ISSN 2398-2942      

Enzyme linked immunosorbent assay (ELISA)


Michael Day

Helen Milner

Synonym(s): ELISA


  • There are two broad categories of microplate-based ELISA:
    • Those designed to demonstrate the presence of antibody.
    • Those designed to demonstrate the presence of antigen.
  • In the former test, antigen is fixed to the surface of the plastic wells of the microtiter plate, and the sample (generally serum or other body fluids) is incubated in this antigen-coated well.
  • If the sample contains antibody, this will bind to the relevant antigenic epitopes.
  • Binding of this primary antibody is detected by the use of a secondary antibody specific for one or more immunoglobulins of the species from which the sample was derived, eg for detecting a canine antibody, a rabbit anti-dog IgG sera may be employed.
  • This secondary antibody is chemically conjugated to an enzyme; usually alkaline phosphatase Blood biochemistry: alkaline phosphatase (ALP) or horseradish peroxidase.
  • In the final stage of the ELISA, an appropriate substrate for the enzyme is added to each well of the plate.
  • Where secondary antibody has bound, the enzyme will act on the substrate to generate a color change that can be quantified spectrophotometrically.
  • In the antigen-detection ELISA, the wells of the microtiter plate are coated with a capture antibody specific for the antigen under consideration.
  • Samples are loaded to the wells, and if antigen is present it will be bound (captured) by the antibody.
  • Detection of this binding is accomplished by the subsequent addition of a second detecting antibody specific for the antigen (often specific for a different epitope to the capture antibody), which is enzyme labeled. Addition of substrate enables determination of positive reactions.
  • The principle of the ELISA has also been used in the generation of the range of simple in-practice serological test kits that detect either antigen or antibody, eg ImmunoComb® or SNAP® tests. In these kits, the antigen, or capture antibody, is affixed to a support membrane and the reaction occurs locally (and rapidly) within a designated area of the strip.


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  • Most commercial clinical pathology laboratories will offer a range of ELISA-based techniques for a variety of purposes.
  • The general methodology for performing an ELISA test is described above. Each ELISA must be developed and standardized by determining the optimum combination of the various components to be used (eg antigen, primary antibody, secondary antibody, substrate), the optimum buffers to dilute these in, the optimum periods and temperatures of incubation, the optimum period of color development.
  • Between each stage of the test, the wells of the microtiter plate will be washed to remove unbound reactants this washing is relatively stringent and is usually performed with buffer containing a detergent substance.
  • The process of defining the conditions for performing the ELISA is known as chequerboarding where one reactant is varied (titrated) whilst the others remain constant.
  • The assays will generally involve an initial blocking step, where a source of irrelevant protein (for example, skim milk powder) is added to the wells to block any free plastic surface to which the specific antigen has not adhered. This prevents the subsequent non-specific binding of other reactants.
  • Each ELISA protocol, including in-practice ELISA kits, must be closely adhered to for optimum results.
  • In a laboratory setting, the color that develops following the enzyme-substrate reaction is quantified by the use of a purpose-designed spectrophotometer (an ELISA plate reader). These are often linked to computer for very precise calculation of test samples versus a standard curve.
  • A standard curve should be run on each ELISA plate, and in the best ELISAs each test sample will be fully titrated to allow comparison of the slope of the standard curve with that obtained from the test sample.
  • Alternatively, the test samples may be tested at a single dilution (usually in triplicate) in a one point assay.
  • For kits designed for in-practice use, such precision is not possible; the company will generally provide some indication of how to score the relative intensity of color development and how to interpret this score.


  • Any one ELISA should be carefully validated by the laboratory or company providing the test.
  • This requires validation of the component reagents, and validation of the performance of the test in a field situation, with determination of sensitivity and specificity.
  • Most ELISA tests used in veterinary medicine are subject to this careful validation and are supported by published literature.
  • The reproducibility of an ELISA is generally assessed by establishing the intraplate and interplate coefficients of variation, which optimally would be under 5%.


  • ELISA tests are widely performed by commercial laboratories, and in-practice test kits of a variety of types are available.



  • ELISA is a highly sensitive test capable of detecting very small quantities of the target molecule (at least in the order of ug/ml).


  • Like all serological tests, the specificity of the ELISA is in part determined by the specificity of the reagents (antisera) used within it.
  • Many ELISAs are rendered highly specific by the use of monoclonal (rather than polyclonal) antibodies and by the use of highly purified recombinant proteins as target antigens.
  • However, as in all serological tests the discriminatory ability of some ELISAs may be less than optimal, eg if two related microbes share common antigenic epitopes, infection with one may lead to generation of antibody that cross-reacts with both organisms and may give a false-positive result in the ELISA.

Predictive value

  • This will be calculated for each individual assay.

Result Data

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Further Reading


Refereed papers

  • Recent references from VetMedResource and PubMed.
  • Harrus S et al (2002) Comparison of three enzyme-linked immunosorbant assays with the indirect immunofluorescent antibody test for the diagnosis of canine infection with Ehrlichia canis. Vet Microbiol 86, 361-368.
  • Lucero N E et al (2002) Sensitivity and specificity of an indirect enzyme linked immunoassay for the diagnosis of Brucella canis infection in dogs. J Med Microbiol 51, 656-660.
  • Lurye J C, Behrend E N & Kemppainen R J (2002) Evaluation of an in-house enzyme-linked immunosorbent assay for quantitative measurements of serum total thyroxine concentration in dogs and cats. JAVMA 221, 243-249.
  • Reithinger R et al (2002) Rapid detection of Leishmania infantuminfection in dogs: comparative study using an immunochromatographic dipstick test, enzyme-linked immunosorbent assay and PCR. J Clin Microbiol 40, 2352-2356.
  • Scalone A, De Luna R, Oliva G et al(2002) Evaluation of the Leishmania recombinant K39 antigen as a diagnostic marker for canine leishmaniasis and validation of a standardized ELISA. Vet Parasitol 104, 275-285.
  • Garcia M E, Caballero J, Cruzado M et al (2001) The value of the determination of anti-Aspergillus IgG in the serodiagnosis of canine aspergillosis: comparison with galactomannan detection. J Vet Med 48, 743-750.
  • Curtis C F (2001) Evaluation of a commercially available enzyme-linked immunosorbent assay for the diagnosis of canine sarcoptic mange. Vet Rec 148, 238-239.
  • Hall S A (2001) ELISA for the diagnosis of canine sarcoptic mange. Vet Rec 148, 420.
  • Lower K S et al (2000) Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of sarcoptic mange in dogs. Vet Dermatol 12, 315-320.
  • German A J, Hall E J & Day M J (1998) Measurement of IgG, IgM and IgA concentrations in canine serum, saliva, tears and bile. Vet Immunol Immunopathol 64, 107-121.
  • Shearer D H & Day M J (1997) The humoral immune response to Staphylococcus intermedius in dogs with pyoderma. Vet Immunol Immunopathol 58, 107-120.
  • Day M J (1996) IgG subclasses of canine anti-erythrocyte, anti-nuclear and anti-thyroglobulin autoantibodies. ResVet Sci 61, 129-135.
  • Day M J, Corato A & Shaw S E (1996) Subclass profile of allergen-specific IgG antibodies in atopic dogs. Res Vet Sci 61, 136-142.


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