Production, crystallization and diffraction to atomic resolution of an antibody Fv specific for the blood-group A oligosaccharide antigen

The histoblood‐group ABO carbohydrate antigens are well known as important factors in blood transfusions, but they can also act as receptors for infectious agents and have been implicated in susceptibility to certain carcinomas. A single‐chain variable‐domain antigen‐binding fragment (scFv) gene bas...

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Bibliographic Details
Published in:Acta crystallographica. Section D, Biological crystallography. Biological crystallography., 1998-11, Vol.54 (6-2), p.1456-1459
Main Authors: Patenaude, Sonia I., MacKenzie, C. Roger, Bilous, Doris, To, Rebecca J., Ryan, Stephen E., Young, N. Martin, Evans, Stephen V.
Format: Article
Language:English
Subjects:
ABO Blood-Group System - immunology
Acetylgalactosamine - chemistry
Acetylgalactosamine - immunology
Animals
Antigen-Antibody Complex - chemistry
Carbohydrate Sequence
Cattle
Cloning, Molecular
Crystallization
Crystallography, X-Ray
Hemagglutinins - chemistry
Hemagglutinins - genetics
Hemagglutinins - isolation & purification
Immunoglobulin Fragments - chemistry
Immunoglobulin Fragments - genetics
Immunoglobulin Fragments - isolation & purification
Molecular Sequence Data
Oligosaccharides - immunology
Oligosaccharides - metabolism
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - isolation & purification
Serum Albumin, Bovine
Surface Plasmon Resonance
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Summary:The histoblood‐group ABO carbohydrate antigens are well known as important factors in blood transfusions, but they can also act as receptors for infectious agents and have been implicated in susceptibility to certain carcinomas. A single‐chain variable‐domain antigen‐binding fragment (scFv) gene based on the known sequence of an anti‐blood‐group‐A monoclonal antibody (AC1001) has been synthesized and expressed in Escherichia coli. The purified scFv preparation existed primarily in the monomeric form but also contained large amounts of dimeric and higher oligomeric forms. The corresponding variable‐domain antigen‐binding fragment (Fv) was generated by cleaving the VL–VH linker with subtilisin, and its activity was demonstrated by surface plasmon resonance with an immobilized bovine serum albumin A–trisaccharide conjugate (KD = 290 µM). AC1001 Fv crystals grown in the presence of N‐acetylgalactosamine diffracted to 0.93 Å resolution. This is the first reported example of a crystal of an antibody antigen‐binding fragment diffracting to atomic resolution.
ISSN:1399-0047
0907-4449
1399-0047
DOI:10.1107/S0907444998005824