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. 2014 May 13:5:208.
doi: 10.3389/fmicb.2014.00208. eCollection 2014.

Wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody

Affiliations

Wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody

Satoko Matsunaga et al. Front Microbiol. .

Abstract

Human parainfluenza virus 3 (HPIV3) commonly causes respiratory disorders in infants and young children. Monoclonal antibodies (MAbs) have been produced to several components of HPIV3 and commercially available. However, the utility of these antibodies for several immunological and proteomic assays for understanding the nature of HPIV3 infection remain to be characterized. Herein, we report the development and characterization of MAbs against hemagglutinin-neuraminidase (HN) of HPIV3. A recombinant full-length HPIV3-HN was successfully synthesized using the wheat-germ cell-free protein production system. After immunization and cell fusion, 36 mouse hybridomas producing MAbs to HPIV3-HN were established. The MAbs obtained were fully characterized using ELISA, immunoblotting, and immunofluorescent analyses. Of the MAbs tested, single clone was found to be applicable in both flow cytometry and immunoprecipitation procedures. By utilizing the antibody, we identified HPIV3-HN binding host proteins via immunoprecipitation-based mass spectrometry analysis. The newly-developed MAbs could thus be a valuable tool for the study of HPIV3 infection as well as the several diagnostic tests of this virus.

Keywords: cell-free protein synthesis; human parainfluenza virus 3; monoclonal antibody; proteomics.

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Figures

FIGURE 1
FIGURE 1
Production of hybridoma cells generating anti-HPIV3-HN antibodies. (A) Schematic diagram of hybridoma cells production generating anti-HPIV3-HN monoclonal antibody (MAb). The recombinant Histidine-tagged recombinant HPIV3-HN (His-HN) protein was produced by wheat germ cell-free system and then purified by nickel-chelated sepharose beads in the presence of 8 M urea. The purified protein was separated by SDS-PAGE and visualized by CBB-staining. Purified His-HN protein was injected into the footpad of Balb/c mice. After 4 weeks, immunized mouse splenocytes were fused with myeloma cells and then 36 hybridoma cells were established. SP6, SP6 promoter sequence; E01, translation enhancer sequence; His, Histidine-tagged sequence; TEV; TEV protease recognized sequence. (B,C) The specificity of MAbs (hybridoma supernatant) evaluated by ELISA. The specificity of 36 MAbs in 2700-fold dilution was determined (B). The black arrows indicate the selected MAbs while the white arrow depicts a selected clone as a negative control (clone no. #30). The selected eight MAbs were diluted at serial points and analyzed by ELISA (C).
FIGURE 2
FIGURE 2
Immunoblotting and immunofluorescent analysis. (A,B) Detection sensitivity of the MAbs for recombinant His-HN (A) or HPIV3-infected cell lysate (B). Recombinant HPIV3-HN (100 ng) was separated using 12.5% SDS-gel and transferred to a PVDF membrane, followed by incubation with MAbs (hybridoma supernatants) at a 1:10 dilution (A). HeLa cells were infected or mock-infected with HPIV3. After 48 h, cells were lysed with SDS-PAGE loading buffer. The total protein was separated in 12.5% SDS-gel and immunobloted with indicated MAbs (B). (C,D) Immunofluoresent analysis of HN (red) in HPIV3-infected HeLa cells. HeLa cells were infected or mock-infected with HPIV3. After 48 h, cells were fixed, and then stained with MAbs (hybridoma supernatant; red) and DAPI (blue). Confocal microscopic analysis was performed at 40× (C) and at 600× magnifications (D).
FIGURE 3
FIGURE 3
Immunoprecipitation and flow cytometry assay. (A) Recombinant GST-HPIV3-HN or GST protein was immnoprecipitated with either #7, #21, #23 MAbs, or IgG (negative control), respectively. Then bound proteins were analyzed with immunoblotting using anti-GST antibody. (B) HPIV3-infected or uninfected HeLa cells were harvested at 4 days post-infection, followed by incubation with indicated MAbs. The cells were then fixed and stained with anti-mouse secondary antibody. The population of stained cells was calculated by flow cytometry. The shaded histogram shows negative hybridoma supernatant and the bold line shows specific MAbs.
FIGURE 4
FIGURE 4
Epitope mapping for generated antibodies. (A) Schematic diagram of seven deletion mutants of HPIV3-HN for epitope mapping. These proteins were produced as N-terminal biotinylated protein by wheat germ cell-free system. (B) Schematic diagram of the AlphaScreen assay used to detect the binding of MAb to full-length or deletion mutants of HPIV3-HN. The interaction between antibodies and recombinant proteins was monitored by AlphaScreen with protein A-conjugated acceptor beads and streptavidin-coated donor. Upon excitation at 680 nm, singlet oxygen molecules were produced from the donor beads, which reacted with the acceptor beads, resulting in light emission that was measured between 520 and 620 nm. (C) In AlphaScreen assay, the binding activity was measured as the level of the AlphaScreen luminescence signal. Error bars represent standard deviations from three independent experiments. (D) The biotinylated-full-length HN, its deletion mutants and GST proteins were separated by SDS-PAGE and transferred to PVDF membrane, followed by immunoblotting with Strepavidin-HRP andibody (left panel) and indicated MAb (right panel). (E) Summary of properties of selected MAbs. Immunoglobulin isotyping was carried out with mouse monoclonal antibody isotyping test kit.
FIGURE 5
FIGURE 5
No evident cross-reactivity of selected MAb to other paramyxoviruses. (A) Schematic diagram of AlphaScreen assay. The biotinylated partial HN proteins derived from several paramyxoviruses containing CT, TM and stalk region were produced by wheat germ cell-free system. (B,C) Specificity of MAbs was varidated by AlphaScreen assay (B) and immunoblotting (C). In AlphaScreen assay, the binding activity was measured as the level of the AlphaScreen luminescence signal (B). Error bars represent standard deviations from three independent experiments. The biotinylated partial HN proteins were separated by SDS-PAGE and transferred to PVDF membrane, followed by immunoblotting with either Strepavidin-HRP andibody (left panel) or anti-HN MAbs (right panel; C).
FIGURE 6
FIGURE 6
Proteomics analysis using selected MAb. (A) Schematic diagram of proteomic analysis for the identification of PIV3-HN-binding protein. HPIV3-infected HeLa cell lysate was immunoprecipitated with #21 MAb. The bound proteins were separated by SDS-PAGE and analyzed by LC-Ms/Ms. (B) The panel shows the list of putative HN-binding proteins identified by mass spectrometry analysis. (C) FLAG-tagged HSP70, HSP90, tubulin alpha 1C, or SERPINA3 proteins were mixed with HPIV3-HN. Samples were pull-down with the streptavidin magnetic beads and the collected proteins were separated by SDS-PAGE. The bound protein detected by immunoblotting analysis with anti-FLAG antibody. The right arrows indicated the position of each protein.

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