Ideas Exchange Column - "Electrolytic Water Analyzer"

Connolly, Kelly

doi:10.1021/ie50562a601

Cited by 1 publication

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“…The model was visualized and adjusted using the programs FRODO (22) and SETOR (23). The molecular surface of the anti-A Fv was generated using MS (24), and the A-trisaccharide antigen was modeled into the Fv-binding site using SETOR. The Fv crystallized in the space group P2 1 2 1 2 1 with cell dimensions a ϭ 51.7, b ϭ 58.0, and c ϭ 83.6 Å.…”

Section: Methodsmentioning

confidence: 99%

Structure of an Anti-blood Group A Fv and Improvement of Its Binding Affinity without Loss of Specificity

Thomas¹,

Patenaude²,

MacKenzie³

et al. 2002

Journal of Biological Chemistry

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The specificity of antibody recognition of the ABO blood group trisaccharide antigens has been explored by crystal structure analysis and mutation methods. The crystal structure of the Fv corresponding to the antiblood group A antibody AC1001 has been determined to 2.2-Å resolution and reveals a binding pocket that is complementary to the blood group A-trisaccharide antigen. The effect of mutating specific residues lining this pocket on binding to the A and B blood group oligosaccharide antigens was investigated through a panel of single point mutations and through a phage library of mutations in complementarity determining region H3. Both approaches gave several mutants with improved affinity for antigen. Surface plasmon resonance indicated up to 8-fold enhancement in affinity for the Apentasaccharide with no observable binding to the blood group B antigen. This is the first example of single point mutations in a carbohydrate-binding antibody resulting in significant increases in binding affinity without loss of specificity.The affinity of anti-carbohydrate antibodies for their antigens is commonly observed to be 3-5 orders of magnitude lower than affinities of anti-protein or anti-peptide antibodies for their antigens, yet there is no clear mechanism to explain this phenomenon. One means of exploring this question is to attempt to generate mutant anti-carbohydrate antibodies with higher affinities, either by design of site-directed mutants or by randomizing selected codons in a phage library. However, the production of antibodies with improved affinities that maintain antigen specificity has proven challenging (1-4). For example, in an exhaustive site-directed mutagenesis study of CDR 1 H3 of an anti-Salmonella Fab, Brummell et al. (1) found that all of the 90 mutant Fabs produced showed similar or decreased binding affinity for the O-antigen. Mutants of the anti-Lewis Y antibody BR96 were obtained as phage-displayed scFv; although the mutant scFv binding affinity had increased by up to 6-fold versus the native antibody, its specificity was altered (2, 3). More recently, higher affinity antibodies against levan, a model for the polysaccharide capsules of bacteria, were obtained by mutational analysis though their fine specificity varied from that of the parent antibody (5). In contrast, significant enhancements have been achieved with antibodies to protein antigens. For example, a mutant scFv of an anti-c-erbB-2 tumor antigen obtained by chain shuffling and phage display had a 5-6-fold increase in affinity (4). In a later study by the same group (6) sequential mutation of light and heavy chains of the scFv yielded 16-and 9-fold increases in affinity for the protein antigen. Yang et al. (7) obtained mutant anti-HIV-1 Fab by phage-display techniques with a 96-fold increase in binding, even though the native antibody fragments already possessed high affinity for the protein antigen. Though phage-display techniques have been reported to yield higher binding mutant scFvs, the resulting antibodies often contain mult...

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Section: Methodsmentioning

confidence: 99%