Detection of Brucella abortus by immunofluorescence assay using anti outer membrane protein of 19 kDa antibody

Background. 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.


Introduction
Brucella spp.are intracellular Gram-negative bacteria that cause human disease and significant economic losses worldwide due to livestock infection. 1Lipopolysaccharide (LPS) of Brucella spp.2][3][4] In serologic tests, LPS of the bacterial cell membrane has been mainly used to identify particular antibodies.4][15][16][17][18] The Brucella cell wall consists of a peptidoglycan (PG) layer firmly connected with the external layer consisting of at least 75 proteins with several OMPs. 19These include the major OMPs of group 2 (porin, 34-40 kDa), group 3 (25-30 kDa) that was described initially by Dubray and Bezard 20 and Verstreate et al., 21 the lipoprotein covalently bound to PG, 22,23 and the minor OMPs of group 1 (88-94 kDa).The OMPs of Brucella spp.have been widely considered as potentially defensive and immunogenic antigens. 24,25onoclonal 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. 26hysicochemical and practical examination has recently confirmed that OMP10, OMP16 and OMP19 are lipoproteins that are uncovered at the cell surface. 27It has been proven that these lipoproteins are present in several Brucella strains.These strains represent all 6 Brucella species and all their biovars.][30][31][32][33] The appearance of OMP19 has been proven to be necessary for the acceptance of a defensive reaction by the vaccine strain B. abortus S19, since the abrogation of its gene in this strain leads to the destruction of its protective ability in heifers, 34 showing that OMP19 should be a key component of a subunit vaccine against brucellosis.Furthermore, we have previously reported that recombinant OMP19, when injected with the mucosal adjuvant cholera poison, is a protective mucosal antigen that confers protection against an oral challenge with virulent Brucella. 30or this reason, in this research, we expressed OMP19 for the creation of a polyclonal antibody to use it in an immunofluorescence assay (IFA).
The study was endorsed by Animal Experimentation Ethics Committee of Kerman University of Medical Sciences, Iran.

Cloning and expression
The OMP19 gene was synthesized based on the existing gene sequence of B. abortus (accession No. U35742) and cloned into the pET-28a expression vector by Genray Biotechnology Company (Shanghai, China).The pET-28a vector allowed the expression of the cloned gene as a fusion protein with a 6-histidine residue at the amino terminus.The recombinant plasmid was transformed into an E. coli strain BL21 (DE3) competent cell by the heat shock method (42°C for 90 s), according to standard protocols. 35The transformant bacterium was chosen from Luria Bertani (LB) agar medium containing 30 µg/mL of kanamycin.The transformant was grown in 10 mL LB containing 50 mg/mL kanamycin at 37°C, shaken at 200 rpm overnight.Then, 1 mL of the medium was supplemented to 100 mL of fresh LB.The transformant was grown at 37°C until the optical density (OD) at 600 nm achieved 0.6, and after being prompted by including Isopropyl-β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM and incubation at 25°C overnight with shaking.The transformant was amassed by centrifugation at 5000 × g for 10 min, then the cell was resuspended in the (5-pellet volume) lysis buffer pH 8.0 (300 mM NaCl, 10 mM Imidazole, 50 mM NaH 2 PO 4 ) and incubated at 4°C for 30 min.The lysate was also sonicated (10 s pulse on followed by 10 s pulse off, total time: 4 min).The resulting lysate was centrifuged at 5000 × g for 30 min at 4°C.The supernatant including the recombinant protein was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 5% stacking and 15% resolving gels, dot blot and western blot analyses.The recombinant OMP19 was purified using metal chelate affinity chromatography by Ni-NTA column (Qiagen, Hilden, Germany).

Immunization
A New Zealand white female rabbit was immunized intramuscularly with the purified recombinant OMP19 protein (0.5 mg/mL) mixed with Freund's complete adjuvant (Sigma-Aldrich, St. Louis, USA) (1:1) administered into the back legs of the rabbit, followed by 2 injections emulsified with Freund's incomplete adjuvant and without adjuvant, respectively, at 2-week intervals.

