The full-length WDV CP gene sequence with 783 nucleotides was PCR-amplified. After double digestion with BamH Ⅰ and Hind Ⅲ restriction enzymes, the PCR fragment was inserted into the expression vector pET-32a to produce pET-32a-CP. DNA sequencing was performed to confirm the CP gene nucleotide sequence and orientation. A correct recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells to express recombinant WDV CP. After IPTG induction, the E. coli BL21 (DE3) cells harboring the pET-32a-CP vector accumulated a 50 kDa fusion protein (Fig. 1A). E. coli BL21 (DE3) cells transformed with the parental pET-32a vector produced an approximately 20 kDa protein, similar to the molecular mass of the thioredoxin-tag. The non-denatured recombinant CP fusion protein was purified using the Ni–NTA agarose method (Qiagen, MD, USA) as described previously (Liu et al. 2017). The expressed recombinant WDV CP protein was later confirmed by Western blot using an anti-His tag MAb (Fig. 1B).
Figure 1. SDS-PAGE (A) and Western blot (B) analyses of the recombinant WDV CP protein. Lane M, protein molecular weight marker. Lanes 1 and 2, E. coli BL21 (DE3) harboring pET-32a induced with and without 0.5 mmol/L IPTG. Lane 3, E. coli BL21 (DE3) harboring pET-32a-CP induced with 0.5 mmol/L IPTG. Lane 4, Purified recombinant WDV CP.
BALB/c mice were immunized with purified recombinant WDV CP. After the fourth immunization, four hybridoma lines (18G10, 9G4, 23F4 and 22A10) secreting anti-WDV CP MAbs were obtained through four time cell fusions, antibody specificity and sensitivity analyses, and cell limiting dilution cloning. Ascitic fluids with MAbs were produced by intraperitoneal inoculations of hybridoma cells to pristane-primed BALB/c mice. IgG of WDV specific MAb was precipitated from different ascitic fluids with saturated ammonium sulfate. Isotypes of the four MAbs were determined to be IgG1, κ light chain. Yield of IgG in ascites was determined at 5.87 to 10.14 mg/mL, and the titers of the four MAbs ranged from 10-6 to 10-7 as determined by an indirect ELISA (Table 1).
Table 1. Properties of the obtained anti-WDV monoclonal antibodies.
Western blot was then used to determine the specificity of the anti-WDV MAbs. Results of the assays indicated that the four MAbs reacted strongly and specifically with approximately 30 kDa WDV CP in the WDV-infected wheat samples as well as the 50 kDa recombinant WDV CP fusion protein (Fig. 2). As expected, no visible protein bands were seen in the lane loaded with an extract from a healthy wheat plant (Fig. 2).
Figure 2. Specificity analyses of anti-WDV MAbs by Western blot. All the SDS-PAGE gels had the same protein loadings but were probed with different MAbs. Lane 1, protein from a healthy wheat plant. Lane 2, protein from a WDV-infected wheat plant. Lane 3, purified recombinant WDV CP fusion protein. Lane M, protein molecular markers. Names of the MAbs are indicated below the figures.
The optimal working dilutions of MAbs and the AP-conjugated goat anti-mouse IgG for the ACP-ELISA were determined by the phalanx tests. Results of the phalanx tests indicated that WDV could be reliably detected in crude extracts from WDV-infected wheat plant tissues using MAb 22A10, 23F4, 18G10 or 9G4, diluted at 1:6, 000, 1:6, 000, 1:5, 000 and 1:5, 000 (v/v). The optimal dilution of AP-conjugated goat anti-mouse IgG was determined at 1:8, 000 (v/v). Using the optimal working dilutions described above, an ACP-ELISA for WDV detection was developed.
The specificity assay using the developed ACP-ELISA protocol demonstrated that WDV could be reliably detected in the WDV-infected wheat samples but it had a negative reaction with WYMV-, BYDV PAV-, BYDV GAV-, BYDV GPV-, BaYMV-or CWMV-infected, or the healthy wheat sample (Fig. 3A).
