ELISA

ELISA

• 1.
  ELISA Presented by: FATIMAH SINDHBIOTECHNOLOGY ASSOCIATON
• 2.
  ELISA  ELISA (Enzymelinked immuno-sorbent assay) is one of the immunoassay method used to detection of 1) ANTIBODIES 2) PROTEINS 3) PEPTIDES 4) BIOMOLECULES
• 3.
  WHAT IS IMMUNOASSAY? •Theterm “immunoassay” is a combined term of “immuno”=(practically antigen- antibody reaction) and • “Assay”=(Determination of purity of substance or the amount of any constituent of a mixture.
• 4.
  WHY KNOWN AS…? EnzymeLinked Immuno-sorbent Assay 1.Antigen/Antibody of interest is absorbed on to plastic surface (‘Sorbent’) 2.Antigen is recognized by specific antibody (‘Immuno’) 3.This antibody recognized by second antibody (‘immuno’) which has enzyme attached(‘enzyme linked’) 4.Substrate react with enzyme to produce product usually colored.
• 5.
  HISTORY OF ELISA Radioimmunoassay was first described in a scientific paper by Rosalyn Sussman Yalow and Solomon Berson Published in 1960.  In 1971, Peter perlmann and Eva Engvall stockhlom University in sweeden,and anton schuurs and Bauke van Wccmen in the Netherlands independently published papers that synthesized this knowledge into methods to perform EIA/ELISA.
• 6.
  Important components in immunoassay 1.AntiBody (Anti-Serum) 2. Antigen 3. Labeling Materials
• 7.
  1.ANTIBODY(ANTISERUM)  Antibody:proteins producedby the immune system which help defend against antigens Symbol for antibody  The variable regions are though to be the place for recognition and binding with antigen.
• 8.
  2.ANTIGEN • Any moleculesthat induces production of antibodies when itroduced in the body is called antigen, OR • Any “thing” foreign to the immune system eg.bacteria viruses,(or their parts), pollen..etc.
• 9.
  3.LABELLING MATERIALS In immunoassay,it is necessary to use any marker to know the antigen-antibody binding.for such purpose, we label either antigen or antibody with some materials that do not interfere with the binding. eg.Horseradishperoxidase ezyme Substrate :trimethylbenzidine
• 10.
  ELISA…. ELISA KIT ELISA READER
• 11.
  Basic Principle ofELISA  Use an enzyme to detect the binding of antigen(Ag) antibody(Ab)  The enzyme convert a colourless substrate (chromogen)to coloured product,indicating the presence of Ag-Ab binding.  An elisa can be used to detect either the presence of antigen or antibodies in a sample depending how the test is designed  Elisa was developed in 1970 and become rapidly accepted
• 13.
  ELISA Quantitative/Qualitative  Qualitative determinesantigen or antibody is present or absent  Quantitative o determine the quantity of antibody o titer o the highest dilution of the specimen usually serum which gives positive reaction in the test
• 15.
  ADVANTAGES OF ELISA Reagents are relatively cheap and have long shelf life  ELISA is highly specific and sensitive  No radiations hazards occur during labelling or disposal of waste.  Easy to perform and quick procedures  Equipment can be inexpensive and widely available.  ELISA can be used to variety of infections.
• 16.
  DISADVANTAGES OF ELISA Measurement of enzyme activity can be more complex than measurement of activity of some type of radioisotopes.  Enzyme activity may be affected by plasma constituent.  Kits are commercially available ,but not cheap  Very specific to a particular antigen. Won’t recognize any other antigen.  False positive/negative possible, especially with mutated/altered antigen
• 17.
  TYPES OF ELISA 1.COMPETATIVEELISA: 2.NON COMPETATIVE ELISA: a)SANDWICH ELISA b)DIRECT ELISA c)INDIRECT ELISA
• 18.
  Common Steps… 1.Collection andprocessing of serum 2.Coating of wells antibodies/antigens 3.Washing 4.Incubation with the test sample 5.Washing 6.Incubation with enzyme conjugated antibodies 7.Washing again 8.Colour development by adding chromogenic substrate 9.Stopping the color development 10.Reading of results
• 20.
  APPLICATIONS OF ELISA.. 1.Todetect Viral Contamination (HIV, Hepatitis C) 2.Hormone levels (HGH, Testosterone, ACTH) 3.Various Infections (Dengue, Human Papilloma Virus) 4.Specific disease factors (Clotting factor VII, Lyme’s disease) 5.Drugs (Narcotics, Antipsychotics, Antidepressants) 6.Allergens in food (egg protein, milk protein) Residues in food (Antibiotic, lupine) 7.Toxins in food (Pesticides, botulinum) 8.Others (Pregnancy Test) 9.Preclinical screening
• 21.
  THANK YOU FOR YOUR PATIENCE