Assessment of determinants of optimum performance of the CAST-2000 ELISA procedure

The Cellular Antigen Stimulation Test (CAST), a recent innovation in the laboratory detection of allergen sensitivity, is a functional assay based on the release of sulphidoleukotrienes (LTs) from allergen-activated circulating basophils. The current study was undertaken to establish optimum determinants of this procedure. The results demonstrate that treatment of blood with preservative-free heparin in polypropylene containers, and removal of erythrocytes by selective lysis with 0.83% ammonium chloride are excellent alternatives to the use of CAST venepuncture tubes and dextran, respectively. Similarly, decreasing and increasing the leucocyte concentration by 0.5- and 4-fold, respectively, as well as omission of interleukin-3 (IL-3) were also without significant effect. However, treatment of leucocyte suspensions with bacterial endotoxin (>1 microg/ml) activated the release of sulphidoleukotrienes from basophils and/or other types of leucocytes. The findings of this study are clearly relevant, both with respect to cost and performance, when implementing the CAST in clinical immunology laboratories.