| Assay Method Information | |
| | HLA-DR4 HTRF Assay |
| Description: | In this assay, anti-human Fc antibody labeled with Tb-Cryptate probe was used as the donor (commercially available from Cisbio—PAb Anti-human IgG-Tb Cryptate, Cat #61HFCTAB) and high affinity peptide Citrullinated CII (cit-CII) labeled with Alexa 647 fluorophore was utilized as the acceptor. Inhibitors of the exchange of the pre-cleaved CLIP peptide for the high affinity cit-CII1236-1249 peptide onto HLA-DRB1*04:01 caused a decrease in HTRF signal as measured by the ratio of the 665 nm/620 nm emitted signal. Screening compounds were robotically pre-dispensed (150 nL, Hummingbird, DigiLab/CyBio AG) onto black, round bottom, nontreated, 1536-well assay plates (Corning #3939), were incubated with 3 nM HLA-DR4 (with CLIP cleaved) plus 1 nM Human IgG-Tb (anti-Fc-Tb) Antibody for 45 min prior to addition of 40 nM Cit-CII peptide labeled with Alexa-647. The negative control was DMSO plus 1 nM Human IgG-Tb (anti-Fc-Tb) Antibody, incubated for 45 minutes before addition of 40 nM Cit-CII peptide labeled with Alexa-647. The positive control was DMSO plus 3 nM HLA-DR4 (with CLIP cleaved) plus 1 nM Human IgG-Tb (anti-Fc-Tb) Antibody with 40 nM Cit-CII peptide labeled with Alexa-647 added after a 45-minute incubation. Plates were centrifuged at 1000 rpm for 1 minute and then incubated at 37° C., at 90% relative humidity for 90 minutes. After incubation, plates were read on a spectrophotometer, the Pherastar FS (BMG Labtech), in HTRF mode. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |