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Assays for anti-IFN binding antibodies (BAb)
Methods for the detection of BAb to type I IFN species include immunometric assays, radioligand assays and blotting techniques. Since BAb bind to both conformational and sequence-restricted (linear) epitopes on proteins, immunometric assays and immunoblotting techniques using a more or less denatured cytokine in the capture phase may hamper detection of BAb restricted to the native conformation of the molecules. These techniques may also show binding of Ab even in cases where the affinity is low; see Ab detection methods. As this binding is often non-saturable and hence non-specific, these Ab cannot be categorized as true BAb.

We prefer to use radioimmunoassays (RIA) for direct binding in solution to 125I-labeled cytokines. There are several reasons for this:
- The tracer can be validated and tested for preserved bioactivity.
- The tracer preserves as closely as possible the natural configuration of the cytokine and allows detection of BAb directed against conformational epitopes.
- High specific activity of the radiolabeled cytokine and therefore high assay sensitivity may be achieved.
- The specificity, kinetics, avidity and capacity of the IFN-BAb binding may be calculated.
- Cross-binding to non-Ab factors in serum or in other biological fluids can be assessed.
- The tracer may be used to test binding to, and interference with, target cell receptors.

We routinely radioiodinate human IFN-alpha and IFN-beta with preserved bioactivity to 2-5 x 10^5 cpm/ng, and tracers are always purified and validated for specific activity, stability and immunoreactivity before use. Serum samples are pre-incubated with 125I-IFN-alpha or 125I-IFN-b, and the amounts of free and IgG-bound tracer are determined by protein G Sepharose affinity chromatography. Steps are included to control for saturability of binding and for factors other than BAb that may interfere with IgG binding.

It is important to note that many serum proteins may influence BAb assays; these include soluble receptors and pre-existing cytokines. BAb in immune complexes may also escape detection because of a low exchange rate between BAb-bound unlabeled IFN and radiolabeled IFN during incubation. This may also occur if the tracer and BAb form complexes with high dissociation rates allowing them to dissociate during the (brief) time it takes to separate bound from free tracer.

We have optimized the RIA assay to measure BAb using 754 Danish MS patients receiving human IFN-beta. The figure shows the number of Bab-positive MS patients during therapy with different IFN-beta preparations and dose schedules.

Adapted from Ross et al. Ann Neurology 2000;48:706-12.

KB&CR 2000
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