分离。其次，使用兔抗小鼠IgG抗血清和125I标记的亲和纯化四瘤抗体，通过特异性放射免疫测定法测量每个级分（双特异性、抗END、抗HRP和无活性）中的抗体浓度。然后，将实验获得的抗体分布与四瘤细胞中IgG链重组不同模型（随机H/L配对、优先同源H/L缔合）的预测抗体分布进行比较。从这些模型可以看出，在随机H/L重组中，四瘤产生的IgG中双特异性抗体的产量不能超过12.5%，双特异性抗体与无活性抗体的比例不能超过0.5。在所分析的克隆中，双特异性抗体的产量约占总IgG的30%，双特异性抗体与无活性抗体的比例约为5 - 8，有力地证明了这些细胞中存在优先同源H/L缔合。抗HRP和抗END抗体的比例约为10:1，表明四瘤细胞中亲本IgG链的产生不相等。四瘤IgG定量分析的结果进一步得到了四瘤IgG亲和纯化级分和两种亲本单克隆抗体的二维凝胶分析的支持。