Figure 3. Specificity (A) and sensitivity (B) analyses of the developed ACP-ELISA using anti-WDV MAbs. CK-, an extract from a healthy wheat plant sample.
Sensitivity assay showed that the developed ACPELISA methods based on MAb 23F4, 22A10, 9G4 or 18G10, could detect the virus in WDV-infected wheat plant crude extracts diluted at 1:163, 840, 1:163, 840, 1:81, 920 or 1:81, 920 (w/v, g/mL), respectively (Fig. 3B). These results showed that the newly established ACP-ELISA is a highly sensitive and specific method for detection of WDV in wheat samples.
Phalanx tests were also performed to determine the optimal working dilutions of the four MAbs and the AP-conjugated goat anti-mouse IgG for dot-ELISA. Results of the tests showed that the optimal working dilutions for MAb 23F4, 22A10, 9G4 and 18G10 were all 1:5, 000 (v/v). For the APconjugated goat anti-mouse IgG, the optimal working dilution was found to be 1:8, 000 (v/v). Using this newly developed dot-ELISA, WDV was reliably detected in the dot of crude extract from a WDV-infected wheat plant or in the dot of purified recombinant WDV CP (Fig. 4, Column 1 and 2). No detection signal was observed in the dots of crude extracts from a WYMV-, BYDV PAV-, BYDV GAV-, BYDV GPV-, BaYMV-or CWMV-infected wheat plant (Fig. 4A, Column 3–8) or from a healthy wheat plant (Fig. 4A, Column 9). Further assays indicated that dotELISAs based on MAb 23F4, 22A10, 18G10 or 9G4, could be used to detect the virus in WDV-infected wheat plant crude extracts diluted at 1:5, 120, 1:1, 280, 1:1, 280 and 1:640 (w/v, g/mL), respectively (Fig. 4B).
Figure 4. Specificity (A) and sensitivity (B) analyses of the developed dot-ELISA using anti-WDV MAbs. A Specificity test of the dotELISA. Columns 1 and 2 have dots of an extract from a WDVinfected wheat plant sample and from the purified recombinant WDV CP, respectively. Columns 3–8 have dots of an extract from a WYMV-, CWMV-, BYDV GAV-, BYDV GPV-, BYDV PAV-or BaYMV-infected wheat plant sample. Column 9 has the dot of an extract from a healthy wheat plant. Each treatment has two dots. MAbs used for the treatments are indicated on the left side. B Sensitivity test of the dot-ELISA. Dots in each panel represent a WDV-infected or a healthy wheat plant. Dilutions of the wheat plant extracts are indicated on the top of the figures and the names of the MAbs are indicated on the right side.
According to the results from the sensitivity and specificity assays, MAb 23F4 was selected to detect WDV in fieldcollected wheat samples through ACP-ELISA and dotELISA. To further confirm the detection results, all the samples were retested by PCR using WDV specific primers.
For this study, a total of 128 wheat plant samples were collected from wheat fields in Hancheng city of Shaanxi Province, and Xining city of Qinghai Province, and tested for WDV infection. Results of the two serological assays indicated that 97 of the 128 samples were tested positive for WDV infection (Fig. 5). Further validation using PCR was agreed with the results obtained by the two serological detection methods (Fig. 5). Sequencing of the resulting PCR products followed by sequence alignment showed that the WDV isolates detected in the field samples sheared 96.5%–97.7% sequence similarity with the published wheat dwarf virus isolate SXHC-2 sequence (GenBank Accession No. JQ836568). Taken together, we have shown that the newly developed ACP-ELISA and dot-ELISA methods using MAb 23F4 is highly sensitive and accurate for detection of WDV in field-collected wheat plant samples.
Figure 5. Detection of WDV in field-collected wheat samples by dotELISA, ACP-ELISA and PCR. A Detection of WDV in fieldcollected wheat samples by dot-ELISA. A representative blot was showing sample detection result. The dots labeled CK + and CKrepresent a WDV-infected and a healthy wheat control sample, respectively. Purple colored dots indicate WDV-infected wheat samples. The same samples used in (A) were tested again by ACPELISA (B) and PCR (C).