Enzyme‐Linked Immunosorbent Assays (ELISA)

Peter Hornbeck1, Scott E. Winston2, Steven A. Fuller3

1 University of Maryland, Baltimore, Maryland, 2 Univax Biologics, Rockville, Maryland, 3 Allelix Inc., Mississauga, Ontario
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.2
DOI:  10.1002/0471142727.mb1102s15
Online Posting Date:  May, 2001
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Abstract

This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell‐surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid‐phase reactants. In the first four protocols, solid‐phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid‐phase reactants are cell‐associated molecules. In all protocols, the solid‐phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or with a microtiter plate reader. The amount of product generated is proportional to the amount of analysate in the test mixture. Support protocols are provided for optimizing the different ELISAs and preparing lysates for use as test antigen from bacterial cultures containing expressed protein.

     
 
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Table of Contents

  • Basic Protocol 1: Indirect ELISA to Detect Specific Antibodies
  • Alternate Protocol 1: Direct Competitive ELISA to Detect Soluble Antigens
  • Alternate Protocol 2: Antibody‐Sandwich ELISA to Detect Soluble Antigens
  • Alternate Protocol 3: Double Antibody–Sandwich ELISA to Detect Specific Antibodies
  • Alternate Protocol 4: Direct Cellular ELISA to Detect Cell‐Surface Antigens
  • Alternate Protocol 5: Indirect Cellular ELISA to Detect Antibodies Specific for Surface Antigens
  • Support Protocol 1: Criss‐Cross Serial Dilution Analysis to Determine Optimal Reagent Concentrations
  • Support Protocol 2: Preparation of Bacterial Cell Lysate Antigens
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Indirect ELISA to Detect Specific Antibodies

  Materials
  • Developing reagent: protein A–alkaline phosphatase conjugate ( Sigma #P9650), protein G–alkaline phosphatase conjugate ( Calbiochem #539304), or anti‐Ig‐alkaline phosphatase conjugate (unit 11.1)
  • recipeAntigen solution
  • PBS ( appendix 22) containing 0.05% NaN 3 (PBSN)
  • Water, deionized or distilled
  • recipeBlocking buffer
  • recipeTest antibody samples
  • 4‐methylumbelliferyl phosphate (MUP) orp‐nitrophenyl phosphate (NPP) substrate solution
  • 0.5 M NaOH (optional)
  • Multichannel pipet and disposable pipet tips
  • Immulon 2 ( Dynatech #011‐010‐3450), Immulon 4 ( Dynatech #011‐010‐3850), or equivalent microtiter plates
  • Plastic squirt bottles
  • Microtiter plate reader (optional)—spectrophotometer with 405‐nm filter or spectrofluorometer ( Dynatech #011‐970‐1900) with 365‐nm excitation filter and 450‐nm emission filter

Alternate Protocol 1: Direct Competitive ELISA to Detect Soluble Antigens

  Additional Materials
  • Specific antibody–alkaline phosphatase conjugate (unit 11.1)
  • Standard antigen solution
  • recipeTest antigen solutions
  • Round‐ or cone‐bottom microtiter plates

Alternate Protocol 2: Antibody‐Sandwich ELISA to Detect Soluble Antigens

  Additional Materials
  • Specific antibody or immunoglobulin fraction from antiserum or ascites fluid, or hybridoma supernatant (unit 10.11), or bacterial lysate (second protocol 8support protocol)

Alternate Protocol 3: Double Antibody–Sandwich ELISA to Detect Specific Antibodies

  Additional Materials
  • Capture antibodies specific for immunoglobulin from the immunized species
  • Specific antibody–alkaline phosphatase conjugate

Alternate Protocol 4: Direct Cellular ELISA to Detect Cell‐Surface Antigens

  Additional Materials
  • Cell samples
  • Specific antibody–alkaline phosphatase conjugate (see second protocol 8support protocol)
  • recipeWash buffer, ice‐cold
  • Cone‐ or round‐bottom microtiter plates
  • Sorvall H‐1000B rotor (or equivalent)

Alternate Protocol 5: Indirect Cellular ELISA to Detect Antibodies Specific for Surface Antigens

