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ELISA

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Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal.

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ELISA

  1. 1. Isfahan University of Medical Science, School of Pharmacy Immunochemistry ELISA Department of Clinical Biochemistry Created by:June 26, 2012 A.N. Emami115 Total slides : Razavi 1
  2. 2. Enzyme LinkedImmunosurbent Assay ELISA
  3. 3. Immunochemistry ELISA Outlines  What is ELISA  History  Application  Mechanism & Reagents  Types of ELISA  Methods  Quality control  HIV testJune 26, 2012 Total slides : 115 3
  4. 4. What is ELISA? ELISA
  5. 5. Immunochemistry ELISA  Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal.June 26, 2012 Total slides : 115 5
  6. 6. Immunochemistry ELISA  Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity.June 26, 2012 Total slides : 115 6
  7. 7. Immunochemistry ELISA Enzyme-linked immunosorbent assay Name suggests three components  Antibody  Allows for specific detection of analyte of interest  Solid phase (sorbent)  Allows one to wash away all the material that is not specifically captured  Enzymatic amplification  Allows you to turn a little capture into a visible color change that can be quantified using an absorbance plate readerJune 26, 2012 Total slides : 115 7
  8. 8. Histor yELISA
  9. 9. Immunochemistry ELISA  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. In radioimmunoassay, the radioactivity provides the signal which indicates whether a specific antigen or antibody is present in the sample. Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.June 26, 2012 Total slides : 115 9
  10. 10. Immunochemistry ELISA  The American physicist Rosalyn S.Yalow (born 1921) made her most outstanding contribution to modern medicine in developing radioimmunoassay (RIA), for which she received a Nobel Prize in physiology/medicine (1977).June 26, 2012 Total slides : 115 10
  11. 11. Immunochemistry ELISA Dr. Solomon A. Berson, M.D. 45 (1919-1972) and Dr. Rosalyn Sussman Yalow (1921- ) co-developed the radioimmunoassay (RIA) in 1959.June 26, 2012 Total slides : 115 11
  12. 12. Immunochemistry ELISA  Because radioactivity poses a health threat, a safer alternative was sought. A suitable alternative to radioimmunoassay would substitute a non-radioactive signal in place of the radioactive signal. When certain enzymes (such as peroxidase) react with appropriate substrates (such as ABTS or 3,3’,5,5’- Tetramethylbenzidine), they can result in changes in color, which can be used as a signal. However, the signal has to be associated with the presence of antibody or antigen, which is why the enzyme has to be linked to an appropriate antibody. This linking process was independently developed by Stratis Avrameas and G.B. Pierce.June 26, 2012 Total slides : 115 12
  13. 13. Immunochemistry ELISA  Since it is necessary to remove any unbound antibody or antigen by washing, the antibody or antigen has to be fixed to the surface of the container, i.e. the immunosorbent has to be prepared. A technique to accomplish this was published by Wide and Porath in 1966  In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden, as well as Anton Schuurs and Bauke van Weemen in The Netherlands, independently published papers which synthesized this knowledge into methods to perform EIA/ELISAJune 26, 2012 Total slides : 115 13
  14. 14. Immunochemistry ELISA  Eva Engvall is one of the scientists who invented the ELISA test in 1971. She is shown at her home near Buellton, Calif. Engvall is currently a professor at the Burnham Institute in La Jolla, Calif.June 26, 2012 Total slides : 115 14
  15. 15. ApplicationsELISA
  16. 16. Immunochemistry ELISA  Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test or West Nile Virus) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs.June 26, 2012 Total slides : 115 16
  17. 17. Immunochemistry ELISA What is it used for?  Measure antibody levels (allergies, vaccines)  Detect viruses (hepatitis, HIV, venereal diseases)  Detect hormonal changes (pregnancy)  Detect circulatory inflammatory markers (cytokines)June 26, 2012 Total slides : 115 17
