ISSN 2398-2977      

Enzyme linked immunosorbent assay (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 an equine antibody, a goat anti-equine IgG sera may be employed. 
  • This secondary antibody is chemically conjugated to an enzyme, usually alkaline phosphatase 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, eg 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 <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 µg/ml). 
  • A low/negative serology result may be obtained in the early stages of infection and paired serum samples may be required to identify recent exposure/infection.


  • 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. 
  • Seropositivity indicates exposure a particular antigen but does not confirm disease; results must always be interpreted in relation to the clinical presentation and other diagnostic tests.

Predictive value

  • This will be calculated for each individual assay.

Result Data

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Further Reading


Refereed papers

  • Recent references from PubMed and VetMedResource.
  • Knowles E J, Mair T S, Butcher N, Waller A S & Wood J L (2010)Use of a novel serological test for exposure to Streptococcus equi subspecies equi in hospitalised horses. Vet Rec 166 (10), 294-297 PubMed.  
  • Medina-Torres C E, Weese J S & Staempfli H R (2010) Validation of a commercial enzyme immunoassay for detection of Clostridium difficile toxins in feces of horses with acute diarrhoea.  J Vet Intern Med 24 (3), 628-632 PubMed.  
  • Waggett B E, McGorum B C, Wernery U, Shaw D J & Pirie R S (2010) Prevalence of Clostridium perfringens in faeces and ileal contents from grass sickness affected horses: comparisons with 3 control populations.  Equine Vet J 42 (6), 494-499 PubMed.  
  • Duthie S, Mills H & Burr P (2008) The efficacy of a commercial ELISA as an alternative to virus neutralisation test for the detection of antibodies to EAV. Equine Vet J 40 (2), 182-183 PubMed.    
  • Cullinane A, Quinlivan M, Nelly M, Patterson H, Kenna R, Garvey M, Gildea S, Lyons P, Flynn M, Galvin P, Neylon M & Jankowska K (2007) Diagnosis of equine infectious anaemia during the 2006 outbreak in Ireland. Vet Rec 161 (19), 647-652 PubMed.  
  • Thomas G W, Bell S C & Carter S D (2005)Immunoglobulin and peripheral B-lymphocyte concentrations in Fell pony foal syndrome.  Equine Vet J 37 (1), 48-52 PubMed.  
  • Matthews J B, Hodgkinson J E, Dowdall S M & Proudman C J (2004) Recent developments in research into the Cyathostominae and Anoplocephala perfoliata. Vet Res 35 (4), 371-381 PubMed
  • Watson E D, Heald M, Leask R, Groome N P & Riley S C (2002) Detection of high circulating concentrations of inhibin pro- and -alphaC immunoreactivity in mares with granulosa-theca cell tumours.  Equine Vet J 34 (2), 203-206 PubMed.  
  • Sheoran A S, Timoney J F, Holmes M A, Karzenski S S & Crisman M V (2000) Immunoglobulin isotypes in sera and nasal mucosal secretions and their neonatal transfer and distribution in horses. Am J Vet Res 61 (9), 1099-1105 PubMed.


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