ELISA ( Enzyme-linked Immunosorbent assay)

What is ELISA :-

The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples.

- An ELISA test may be used to diagnose: HIV, which causes AIDS. 

Type of ELISAs :-

The four main types of ELISAs are -

1) Direct, 

2) Indirect, 

3) Sandwich, 

4) Competitive.

1)Direct ELISA :-

-In a direct ELISA, an antigen or sample is immobilized directly on the plate and a conjugated detection antibody binds to the target protein. 

-Substrate is then added, producing a signal that is proportional to the amount of analyte in the sample. 

-Since only one antibody is used in a direct ELISA, they are less specific than a sandwich ELISA.

When to Use: Assessing antibody affinity and specificity. Investigating blocking/inhibitory interactions.

2) Indirect ELISA :-

-An indirect ELISA is similar to a direct ELISA in that an antigen is immobilized on a plate, but it includes an additional amplification detection step. 

-First, an unconjugated primary detection antibody is added and binds to the specific antigen. 

-A conjugated secondary antibody directed against the host species of the primary antibody is then added. 

-Substrate then produces a signal proportional to the amount of antigen bound in the well.

When to Use: 

Measuring endogenous antibodies.

3) Sandwich ELISA:-

-Sandwich ELISAs are the most common type of ELISA. Two specific antibodies are used to sandwich the antigen, commonly referred to as matched antibody pairs. 

-Capture antibody is coated on a microplate, sample is added, and the protein of interest binds and is immobilized on the plate. 

-A conjugated-detection antibody is then added and binds to an additional epitope on the target protein.

- Substrate is added and produces a signal that is proportional to the amount of analyte present in the sample. Sandwich ELISAs are highly specific, since two antibodies are required to bind to the protein of interest.

When to Use: 

Determining analyte concentration in a biological sample.

4) Competitive ELISA:-

- Competitive ELISAs are commonly used for small molecules, when the protein of interest is too small to efficiently sandwich with two antibodies.

- Similar to a sandwich ELISA, a capture antibody is coated on a microplate. Instead of using a conjugated detection antibody, a conjugated antigen is used to complete for binding with the antigen present in the sample. -The more antigen present in the sample, the less conjugated antigen will bind to the capture antibody. Substrate is added and the signal produced is inversely proportional to the amount of protein present in the sample.

When to Use:

Determining concentrations of a small molecules and hormones.

Application of ELISA :-

1) The presence of antibodies and antigens in a sample can be determined.

2)It is used in the food industry to detect any food allergens present.

3) To determine the concentration of serum antibody in a virus test.

4) During a disease outbreak, to evaluate the spread of the disease, e.g. during recent COVID-19 outbreak, rapid testing kits are being used to determine presence of antibodies in the blood sample.