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

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.

Lens protein phylogeny: immunocrossreactivity of vertebrate lens antigens to anti-shark crystallin antibody.

Antisera prepared against total water-soluble lens proteins of the shark, Scoliodon sorrakowah were reacted with homologous antigen and analysed reaction products by immunoelectrophoresis (IE) and two dimensional crossed antigen-antibody electrophoresis (2D-CE). On IE, shark antigens formed 5 precipitin lines including 1 alpha, 3 beta and 1 gamma crystallins and on 2D-CE 3 alpha, 6 beta and 6 gamma peaks accounting for 8%, 27% and 65% antigen in the respective group were obtained from the total crystallins. Using anti-shark antisera, the immunocrossreactivity of lens proteins from 6 Chondropterygii, 23 teleosts and 16 higher vertebrates was examined by IE. It is found that beta crystallins are the most conserved and crossreact with all vertebrate classes, whereas gamma crystallin crossreactivity is specific to the class Pisces and alpha crystallins are least conserved and their crossreactivity is confined to subclass Chondropterygii. Based on IE patterns, a phylogenetic tree is constructed which demonstrates the intrafamily closeness except in case of adaptive radiation.

Antibodies to lens crystallins after endocapsular cataract surgery

Levels of serum antibodies against lens alpha, beta H and beta L crystallins were determined in 15 patients before and after endocapsular cataract extraction. The most abundant antibody at the time of surgery was anti-beta L, followed by anti-beta H and anti-alpha. An increase in the basal levels of these antibodies was seen after surgery in 93 percent of the cases. Relative increase was highest for anti-alpha, followed by anti-beta H and anti-beta L. There were individual variations both in the shape of the time-dependent antibody response after surgery and in the relative intensity of the response to the different antigens. The endocapsular technique therefore leads to the release of crystallins into the anterior chamber in sufficient amounts to provoke in many patients a humoral immune response