Preliminary study for the detection of pig specific antibodies against peptides obtained from PRRSV envelope proteins using Dot blot assay.
Estudio preliminar para la detección de anticuerpos específicos en cerdos contra péptidos obtenidos a partir de proteínas de envoltura del virus PRRS mediante un ensayo de Dot blot.
Zuniga Sanchez R1., Czub M1., Waeckerlin R1., Mansilla Ojeda R2., Camacho F2., Ruiz Garrido A3.
Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) is found worldwide being responsible for one the largest economic losses in the swine industry. Currently, different immunological assays are being used to detect specific anti-PRRSV antibodies, however they have some deficiencies. As an example of this is the ELISA test that usually does not work well with small molecules because it generally absorbs peptides larger than 15-20 residues in length. This study was conducted for the detection of specific antibodies in field pig sera against PRRSV using small synthetic peptides by Dot Blot assay. Those peptides were obtained from the envelope protein GP5 of PRRSV type 1 and type 2. Each sera sample was diluted using a range from 0.000625 to 0.005. The level of concordance between the results obtained by Dot-blot and commercial ELISA for pig categories oscillated from discrete to moderate against peptides. Sows showed higher antibody titers than younger pigs towards all peptides, being more noticeable against GP5 P1.
It was demonstrated that Dot-blot assay is a valuable technique for detecting specific antibodies against small synthetic peptides.
Keywords: PRRSV, pigs, Dot-blot, antibodies, synthetic peptides, envelope proteins.
El Síndrome reproductivo respiratorio porcino conocido como PRRSV, posee una distribución mundial y genera una de las mayores pérdidas económicas dentro de la industria porcina. Actualmente diferentes ensayos inmunológicos se utilizan para la detección de anticuerpos específicos contra el virus de PRRS, sin embargo, estos presentas diversas deficiencias. Un ejemplo, es el test de ELISA, en el cual moléculas pequeñas no se adhieren correctamente a la placa, debido a que esta última absorbe péptidos que posean entre 15-20 residuos de aminoácidos. Este estudio fue realizado con el objetivo de detectar anticuerpos específicos en cerdos contra péptidos de pequeño tamaño, mediante un ensayo de Dot blot. Estos péptidos fueron sintetizados en base a la proteína GP5 del virus PRRS tipo 1 y 2. Los sueros fueron diluidos en un rango desde 0.000625 hasta 0.005. El nivel de concordancia entre los resultados obtenidos mediante Dot blot y ELISA comercial por cada categoría animal fue de discreto a moderado contra los péptidos. Las cerdas mostraron mayores títulos de anticuerpos hacia todos los péptidos, siendo esto más marcado para el péptido GP5 P1. Se demostró que el ensayo de Dot blot es una valiosa técnica, la cual permite identificar anticuerpos específicos en cerdos, utilizando péptidos de pequeño tamaño.
Palabras Claves: PRRSV, cerdos, Dot-blot, anticuerpos, péptidos sintéticos, proteínas de envoltura.
In this work, it was identified specific antibodies against small peptides that were synthetized based on the sequence of epitope B of protein GP5 from North American and European strain of PRRSV using a Dot blot assay. The antibody response from different pig categories were analyzed toward each peptide and those results were compared to commercial ELISA.
Peptides from GP5 were synthesized based on published sequences for the genes encoding PRRSV structural glycoproteins (Table 1). These sequences were collected into a database (Geneious Pro 5.5, Biomatters, New Zealand), homology was determined on a nucleotide and amino acid level, and conserved sites in the PRRSV structural glycoproteins were selected. Their exposure to the immune system was predicted in silico by the 3D protein structure (HHPred, Max-Planck-Institute for Developmental Biology, Tübingen, Germany). Each peptide sequence (9 to 10-mer peptides) representing potentially immunogenic epitopes were chosen and were commercially synthesized (CanPeptide, Pointe-Claire, QC, Canada).
Table 1. Summary of synthetic peptides. NA: North American strain EU: European strain.
Twenty-eight pig serum samples with known PRRS-serostatus with a commercial ELISA (IDEXX, Westbrook, ME) belonging to 4 pig farms from the province of Alberta, Canada were screened against the peptides using a Dot-blot assay (Table 2). The sera came from three different pig categories: 20 from sows (6 PRRS-positive and 4 negative), 7 grower pigs (5 PRRS-positive and 2 negative) and 9 finisher pigs (6 PRRS-positive and 3 negative). A positive-PRRSV grower pig was used as positive control and serum sample from a newborn piglet belonging to negative farm was selected as the negative control.
Table 2. Individual serum samples organized based on pig category (sows, grower and finisher pigs) and antibody PRRSV status (positive and negative).
Swine sera screening against peptides through Dot blot assay.
