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N. M. Kalariya, H. H. Herce, K. V. Ramana, S. K. Srivastava, F. J. G. M. Van Kuijk; Immunochemical Detection of Lutein Aldehydes-Protein Adducts in Human Donor Retinal Tissues. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3430.
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© ARVO (1962-2015); The Authors (2016-present)
Lutein-derived aldehydes (L-CDA) generated in the retinal tissues have been shown to bind with proteins through schiff’s base reaction and thioether linkage. Therefore, our aim was to develop an immunochemical method to detect of protein-Lutein aldehyde conjugates in human donor retinal tissues.
Proteins (KLH, BSA, GAPDH & G6PDH) were incubated with L-CDA to prepare conjugates. The conjugation was confirmed by performing MALDI-TOF, SDS-PAGE and amino acid analysis. Specific KLH-L-CDA conjugate antibodies were raised in rabbits and used immunochemically to detect the levels of L-CDA-protein conjugates in L-CDA treated ARPE-19 cells as well as in non-AMD and AMD donor eyes.
MALDI-TOF and SDS-PAGE analysis of L-CDA-protein conjugates revealed increase in the protein mass corresponding to protein adduct formation with L-CDA. Amino acid analysis of L-CDA-Protein conjugates revealed possible interaction of L-CDA with lysine and cysteine. The immuno-blot analysis revealed that L-CDA antibodies specifically bind to proteins conjugated with L-CDA. ARPE-19 cells treated with L-CDA exhibited presence of L-CDA-Protein conjugates. The retinal tissues from non-AMD and AMD donor eyes revealed presence of L-CDA-Protein conjugates.
Our results suggest that L-CDA could efficiently conjugate with protein. The antibodies developed against L-CDA-Protein efficiently recognized the adduct formation between protein and L-CDA. Using these antibodies, we introduce an immunochemical method to identify L-CDA-Protein conjugate in human retinal tissues. Based on these observations further studies are required to understand the delicate balance between beneficial and/or harmful effects of carotenoids as the therapy for AMD.
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