ELISA stands for “enzyme-linked immunosorbent assay” that is an immunochemical test involving an enzyme and an antibody or antigen. ELISA combines the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily assayed enzyme. ELISA tests are utilized to detect substances that have antigenic properties, primarily proteins. Some of these include hormones, bacterial antigens and antibodies.
In ELISA an unknown amount of antigen is affixed to a surface and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added so that the enzyme can convert to some detectable signal. In the case of fluorescence ELISA, when light of the appropriate wavelength is projected upon the sample, it will glow and thus detected.
The enzymeImmunoassay (EIA) and Enzyme–linkedImmunosorbentAssay (ELISA) were developed independently and simultaneously by the research group of PeterPerlmann and EvaEngvall at Stockholm University, Sweden and by the research group of Anton Schuurs and Bauke van Weemen in The Netherlands. Both techniques are based on the principle of immunoassay with an enzyme rather than radioactivity as the reporter label.
In the early 1970s, the idea of using enzyme labels was met with scepticism and incredulity. How could so bulky and large a molecule as an enzyme be attached to an antigen or antibody without hindering the immunochemical reaction between the antigen and antibody? However, between 1966 and 1969, the group in Villejuif reported their successful results of coupling antigens or antibodies with enzymes such as alkaline phosphatase glucose oxidase etc. Their purpose was to use the enzyme-labelled antigens and antibodies to detect antibodies or antigens by immunofluorescence.
Engvall and Perlmann published their first paper on ELISA in 1971 and demonstrated quantitative measurement of IgG in rabbit serum with alkaline phosphatase as the reporter label. Perlmann’s further research included cytotoxicity of human lymphocytes and immunogen selection and epitope mapping for malaria vaccine development. Engvall’s group applied the ELISA measurement tool to malaria and trichinosis, microbiology and oncology.
Engvall then focussed her scientific interests on the biochemistry of fibronectin, laminin, integrins and muscular dystrophies. In 1976, Organon Teknika developed and marketed a highly successful EIA system for the hepatitis B surface antigen featuring a 96-well microtiter plate format. This test became the first commercially available EIA. Other microbiological and virologic diagnostic tests soon followed, e.g., for hepatitis B e-antigens, Rubella antibodies, Toxoplasma antibodies, and in 1980s for detection of human immunodeficiency virus antibodies.
In the rabies field, ELISA was initially developed for the titration of rabies virus-neutralizing antibodies. The technique was applied to the quantification of rabies antigen by Atanasiu et al. using fluorescein-labelled IgG to the purified nucleocapsid. Subsequently, Perrin et al. developed an ELISA called rapid rabies enzyme immunodiagnosis (RREID), which was based upon the detection of rabies virus nucleocapsid antigen in brain tissue. In this test microplates were coated with purified IgG and an IgG-peroxidase conjugate was used to react with immunocaptured antigen.
1. Coating of Wells with Antibody
100 µL of antibody diluted in buffer A is added to each well. Pipette 0.2 ml of the diluted capture antibody to each well of a microtiter plate. The antibody should be directed against the antigen to be determined. Cover the plate with plastic film or aluminium foil and incubate at 4°C overnight. Use the immunoglobulin fraction of the antiserum for coating, not whole, unprocessed antiserum. The protein concentration of diluted antibody should be about 10 mg per L. Capture antibodies are typically plated at 0.2-10.0 µg/ml. Incubate the plate for 1 hour at 37 °C.
Wash the wells 4 times with buffer B using a micro plate washer. Alternatively, wash manually. The 0.1% Tween 20 in the washing buffer reduces the background and should be preferred for 0.05% Tween 20, which is commonly used.
Empty the plate by inversion over a sink. Tap the inverted plate against some layers of soft paper tissue to remove residual liquid. Wash the plate by filling the wells by immersion in buffer B. Leave on the table for 3 minutes. Empty the plate as described above and repeat washing two more times.
3. Incubation with Test Samples
100 µL of test sample or standard diluted in buffer B is added per well. Cover the plate and incubate at room temperature for 2 hours. Generally, biological samples should be diluted at least 1+1 to reduce the risk of non-specific reactions. Using the given general procedure, the detection limit is about 1 to 5 µg of antigen per litre of test sample. The sensitivity of a given antigen/antibody system might be improved if the sample is made 3% in polyethylene glycol. Further, the sensitivity will be improved by prolonging the incubation time.
4. Wash as described in step 2.
5. Incubation with Peroxidase-Conjugated Antibody
100 µL of peroxidase-conjugated antibody diluted in buffer B is added to each well. Cover the plate and incubate at room temperature for 1 hour. The peroxidase-conjugated antibody should be directed against the antigen to be determined. It might speed up the reaction and improve the sensitivity if the peroxidase-conjugated antibody is made 3% in polyethyleneglycol. In addition, the sensitivity might be improved if the incubation time is prolonged to 1 or 2 hours.
6. Wash as described in step 2.
When washing manually it is mandatory at this step that the washing buffer in the reservoir is totally exchanged after the first of the 3 washes. If this is not done, a high background staining will occur.
7. Colour Development
100 µL of chromogenic substrate C is added to each well. Cover the plate and incubate for 15 minutes, or until a suitable colour has developed. The plate should preferably be protected against light during this incubation.
Popular enzymes are those that convert a colourless substrate to a coloured product, e.g., pnitrophenylphosphate (pNPP), which is converted to the yellow p-nitrophenol by alkaline phosphatase. Substrates used with peroxidase include 2,2’-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) and 3,3’5,5’- tetramethylbenzidine base (TMB), which yield green, orange and blue colours, respectively.
Alkaline phosphatase yellow (pNPP) liquid substrate can be used as dye for the ELISA . This product is supplied as a ready-to-use buffered alkaline phosphatase substrate p-nitro-phenylphosphate (pNPP). Prior to the reaction with alkaline phosphatase, the substrate should appear as a colourless to pale yellow solution. It will develop a yellow reaction product when reacted with alkaline phosphatase in microwell applications and can be read at 405 nm. For the end-point assays, the reaction can be stopped with 0.05 ml/well of 3 N NaOH for every 0.2 ml of substrate reaction
8. Stopping the Colour Development
The reaction can be stopped by adding reagent D (100ul 0.5 M H2SO4) to each well.
9. Reading of Results
Read results directly through the bottom of the microwell plate using an automated or semi automated ELISA-reader. Read the plate at 490 nm. The subtraction of the absorbance at a reference wavelength (between 620 and 650 nm) is recommended, but not essential. Alternatively, read results within 3 hours in a photometer at 490 nm using a cuvette requiring no more than a 200 µL volume. The cuvette can conveniently be emptied by a piece of plastic tubing connected via a reservoir to a vacuum pump or to water suction.
An ELISA plate reader is used to measure the intensity of the colour formed in each well. A high-intensity lamp passes light to the microtiter well and the light emitted by the reaction happening in the microplate well is quantified by a detector. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization.
10. Plot the standard curve on semi logarithmic paper with A490 nm as ordinate and log10 concentration o standard as abscissa.