Comparison of two functional kappa light-chain transcripts amplified from a hybridoma.

Three heavy-chain and three kappa (κ)-chain transcripts were amplified from hybridoma cells secreting a monoclonal antibody (mAb) against transferrin receptor. Sequence analysis via IMGT/V-QUEST yielded the functional/aberrant prediction. Two functional κ-chain transcripts, Vκ2 and Vκ3, and one functional VH1 were revealed. Comprehensive bioinformatics analyses including sequence alignment, phylogenetic tree, somatic hypermutation prediction, and three-dimensional-molecular structure modeling were used to predict the origin of the two κ-chain transcripts. The results of bioinformatics analysis suggest that Vκ3 is derived from the myeloma partner of the hybridoma; Vκ2 is derived from B-cell. Functional transcripts VH1 and Vκ2 and Vκ3 were then used to construct two chimeric antibodies chi-C2 (Vκ2-VH1) and chi-C3 (Vκ3-VH1), respectively. Antigen-binding experiments showed that only chi-C2 remained the same affinity as its parental mAb. Possible explanations for the coexistence of two functional κ-chain transcripts and the different affinity of the two chimeric antibodies are discussed.