Feb 14, 2004

Alteration of amino acid residues at the L-chain N-terminus and in complementarity-determining region 3 increases affinity of a recombinant F(ab) for the human N blood group antigen

Transfusion
Shuh-Chyuny SongS L Spitalnik

Abstract

To examine the fine specificity of glycopeptide-specific antibodies, this study focused on the human MN blood group system. F(ab) phage display methods were previously used to construct an F(ab) family in which the H-chain Fd fragment was held constant whereas the L chains were "shuffled." This yielded two related F(ab), N92 and NNA7, with low and high affinity for N, respectively. Although their L-chain sequences are very similar, sharing 92 percent amino acid identity, there are intriguing differences at the N-terminus and in complementarity-determining region 3 (CDR3) at positions 89, 91, 92, and 96. Site-directed mutagenesis, ELISA, and hemagglutination were used to examine the contributions of these variations to antibody affinity. Studies with the N92-S91G and NNA7-G91S mutants demonstrated that the Gly at position 91 was critically important for ensuring high affinity. Indeed, the affinity of N92-S91G was almost as high as N92TM, in which all four CDR3 residues were changed to match NNA7. N-terminal L-chain differences were surprisingly important in determining affinity. For example, when the N-terminus of N92 was changed to match that of NNA7, affinity increased approximately 30-fold. Specific residues at the L-chain N-...Continue Reading

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Mentioned in this Paper

Transfection
Mutagenesis, Site-Directed
Hemagglutination
Phage Display
Tertiary Protein Structure
Chinese Hamster Ovary Cell
Indirect Immunofluorescence
CA9 protein, human
Fab Immunoglobulins
Antigens

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