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Elisa from A to Z


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Published in: Health & Medicine

Elisa from A to Z

  1. 1. ELISA From A~Z
  2. 2. ELISA ELISA (Enzyme-linked immunosorbent assay) is one of immunoassay method used to detection of 1-Antibodies 2-Proteins 3-Peptides 4-Biomolecules
  3. 3. What is immunoassay? The term “immunoassay” is a combined term of “immuno”(= immunological, practically immunochemical antigen-antibodyreaction) and “assay” (= determination of the purity of a substance or the amount of any constituent of a mixture.
  4. 4. 1. Antigen/antibody of interest is absorbed on to plastic surface („sorbent‟). 2. Antigen is recognised by specific antibody („immuno‟). 3. This antibody is recognised by second antibody („immuno‟) which has enzyme attached („enzyme-linked‟). 4. Substrate reacts with enzyme to produce product, usually coloured.
  5. 5. 1-Antibody (antiserum) 2-Antigen 3-Labeling materials
  6. 6. 1-Antibody (antiserum)  Antibody: proteins produced by 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 the antigen.
  7. 7. 2-Antigen Any molecule that induces production of antibodies when introduced in the body is called antigen. OR  Any “thing”, foreign to the immune system. e.g. bacteria, viruses, (or their parts), pollen, etc. SYMBOL FOR ANTIGEN
  8. 8. abeling 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 interefere with the binding. e.g:horseradishperoxidase enzyme substrate: trimethylbenzidine
  10. 10. Components of Kit  Pre-Coated, Stabilized 96-well Microtiter       Plate. Sample Diluent Standards and controls Conjugated Detection Antibody 10X Wash Solution Substrate Stop Solution
  11. 11. Advantages of ELISA  Reagents are relatively cheap & have a long shelf life  ELISA is highly specific and sensitive  No radiation 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 a variety of infections.
  12. 12. 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 constituents. Kits are commercially available, but not cheap Very specific to a particular antigen. Won’t recognize any other antigen False positives/negatives possible, especially with mutated/altered antigen
  13. 13. 1-Direct ELISA 2-Indirect ELISA 3-Sandwich ELISA 4-Competitive ELISA 5-Ogives ELISA
  14. 14. The direct detection method uses a labeled primary antibody that reacts directly with the antigen. Direct detection can be performed with antigen that is directly immobilized on the assay plate . Direct detection is not widely used in ELISA but is quite common for immunohistochemical staining of tissues and cells.
  15. 15. The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody.The secondary antibody has specificity for the primary antibody
  16. 16. The sandwich measures the amount of antigen between two layers of antibodies. Sandwich are especially useful if the concentration of antigens is low or they are contained in a mix of high concentrations of contaminating protein To utilize this assay, one antibody (capture) is bound to a microtiter plate well. Antigen is then added and bound to the antibody. Unbound products are then removed, and 2ry antibody is added (detection), then add the 3rd labeled antibody to complete the sandwich Major advantages of this technique are that the antigen does not need to be purified prior to use, due to its high specificity.
  17. 17. In this Unlabeled antibody is incubated in the presence of its antigen. These bound antibody/antigen complexes are then added to an antigen coated well. The plate is washed unbound antibody is removed. The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal. For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.
  18. 18. A newer technique uses an solid phase made up of an immuno-sorbent polystyrene rod with 8-12 protruding ogives. The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous ) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents
  19. 19. The method
  20. 20. APPLICATIONS : 1-HIV-1 and HIV-2 (presence of anti-HIV antibodies). hepatitis C (presence of antibodies). 2-hepatitis B (testing for both antibodies and a viral antigen) . 3-Measuring hormone levels HCG (as a test for pregnancy). 4-LH (determining the time of ovulation). TSH, T3 and T4 (for thyroid function).
  21. 21. 1. Coating of Wells with Antibody 100 μL of antibody diluted in buffer is added to each well. Cover the plate and incubate at 4 °C overnight. 2. Washing wash manually 3 times as follows:Empty the plate by inversion over a sink. Tap the inverted plate against some layers of soft paper tissue to remove residual liquid. Wash the plate by filling the wells by immersion in buffer B. Leave on the table for 3 minutes. Empty the plate as described above and repeat washing two more times.
  22. 22. 2-A concentrated solution of non-interacting protein, such as bovine serum albumin (BSA) or casein, is added to all plate wells.This step is known as blocking,because the serum proteins block nonspecific adsorption of other proteins to the plate.
  23. 23. 3. Incubation with Test Samples. 100 μL of test sample or standard diluted in buffer is added per well. Cover the plate and incubate at room temperature for 2 hours. 4. Wash as described in step 2. 5. Incubation with enzyme- Conjugated Antibody. 100 μL of enzyme-conjugated antibody diluted in buffer is added to each well. Cover the plate and incubate at room temperature for 1 hour. The enzyme-conjugated antibody should be directed against the antigen to be determined.