  18. 18. Immunochemistry ELISA Advantages of ELISA  Sensitive: nanogram levels or lower  Reproducible  Minimal reagents  Qualitative & Quantitative  Qualitative  Eg. HIV testing  quantitative assays  Eg. Drug Monitoring  Wells can be coated with Antigens OR Antibodies  Suitable for automation high speed  NO radiation hazardsJune 26, 2012 Total slides : 115 18
  19. 19. Immunochemistry ELISA Sensitivity Relative sensitivities of tests (approx) Usual operating range [Ab] or [Ag] precipitation immunoelectrophoresis 10 µg/ml - 1 mg/ml double/radial diffusion immunofluorescence 0.1 - 10 µg/ml ELISA (colour) 0.1 - 10 ng/ml (chemiluminescence) 0.01 - 10 ng/ml radioimmunoassay 0.01 - 10 ng/mlJune 26, 2012 Total slides : 115 19
  20. 20. Immunochemistry ELISA Enzymes with Chromogenic Substrates  High molar extinction coefficient (i.e., strong color change)  Strong binding between enzyme and substrate (low KM)  Linear relationship between color intensity and [enzyme]June 26, 2012 Total slides : 115 20
  21. 21. Immunochemistry ELISA Limitations  Results may not be absolute  False positive possible  Falsenegative possible  Antibody must be availableJune 26, 2012 Total slides : 115 21
  22. 22. Immunochemistry ELISA Materials needed  Testing sample  Antibody (1st, 2nd) / Antigen  Polystyrene microtiter plate  Blocking buffer  Washing buffer  Substrate  EnzymeJune 26, 2012 Total slides : 115 22
  23. 23. Mechanism & Reagents ELISA
  24. 24. Immunochemistry ELISA Mechanism  The basic mechanism involved in these test utilizes absorption of antigen to a solid surface which is placed in contact with a dilution of serum. The reaction which detects and quantifies the binding of antibody uses an antibody labeled with an enzyme followed by the addition of an appropriate substrate on which the enzyme can act to produce a colour reaction. Two distinct test mechanisms are “noncompetitive " and "competitive".June 26, 2012 Total slides : 115 24
  25. 25. Immunochemistry ELISA Reagents  Antigens may be produced using any of the standard techniques. They may be purified by precipitation and ultra- centrifugation or by column chromatography before being absorbed onto the surface. An alternative method which allows the use of relatively impure antigens is to bind specific antibody to the surface and allow it to absorb the antigen from the preparation. Since the latter approach usually adds an extra stage to the test this is not the preferred approach for commercial test kits. In most cases the antigen is delivered pre-absorbed onto plates, though they need to be carefully dried and packed to maintain their stability at refrigerator temperature for a reasonable period.June 26, 2012 Total slides : 115 25
  26. 26. Immunochemistry ELISA Reagents  The labeled anti-globulin used in the standard test is included in the test kits as is the appropriate substrates and stop-solutions for use with it. All of these reagents may be purchased separately from a number of sources however their titration to a standard level of activity for a specific purpose is rarely worthwhile for a commercial laboratory carrying out routine serology.June 26, 2012 Total slides : 115 26
  27. 27. Immunochemistry ELISA Reagents  Finally, and most importantly, standard negative sera and positive sera of known potency are required. These are even more important than those used in other serological tests since it is common practice to express the results of the test sera by comparing them with those of the control sera (serum-to-positive or serum-to-negative ratios). The objective of this is to remove some of the variation due to operator, environment and plate effects.June 26, 2012 Total slides : 115 27
  28. 28. Types of ELISA ELISA
  29. 29. Immunochemistry ELISA  Noncompetitive binding assay or Sandwich method  Antigen measuring system [Titrewells coated with antibodies ; Enzyme labelled antibodies]  Antibody measuring system [Titrewells coated with antigens ; Enzyme labelled antiantibodies]  Competitive binding assay  Titrewells coated with antibodies ; Enzyme labelled antigensJune 26, 2012 Total slides : 115 29
