The post-challenge SN titers for the 156 samples are summarized in Table 1. For the different SN titers, the range of proportions of the 156 samples was 8.3% to 19.2%. More than a quarter (26.4%) of the samples had an SN titer > 1:128. In addition, the results showed that there were 123 positive sera (SN titer > 1:8) and 33 negative sera (SN ≤ 1:8) ( Table 1). Further, the serum samples of different SN titers were used to determine the correlation coefficients of the correlations between the SN titers and ELISA results (involving the recombinant S fragments and the virus antigen). As ROC curves can be used to evaluate the performance of binary classifiers, they have been widely utilized in medical science to determine optimum diagnostic threshold values and to compare the ability of different diagnostic methods to recognize viruses. To determine the reliability of each type of ELISA, a ROC analysis was used for both the positive and negative samples.
SN titer* Number of samples Proportion ≥ 256 21 13.5% (21/156) 128 20 12.9% (20/156) 64 28 17.9% (28/156) 32 24 15.4% (24/156) 16 30 19.2% (30/156) 8 20 12.8% (20/156) ≤ 4 13 8.3% (13/156) Note: * SN titers are expressed as the reciprocal dilution of the serum samples.
Table 1. SN titers for 156 sow serum samples from four major swine-rearing provinces in China.
The recombinant proteins were expressed and purified after IPTG induction (Figure 2A). Compared with non-induced cell lysate, all the constructs produced antigenic proteins, but they did so to different extents. After purification using Ni-based chromatography, proteins of the predicted sizes were purified (Figure 2B) and the proteins were further confirmed using a western blotting analysis with anti-His-tag antibodies (Figure 2C).
Figure 2. Expression, purification, and identification of the recombinant S protein fragments. (A) Expression of the seven S fragments with or without isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. (B) Recombinant proteins purified using Ni-resin affinity chromatography. (C) Western blotting analysis of the seven recombinant S peptides detected using an anti-His-tag monoclonal antibody.
The checkerboard titration method was used to determine the optimum ELISA conditions regarding the coating antigen and detection antibody. The optimum amount of coating antigen was 4.13 μg of recombinant protein per well and the optimum detection antibody dilution ratio was 1:1000. The 123 positive and 33 negative serum samples (as determined based on the SN titers) were used to evaluate the performance of the recombinant S fragment ELISAs.
In addition, the GDS01 virus antigen was prepared as described above. The purified protein band between two concentrations of sucrose was collected with a syringe and used as the ELISA coating antigen. Based on the results of the checkerboard titration method, the optimum dilution ratio for the coating antigen and detection antibody was 1:100 and 1:1000, respectively. The field serum samples were also tested using the virus-based ELISA.
The correlation coefficients (r) of the correlation between recombinant S fragment ELISA results and the SN titer were determined, as shown in Figure 3. The r value for SP4 was 0.6113, which is higher than that for SP1, SP2, SP3, SP5, SP6, and SP7 (0.3810, 0.5151, 0.4663, 0.3596, 0.5384, and 0.2660, respectively).
Figure 3. Correlation between the OD450 values (obtained from recombinant S fragment and virus-based ELISAs) and SN titers. Each closed circle represents the OD450 value of a field serum sample plotted against the SN titer. r represents the correlation coefficient
The virus-based ELISA results were also correlated with the SN titer, with an r value of 0.4229. This result was similar to the r value for SP3. In general, when an r value is > 0.4000, the correlation is considered relatively strong. Thus, the correlation was relatively strong between the ELISA results involving SP2, SP3, SP4, SP6, and virus antigen and the SN titer. The r value for SP4 was greater than those for the other recombinant proteins or the virus antigen.
The performance of the ELISAs involving the recombinant antigens was analyzed using ROC curves (Figure 4A). The results revealed that the ELISA based on the SP4 antigen (AUC = 0.8538) could differentiate between positive specimens (positive for PEDV neutralizing antibody) and negative specimens (negative for PEDV neutralizing antibody) more precisely than the ELISAs based on the SP1, SP2, SP3, SP5, SP6, and SP7 antigens (AUC = 0.6863, 0.7249, 0.7379, 0.5729, 0.7262, and 0.6085, respectively).
Figure 4. Testing of field serum samples (from sows diagnosed based on SN titers) using recombinant S fragments ELISAs. (A) Graph of the sensitivity versus the false-positive rate (1 - specificity). A ROC analysis was used to determine the discriminatory accuracies of the ELISAs and the AUC was used to evaluate the diagnostic test performance. OD450 values for (B) low-titer PEDV serum samples (n = 33) and (C) high-titer PEDV serum samples (n = 123).
The optimal OD450 cutoff values for the recombinant S fragment ELISAs were determined according to the ROC analyses, as shown in Table 2. Regarding the recombinant S fragment ELISA results, the OD450 values for the negative serum samples (Figure 4B) were lower than those of the positive serum samples (Figure 4C). The sensitivity of each recombinant S fragment ELISA was calculated based on the cutoff value. The ELISA specificities were as follows: SP1: 96.97%; SP2: 96.97%; SP3: 90.91%; SP4: 93.94%; SP5: 90.91%; SP6: 96.97%; and SP7: 96.96%. The ELISA sensitivities were as follows: SP1: 13.01%; SP2: 12.2%; SP3: 39.84%; SP4: 69.11%; SP5: 21.24%; SP6: 26.83%; and SP7: 25.2% (Table 3). Further, the mean OD450 values for the 20 negative sera were as follows: SP1: 0.185; SP2: 0.307; SP3: 0.180; SP4: 0.353; SP5: 0.166; SP6: 0.256; and SP7: 0.206. The sensitivity was 100% when a cutoff value of 0.353 was used for the SP4 ELISA. However, the specificity was too low for this cut-off value to be considered. Therefore, a final cutoff value of 0.774 was selected based on the ROC analysis and samples with OD450 values between 0.353 and 0.774 were considered to be indeterminate.
