Development of an immunoanalytical method for the detection of beta- and gamma-crystallins and anti-crystallin antibodies. A molecular biomarker for cataract.

ABSTRACT

PURPOSE: To develop and evaluate an immunoanalytical method for the detection of beta- and gamma-crystallins and anti-crystallin antibodies. MATERIALS AND METHODS: Beta and gamma-crystallins isolated from rat lens were used as immunogens to raise polyclonal antibodies in rabbits. Antibody capture assay and western blot analysis showed that the antibodies to beta- and gamma-crystallins were specific. An indirect competitive enzyme linked immunosorbent assay (ELISA) developed to quantitate beta- and gamma-crystallin showed an IC50 value of 70 ng and 65 ng, respectively, based on regression analysis. Spiking studies with purified beta-crystallin antibodies showed that 33 ng of the purified antibody gave an absorbance of 1.1 at 450 nm, indicating the sensitivity of the method. RESULTS: Antibodies to beta- and gamma-crystallins were not detected in serum samples of the cataractous CFY/NIN rats (used as an animal model for induction of experimental cataract by feeding high galactose diet). However, the cataractous rat serum samples effectively displaced beta- and gamma-crystallin antibodies, indicating that these crystallins leak during cataract formation. The concentration of beta- and gamma-crystallins in the rat serum, as analysed by indirect competitive ELISA, was found to be in the range of 17.6-81.6 micrograms/ml [corrected] and 12.4-19.6 micrograms/ml, respectively. CONCLUSIONS: The methodology developed in the present study may find application as a biochemical tool in molecular epidemiology of cataract.