  30. 30. Immunochemistry ELISA Noncompetitive or Sandwich Assay  Antigen measuring system  Titre wells coated with suitable antibody  Add patient sample containing the antigen  Incubate: till antigen antibody reaction is complete  Wash remove unbound antigen  Add Antibody labelled with Enzyme  Incubate till antigen binds labelled antibody  Wash  remove unbound labelled antibody  Add substrate ; incubate  Enzyme + Substrate  Product  measure colour  Colour proportional to antigen in patient sampleJune 26, 2012 Total slides : 115 30
  31. 31. Immunochemistry ELISA Noncompetitive or Sandwich Assay  Antibody measuring system  Titre wells coated with suitable antigen  Add patient sample containing the antibody  Incubate: till antigen antibody reaction is complete  Wash remove unbound antibody  Add Antiantibody labelled with Enzyme  Incubate till labelled antiantibodies binds antigen- antibody complex  Wash  remove unbound labelled antiantibody  Add substrate ; incubate  Enzyme + Substrate  Product  measure colour  Colour proportional to antibody in patient sampleJune 26, 2012 Total slides : 115 31
  32. 32. Immunochemistry ELISA Competitive binding assay  Titrewells coated with antibodies  Known quantities of patient sample containing antigen + antigen labelled with enzyme  Incubate: till antigen antibody reaction is complete  Wash remove unbound antigens  Add substrate ; incubate  Enzyme + Substrate  Product  measure colour  Colour inversely related to antigen in patient sampleJune 26, 2012 Total slides : 115 32
  33. 33. Immunochemistry ELISA Enzyme labels  Enzyme labels should have high specific reactivity  Should be easily coupled to ligands & the labelled complex must be stable  The reactivity should be retained after linking of the enzyme to the antigen/antibody  The chosen enzymes should not be normally present in the patient samples  Examples of enzyme labels  Horse radish peroxidase, Alkaline phosphatase, Glucose oxidaseJune 26, 2012 Total slides : 115 33
  34. 34. Immunochemistry ELISA Enzyme-Mediated Detection Enzyme Horseradish Peroxidase Substrate Abbrev. Color Diaminobenzidine DAB Brown Enzyme Alkaline Phosphatase (AP) Substrate Abbrev. Color BromochloroindolylphosphateNitr BCIP (AP substrate) Purple o Blue Tetrazolium NBT (Enhance color)June 26, 2012 Total slides : 115 34
  35. 35. MethodsELISA
  36. 36. Immunochemistry ELISA BASIC FORMAT Solid phase = 96 / 384-well microplateJune 26, 2012 Total slides : 115 36
  37. 37. Immunochemistry ELISA 96 Well 350µL Polypropylene PlatesJune 26, 2012 Total slides : 115 37
  38. 38. Immunochemistry ELISA 96 Well 500µL Polypropylene PlatesJune 26, 2012 Total slides : 115 38
  39. 39. Immunochemistry ELISA 96 Well 600µL Polypropylene PlatesJune 26, 2012 Total slides : 115 39
  40. 40. Immunochemistry ELISA 96 Well 1mL Polypropylene PlatesJune 26, 2012 Total slides : 115 40
  41. 41. Immunochemistry ELISA 96 Well 2mL Polypropylene PlatesJune 26, 2012 Total slides : 115 41
  42. 42. Immunochemistry ELISA 384 Well Polystyrene Assay PlatesJune 26, 2012 Total slides : 115 42
  43. 43. Immunochemistry ELISA 384 Well Polypropylene Microtiter PlatesJune 26, 2012 Total slides : 115 43
  44. 44. Immunochemistry ELISA 1. Coat solid phase with antigen when analysing antibody antibody when analysing antigen Analyte = antibody Analyte = antigen Incubate, washJune 26, 2012 Total slides : 115 44
  45. 45. Immunochemistry ELISA 2. Block free binding sites. Incubate. Wash. Analyte = antibody Analyte = antigenJune 26, 2012 Total slides : 115 45
  46. 46. Immunochemistry ELISA 3. Add sample. Incubate. Wash Analyte = antibody Analyte = antigenJune 26, 2012 Total slides : 115 46
  47. 47. Immunochemistry ELISA 4. Add conjugate. Incubate. Wash. E E E E Analyte = antibody Analyte = antigenJune 26, 2012 Total slides : 115 47
  48. 48. Immunochemistry ELISA 5. Add substrate 6. Incubate, stop, measure colour change ENZYME Colourless OD CONCENTRATIONJune 26, 2012 Total slides : 115 48
