Vibriocidal Assays to Determine the Antibody Titer of Patient Sera Samples

Mike S. Son1, Ronald K. Taylor1

1 Dartmouth Medical School, Hanover, New Hampshire
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6A.3
DOI:  10.1002/9780471729259.mc06a03s23
Online Posting Date:  November, 2011
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The vibriocidal titer assay can be used to detect antibodies against Vibrio cholerae in serum samples, serving as an indicator of prior infection and potential protection against cholera. The assay can be utilized in research and clinical settings to test the effectiveness of vaccines, and also in epidemiological studies relevant to cholera transmission and surveillance. This unit outlines the steps involved in conducting an easily interpreted colorimetric vibriocidal titer assay with a relatively short turnaround time for results of around 8 hr, with final result observations in 24 hr. The assay can also be easily scaled up or down to accommodate as many or as few serum samples available and is not V. cholerae strain specific. Curr. Protoc. Microbiol. 23:6A.3.1‐6A.3.9. © 2011 by John Wiley & Sons, Inc.

Keywords: Vibrio cholerae; vibriocidal titer assay; serum; antibody titer

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Table of Contents

  • Introduction
  • Basic Protocol 1: Colorimetric Determination of Vibriocidal Activity of Antibodies or Sera Samples
  • Alternate Protocol 1: Colony Counting Method to Determine Titer
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Colorimetric Determination of Vibriocidal Activity of Antibodies or Sera Samples

  • Overnight cultures of appropriate Vibrio cholerae strain(s)
  • Luria‐Bertani broth (see recipe)
  • 1× PBS (see recipe), ice cold
  • Guinea pig complement serum (see recipe)
  • Neotetrazolium chloride/sodium succinate solution (see recipe)
  • Glass culture tubes (Fisher, cat. no. 14‐961‐32)
  • Spectrophotometer (Milton Roy, model no. Genesys5)
  • Spectrophotometer cuvettes (Sarstedt, cat. no. 67.742)
  • Microcentrifuge (Eppendorf, Model no. 5415D)
  • 2‐ml microcentrifuge tubes (USA Scientific, cat. no. 1620‐2700)
  • Ice bucket with Ice
  • 96‐well U‐bottom plates (Falcon, cat. no. 353911)
  • 20‐ to 200‐µl multichannel pipettor (Rainin, cat. no. L12‐200), optional
  • Lids for U‐bottom plates (Falcon, cat. no. 353913)
  • 37°C incubator
  • Humidified chamber with lid (any container that has a lid and can hold a 96‐well plate with ample room)
  • Aluminum foil

Alternate Protocol 1: Colony Counting Method to Determine Titer

  • Luria‐Bertani agar plates (twelve plates for each strain; see recipe)
  • 650‐µl microcentrifuge tubes
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Literature Cited

Literature Cited
   Attridge, S.R., Johansson, C., Trach, D.D., Qadri, F., and Svennerholm, A.‐M. 2002. Sensitive microplate assay for detection of bactericidal antibodies to Vibrio cholerae O139. Clin. Diagn. Lab. Immunol. 9:383‐387.
   Attridge, S.R., Qadri, F., Albert, M.J., and Manning, P.A. 2000. Susceptibility of Vibrio cholerae O139 to antibody‐dependent, complement‐mediated bacteriolysis. Clin. Diagn. Lab. Immunol. 7:444‐450.
   Benenson, A., Saad, A., and Mosley, W.H. 1968. Serological studies in cholera: 2. The vibriocidal antibody response of cholera patients determined by a microtechnique. Bull. World Health Organ. 38:277‐285.
   Boutonnier, A., Dassy, B., Duménil, R., Guénolé, A., Ratsitorahina, M., Migliani, R., and Fournier, J.‐M. 2003. A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139. J. Microbiol. Methods 55:745‐753.
   Carroll, M.C. 2004. The complement system in regulation of adaptive immunity. Nat. Immunol. 5:981‐986.
   Chernyak, A., Kondo, S., Wade, T.K., Meeks, M.D., Alzari, P.M., Fournier, J.‐M., Taylor, R.K., Kovac, P., and Wade, W.F. 2002. Induction of protective immunity by synthetic Vibrio cholerae hexasaccharide derived from V. cholerae O1 Ogawa lipopolysaccharide bound to a protein carrier. J. Infect. Dis. 185:950‐962.
   Dharmasena, M.N., Krebs, S.J., and Taylor, R.K. 2009. Characterization of a novel protective monoclonal antibody that recognizes an epitope common to Vibrio cholerae Ogawa and Inaba serotypes. Microbiology 155:2353‐2364.
   Finkelstein, R.A. 1962. Vibriocidal antibody inhibition (VAI) analysis: A technique for the identification of the predominant vibriocidal antibodies in serum and for the detection and identification of Vibrio cholerae antigens. J. Immunol. 89:264‐271.
   Holmgren, J., Svennerholm, A.‐M., and Ouchterlony, O. 1971. Quantitation of vibriocidal antibodies using agar plaque techniques. Acta Pathol. Microbiol. Scand. B 79:708‐714.
   Oda, T. and Okazaki, H. 1958. An analytical study on the reduction of neotetrazolium chloride by the terminal electron transport system. Acta Medica Okayama 12:193‐204.
   Okui, S., Suzuki, Y., Momose, K., and Ogamo, A. 1963. Chemical properties and enzymatic reduction of neotetrazolium chloride. J. Biochemistry 5:500‐502.
   Slater, T.F. 1963. Studies on a succinate‐neotetrazolium reductase system of rat liver: Points of coupling with the respiratory chain. Biochim. Biophys. Acta 77:365‐382.
   Yang, J.S., Kim, H.J., Yun, C.‐H., Kang, S.‐S., Im, J., Kim, H.‐S., and Han, S.H. 2007. A semi‐automated vibriocidal assay for improved measurement of cholera vaccine‐induced immune responses. J. Microbiol. Methods 71:141‐146.
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