Recombinant PEDV S proteins PEDV-S-SP1 PEDV-S-SP2 PEDV-S-SP3 PEDV-S-SP4 PEDV-S-SP5 PEDV-S-SP6 PEDV-S-SP7 + – + – + – + – + – + – + – PEDV-positive sow sera (n = 123)a 16 107 15 108 49 74 85 38 26 97 32 91 32 92 PEDV-negative sow sera (n = 33)b 1 32 1 32 3 30 2 31 3 30 1 32 1 32 OD450 cutoff value for ELISA 1.448 1.365 1.106 0.774 0.717 1.123 0.9465 Sensitivity (%)c 13.01 12.2 39.84 69.11 21.24 26.83 25.2 Specificity (%)c 96.97 96.97 90.91 93.94 90.91 96.97 96.97 Note: a 123 serum samples from sows that were diagnosed as PEDV-positive based on the SN titer. The cutoff value was 1:8; b 33 serum samples from sows that were diagnosed as PEDV-negative based on the SN titer. The cutoff value was 1:8;c The sensitivity and specificity for each recombinant protein was calculated based on an OD450 cutoff value determined using a ROC analysis.
Table 2. Comparison of the recombinant S fragment ELISA results using 156 field serum samples and SN titers
Samples Mean OD450 ± SD Intra-assay CV*(%) Inter-assay CV*(%) Intra-assay Inter-assay Negative sera 0.206 ± 0.005 0.226 ± 0.014 2.22 6.11 0.308 ± 0.011 0.344 ± 0.038 3.48 11.09 0.279 ± 0.014 0.267 ± 0.045 4.90 16.90 0.381 ± 0.010 0.377 ± 0.047 2.55 12.43 0.280 ± 0.007 0.302 ± 0.018 2.38 5.99 0.250 ± 0.009 0.248 ± 0.017 3.46 6.94 Positive sera 0.838 ± 0.040 0.877 ± 0.050 4.71 5.73 0.673 ± 0.005 0.657 ± 0.047 0.73 7.22 0.760 ± 0.032 0.750 ± 0.040 4.18 5.37 1.065 ± 0.041 1.038 ± 0.115 3.82 11.063 0.938 ± 0.023 0.955 ± 0.051 2.41 5.34 0.548 ± 0.030 0.559 ± 0.047 5.39 8.46 Note: CV*: Coefficient of variation.
Table 3. Repeatability of the SP4 ELISA for intra- and inter-assays (with three replications) using neutralizing antibody-negative and -positive serum samples
To assess the newly isolated PEDV virus-based ELISA under field conditions, a panel of the 156 sow serum samples was used. The AUC of the virus-based ELISA was 0.7134. Using a ROC analysis, the cutoff value was 1.233, and the corresponding specificity was 93.94% and the sensitivity was 36.59%. Among the seven S peptides and the virus antigen, the SP4 antigen had the highest sensitivity and specificity.
The repeatability of the SP4 ELISA was assessed using six neutralizing antibody-positive samples and six neutralizing antibody-negative samples. All the samples were randomly selected. Three repeats were conducted for each sample for the intra-assays. The repeatability for the inter-assays was also assessed. Finally, the SD and coefficient of variation were calculated to analyze the reliability of the SP4 ELISA. The results revealed that the assay was reliable (Table 3).
Furthermore, we compared the SP4 amino acid sequence of different strains (Supplementary Figure S1). The results showed that the new epidemic strains were highly homologous in terms of the SP4 amino acid sequences. These data suggest SP4 could be used as a promising antigen.
Table S1. Primers used for polymerase chain reaction (PCR) involving the seven recombinant S peptides
Figure S1. Alignment of the deduced amino acid sequence of the partial S2 subunit. Amino acid residues 791–954 of the S2 subunit of the PEDV GDS01 strain were aligned with those of other PEDV strains (including vaccine strains, which are marked with asterisks, and newly isolated strains from various areas). The dots indicate the regions where the sequences are identical to the consensus sequence
Table S2. Comparison of SP4 ELISA results with SN titers based on testing 156 field sow serum samples
Evaluation of purified recombinant spike fragments for assessment of the presence of serum neutralizing antibodies against a variant strain of porcine epidemic diarrhea virus
- Received Date: 06 March 2017
- Accepted Date: 14 June 2017
- Published Date: 20 July 2017
Abstract: Since 2010,variant strains of porcine epidemic diarrhea virus (PEDV) have caused disasters in the pork industry.The spike (S) protein,as the major immunity-eliciting antigen,has previously been used for serological testing and has been found to correlate significantly with the results of the serum neutralization (SN) test.However,further evaluation of this method is needed as new epidemic strains of PEDV emerge.Hence,the main objective of this study was to assess sow sera and determine the correlation between enzyme-linked immunosorbent assay (ELISA) results (involving a newly isolated GDS01 virus-based ELISA and ELISAs based on seven recombinant fragments comprising overlapping S1 and partial S2 sequences) and SN titers.Furthermore,we determined the reliability of the ELISAs based on receiver operating characteristics (ROC) curve analyses.For the most promising ELISA,i.e.,the SP4 ELISA,the correlation coefficient (r) and the area under curve (AUC) were determined to be 0.6113 and 0.8538,respectively.In addition,we analyzed the homology of the SP4 sequences obtained from different strains (including vaccine strains) and found that various strains showed a high degree of homology in this region.Thus,we conclude that SP4 is a promising serological testing protein for use in the field.