Elisa Technique

Introduction to Elisa

Enzyme-linked immunosorbent assay is shortly known as ELISA. It is one of the most important immunoassays tests. It can identify the presence of some substances in the blood using laboratory technique that includes antibodies, antigens, proteins, glycoproteins, and hormones.

In this process, the antibodies and antigens are combined to get a significant result. The antibody is the production of a type of protein by the immune system of an individual. This protein type bind to specific regions of target antigens. In the case of certain infectious diseases, we can detect the presence of antibodies in the body. But this can be successful if the antigen-antibody bonding is successful.

Materials required for ELISA

ELISA tests are generally performed in 96 well plates. The main aim is to use 96 well plates as multiple samples can be tested in one experiment. The main characteristics of the absorbent plates are that antigens and antibodies can stick only on the surface. Each ELISA techniques measure the specific antigen. The steps are-

• There are two sets of antibodies are detects selected products like cytokines. ELISA plates should be coated to capture antibodies. In case of excess antibodies are present, the unbound antibodies should be washed from the plate.

• The sample is put on the plate. The capture antibody that is already coated in the plate is attached to any antigen of the samples. The samples have different concentrations. The samples that are present in excess would be washed out from the plate.

• The next step is the addition of a detection antibody. Either horse radish peroxidase or alkaline phosphatase enzymes are mixed with detection antibodies. This type of antibody holds together with any target antigen. This followed by the substrate should be added to the plates. In the ELISA assays process, they transform the substance into a coloured product.

• Using optical density the antigen concentration can be measured.

Antibody-antigen binding

In solitary antigenic places, the strength of interaction between antibody and antigen occurred. The distinct antibody arm link antigen at several sites within each antigen site. Generally, the antibodies interact through weak covalent force with antigen. A stronger affinity can be seen with them if the interaction between them is strong. Antigen and antibody stability and strength are controlled by three major factors. While its interactions are influenced by numerous factors, including pH, temperature, and ionic strength.

Principles

The basic principles of ELISA techniques are as follows

• ELISA process is carried out on a solid surface of a multi-well plate.

• The solid surface is present for the immobilization of the antigen.

• This can be done directly or indirectly by a captured antibody that prevents its movement on the surface by itself.

• The antigen is spilt up from other components that are present in the sample. This is facilitated by the analyst

• In ELISA technique, multiple samples can test easily.

Types of ELISA

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Direct ELISA

• It helps to find out the existence of antibodies in samples.

• Plates are coated with antigen

• In the sample plates, the antigen should be added and it is the first binding step.

• The plates should be incubated for a night.

• After the completion of incubation excess antibodies should wash away.

• Buffer is added to the plates and plates are rewashed.

• For conjugation of the enzyme, a specific primary detection antibody is put there.

• A selected enzyme-conjugated primary detection antibody is added.

• The absorbance quantity of coloured products is calculated with the help of spectrophotometry.

Indirect ELISA

• It helps to find out the existence of antibodies in samples.

• In this method, antibodies tie up on the well of the sample plate.

• Then antibodies from the sample are mixed with wells which are coated with antigen so that they can bind with each other.

• The free primary antibody would be washed away.

• By adding secondary antibodies that are conjugated with enzymes the detection of the antigen-antibody complex is successful.

• The next step is to wipe out the free secondary antibody.

• To get a coloured product specific substance is added.

• The absorbance quantity of coloured products is calculated with the help of spectrophotometry.

Sandwich Elisa

• In the sample appearance of antigen is detected by this method.

• The flat plate will multiple well is overlaid with antibody.

• In the good antigen from the sample is added. The free antigen was washed away.

• An enzyme bond with a secondary antibody that also binds to another antigen is added.

• The free secondary antibodies were washed away.

• To get a coloured product enzyme specific substance is added to the plate.

• With the help of spectrophotometry, the colour product absorbent quantity is measured.

Competitive ELISA

• In the sample concentration of antigen is detected by this method.

• The flat plate with multiple well is overlaid with antigen in this method.

• Then antibody is incubated. It is done by keeping the antigen in a solution that is rich in antigen.

• Then antigen is incubated in a solution that contains the antigen

• Then in the microtitre well should put the solution of antibody-antigen.

• An excess amount of antibodies was washed away.

• The secondary antibody that is bound with an enzyme is added that can identify the primary antibody numbers available in the well.

• The absorption quantity for the same is set up with the help of spectrophotometry

Diseases diagnosed using ELISA

• HIV.

• Lyme disease.

• COVID-19.

• Pernicious anaemia.

• Rocky Mountain spotted fever.

• Squamous cell carcinoma.

• Syphilis.

• Pregnancy test

Advantages of ELISA

• ELISA technique is highly effective.

• As in this method, antibodies have used the detection is done in a certain mechanism

• It has an elevated turnout. The ELISA kits that are commercially available are found in the format of 96 well plates The assay is effective against 384well plated.

• It is easy to perform. The steps involved in this process require less time and are very easy to understand.

• The antigen concentration in a sample is determined by this process.

• It can test different samples of serum, plasma, cellular, tissue extracts, urine, saliva, etc.

Application of ELISA

• In the food industry, this method is used to identify the existence of allergens in the food.

• In a virus test, the concentration of serum antibodies can be measured.

• It can determine the presence of antibodies and antigens in a sample.

• It can evaluate the spread of disease during disease outbreaks.

Conclusion

ELISA is one of the most specific and important immunoassays tests. It can identify the presence of some substances in the blood using laboratory technique that includes antibodies, antigens, proteins, glycoproteins, and hormones.

FAQs

Q1. What is an immunoassay?

Ans. Immunoassay is a biochemical test that detects disease and works on the principle of antigen-antibody reaction.

Q2. What is Lyme disease?

Ans. Bacteria are responsible for Lyme disease. It is spread through the bites of infected black-legged ticks.

Q3. What is the full name of HIV?

Ans. The full form of HIV is Human Immuno Deficiency Virus.

Q4. Name the viral disease that outbreaks in 2019?

Ans. Covid 19 is a viral disease that outbreaks in 2019.