Antiserum collection
The rabbit was bled 4 times (before each injection and 2 weeks after the 3 rd injection).Blood was obtained from the marginal vein of the ear and kept at 4°C overnight.Serum was isolated by centrifugation at 3000 × g for 10 min and put away at −20°C until utilized.
To evaluate the serum titer, 2 enzyme-linked immunosorbent assay (ELISA) tests were carried out.In the 1 st ELISA test, the reaction of serum during immunization against the purified recombinant protein was tested.In the 2 nd ELISA test, final serum was diluted from 1/100 until 1/100000 to determine the serum titer.

Enzyme-linked immunosorbent assay
Sera were assessed by ELISA for antibody reactivity vs the recombinant OMP19 as follows: 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark) were covered with the recombinant OMP19 (100 µL per well) in phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, USA) at a density of 25 µg/mL and incubated overnight at 4°C.The wells were washed 3 times with PBS and blocked with 200 µL of blocking buffer PBS containing 3% bovine serum albumin (BSA) (Sigma-Aldrich) for 2 h at 37°C.After 3 washes, the diluted sera were added and the plates were incubated for 1 h at 37°C.The plates were then washed 3 times with 0.1% (v/v) Tween-20 in PBS (PBST).Antibodies attaching to OMP19 were visualized using 100 µL polyclonal goat peroxidase-conjugated anti-rabbit IgG (Bio-Rad Laboratories, Inc., Hercules, USA) diluted 15,000-fold in blocking buffer.After 1-h incubation at 37°C, the plates were washed with PBST and developed with 100 µL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (1 mL TMB, 9 mL of citrate-phosphate buffer, pH 5, and 3 µL of 30% H 2 O 2 ).The reaction was stopped after 15 min by the addition of 50 µL 1.0 M H 2 SO 4 to each well and read at 450 nm by the ELISA plate reader.

Immunofluorescence test
The polyclonal antibody was purified using the Proteus Protein Mini Purification Spin Column Pack (Bio-Rad Laboratories, Inc., Hercules, USA).The antibody was conjugated to fluorescein isothiocyanate (FITC) using standard protocols.Briefly, 40 µL of antibody (100 µg/mL) was mixed with 80 µL of borate buffer (0.1 M).Then, 4 µL of FITC (1 mg/mL) was added to 120 µL of the mixture and incubated at 37°C for 30 min.The conjugated antibody was stored at 4°C until use.
The detection of B. abortus by IFA: The amount of 500 µL of culture medium, which included B. abortus, E. coli, Salmonella, and Klebsiella (negative controls), was centrifuged at 4000 × g for 10 min.The resulting pellets were washed 3 times with PBS and suspended in 200 µL of PBS.Then, 20 µL of the conjugated antibody was added and the suspensions were incubated at room temperature overnight with shaking.Subsequently, the suspensions were centrifuged, washed 3 times with PBS, and then 10 µL of the samples were examined under a fluorescence microscope.
In this study, 29 bacterial isolates including 19 Brucella isolates, confirmed with specific primers in the polymerase chain reaction (PCR), and 10 non-Brucella isolates without a specific amplicon in the PCR, were used to determine the sensitivity and specificity of the above-mentioned immunofluorescence (IF) test, using the anti-OMP19 antibody.

Expression and purification
The induced transformant was examined using SDS-PAGE and immunoblotting.The SDS-PAGE examination of extracted total proteins indicated that OMP19 was expressed successfully with an estimated molecular weight of 19 kDa (Fig. 1, 2).The protein was transferred to the nitrocellulose membrane and the specificity of the expressed 6×His-tagged recombinant OMP19 protein was determined using a commercial anti-His-tag antibody (Sigma-Aldrich, St. Louis, USA) by an immunoblot assay (Fig. 3, 4).The recombinant protein was purified with an affinity chromatography and subsequently used for the immunization of the rabbit.

Production of a polyclonal antibody
The immunization of a New Zealand white female rabbit led to the production of a specific polyclonal antibody against the OMP19 protein.As Fig. 5 shows, during the immunization procedure the titer of the antibody against this protein rose gradually.Figure 6 indicates that a high affinity antibody with a strong reaction was achieved after the last immunization.

