Rabbit polyclonal antibody against Erns protein for the detection of Classical Swine Fever Virus

The protein was extracted from Escherichia coli BL21 DE3 cells transformed with pET22b-Erns, which expressed a truncated form of Erns fused to a 6-histidine tag at the C-terminal end and to pelb leader sequence by the N-terminal end. Chromatography was performed by metal chelate affinity chromatography (IMAC) under denaturing conditions, using 6.5 mL of Chelating Sepharose ^TM Fast Flow matrix charged with Ni²⁺ and packed in a C10/10 column (GE Healthcare). The column was equilibrated with binding buffer (20 mM Tris-HCl, 200 mM NaCl, 2 mM Imidazole, 8 M Urea, pH 6) at a volumetric flow rate of 0.6 mL/min. Purification processes included a washing with 50 mM Imidazole, and protein elution was performed with 200 mM Imidazole.

Protein electrophoresis was performed on a 12 % polyacrylamide denaturing gel with 2 % Sodium Dodecyl Sulfate (SDS). Samples were diluted in loading buffer (125 mM Tris-HCl, pH 6.8, 1 % (w/v) SDS, 5 % (v/v) glycerol, 10 mM Dithiothreitol, 0,005 % (w/v) Blue Bromophenol) and boiled for 10 minutes. The samples were run for about 45 minutes at a constant voltage of 200 V in running buffer (25 mM Tris-HCl, pH 8.8; 192 mM Glycine; 35 mM SDS). The gel was stained with a 0.1 % (w/v) Coomassie R-250 solution, 40 % (v/v) methanol, 10 % (v/v) acetic acid for 20 minutes and the bands were visualized after gel destaining with a 10 % (v/v) methanol and 10 % (v/v) acetic acid.

For western blotting assay, proteins were transferred (100 V, for 1 h) from the polyacrylamide gel to a PVDF membrane (Roche Diagnostics, Germany). As the primary antibody, the purified anti-Erns polyclonal antibody was used (5 μg/mL) in skimmed milk powder (1 %) - PBS-1X-Tween 20 (0.05 %). As the secondary antibody, an anti- rabbit IgG conjugated to alkaline phosphatase diluted 1: 20000 (Sigma Aldrich, USA) was used. Colorimetric detection was performed with 5-bromo-4-dichloro-indolyl phosphate and nitroblue tetrazolium (Promega, USA).

Two white New Zealand male rabbits weighing approximately 1.5 kg, supplied by the National Laboratory Animal Production Center (CENPALAB), were immunized subcutaneously with 217 μg of purified Erns each one. The protein was previously diluted until the urea decreased from 8 M to 2 M, to avoid toxic effects for immunized animals. For the first dose, the antigen was emulsified with a 1:1 ratio in complete Freund's adjuvant (CFA). Six subsequent immunizations were performed every 28 days; the first two with 100 μg and the rest with 50 μg of antigen emulsified with a 1:1 ratio in incomplete Freund's adjuvant (IFA). Blood samples were taken by puncture of the ear vein to sera titration. At the end of the immunization scheme, exsanguination was performed by cardiac puncture. Collected blood was incubated for 30 min at 37°C and then for 30 min at 4°C. The serum was extracted by centrifugation at 600 x g during 20 min. Animal experiments were carried out in accordance with the legal requirements of the national authorities.

A volume of 10 mL of immunized rabbit serum was filtered through glass wool and precipitated by 50 % (w/v) of (NH₄)₂SO₄. The resulting pellet was resuspended into 3 volumes of binding buffer (1.5 M Glycine, 3 M NaCl, pH 8.9) and charged to an nProtein A Sepharose Fast Flow (GE Healthcare) matrix packaged in a C10/10 column (GE Healthcare) with a height of 8.5 cm and equilibrated with the same buffer. Elution was performed at acidic pH with 0.1 M citrate buffer pH 3.0. The flow was kept constant at 0.6 mL/min throughout the process. The chromatographic profile was monitored by measuring absorbance at 280 nm. The eluate was neutralized with 2 M Tris-HCl and dialyzed on 20 mM Tris-HCl, 150 mM NaCl pH 7 for 16 h, and then it was filtered by 0.2 μm membranes. Thimerosal was added as a preservative at a final concentration of 0.02 % (v/v). The purity was evaluated by SDS-PAGE under reducing conditions, and the protein concentration was determined by Lowry´s method using bovine serum albumin as standard (9).

Immunoenzymatic assays were performed on a 96-well polystyrene microtiter plate (Greiner, Microlon, and High Binding Germany). The coating was performed in 0.1 M carbonate buffer pH 9.5 during 1h at 37°C with purified Erns (10 µg/mL) in titration ELISAs and anti-Erns polyclonal antibody in all antigen capture ELISAs. In blocking step, skimmed milk powder 3 % (Oxoid, UK) (w/v) in PBS was used for 1 h at 37°C. In the case of the conjugate in indirect ELISA, an anti-rabbit IgG antibody coupled to peroxidase produced in sheep was used, according to the supplier (CIGBSS, Cuba). In the direct ELISAs, the obtained polyclonal antibody, conjugated to peroxidase, was used. All washing steps were performed with wash solution (50 mM Tris, 140 mM NaCl, 0.05 % Tween 20). Dilutions of the samples and antibodies were performed in a 1 % (w/v) skimmed milk powder solution in PBS buffer, and incubations were performed at 37°C in a wet chamber. In all assays 100 μL were added per well except the blocking step which was carried out with 300 μL. The revealed reaction was developed with a solution of Ortho-phenylenediamine (0.5 mg/mL, dissolved in 0.0243 M citric acid with 0.0514 M disodium hydrogen phosphate and 0.015 % hydrogen peroxide). The development time was of 15 minutes in all cases and reaction was stopped with 2 M H₂SO₄. The reading was carried out at 492 nm in a plate reader (Multiskan Titertek).

