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K-I Endo, S Kawasaki, S Kinoshita; An IGG FC Binding Protein in Human Corneal Epithelium . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1670.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Rabbit IgG has a binding behavior toward human corneal epithelium that, in rabbit IgG used as negative control, has been a cause of trouble in corneal immunohistochemistry. This study was carried out in order to elucidate the binding mechanism. Methods: Sections of human cornea were respectively immunostained with negative control grade rabbit IgG, F(ab')2 (fragment of IgG yielded by pepsin digestion) and Fc fragment. Gene expression of IgG binding proteins, including Fc gamma receptors (CD16, CD32 and CD64), on human corneal epithelium was investigated by RT-PCR; the corneal sections were also respectively stained with antiCD16, CD32 and CD64 antibodies. Through use of a rabbit IgG-linked affinity column, the binding materials were selectively collected from cell lysate of human corneal epithelial cells and resolved in SDS-PAGE. Results: With negative control grade rabbit IgG, intracellular staining was observed in all layers of the epithelium; Fc fragment also produced a similar immunohistochemical result. However, almost all of the positive staining disappeared with use of the F(ab')2 fragment instead of IgG. All Fc gamma receptor mRNAs were detected from human corneal epithelium. Although all the proteins were also detected on the cornea immunohistochemically, all the immunostaining patterns by antiCD16, CD32 and CD64 antibodies were scattered and faint, and differed from that of rabbit IgG. With the rabbit IgG-linked affinity column, a binding protein of molecular weight approximately 50kDa and several minor proteins were successfully obtained from human corneal epithelial cells. Conclusion: These data indicate that rabbit IgG bound to human corneal epithelium via the Fc region of the molecule, suggesting that unique Fc binding proteins in corneal epithelium, apart from the Fc gamma receptors are involved in the binding mechanism.
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