  49. 49. Immunochemistry ELISA COATING THE PLATE  Dilute the Ag in PBS containing 0.02% NaN3 (5-10 ug/ml) and despense 50 ul into each well of 96-well microtiter plate using a multipipette/dispenser. (Micro plates vary in their ability to bind Ag. So each resercher should test for the plates of choice for their specific Ag). Seal plates with adhesive plate sealer and incubate overnight at 4° C or 2 to 3 h at 37° C (coated plates can be prepared and stored in the refrigerator for several weeks).June 26, 2012 Total slides : 115 49
  50. 50. Immunochemistry ELISA WASHING THE PLATE Wash the plate three time with PBS using the Nunc- Immuno wash device (this device simultaneously delivers and aspirates fluid)June 26, 2012 Total slides : 115 50
  51. 51. Immunochemistry ELISA BLOCKING THE PLATE block any residual binding capacity by adding 50 ul of a blocking buffer (PBS containing 5% BSA, 0.02% NaN3) to each well. Incubate the plate for 1 h at room temperature.June 26, 2012 Total slides : 115 51
  52. 52. Immunochemistry ELISA SAMPLE  Dilute in buffer-Tween 20  Include known positive and negative samples  Standards……. recombinant protein International standard antibody Double-dilute from 10 pg/ml - 10 ng/ml  100 µl/well, duplicates  2 - 4 hours 20/37oC or overnight 4oC  3 - 6 washes with buffer-Tween 20June 26, 2012 Total slides : 115 52
  53. 53. Immunochemistry ELISA CONJUGATE  For assays of (human) antibodies use anti- (human) Ig-enzyme  For assays of antigens use enzyme-conjugated antibodyJune 26, 2012 Total slides : 115 53
  54. 54. Immunochemistry ELISA Conjugating antibodies to Alkaline Phosphatase  Centrifuge 5 mg of alkaline phosphatase suspension (supplied as a suspension in 65% (NH4)SO4) in a microfuge for 5 to 10 min. resuspend the enzyme pellet in a total volume of 1 mg of the antibody to be labeled.  Dialyzed overnight against 0.1 M sodium phosphate buffer, pH 6.8 to remove any contaminating free amino groups.  In a fume hood, slowly add 50 ug 1% glutaraldehyde solution while stirring for 5 min. Incubate at room temperatyre for 2 to 3 h, add 100 ul of 1 M ethanolamine, pH 7, and then incubate for additional 2 h at room temperature.  Dialyze overnight against PBS and remove any insoluble materials by centrifugation at 40,000 x g for 30 min.  Store the conjugate (supernatant) in the presence of 50% glycerole,1mM MgCl2 1mM ZnCl2, and 0.02% NaN3 at 4ºCJune 26, 2012 Total slides : 115 54
  55. 55. Immunochemistry ELISA AMPLIFICATION E Directly conjugated developing antibody may give weak signalJune 26, 2012 Total slides : 115 55
  56. 56. Immunochemistry ELISA amplify with E E unlabelled (rabbit) anti-(human) Ig followed by anti-(rabbit) Ig-enzymeJune 26, 2012 Total slides : 115 56
  57. 57. Immunochemistry ELISA or E S E-S B S-E Biotin-labelled anti-Ig followed by streptavidin-enzymeJune 26, 2012 Total slides : 115 57
  58. 58. Immunochemistry ELISA SUBSTRATES See Sigma catalogue for list of conjugates and substrates Orthophenylene diamine Tetramethyl hydrochloride (OPD) benzidine (TMP) Horse radish peroxidase (HRP) Orange, 490 nm Yellow, 450 nm SpectrophotometerJune 26, 2012 Total slides : 115 58
  59. 59. Immunochemistry ELISA Paranitrophenyl phosphate (PNP) Methyl umbelliferol phosphate Alkaline phosphatase Yellow, 405 nm Methyl umbelliferone Spectrophotometer 365 nm 445 nm FluorimeterJune 26, 2012 Total slides : 115 59
  60. 60. Immunochemistry ELISAJune 26, 2012 Total slides : 115 60
  61. 61. Immunochemistry ELISAJune 26, 2012 Total slides : 115 61
  62. 62. Immunochemistry ELISAJune 26, 2012 Total slides : 115 62
  63. 63. Immunochemistry ELISA  Micro-titre plate with a standard Elisa test seen from below. The first 2 wells at top right are the negative controls. The following 2 are positive controls. The remaining sera are field test sera. Usually at least 2 wells with a known laboratory positive control are included on each plate.June 26, 2012 Total slides : 115 63
  64. 64. Immunochemistry ELISA INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES • screening hybridoma supernatants • detecting clinically important antibodies - autoantibodies - anti-pathogens - anti-allergens 1. AntigenJune 26, 2012 Total slides : 115 64
  65. 65. Immunochemistry ELISA INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES 2. Sample (human) antibody 1. AntigenJune 26, 2012 Total slides : 115 65
