The 672-bp major CP gene of a CTV stem pitting isolate collected and identified from a citrus grove in Chongqing, China (Zhou et al., 2007) was amplified by RT-PCR and then inserted into the prokaryotic expression vector pET-28a. Sequencing analysis confirmed that the nucleotide sequence and the orientation of the ORF of the major CP in the recombinant expression vector pET-28a-CP were correct. E. coli BL21(DE3) cells harboring pET-28a-CP expressed an approximately 29-kDa recombinant fusion protein with IPTG induction, which coincided with the predicted molecular weight of the 6×His-tagged major CP (Figure 1A). Western blot analysis using an anti-His MAb (Roche, Indianapolis, IN, USA) as the primary antibody revealed a 29-kDa protein band in lanes with ly-sate from E. coli BL21(DE3) cells harboring the pET-28a-CP and purified recombinant protein (Figure 1B), indicating that the major CP recombinant protein was successfully expressed in E. coli BL21(DE3) and purified.
Figure 1. SDS-PAGE (A) and Western blot (B) anal-yses of the expressed major CP of CTV. Lane M: protein molecular weight marker. Lane 1: lysate of E. coli BL21 (DE3) harboring pET-28a with 0.5 mmol/L IPTG induction. Lanes 2 and 3: lysates of E. coli BL21 (DE3) harboring pET-28a-CP without or with 0.5 mmol/L IPTG induction, respectively. Lane 4: purified recombinant protein. An anti-His MAb was used to probe for the major CP recombinant protein in Western blot.
Four hybridoma lines (14B10, 14H11, 20D5, and 20G12) secreting MAbs specific for CTV were obtained after three cell fusion experiments and three cell cloning. Each hybridoma line was injected into pristane-primed BALB/c mice to produce ascitic fluid containing MAb. Isotypes and subclasses of all four MAbs belonged to IgG1, κ light chain. The titers of four MAbs determined by indirect ELISA ranged from 10-6-10-7, and IgG yields of MAbs in ascitic fluids ranged from 8.82 to 12.63 mg/mL (Table 1).
Table 1. Properties of CTV-specific monoclonal antibodies
The specificity of MAbs was first determined by Western blot. All four MAbs reacted strongly with the 25-kDa major CP of CTV in the crude extract from CTV-infected citrus leaf tissues but not with the crude extract from healthy citrus leaf tissues (Figure 2). An approximately 50-kDa protein band observed in CTV-infected tissues (Figure 2) was predicted to be a dimer of the major CP. The specificity of the MAbs with CTV-, CTLV-, and CYVCV-infected and healthy citrus leaf tissues was further tested by TAS-ELISA. All four MAbs reacted strongly with the crude extract from CTV-infect-ed citrus leaf tissues but not with the crude extracts from CTLV-, CYVCV-infected or healthy citrus leaf tissues (Figure 3), indicating that all four MAbs were specific for CTV.
Figure 2. Specificity analyses of four MAbs against CTV by Western blot. M: protein molecular weight marker. Lanes 1 and 2: CTV-infected and healthy citrus leaf tissues, respectively. Each lane was loaded with 10 μg total protein.
Figure 3. Specificity analyses of four MAbs by TAS-ELISA. Samples were diluted 1:20 (w/v, g/mL) and probed with MAbs diluted 1:5000 followed by AP-labeled goat anti-mouse IgG diluted 1:8000. Absor-bance value indicate the mean [±SD] from three independent assays at 30 min after adding the substrate at room temperature.
TAS-ELISA was also used to analyze the sensitivities of MAbs. The results of triplicate phalanx tests indicated that the PAbs (1:2000), the MAb (1:5000), and AP-conjugated goat anti-mouse IgG (1:8000) were optimal for TAS-ELISA. All four MAbs (14B10, 14H11, 20D5, and 20G12) could detect viruses in crude extracts of infected leaf tissue diluted up to 1:10, 240(w/v, g/mL), indicating that the MAbs and developed TAS-ELISA were very sensitive for CTV detection (Figure 4).
Figure 4. Sensitivity analyses of MAbs by TAS-ELISA. CTV-infected leaf crude extracts and healthy leaf crude extracts (CK-) were serially two-fold diluted in PBS from 1:40 to 1:40, 960 (w/v, g/mL) and probed with MAbs diluted 1:5000 followed by AP-labeled goat anti-mouse IgG diluted 1:8000. The OD405 absorbance values were measured 30 min after incubation with the substrate at RT. The OD405 values in figure indicate the mean of three independent assays.
Analytical phalanx tests demonstrated that 1:5000 and 1:8000 dilutions of MAb 14B10 and AP-conjugated goat anti-mouse IgG, respectively, were optimal in dot-ELISA for CTV detection. The developed dot-ELISA specifically detected CTV in infected citrus leaf tissues and was negative for CTLV-infected, CYVCV-infected, or healthy citrus leaf tissues (Figure 5A). Sensitivity anal-ysis revealed that the established dot-ELISA could detect viruses in infected citrus leaf extracts diluted up to 1:2560(w/v, g/mL) (Figure 5B).
Figure 5. Specificity (A) and sensitivity (B) analyses of dot-ELISA. CTV-, CTLV-, and CYVCV-infected and healthy (CK-) citrus leaf tissue extracts were tested by dot-ELISA. 1-10: CTV-infected and healthy (CK-) citrus leaf tissue extracts were serially two-fold diluted from 1:10 to 1:5120 (w/v, g/mL), respectively. Purple indi-cates positive reactions, and green or no color indi-cates negative reactions. Samples 1 and 2 are two differ-ent CTV-infected citrus leaf tissues.
The working dilutions of MAb 14B10 and AP-conju-gated goat anti-mouse IgG in tissue print-ELISA were also determined by the phalanx test; the optimal dilutions of two antibodies in Tissue print-ELISA for CTV detection were 1:4000 and 1:8000, respectively. To identify a suitable tissue for CTV detection by Tissue print-ELISA, young fully expanded leaves and young stems of CTV-infected or healthy citrus trees were sectioned and printed on a nitrocellulose membrane. Purple spots were observed in prints of leaves and young stems from CTV-infected citrus trees, whereas green spots were observed in those from healthy citrus trees (Figure 6). Thus, both young leaves and stems of citrus trees were suitable for Tissue print-ELISA.
Figure 6. Detection of CTV in infected citrus tissues by Tissue print-ELISA. Young leaves and stems from CTV-infected citrus trees (a and b) or from healthy citrus trees (c and d) were tested. CTV in infected citrus tissues was probed with MAbs diluted 1:4000 followed by AP-labeled goat anti-mouse IgG diluted 1:8000. Purple and green colors indicate positive and negative reactions, respectively.
We screened 219 citrus samples collected from the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China during 2013-2014 for the presence of CTV using the developed serological assays. Sixty-eight of the 219 citrus samples were tested positive for CTV by Tissue print-ELISA, dot-ELISA, and TAS-ELISA (Table 2). The results from the three serological me-thods were consistent with those from RT-PCR except for one citrus sample collected in Zhejiang Province, which was tested negative in the serological assays but positive in RT-PCR (Table 2). DNA sequencing and alignment results confirmed that the PCR product was indeed amplified from the CTV genome. But, the coincidence rate of serological and RT-PCR test results reached more than 99.5%. Based on the survey results, we can infer that the MAbs we prepared can target a broad spectrum of CTV isolates in China.
Table 2. Detection of CTV in citrus groves