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Introduction. Brucellosis is an important bacterial zoonosis, re-emerging as a serious public health concern in developing countries. Two major species, Brucella melitensis and Brucella abortus , cause recurrent facile infection in human. Therefore, rapid and accurate diagnosis for early disease control and prevention is needed in areas with low disease burden. Hypothesis. This study evaluated the sandwich enzyme-linked immunosorbent assay (ELISA) (S-ELISA) immunoassay for potential use of whole-cell (WC) and recombinant outer-membrane protein (rOmp28)-derived IgG polyclonals in sensitive detection of Brucella . Aim. Immunoassay-based WC detection of Brucella species in important sub-clinical matrices at lower limits of detection. Methodology. We purified recombinant rOmp28 with Ni-NTA gel affinity chromatography and produced IgG polyclonal antibodies (pAbs) using BALB/c mice and New Zealand white female rabbits against different antigens (Ags) of Brucella . Checkerboard sandwich ELISA and P/N ratio (optical density of 'P' positive test sample to 'N' negative control) were used for evaluation and optimization of the study. The pAbs were characterized using Western blot analysis and different matrices were spiked with WC Ag of Brucella . Results. Double-antibody S-ELISA was developed using WC Ag-derived rabbit IgG (capture antibody at 10 µg ml -1 ) and rOmp28-derived mice IgG (detection antibody at 100 µg ml -1 ) with a detection range of 10 2 to 10 8 cells ml -1 and a limit of detection at 10 2 cells ml -1 . A P/N ratio of 1.1 was obtained with WC pAbs as compared to 0.6 and 0.9 ratios with rOmp28-derived pAbs for detecting B. melitensis 16M and B. abortus S99, respectively. An increased P/N ratio of 4.4 was obtained with WC Ag-derived rabbit IgG as compared to 4.2>4.1>2.4 ratios obtained with rabbit IgGs derived against cell envelope (CE), rOmp28 and sonicated antigen (SA) of Brucella with high affinity for rOmp28 Ag analysed on immunoblots. The rOmp28-derived mice IgG revealed two Brucella species at P/N ratios of 11.8 and 6.3, respectively. Upon validation, S-ELISA detected Brucella WCs in human whole blood and sera samples with no cross-reactivity to other related bacteria. Conclusion. The developed S-ELISA is specific and sensitive in early detection of Brucella from different matrices of clinical and non-clinical disease presentation.

Brucella is alpha-2 Proteobacteria mainly responsible for multi-factorial bacterial zoonotic disease brucellosis with low concentration (10-100 CFU) required to establish the infection. In this study, we developed sandwich ELISA with detection range of 10 2 to 10 8 cells mL -1 and limit of detection at 10 3 cells mL -1 by employing polyclonal rabbit IgG (capture antibody, 10 µg mL -1 ) and mice IgG (detection antibody, 50 µg mL -1 ) antibody for its detection. Surface Plasmon Resonance evaluated the interaction of detection antibody with whole cell spiked serum samples at LOD of 10 2 cells mL -1 along with non co-operative interaction of protein albumin. Further, kinetic evaluation study using detection antibody against cell envelope antigen was performed whereby, Equilibrium Dissociation Constant (K D ) and Maximum Binding Capacity (B max ) were found to be 16.48 pM and 81.67 m° for Brucella abortus S99 and 0.42 pM and 54.50 m° for Brucella melitensis 16 M, respectively. During interference study, sandwich ELISA assay cross-reacted with either of the polyclonal antibody of above Brucella species. Upon validation, no cross-reactivity observed with bacteria-closely related to Brucella. In conclusion, developed semi-quantitative sandwich immunoassay is sensitively rapid in whole cell detection of Brucella and will be useful in development of detection assays from environmental and clinical matrices.