Protran BA79 Nitrocellulose Blotting Membrane 0.1µm (GE Healthcare, Pittsburgh, PA) assembled in Bio-Dot Microfiltration System (Bio-Rad, Hercules, CA) was coated with 100 ng/well of peptides and incubated at 4°C overnight. Membrane was blocked adding bovine albumin serum (BSA) 1% in TBS buffer for 1 hour (h) at room temperature (RT). Swine sera from all pig categories were diluted in Log2 serial dilutions from 1:200 to1:1600 and pre-absorb in BSA 5% during 1 h at RT. Consequently, wells were incubated with 100ul of the pig sera at 37°C during 1 h. Rabbit anti-pig IgG (Sigma-Aldrich, St Louis. MO 63103 USA) was diluted 1:4000 in PBS buffer and 100 ul/well of the conjugated antibody was added to the membrane and incubated at 37°C for 1 h. ECLTM was added for color development with an incubation time of 5-10 minutes. Color intensity was measured using VersaDocTM MP 4000Imaging System, (Bio-Rad, Hercules, CA), and the analysis was performed using software Quantity One®.
Data obtained from software Quantity One® was analyzed to calculate the cut-off value for the samples based on the negative control. It was established that samples with intensity values (intensity*mm2) 3.5 times greater or equal than negative control were considered positive. On the other hand, concordance level between Dot blot and commercial ELISA was determined by calculating Kappa index, through the program VassarStats.
The results showed that sows category exhibited a higher level of concordance (kappa index between 0.55 to 0.60) with commercial ELISA results, in comparison to the other two pig categories which obtained a level of concordance between discrete to moderate. Finisher pigs showed the lowest level of concordance with a Kappa index of 0.25 (Table 3).
Table 3. Level of concordance between commercial ELISA results and Dot-blot results per animal category against GP5 P1, GP5 P3, GP5 P12 and GP5 P29. + (Positive); - (Negative); * (positive and negative concordant samples in both tests). Kappa: < 0 (No concordance); > 0.00 - 0.20 (Insignificant); 0.21 - 0.40 (Discrete); > 0.41 - 0.60 (Moderate); 0.61 - 0.80 (Substantial); 0.81 - 1.00 (Almost perfect).
On the other hand, there was not a significant difference between the level of concordance in sows per peptide. In grower pigs the lowest concordance was observed for peptide GP5 P3 and finisher pigs exhibited the same level of concordance for all peptides (Table 3).
The results for GP5 P1described that sows showed higher antibody titers than grower and finisher pigs because it was the only pig category from which 50% of the samples reacted positively to the peptide from the lowest concentration of serum tested (0.000625). Also, it was observed that only against GP5 P1, the percentage of positive sows decreased when swine sera were more concentrated therefore may be the detection capacity of the Dot-blot assay reaches a point from where the system is saturated because of a high concentration of GP5 P1 or the high concentration of sera (Figure 1a).
Figure 1. Percentage of positive pigs to the lowest ratio of pig sera/buffer concentration from different age groups tested against GP5 P1 (a), GP5 P3 (b), GP5 P12 (c) and GP5 P29 (d).
In contrast, the results for GP5 P3 showed that not only sows reacted positively from concentrations of serum of 0.000625, but also grower and finisher pigs were positive against the peptide. These group of pigs also exhibited higher antibody titers as well as the sows. Nevertheless, the percentage of sows that were positive from that concentration is still higher than the younger pigs (Figure 1b).
In GP5 P12 it was found that 50% of positive sows exhibited higher antibody titers than grower and finisher pigs. Half of the grower pigs (50%) and most finisher pigs (57.14%) were positive to that peptide from higher concentrations. In addition, there was none grower and finisher pig that reacted positively from concentration of serum of 0.000625 to this peptide (Figure 1c).
Finally, results for GP5 P29 showed that sows exhibited higher antibody titers than the other pig categories. Forty percent of positive sows reacted positively against GP5 P29 from concentration of serum of 0.000625 and a small percentage of grower pigs were positive from the same concentration (Figure 1d).
In general, at least for all peptides it was found a positive sow from the lowest concentration of serum tested (0.000625). Sows exhibited higher antibody titers than younger pigs toward all peptides.
The level of concordance between Dot blot and commercial ELISA results for sows was higher than those obtained in the other pig’s categories. Furthermore, specific antibodies against the peptides GP5 P1, GP5 P3, GP5 P12 and GP5 P29 were detected using Dot-blot assay, nevertheless those results require also be compared with other assays. Finally, as sows needed less concentration of serum to be positive toward all peptides, it may suggest that they presented higher antibody titers than younger pigs. However, due to the small number of samples used in this study it will be considered to assay a higher n in a future work.
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1 Virology, and Emerging Infectious Diseases, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada, T2N 4Z6.
2 Pharmacology Department, Faculty of Biological Sciences, Universidad de Concepcion, Concepcion, Chile, 4030000.
3 Pathology and Preventive Medicine, Faculty of Veterinary Medicine, Universidad de Concepcion, Chillan, Chile, 3820000.