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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol: Colorimetric Determination of Vibriocidal Activity of Antibodies or Sera Samples
  • Alternate Protocol: Colony Counting Method to Determine Titer
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol: Colorimetric Determination of Vibriocidal Activity of Antibodies or Sera Samples

 Materials
  • 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: Colony Counting Method to Determine Titer

 Additional Materials (also see Basic Protocol)
  • Luria-Bertani agar plates (twelve plates for each strain; see recipe)
  • 650-µl microcentrifuge tubes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •  FigureFigure 6A.3.1 Sample plates of positive and negative vibriocidal assay results for three different Vibrio cholerae strains (A) O1 classical biotype wild-type strain O395, (B) O1 El Tor biotype wild-type strain N16961, and (C) O1 El Tor biotype strain BAA-2163 (representative strain from the 2010 Haitian cholera outbreak). For each strain, three serum samples were tested, individual A (top row – Ind. A), individual B (second row – Ind. B), and individual C (third row – Ind. C) along with one no serum negative control, BLANK (bottom row). Serial dilutions were performed from left to right (column 1 is undiluted; column 12 is 1:2048). Titers were determined as the reciprocal of the last clear well (due to cell lysis) for each row, resulting in no reduction of neotetrazolium chloride (NTC), which gives the characteristic purple coloration. For example, for (A) O1 classical biotype wild-type strain O395, the last clear well for individual A is in column 3 (dilution of 1:4) and so the titer would be 4. For individual B, the last clear well is in column 5 (dilution of 1:16), and so the titer is 16. Individual C does not have any clear wells, indicating that this serum sample does not contain any antibodies against V. cholerae. As expected, the bottom row (BLANK) did not have any clear wells. Note that when testing serum samples against El Tor biotype strains, the color development is much more dramatic (B and C) than when testing against classical biotype strains (A). Classical O395 (A) showed slight purple color formation and settling of the reduced NTC, while both El Tor strains (B and C) produced a darker purple color, but still showed evidence of reduced NTC settling. This may reflect the hardiness of the strain being tested, as the longer the cells are alive and metabolizing, the more NTC will be reduced, resulting in stronger color development, as observed in the El Tor strains (B and C).
    View Image

Videos

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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library