Conjugation was performed by the periodate method (10). Briefly, 1 mL of purified antibody (8 mg/mL) in carbonate buffer 10 mM (pH 9.5) was added to the activated enzyme and it was put on the stirrer for 2h at 25ºC. Then, 0.4 mg of sodium borohydride was added to the conjugated solution. Precipitation by ammonium sulfate was carried out and pellet was resuspended in 1.5 volumes of PBS. The conjugated solution was dialyzed in PBS buffer, and bovine serum albumin (BSA) was added at a concentration of 10 mg/mL. After BSA was completely hydrated, an equal volume of 99 % glycerol was added along with thimerosal solution to a final concentration of 0.01 %.

In order to clarify the serum, it was filtered by glass wool to contribute to delipidation and avoid unwanted interactions with the matrix. The purification of anti-Erns antibodies was carried out by Protein A affinity chromatography. An eluate with 32 mg of total IgG (4 mg of IgG/mL of Protein A matrix) was collected with purity higher than 90 %. One of the most frequent contaminants in antibody purification is albumin, due to its high concentration in animal sera. In order to eliminate part of the total albumin into the clarified serum, it was precipitated with ammonium sulfate. Regarding the chromatogram of the process (Fig. 1A), it could be seen that the peak of the elution corresponded to a high purity fraction of the polyclonal antibody (Fig. 1B). The absorbance corresponding to the non-absorbed fraction indicated that the clarification of the serum with glass wool and the ammonium sulfate precipitation contributed to the purity of eluted antibody as they prevented the overloading of the matrix. However, a band of approximately 70 kDa was observed in the not-bound fraction corresponding to albumin, which gave an idea of its high amounts in serum. (Figure 1B).

To determine the reactivity of the purified polyclonal antibody with the corresponding protein, a western blot assay was performed using the previously purified protein. The antibody could recognize a band of approximately 19 kDa, corresponding to the truncated Erns (Fig. 2B). This implied that the immunization procedure was efficient.

The reactivity of the antibody against other unrelated proteins was assessed by an indirect ELISA (Fig. 2A). The antibody showed no reactivity against any of the adjuvants used in immunization (complete and incomplete Freund's adjuvants). As unrelated protein, the recombinant p24 (belonging to the human immunodeficiency virus) was used. This protein had a 6 histidine tag as well as the truncated Erns used in immunization. No reaction indicated that there were not antibodies in the serum capable of recognizing this fragment used in the production of chimeric proteins. Another negative control used was the extract of Escherichia coli BL21-DE3 transformed with the PET-22 plasmid, but without the insert containing the sequence encoding the truncated Erns.

The reactivity of the antibody was observed against proteins of the strain used for the production of the antigen. However, the antibody (10 μg/mL) had a 5-fold higher affinity for the truncated Erns than for E. coli contaminants and the monoclonal antibody evaluated as unrelated protein. Besides, upon dilution of the antibody up to 1250 ng/mL, the corresponding absorbance was greater than 1 and no cross-reactivity was observed at this concentration. This cross-reactivity was attributed to small concentrations of contaminants that may have been more immunogenic than the truncated Erns, but also other interactions (not antigen-antibody) could have influenced the assay.

In order to reduce the incubation steps in the immuno-enzymatic assays for Erns detection, the antibody was conjugated to peroxidase. Direct ELISA was developed with coated Erns (10 μg/mL) to evaluate working dilution. The antibody was added at an initial dilution of 1:500 and serial 1:2 dilutions were performed. The optimal dilution work was taken when the observed absorbance was approximately 1 because with it, the signal over-saturation would be avoided in the plate reader. As shown in Figure 3, absorbance was about 1 with a dilution of 1: 64000, so this could be taken as working dilution. This conjugated antibody could also be used in other assays such as western blot and dot-blot, but for these kinds of assays, the optimal dilution and substrate conditions must have been established to ensure the best performance for each technique.

Four coating concentrations were evaluated with the antibody to determine the optimal capture conditions. Eight dilutions of the used samples were evaluated: purified recombinant Erns as positive control, P24 as a negative control and a mixture of sera from healthy pigs to evaluate the possible recognition of nonspecific proteins in sera (Fig. 4). The assay demonstrated that coating at 10 μg/ mL was the condition at which higher Erns was captured. It was also observed that the antibody did not capture proteins from healthy pig sera.

The best coating concentration (10 µg/mL) was used in a Sandwich ELISA (Fig. 5). The polyclonal antibody could recognize Erns from the sera of individual animals positive to CSFV and from a pool of sera which belonged to the animals vaccinated with the Chinese strain of CSFV. Other authors have also detected viral Erns in sandwich ELISA for the diagnosis of CSF and they have obtained successful results when compared with more specific techniques such as RT-PCR (5). Nevertheless, in some cases, good results have not been obtained with the antigen-capture ELISAs for the diagnosis of CSF (4).

Negative samples (serum from animals vaccinated with Porvac®) were evaluated in the same ELISA. The fact that it did not recognize negative sera, including healthy animals immunized with E2, supported the hypothesis that the antibody could be a useful tool to distinguish vaccinated animals from those infected with CSFV. Besides, the development of an antigen capture technique by ELISA could work like a complement in the diagnosis of CSF in passively immunized animals and in persistently infected pigs a few days after birth with moderately virulent strains of CSFV that did not rise up an immune response. Unlike the direct ELISA, where the antibody detected E. coli BL21DE3-Pet22b proteins coated at 10 μg/mL (Fig. 2A), this non-specific reactivity was not observed in the sandwich ELISA. In addition, there was no response against the sera from animals immunized with E2.