Measurement of neutralising antibodies to type I interferons by gene expression assays specific for type 1 interferon-inducible 6-16 mRNA.
Human type I interferon products have been approved for the treatment of several diseases, though neutralising antibodies against them may develop and reduce therapeutic efficacy. Traditionally, potencies of human interferons (IFNs) and of neutralising antibodies (NAbs) against them are quantified by antiviral assays. These are being increasingly replaced by less cumbersome and faster bioassay methods. Since IFNs exert their biological effects by binding to receptors on target cells and stimulating the expression of IFN-inducible genes, measurement of transcribed mRNAs can form the basis of functional bioassays. In this study we have used two approaches, quantitative reverse transcription-polymerase chain reaction (qPCR) and branched DNA (bDNA), to develop efficient, sensitive and robust non-viral assays to quantify type I IFNs per se and NAbs in sera from patients treated with either IFNbeta or IFNalpha2a. We show the rapid (4h) induction of the type I IFN-inducible 6-16 mRNA in A549 lung carcinoma cells is sensitively and reproducibly concentration-dependent for both IFNbeta and IFNalpha2a stimulation, is quantifiable by either approach, and is readily adaptable for the detection and measurement of NAbs against type I IFNs. Quantitative neutralisation of IFN-stimulated 6-16 mRNA expression was achieved in both assays when sera from patients receiving IFNbeta or IFNalpha2a therapy known to contain NAbs against these IFNs were tested. Their rapid and potentially automatable performance strongly suggests these assays could be used in a clinical setting to monitor the development of neutralising antibodies in patients receiving IFN therapy.
Moore
M
M
National Institute for Biological Standards and Control, Potters Bar, Herts, EN6 3QG, UK. mmoore@nibsc.ac.uk
Meager
A
A
Wadhwa
M
M
Burns
C
C
D000906:Antibodies / Q000097:blood* / Q000276:immunology; D000998:Antiviral Agents / Q000627:therapeutic use; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use; D050091:Dendrimers; D015870:Gene Expression; D006801:Humans; D007118:Immunoassay; D007370:Interferon Type I / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000276:immunology / Q000627:therapeutic use; D016899:Interferon-beta / Q000276:immunology / Q000627:therapeutic use; D008175:Lung Neoplasms / Q000276:immunology / Q000473:pathology; D009500:Neutralization Tests / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D055752:Small Cell Lung Carcinoma / Q000276:immunology / Q000473:pathology; D013997:Time Factors
D000906:Antibodies; D000998:Antiviral Agents; D050091:Dendrimers; D007370:Interferon Type I; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016899:Interferon-beta; D004247:DNA