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Clinical and Diagnostic Laboratory Immunology, November 2004, p. 1085-1088, Vol. 11, No. 6
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.6.1085-1088.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Improved Affinity of a Human Anti-Entamoeba histolytica Gal/GalNAc Lectin Fab Fragment by a Single Amino Acid Modification of the Light Chain

Departments of Infectious Diseases,¹ Molecular Life Sciences,³ Laboratory for Molecular Science Research, Tokai University School of Medicine, Isehara, Kanagawa, Japan²

Received 18 August 2004/ Returned for modification 2 September 2004/ Accepted 9 September 2004

We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose- and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica. To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser⁹¹ and Arg⁹⁶ in the third complementarity-determining region of the light chain with other amino acids. The screening of 200 clones of each exchange by an indirect fluorescent antibody test showed that 14 clones for Ser⁹¹ and nine clones for Arg⁹⁶ reacted strongly with E. histolytica trophozoites. Sequence analyses revealed that the substituted amino acids at Ser⁹¹ were Ala in five clones, Gly in three clones, Pro in two clones, and Val in two clones, while the amino acid at position 96 was substituted with Leu in three clones. The remaining eight clones exhibited no amino acid change at position 91 or 96. These mutant Fab fragments were purified and subjected to a surface plasmon resonance assay to measure the affinity of these proteins to the cysteine-rich domain of lectin. Pro or Gly substitution for Ser⁹¹ caused an increased affinity of the Fab, but substitution with Ala or Val did not. The replacement of Arg⁹⁶ with Leu did not affect affinity. These results demonstrate that modification of antibody genes by recombination PCR is a useful method for affinity maturation and that amino acid substitution at position 91 yields Fabs with increased affinity for the lectin.