Elisa test

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1. ELISA TEST by Neelma nayab

2. CONTENT INTRODUCTION NOMENCLATURE OF ELISA BASIC PRINCIPLE OF ELISA MATERIAL REQUIRED FOR ELISA TEST TYPES OF ELISA APPLICATIONS OF ELISA

3. INTRODUCTION ELISA(Enzyme Linked ImmunoSorbent assay) is a widely used technique for detection of antigen (Ag) or antibody(Ab). The technique was developed in 1971 by Peter Perlmann and Eva Engvall at Stockholm University, Sweden. A technique to prepare something like immunosorbent to fix antibody or antigen to the surface of a container was published by Wide and Jerker Porath in 1966 Eva Engvall Peter Perlmann

4. EL: The second antibody is attached to an enzyme (‘enzyme-linked’) I: Antigen is recognized by specific antibody (‘ immuno ’) S: Antigen of interest is absorbed on to plastic surface (‘sorbent’) A: Substrate reacts with enzyme to produce a product, usually colored, which can be assessed (‘assay’) ELISA NOMENCLATURE

5. BASIC PRINCIPLE OF ELISA Enzyme is used to detect the binding of Antibody – Antigen Enzyme converts colorless substrate into colored product, indicating the presence of Antibody - Antigen complex ELISA can be used to detect either presence of Antigens or Antibodies

6. ELISA: MATERIAL REQUIRED substrate Antibody (1 st and 2 nd )Antigen Microtiter well Blocking buffer TESTING SAMPLE Washing buffer

7. Types of ELISA DIRECT ELISA INDIRECT ELISA SANDWICH ELISA COMPETENT ELISA

8. DIRECT ELISA Direct ELISA detects the presence of antigen in a sample Microtiter wells are initially coated with antigen to be detected which is followed by an antibody linked to an enzyme conjugate. This follows the addition of substrate which produces colour detected using ELISA detector.

9. INDIRECT ELISA It is used for detection of an antibody in the given sample. Microtiter wells are initially coated with antigen specific for antibody to be detected, followed by the addition of sample. Enzyme conjugated Secondary Antibody is added followed by the substrate which forms a coloured reaction product.

10. SANDWICH ELISA It is used for detecting an antigen in the given sample. Microtiter wells are initially coated with monoclonal antibodies(called capture antibody) raised against antigen to be detected, followed by addition of sample. Any trace of antigen is detected by adding primary antibody (a MAb ),followed by enzyme conjugated secondary Ab and a chromogenic substrate; or by directly adding an enzyme conjugated primary Ab.

11. COMPETENT ELISA This variation of ELISA is used to quantitatively estimate the amount of antigen in the given sample Ag and Ab are initially incubated so that they form Ag-Ab complex. This mixture is then added to microtiter wells coated with synthetic analogue of antigen to be detected, any free antibody binds to these antigens . This complex is estimated by enzyme conjugated secondary antibody by chromogenic detection .More the amount of antigen in the sample, lesser is the antibody available to bind to microtiter wells.

12. COMPETENT ELISA

13. APPLICATIONS OF ELISA TEST ELISA can detect both antigen and antibody it is a useful tool for determining serum antibody concentrations . It has also found applications in the food industry in detecting potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs. The other uses of ELISA include: A. Detection of Mycobacterium antibodies in tuberculosis B. Detection of hepatitis B markers in serum C. Detection of enterotoxin of E. coli in feces D. Detection of HIV antibodies in blood samples