Immunology

Direct ELISA

Direct Enzyme Assay

Direct ELISA refers to an ELISA technique in which only a labelled primary antibody is used. It is a variant of enzyme assay where only one antibody is used which is conjugated directly to the detection enzyme for determining the presence and quantity of an antigen.

When the presence of an antigen is analyzed, the name using only a labelled primary antibody, the enzyme assay is termed as Direct ELISA.

 

Direct Enzyme Assay

 

It is simplest ELISA technique requiring an antigen and an enzyme-conjugated antibody specific to antigen and is best for analyzing immune response to an antigen.

In this technique only primary antibodies are applied and they are conjugated with an enzyme which produces a colored product when a suitable substrate is added.

 

However on using single antibody act of attaching an enzyme to antibody can interfere with locations on antibody that should be reacting with antigen which can decrease reactivity of antibody.

This can be used to test specific antibody- antigen reactions, and helps to eliminate cross-reactivity between other antibodies. It is typically used when the immune response to an antigen needs to be analyzed.

 

Principle

Initially antigen is adsorbed to a plastic plate, then an excess of another protein (normally bovine serum albumin) is added to block all other binding sites. While an enzyme is linked to an antibody in a separate reaction, enzyme-antibody complex is applied to adsorb antigen. After excess enzyme-antibody complex is washed off, enzyme-antibody bound to antigen is left. By adding enzyme’s substrate, enzyme is detected illustrating the signal of antigen.

 

Direct ELISA

 

A direct ELISA involves coating the plates with a mixture containing the target antigen which is detected using conjugated anti-albumin antibody. Alternatively enhance the signal an unconjugated anti-albumin antibody followed by a secondary, enzyme-conjugated detection antibody can be used.

 

Advantages:

  • Faster as this technique has fewer steps.
  • Less prone to error as less reagents and fewer steps are required
  • Only one antibody is needed and it requires less time to complete.
  • Potential for non-specific cross-reactivity of the secondary antibody is completely eliminated.

 

Disadvantages:

  • Less flexible as each target protein needs a specific conjugated primary antibody.
  • Less flexibility in choice of enzyme-substrate detection system.
  • Lack of signal amplification reduces assay sensitivity.
  • Antigen immobilization is not specific.

 

Additionally labeled antibody must be created for each run. The primary antibody must be labeled individually, which can be time-consuming and inflexible when performing multiple experiments.

 

Download Link: Direct ELISA.pdf

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