The Brucellin Skin Test (BST) detects brucellosis in animals through a cell-mediated immune response to a protein extract from B. melitensis strain 115, which is almost free of lipopolysaccharide. It is highly specific and used to confirm suspected false positives in small ruminants and swine, but not recommended for screening due to low sensitivity. Despite its diagnostic significance, the protein composition of brucellin has not been fully characterized. This study used nLC-ESI-MS/MS analysis and bioinformatics tools to evaluate brucellin's protein composition and identify immunoreactive proteins. An allergen suspension of purified proteins (free of S-LPS) of EU Standard Brucellin (batch 2011-01), produced by ANSES was used. A total of 248 proteins were identified 206 of which are immunogenic. Key proteins identified include the L7/L12 ribosomal protein, bacterioferritin, outer membrane protein BP26/OMP28, and EipB, which are crucial in inducing DTH and for Brucella pathogenesis. The L7/L12 protein, induces strong delayed-type hypersensitivity (DTH), dependent on post-translational modification. It is also a major component of tuberculin purified protein derivative (PPD) and is a major heat-resistant protein inducing strong delayed-type hypersensitivity Additionally, proteins such as chaperonin, superoxide dismutase, DNA-binding proteins, and ABC transporter substrate-binding proteins are known for their immunogenic roles in cellular functions and stress responses These findings underscore the potential of these proteins as candidates for detecting cellular immunity to Brucella. Developing recombinant Brucella-allergenic proteins could help in standardizing skin tests, providing reliable allergens and reducing exposure risks. Moreover, a serological test using these recombinant proteins could improve sensitivity and specificity in Brucella infections diagnostic, eliminating false positivities related to LPS-based diagnostics.

Bachrach G, Banai M, Bardenstein S, Hoida G, Genizi A, Bercovier H. Brucella ribosomal protein L7/L12 is a major component in the antigenicity of brucellin INRA for delayed-type hypersensitivity in brucella-sensitized guinea pigs. Infect Immun. 1994 Dec;62(12):5361-6. doi: 10.1128/iai.62.12.5361-5366.1994. PMID: 7960115; PMCID: PMC303276

Nandini P, Jakka P, Murugan S, Mazumdar V, Kumar D, Prakash R, Barbuddhe SB, Radhakrishnan G. Immuno-profiling of Brucella proteins for developing improved vaccines and DIVA capable serodiagnostic assays for brucellosis. Front Microbiol. 2023 Oct 4;14:1253349. doi: 10.3389/fmicb.2023.1253349. PMID: 37860136; PMCID: PMC10582347

He CY, Yang JH, Ye YB, Zhao HL, Liu MZ, Yang QL, Liu BS, He S, Chen ZL. Proteomic and Antibody Profiles Reveal Antigenic Composition and Signatures of Bacterial Ghost Vaccine of Brucella abortus A19. Front Immunol. 2022 Apr 22;13:874871. doi: 10.3389/fimmu.2022.874871. PMID: 35529865; PMCID: PMC9074784.