  66. 66. Immunochemistry ELISA INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES E E E 3. Anti-(human) Ig-enzyme 2. Sample (human) antibody 1. AntigenJune 26, 2012 Total slides : 115 66
  67. 67. Immunochemistry ELISA INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES 4. Substrate E E E 3. Anti-(human) Ig-enzyme 2. Sample (human) antibody 1. AntigenJune 26, 2012 Total slides : 115 67
  68. 68. Immunochemistry ELISA ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES Useful when pure antigen not available or antigen coats poorly 1. Specific antibodyJune 26, 2012 Total slides : 115 68
  69. 69. Immunochemistry ELISA ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES 2. Impure antigen eg tissue homogenate 1. Specific antibodyJune 26, 2012 Total slides : 115 69
  70. 70. Immunochemistry ELISA ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES 3. Wash → pure antigen 2. Impure antigen 1. Specific antibodyJune 26, 2012 Total slides : 115 70
  71. 71. Immunochemistry ELISA ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES 4. Sample (human antibody) 3. Wash → pure antigen 2. Impure antigen 1. Specific antibodyJune 26, 2012 Total slides : 115 71
  72. 72. Immunochemistry ELISA ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES 5. Anti-human Ig-enzyme E E 4. Sample (human antibody) 3. Wash → pure antigen 2. Impure antigen 1. Specific antibodyJune 26, 2012 Total slides : 115 72
  73. 73. Immunochemistry ELISA ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES 6. Substrate 5. Anti-human Ig-enzyme 4. Sample (human antibody) 3. Wash → pure antigen 2. Impure antigen 1. Specific antibodyJune 26, 2012 Total slides : 115 73
  74. 74. Immunochemistry ELISA ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS eg. hormones drugs tumour antigens cytokines 1. Anti-analyteJune 26, 2012 Total slides : 115 74
  75. 75. Immunochemistry ELISA ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS 2. Sample 1. Anti-analyteJune 26, 2012 Total slides : 115 75
  76. 76. Immunochemistry ELISA ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS E E 3. Anti-analyte-enzyme 2. Sample 1. Anti-analyteJune 26, 2012 Total slides : 115 76
  77. 77. Immunochemistry ELISA ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS 3. Or: E E anti-analyte-biotin S S followed by E-S B S-E E-S B S-E streptavidin-enzyme 2. Sample 1. Anti-analyteJune 26, 2012 Total slides : 115 77
  78. 78. Immunochemistry ELISA ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS 4. Substrate 3. Or: anti-analyte-biotin followed by streptavidin- enzyme 2. Sample 1. Anti-analyteJune 26, 2012 Total slides : 115 78
  79. 79. Immunochemistry ELISA COMPETITION ELISA TO DETECT ANTIGENS (antigen-coated plate) 1. AnalyteJune 26, 2012 Total slides : 115 79
  80. 80. Immunochemistry ELISA COMPETITION ELISA TO DETECT ANTIGENS Low [analyte] High [analyte] E E E E E E E E E 2. Anti-analyte-E E + sample 1. AnalyteJune 26, 2012 Total slides : 115 80
  81. 81. Immunochemistry ELISA COMPETITION ELISA TO DETECT ANTIGENS Low [analyte] High [analyte] 3. Wash E E E 2. Anti-analyte-E E + sample 1. AnalyteJune 26, 2012 Total slides : 115 81
  82. 82. Immunochemistry ELISA COMPETITION ELISA TO DETECT ANTIGENS Low [analyte] High [analyte] 4. Substrate 3. Wash 2. Anti-analyte-E + sample 1. AnalyteJune 26, 2012 Total slides : 115 82
  83. 83. Immunochemistry ELISA COMPETITION ELISA TO DETECT ANTIGENS (antibody-coated plate) 1. Anti-analyteJune 26, 2012 Total slides : 115 83
  84. 84. Immunochemistry ELISA COMPETITION ELISA TO DETECT ANTIGENS Low [analyte] High [analyte] 2. Analyte-E + sample 1. Anti-analyteJune 26, 2012 Total slides : 115 84
  85. 85. Immunochemistry ELISA COMPETITION ELISA TO DETECT ANTIGENS Low [analyte] High [analyte] 3. Wash 2. Analyte-E E E E E E + sample E E 1. Anti-analyteJune 26, 2012 Total slides : 115 85
  86. 86. Immunochemistry ELISA COMPETITION ELISA TO DETECT ANTIGENS Low [analyte] High [analyte] 4. Substrate 3. Wash 2. Analyte-E + sample 1. AnalyteJune 26, 2012 Total slides : 115 86
  87. 87. Immunochemistry ELISA CYTOKINES Type 1 Type 2 IL2 HEALTH IL4 IL12 IL5 IFNγ IL6 TNFα IL10 RHEUMATOID ARTHRITIS CANCER MULTIPLE SCLEROSIS VIRUSES DIABETES MYCOBACTERIA 2 1 ASTHMA, ALLERGY LUPUS 1 2June 26, 2012 Total slides : 115 87
