Quantification of neutralizing antibodies to human type I interferons using division-arrested frozen cells carrying an interferon-regulated reporter-gene.
Development of neutralizing antibodies (NAbs) to interferons (IFNs) can reduce the clinical response to IFN therapy. As current cell-based assays for quantifying NAbs have limitations, a highly sensitive and reproducible assay was developed, using division-arrested frozen human U937 cells transfected with the luciferase reportergene controlled by an IFN-responsive chimeric promoter, which allows IFN activity to be determined with precision within hours. Assay-ready PIL5 cells can be stored frozen for >3 years without loss of IFN sensitivity or the need for cell propagation. The assay is highly IFN sensitive (detecting <1.0 IU/mL), reproducible (SE +/- 15%) over concentrations from <1.0 to 100 IU/mL and able to measure different IFN subtypes and their pegylated variants. The use of this assay has shown that NAbs from patients treated with IFN-alpha2 exhibited markedly lower titers against 10 LU/mL of low specific activity IFNs, namely, IFN-alpha1, PEG-Intron(TM) (Schering-Plough, Levallois-Perret,France), or Pegasys(TM) (Hoffmann-La Roche, Neuilly-sur-Seine, France, than against 10 LU/mL IFN-alpha2. Similarly, NAbs from patients treated with IFN-beta1a exhibit lower titers against 10 LU/mL of low specific activity IFN-beta1b than against IFN-beta1a. The combination of the use of division-arrested, IFN-responsive human cells transfected with the luciferase reporter-gene makes the rapid PIL5 assay for NAbs highly advantageous.
Lallemand
C
C
Laboratory of Viral Oncology, CNRS FRE, Institut André Lwoff, Villejuif, France.
Meritet
J-F
JF
Erickson
R
R
Grossberg
S E
SE
Roullet
E
E
Lyon-Caen
O
O
Lebon
P
P
Tovey
M G
MG
D000906:Antibodies / Q000097:blood / Q000276:immunology*; D002455:Cell Division; D000939:Epitopes; D017930:Genes, Reporter; D006801:Humans; D007118:Immunoassay; D016233:Immunotherapy, Active; D007370:Interferon Type I / Q000276:immunology*; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000276:immunology* / Q000627:therapeutic use; D008156:Luciferases; D011092:Polyethylene Glycols; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins; D020298:U937 Cells
D000906:Antibodies; D000939:Epitopes; D007370:Interferon Type I; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D008156:Luciferases; C417083:peginterferon alfa-2b; C100416:peginterferon alfa-2a