Novel approach to activity evaluation for release-active forms of anti-interferon-gamma antibodies based on enzyme-linked immunoassay

Abstract

Selection of a suitable assay to measure the activity of drug agents based on release-active forms of anti-interferon-gamma antibodies (RA forms of Abs) is an important step forward in the investigation of such agents. In this study, the enzyme-linked immunosorbent assay was utilized to examine the effect of RA forms of Abs specific for human interferon gamma on the interaction between monoclonal anti-interferon gamma antibodies and recombinant human interferon gamma. The experimental data and the results obtained by using relevant mathematical analysis showed that such RA forms of Abs are able to modulate the monoclonal antibody interaction with both soluble and immobilized (to the assay plate well) interferon gamma. These data demonstrated the importance of using relatively low concentrations of both soluble and plate-immobilized interferon gamma to detect the effects of RA forms of Abs to interferon gamma on the binding of monoclonal antibodies to interferon gamma. It has been suggested that the observed influence of RA forms of Abs on 'antibody-antigen' interaction could be used to detect and analyze the activity of drugs containing RA forms of Abs.

Figure 1. Linear relationship between mAbs to IFN-gamma dilutions and optical density.

The concentration of IFN-gamma absorbed on the plate is 1 µg/ml. Dilutions of mAbs are on X axis (where 0.00001 means dilution of starting antibodies to 1∶100000 times, 0.00002 means dilution of starting antibodies to 1∶50000 times etc.). Optical densities are average values of three measurements. The error bars are the standard deviations.

Figure 2. The influence of absorbed IFN-gamma concentration on detection of RA forms of anti-IFN-gamma antibodies.

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.

Figure 3. Effect of RA forms of anti-IFN-gamma antibodies on antigen (soluble form)-antibody interactions.

The figure displays the effect of RA forms of Abs to IFN-gamma (RA Abs to IFNg) on the ability of soluble IFN-gamma to bind anti-IFN-gamma mAbs. The amount of mAbs which have not been bound by the respective antigen after reaching the dynamic equilibrium condition was measured by adding mAbs samples with different concentrations of antigen (shown on X axis of A where 0.00025 means dilution of starting interferon-gamma to 1∶4000 times, 0.0005 means dilution of starting interferon-gamma to 1∶2000 etc.) into antigen-coated wells of the assay plate for 45 min. (A): the influence of mAbs (1∶50000 dilution) in the mixture with IFN-gamma in different concentrations in presence of RA forms of Abs to IFN-gamma or control after equilibration. The error bars represent the standard deviations of the measurements. The sample RA Abs to IFNg prepared by procedure 1 (RA Abs to IFNg -1) was mixed with mAbs initially. IFN-gamma in different concentrations was added 45 minutes later. RA Abs to IFNg prepared by procedure 2 (RA Abs to IFNg-2) was mixed with IFN-gamma at different concentrations and mAbs was added 45 min later. It was shown that the order of reagent addition during pre-incubation is not important. RA samples are significantly different from control (p<0.0001) for each RA sample. RA Abs to IFNg-1 and RA Abs to IFNg-2 do not differ significantly from each other (p = 0.2). (B): there are linear relationships from 1/l_i calculated from “A” curves and the equations for these linear functions where the slopes are equal to the dissociation constants for antigen-antibody reaction.

Figure 4. Effect of RA forms of anti-IFN-gamma antibodies on antigen (absorbed form)-antibody interactions.

The figure shows changes in presence of control or RA forms of Abs to IFN-gamma in mAbs binding with interferon gamma adsorbed onto the plate (0,5 ug/ml); the error bars are the standard deviations for the groups. (A). Using the curves presented in Figure (A) and the method described in Materials and Methods, P _∞ and k were determined for mAbs in presence of control (B and C) or RA forms of Abs to IFN-gamma (RA Abs to IFNg) (D and E). RA sample is statistically different from control (F15/31 = 72.6; p<0.0001). (B), (C), (D) and (E) show equations of linear relationships obtained using MS Excel.