Identification of major factors influencing ELISpot-based monitoring of cellular responses to antigens from Mycobacterium tuberculosis.

A number of different interferon-gamma ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity. Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-gamma spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen.

On the contribution of stereochemistry to human ITPK1 specificity: Ins(1,4,5,6)P4 is not a physiologic substrate

Ins(1,4,5,6)P4, a biologically active cell constituent, was recently advocated as a substrate of human Ins(3,4,5,6)P4 1-kinase (hITPK1), because stereochemical factors were believed relatively unimportant to specificity [Miller, G.J. Wilson, M.P. Majerus, P.W. and Hurley, J.H. (2005) Specificity determinants in inositol polyphosphate synthesis: crystal structure of inositol 1,3,4-triphosphate 5/6-kinase. Mol. Cell. 18, 201-212]. Contrarily, we provide three examples of hITPK1 stereospecificity. hITPK1 phosphorylates only the 1-hydroxyl of both Ins(3,5,6)P3 and the meso-compound, Ins(4,5,6)P3. Moreover, hITPK1 has >13,000-fold preference for Ins(3,4,5,6)P4 over its enantiomer, Ins(1,4,5,6)P4. The biological significance of hITPK1 being stereospecific, and not physiologically phosphorylating Ins(1,4,5,6)P4, is reinforced by our demonstrating that Ins(1,4,5,6)P4 is phosphorylated (K(m) = 0.18 microM) by inositolphosphate-multikinase.

Monitoring SO2 emission at the Soufriere Hills Volcano: implications for changes in eruptive conditions

info:eu-repo/semantics/published