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M. Kitamura, P. G. Sreekumar, S. Sonoda, C. Spee, S. J. Ryan, R. Kannan, D. R. Hinton; Quantification of Expression of B-Crystallin in Mitochondria in Human Retinal Pigment Epithelial Cells and Its Secretion Using Enzyme-Linked Immunosorbent Assay (ELISA). Invest. Ophthalmol. Vis. Sci. 2010;51(13):1414. doi: https://doi.org/.
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The aim of the present study was to a) standardize and validate an enzyme-linked immunosorbent assay (ELISA) for quantification of αB-crystallin and b) to apply the assay for measurement of αB-crystallin in mitochondria and secretion into extracellular media of human cultured fetal RPE.
Early passage, confluent, cultured fetal human RPE cells and polarized RPE cells were used for the study. RPE cells were switched to serum free medium for 16 h and were then exposed to 150 µM tert-butylhydroperoxide (tBH) for 2-4 h. Cytosol and mitochondria were isolated using a Mitochondria/Cytosol Fractionation Kit for αB-crystallin determination by ELISA using reagents from Stressgen. In separate experiments, 24 h secretion of αB crystallin into extracellular medium was measured in polarized and non-polarized RPE under unstimulated conditions.
The ELISA standard curves were linear in the range 1.25 ng/ml-40 ng/ml (r2=0.9996). The levels of αB-crystallin (ng/mg protein) in cytosol and mitochondria of non-polarized RPE were 2522 ± 61 and 162 ± 4.7 respectively. tBH-induced oxidative stress caused an increase in mitochondrial expression of αB-crystallin while the cytosolic pool decreased. The 24 h secretion of αB-crystallin in non polarized RPE was 2.22 ± 0.55 ng/106 cells. αB-crystallin was selectively secreted to the apical side with negligible secretion to the basolateral side.
Our data show that αB-crystallin is expressed in mitochondria and is regulated by oxidative stress. Using polarized RPE, we could also obtain quantitative evidence for preferential secretion of αB-crystallin to the apical surface.
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