Detection of spontaneous and antigen-induced human interleukin-4 responses in vitro: comparison of ELISPOT, a novel ELISA and real-time RT-PCR.
Interleukin-4 (IL-4) is an important T-helper cell type 2 (Th2) cytokine in man, driving Th2 polarisation and exerting the most antagonistic effects to the Th1 cytokine interferon-gamma (IFN-gamma). Nevertheless, few data on spontaneous and antigen-specific secretion of IL-4 in man are available, mainly due to difficulties in the detection of IL-4. In this study, we compared three assays that can detect antigen-induced IL-4 responses; ELISPOT, ELISA after blocking the IL-4 receptor during cell culture, and real-time reverse transcription polymerase chain reaction (RT-PCR). Spontaneous, antigen- and allergen-induced responses were analysed in peripheral blood mononuclear cells from three groups with different secretion patterns for IL-4: atopic individuals, nonatopic individuals and pregnant women. ELISPOT displayed the highest sensitivity and was the only assay that could detect spontaneous secretion of IL-4 in all analysed samples. The IL-4 receptor blocking ELISA was considered best for the detection of in vitro antigen- and allergen-induced responses, since the results obtained from the ELISPOT and real-time RT-PCR displayed lower specificity, possibly because of seemingly aberrant IL-4 responses in the group of pregnant women. The real-time RT-PCR for detection of IL-4 mRNA proved to be sensitive, but expression of IL-4 mRNA was not correlated with the secretion of IL-4.
Ekerfelt
Christina
C
Clinical Research Centre, Faculty of Health Sciences, University Hospital, S-581 85, Linköping, Sweden. Christina.Ekerfelt@kfc.liu.se
Ernerudh
Jan
J
Jenmalm
Maria C
MC
D000328:Adult; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology; D015847:Interleukin-4 / Q000032:analysis* / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D011247:Pregnancy / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018417:Th1 Cells / Q000276:immunology; D018418:Th2 Cells / Q000276:immunology
D015847:Interleukin-4