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. 2000 Oct 2;192(7):1027–1034. doi: 10.1084/jem.192.7.1027

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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The binding of PD-1 to PD-L1. (A) CHO cells stably transfected with human or murine PD-L1 or vector alone were stained with hPD-1.Ig(γ2a), mPD-1.Ig(γ1), hCTLA-4.Ig(γ2a), hCD28.Ig(γ1), or hICOS.Ig(γ2a) (species matched), and developed with goat anti–murine IgG2a-PE or anti–human IgG-FITC antisera. (B) PD-L1.Ig was tested for binding to immobilized mPD-1.Ig(γ1) (gray bars), hPD-1.Ig(γ1) (black bars), and hCTLA-4.Ig(γ1) (white bars) using surface plasmon resonance on a BIAcore 2000 instrument. Receptor-Fc fusion proteins were immobilized on a CM5 dextran chip by amine coupling with normal human serum/N-ethyl-N′-(dimethylamino)propyl I carbodiimide hydrochloride (EDC) in 10 mM sodium acetate, pH 4.0, as described (reference 27). The amounts of protein immobilized were 5,383 response units (RU) for mPD-1.Ig(γ1), 5,416 RU for hPD-1.Ig(γ1), and 11,493 RU for hCTLA-4.Ig(γ1). Concentrated COS-conditioned medium from hPD-L1.Ig(γ2a)–transfected cells was analyzed with (+) or without (−) coinjection of 100 μg/ml of soluble mPD-1.Ig, hPD-1.Ig, or hCTLA-4.Ig for competition. Binding was quantified as an increase in RU at 60 s after the end of injection compared with a baseline established 20 s before injection.

The binding of PD-1 to PD-L1. (A) CHO cells stably transfected with human or murine PD-L1 or vector alone were stained with hPD-1.Ig(γ2a), mPD-1.Ig(γ1), hCTLA-4.Ig(γ2a), hCD28.Ig(γ1), or hICOS.Ig(γ2a) (species matched), and developed with goat anti–murine IgG2a-PE or anti–human IgG-FITC antisera. (B) PD-L1.Ig was tested for binding to immobilized mPD-1.Ig(γ1) (gray bars), hPD-1.Ig(γ1) (black bars), and hCTLA-4.Ig(γ1) (white bars) using surface plasmon resonance on a BIAcore 2000 instrument. Receptor-Fc fusion proteins were immobilized on a CM5 dextran chip by amine coupling with normal human serum/N-ethyl-N′-(dimethylamino)propyl I carbodiimide hydrochloride (EDC) in 10 mM sodium acetate, pH 4.0, as described (reference 27). The amounts of protein immobilized were 5,383 response units (RU) for mPD-1.Ig(γ1), 5,416 RU for hPD-1.Ig(γ1), and 11,493 RU for hCTLA-4.Ig(γ1). Concentrated COS-conditioned medium from hPD-L1.Ig(γ2a)–transfected cells was analyzed with (+) or without (−) coinjection of 100 μg/ml of soluble mPD-1.Ig, hPD-1.Ig, or hCTLA-4.Ig for competition. Binding was quantified as an increase in RU at 60 s after the end of injection compared with a baseline established 20 s before injection.