Development of Monoclonal Antibodies and Antigen-Capture ELISA for Human Parechovirus Type 3

Figures

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  Figure 1. Production of anti-HPeV3 mAbs using wheat germ cell-free synthesized VP0 antigen. (a) Schematic representation of hybridoma cell production to generate anti-HPeV3 mAbs using the recombinant Histidine-tagged HPeV3-VP0 (His-VP0) protein produced by the wheat germ cell-free system, and then purified using nickel-chelated Sepharose beads. Each protein fraction was analyzed by SDS–PAGE and visualized by CBB staining. Red dots and arrow indicate the target protein. T—total fraction; S—supernatant; P—precipitate; FT—flow-through; and purified fractions 1–4. Purified His-HPeV3-VP0 antigen was injected into BALB/c mice. After 4 weeks, the spleen cells of immunized mice were fused with myeloma cells and 48 hybridoma clones were established. (b) Out of these 48 clones, nine exhibited relatively high reactivity to antigen proteins, as revealed by ELISA. We used HPeV1 and HPeV3 VP0 proteins as antigens. (c) Isotyping of selected mAbs-producing hybridoma clones.

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  Figure 2. Epitope mapping of mAbs. (a,b) Deletion mutants of VP0 were produced by wheat germ cell-free protein synthesis. Reactivity of each mAb to the deletion mutants was evaluated by immunoblotting. + and – indicate positive and negative detection, respectively. (c) Position of epitopes in a structural model of HPeV3 VP0 (PDB ID. 6GV4). Epitope localizations of the various mAbs are highlighted in different colors. (d) Multiple alignment of sequences in HPeV VP0 proteins in the epitope region.

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  Figure 3. Cross-reactivity of anti- HPeV3-VP0 mAbs. Cross-reactivity of selected mAbs to related HPeVs. His-tagged VP0 proteins of HPeVs were synthesized by a wheat germ cell-free system and the reactivity of mAbs was then assessed by immunoblotting analysis. The numbers associated with lanes showing positive bands are highlighted in red.

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  Figure 4. Development of antigen-capture ELISA for HPeV3 VP0. (a) Thirty-six mAb pairs were tested for sandwich ELISA. Signal-to-noise (S/N) ratio for antigen detection by each of the 36 combinations was calculated in the presence of 8 ng antigen vs. blank. (b) KD value, kon, and kdis of #8 and #38 mAbs. (c) Detection limit of ELISA for HPeV3 recombinant VP0 proteins (left) and viral particles (right). (d) Cross-reactivity with related viruses of the HPeV3 ELISA system.