Recognition of Brucella with a fluorescence microscope
The anti-OMP19 polyclonal antibody conjugated to FITC was able to properly identify B. abortus, while the negative controls were not identified (Fig. 7).In evaluating sensitivity and specificity, 16 of 19 Brucella isolates and 5 of 10 non-Brucella isolates were IF-positive in the test.Therefore, the sensitivity and specificity of this test were 84.2% and 50%, respectively.

Discussion
The diagnosis of brucellosis by serological examinations on the basis of the detection of antibodies against LPS of bacterial cell membranes causes false positive results in some cases because of cross-reactivity with other Gram-negative microorganisms.The OMPs of Brucella spp.have been widely considered as potential immunogenic and protective antigens.
In this research, 1 OMP of Brucella, a non-LPS group of immunogens, was used.The OMP19 was expressed as a fusion protein containing 6×His-tag.The recombinant OMP19 reacted positively with the antibody against Histag in blotting assays.The immunization of a New Zealand white female rabbit by the recombinant OMP19 led to a high-titer polyclonal antibody, as showed in ELISA.The high-titer polyclonal antibody can be utilized for experimental biology, medicine, biomedical research, the development of a vaccine and improved diagnostic tests.Some other investigators have previously produced particular antibodies vs several OMPs of Brucella.For example, several mAbs to 7 Brucella outer membrane proteins were considered by Cloeckaert. 14These antibodies were achieved by vaccinating mice with sodium dodecyl sulfateinsoluble (SDS-I) portions, cell walls, or whole bacterial cells of B. melitensis or B. abortus.The OMPs detected by these antibodies were the proteins with a molecular weight of 25-27 kDa and 36-38 kDa (porin) (major proteins), and the proteins with a molecular weight of 10, 16.5, 19, 31-34, and 89 kDa (minor proteins). 26he production of an mAb against OMPs with a molecular mass of 25-27 kDa (Ba-4, Ba-5), 36-38 kDa (Ba-6) of B. abortus S99 and rOMP31 of goat Brucella was performed by Farshad et al. and Zheng et al. 36,37 It was proven by Ghasemi et al. that the immunized serum was achieved from a rabbit that was inoculated with attenuated B. melitensis.This serum contained antibodies against recombinant OMP31 (rOMP31). 38The production of antibodies against OMPs of B. ovis in rams was performed by Riezu-Boj et al. 39 The production of immunoglobulins was investigated by Sung et al. in BALB/c immunized with Cu/Zn superoxide dismutase (SOD1) and OMP2b porin of B. abortus. 40The production of an antibody against the minor 89-kDa OMP of Brucella in cattle was performed by Limet et al. 41 Western immunoblot analysis using antisera raised against 3 strains of B. abortus, i.e., RB 51, S19 and a local field isolate of biotype 1 in buffaloes, indicated the production of antibodies against OMPs in a study by Munir et al. 42 On the basis of previous studies, it can be stated that OMPs of Brucella are capable of inducing an immune response and antibody production.These antibodies can be utilized for the improvement of vaccine candidates and diagnostic tests of brucellosis.
The antibody produced against the recombinant OMP19 had a high titer.Therefore, it could be utilized for diagnostic tests of brucellosis and the improvement of vaccine candidates for humans and animals.Moreover, this antibody conjugated to FITC was able to properly identify B. abortus.Therefore, it could be used for diagnostic tests of brucellosis in humans and animals.Although the sensitivity of antibodies was high, their specificity was moderate in an IF test.This could be due to some cross-reactions of the polyclonal antibody.

Fig. 1 .
Fig. 1.SDS-PAGE analysis of total cell lysate of E. coli producing the OMP19 protein OMP19 -the recombinant outer membrane protein 19 kDa; lane 1 -OMP19; lane M -protein prestained ladder; the arrowhead shows the location of the Brucella recombinant protein.

Fig. 5 .
Fig. 5.The production of the polyclonal antibody against the recombinant outer protein 19 kDa (OMP19) of Brucella spp.during the immunization of a rabbit by the enzyme-linked immunosorbent assay (ELISA) OD -optical density.

Fig. 6 .
Fig. 6.Determination of the titer of the polyclonal antibody by the enzyme-linked immunosorbent assay (ELISA) OD -optical density.