Figure 1

Quantitative binding of mAb to hCD200R-CD4d3+4. (A). Scheme illustrating a reverse phase microarray in which purified CD200R proteins were immobilized and then screened with fluorescent mAb specific for hCD200R or for the antigenic rCD4 tag (OX68) of the hybrid recombinant protein.(B). Typical microarray shows binding of OX68 mAb (red) to control proteins or the overlapping binding of hCD200R mAb and OX68 (yellow) to immobilized human CD200R.(C). Shows the green fluorescence intensity of each spot for all four replicates (mean ± SEM).(D). Shows red fluorescence intensity due to binding of OX68 mAb using data in left panel for DX147 (similar levels were found with the other mAb). Serial two-fold dilutions of purified, soluble, recombinant human and mouse CD200R-CD4d3+4 proteins, and of control rat CD4d3+4 were arrayed onto epoxy-coated microscope slides. Each protein dilution series was arrayed in 3 rows of 4 spots, ranging in concentration from 40 μg ml^-1 (first spot) to 0.08 μg ml^-1 (spot 10), with control spotting buffer containing 0.5 mg ml^-1 BSA in the last two spots. All arrays were performed in quadruplicate and a representative set is shown in (B). Each slide was incubated for 16 h at 4°C with a mixture of hCD200R mAb (DX147, DX136 or OX108) labelled with Alexa-555 (indicated as green fluorescence measured at 532 nm) and rCD4 mAb (OX68, detecting the antigenic tag and allowing for measurement of recombinant protein concentration) labelled with Alexa-647 (red fluorescence measured at 635 nm). At the highest concentrations, the hCD200R spots appear either white (saturating conditions) or yellow, due to the combination of green and red signals given by the specific binding of the Alexa-555-mAb to hCD200R and Alexa-647-OX68 mAb respectively. Quantitative measurements are expressed as mean fluorescence units at 532 nm (green) and 647 nm (red) versus amount of protein arrayed.