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Assays for neutralising anti-IFN antibodies (NAb)Antiviral neutralisation bioassay (ANB) - viral cytopathic effect (CPE) assay |
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| Methods for the detection of NAb to type I IFN include antiviral neutralisation assays and tests for inhibition of cellular effects induced by these cytokines, for example MxA. We have developed an antiviral neutralisation assay which is convenient and sufficiently cost-effective to allow for the screening of NAb in large numbers of MS patients (1).
The test consists of a modified IFN bioassay testing the ability of serum samples to counteract the protective effect of IFN when MC-5 cells (a subclone of A549 cells) are exposed to encephalomyocarditis virus; see figure. The test directly measures the IFN neutralising capacity of a test sample which eliminates the need for measurements of serially diluted samples in order to provide a titer of anti-IFN activity. This makes the assay well suited for clinical purposes and reduces the cost of screening considerably.
To avoid false positive and false negative results, separate experiments are included in each analysis to control for serum toxicity and endogenous antiviral activity; the latter can usually be avoided if blood is drawn at least 24 hours after the last injection of IFN. The test may be carried out at several sensitivity levels depending upon the amounts of added IFN. For screening purposes, human IFN preparations are added to the test at concentrations of 3 and 10 LU/ml, corresponding to 0.9 and 3 IU IFN/ml. Other levels of exogenous IFN may be used for more elaborate assays, but NAb measured by tests using 3 and 10 LU/ml have been validated in MS patients and found to correlate with reduced clinical effect of IFN-beta preparations (2). The data depend highly upon the amount of IFN-beta added to ANB; adding > 10 LU/ml usually masks anti-IFN-beta NAb detection; see figure below.
Adapted from Ross et al. Ann Neurology 2000;48:706-12. Cited references: 1. Ross C, Clemmesen KM, Svenson M, Sorensen PS, Koch-Henriksen N, Skovgaard GL, Bendtzen K. Immunogenicity of interferon-b in multiple sclerosis patients: influence of preparation, dosage, dose frequency, and route of administration. Danish Multiple Sclerosis Study Group. Ann Neurology 2000;48:706-12. 2. Sorensen PS, Ross C, Clemmesen KM, Bendtzen K, Frederiksen JL, Jensen K, Kristensen O, Petersen T, Rasmussen S, Ravnborg M, Stenager E, Koch-Henriksen N, the Danish Multiple Sclerosis Study Group. Clinical significance of neutralising antibodies against interferon-beta in patients with relapsing-remitting multiple sclerosis. Lancet 2003; (in press). KB&CR 2000 |
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