ELISPOT Assay for Measurement of Antigen‐Specific and Polyclonal Antibody Responses

Nils Lycke1, Richard Coico2

1 Department of Microbiology and Immunology, University of Göteborg, Göteborg, Sweden, 2 Department of Cell Biology, SUNY Downstate College of Medicine, Brooklyn, New York
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.14
DOI:  10.1002/0471142735.im0714s108
Online Posting Date:  February, 2015
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Abstract

The enzyme‐linked immunospot (ELISPOT) assay for detection of antigen‐specific and polyclonal antibody responses by single antibody‐secreting cells has become the method of choice due to its cell‐based quantitative value. Antigen stability and specificity and the diversity of antigens that can be used in the assay have contributed to the translational application of ELISPOT as demonstrated by many FDA‐approved clinical tests that employ this technique. In addition, the ELISPOT assay can be used to detect two antigenically different secreted antibodies simultaneously by two‐color analysis and offers the unique possibility of quantifying the number of antibody molecules secreted per cell. © 2015 by John Wiley & Sons, Inc.

Keywords: ELISPOT; ELISA; antibody‐secreting cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: ELISPOT Assay of Antibody‐Secreting Cells in Petri Dishes or Microtiter Plates
  • Alternate Protocol 1: ELISPOT Assay for Polyclonal or Antigen‐Specific Antibody in Manifold Apparatus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: ELISPOT Assay of Antibody‐Secreting Cells in Petri Dishes or Microtiter Plates

  Materials
  • Antigen in coating buffer (unit )
  • PBS ( )
  • 5% (w/v) FBS (heat‐inactivated 30 min at 56°C; ) in PBS or 1% (w/v) BSA in PBS, freshly prepared
  • Antibody‐secreting cells, such as peripheral blood mononuclear cells (PBMC; unit ) or spleen cells
  • Complete IMDM‐5 medium ( )
  • Gentamicin, optional
  • Tween/PBS: 0.05% (v/v) Tween 20 in PBS
  • Enzyme‐conjugated, affinity‐purified developing antibody (see recipe)
  • 0.1% (w/v) BSA in PBS (BSA/PBS)
  • Gel substrate (if using polystyrene dish) or soluble substrate (if using nitrocellulose membrane), for horseradish peroxidase (HRPO)– or alkaline phosphatase–conjugated antibody (see reciperecipes)
  • 40‐ to 60‐mm‐diameter polystyrene petri dishes, 6‐, 24‐, 48‐, or 96‐well polystyrene microtiter plates (tissue‐culture grade; Nunc), nitrocellulose membrane in 96‐well Millititer HA plate (Millipore), or equivalent
  • Humidified chamber (see Fig. ), 4°C
  • Stereomicroscope

Alternate Protocol 1: ELISPOT Assay for Polyclonal or Antigen‐Specific Antibody in Manifold Apparatus

  Additional Materials (also see protocol 1Basic Protocol)
  • 1% (w/v) bovine serum albumin (BSA) in phosphate‐buffered saline (PBS; )
  • Nitrocellulose membrane (Millipore)
  • 100‐mm petri dish or lid of 96‐well microtiter plate (or equivalent)
  • Aspirator
  • 96‐well format blotting manifold (e.g., Minifold 96‐Well 1 System, Schleicher & Schuell; Bio‐Dot, Bio‐Rad; or equivalent; see Fig. )
  • Parafilm, optional
  • 37°C, 5% CO 2 humidified incubator
  • Light microscope
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Figures

Videos

Literature Cited

Literature Cited
   Alexander, N. , Fox, A. , Lien, V.T. , Dong, T. , Lee, L.Y. , Hang , Nle, K. , Mai, le Q. , and Horby, P. 2013. Defining ELISpot cut‐offs from unreplicated test and control wells. J. Immunol. Methods 392:57‐62.
   Bland, M.J. and Altman, D.G. 1986. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1:307‐311.
   Czerkinsky, C. , Nilsson, L.Å. , Nygren, H. , Ouchterlony, Ö. , and Tarkowski, A. 1983. A solid‐phase enzyme‐linked immunospot (ELISPOT) assay for enumeration of specific antibody‐secreting cells. J. Immunol. Methods 65:109‐121.
   Czerkinsky, C. , Moldeveanu, Z. , Mestecky, J. , Nilsson, L.Å. , and Ouchterlony, Ö. 1988a. A novel two‐color ELISPOT assay. I. Simultaneous detection of distinct types of antibody‐secreting cells. J. Immunol. Methods 115:31‐37.
   Czerkinsky, C. , Nilsson, L.Å. , Tarkowski, A. , Koopman, W.J. , Mestecky, J. , and Ouchterlony, Ö. 1988b. The enzyme‐linked immunospot assay for detection of specific antibody‐secreting cells: Methodology and applicability. In Theoretical and Technical Aspects of ELISA and Other Solid Phase Immunoassays ( D.M. Kemeny and S.J. Challacombe , eds.) pp. 217‐239. John Wiley & Sons, New York.
   Jahnmatz, M. , Kesa, G. , Netterlid, E. , Buisman, A. , Thorstensson, R. , and Ahlborg, N. 2013. Optimization of a human IgG B‐cell ELISpot assay for the analysis of vaccine‐induced B‐cell responses. J. Immunol. Methods 391:50‐59.
   Franci, C. , Ingles, J. , Castro, R. , and Vidal, J. 1986. Further studies on the ELISA‐spot technique. Its application to particulate antigens and potential improvement in sensitivity. J. Immunol. Methods 88:225‐232.
   Lycke, N. 1986. A sensitive method for the detection of specific antibody production in different isotypes from single lamina propria plasma cells. Scand. J. Immunol. 24:393‐403.
   Lycke, N. , Kilander, A. , Nilsson, L.Å. , Tarkowski, A. , and Werner, N. 1989. Production of antibodies to gliadin in intestinal mucosa of patients with coeliac disease: A study at the single cell level. Gut 30:72‐77.
   Möller, S.A. and Borrebaeck, C.A.K. 1985. A filter immuno‐plaque assay for the detection of antibody‐secreting cells in vitro. J. Immunol. Methods 79:195‐204.
   Sedgwick, J.D. and Holt, P.G. 1983. A solid‐phase immunoenzymatic technique for the enumeration of specific antibody‐secreting cells. J. Immunol. Methods 57:301‐309.
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