Abstract Activation of Tcells results in intracellular expression and secretion of cytokines such as interferon (IFN)‐γ. Here we have used three different assays for determination of IFN‐γ in tetanus toxoid‐ or mitogen‐activated human T cell cultures. Two of these assays [intracytoplasmic immunofluorescence and enzyme‐linked immuno spot assay (ELISPOT)] determined the expression and secretion of IFN‐γ at the single‐cell level while the third assay enzyme‐linked immunosorbent assay (ELISA) measured IFN‐γ secreted into the culture supernatant. Comparison of all three tests revealed a good correlation between the ELISPOT assay and the ELISA, whereas expression of intracellular IFN‐γ showed a qualitative but not a quantitative correlation with the latter. Both the immunospot assay and the immunofluorescence may be used to detect approximate numbers of specific T cells even when present at low frequencies. With the use of the immunospot assay antigen‐specific T cells could be detected even in the absence of detectable IFN‐γ in the culture supernatants. However, the ELISA assay should be more convenient for screening large clinical material.
The major functions of the immune system are to combat infections and to control that normal auto-immunity does not turn into auto-aggression. Antibodies, together with T cell receptors and MHC mol ...
Four different mouse monoclonal antibodies to human interferon-α (IFN-α) were evaluated for application in quantitative and comparative analysis of natural IFN-α mixtures. Binding to IFN-α subtypes in solution revealed individual reactivity patterns. These patterns changed if the IFN-α molecules were immobilized either passively to a surface or bound by another antibody. Also, substitution of a single amino acid in IFN-α2 affected the binding, apparently by altering the conformation. Isoelectric focusing of three natural IFN-α preparations from different sources, followed by immunoblotting, resulted in individual patterns with each of the four m Abs and also demonstrated variation in the composition of the IFN-α preparations. None of the mAbs was subtype specific, but by combining the different mAbs, and also applying polyclonal anti-human IFN-α antibodies, it was possible to design sensitive sandwich ELISAs with broad or more limited IFN-α subtype specificity.
Abstract The enzyme‐linked immunospot (ELISPOT) assay has been proven to be an efficient and sensitive method for the enumeration of single cells secreting antibodies or cytokines. Here we have used this method to determine the number of interleukin‐4 (IL‐4)‐ and interferon‐γ (IFN‐γ)‐producing cells in in vitro secondary responses to tetanus toxoid (TT) and the mycobacterial antigen (purified protein derivative; PPD) or the mitogen phytohemagglutinin (PHA). PHA‐induced IL‐4 and IFN‐γ secretion was well correlated suggesting polyclonal activation of cells. This was not the case with the specific antigens, where PPD preferentially induced IFN‐γ‐ and very few IL‐4‐producing cells, while TT‐induced both IL‐4 and IFN‐γ. These differences are probably a reflection of the types of immunity the two antigens induce, mycobacteria preferentially inducing a cell‐mediated T helper type 1 (Th 1) type of immunity, while immunity to tetanus is an antibody‐dependent, Th 2 type of response. In individuals recently boosted with TT, a significant increase in both IL‐4‐ and IFN‐γ‐producing cells in response to TT was seen at day 7 after boost, followed by decline. This was in contrast to what was seen in response to PPD where an increase of IFN‐γ‐producing cells after the TT boost at day 7 persisted for at least 14 days. These results suggest that after an in vivo boost both antigen‐specific and nonspecific T cells are activated and that antigen‐specific cells home to other organs and therefore may be difficult to demonstrate in the circulation. Our data show that the ELISPOT assay is a powerful tool for determining the frequency of cells secreting cytokines. The assay has several advantages over other assays since it is sensitive, measures the number of actually secreting cells, and avoids the problems of binding of cytokines to their cell‐bound or soluble receptors.
We have measured cellular and humoral immune responses to short synthetic peptides representing epitopes of the malaria vaccine candidate antigen Pf155/RESA in a longitudinal, prospective study of clinical immunity to Plasmodium falciparum malaria in a cohort of 354 Gambian children aged 3–8 years. A significant association was observed between presence of antibodies to the 3′ repeat region peptide (EENV)6 and resistance to clinical malaria. The prevalence of protective antipeptide antibodies varied significantly between different ethnic groups, suggesting that immune recognition of some Pf155/RESA epitopes may be genetically regulated. There was no obvious association between proliferative or interferon γ responses to T cell epitopes of Pf155/RESA and resistance to malaria infection or disease. At an individual level, the presence of peptide-binding antibodies was associated with the induction of interleukin 4 messenger ribonucleic acid expression in T cells activated with the overlapping T cell epitope EENVEHDA(EENV)2. This suggests that measurement of interleukin 4 production by T cells may represent a functional assay for T helper activity.
Interleukin 6 (IL-6) is a cytokine with many biological functions. It is produced by different tissues in response to infection and is secreted into the local body fluids. The aim of this study was to find a suitable assay to measure IL-6 in human urine. IL-6 was quantitated by a bioassay and by immunoassays based on neutralizing or nonneutralizing antibodies. The effect of human urine on the quantitation of IL-6 by these assays was analyzed using pooled human urine with added recombinant or natural human IL-6. Urine was found to disturb the growth of the B9 cells. When fractions from gel-filtered human urine were tested, a fraction corresponding to a protein molecular weight range of 10,000–1,000 was found to have a strong inhibitory effect in the B9 assay. In contrast, the low molecular weight fractions containing salts and pigments were not found to disturb the assay. The inhibitory effect of urine was avoided by diluting the samples > 80 times (final dilution in the test plate) or by dialysis. Furthermore, we analyzed IL-6 in urine samples from patients with urinary tract infection and supernatants from epithelial cells stimulated with bacteria in vitro. The B9 assay and the immunoasay based on non-neutralizing anti-IL-6 antibodies were more sensitive than the immunoassays based on neutralizing antibodies. While most of the B9 activity in the urine samples and supernatants could be neutralized by anti-IL-6 antibodies, some samples contained unneutralizable activity. These components remain to be defined. The results demonstrate considerable variation between assays used to quantitate natural IL-6.
Abstract Myelin basic protein (MBP)‐autoreactive T cells have a crucial pathogenetic role in experimental allergic encephalomyelitis (EAE) and certain MBP epitopes may be immunodominantly recognized. The heterogeneity and quantity of the T cell response to different epitopes of MBP in multiple sclerosis (MS) and non‐MS controls is not so clearly defined. We now study T cell reactivity to six different peptides of MBP in MS compared to controls in short‐term cultures of blood mononuclear cells by measuring numbers of T cells that secrete interferon‐γ in response to antigen. In comparison with controls, MS patients showed dramatically increased numbers of MBP peptide‐reactive T cells with mean values varying between 10.4 and 22.5 per 10 5 blood mononuclear cells. Among those MBP peptides examined (amino acid 1–20, 63–88, 89–101, 96–118, 110–128 and 148–165), no single peptide is preferentially recognized. Neither is any preferential response apparent after subdivision of the MS patients according to their HLA‐DR genotype. Our findings suggest that a quantitative increase of a broad repertoire of myelin‐autoreactive T cells with capacity to secrete IFN‐γ can be important for the pathogenesis of MS.