  Additional Materials
  • Positive‐control antibodies (i.e., those that react with the experimental cells and are from the immunized species)
  • Negative‐control antibodies (i.e., those that do not react with the experimental cells)
  • recipeTest antibody solution
  • Antibody or F(ab′) 2 (against immunoglobulin from the immunized species) conjugated to alkaline phosphatase
  • Cone‐ or round‐bottom microtiter plates

Support Protocol 1: Criss‐Cross Serial Dilution Analysis to Determine Optimal Reagent Concentrations

  Additional Materials
  • Coating reagent
  • Secondary reagent
  • Developing reagent
  • 17 × 100–mm and 12 × 74–mm test tubes

Support Protocol 2: Preparation of Bacterial Cell Lysate Antigens

  Materials
  • Escherichia coli culture in broth or agar (units 1.2 & 1.3)
  • recipeCell resuspension buffer
  • recipeLysozyme solution
  • Tris/EDTA/NaCl (TEN) buffer (unit 11.1)
  • 10% sodium dodecyl sulfate (SDS)
  • 8 M urea (optional)
  • Nylon‐tipped applicator (Falcon #2069, Becton Dickinson)
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Figures

Videos

Literature Cited

Literature Cited
   Bartlett, W.C. and Noelle, R.J. 1987. A cell‐surface ELISA to detect interleukin 4–induced class II MHC expression on murine B cells. J. Immunol. Methods 105:79‐85.
   Beatty, J.D., Beatty, B.G., and Vlahos, W.G. 1987. Measurement of monoclonal affinity by noncompetitive immunoassay. J. Immunol. Methods 100:173‐179.
   Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M., and Strober, W., eds. 1991. Current Protocols in Immunology, Chapter 5: Immunofluorescence and cell sorting. Greene Publishing and Wiley‐Interscience, New York.
   Engvall, E. and Perlman, P. 1971. Enzyme‐linked immunosorbent assay (ELISA): Quantitative assay of immunoglobulin G. Immunochemistry 8:871‐879.
   Feit, C., Bartal, A.H., Tauber, G., Dymbort, G., and Hirshaut, Y. 1983. An enzyme‐linked immunosorbent assay (ELISA) for the detection of monoclonal antibodies recognizing antigens expressed on viable cells. J. Immunol. Methods 58:301‐308.
   Harada, N., Castle, B.E., Gorman, D.M., Itoh, N., Schreurs, J., Barrett, R.L., Howard, M., and Miyajima, A. 1990. Expression cloning of a cDNA encoding the murine interleukin 4 receptor based on ligand binding. Proc. Natl. Acad. Sci. U.S.A. 87:857‐861.
   Jitsukawa, T., Nakajima, S., Sugawara, I., and Watanabe, H. 1989. Increased coating efficiency of antigens and preservation of original antigenic structure after coating in ELISA. J. Immunol. Methods. 116:251‐257.
   Kurstak, E. 1986. Enzyme Immunodiagnosis. Academic Press, San Diego.
   Linscott's Directory of Immunological and Biological Reagents, Santa Rosa, Calif.
   Macy, E., Kemeny, M., and Saxon, A. 1988. Enhanced ELISA: How to measure less than 10 picograms of a specific protein (immunoglobulin) in less than 8 hours. FASEB J. 2:3003‐3009.
   Maggio, E.T. 1981. Enzyme Immunoassay. CRC Press, Boca Raton, Fla.
   Quinn, A., Harrison, R., Jehanli, A.M.T., Lunt, G.G., and Walsh, S.S. 1988. An ELISA for the detection of anti‐acetylcholine receptor antibodies using biotinylated α‐bungarotoxin. J. Immunol. Methods 107:197‐203.
   Rubenstein, K.E., Schneider, R.S., and Ulmann, E.L. 1972. Homogeneous enzyme immunoassay: A new immunochemical technique. Biochem. Biophys. Res. Commun. 47:846.
   Schots, A., Van der Leede, B.J., De Jongh, E., and Egberts, E. 1988. A method for the determination of antibody affinity using a direct ELISA. J. Immunol. Methods 109:225‐233.
   Wang, K.C. and Leung, B.S. 1985. Fluorometric ELISA methods for rapid screening of anti‐estrogen receptor antibody production in hybridoma cultures. J. Immunol. Methods 84:279.
Key Reference
  Linscott's Directory. See above.
  Highly recommended publication listing sources of immunological reagents, kits, and cells/organisms, including addresses and phone numbers of commercial suppliers (updated quarterly).
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