The Enzyme Linked Immunosorbent Assay (ELISA), (Enzyme Immunoassay (EIA) or Solid-Phase Immuosorbent Assay (SPIA)) is a sensitive laboratory method used to detect the presence of antigens (Ag) or antibodies (Ab) of interest in a wide variety biological samples.

Many variations in the methodology of the ELISA have evolved since its develeopment in the 1960s but the basic concept is still the imunological detection and quantitation of single or multiple Ag or Ab in a pateint sample (usually serum). The following pages should provide some general background to the variation available in ELISAs today.

Types of ELISA

Direct ELISA

Direct ELISA is the most basic of ELISA configurations. It is used to detect an Ag (red triangle; virus/bacteria/fungus, recombinant peptide/protein, or another Ab) after it has been attached to the solid phase (eg. a membrane or polystyrene microwell or dipstick).
An Ab (green), conjugated with a label (yellow star; eg. HRPO, AP, FITC) is then incubated with the captured antigen. After washing off excess conjugate and incubating with a substrate and chromogen, the presence of an expected colour indicates a specific Ab-Ag interaction.
The conjugate could be a commercial prepartion specific for the Ag of interest, or an in-house conjugated monoclonal or polyclonal Ab, or even patient serum.
This format is identical to the Direct Fluorecent Assay (DFA) except this is performed on pateint cells, usually witrh a glass slide as the solid phase and an FITC or TR conjugate.

Indirect

Once again an Ag is adsorped onto a solid phase. The first, or primary Ab (green) is incubated with the Ag, then the excess is washed off. A second or secondary Ab (blue), the conjugate, is then incubated with the samples.
The excess is again removed by washing.
For colour to develop, a primary Ab that is specific for the Ag must have been present in the sample (eg. human serum, CSF or saliva or the supernatant from a hybrodoma culture).
This indiactes a positive reaction.
It is important, during assay optimisation, to ensure that the secondary Ab does not bind non-specifically to the Ag preparation or impurities within it, nor to the solid phase.

Capture ELISAs

Antigen Capture

In this, more specific approach, a capturing Ab (orange) is adsorped onto the solid phase. The capture antibody may be the reagent to be tested (eg. the titre of a patients immune response to a known Ag). However, the Ab may be a standard reagent and the antigen the unknown(as when a patients serum is being investigated for the presence of a microbial infection).
The same stringent optimisation is required as for Indirect ELISA. This will ensure that the Ab do not cross-react in the absence of Ag, or non-specifically bind to the solid phase.
It is also important, when detecting the Ag, to use Ab from different animal species to prevent same-species Ab binding (eg. a polyclonal rabbit capture Ab will capture a monoclonal conjugate if it was raised in rabbits. This will produce a positve result in the absence of Ag).
Antibody Capture
In this approach, a capturing Ab (orange) is adsorped onto the solid phase. The Ab is designed to capture a class of human Ab (green; eg. IgG, IgA or IgM). Next the sample is applied, containing the Ab under investigation. After washing, an Ag (red) specific for the Ab is added and finally an anti-Ag conjugate (blue) provides the signal.

Competitive or Blocking ELISA

In a competitive ELISA, a patient serum and an Ag-specific conjugate (pink) are co-incubated with a captured Ag.
The amount of colour developed is inversely proportional to the amount of Ag-specific patient Ig present. Careful standardisation is required to interpret the results.

 

 

In a blocking ELISA, the patient serum is added first, incubated and the excess washed off.
Next an Ag-specific conjugate is added and the results interpreted as above. Titres here may be lower if the conjugate is of a high enough titer to displace patient Ab.

 

 

 

 

 

 

In a variation of this format, a conjugated Ag is used the competitor.

Coating Of Antigen/Antibody To The Solid Phase


The conditions used for the coating of the solid phase with Ag or Ab can seriously effect the outcome of the assay. Some of the variables to be considered include the (i) temperature used for coating, the (ii) concentration of Ag, the (iii) type of buffer, the (iv) time of incubation of the antigen with the solid phase and the (v) type of solid phase used for the ELISA.


(i) Coating Temperature
It has been reported for some antigens that a period of incubation at 37'C followed by incubation overnight at 4'C provided the best results. Higher incubation temperatures (up to 56'C) can provide more beneficial adsorption of some hydrophobic antigens, in a shorter period of time however deterioration of structure can occur at higher temperatures. Overnight incubation at 4'C is common and usually provides an adequate coating for non-commercial assays. For urgent testing, coating for 2 hours at 37'C usually provides an adequate result. Ensure your anitigen preparation is free or protected from proteases and yeast in this case.
Antigens used for the production of commercial kits must also be prepared in such a way that they will retain their antigenicity following any steps needed to increase the shelf-life of the kits. These steps include dehydration of the plates after coating, and the addition of anti-bacterial/anti-fungal or chemical coupling (an extra step in bonding the Ag to the plate) agents.

(ii) Concentration of the Antigen
The assay is ideally designed if the maximum number of binding sites on the solid phase are taken up by an Ag which is in the correct conformation to optimally bind Ab. However, as the concentration of Ag in the coating buffer increases, so does the rate of desorption of Ag from the matrix, during the assay. The steric conformation of the Ag may also be altered at high Ag concentrations, thus reducing Ab binding and increasing the rate of Ag desorption during the assay.
The optimal Ag concentration must therefore be determined through the use of a chequerboard titration of diluted Ag vs known positive and negative reference sera.

(iii) Type of Buffer
The buffer composition is usually based on carbonate buffer (e.g. 10-50mM, pH 9.6) Tris.HCl (pH 8.5) or simply PBS (pH 7.2).

(iii) Conjugates and Related Reagents
Horseradish peroxidase (HRPO or HRP) is the most common enzyme conjugate to an antibody in ELISA. Alkaline phosphatase (AP) is also common, and less often ß-galactosidase, urease and glucose oxidase have been used.

(iv) Reaction Substrates
The substrate An ideal substrate should have a defined absorbance peak, be non-carcinogenic, produce a maximal colour in a minimal amount of time and should remain stable during periods of storage - especially useful for commecial kits.
Examples of HRPO substrates include TMB, ABTS, o-toluidine, and OPD


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