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


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.
Nayak
Sujatha
S
Department of Biochemistry, University College of Science, Osmania University, Hyderabad-500 007, India.
Sashidhar
R B
RB
Bhat
K Seetharam
KS
D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D002386:Cataract / Q000175:diagnosis / Q000209:etiology / Q000276:immunology*; D003459:Crystallins / Q000276:immunology* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D007908:Lens, Crystalline / Q000737:chemistry / Q000276:immunology; D008297:Male; D023421:Models, Animal; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar
D001323:Autoantibodies; D015415:Biomarkers; D003459:Crystallins