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introduction to elisa

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  1. 1. ELISA TO Ms.Sannia By Urooj Sabar 4318 5 th Semester (Morning) Bioinformatics & Biotechnology
  2. 2. Enzyme-Linked Immuno sorbent Assay <ul><li>INTRODUCTION </li></ul><ul><li>Is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. </li></ul><ul><li>first and most basic test to determine if an individual is positive for a selected pathogen, such as HIV. </li></ul>
  3. 3. 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter.
  4. 4. What To Detect or Measure? <ul><li>ELISA provide a useful measurement of antigen or antibody concentration. </li></ul><ul><li>detect the presence of antigens that are recognized by an antibody </li></ul><ul><li>test antibodies that recognize an antigen. </li></ul>
  5. 5. An ELISA is a five-step procedure Coat the micro titer plate wells with antigen or Antibody.
  6. 6. Second step <ul><li>block all unbound sites to prevent false positive results using the blocking solution. </li></ul>
  7. 7. 3 rd step <ul><li>Add sample to the wells. </li></ul>
  8. 8. <ul><li>add anti-mouse IgG (antibody) conjugated to an enzyme. </li></ul><ul><li>enzyme – alkaline phosphatase is used commonly </li></ul>4 th step
  9. 9. 5 th step <ul><li>reaction of a substrate with the enzyme </li></ul><ul><li>to produce a colored product, thus indicating a positive reaction. </li></ul>
  10. 10. Types Of ELISA <ul><li>Direct ELISA </li></ul><ul><li>Indirect ELISA </li></ul><ul><li>Sandwich ELISA </li></ul><ul><li>Competitive ELISA </li></ul>
  11. 11. Direct ELISA <ul><li>Used to test the presence of a specific antigen in sample. </li></ul>
  12. 12. Indirect ELISA <ul><li>Used to test the presence of a specific antibody in a sample. </li></ul>
  13. 13. Direct Sandwich ELISA
  14. 14. Indirect Sandwich ELISA
  15. 15. Competitive ELISA <ul><li>The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal). </li></ul>
  16. 17. Applications <ul><li>Serum Antibody Concentrations </li></ul><ul><li>Detecting potential food allergens </li></ul><ul><li>(milk, peanuts, walnuts, almonds and eggs) </li></ul><ul><li>Disease outbreaks- tracking the spread </li></ul><ul><li>of disease </li></ul><ul><li>e.g. HIV, bird flu, common, colds, cholera, </li></ul><ul><li>STD etc </li></ul><ul><li>Detections of antigens </li></ul><ul><li>e.g. pregnancy hormones, drug allergen, </li></ul><ul><li>GMO, mad cow disease </li></ul><ul><li>Detection of antibodies in blood sample for </li></ul><ul><li>past exposure to disease </li></ul><ul><li>e.g. Lyme Disease, trichinosis, HIV, bird flu </li></ul>
  17. 18. Any Question?