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Updated: Jul 16, 2026
Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α
Published on: June 14, 2018
Development and validation of a real time PCR-based bioassay for quantification of neutralizing antibodies against
A Bertolotto1, A Sala, M Caldano
1Centro Riferimento Regionale Sclerosi Multipla (CRESM) & Neurobiologia Clinica, Ospedale Universitario S. Luigi Gonzaga, Regione Gonzole 10, I-10043, Orbassano, Torino, Italy. sclerosi.multipla@sanluigi.piemonte.it
Insights
A new real-time PCR bioassay accurately detects neutralizing antibodies (NAbs) against interferon-beta (IFNbeta) in multiple sclerosis patients. This MxA gene-expression assay (MGA) offers reliable and reproducible results comparable to existing methods.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- Interferon-beta (IFNbeta) is a crucial therapy for multiple sclerosis (MS).
- Detection of neutralizing antibodies (NAbs) against IFNbeta is essential for monitoring treatment efficacy.
- Current NAb detection methods, like cytopathic effect assay (CPE) and MxA protein assay (MPA), have limitations.
Purpose of the Study:
- To describe and validate a novel real-time PCR-based bioassay for quantifying NAbs against IFNbeta.
- To compare the performance of this new assay with established MPA and CPE methods.
- To assess the reliability, reproducibility, and robustness of the MxA gene-expression assay (MGA).
Main Methods:
- Developed a real-time PCR bioassay measuring MxA gene mRNA induction by IFNbeta in cultured human cells.
- Tested serum samples from 104 MS patients treated with IFNbeta.
- Validated the assay using commercial IFNbeta preparations and compared results with MPA and CPE assays.
Main Results:
- The real-time PCR bioassay demonstrated a calibration range of 0.125-30 LU/mL with excellent intra- and inter-assay reproducibility (CVs < 5%).
- The assay showed robust performance across varying cell densities and produced dose-response curves parallel to the NIH reference IFNbeta.
- NAb titers obtained by MGA closely correlated with those from MPA (r=0.899) and CPE (r=0.790) assays.
Conclusions:
- The MxA gene-expression assay (MGA) is a reliable and reproducible method for detecting NAbs against IFNbeta.
- MGA offers significant advantages in terms of accuracy and consistency compared to traditional CPE and MPA assays.
- This assay utilizes accessible equipment and methodologies, making it suitable for many clinical laboratories despite higher initial costs.
Abstract:
There are two commonly employed types of bioassays for the detection of neutralizing antibodies (NAbs) against interferon-beta (IFNbeta): 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 IFNbeta. Serum samples from 104 patients with multiple sclerosis (MS) treated with IFNbeta 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 IFNbeta-1a and -1b were measured showing dose-response curves parallel to that of the NIH reference IFNbeta (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 12x10(3)/well to 384x10(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.
