Isolation of an IgG anti-B from a human Fab-phage display library.
BACKGROUND
ABO incompatibility is a common cause for mild hemolysis in the newborn, ranging from 1 in 30 to 1 in 150 births. Fortunately, hemolysis requiring transfusion is rare and restricted to blood group O mothers, because blood group A and B individuals make poor IgG anti-B and anti-A responses. No human IgG ABO antibody sequences have been reported, in part because of the difficulty in obtaining human IgG hybridomas. Phage-display technology may be able to circumvent these difficulties, but its application to carbohydrate antigens is poorly studied.


METHODS
A human IgG1 phage-display Fab library was constructed from splenocytes derived from a nonhyperimmunized blood group O person, and panned against group B RBCs.


RESULTS
After five rounds of panning, essentially all phage bound to group B RBCs. Nucleotide sequence analysis of a single monoclonal IgG1lambda phage, FB5.7, revealed a highly mutated VH4 family heavy chain, and a nearly germline VL7 family lambda light chain. The Fab agglutinated group B, but not group A, random-donor RBCs. However, group B ELISA reactivity could be inhibited by soluble B-trisaccharide, soluble A-trisaccharide, galactose, and N-acetyl galactosamine. Similarly, galactose and N-acetyl galactosamine were able to inhibit group B RBC agglutination.


CONCLUSIONS
FB5.7 is the first human IgG ABO MoAb described. Alhough it behaves serologically like a group B-specific antibody, it demonstrates interaction with both the A and B epitopes. Phage-display technology can be used to better define the relationship between antibody genotype and phenotype in anti-carbohydrate responses in nonhyperimmunized hosts, and thus to improve our understanding of the composition of the antibody repertoire.
Chang
T Y
TY
Department of Pathology and Laboratory Medicine, University of Rochester, Rochester, New York, USA.
Siegel
D L
DL
D000017:ABO Blood-Group System / Q000276:immunology*; D000328:Adult; D000595:Amino Acid Sequence / Q000235:genetics; D000918:Antibody Specificity; D018095:Germ-Line Mutation / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007146:Immunoglobulin lambda-Chains / Q000235:genetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D017386:Sequence Homology, Amino Acid
D000017:ABO Blood-Group System; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D007146:Immunoglobulin lambda-Chains; D019151:Peptide Library