抗CD8モノクローナル抗体VH領域の解析

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  • Analysis of the Immunoglobulin Heavy Chain Variable Region of Hybridomas Producing anti-CD8 Monoclonal Antibodies.

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We analyzed the nucleotide sequence of immunoglobulin （Ig） heavy chain variable regions （VH） of seven hybridomas which produce anti-CD8 monoclonal antibodies. These seven antibodies were classified into four groups according to the results of a cell binding inhibition assay. The assay also indicated that CD8 antigen expressed at least four different epitopes. The VH nucleotide sequences were classified in three families. Three clones belonged to VH subgroup I（B）, and two to VH subgroup V（A）. Sequences in members of each family showed identical V-D-J recombinations, suggesting that the clones of each family arose from common ancestral clones. Some somatic mutations were observed in both the framework segments and the complementarity determining regions （CDR） of the I（B） clones, and only one amino acid substitution was observed. Two clones of the V（A） subgroup possessed an identical VH sequence. Although the clones of each family had originated from single ancestral clones, reactivity within families with CD8 antigen was found to vary widely. At the same time, two clones derived from different fusions, on the basis of their equal reactivities with CD8, were classified into subgroup II（A） ; subsequently, they were shown to share the same V-D-J combination. The speeds of reaction of the antibodies with CD8 antigen were investigated by flow cytometry. The observed rates of increase of brightness and final saturated brightness with these antibodies were quite different. Those results indicated that somatic mutations occurring in the VH gene of the anti-CD8 antibodies cause differences in their affinity with epitopes. Two separately originated clones that were induced by the same CD8 antigen showed identical paratopes and VH usage.

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