ELISA for Molluscum Contagiosum Virus

Subuhi Sherwani1, Mohammed Chowdhury1, Joachim J. Bugert2

1 Department of Microbiology, Cardiff University School of Medicine, Cardiff, 2 Institut für Mikrobiologie der Bundeswehr, München
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14A.6
DOI:  10.1002/cpmc.42
Online Posting Date:  November, 2017
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Abstract

Molluscum contagiosum virus (MCV) is a common skin pathogen of children and young adults. Infection with MCV causes benign skin tumors in children and young adults and is mostly self‐limiting. In contrast to orthopoxviruses, MCV infections tend to take a subacute clinical course but may persist for up to 12 months. Current numbers for MCV seroprevalence in different geographical areas are based on a variety of historical serological methods from complement fixation assays to MCV ELISAs based on purified MCV virions and MC133 antigen expressed in a Semliki Forest Virus expression system. A standardized ELISA for the assessment of MCV seroprevalence would be useful to determine global MCV seroprevalence. The methods described show that polypeptides derived from MCV open reading frames MC084 (residues V123 to R230 and V33 to G117), mc133 (residues M1 to N370), and glutathione S‐transferase (GST)‐H3L (residues I142 to W251) expressed in E. coli RIL+ as GST fusion proteins can be used to assess antibody binding in a GST capture ELISA. We show how the ELISA can be used to screen a panel of patient sera previously characterized with the mc084 V123‐R230 ELISA. © 2017 by John Wiley & Sons, Inc.

Keywords: E. coli BL21 RIl+ expression system; molluscum contagiosum virus; GST fusion proteins; MC084; MC133; H3L; ELISA

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Antigen Production in E. coli
  • Basic Protocol 2: Purification and Elution of GST Fusion Proteins
  • Basic Protocol 3: GST Fusion ELISA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Antigen Production in E. coli

  Materials
  • E. coli strain BL21 RIL+ (BL21‐CodonPlus [DE3]‐RIL Competent Cells; e.g., Agilent Technologies)
  • Expression plasmids
  • LB agar plates containing 100 µg/ml ampicillin (see appendix 4A; Stevenson, )
  • LB medium (see appendix 4A; Stevenson, )
  • 1 M IPTG stock solution (e.g., Sigma‐Aldrich)
  • Lysis buffer (see recipe)
  • 4% to 12% Bis‐Tris gels (e.g., NuPAGE, Thermo Fisher Scientific)
  • Coomassie brilliant blue dye
  • Spectra Multicolor Broad Range Protein Ladder (e.g., Thermo Fisher Scientific)
  • Culture flasks
  • Variable temperature shaking incubator
  • Centrifuge tubes
  • Centrifuge
  • Probe sonicator
  • Ice and ice bucket
  • Additional reagents and equipment for SDS‐PAGE (see appendix 3M; Manns, )

Basic Protocol 2: Purification and Elution of GST Fusion Proteins

  Materials
  • Glutathione sepharose 4B beads (e.g., GE Healthcare)
  • Phosphate‐buffered saline (PBS) with and without Triton X‐100 (see recipe)
  • Protein lysate (see protocol 1)
  • 10 mM reduced glutathione (e.g., Sigma‐Aldrich, cat. no. G4251)
  • 50 mM Tris⋅Cl, pH 8.0 (see appendix 2A)
  • Centrifuge
  • 4°C incubator with agitation
  • Dialyzer
  • Additional reagents and equipment for determination of protein concentration (see appendix 3A; Lovrien & Matulis, )

Basic Protocol 3: GST Fusion ELISA

  Materials
  • 1 µg/ml GST solution
  • GST‐tagged antigen eluates from glutathione sepharose 4B beads (see protocol 2)
  • Phosphate buffered saline (PBS) with and without Tween (PBS‐T; see recipe)
  • Sera to be tested
  • Secondary antibody: horseradish peroxidase conjugated anti‐human IgG (e.g., Promega)
  • ELISA blocking buffer (dilution buffer; see recipe)
  • BD OptEIA substrate reagents A+B (e.g., BD Biosciences)
  • 0.25 M H 2S0 4
  • 96‐well Nunc Immobilizer glutathione ELISA plates (e.g., Thermo Fisher Scientific)
  • 37°C incubator
  • Microplate reader (e.g., BMG Labtech FLUOstar Optima microplate reader)
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Figures

Videos

Literature Cited

Literature Cited
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  Chowdhury, M. (2015). Construction of pGEX6P1‐GST‐H3L (I142‐W251) fusion vector plasmid and evaluation as a GST fusion protein in the MCV ELISA (Unpublished thesis). Cardiff University, Cardiff, United Kingdom.
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  Manns, J. M. (2011). SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) of proteins. Current Protocols in Microbiology, 22, A.3M.1–A.3M.13. doi: 10.1002/9780471729259.mca03ms22.
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  Sherwani, S., Farleigh, L., Agarwal, N., Loveless, S., Robertson, N., Hadaschik, E., … Bugert, J. J. (2014). Seroprevalence of Molluscum contagiosum virus in German and UK populations. PLoS One, 9(2), e88734. doi: 10.1371/journal.pone.0088734.
  Sherwani, S. (2013). Investigation of the infectious cycle of molluscum contagiosum virus in human skin and the nature of MCV induced immunity (Unpublished doctoral dissertation). Cardiff University, Cardiff, United Kingdom. doi: Orca.cf.ac.uk/57429/.
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