Whole-blood microassay for immunodetection of antigen specific cell mediated immunity using bromodeoxyuridine incorporation.

ABSTRACT

The study evaluates a novel version of the bromodeoxyuridine (BrdU) incorporation assay for in vitro proliferative responses, which works with finger-prick blood specimens. It was developed primarily for field-work involving children in Africa and elsewhere. Heparinized blood specimens from healthy volunteers were diluted 115 and cultured with purified protein derivative, tetanus toxoid, concanavalin A or medium alone for four-seven days and pulsed during the last 24 hours with bromodeoxyuridine. Thereafter, white and red cells were separated by Ficoll-Pacque centrifugation. The white cells were made to adhere to Multitest slides pre-treated with poly-L-lysin. After fixation with paraformaldehyde, the cells were incubated with mouse monoclonal antibodies to a surface marker for activated T cells (CD25, interleukin-2 receptor) and the reaction visualized with anti-mouse alkaline phosphatase conjugated antibodies and a Fast Red substrate. After treatment with cold acetone, the cells were covered with formamide and heated to 70 degrees C, without loss of surface staining. The incorporation of bromodeoxyuridine was visualized in the UV-microscope with mouse anti-bromodeoxyuridine monoclonal antibodies and a rabbit anti-mouse fluorescein conjugate. Both the surface staining and the nuclear fluorescence can be seen in UV-light and the cells be classified as single- or double-positive or negative. Parallel experiments on the same blood-sample from seven donors allowed for at least 42 paired comparisons between the proportion of BrdU positive cells and thymidine incorporation stimulation indices. The latter assay was carried out both in the conventional version and in a whole-blood micro-version. The rank order correlations were 0.84 and 0.91 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)