  88. 88. Immunochemistry ELISA Detection of cytokines by ELISA  Plasma or supernatant of cultured mononuclear cells  Coat plate with anti-CK (Pharmingen) 0.5 µg/ml in bicarbonate buffer, 4o overnight  Wash x 2 with PBS-T  Block with PBS + 10% FCS, 2 hours RT  Wash x2 with PBS-TJune 26, 2012 Total slides : 115 88
  89. 89. Immunochemistry ELISA  Add standards (recombinant CK 10 pg-10 ng /ml), controls and samples  4o overnight  Wash x3 with PBS-T  Add biotinylated anti-CK (Pharmingen) 0.5 µg/ml  RT 60 minutes  Wash x6 with PBS-T  Add streptavidin-peroxidase  RT 30 minJune 26, 2012 Total slides : 115 89
  90. 90. Immunochemistry ELISA  Wash x8 with PBS-T 2  Add OPD substrate  15 min RT, dark 1  Stop with N H2SO4  Read A490 0 [rCK]June 26, 2012 Total slides : 115 90
  91. 91. HIV TestELISA
  92. 92. Immunochemistry ELISA HIV  The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.June 26, 2012 Total slides : 115 92
  93. 93. Immunochemistry ELISA HIV  An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the first and most basic test to determine if an individual is positive for a selected pathogen, such as HIV. The test is performed in a 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.June 26, 2012 Total slides : 115 93
  94. 94. Immunochemistry ELISA An ELISA plateJune 26, 2012 Total slides : 115 94
  95. 95. Immunochemistry ELISA The ELISA Method  Partially purified, inactivated HIV antigens pre- coated onto an ELISA plate  Patient serum which contains antibodies. If the patient is HIV+, then this serum will contain antibodies to HIV, and those antibodies will bind to the HIV antigens on the plate.  Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies.  Chromogen or substrate which changes color when cleaved by the enzyme attached to the second antibody.June 26, 2012 Total slides : 115 95
  96. 96. Immunochemistry ELISA  Negative ELISA  PositiveELISA Test TestJune 26, 2012 Total slides : 115 96
  97. 97. Immunochemistry ELISA ELISA data from three patients Positive Control Negative Control Patient A Patient B Patient C Assay Control 1.689 0.153 O.055 0.412 1.999 0.123  Above is ELISA data from three patients. Numbers are expressed as optical density at 450 nm. The cutoff value indicating a positive result is 0.500. Optical densities of 0.300 to 0.499 are indeterminate and need to be retested. Values below 0.300 are considered to be negative. In most cases, a patient will be retested if the serum gives a positive result. If the ELISA retests are positive, the patient will then be retested by western blotting analysis.June 26, 2012 Total slides : 115 97
  98. 98. Immunochemistry ELISA Sources of Error for HIV EIA TestsJune 26, 2012 Total slides : 115 98
  99. 99. Immunochemistry ELISA Sources of False Negative Results  Early Seroconverters  the window between infection and an antibody response to the virus  Operator Error  Fail to add serum or reagent to the correct well  Reagent diluted in wrong diluvent or in wrong dilution  Equipment ErrorJune 26, 2012 Total slides : 115 99
  100. 100. Immunochemistry ELISA Source of False Positive Results  MULTIPLE PREGNANCY  MULTIPLE TRANSFUSION  AUTO IMMUNE DISORDER  CHRONIC HEPATITIS,  CHRONIC ALCOHOLIC  HBV VACCINATION  ANTIBODY TO POLYSTERENEJune 26, 2012 Total slides : 115 100
  101. 101. Immunochemistry ELISA Trouble shooting EIA  Kit integrity  Controls (kit and in-house)  Equipment  Cross contamination  Sample quality  Personnel training  Correct validation and interpretation of resultsJune 26, 2012 Total slides : 115 101
  102. 102. Immunochemistry ELISA Kit integrity  Was cold chain maintained during kit transport?  Has the kit expired?  Has the kit been stored properly in your lab?June 26, 2012 Total slides : 115 102
  103. 103. Immunochemistry ELISA Controls  Kit Controls should be run on each EIA plate.  Indicates whether the kit components are functioning.  Used to validate EIA run, calculate cut off  In-house controls  Kit controls are designed to be quite robust and do not reflect subtle changes in testing.  In-house controls should be calibrated to test as a low positive (above cut-off, below maximum)June 26, 2012 Total slides : 115 103