  24. 24. 6. Wash as described in step 2. 7. Colour Development 100 μL of chromogenic substrate is added to each well. Cover the plate and incubate for 15 minutes, or until a suitable colour has developed. The plate should preferably be protected against light during this incubation. 8. Stopping the Colour Development Stop the reaction by adding 100 μL 0.5 M H2SO4 to each well. 9. Reading of Results Read results directly through the bottom of the microwell plate using an automated or semiautomated photometer (ELISA-reader). The subtraction of the absorbance at a reference wavelength (between 620 and 650 nm) is recommended.
  25. 25. Fundamental techniques for performing ELISA
  26. 26. Fundamental techniques for performing ELISA 5. Structure of antibody-coated microplate and treatment
  27. 27. Fundamental techniques for performing ELISA 6. How to wash a microplate
  28. 28. 7-HOW TO TREAT WITH THE REAGENTS? Use reservoir for each reagent Label the reservoi
  29. 29. Don‟t use the same reservoir for multiple regents Don‟t return the reagents to the stock
  30. 30. 8. Shaking of the well-plate for mixing Place the plate on the flat and smooth surface of a laboratory table, hold the plate and move the plate roundly to draw circles rapidly for approx. 10 seconds while lightly pressing the plate on the surface. Repeat 3 times.
  31. 31. Important points in performing ELISA and improvement of assay performance 1-Sample treatment. 3-Stability of assay samples. 2-Infleunce of humidity and air stream.
  32. 32. Important points in performing ELISA and improvement of assay performance 1. Sampling and treatments of samples Serum or plasma
  33. 33. sampling In general, we recommend using When getting heparin is most often used as an anticoagulant Use of fluoride must be avoided because fluoride ion is a potent inhibitor of peroxidase.
  34. 34. An important phenomenon with frozen plasma is that an insoluble substance (fibrin) will be formed when thawed. In this case, the sample must be mixed and centrifuged, then the insoluble cluster flowing in the plasma should be taken out by a thin wire needle sharply bent at an end. If such fibrin remains in the sample, it may clog the tip of a pipette and influences assay variability
  35. 35. Hemolysis and Lipemia
  36. 36. pH Of the sample Serum or plasma, when fresh, shows pH near neutral, however, it very quickly goes to alkaline more than pH 8 by losing CO2. In alkaline pH, the antigenantibody reaction is interfered. resulting in cancellation of the assay or giving inaccurate assay values.
  37. 37. Storage temperature and freezingthawing. Sample storage temperature is better to be lower than -35 C. Ultra-low temperature such as -80 C is recommended for a long-term storage. Repeated freezing and thawing is also harmful to the protein, and may cause inactivation.
  38. 38. When samples are taken out from the freezer and thawed, never forget to mix these samples because the solution after thawing is not homogeneous, and the bottom area contains more solute
  39. 39. 2-Stability of assay samples. In assay, the problem of sample stability, i.e. how long the substance to be measured can keep its immunoreactivity, in serum or plasma, is very important. Blood samples also contain enzymes to destroy peptides or proteins, and stability against those enzymes differs from substance to substance. Freezer of –20C is not trustable for the constancy of temperature but use of a freezer of –35 C or lower temperature is recommended.
  40. 40. Avoid air fans Avoid sunlight
  41. 41. 3-infeluence of humidity and air stream During all the incubation process, the well-plate should be covered using the attached plate cover. Plate cover is effective only under the most suitable condition, i.e. room temperature, humidity more than 50%, and air stream of less than 0.2m/sec. N.B It is recommend to get a small semi-transparent plastic box, and put moistened paper towel on the bottom .
  42. 42. Trouble shooting in ELISA
  43. 43. 1-Poor or no coloration after the last step 1) The standard or samples might not be added. 2) Reagents necessary for coloration might not be added. 3) Wrong reagents related to coloration might have been added. 4) Influence of the temperature under which the kits had been stored. 5) Excessive hard washing of the well plate.
  44. 44. 2)The standard curve obtained was not smooth. There might be some mistake in the serial dilution of the original standard solution. 3)Flat standard curve. Standard solutions are not added.
  45. 45. 4-Big variation between two wells in duplicated assay was observed. 1) Scratching the bottom of the well by aspirator tip during aspiration of washing buffer. 2) Scratching the bottom of the well by pipette tip during addition of standards, samples, or reagents. 3)Assay might be started while the well-plate was still cooler than room temperature.
  46. 46. 4) Air stream, warmer or cooler than room temperature 5) Air stream from air conditioner or other instruments might dry wells. 6) Insufficient removal of washing buffer from the wells might dilute reagent solution added in the following step of the procedure. 7)Big variation would be obtained if the sample is not homogeneous.
  47. 47. Shapes of standard curves depending on scales in X-and Y-axes. Standard curve of ELISA prepared by plotting standard concentration on X-axis and absorbance on Y-axis, both in normal scale, looks like a linear line except for lower concentration area.