Figure 2

Analysis of mAb reactivity with hCD200R by orientation via antibody immobilization. (A). Scheme of one forward phase microarray in which purified human CD200R protein was immobilized via OX68 mAb and detected with the DX136 hCD200R mAb (green fluorescence).(B). Typical microarray shows binding of the hCD200R mAb (green) and OX68 mAb (red) to hCD200R immobilized via four different capture mAb.(C). Shows the mean fluorescence intensity ± SEM for each spot of all replicates. Serial two-fold dilutions of capture human CD200R mAb OX108, DX136 and DX147 and control rat CD4 mAb OX68 were arrayed onto epoxy-coated microscope slides. Each mAb dilution series was arrayed in quadruplicate of 2 rows of 6 spots, ranging in concentration from 80 μg ml^-1 (first spot) to 0.16 μg ml^-1 (spot 10), with control spotting buffer containing 0.5 mg ml^-1 BSA in the last two spots. The whole array was repeated on the slide for a total of 8 replicates per spot. Each slide was incubated for 2 h with 20 μg ml^-1 of purified recombinant hCD200R-CD4d3+4 protein, prior to incubation with Alexa-555-labeled CD200R mAb (DX147, DX136 or OX108) or Alexa-647 control rCD4 mAb (OX68). Quantitative measurements are expressed as mean fluorescence units at 532 nm (green) and 635 nm (red) versus amount of capture mAb arrayed.