Production and characterization of monoclonal antibodies against non-A1c glycated hemoglobin.

Hybridomas secreting monoclonal antibodies specific for hemoglobin nonenzymatically glycated in the non-A1c position were produced by fusion of SP 2/0 myeloma cells with spleen cells from BALB/c mice immunized with nonenzymatically glycated hemoglobin prepared from human erythrocytes. Wells containing hydridomas secreting antibodies against glycohemoglobin were identified by binding, in an enzyme-linked immunosorbent assay, to purified glycated hemoglobin. The colony designated E85, which secreted antibodies discriminating between glycated versus unglycated hemoglobin, was cloned at least four times by limiting dilution and used for further study, performed with purified monoclonal antibody. Specificity of E85 was demonstrated by immunoblotting and by ELISA, wherein the monoclonal antibody reacted with glycated hemoglobin but not with hemoglobin A1c or with unglycated hemoglobin. Immunoblotting of human plasma with E85 on nitrocellulose yielded no reactive proteins, indicating site specificity for glycated epitopes residing in hemoglobin but not in other nonenzymatically glycated proteins present in plasma. E85 differs from other antibodies raised against glycated hemoglobin and other glycated proteins, which recognize hemoglobin glycated at the N terminal valine of the beta chain (HbA1c) or which recognize glycated residues only after reductive conversion to glucitollysine and which do not discriminate between different glycated proteins. Thus, this report describes the establishment of the first hybridoma secreting monoclonal antibody raised against a physiologic (unreduced) form of non-A1c glycohemoglobin, and for the glycated epitope when it resides in glycohemoglobin but not in other proteins or in hemoglobin A1c.

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.