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Abstract
Coxsackievirus B3 (CVB3) is the nonenveloped virus containing a single-stranded positive-sense RNA as a genome. CVB3 infection can induce acute myocarditis and dilated cardiomypathy. CVB3 of icosahedral symmetry has four capsid proteins called VP1, VP2, VP3, and VP4. Although VP1 is a major antigenic determinant, VP2 is also an important protein for viral physiology, such as maturation cleavage and attenuation. However, VP2 study has been hampered, partly because VP2 antibody is not available. In this study, we developed peptide-based polyclonal VP2 antibody and analyzed its potency by Western blotting analysis and immunofluorescent assay. Purified B3–1 antibody (VP2 peptide antibody developed in here) showed the sensitivity and specificity, similar to VP1 monoclonal antibody which is commercially available. Moreover, this peptide antibody may be useful for double-staining with other antibodies derived from mouse. Therefore, the VP2 antibody may allow us to study CVB assembly and understand VP2 function in depth.
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Figure 1.
CVB3 VP2 region for peptide antibody. (A) Hydrophilicity plot, Antigenic index and Surface probability plot of CVB3 VP2. It derived by DNASTAR programme. (B) Alignment of amino acid sequence of various Enteroviruses, which is used for developing VP2 peptide antibody.
Figure 3.
ELISA results from rabbit 1 (A) and rabbit 2 (B). B3-1 peptide was used as antigen and week 9 serum derived from two rabbits immunized with B3–1 peptide immunization were used as primary antibodies.
Figure 4.
SDS-PAGE results of purified B3–1 peptide antibody. (A) The primary step is done by processing antigen specific affinity purification. (B) The second step is done by Protein A purification.
Figure 5.
Comparison of Western blotting results. (A) Various enteroviruses-infected cell lysates are used as antigen for Western blot and purified B3–1 peptide antibody was used as primary antibody. (B) Summary table for Western blot data.
Figure 6.
Comparison of Immunofluorescent assay (IFA) results. (A) Various enteroviruses-infected cells cultured on cover slip are used as antigen for IFA and purified B3–1 peptide antibody was used as primary antibody. (B) Summary table for IFA data. NC indicates negative control which means non-infected cells.
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