Abstract
Brucellosis in humans is one of the most prevalent zoonotic diseases around the world with more than 500,000 new cases per year. It is a weakening disease that requires long-term antibiotic treatment, often resulting in permanent and disabling consequences. Outer membrane proteins (OMPs) of Brucella, which are non-lipopolysaccharide (LPS) antigens, have been used for the diagnostic kits of brucellosis and vaccine design. The aim of this study was to identify Brucella abortus with an immunofluorescence (IF) test using an antibody against recombinant outer membrane protein (OMP) of 19 kDa of this bacterium. The OMP19 gene of Brucella spp. was synthesized, cloned and expressed in Escherichia coli cells. The OMP19 protein was purified by metal chelate affinity chromatography and subsequently used for the immunization of rabbits to produce a polyclonal antibody. Then, this antibody was conjugated to fluorescein isothiocyanate (FITC) and used for the detection of Brucella by an IF test. Also, the sensitivity and specificity of this antibody for the diagnosis of clinical isolates was calculated. Outer membrane protein 19 was expressed well and reacted with a commercial antiserum against His-tag in an immunoblot assay. Polyclonal antibodies obtained from the serum of rabbits immunized with the purified protein showed strong reactivity in the enzyme-linked immunosorbent assay (ELISA). Moreover, the polyclonal antibody conjugated to FITC was able to properly identify Brucella abortus. Sensitivity and specificity of this IF test in comparison with a polymerase chain reaction (PCR) assay was 84.2% and 50%, respectively. This high-titer antibody could potentially be valuable for the specific diagnostic test of brucellosis.
Highlights
Brucella spp. are intracellular Gram-negative bacteria that cause human disease and significant economic losses worldwide due to livestock infection.[1]
Polyclonal antibodies obtained from the serum of rabbits immunized with the purified protein showed strong reactivity in the enzyme-linked immunosorbent assay (ELISA)
The polyclonal antibody conjugated to fluorescein isothiocyanate (FITC) was able to properly identify Brucella abortus
Summary
Brucella spp. are intracellular Gram-negative bacteria that cause human disease and significant economic losses worldwide due to livestock infection.[1]. The OMPs of Brucella spp. have been widely considered as potentially defensive and immunogenic antigens.[24,25] Monoclonal antibodies (mAbs) against group 1, 2 and 3 OMPs, as well as mAbs to minor surface-exposed OMPs with molecular masses (MMs) of 10, 16, 19, and 31–34 kDa (the latter is a major OMP in B. melitensis strains but is less abundant in B. abortus), have been produced.[26] Physicochemical and practical examination has recently confirmed that OMP10, OMP16 and OMP19 are lipoproteins that are uncovered at the cell surface.[27] It has been proven that these lipoproteins are present in several Brucella strains. Outer membrane proteins (OMPs) of Brucella, which are non-lipopolysaccharide (LPS) antigens, have been used for the diagnostic kits of brucellosis and vaccine design
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