Enzyme‐Linked Immunosorbent Assays

Peter V. Hornbeck1

1 Cell Signaling Technology, Danvers, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.1
DOI:  10.1002/0471142735.im0201s110
Online Posting Date:  August, 2015
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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. One of the support protocols can be used to optimize the different ELISAs. A second support protocol presents a method for preparing alkaline phosphatase conjugates. © 2015 by John Wiley & Sons, Inc.

Keywords: enzyme‐linked immunosorbant assay; ELISA; solid‐phase reactants; antibody; antigen

     
 
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Table of Contents

  • Assays for Antibody Production
  • Introduction
  • 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 Antibody–Alkaline Phosphatase Conjugates
  • 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, cat. no. P9650), protein G–alkaline phosphatase conjugate (Calbiochem, cat. no. 539304), or anti‐Ig‐alkaline phosphatase conjugate ( protocol 8)
  • Antigen solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 0.05% NaN 3 (PBSN)
  • Water, deionized or distilled
  • Blocking buffer (see recipe)
  • Test antibody samples
  • 4‐methylumbelliferyl phosphate (MUP) or p‐nitrophenyl phosphate (NPP) substrate solution (see recipe)
  • 0.5 M NaOH (optional)
  • Multichannel pipet and disposable pipet tips
  • Immulon 2 (Dynatech, cat. no. 011‐010‐3450), Immulon 4 (Dynatech, cat. no. 011‐010‐3850), or equivalent microtiter plates
  • Plastic wrap
  • 37°C incubator, optional
  • Plastic squirt bottles
  • Paper towels
  • Vortex mixer
  • Microtiter plate reader (optional)—spectrophotometer with 405‐nm filter or spectrofluorometer (Dynatech, cat. no. 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 (also see protocol 1Basic Protocol)
  • Specific antibody–alkaline phosphatase conjugate ( protocol 8)
  • Standard antigen solution
  • Test antigen solutions (see recipe)
  • Round‐ or cone‐bottom microtiter plates

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

  Additional Materials (also see protocol 1Basic Protocol)
  • Specific antibody or immunoglobulin fraction from antiserum (units 2.7 & 2.9) or ascites fluid, or hybridoma supernatant (unit 2.6)

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

  Additional Materials (also see protocol 1Basic Protocol)
  • Capture antibodies specific for immunoglobulin from the immunized species
  • Specific antibody–alkaline phosphatase conjugate ( protocol 8)

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

  Additional Materials (also see protocol 1Basic Protocol)
  • Cell samples
  • Specific antibody–alkaline phosphatase conjugate (see second support protocol)
  • Wash buffer, ice‐cold
  • Benchtop centrifuge
  • Sorvall H‐1000B rotor (or equivalent)
  • 15‐ to 50‐ml centrifuge tubes
  • Cone‐ or round‐bottom microtiter plates
  • Vortex mixer or microtiter plate shaker
  • Additional reagents and equipment for counting the cells ( appendix 3A)

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

  Additional Materials (also see protocol 1Basic Protocol)
  • 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)
  • Test antibody solution
  • Ice‐cold wash buffer (see recipe)
  • Antibody– or F(ab′) 2 (against immunoglobulin from the immunized species)– alkaline phosphatase conjugate ( protocol 8)
  • Cone‐ or round‐bottom microtiter plates
  • Vortex mixer

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

  Additional Materials (also see protocol 1Basic Protocol)
  • Coating reagent
  • Secondary reagent
  • Developing reagent
  • 17 × 100‐mm and 12 × 74‐mm test tubes
  • Test tube rack

Support Protocol 2: Preparation of Antibody–Alkaline Phosphatase Conjugates

  Additional Materials (also see protocol 1Basic Protocol)
  • >0.2 mg/ml antibody in phosphate‐buffered saline (PBS)
  • Alkaline phosphatase in NaCl solution (Sigma, cat. no. P0905)
  • 25% glutaraldehyde, EM grade (Sigma, cat. no. G5882)
  • Phosphate‐buffered saline (PBS) containing 100 mM lysine and 100 mM ethanolamine (PBSLE)
  • Blocking buffer containing 2.5 mM MgCl 2
  • Sephadex G‐25 column
  • 0.2‐μm filter
  • Additional reagents and equipment for column chromatography ( appendix 3I)
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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.
  Engvall, E., Jonsson, K., and Perlmann, P. 1971. Enzyme‐linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme‐labelled antigen and antibody‐coated tubes. Biochim. Biophys. Acta 251:427‐434.
  Engvall, E. and Perlmann, P. 1971. Enzyme‐linked immunosorbent assay (ELISA): Quantitative assay of immunoglobulin G. Immunochemistry 8:871‐879.
  Engvall, E. and Perlmann, P. 1972. Enzyme‐linked immunosorbent assay, Elisa. III. Quantitation of specific antibodies by enzyme‐labelled anti‐immunoglobulin in antigen‐coated tubes. J. Immunol. 109:129‐135.
  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. http://www.linscottsdirectory.com.
  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. 47:846‐851
  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.
  Stenken, J.A. and Pschenrieder, A.J. 2015. Bioanalytical chemistry of cytokines: A review. Anal. Chim. Acta 853:95‐115.
  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 84:279‐290.
Key References
  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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