The diagnosis of brucellosis is mainly based on the detection of anti-LPS antibodies. High temperature kills Brucella cells by causing lysis of the membrane, so the phenol-heat killed brucella antigen may lake specificity as a result of destruction the majority of proteins in the cell wall. Accordingly, attention was directed to produce antigen using binary ethylene imine as an inactivator. The produced antigen showed high specificity in detecting Brucella abortus and Brucella melitensis-infected animals, but sensitivity was not affected in comparison with the standard Rose Bengal antigen. In Enzyme immunotransfer blot (EITB), phenol-heat killed brucella cells showed only 3 bands (37.375, 23.47 and 7.83 kDa) that denotes denaturation for at least 6 bands whereas binary inactivated brucella cells showed similarity with non-treated ones.