Enzyme-linked Immunosorbent Assay
(Dr. Girish Chandra)
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 enzyme Immunoassay (EIA) and Enzyme-linked Immunosorbent Assay (ELISA) were developed independently and simultaneously by the research group of Peter Perlmann and Eva Engvall 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.
An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the most basic test to determine if an individual is positive for a selected pathogen, such as HIV. The test is performed in an 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which is about 1 cm high and 0.7 cm in diameter.
When the body is infected with a virus, the immune system responds by producing antibodies–proteins that circulate in the blood and attempt to destroy the virus. If antibodies against HIV are present in a blood sample, they will stick to the plate coated with fragments of the virus. The ELISA reagent will detect the presence of the bound antibodies and will change colour giving a positive result.
Partially purified, inactivated HIV antigens are coated onto an ELISA plate. Patient’s blood serum which contains antibodies to HIV is added and those antibodies will bind to the HIV antigens on the plate. Anti-human immunoglobulin is coupled to an enzyme. This is the second antibody that binds to human antibodies. Chromogen or substrate which changes colour when cleaved by the enzyme is attached to the second antibody.
In most cases if the ELISA tests are positive, the patient will be retested by the western blotting analysis again for confirmation.
TYPES OF ELISA
Generally there are 5 types of ELISA: Direct ELISA; Indirect ELISA; Sandwich ELISA; Competitive ELISA and Multiplex ELISA. The two common types of ELISA are indirect ELISA (that detect antibodies in sample) and sandwich ELISA (that detect antigen in sample).
The direct ELISA uses the method of directly labelling the antibody itself. Microwell plates are coated with a sample containing the target antigen, and the binding of labelled antibody is quantitated by a colorimetric, chemiluminescent or fluorescent end-point. Since the secondary antibody step is omitted, the direct ELISA is relatively quick, and avoids potential problems of cross-reactivity of the secondary antibody with components in the antigen sample.
The indirect ELISA is a two-step method that uses a labelled secondary antibody for detection. Apply a sample of known antigen of known concentration to the well of a microtiter plate. A concentrated solution of non-interacting protein, such as bovine serum albumin or casein, is added to all plate wells for blocking, because the serum proteins block non-specific adsorption of other proteins to the plate. The plate wells are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins. Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
View the result using a spectrophotometer, spectrofluorometer, or other optical or electrochemical devices.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules.
The sandwich ELISA measures the amount of antigen between two layers of antibodies. The antigens to be measured must contain at least two antigenic sites, capable of binding to the antibodies, since at least two antibodies act in the sandwich. To utilize this assay, one antibody (the “capture” antibody) is purified and bound to a solid phase typically attached to the bottom of a plate well. Antigen is then added and allowed to complex with the bound antibody. Unbound products are then removed with a wash and a labelled second antibody (the “detection” antibody) is allowed to bind to the antigen, thus completing the “sandwich”.
Prepare a surface to which a known quantity of capture antibody is bound. Block any non specific binding sites on the surface by adding serum proteins. Apply the antigen-containing sample to the plate. Wash the plate, so that unbound antigen is removed. Apply primary antibodies that bind specifically to the antigen. Apply enzyme-linked secondary antibodies which are specific to the primary antibodies. Wash the plate, so that the unbound antibody-enzyme conjugates are removed. Apply a chemical which is converted by the enzyme into a colour or fluorescent or electrochemical signal. Measure the absorbance or fluorescence or electrochemical signal of the plate wells to determine the presence and quantity of antigen.
When two “matched pair” antibodies are not available for a target, another option is the competitive ELISA. The advantage of competitive ELISA is that non-purified primary antibodies may be used. Although there are several different configurations for competitive ELISA, one reagent must be conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may be linked to either the antigen or the primary antibody. In this type of ELISA, there is an inverse relationship between the signal obtained and the concentration of the analyte in the sample, due to the competition between the free analyte and the ligand-enzyme conjugate for the antibody coating the microplate, i.e. the more analyte the lower the signal.
An unlabelled purified primary antibody is coated onto the wells of a 96 well microtiter plate. This primary antibody is then incubated with unlabeled standards. After this reaction is allowed to go to equilibrium, conjugated antigen is added. This conjugate will bind to the primary antibody wherever its binding sites are not already occupied by unlabelled antigen. Thus, the more unlabelled antigens in the sample or standard, the lower the amount of conjugated antigen bound. The plate is then developed with substrate and colour change is measured.
In this ELISA a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. Antigens are measured by coating or printing capture antibodies in an array format within a single well to allow for the construction of "sandwich" ELISA quantification assays. Generally, multiplex ELISA can also be achieved through antibody array, where different primary antibodies can be printed onto the glass plate to capture corresponding antigens in a biological sample such as plasma, cell lysate, or tissue extracts. Detection method can be direct or indirect, sandwich or competitive, labelling or non-labelling, depending upon antibody array technologies.