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  1. 1. Enzyme Linked Immunosorbent Assay ELISA
  2. 2. Enzyme-linked Immunosorbent Assay
  3. 3. ELISA Kits for Antibody Detection  Commercial ELISA test kits are available to detect  Avian influenza virus antibody in chicken serum  Newcastle disease virus antibody in chicken serum  Newcastle disease virus antibody in turkey serum
  4. 4. ELISA Kits for avian Detection Ab IDEXX SYNBIOTICS
  5. 5. Definition of ELISA  A sensitive immunoassay that uses an enzyme linked to an antibody or antigen as a marker for the detection of a specific protein, especially an antigen or antibody. It is often used as a diagnostic test to determine exposure to a particular infectious agent (technique involving the reaction of the antigen or antibodies in vitro )
  6. 6. HISTORY  Prior to the development of the EIA/ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively-labeled antigens or antibodies.  Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in .1960
  7. 7. ELISA objective  To detect the presence of an antigen or antibodies in a sample and to use it as a diagnostic tool in medicine.  To detect potential food allergens.  To be used in toxicology as a rapid presumptive screen for certain classes of drugs
  8. 8. Principle of ELISA
  9. 9. Principle of ELISA  Antibody is immobilized on micro-plate wells  Competition between in sample and labeled enzyme for antibody binding sites  The unbound material is washed out  Chromogenic substrate added to develop color  Resulting color is read
  10. 10. Component of ELISA  Antigen  Primary antibodies  Secondary antibodies  Enzyme  Substrate  Stop solution
  11. 11. • • • • • • • Equipments are widely available. No radiation hazards. Reagents are cheap with long shelf life. Adaptable to automation and high speed. Qualitative and quantitative. Reproducible. ELISA can be used on most types of biological samples, such as plasma, serum, urine, and cell extracts • Sensitive assay
  12. 12. Types of ELISA  Direct method  In direct method  Sandwich method  Competitive method
  13. 13. Direct ELISA
  14. 14. Direct ELISA  The direct Enzyme-Linked Immunoabsorbent Assay (ELISA) is a method for detecting and measuring antigen concentration in a sample. Using a capture monoclonal antibody, the presence of a particular antigen in a sample is detected.
  15. 15. Advantage of direct ELISA This type of ELISA has two main advantages:  It is faster, since fewer steps are required  It is less prone to error, since there are fewer steps and reagents
  16. 16. Indirect ELISA  Indirect ELISA is a two-step method that uses a primary antibody and labeled secondary antibody. The primary antibody is incubated with the antigen followed by the incubation of the secondary antibody. But, this may give a nonspecific signal result because crossreaction with the secondary antibody may occur
  17. 17.  Antigen coated to a polystyrene multiwell plate is detected in two stages or layers. First an unlabeled primary antibody, which is specific for the antigen, is applied. Next, an enzyme-labeled secondary antibody is bound to the first antibody. The secondary antibody is usually an anti-species antibody and is often polyclonal.
  18. 18. Advantage of indirect method This method has several advantages:  Increased sensitivity, since more than one labeled antibody is bound per primary antibody  Flexibility, since different primary detection antibodies can be used with a single labeled secondary antibody  Cost savings, since fewer labeled antibodies are required
  19. 19. ELISA Results  Results should be recorded by reading the optical densities of the plates in a plate reader at the correct absorbance:
  20. 20. ELISA Results  The status of a sample are evaluated by the sample to positive ratio (S/P ratio):  Sample mean - negative control mean positive control mean - negative control mean  (mean of optical absorbance) With the IDEXX kit S/P ratios of greater than 0.5 are considered positive With the IDEXX kit S/P ratios of greater than 0.5 are considered positive
  21. 21. ELISA Results  Example:    Sample mean= 0.820 Negative control mean=0.053 Positive control mean=0.563 ELISA titer =(1.642xlog10 SP)+3.568 Values are relatively quantitative: a higher value indicates more antibody.
  22. 22. ELISA Laboratory  Materials Needed  ELISA plate  Record Sheet  Test samples  Dilution Tubes  Pipets and tips
  23. 23. Materials Needed  The materials for your kit        ELISA plate Positive control Negative control Dilution Buffer (already in dilution tubes) Conjugate (secondary antibody) TMB Substrate Stop solution
  24. 24. ELISA Laboratory 1     Label dilution tubes Add 1ml of diluent to dilution tubes (done) Add 2μl of test serum to a dilution tube Do NOT dilute controls
  25. 25. ELISA Laboratory 2 1 2 3 4 5 6 7 8 9 10 11 12 A + - + 1 2 3 4 5 6 7 8 9 B - + - C D E F G H
  26. 26. Add 100μl of diluted test serum to the plate according to your record sheet 1 2 3 4 5 6 7 8 9 10 11 12 A + - + 1 2 3 4 5 6 7 8 9 B - + - C D E F G H Incubate for 30 minutes
  27. 27. ELISA Laboratory 3  Wash with 350 μl distilled water (three times)  Add 100 μl of conjugate to test wells on your plate  Incubate for 30 minutes
  28. 28. ELISA Laboratory 4  Wash with distilled water (3 times)  Add 100 μl of TMB substrate to each well  Incubate for 15 minutes  Add 100 μl of stop solution to each well  Read results
  29. 29. Interpretation of Results  Negative control = 0.150 or less  The difference between the positive and negative control means must be greater than 0.075  Example: if negative control mean = 0.100, the positive control mean must be 0.176 or greater
  30. 30. Calculation of Results  Average the 2 negative control wells  Average the 2 positive control wells  Average 2 wells for each sample
  31. 31. Calculation of Results  Example:    Sample mean= 0.820 Negative control mean=0.053 Positive control mean=0.563 S/P ratios of greater than 0.5 are considered positive (Positive values will be different for each kit)
  32. 32. Sensitivity  ELISAs are one of the most sensitive immunoassays available. The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular characteristics of the antibody – antigen interaction. In addition, some substrates such as those yielding enhanced chemiluminescent or fluorescent signal, can be used to improve results. As mentioned earlier, indirect detection will produce higher levels of signal and should therefore be more sensitive.
  33. 33. If the negative controls are giving positive results:  Contamination of the substrate solution, enzyme-labelled antibody, control themselves.  Inadequate rinsing of plates.  Inadequate blocking of plates.
  34. 34.  If no colour has developed for the positive controls or for the samples: a. Check all reagents for dating and storage conditions. b. Microwell plates not coated properly. c. Reagents applied in wrong order or step omitted. d. Enzyme conjugate defective or inhibited by contaminant.
  35. 35.  If very little colour has developed for positive controls and the test samples: a. Check the dilution of the enzyme labelled antibody. b. The concentration of the substrate. c. Wash buffer not adequately drained after every wash step. d. Inadequate incubation times. e. Enzyme conjugate defective or inhibited by contaminant, Substrate defective or contaminated, f. Micro well plates poorly coated.
  36. 36.  If colour has developed for the test samples but not the positive controls:  Check the source of positive controls, their expiry date and storage.  If the colour can be seen, but the absorbance is not high as expected, check the wave length.