An enzyme immunoassay for bovine brucellosis using a monoclonal antibody specific for field strains of Brucella abortus.

The success of various attempts to discriminate serologically between vaccinated cattle and those infected with field strains of Brucella abortus has been limited. A reliable assay that has this discriminating ability would greatly benefit the Australian Brucellosis Eradication Scheme. Studies of similar diagnostic problems have shown that species and strain-specific monoclonal antibodies with diagnostic potential can be produced. Therefore, the production of monoclonal antibodies specific for Br. abortus field strains was attempted. Spleen cells from Balb/C mice immunized with formalin-killed Br. abortus strain 544 were fused with P3/NSI-1 Ag4-1 cells using polyethylene glycol 4000. The resulting hybrid cells were screened by ELISA using soluble fractions of sonicated, heat-killed (60 degrees C, 1 h) bacteria as antigen. More than 200 cultures of hybrid cells contained detectable amounts of antibody to both field and vaccine strains of Brucella. However, one monoclonal antibody, Br 25, was found to bind field strains of Br. abortus biotypes 1 and 2, but not strain 19, Br. suis, Br. melitensis, or Br. ovis. The titre of Br 25 hybridoma cell culture supernatant is over 2 000, Br 25 cells injected into mice produce ascites fluid having titres of more than 100 000. A sandwich ELISA incorporating successive incubations of Br 25, soluble Brucella antigen, bovine sera and labelled anti-bovine immunoglobulin was developed. Sera from cattle that had been either vaccinated with strain 19 and infected were tested in the ELISA. In this ELISA most infected cattle were positive, whether vaccinated or not, whereas uninfected and vaccinated cattle were negative.