  104. 104. Immunochemistry ELISA Equipment : Pipettes  Single and Multi channel Pipettes should be calibrated on a monthly basis.June 26, 2012 Total slides : 115 104
  105. 105. Immunochemistry ELISA Equipment : Microplate Washers  Daily  Prime the washer with wash solution before running sample plates  Set the washer to wash the recommended number of times (with correct volume)  Check for accurate dispensing and complete aspiration in each plate well, if not clean the washer head  Listen for changes in the sound the washer makes, this can indicate a vacuum leak  At the end of the day prime the washer with DI waterJune 26, 2012 Total slides : 115 105
  106. 106. Immunochemistry ELISA Equipment : Microplate Washers  Weekly  If a washer is not used during the week rinse it out with DI water to reduce microbial growth.  Monthly  Run a 10% solution of ethanol through the washer to disinfect. This can also be done if the washer exhibits signs of contamination (high background).  Thoroughly rinse the washer after alcohol is used.June 26, 2012 Total slides : 115 106
  107. 107. Immunochemistry ELISA Equipment : Micro-plate Reader  Daily  Each time a reader is turned on it runs a self test, it will then report any errors.  Weekly  Run a control plate weekly. Variations in positive or negative specimens could be a sign of a bad diode or a spill on a diode.June 26, 2012 Total slides : 115 107
  108. 108. Immunochemistry ELISA Cross contamination  Can be caused by:  Reusing pipette tips (contaminated with + plasma)  Splashes from one well to another during removal of plate coversJune 26, 2012 Total slides : 115 108
  109. 109. Immunochemistry ELISA Sample Quality  Properly collected (no haemolysis)  Transport conditions  Storage conditions  Number of freeze/thaw cycles  Age of sampleJune 26, 2012 Total slides : 115 109
  110. 110. Immunochemistry ELISA Validation and Interpretation of Results  Positive and Negative controls must fall within a certain range.  Controls are used to calculate a cut-off.  Samples below cut-off are negative, those above are positiveJune 26, 2012 Total slides : 115 110
  111. 111. Immunochemistry ELISA Test your self 1. What does ELISA measure? 2. What if serum were left out? 3. Omission of the wash step 4. Which patient is HIV positive? Use these problems to test your understanding of this topic.June 26, 2012 Total slides : 115 111
  112. 112. Immunochemistry ELISA Problem 1 What is the ELISA test intended to measure? A Antibody to HIV only B  Antigen to HIV only C  Presence of free, circulating virus in the patient D  Antibodies directed against HLA moleculesJune 26, 2012 Total slides : 115 112
  113. 113. Immunochemistry ELISA Problem 2  What would happen if serum were omitted from the ELISA, but all other steps remained the same and were performed properly? A Anti-human Ig-conjugate would not bind and be washed away. B Anti-human Ig-conjugate would bind non-specifically to the ELISA plate. C The O.D. values would be nearly the same as the assay control. D Both A and C.June 26, 2012 Total slides : 115 113
  114. 114. Immunochemistry ELISA Problem 3  What would happen if the anti-human Ig-conjugate were not washed free of the well before the substrate was added? A The ELISA would not develop when the substrate was added. B The ELISA would develop normally. C All wells would show uniform over development due to unbound and excess anti-human Ig enzyme conjugate. D Both A and B.June 26, 2012 Total slides : 115 114
  115. 115. Immunochemistry ELISA Problem 4  From the ELISA data, which patient is seropositive for HIV? Positive Control Negative Control Patient A Patient B Patient C Assay Control 1.689 0.153 O.055 0.412 1.999 0.123 A Patient A C Patients A and B B Patient B D Patient CJune 26, 2012 Total slides : 115 115
  116. 116. Thank you Questions
  117. 117. Immunochemistry ELISA A Antibody to HIV only Correct! Antibody from patient serum made in response to various HIV proteins binds to antigen.June 26, 2012 Total slides : 115 117
  118. 118. Immunochemistry ELISA B Antigen to HIV only Incorrect! HIV antigens are already coated onto the plate. Antibody from the patient binds to the HIV antigens.June 26, 2012 Total slides : 115 118
