ELSIA or Enzyme linked Immunosorbent Assays combines the sensitivity of simple enzyme assays with specificity of the antibodies by using antigens or antibodies couples perfectly to any easily-assayed enzyme. This is done to measure the antigen or antibody concentration in the blood.
Two distinctive variations of the ELISA Test
The method comes with two variations:
- ELISA test could be conducted to figure out the presence of antigens being recognized by some specific antibody
- ELISA can be also conducted for testing the antibodies to recognize some specific type of antigen
Steps of ELISA Process
In general, the ELISA Process comprises of 5 steps:
- Coating the micro-titer plate wells with the recommended antigen
- Blocking every necessary unbound sites that would help in preventing the false positivity in results
- Adding primary antibody (example: RPL13) to the wells
- Adding secondary antibody (example: IG5) conjugated to the enzyme
- Reaction that takes place when a substrate combines with the enzyme
Sandwich ELISA
There are several kinds of ELISA. However, one of the major types to mention is Sandwich ELISA.
Sandwich ELISA test is considered to be extremely effective in detecting sample antigen in the blood. These days, there are several ELISA pair sets available that feature the necessary apparatus for Sandwich ELISA test and analysis.
Sandwich ELISA basically quantifies the antigens between capture and detection layer of antibody. Here, it must be remembered that the antigen to be measure should contain at least a couple of antigenic epitopes required to bind the antibody. Since the mode is Sandwich, the presence of a minimum two antibodies would be required. Either polyclonal or monoclonal antibodies could be used to act as detection and capture antibodies in the Sandwich ELISA systems.
Monoclonal Antibodies
Monoclonal Antibodies usually recognize a single epitope for detection and quantification of the minor differences in an antigen.
Polyclonal Antibodies
Polyclonal Antibodies would be used most commonly as capture antibody for pulling down the maximum amount of antigen possible.
Steps for Sandwich ELISA Testing Process
Have a look at the steps to conduct the Sandwich ELISA testing process
- First of all, prepare the surface accordingly for placing the captured antibody.
- Block any and every nonspecific binding sites available on this surface.
- Now, the antigen containing samples needs to be applied to the plate.
- Wash the plate thoroughly in order to remove the unbound antige.
- Add the specific antibody and then bind it to the antigen.
- Then, include secondary antibody (must be enzyme linked). This would act as detection antibody that would also help in binding the antibody’s Fc region.
- Again, wash the plate thoroughly to remove unbound antibody enzyme conjugates.
- Now, apply a certain chemical that would get converted by the enzyme to form some kid electrochemical signal or coloring impact.
- Try to measure the impact of the plate wells in order to determine the presence of antigen along with the total percentage.
Advantages of using Sandwich ELISA
The most distinctive advantage of using Sandwich ELISA is that samples collected would never require any kind of purification prior to the analysis. More importantly, the assay could also be highly sensitive when compared to direct or indirect ELISA.
Difficulties concerning the Sandwich ELISA process
Sandwich ELISA testing process could be somewhat difficult to get optimized. It is recommended to use only the tested Match Pair antibodies to get optimum results. This would also ensure that the antibodies are detecting different types of epitopes on target protein level while allowing non-interference with any of the binding of other antibody.
Author Bio: Simone Douglas is a noted medical expert and journalist who have been working on the impacts of effectiveness of Sandwich Elisa test for the last two years. He is also researching on VAC14 Antibody. The article provides some lights to the process.