Immunochemical detection of glycated lens crystallins and their circulating autoantibodies in human serum during aging

PURPOSE The aim of this investigation was to exploit lens-specific glycated crystallins as an immunogen to detect human glycated crystallins and their circulating autoantibodies in human serum during aging in relation to the development of cataract. METHODS Polyclonal antibodies were produced against human total lens proteins (40-80 years) in rabbits. The specificity of the antibodies produced were determined by antibody capture assay using purified human lens crystallins (high molecular weight fraction [HMW]+alpha, HMW+alpha-glycated, beta, beta-glycated, gamma, and gamma-glycated) as antigens. The cross-reactivity of these lens specific antibodies against rat beta-, beta-glycated, gamma-, and gamma-glycated lens crystallins was also analyzed. A non-competitive enzyme linked immunosorbent assay (ELISA) methodology was developed for the detection of circulating lens crystallins in human sera using HMW+alpha, HMW+alpha-glycated, beta-, and beta-glycated crystallins from humans and gamma- and gamma-glycated crystallins from rats as immobilized antigens. Circulating autoantibodies were also detected in human sera by antibody capture assay. The methodology was validated by evaluating 60 human serum samples collected from cataract patients and 30 human serum samples from apparently normal subjects belonging to the same age group. RESULTS The polyclonal antibodies raised against human total lens proteins showed 90% and 65% cross-reactivity with rat gamma- and beta-crystallins, respectively, by ELISA. Further, these polyclonal antibodies were capable of detecting both native and in vitro synthesized glycated crystallins. Their IC50 values were observed to be (i) human total lens proteins (55 ng), (ii) human HMW+alpha (16.45 ng), (iii) human HMW+alpha-glycated (273 ng), (iv) human beta- (37.82 ng), (v) human beta-glycated (260 ng), (vi) rat gamma- (105.34 ng), and (vii) rat gamma-glycated (313 ng). The immunochemical analysis of human serum indicated a significant change (p<0.001) in the levels of circulating beta-glycated and gamma-glycated crystallins in the age group of 40-80 years with respect to their control groups. However, there was no statistically significant change in the levels of HMW+alpha-glycated crystallins in the age group of 40-80 years as compared to their age-matched controls. Notably, the levels of serum gamma-glycated crystallins were found to be threefold higher than that of HMW+alpha-glycated and beta-glycated crystallins in the age group of 70-80 years. Circulating autoantibodies to HMW+alpha-glycated, beta-glycated, and gamma-glycated crystallins were detected in the serum of both apparently normal and cataract patients in the age group of 40-80 years by antibody capture assay. The levels of these autoantibodies were significantly higher at every time point compared to their respective controls. Autoantibodies to gamma-glycated crystallins were found to be twofold and 3.2 fold higher as compared to the levels of autoantibodies to beta-glycated and HMW+alpha-glycated crystallins, respectively. Western blot and immunohistochemical analysis substantiated the observations made in non-competitive ELISA. CONCLUSIONS During the course of aging, leakage of lens crystallins (HMW+alpha, HMW+alpha-glycated, beta, beta-glycated, gamma, and gamma-glycated) elicit an immune response resulting in the formation of autoantibodies in cataract patients (40-80 years) as compared to age matched controls. This is the first experimental report where polyclonal antibodies raised against lens-specific glycated crystallins were capable of detecting the early leakage of glycated crystallins in human subjects. This immunochemical approach has implications in the early detection of senile cataract.