Sandwich ELISA Technique: Learn All About the Process and Its Application
When researchers are working with a completely unknown sample, they use the sandwich ELISA technique to detect the presence of target antigens and measure their concentration in the sample.
This is because the sandwich ELISA technique offers superior sensitivity and greater durability, which together make it an excellent diagnostic tool for medical processes. This technique is particularly useful in the process of testing for drugs and identifying food allergens.
Sandwich ELISA is simply one of many options in this assay category. Some of the other types of ELISA assays include direct, indirect, and competitive ELISA. Among these, the sandwich ELISA technique is considered best for quantifying the antigens that are ‘sandwiched’ between detection antibody and capture antibody.
In terms of sensitivity, sandwich ELISA is anywhere between two to five times superior to indirect, direct, and competitive ELISA. Hence, sandwich ELISA can be used to quickly and accurately detect antigens in unknown samples.
In the sandwich ELISA technique, antigens don’t need to be purified before the measurement. Hence, it is ideal for complex samples and is also known for being very flexible. Usually, monoclonal and polyclonal antibodies are used for capture and detection in this technique.
The Basics of Sandwich ELISA
Quantifying antigen concentration in previously untested samples is one of the primary uses of the sandwich ELISA technique. For the antigen to be detected and measured through this technique, it needs to have a couple of non-overlapping antigenic epitopes, at a minimum.
This is because sandwich ELISA uses at least two antibodies, so it requires a sample that contains at least two antigenic epitopes capable of connecting with the antibodies.
As mentioned above, the capture and detection antibodies in this process are usually of the monoclonal and polyclonal variety. Monoclonal antibodies allow for accurate detection and quantification, as a result of which they are often used as detection antibodies.
On the other hand, polyclonal antibodies are great at binding antigens, so they are typically used as the capture antibody in the sandwich ELISA process.
To ensure that the antibodies being used do not end up interfering with one another’s binding capabilities, researchers typically use only match-paired antibodies for the sandwich ELISA technique.
This allows the antibodies to bind properly with separate epitopes on the target protein. The only drawback of this process is that, depending on the availability of the antibodies and antigens, the development of a sandwich ELISA assay usually requires two months at the very least.
The General Protocol for Conducting a Sandwich ELISA Assay
To successfully undertake the sandwich ELISA technique, researchers need to follow certain rules and protocols. Some of the most important steps involved in this process have been listed below.
1. Microtiter Plate Coating
The first step in the sandwich ELISA technique involves coating the well of a microtiter plate with the capture antibody. This will render the antibody immobile and help enhance assay efficacy.
Any unbound antibodies will then need to be washed off with PBS, to increase the sensitivity of the assay. Before the washing, however, the assay must be incubated overnight at 4oC temperature.
2. Blocking Buffer
A blocking buffer made from 5% non-fat dry milk or PBS will have to be used to block the coated wells and any residual protein-binding sites.
Coating the plate well with a blocking buffer will also help reduce non-specific and background binding. After the blocking buffer has been applied, the plate will just need to be incubated for a couple of hours at room temperature, before use.
3. Sample Application
The third step involves adding diluted samples to each of the microtiter plates. The samples and standards should be run in duplicates, to ensure accuracy. Each sample must also be incubated for 90 minutes so that the target antigens will have sufficient time to bind with the capture antibodies immobilized in their wells. At this point, the samples can be removed and rinsed with PBS to get rid of all the remaining unbound antigens.
4. Detection Antibodies
Detection antibodies now need to be applied for the sandwich ELISA technique to move forward. The plates containing diluted antibodies must be covered with adhesive plastic and incubated for two hours at room temperature. The plates must then be rinsed with PBS repeatedly to ensure that everything apart from the antibody-antigen complexes is washed away.
5. Secondary Antibodies
The addition of enzyme-linked secondary antibodies to the plates is the final step of the process. These secondary antibodies must then be incubated for about two hours at room temperature. After being diluted, they will play the role of the detection antibody. Later, the plate will have to be washed with PBS to remove all unbound antibody-enzyme conjugates.
Now that you know the importance as well as the process of the sandwich ELISA technique, all that remains is to find a reliable laboratory that can perform this essential assay on your behalf. Hiring the services of a good laboratory can save you a lot in terms of time, money, and labor, while allowing you to take advantage of effective assays and other diagnostic tools.