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.
Van Rooyen
Cathrine
C
Department of Medical Virology, Faculty of Health Sciences, University of Pretoria and Tshwane Academic Division of the National Health Laboratory Service, P.O. Box 2034, Pretoria 0001, South Africa. cvrooyen@medic.up.ac.za
Anderson
Ronald
R
D000328:Adult; D000643:Ammonium Chloride; D000906:Antibodies / Q000032:analysis / Q000276:immunology*; D000941:Antigens / Q000276:immunology*; D003911:Dextrans; D004731:Endotoxins; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006493:Heparin; D006801:Humans; D006967:Hypersensitivity / Q000145:classification / Q000175:diagnosis*; D007377:Interleukin-3; D007962:Leukocytes; D008297:Male
D000906:Antibodies; D000941:Antigens; D003911:Dextrans; D004731:Endotoxins; D007377:Interleukin-3; D000643:Ammonium Chloride; D006493:Heparin