Immunological studies on glycated human IgG.
OBJECTIVE
To study the immunogenicity of advanced glycation end product (AGE) modified IgG (AGE-IgG) in experimental animals.


METHODS
Human IgG was subjected to in vitro glycation with glucose and the formation of N(ε)-(carboxymethyl)lysine (CML) was evaluated by high performance liquid chromatography (HPLC). The immunogenicity of native and AGE-IgG was investigated by raising polyclonal antibodies against them in rabbits. The induced antibodies were purified on a Protein-A agarose affinity column. Specific binding of antibodies was screened by competitive inhibition assay and band shift assay. Cross reactions of induced antibodies with various proteins or amino acids and their glycated conformers were ascertained by competitive inhibition ELISA.


RESULTS
We detected the CML formation in AGE-IgG. The AGE-IgG was found to be highly immunogenic due to the generation of neo-epitopes on it. Affinity purified antibodies exhibited high degree of specific binding with AGE-IgG in comparison to the native IgG. Antibodies against AGE-IgG exhibited diverse antigen binding characteristics and the glycated conformers of various proteins and amino acids were found to be effective inhibitors of antibody-immunogen interaction in cross reaction studies. Band shift assay reiterated the results obtained by direct binding and competitive inhibition assay.


CONCLUSIONS
The induced antibodies against AGE-IgG resembled the diverse antigen binding characteristics of autoantibodies found in rheumatoid arthritis (RA). IgG modified by AGEs under oxidative stress presents unique neo-epitopes which may be one of the factors for the induction of autoantibodies in RA patients.
Department of Biochemistry, J.N. Medical College, Faculty of Medicine, Aligarh Muslim University, Aligarh, India.
Saman
S
Ahmad
Moinuddin
Asif
A
Ali
D000818:Animals; D001666:Binding Sites, Antibody; D001667:Binding, Competitive / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D017127:Glycation End Products, Advanced / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics* / Q000378:metabolism*; D011817:Rabbits
D017127:Glycation End Products, Advanced; D007074:Immunoglobulin G