Development of a migration inhibitory factor assay under agarose of bovine mononuclear leukocytes, using an antigen of Brucella abortus.

A study was conducted to develop a migration inhibitory factor assay under agarose of bovine mononuclear leukocytes, with an antigen of Brucella abortus. Different concentrations of mononuclear leukocytes were prepared by the Ficoll-Hypaque technique from the blood of nonvaccinated calves and from calves previously vaccinated with strain 19. Concentrations of 0.5, 1, 2, 3, 4, and 5 x 10(6) leukocytes were suspended in RPMI-1640 medium and various dilutions (20, 10, 1, and 0.1 microgram) of B abortus-soluble antigen, dispensed in triplicate wells cut in 1% agarose containing minimal essential medium and 10% bovine fetal serum. These agarose plates were incubated for 4-, 8-, 12-, 16-, 20-, and 24-hour periods and then were fixed; leukocytes were stained with Wright's stain. Migration distances were measured, and statistical analyses of the data revealed a concentration of 2 x 10(6) cells/well and an antigen concentration of 10 microgram/well. An incubation period of 20 hours was optimal for the assay.