Brucellosis is a zoonotic disease caused by various species of the genus Brucella. The control of this disease mainly depends on its accurate and early diagnosis. Culture methods employed for diagnosis are time consuming and require well equipped biosafety level 3 laboratories and hence serological tests are favored alternative for brucellosis diagnosis. At present serological diagnosis is based on LPS (lipopolysaccharide) which is less specific as it shows cross reactivity with other gram-negative bacteria. There is a need to develop serological diagnostic assay based on purified recombinant antigen of Brucella. T4SS (Type IV Secretion System) is an important virulent factor of Brucella and required for infection suggesting their expression in vivo and can be targeted as serological marker for infection. To test this concept, the present study is designed to clone, express and purify virB10 gene of Brucella T4SS under denaturing conditions and to evaluate its use as serological marker of Brucella infection. The immunoreactivity of this recombinant antigen was checked with antisera collected after experimental infection in Balb/C mice with B. melitensis 16M, BR31 (human clinical isolate) and Y. enterocolitica O:9. The recombinant protein was also tested against a panel of 46 bovine sera samples collected from Leh, India. Antibody response against VirB10 was detected in experimental and natural host suggesting that it can be explored as potential target for serodiagnosis of Brucella infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Surface plasmon resonance (SPR) immunosensor using 4-mercaptobenzoic acid (4-MBA) modified gold (4-MBA/Au) SPR chip was developed first time for the detection of Brucella melitensis (B. melitensis) based on the screening of its complementary DNA target by using two different newly designed DNA probes of IS711 gene. Herein, interaction between DNA probes and target molecule are also investigated and result revealed that the interaction is spontaneous. The kinetics and thermodynamic results derived from the experimental data showed that the interaction between complementary DNA targets and probe 1 is more effective than that of probe 2. Equilibrium dissociation constant (K D ) and maximum binding capacity of analyte (B max ) values for the interaction of complementary DNA target with the immobilized DNA probes were calculated by using kinetic evaluation software, and found to be 15.3 pM (K D ) and 81.02m° (B max ) with probe 1 and 54.9pM and 55.29m° (B max ), respectively. Moreover, real serum samples analysis were also carried out using immobilized probe 1 and probe 2 with SPR which showed the applicability of this methodology and provides an alternative way for the detection of B. melitensis in less than 10min. This remarkable sensing response of present methodology offer a real time and label free detection of biological warfare agent and provide an opportunity to make miniaturized sensor, indicating considerable promise for diverse environmental, bio-defence, clinical diagnostics, food safety, water and security applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background & Objectives: Brucellosis is endemic in the southern part of India. A combination of biochemical, serological and molecular methods is required for identification and biotyping of Brucella. The present study describes the isolation and biochemical, molecular characterization of Brucella melitensis from patients suspected for human brucellosis., Methods: The blood samples were collected from febrile patients suspected to have brucellosis. A total of 18 isolates were obtained from 102 blood samples subjected to culture. The characterization of these 18 isolates was done by growth on Brucella specific medium, biochemical reactions, CO2 requirement, H2S production, agglutination with A and M mono-specific antiserum, dye sensitivity to basic fuchsin and thionin. Further, molecular characterization of the isolates was done by amplification of B. melitensis species specific IS 711 repetitive DNA fragment and 16S (rRNA) sequence analysis. PCR-restriction fragment length polymorphism (RFLP) analysis of omp2 locus and IS711 gene was also done for molecular characterization., Results: All 102 suspected samples were subjected to bacteria isolation and of these, 18 isolates could be recovered on blood culture. The biochemical, PCR and PCR-RFLP and 16s rRNA sequencing revealed that all isolates were of B. melitensis and matched exactly with reference strain B. melitensis 16M., Interpretation & Conclusions: The present study showed an overall isolation rate of 17.64 per cent for B. melitensis. There is a need to establish facilities for isolation and characterization of Brucella species for effective clinical management of the disease among patients as well as surveillance and control of infection in domestic animals. Further studies are needed from different geographical areas of the country with different level of endemicity to plan and execute control strategies against human brucellosis.

The high level expression of recombinant proteins in Escherichia coli often leads to the formation of inclusion bodies that contain most of the expressed protein held together by non-covalent forces. The inclusion bodies are usually solubilized using strong denaturing agents like urea and guanidium hydrochloride. In this study recombinant Omp28 (rOmp28) protein of Brucella melitensis was expressed in two different vector systems and further efficient purification of the protein was done by modification in buffers to improve the yield and purity. Different concentrations of Triton X-100 and β-mercaptoethanol were optimized for the solubilization of inclusion bodies. The lysis buffer with 8M urea alone was not sufficient to solubilize the inclusion bodies. It was found that the use of 1% Triton X-100 and 20mM β-mercaptoethanol in lysis and wash buffers used at different purification steps under denaturing conditions increased the yield of purified rOmp28 protein. The final yield of purified protein obtained with modified purification protocol under denaturing conditions was 151 and 90mg/l of the culture or 11.8 and 9.37mg/g of wet weight of cells in pQE30UA and pET28a(+) vector respectively. Thus modified purification protocol yielded more than threefold increase of protein in pQE30UA as compared with purification by conventional methods., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Brucellosis is a disease caused by Gram-negative, facultative, intracellular bacteria belonging to the genus Brucella. It is an emerging zoonosis, and an economically important infection of humans and livestock with a worldwide distribution. Human infection is known to occur through consumption of infected raw milk, milk products and undercooked or raw meat. Serodiagnosis of brucellosis is carried out by detection of antibodies generated against LPS or whole-cell bacterial extracts by ELISA or agglutination tests using colorimetry. The present study was designed to develop a highly sensitive and specific indirect ELISA in both a microtitre plate and dot-blot format employing the recombinant outer-membrane protein 28 (rOmp28). Cloning and expression of Brucella melitensis Omp28 protein, which is a group 3 antigen, was accomplished by PCR amplification and cloning of the gene in a pET-28a expression system, followed by Ni-NTA affinity chromatography purification of the His-tagged recombinant protein. An indirect ELISA in both a microtitre plate and dot-blot format was optimized with sera collected from three groups: culture-confirmed cases, clinically suspected cases and healthy individuals. The rOmp28 protein reacted only with the culture-confirmed positive samples and no reaction was observed with culture-negative samples, confirming the immunoreactivity of the recombinant protein. The test in both formats had a correlation of approximately 90 % with the Rose Bengal plate agglutination test (RBPT) and a standard tube agglutination test, assays that are routinely performed for the serodiagnosis of brucellosis. The sensitivity and specificity of the assay in the plate format were 97.50 and 85.59 %, and in the dot-blot format were 82.05 and 92.43%, respectively, in comparison with RBPT. The specificity of this assay was further confirmed by testing samples that were positive for malaria and typhoid, which gave negative results. This ELISA system in microtitre plates and a dot-blot format will be useful for the rapid screening of large numbers of samples for the diagnosis of human brucellosis in endemic areas.