1. Test for IFN-binding Ab (BAb), often as a screening.
2. Tests for IFN-neutralising Ab (NAb), reported by
- a percentage of neutralising capacity, using 10 LU/ml of added IFN.
- a titer, using the 10-fold reduction unit method of Kawade-Grossberg.
Technical details are in italic.

BINDING ANTIBODIES (BAb)

Serum, usually at 25% final concentration, is incubated with 125I-human recombinant IFN-a/IFN-beta. The formed antigen-Ab complexes are passed through a protein-G column, whereby IgG and IgG-complexes are being isolated. The amount of immobilized tracer is measured, corresponding to the amount of anti-IFN-alpha (or anti-IFN-b) IgG Ab in the serum sample (BAb of other Ig classes are extremely rare, and in those cases found in only small amounts).

The amount of anti-IFN IgG BAb in the sample is expressed as % bound of the amount of tracer added.
Since the background in this test +3SD is 16%, positive samples are those with BAb > 16%. Max binding is usually around 85%.
Note: If this sample is negative (BAb <= 16%), there is neither BAb nor NAb in appreciable quantities in the sample. On the other hand, a positive test (BAb > 16%) does not necessarily mean that the sample contains neutralising Ab (NAb) - in these cases, a set of bioassays for NAb must be carried out.

NEUTRALISING ANTIBODIES (NAb):

A selected IFN-sensitive cell line (MC-5) is infected with EMC virus -/+ addition of a preselected appropriate amount of human recombinant IFN-alpha or IFN-beta. This will protect the cells against virus-induced killing. The serum sample to be tested for NAb, usually at 5% final concentration, is added to cells cultured in parallel. If the sample contains NAb, the added IFN-alpha / IFN-beta will not be able to protect the cells against the virus attack - they will die dependent upon the amount of NAb in the added serum sample (antiviral neutralisation).

Note: Appropriate controls for the presence of "intrinsic" IFN activity and cell-toxic components are always included making the assay complex and difficult to interpret by non-specialists.

For screening purposes, we recommend the neutralising capacity method (see below), as this is economical, sensitive and clinically relevant (the more laborious 10-fold reduction unit method is used if specifically requested; see details here).

The amount of anti-IFN NAb in the sample is expressed as % neutralisation of IFN bioactivity with a conclusive statement, see below.
All analyses are usually performed with addition of 10 LU/ml of recombinant IFN, thus creating a 'medium sensitivity' assay. Lower or higher amounts may be added to create 'high-' and 'low sensitivity' assays, respectively. It is a specialists task to interpret the data, and these are usually accompanied by one of three conclusions: "Negative", " Positive, but the low level makes the clinical implication uncertain", or "Positive with expected clinical response failure". If no endogenous IFN activity and no toxicity is found, samples with less than 20% IFN neutralisation are usually considered "Negative".

Further information can be found in the following references.

KB 2003