title: Improved affinity of a human anti-Entamoeba histolytica Gal/GalNAc lectin Fab fragment by a single amino acid modification of the light chain.
abstract: 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.
authors.affiliation: Department of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan. htachiba@is.icc.u-tokai.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tachibana
authors.forename: Naohisa
authors.initials: N
authors.lastname: Matsumoto
authors.forename: Xun-Jia
authors.initials: XJ
authors.lastname: Cheng
authors.forename: Hideo
authors.initials: H
authors.lastname: Tsukamoto
authors.forename: Eisaku
authors.initials: E
authors.lastname: Yoshihara
mesh_terms: D000116:Acetylgalactosamine / Q000276:immunology; D019943:Amino Acid Substitution / Q000235:genetics* / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D000913:Antibodies, Protozoan / Q000235:genetics* / Q000276:immunology; D000915:Antibody Affinity / Q000235:genetics* / Q000276:immunology; D000953:Antigens, Protozoan / Q000276:immunology; D004748:Entamoeba histolytica / Q000276:immunology*; D005690:Galactose / Q000276:immunology; D037161:Galectins / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D037161:Galectins; D007140:Immunoglobulin Fab Fragments; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; D000116:Acetylgalactosamine; D005690:Galactose