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ORIGINAL ARTICLE
Year : 2002  |  Volume : 50  |  Issue : 1  |  Page : 41-48

Development of an immunoanalytical method for the detection of β- and γ- Crystallins and anti-crystallin antibodies. A molecular biomarker for cataract


Department of Biochemistry, University College of Science, Osmania University, Hyderabad-500 007, India

Correspondence Address:
S Nayak
Department of Biochemistry, University College of Science, Osmania University, Hyderabad-500 007
India
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Source of Support: None, Conflict of Interest: None


PMID: 12090086

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Purpose: To develop and evaluate an immunoanalytical method for the detection of β - and g-crystallins and anti-crystallin antibodies. Materials and Methods: Beta and g-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 β - and g-crystallins were specific. An indirect competitive enzyme linked immunosorbent assay (ELISA) developed to quantitate β - and g-crystallin showed an IC50 value of 70 ng and 65 ng, respectively, based on regression analysis. Spiking studies with purified β -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 β - and g-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 β - and g-crystallin antibodies, indicating that these crystallins leak during cataract formation. The concentration of β - and g-crystallins in the rat serum, as analysed by indirect competitive ELISA, was found to be in the range of 17.6 - 81.6 mg/l and 12.4- 19.6 g/ml, respectively. Conclusions: The methodology developed in the present study may find application as a biochemical tool in molecular epidemiology of cataract


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