Detection of Borreliacidal Antibodies by Flow Cytometry

Steven M. Callister1, Dean A. Jobe1, Ronald F. Schell2

1 Gundersen Lutheran Medical Center and Microbiology Research Laboratory and Gundersen Lutheran Medical Foundation, La Crosse, Wisconsin, 2 Wisconsin State Laboratory of Hygiene and Department of Medical Microbiology and Immunology, Madison, Wisconsin
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.5
DOI:  10.1002/0471142956.cy1105s26
Online Posting Date:  November, 2004
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Abstract

Lyme disease is a multisystem disorder that usually begins with a skin lesion called erythema migrans and with constitutional symptoms. If the disease is left untreated or treated inappropriately, dissemination of the organism can lead to more severe sequelae, including nervous system disorders or arthritis. Vaccinations with B. burgdorferi or several individual B. burgdorferi proteins induce borreliacidal antibodies that provide protection against infection by inducing a complement cascade that kills the spirochetes without the necessity of scavenging by phagocytic cells. Detection of borreliacidal antibodies is therefore useful for serodiagnosing Lyme disease and monitoring immune status after vaccination. This unit provides a technique for detecting anti‐B. burgdorferi antibodies, as well as for preparing and determining the quality of Barbour‐Stoenner‐Kelly (BSK medium) and complement. In addition, methods are provided for preparation of a B. burgdorferi stock and Mueller‐Hinton agar containing Bacillus subtilis spores.

Keywords: Borrelia burgdorferi; Lyme disease; borreliacidal antibodies; BSK medium; Mueller‐Hinton agar; Bacillus subtilis

     
 
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Table of Contents

  • Basic Protocol 1: Flow Cytometric Detection of Anti–B. burgdorferi Antibodies
  • Support Protocol 1: Preparation of Barbour‐Stoenner‐Kelly (BSK) Medium
  • Support Protocol 2: Quality Control of Barbour‐Stoenner‐Kelly (BSK) Medium
  • Support Protocol 3: Preparation of B. burgdorferi Stock Culture
  • Support Protocol 4: Preparation of Mueller‐Hinton Agar Containing Bacillus subtilis Spores
  • Support Protocol 5: Rehydration of Lyophilized Complement
  • Support Protocol 6: Quality Control of Complement
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Flow Cytometric Detection of Anti–B. burgdorferi Antibodies

  Materials
  • Borrelia burgdorferi stock culture aliquots (see protocol 4) in BSK medium
  • Barbour‐Stoenner‐Kelly (BSK) medium (see protocol 2 and protocol 3)
  • Mueller‐Hinton agar plates containing B. subtilis spores (see protocol 5)
  • Serum to be tested for anti–B. burgdorferi borreliacidal antibodies
  • Normal antibiotic‐free control serum from the same species as serum sample to be tested
  • Normal control serum containing 5 µg/ml doxycycline (see recipe)
  • Amberlite XAD‐16 nonionic polymeric resin (Sigma)
  • Phosphate buffered saline (PBS; appendix 2A), pH 7.2, filter‐sterilized
  • Penicillinase (optional; Sigma)
  • Guinea pig complement (see protocol 6), tested for activity (see protocol 7)
  • Acridine orange working solution (see recipe)
  • 50‐ml screw‐cap centrifuge tubes (e.g., Fisher)
  • Filter paper
  • 0.2‐µm microcentrifuge spin‐filter tubes (1.5 ml; Costar)
  • 56°C water bath
  • Petroff‐Hausser counting chamber (Fisher)
  • Dark‐field microscope
  • 12 × 75–mm polypropylene or polystyrene tubes as required for flow cytometer
  • Flow cytometer with a 488‐nm argon laser and 530/30 band‐pass filters

Support Protocol 1: Preparation of Barbour‐Stoenner‐Kelly (BSK) Medium

  Materials
  • HEPES (Sigma)
  • Neopeptone (Difco)
  • Sodium citrate (Sigma)
  • Glucose (Sigma)
  • Sodium bicarbonate (Sigma)
  • TC yeastolate (Difco)
  • Pyruvic acid (Sigma)
  • N‐acetyl glucosamine (Sigma)
  • Bovine serum albumin (Sigma)
  • Gelatin (microbiological grade; Difco)
  • 5 N NaOH
  • 10× CMRL 1066 medium with L‐glutamine and without sodium bicarbonate (ICN Biomedicals)
  • Rabbit serum (Life Technologies), heat‐inactivated 45 min at 56°C.
  • 56°C water bath
  • Positive‐pressure pump
  • Millipore filter manifold
  • Prefilter (124 mm)
  • 0.2‐, 0.45‐, and 0.8‐µm filters (142‐mm diameter)
  • 0.2‐µm bell filters
  • Sterile 100‐ml containers
  • Dark‐field microscope

Support Protocol 2: Quality Control of Barbour‐Stoenner‐Kelly (BSK) Medium

  Materials
  • BSK medium, sterile (see protocol 2)
  • B. burgdorferi culture in logarithmic growth phase (see protocol 4)
  • 13 × 100–mm sterile culture tubes
  • Dark‐field microscope

