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 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.
Uses
Alone
The applications for ELISA are widespread in veterinary medicine.
One of the most common applications is to infectious disease serology ' for detection and quantification of specific antibody that indicates previous or current exposure to an organism.
ELISA may also be used to detect antigenemia as evidence of current infection.
ELISA may be used to quantify a range of biological molecules, eg hormones, immunoglobulins, in a variety of biological fluids, eg for example quantification of IgA in CSF of dogs with steroid-responsive meningitis-arteritis .
ELISA may be used to detect and quantify the presence of autoantibodies, eg anti-thyroglobulin antibody, in serum or other body fluids.
ELISA for detection of allergen-specific IgE or IgG antibodies has become an important diagnostic tool in cases of suspected hypersensitivity dermatitis .
ELISA can be used to help determine response to therapies by assessing changes in antigen or antibody titers.
ELISA can be used to determine immuno-response to vaccination policies.
ELISA can be used to help epidemiological studies both within and between species, including having important human health implications.
Other points
ELISA is a highly sensitive means of detecting relatively low levels of molecules within a biological fluid. For this reason, ELISA may be applied to a wide variety of samples from different body fluids.
Most ELISA testing is performed on serum samples (from clotted blood).
Those designed to detect serum antibody do not require stringent sample handling as the antibody is robust and should survive periods at room temperature, eg when shipping to a laboratory for analysis.
Samples destined for detection of circulating antigen or other heat-labile substances may have special handling requirements and the laboratory that is receiving the sample should be consulted.
The exquisite sensitivity of ELISA means that samples are often highly diluted in assays. Therefore, minimal volumes of starting sample are required.
For example, a 1ml volume of serum is usually adequate for ELISA. However, if there is any doubt, the laboratory that is receiving the sample should be consulted.
Quality control
Precautions
Normal levels of care should be applied to the handling of blood that is to be submitted for testing where a disease with zoonotic potential is considered.
Sample storage
As for most serological assays, serum samples for analysis may be stored in the refrigerator (4°C) if testing is to be performed within one week. Otherwise, serum samples should be frozen (after harvesting serum from the blood clot) at either -20°C or -70°C (usually only available in research institutes). Such samples will be adequately preserved for many years, and could be analysed for retrospective seroepidemiological studies.
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.
Control
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%.
Availability
ELISA tests are widely performed by commercial laboratories, and in-practice test kits of a variety of types are available.
Validity
Sensitivity
ELISA is a highly sensitive test capable of detecting very small quantities of the target molecule (at least in the order of ug/ml).
Specificity
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.
The interpretation of an ELISA will depend entirely upon the nature of the test and the method of read-out.
Laboratory assays performed in a microtiter system with an automated plate-reader will enable precise relationship of the unknown samples to the standard curve that should be generated on each plate.
The testing laboratory should provide an interpretation of the clinical significance of the test result.
With commercially produced test kits, the instructions should be such that interpretation of a positive result is clear.
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Vetstream contributor(s)
Professor Michael J Day BSc BVMS PhD FASM DipECVP MRCPath FRCVS , Division of Veterinary Pathology, Infection and Immunity, Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU, UK.
Dr Helen R Milner BVSc(Dist) PhD CertVR MRCVS , PO Box 8730, Riccarton, Christchurch, New Zealand.
