Journal: PLoS ONE

Article Title: Human and Mouse CD137 Have Predominantly Different Binding CRDs to Their Respective Ligands

doi: 10.1371/journal.pone.0086337

Figure Lengend Snippet: (A) Diagram of hCD137 entire extracellular region and hE, hE2, hE3, hE4, hE300, hE3003 and hE3004 represent the expressed hCD137 segments covering different CD137 CRDs used in this study. (B) Identification of purified expressed series of Fc-fusion proteins with Ponceau S staining of SDS-PAGE gel (left top panel) and ECL (right panel). hE, hE2, hE3, hE4 and hCD137L-hFc (hL-hFc ) were expressed by pCDH and pCDH-L respectively.hE300, hE3003, hE3004 and hCD137L-mFc ( hL-mFc ) were expressed by pCDM-L. (C) Binding of hCD137L to various recombinant hE and subdomains by ELISA assay. Affinity-purified hCD137L-mFc (1 µg/ml) was coated on plastic, followed by addition various hCD137 fragments expressed supernatant, then detected by HRP-goat-anti-human IgG. (D) hCD137L-hFc (1 µg/ml) was coated, followed by addition hE300, hE3003 and hE3004 fragments expressed supernatant and also an unrelated epithelial cell adhesion molecule (Epcam) protein with mFc, the binding detected by HRP-goat-anti-mouse IgG., Similar results were obtained in all of 3 individual experiments. *P/N>2.1.

Article Snippet: In brief, anti-human CD137 mAb h4-1BB-M127 (BD Pharmingen, San Diego, CA, USA) was used as a capture antibody, and a serially diluted solution of hCD137-hFc fusion proteins (Sino Biological Inc, 10041-H03H, China) with a purity over 95% was used as reference standard.

Techniques: Purification, Staining, SDS Page, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Affinity Purification

Journal: PLoS ONE

Article Title: Human and Mouse CD137 Have Predominantly Different Binding CRDs to Their Respective Ligands

doi: 10.1371/journal.pone.0086337

Figure Lengend Snippet: hE3, which is hIgG1-Fc fusion protein expressed by pCDH, containing hCD137 CRD I+II. hCD137L-mFc was coated at 1 µg/ml and a series of decreasing concentration of hE3, 10 µg/ml, 5 µg/ml, 1 µg/ml and 0.2 µg/ml were added, followed by detecting with HRP-goat-anti-human IgG. The experiment was repeated 3 times with different batch supernatants.

Article Snippet: In brief, anti-human CD137 mAb h4-1BB-M127 (BD Pharmingen, San Diego, CA, USA) was used as a capture antibody, and a serially diluted solution of hCD137-hFc fusion proteins (Sino Biological Inc, 10041-H03H, China) with a purity over 95% was used as reference standard.

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Human and Mouse CD137 Have Predominantly Different Binding CRDs to Their Respective Ligands

doi: 10.1371/journal.pone.0086337

Figure Lengend Snippet: mCD137 extracellular region (mE) and hCD137 extracellular region (hE) were run on 10% SDS-PAGE gel without 2-ME, then stained by Ponceau S(left panel) and followed by Western blotting with hCD137L-mFc and HRP-goat-anti-mouse IgG for detection (ECL,right panel).

Article Snippet: In brief, anti-human CD137 mAb h4-1BB-M127 (BD Pharmingen, San Diego, CA, USA) was used as a capture antibody, and a serially diluted solution of hCD137-hFc fusion proteins (Sino Biological Inc, 10041-H03H, China) with a purity over 95% was used as reference standard.

Techniques: SDS Page, Staining, Western Blot

Journal: PLoS ONE

Article Title: Human and Mouse CD137 Have Predominantly Different Binding CRDs to Their Respective Ligands

doi: 10.1371/journal.pone.0086337

Figure Lengend Snippet: Panel A shows the binding of the two mAbs to hE, hE2, hE3, hE4 and control. Panels B, C and D shows an experiment where human CD137L-mFc (hL) was coated (1 µg/ml) on an ELISA plate, blocked with milk and followed by adding hE(1 µg/ml) which had been preincubated with h41BB-M127 (B), 4B4-1 (C) and an anti-human CD3, OKT3 (D) at a concentration of 5 µg/ml or 25 µg/ml. (E) Inhibition of the binding of hCD137L to hCD137 at a series of concentrations of anti-human CD137mAbs.

Article Snippet: In brief, anti-human CD137 mAb h4-1BB-M127 (BD Pharmingen, San Diego, CA, USA) was used as a capture antibody, and a serially diluted solution of hCD137-hFc fusion proteins (Sino Biological Inc, 10041-H03H, China) with a purity over 95% was used as reference standard.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Inhibition