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 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.
Bertolotto
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Centro 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
Sala
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Caldano
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Capobianco
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Malucchi
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Marnetto
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Gilli
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F
D000906:Antibodies / Q000097:blood* / Q000276:immunology; D001681:Biological Assay / Q000379:methods* / Q000592:standards; D002138:Calibration; D045744:Cell Line, Tumor; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004306:Dose-Response Relationship, Immunologic; D004686:Encephalomyocarditis virus / Q000187:drug effects / Q000472:pathogenicity; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007155:Immunologic Factors / Q000276:immunology* / Q000494:pharmacology / Q000627:therapeutic use; D000068556:Interferon beta-1a; D000068576:Interferon beta-1b; D016899:Interferon-beta / Q000276:immunology* / Q000494:pharmacology / Q000627:therapeutic use; D009103:Multiple Sclerosis / Q000188:drug therapy / Q000276:immunology*; D064547:Myxovirus Resistance Proteins; D009500:Neutralization Tests / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects
D000906:Antibodies; D007155:Immunologic Factors; C579071:MX1 protein, human; D064547:Myxovirus Resistance Proteins; D012333:RNA, Messenger; D000068576:Interferon beta-1b; D016899:Interferon-beta; D019204:GTP-Binding Proteins; D000068556:Interferon beta-1a