The influence of absorbed IFN-gamma concentration on detection of RA forms of anti-IFN-gamma antibodies.

<p>The figure shows the influence of the concentration of IFN-gamma adsorbed onto microtiter plates on the mAbs dilution curves observed in the presence of control or RA-antibodies to IFN-gamma (RA Abs to IFNg). Test samples prepared of mAbs, pre-diluted up to 1∶5000–1∶135000 in TBS, and either RA forms of Abs to IFN-gamma (RA Abs to IFNg) or control, mixed at a ratio 1∶4 v/v respectively. 100 µL of each sample was transferred into the wells (3 wells for each sample) and was incubated for 45 minutes at room temperature while shaking (shaker-incubator Dynatech, USA). The remainder of the experiment was conducted in accordance with the standard ELISA protocol. Dilutions of mAbs on X axis are those observed after mixing with RA forms of Abs to IFN-gamma or control (where 0.00001 means dilution of starting antibodies to 1∶100000 times, 0.00002 or 2E-05 means dilution of starting antibodies to 1∶50000 times etc.). The Y-axis displays 490 nm optical densities. The error bars represent the standard deviations of the measurements. The concentrations of IFN-gamma adsorbed on the plate are: A 6 µg/ml B - 2 µg/ml C - 0.7 µg/ml; D - 0.25 µg/ml. RA samples are significantly different from the control (F31/60 = 93.4; p<0.0001) in the case of the C and D curves.