ELISA (Enzyme-linked immunosorbent assay) remains one of the most applicable immunoassays both in diagnostic and research laboratories. The most popular ELISA variants you can employ as immunoassay techniques in immunology are direct, indirect, sandwich, and competitive ELISA.
Direct ELISA is a method in which an antigen from the sample is immobilized on the wells of the plate. A commercially procured enzyme-conjugated antibody is then added to the wells. The mix is incubated and then washed to remove any unbound material.
If the enzyme in the conjugate is horseradish peroxidase (HRP), then Tetramethylbenzidine(TMB) is added and the mix is incubated as appropriate (According to the manufacturer’s instructions). The plate is washed to remove any unbound substrate.
The action of HRP on TMB makes a blue substance that changes to yellow when the reaction is stopped using a strong acid like sulphuric acid (H2SO4). The absorbance signal (at a specific wavelength like 540nm) is read off from the plate reader and recorded.
To determine the concentration of the antigen in the sample, the absorbance is compared with those of the standards of the antigen using a standard curve.
The advantage of this method is that it is easy to perform because the protocol is short. However, this method is not as specific as the sandwich ELISA and this is a disadvantage.
In this variant of ELISA, two antibodies are used for it to work. The first antibody is the primary antibody which binds specifically to the antigen that has been immobilized on the plate wells. Then the mix is washed off to remove the unbound material after an appropriate period of incubation.
The secondary antibody is an anti-immunoglobulin raised to bind specifically to the crystallizable fragment (Fc) of the primary antibody. The secondary antibody must be tagged to an enzyme of a fluorophore which is the assay detection system.
The enzyme-substrate is then added. For HRP then TMB is added and a yellow substance indicates a positive reaction which is stopped using sulphuric acid before the absorbance of the colored compound is read off using a computer-attached plate reader.
An advantage of this method is high sensitivity due to the high avidity of the binding between the secondary antibody’s multiple epitopes. This ensures that there is an amplification of the signal.
The other advantage is that enzyme or fluorochrome-labeled secondary antibodies are readily available and can be procured commercially. The disadvantage is the possibility of cross-reaction on the part of the secondary antibody which can give a false-positive reaction.
This type of ELISA uses two antibodies that sandwich the antigen between them. The first antibody is the capture antibody which is immobilized on the surface of the multiple-wells micro-titer plate.
The second antibody is the detection antibody which is tagged to an enzyme or tagged to the fluorophore. The two antibodies are specific for different epitopes on the same antigen. This method tests for the presence of the specific antigen in the sample of interest.
Immobilization of the Capture Antibody
The capture antibody is made to adsorb to the wells of the microtiter plate. Once this is done, the unbound antibody is washed off after incubation at the appropriate time period.
Blocking Unbound Polystyrene Surface
In the plate wells with the capture antibody, a suitable buffer that contains a protein like serum bovine albumin (BSA) is added. The protein will adsorb to the unbound surface of the plate wells (polystyrene). This blocking is meant to ensure that the target antigen does not attach here.
Sample addition phase
The sample suspected to be containing the antigen or the control is added to the plate. The plate is incubated to allow the captured antibody to react and form an immune complex with the antigen in the sample. The plate is then washed to remove the loose unbound material from the sample.
The second antibody which is conjugated to horseradish peroxidase (HRP) is added. The other popular enzyme that can be used here is alkaline phosphatase (ALP). The set-up is now incubated as appropriate.
This antibody will identify and specifically bind to alternative epitopes on the antigen already captured in the [Ag-Ab]. The plate is then washed to remove the unbound material.
The substrate to the HRP which is Tetramethylbenzidine (TMB) is added. The reaction makes the solutions in the micro-titer plate turn to a blue chromogen. The change in color to blue reflects the oxidation of TMB.
The interaction between TMB and HRP causes hydrolysis of hydrogen peroxide to oxygen radicals that show a blue color. A stop solution (H2SO4) is added to stop the reaction and this is indicated by a yellow chromogen appearing.
A micro-titer plate reader is used to measure the absorbance of the solution which is proportional to the concentration of the antigen present in the sample. A standard curve of the standards is used to interpret the results.
This method is highly sensitive and this is its advantage. Another advantage is that for detection purposes there are various conjugates (e.g. fluorophores) that can be used other than an enzyme.
The disadvantage of this type of ELISA is that the detection antibody can potentially bind to the primary capture antibody. This would give a false-positive result.
This technique is also known as inhibition ELISA and measures the signal interface. This method is meant for the determination of the concentration of the unlabeled antigen in the sample.
In this method, there are two antigens of the same type that are involved – the labeled and the unlabeled antigen (from the sample). The two antigens compete with each other for the binding sites on the primary antibody. The capture/labeled antigen is immobilized on the wells of the microtiter plate.
The unlabeled antigen is incubated with the primary antibody specific for this antigen, after which the mixture is added to the plate wells. The more the unlabeled antigen in the sample the less the free primary antibody will be available to bind to the capture/labeled antigen immobilized on the wells.
Then the addition of an enzyme-labeled secondary antibody specific to the Fc region of the primary antibody will be used to quantitate the primary antibody whose concentration will be inversely proportional to the concentration of the unlabeled antigen of interest in the sample.
The microtiter plate wells are first coated with the capture antigen. This is done by adding the antigen, then incubating the plate overnight at 40C to make the antigen immobilized. The following morning, you should flick the plate over the sink to remove all the unbound material.
The unbound surfaces of the plate wells are blocked using a blocking buffer (containing a protein like serum bovine albumin, or BSA). The plate is incubated for about 2 hours at room temperature to complete the process of blocking the naked surfaces of the plate.
The antigen-antibody mixture is prepared by adding the primary antibody to the unlabeled antigen in the sample (e.g. cortisol). This mixture, which is prepared in vial tubes for each sample, is incubated for about 1 hour at 370C to allow the formation of Ag-Ab complexes if the antigen is present in the sample.
The mixture is then added to the plate that already has the capture/labeled antigen immobilized. The plate is flicked over the sink after an appropriate incubation period to remove the unbound material.
The secondary antibody, which is conjugated to an enzyme, is added and incubated as appropriate. The plate is washed to remove the unbound material. An appropriate substrate like Tetramethylbenzidine (TMB) for HRP is added and the mixture is incubated for about 5 minutes at room temperature to allow the enzyme-substrate reaction to occur.
A positive reaction, in this case, will be indicated by a change of color of the well’s contents to blue. With the addition of the stop solution like Sulphuric acid (H2SO4), the color will change to yellow.
Using a microtiter plate reader the absorbance should be read within 30 minutes. In this type of ELISA, the strength of the signal is inversely proportional to the concentration of the unlabeled antigen (e.g. cortisol) in the sample.
From a standard curve that had been prepared using standards of known concentrations, then the concentration of the sample antigen can be determined and compared to the reference values to see if it is within the normal range.
ELISA remains one of the most important immunological techniques in modern medicine. There are four types of ELISA that include direct, indirect, sandwich, and competitive ELISA. They are employed in different situations in the diagnosis of diseases in the laboratory.Follow us on Social Media