Development and validation of a real time PCR-based bioassay for quantification of neutralizing antibodies against human interferon-beta

There are two commonly employed types of bioassays for the detection of neutralizing antibodies (NAbs) against interferonbeta (IFN beta): the cytopatic effect assay (CPE), and the MxA (myxovirus resistance protein A) protein assay (MPA). This article describes a bioassay based on the real time PCR measurement of mRNA that results from the induction, in cultured human cells, of the MxA gene by IFN beta. Serum samples from 104 patients with multiple sclerosis (MS) treated with IFN were tested for NAbs using our real time PCR bioassay. NAbs also were measured in the same specimens by the MPA assay and CPE assay. The calibration range of the real time PCR bioassay is 0.125-30 LU/mL. The range of the intra- and inter-assay variations (coefficients of variation in log(10)) were 4.05% (range 0.88%-7.90%) and 4.42% (range 0.31%-9.15%), respectively. Samples of the three commercial preparations of IFN beta-1a and -1b were measured showing dose-response curves parallel to that of the NIH reference IFN beta (mean SD at the midpoint of the dose-response curve = 5%). In addition, the assay was robust with respect to number of cells plated (i.e., increasing cell densities from 12 x 10(3)/well to 384 x 10(3)/well resulted in 3.03% variability in MxA expression normalized with glyceraldehyde-3 phosphate dehydrogenase). NAbs titers measured were closely comparable to those obtained by the MPA [r(spearman)=0.899; 89% of observed agreements; K=0.779] and the CPE [r(spearman)=0.7899); 86%; K=0.729] assays. Despite the obvious disadvantage of cost, when carried out according to quality assurance guidelines for molecular diagnostics the new MxA gene-expression assay (MGA) has significant advantages over the other methods for testing NAbs: it has excellent reliability and reproducibility, and utilizes equipment and methodologies already accessible in many clinical laboratories. (c) 2007 Elsevier B.V. All rights reserved.