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Specificity and Applicability of Advanced Glycated Albumin in Competitive ELISA-type Assays

Background/Aim The competitive ELISA-type assays for advanced glycation endproduct (AGE) measurement based on anti-AGE-antibody and in vitro prepared AGE-carrier competitive antigen. However, AGEs prepared by letting protein and sugar to react under conditions that, due to differences in e.g. pH values, temperature, sugar concentration or trace amounts of metal ions, resulted in variety of glycated adducts. AGE-human serum albumin (HSA), prepared by a non-enzymatic reaction between protein and glucose, is often used competing antigen and this study aimed to examine the impact of pH on its preparation. Methods AGEs derived from HSA and glucose were analyzed by UV/VIS spectrophotometry, gel filtration chromatography, and isoelectrofocusing. Multiple standard rabbit immunization by AGE-HSA was used in the preparation of AGE-specific antiserum. We developed our own competitive ELISA method by polyclonal AGE-antibodies using in vitro prepared AGE-HSA at pH of 4.5, 6.5, 7.5 and 8.0. Results Gel chromatography revealed marked appearance of albumin dimmers and polymers, and absorbance spectrum showed the appearance of different families within 230-350 nm wavelength range. The absorbance of AGE-HSA set with baseline pH 8.0 increased with the incubation time of 2, 4, 6 and 12 weeks. The results obtained indicated a slight hyperbolic decrease in pH relative to the time of incubation in the samples with initial pH values of 7.5 and 8.0. An acidity increase was evident in all other samples with initial pH values 4.5-6.5 ; however, it was moderate and showed linear dependence on glycation duration. Isoelectrofocusing analysis of glycated HSA in comparison with native HSA shows pronounced changes in surface charge of glycated protein. Initial pH was 8.0 in all samples, but final pH changed for Δ of 0.35 to 1.25 pH units, depending on the glycation period. The measurement of AGE-content in normal and two diabetic pool sera performed in parallel showed highest detection level by the competitive antigen prepared at pH 4.5. The detection limit declined by ~50% when using competitive antigens prepared at pH 6.5, 7.5 or 8.0. This observation confirmed the impact of pH on the glycation adduct formation, which in turn significantly influenced the results of competitive ELISA detection. Conclusion The heterogeneity of AGE-derived competitive antigens significantly influenced the results of competitive ELISA measurements. This might be explained by the fact that glycation reactions at lower pH values occur at a lower rate, resulting in the formation of minimally modified glycated adducts. It may be presumed that these antigens resembled more physiologically derived AGEs and therefore yielded better results in competitive ELISA analysis of human serum proteins. Detection of clinically relevant AGEs should be included as an important diagnostic parameter involved in the risk of diabetes complications. However, as long as there is no standard method of their determination, the results obtained in different laboratories or even the results of long-term patient follow up testing performed at the same laboratory cannot be compared.

advanced glycation endproduct; competitive ELISA-type assays

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