Solid‐Phase Immunoassays

Michael A. Lynes1

1 University of Connecticut, Storrs, Connecticut
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.7
DOI:  10.1002/0471140856.tx1807s23
Online Posting Date:  March, 2005
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Solid‐phase quantitative immunoassays are some of the most commonly used diagnostic tests for both soluble antigen composition and the assessment of cellular functions. These immunoassays (e.g., ELISA, ELISPOT) provide a highly sensitive means to measure the presence of antigen in defined and homogeneous samples such as purified proteins in buffer, as well as in undefined heterogenous biological samples such as cell lysates, tissue culture supernatants, blood, and other clinical samples. The sensitivity of these assays can enable (under optimal conditions) detection of protein concentrations in the picogram range. A recent modification of the basic ELISA immunoassay takes advantage of the phenomenon of grating‐coupled surface plasmon resonance (GCSPR) to provide a label‐free real‐time variant of this solid‐phase immunoassay. Using GCSPR, similar assessments of antigen‐antibody interactions can be done with smaller sample sizes and in a microarray format that enables the simultaneous measurement of large numbers of antibody/antigen interactions on the same sensor chip. These measurements allow for a highly refined and sensitive determination of the effects that toxins can have on biological systems, and they can be applied to a variety of immune and non‐immune protein, cell, and tissue evaluations.

Keywords: Solid‐phase immunoassay; ELISA; Enzyme linked immunosorbent assay; ELISPOT; Grating‐coupled surface plasmon resonance; GCSPR

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Direct Enzyme‐Linked Immunosorbent Assays
  • Alternate Protocol 1: Indirect ELISA
  • Alternate Protocol 2: Competition ELISA
  • Basic Protocol 2: ELISPOT Assay
  • Basic Protocol 3: Grating‐Coupled Surface Plasmon Resonance Immunoassay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Direct Enzyme‐Linked Immunosorbent Assays

  • Antigen diluted in ELISA coupling buffer (see recipe for ELISA coupling buffer)
  • Blocking reagent (Table 18.7.1)
  • ELISA wash buffer (see recipe)
  • Antibody‐enzyme conjugate (e.g., Southern Biotech)
  • Substrate (Table 18.7.2) dissolved in the appropriate substrate buffer (e.g., for alkaline phosphatase‐based assays, the most common substrate is p‐nitrophenyl phosphate, PNPP, Sigma; see recipe)
  • 96‐well flat‐bottom microtiter plates (plates specifically treated to enhance binding of hydrophobic or hydrophyllic proteins, or DNA molecules; e.g., Immulon 2 HB plates, Thermo Electron)
  • 37°C incubator
  • Automated plate washer (optional)
  • Plate sealer (adhesive‐backed mylar sheets, Fisher)
  • Microtiter plate reader with appropriate wavelength filters (e.g., Molecular Devices, Bio‐Tek Instruments, Thermo Electron, and Tecan)
  • Plate shaker

Alternate Protocol 1: Indirect ELISA

  • Unconjugated primary antibody/antiserum reagent

Alternate Protocol 2: Competition ELISA

  • Soluble antigen in purified form
  • Goat anti‐mouse Ig‐alkaline phosphatase conjugate that recognizes both heavy and light chains

Basic Protocol 2: ELISPOT Assay

  • Antigen
  • ELISPOT coating buffer (see recipe)
  • ELISPOT wash buffer (see recipe)
  • RPMI‐1640 supplemented with 10% FBS or ELISPOT blocking buffer (see recipe)
  • Test cells
  • Capture and detection antibodies for the target molecule of interest
  • ELISPOT assay diluent
  • ELISPOT culture medium
  • ELISPOT substrate solution: BCIP (5‐bromo,4‐chloro,3‐indolylphosphate)/NBT (Kirkegaard and Perry)
  • 96‐well filtration plates with sterile surfactant‐free membrane (e.g., Millipore)
  • Automated plate washer (optional)
  • ELISPOT reader (ImmunoBiosys, Zeiss, Sanquin, or A. EL. VIS) or dissecting microscope and digital image analysis software (e.g., ImageJ available as freeware at

Basic Protocol 3: Grating‐Coupled Surface Plasmon Resonance Immunoassay

  • Ethanol
  • Capture antibody
  • Antigen
  • PBS (see recipe)
  • GCSPR blocking reagent
  • Analyte solution
  • MicroCaster System, 8‐pin manual arrayer (Schleicher and Schuell Bioscience), or another spotting system
  • GCSPR sensor chips (Applied Biosystems)
  • 96‐well microtiter plates
  • 37°C incubator and humid box
  • Applied Biosystems 8500 affinity chip analyzer
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Literature Cited

   Amoura, Z., Chabre, H., Koutouzov, S., Lotton, C., Cabrespines, A., Bach, J.F., and Jacob, L. 1994. Nucleosome‐restricted antibodies are detected before anti‐dsDNA and/or antihistone antibodies in serum of MRL‐Mp lpr/lpr and +/+ mice, and are present in kidney eluates of lupus mice with proteinuria. Arthritis Rheum. 37:1684‐1688.
   Brockman, J.M. and Fernández, S.M. 2001. Grating‐coupled surface plasmon resonance for rapid, label‐free, array‐based sensing. American Laboratory 6:37‐40.
   Czerkinsky, C., Nilsson, Langstrom., Nygren, H., Ouchterlony, O., 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.
   Lalvani, A., Pathan, A.A., Durkan, H., Wilkinson, K.A., Whelan, A., Deeks, J.J., Reece, W.H., Latif, M., Pasvol, G., and Hill, A.V. 2001. Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen‐specific T cells. Lancet. 357:2017‐2021.
   Probst, P., Kuntzlin, D., and Fleischer, B. 1995. Th2‐type infiltrating T cells in nickel‐induced contact dermatitis. Cell. Immunol. 165:134‐140.
   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.
Internet Resources
  Molecular Probes is an excellent source for information and materials related to fluorescent immunoassays.
  These companies provide a wide range of antibody/enzyme conjugates.
  Online search engines for identifying sources of antibodies from a range of commercial vendors. Linscott's directory of immunological and biological reagents is available as a CD, or in print and is also available online at
  Sources of antibodies, purified antigens, blocking reagents, and other useful reagents for these techniques.
  A Website supported by many of the vendors of ELISPOT materials and instrumentation.
  Website for GCSPR Flexchip Analyzer.
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