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is an abbreviation for enzyme linked immunosorbent assay ELISA: is a biochemical technique used mainly in immunology
detect the presence of Ab or Ag in a sample also used as a diagnostic tool in medicine and plant pathology quality control check in various industries
General steps and components
The main steps and components involved in ELISA running: 2. Solid phase: represented by microtiter plate which must have certain characteristics like
high level of adsorption of proteinious substances the wells are flat in shape
Coating step: with Ag or Ab
Incubation: to allow the reaction between Ab and Ag substrate and enzyme etc…..
Less time the reaction is not complete More time affect Ab sensitivity
Washing: to remove excess Ab or Ag and remove non well attached Ab or Ag
Soft washing : the excess still in wells and bind conjugate and gives fouls results Aggressive washing: affect binding of Ab and Ag
Conjugates: are Ab of a certain spp against injected Ab from another spp and it is also called antispecies that is covalently bond to Enzyme The main types of enzymes that can be used in this test are:
1. 2. 3. 4.
Horseradish preoxidase Alkaline phosphatase β – Galactosidase Urease
Substrate “chromogene”: produce the color when react with the enzyme 2. Stopping solution: stop the reaction between the enzyme and the substrate 3. Washing buffer: used for washing step 4. Sample buffer: used for making dilution for the samples
Types of ELISA
2. 3. 4. 5.
The main methods of ELISA: Direct ELISA Indirect ELISA Sandwich ELISA competitive ELISA
Direct sandwich ELISA
Indirect sandwich ELISA
Direct competitive ELISA for Ag
Direct Competitive ELISA for Ab