The neutralization of interferons by antibody III. The constant antibody bioassay, a highly sensitive quantitative detector of low antibody levels.

The neutralizing antibodies (NAbs) that develop in patients during interferon (IFN) therapy can reduce its beneficial effects. The universally employed method of NAb measurement currently is the constant IFN method, in which antigen at a single given concentration is mixed with serial dilutions of serum, the lowest final dilution of which (usually 1:20) is constrained by the potential adverse effect of human serum on human cells in culture. The constant antibody (Ab) method described herein uses serum at a certain set dilution (usually 1:20) mixed with a series of IFN concentrations. Theoretical neutralization curves based on the previously presented model of the Ab-IFN reaction are depicted herein in terms of experimentally observable quantities. As predicted by the theoretical studies, the constant Ab method was demonstrated experimentally to extend the lower limits of detection of Ab by a factor of 10-20. The excellent agreement observed between the theoretical prediction and experimental findings reinforces the validity of using as NAb unitage the titer based on 10-fold reduction of IFN activity, reportable as Tenfold Reduction Units (TRU)/mL, as previously recommended. Testing by the constant Ab method of sera previously considered negative (<20 TRU/mL by the constant IFN method) from patients treated with Rebif or Betaseron showed that approximately 50% had detectable NAbs; such sera from Avonex-treated patients had titers of <1 TRU/mL. The constant Ab method can be used as a quantitative, sensitive IFN NAb screening bioassay of any nature, and should be able to detect low levels of NAbs early in the course of IFN therapy. The method may be useful to test monoclonal antibodies for otherwise undetectable NAbs. In principle, the constant Ab method should be applicable to the measurement of NAbs against any cytokine or other protein-effector molecule.