Support Protocol 3: Preparation of B. burgdorferi Stock Culture

  Materials
  • Borrelia burgdorferi
  • Barbour‐Stoenner‐Kelly (BSK) medium (see protocol 2 and protocol 3)
  • 13 × 100–mm sterile culture tubes

Support Protocol 4: Preparation of Mueller‐Hinton Agar Containing Bacillus subtilis Spores

  Materials
  • Mueller‐Hinton agar medium (Difco)
  • Bacillus subtilis (ATCC #6633) spore suspension (Difco)
  • 500‐ml flask
  • Sterile 100 × 15–mm petri plates

Support Protocol 5: Rehydration of Lyophilized Complement

  Materials
  • Lyophilized guinea pig complement (Sigma, Life Technologies, Rockland, or Cedarlane Laboratories)
  • 0.2‐µm and 0.8‐µm syringe‐tip filters or filter units
  • Sterile microcentrifuge tubes, prechilled

Support Protocol 6: Quality Control of Complement

  Materials
  • Normal serum and positive control serum (obtained in the laboratory)
  • Barbour‐Stoenner‐Kelly (BSK) medium (see protocol 2 and protocol 3)
  • B. burgdorferi in logarithmic phase (see protocol 4)
  • Reconstituted complement to be tested (see protocol 6)
  • 0.2‐µm filters
  • 56°C water bath
  • Petroff‐Hausser counting chamber (Fisher)
  • Dark‐field microscope
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Figures

Videos

Literature Cited

Literature Cited
   Callister, S.M., Case, K.C., Agger, W.A., Schell, R.F., Johnson, R.C., and Ellingson, J.L.E. 1990. Effects of bovine serum albumin on the ability of Barbour‐Stoenner‐Kelly medium to detect Borrelia burgdorferi. J. Clin. Microbiol. 28:363‐365.
   Callister, S.M., Schell, R.F., Case, K.L., Lovrich, S.D., and Day, S.P. 1993. Characterization of the borreliacidal antibody response to Borrelia burgdorferi in humans: A serodiagnostic test. J. Infect. Dis. 167:158‐164.
   Callister, S.M., Schell, R.F., Lim, L.C.L., Jobe, D.A., Case, K.L., Bryant, G.L., and Molling, P.E. 1994. Detection of borreliacidal antibodies by flow cytometry: An accurate, highly specific serodiagnostic test for Lyme disease. Arch. Intern. Med. 154:1625‐1632.
   Callister, S.M., Jobe, D.A., Schell, R.F., Pavia, C.S., and Lovrich, S.D. 1996. Sensitivity and specificity of the borreliacidal‐antibody test during early Lyme disease: a “gold standard”? Clin. Diagn. Lab. Immunol. 3:399‐402.
   Callister, S.M., Jobe, D.A., Schell, R.F., Kowalski, T.J., Lovrich, S.D., and Marks, J.A. 2002. Ability of the borreliacidal antibody test to confirm Lyme disease in clinical practice. Clin. Diagn. Lab. Immunol. 9:908‐912.
   Golde, W.T., Piesman, J., Dolan, M.C., Kramer, M., Hauser, P., Lobet, Y., Capiau, C., Desmons, P., Voet, P., Dearwester, D., and Frantz, J.C. 1997. Reactivity with a specific epitope of outer surface protein A predicts protection from infection with the Lyme disease spirochete, Borrelia burgdorferi. Infect. Immun. 65:882‐889.
   Jobe, D.A., Lovrich, S.D., Schell, R.F., and Callister, S.M. 2003. C‐terminal region of outer surface protein C binds borreliacidal antibodies in sera from patients with Lyme disease. Clin. Diagn. Lab. Immunol. 10:573‐578.
   Jobe, D.A., Rawal, N., Schell, R.F., and Callister, S.M. 1999. Detection of borreliacidal antibodies in Lyme borreliosis patient sera containing antimicrobial agents. Clin. Diagn. Lab. Immunol. 6:930‐933.
   Padilla, M.L., Callister, S.M., Schell, R.F., Bryant, G.L., Jobe, D.A., Lovrich, S.D., DuChateau, B.K., and Jensen, J.R. 1996. Characterization of the protective borreliacidal antibody response in humans and hamsters after vaccination with a Borrelia burgdorferi outer surface protein A vaccine. J. Infect. Dis. 174:739‐746.
   Rousselle, J.C., Callister, S.M., Schell, R.F., Lovrich, S.D., Jobe, D.A., Marks, J.A., and Wieneke, C.A. 1998. Borreliacidal antibody production against outer surface protein C of Borrelia burgdorferi. J. Infect. Dis 178:733‐741.
   Steere, A.C. 1989. Medical progress: Lyme disease. N. Engl. J. Med. 321:586‐596.
   Steere, A.C., Grodzicki, R.L., Kornblatt, A.N., Craft, J.E., Barbour, A.G., Burgdorfer, W., Schmid, G.P., Johnson, E., and Malawista, S.E. 1983. The spirochetal etiology of Lyme disease. N. Engl. J. Med. 308:733‐740.
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