Purification of glycated hemoglobin free of hemoglobin A1c and its use to produce monoclonal antibodies specific for deoxyfructosyllysine [correction of deoxyfructosyllsine] residues in glycohemoglobin.
Hemoglobin nonenzymatically glycated at E-amino groups of lysine residues was purified from human erythrocyte lysates and used for immunization of BALB/c mice. Hybridomas secreting monoclonal antibodies for glycated hemoglobin were produced by fusion of mouse spleen cells with SP 2/0 myeloma cells. Immunoblotting with purified monoclonal antibody demonstrated specificity for glycated hemoglobin, with no reaction with HbAO. Glycated hemoglobin was effectively separated from other hemoglobins upon application of erythrocyte lysates to an affinity column of monoclonal antibody immobilized onto Sepharose 4B. A small fraction of purified HbA1c adsorbed to the monoclonal antibody affinity column, indicating that glycation can occur at both E-amino lysine and N-terminal valine positions in the same molecule. HbA1c did not react with the antibody after removal by immunoadsorption of molecules containing glycated lysine, confirming specificity of the antibody for deoxyfructosyl-lysine residues. The findings indicate that these monoclonal antibodies are site specific for glycated lysine amino groups in hemoglobin, and can provide rapid and efficient separation and identification of glycated hemoglobin in human erythrocyte lysates.
Wu
V Y
VY
Exocell, Inc., Philadelphia, PA 19104.
Steward
L A
LA
Cohen
M P
MP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D002846:Chromatography, Affinity; D005260:Female; D005632:Fructose / Q000031:analogs & derivatives* / Q000032:analysis; D006442:Glycated Hemoglobin / Q000276:immunology* / Q000302:isolation & purification; D006441:Hemoglobin A / Q000302:isolation & purification; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives* / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology
D000911:Antibodies, Monoclonal; D005632:Fructose; D006442:Glycated Hemoglobin; D006441:Hemoglobin A; D008239:Lysine