Notes on Immunoprecipitin Reactions with Rinderpest Virus.

Abstract

The standard agar gel immunodiffusion (AGID) test for detecting rinderpest virus antigens with hyperimmune sera (Scott, 1967) has recently been adapted to electrophoretic and micro-systems (Rossiter and Mushi, 1980; Foreman, Rowe and Taylor, 1983). This report summarises some additional findings which may prove useful with these tests. Counterimmunoelectrophoresis (CIEP) was carried out as described (Rossiter and Mushi, 1980) and the effect of temperature and distance between wells examined. Positive antigens were prepared from infected cell cultures and lymph nodes; an antiprotease agent, 2mM phenylmethyl-sulphonylfluoride (PMSF) was added to aliquots of antigen which were then stored on a sunny window-ledge. Some aliquots also received sodium azide (NaN3). Micro-dou}'le diffusion tests were made using the microscope slide-template method (Tyrell, 197 8). Rabbits were hyperimmunised with washed precipitin lines of rinderpest virus antigen and rabbit antibody formed in routine AGID tests. Complexes formed between different ratios of antigen and antibody were used and were administered to rabbits in two intramuscular inoculations of Freund's Complete Adjuvant three weeks apart. Sera were collected four weeks later. Clear positive precipitin lines could be detected within 25 min if the distance between antigen and antibody wells was reduced to 3 mm in the CIEP test. Results from two repeated teries of experiments using a temperature-controlled plate showed that the CIEP test was most sensitive between 30 and 500C, the titres of cell culture and lymph node antigen being two-fold higher than at 20 and 6000. Two precipitin lines were routinely detected up to 300C after which the faster migrating antigen was less easily detectable at 40 to 500C and absent at 6000. Using a portable petro: electricity generator the CIEP test worked at maximum sensitivity in the t 'idday sun at an altitude of 1,000m above sea level in northern Kenya the gel temperature being 45 0C.