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Elissa test

  1. Presented by Daxa Rathwa 4th year B.Sc Nursing
  3.  An enzyme-linked immunosorbent assay, also called ELISA or EIA, is a test that detects and measures antibodies in your blood. This test can be used to determine if you have antibodies related to certain infectious conditions.  IMMUNO- refers to immune response that couses the body to generate antibodies.  ASSAY - refers to test or check amount substant present in sample
  4.  ANTIGEN Antigens are large molecules of proteins, present on the surface of the pathogen- such as bacteria, fungi viruses, and other foreign particles. When these harmful agents enter the body, it induces an immune response in tHe body for the production of antibodies.  ANTIBODY Antibodies are proteins that your body produces in response to harmful substances called antigens.
  5.  ELISA works on the principle that specific antibodies bind the target antigen and detect the presence and quantity of antigens binding. In order to increase the sensitivity and precision of the assay, the plate must be coated with antibodies with high affinity. ELISA can provide a useful measurement of antigen-antibody concentration.
  6.  Microtiter plate  Antibody  Antigen  Washing buffer  Enzyme[ horseradish, alkline phosphate]  Substrate  speectrophotometer
  7. Washing buffer Spectro photometer Substrate
  8.  ELISA tests can be classified into three types depending upon the different methods used for binding between antigen and antibodies, namely:  Direct Elisa  Indirect Elisa  Sandwich elisa  Competitive elisa
  9.  Take a empty microtiter plate. And add patient sample containing antigen.  Do washing to remove unbound antigen.  An enzyme-labeled primary antibody (e.g. HRP- labeled primary antibody) specific for the target antigen is added to the wells and directly binds to the antigen.
  10. Do washing to remove remove unbound antibody.  respective enzyme substrate is added, which upon reaction with the enzyme, produces a visible colorimetric output that can be measured by a spectrophotometer
  11.  Indirect ELISA detects the presence of an antibody in a sample.  The antigen is attached to the wells of the microtitre plate[readymade kit]  A sample containing the antibodies is added to the antigen- coated wells for binding with the antigen.  The free primary antibodies [sample] are washed away and the antigen-antibody complex is detected by adding a secondary antibody [enzyme linked] conjugated with an enzyme that can bind with the primary antibody.
  12. All the free secondary antibodies are washed away. A specific substrate is added which gives a colourd product. The absorbance of the coloured product is measured by spectrophotometry.
  13.  Sandwich elisa helps to detect the presence of antigen in a sample.  coat the well with capture antibody and washed to remove free antibody.  The sample containing the antigen is added to the well and washed to remove free antigen.
  14.  Then an enzyme-linked secondary antibody (detection antibody), which binds to another epitope on the antigen is added. The well is washed to remove any free secondary antibodies. The enzyme-specific substrate is added to the plate to form a coloured product, which can be measured. 2-5 time More sensitive than direct elisa.
  15.  Competitive ELISA helps to detect antigen concentration in a sample.  The microtitre wells are coated with the antibody.  Mixed patient sample containing antigen and secondary antigen that linked with enzyme.  The mixed solution of both antigen is added into the microtitre well. The well is then washed to remove any unbound antigen.
  16.  The enzyme specifi substrate add in well and measure colour product to find out the concentration of antigen  The both specific antigen compitte with each other to bind with antibody  If enzyme linked antigen more bind with antibody that indicate more concentration of enzyme linked antigen and produced more colour and least concentration of antigen in patient sample
  17.  If the sample antigen is more bind with antibody that produced less colour that indicate more concentration of antigen in patient sample.
  18. 1) It can be used to detect the presence of antigen or antibody in a sample. 2) It can be used to determine antigen and antibody concentrations in a sample. 3) It can be used in food industry to detect potential food allergens. 4) It can be used in disease outbreaks to track the spreading of diseases
  19. Screening donated blood for evidence of viral contaminatio by • HIV-1 and HIV-2 (presence of anti-HIV antibodies) Hepatitis C (presence of antibodies) Hepatitis B (testing for both antibodies and a viral antigen) Measuring hormone levels HCG (as a test for pregnancy) LH (determining the time of ovulation) TSH, T3 and T4 (for thyroid function)