Gudrun Andersson

Reverse ELISPOT assay for clonal analysis of cytokine production I. Enumeration of gamma-interferon-secreting cells

A reverse modification of ELISPOT assay using nitrocellulose membranes and epitope-specific monoclonal antibodies is described for the detection of single lymphokine-secreting cells. As a model, the production of gamma-interferon by mitogen stimulated human peripheral blood lymphocytes has been examined. The assay can also be modified to permit microscopic examination of spot-forming cells.

CD8 is critically involved in lymphocyte activation by a T. brucei brucei-released molecule

T. brucei brucei released a lymphocyte triggering factor (TLTF), which triggered purified CD8+, but not CD4+, T cells to interferon gamma (IFN-gamma) mRNA expression and secretion and to [3H]thymidine incorporation. TLTF also induced mRNA for transforming growth factor beta, but not for interleukin-4. The action of this TLTF on mononuclear cell (MNC) cultures was blocked by anti-CD8 antibodies and by soluble CD8. MNCs from a mutant mouse strain lacking CD8 expression were not triggered by TLTF. IFN-gamma provides a growth stimulus for T. brucei brucei, and infected CD8- mice had much lower parasitemia and survived longer than CD8+ mice. The host-parasite interaction in experimental African trypanosomiasis thus involves parasite release of TLTF, which by binding to CD8 triggers CD8+ cells to produce the parasite growth-promoting cytokine IFN-gamma.

Monoclonal antibody two-site ELISA for human IFN-γ: Adaptation for determinations in human serum or plasma

Mouse monoclonal antibodies (mAbs) against human interferon-gamma (IFN-gamma) were produced after immunization with recombinant IFN-gamma. Two mAbs (1-D1K and 7-B6-1) recognizing distinct epitopes on natural IFN-gamma were selected for the development of a two-site ELISA. The sensitivity was similar for IFN-gamma diluted in PBS with 1% bovine albumin, spent culture medium or fetal calf serum but reduced to approximately 50% when diluted in normal human serum. Individual normal human sera were tested and three of 14 gave false reactivities in the ELISA. One serum factor with major impact on the individual variation and the decreased sensitivity could be adsorbed to and eluted from protein A-Sepharose. Based on these observations we established a new ELISA protocol which made it possible to test for low levels of IFN-gamma in human serum and plasma samples. The modifications in this protocol are easy to apply with basic laboratory equipment.

Determination of Interleukin 6 in Human Urine and Epithelial Cell Supernatants

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 immunoassay 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.

Interferon-α: A gene family in therapeutic use☆

Abstract Several variants of interferon-α (IFN-α) were isolated and purified to homogeneity. They differed to various degrees in biological properties. However, three IFN-α2 variants showed only minor differences from a variant called IFN-α88 with regard to their ability to inhibit growth and to bind to specific receptors, tested on Daudi cells. Two monoclonal antibodies were studied, which showed overlapping specificity for at least one peptide obtained after HPLC separation of tryptic digests. The monoclonal antibodies could discriminate between sequence differences to a much higher degree than the receptor on Daudi cells. It is concluded that the receptor is degenerate and binds well to different structural variants of IFN and that for therapeutic use, several of the variants will probably have the same biological potency.