Advanced glycosylation end product quantification: differently produced polyclonal antisera do not share the...

Advanced glycosylation end products (AGE) which are probably involved in the pathogenesis of diabetic complications, comprise a series of related chemical structures. Thus different antisera might recognize particular AGE epitopes rather than the complete range of epitopes. To test this hypothesis, two antisera were raised using different immunization techniques and different AGE-carrier proteins as immunogens. The antisera reactivity towards different AGE-proteins under various experimental conditions was compared. Both antisera recognized all AGE-proteins, although with different binding curves. Following pre-incubation with carboxymethyllysine-BSA (CML-BSA) (an oxidation-derived AGE) one antiserum partially retained its reactivity, suggesting recognition of non-oxidation-derived AGE. This result was confirmed both in the cross-reactivity and the preincubation experiments and when the reactivity of the antisera was tested against antigens incubated under oxidative and non-oxidative conditions. These results confirmed the hypothesis that differently produced antisera may not share the recognition of epitopes of different nature and suggest the necessity to adopt a standardized methodology for the production of antisera for an accurate and reproductible determination of the in vivo AGE concentration.