ELISA (enzyme-linked immunosorbent assay)

Purpose

This test is used for the detection of Ag and Ab.
Basically, it is a colorimetric analysis. In this test, we see color by spectrophotometer. spectrophotometer detects the optical density of the solution. It is based on serology (Ag-Ab reaction)

Advantages

  • This test is easy and we get the result in 30 minutes.
  • There is no need for standardization.
  • It is preferably used over the HI test.

Disadvantage

  • ELISA kit is commercially available but expensive.

Types

There are two main types of ELISA.
  1. Direct ELISA
  2. Indirect ELISA

Direct ELISA


 It is also called a double antibody sandwich ELISA. In this test we detect Ag.

Procedure

  1. Incubate the plate overnight at 4oC by coating NDV Abs.
  2. Wash plate after every step with Tween-20 or PBS.
  3. Add test Ag after coating with NDV Abs (specific to NDV Abs). If it is not specific, it will wash out.
  4. Add another Ab specific to NDV. This Ab is enzyme-linked (enzyme: Horse reddish peroxidase enzyme). These Abs bind to Ag.
  5. In the 4th step, we add a substrate chromogen. When this enzyme binds with a chromogen, it produces color.
  6. If the test is positive, the color will be produced.
  7. If the test is negative, no color will produce.
  8. Check color by spectrophotometer.

Types of substrate

  • Chromogen
  • H2O2
The substrate is a colourless substance, but it produces color when it binds with the enzyme.

Indirect ELISA

Purpose

This test is used for the detection of antibodies.

Procedure

  1. Incubate plate with Ag overnight. Ag is absorbed in the walls of wells.
  2. Add antisera that contain Ab. If it has Ab against NDV, it will bind with Ag.
  3. Add 2nd Ab specific to 1st Ab (It is enzyme coated).
This test is designed by IAEA (International atomic energy agency) and the food and agriculture organization (FAO). This test is also designed for Animal production and health science.

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