  119. 119. Immunochemistry ELISA C Presence of free, circulating virus in the patient Incorrect! Serum antibody is detected, not circulating virus in the serum.June 26, 2012 Total slides : 115 119
  120. 120. Immunochemistry ELISA D Antibodies directed against HLA molecules Incorrect! Although there may be anti-HLA antibodies present in the serum, the test is not designed to measure them.June 26, 2012 Total slides : 115 120
  121. 121. Immunochemistry ELISA A Anti-human Ig-conjugate would not bind and be washed away. Correct!  but there is another correct answerJune 26, 2012 Total slides : 115 121
  122. 122. Immunochemistry ELISA B Anti-human Ig-conjugate would bind non- specifically to the ELISA plate. Incorrect! The anti-human Ig conjugate is specific for human Ig and has very little nonspecific binding activity.June 26, 2012 Total slides : 115 122
  123. 123. Immunochemistry ELISA C The O.D. values would be nearly the same as the assay control. Correct! but there is another correct answer.June 26, 2012 Total slides : 115 123
  124. 124. Immunochemistry ELISA D Both A and C. Correct! Since no serum was added, the anti-human Ig conjugate would not be able to bind to human Ig and the O.D. values would be the same as the assay control.June 26, 2012 Total slides : 115 124
  125. 125. Immunochemistry ELISA A The ELISA would not develop when the substrate was added. Incorrect! The substrate would overdevelop because an excess of enzyme coupled to the anti- human Ig was present.June 26, 2012 Total slides : 115 125
  126. 126. Immunochemistry ELISA B The ELISA would develop normally. Incorrect! The substrate would overdevelop because an excess of enzyme coupled to the anti- human Ig was present.June 26, 2012 Total slides : 115 126
  127. 127. Immunochemistry ELISA C All wells would show uniform over development due to unbound and excess anti- human Ig enzyme conjugate. Correct! Since the enzyme which acts on the substrate is in excess, it would turn all the wells a uniform color whether they were truly positive or not.June 26, 2012 Total slides : 115 127
  128. 128. Immunochemistry ELISA D Both A and B. Incorrect! The substrate would overdevelop because an excess of enzyme coupled to the anti- human Ig was present.June 26, 2012 Total slides : 115 128
  129. 129. Immunochemistry ELISA A Patient A Incorrect! Patient A has an O.D. of 0.055 and would be considered negative.June 26, 2012 Total slides : 115 129
  130. 130. Immunochemistry ELISA B Patient B Incorrect! Patient B would be considered indeterminate and would need to be retested.June 26, 2012 Total slides : 115 130
  131. 131. Immunochemistry ELISA C Patients B and C Incorrect! Patient A has an O.D. of 0.055 and would be considered negative and patient B would be considered indeterminate and would need to be retested.June 26, 2012 Total slides : 115 131
  132. 132. Immunochemistry ELISA D Patient C Correct! Serum from patient C showed an O.D. of 1.999 which is even higher than the positive control O.D. for the assay.June 26, 2012 Total slides : 115 132
  133. 133. Immunochemistry ELISAJune 26, 2012 Total slides : 115 133
  134. 134. Immunochemistry ELISAJune 26, 2012 Total slides : 115 134
  135. 135. Immunochemistry ELISA Extinction coefficient  The extinction coefficient for a particular substance is a measure of how well it scatters and absorbs electromagnetic radiation (EM waves). If the EM wave can pass through very easily, the material has a low extinction coefficient. Conversely, if the radiation hardly penetrates the material, but rather quickly becomes "extinct" within it, the extinction coefficient is high.  A material can behave differently for different wavelengths of electromagnetic radiation. Glass is transparent to visible light, but many types of glass are opaque to ultra-violet wavelengths. In general, the extinction coefficient for any material is a function of the incident wavelength. The extinction coefficient is used widely in ultraviolet-visible spectroscopy.June 26, 2012 Total slides : 115 135

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