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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 ...

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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  • Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation.
  • After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.
  • Dr. Solomon A. Berson, M.D. '45 (1919-1972) and Dr. Rosalyn Sussman Yalow (1921- ) co-developed the radioimmunoassay (RIA) in 1959. The radioimmunoassay technique chemically analyzes human blood and tissue. A simple and relatively inexpensive test that requires just a tiny sample of blood or tissue, RIA revolutionized diagnoses. Among other uses, RIA can screen blood, detect drug use and identify people with high blood pressure or diabetes. For inventing RIA, Yalow won the Nobel Prize in Medicine in 1977 (Yalow accepted for Berson, who died in 1972). Yalow and Berson did not patent the RIA; instead they allowed the common use of RIA to benefit human health. These awards honor the memory of Dr. Berson and his co-development of RIA, a discovery of inestimable value in endocrinology. Despite world acclaim, he remained the true physician - humble, dedicated, productive. The Alumni Achievement Awards have been presented annually since 1954 (Dr. Berson himself was so honored in 1969), and were named in Dr. Berson's memory in 1979.
  • 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 . Beer’s Law states that molar absorptivity is constant (and the absorbance is proportional to concentration) for a given substance dissolved in a given solute and measured at a given wavelength.2 For this reason, molar absorptivities are called molar absorption coefficients or molar extinction coefficients . Because transmittance and absorbance are unitless, the units for molar absorptivity must cancel with units of measure in concentration and light path. Therefore, molar absorptivities have units of M-1 cm-1. Standard laboratory spectrophotometers are fitted for use with 1 cm-width sample cuvettes; hence, the path length is generally assumed to be equal to one and the term is dropped altogether in most calculations. Aλ = ε c L = ε c when L = 1 cm The molar absorption coefficient of a peptide or protein is related to its tryptophan (W), tyrosine (Y) and cysteine (C) amino acid composition. At 280 nm, this value is approximated by the weighted sum of the 280 nm molar absorption coefficients of these three constituent amino acids, as described in the following equation:3,4 ε = ( n W×5500) + ( n Y×1490) + ( n C×125)
  • referance:Basic methods in antibody production and characterization. Gary C. Howard , Dwlia R. Bethell , 2001
  • T=Tween
  • In an ELISA test, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens have been attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to human antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and negative result.
  • False negatives can occur during the window between infection and an antibody response to the virus (seroconversion).
  • Fuls positive results: It is entirely possible that an individual not infected with HIV has antibodies which may give a positive result in the HIV ELISA. This is called a false positive. One reason for this is that people (especially women who have had multiple pregnancies) may possess antibodies directed against human leukocyte antigens (HLA) which are present on the host cells used to propagate HIV. As HIV buds from the surface of the host cell, it incorporates some of the host cell HLA into its envelope.
  • Molar Absorption Coefficients (UV-VIS) These spectral properties can sometimes be found in reference sources. First, some definitions: Molar absorption coefficient (ε) Synonyms: Molar extinction coefficient, Molar absorptivity "The recommended term for the absorbance for a molar concentration of a substance with a path length of l cm determined at a specific wavelength. Its value is obtained from the equation ε = A / cl Strictly speaking, in compliance with SI units the path length should be specified in meters but it is current general practice for centimeters to be used for this purpose. Under defined conditions of solvent, pH and temperature the molar absorption coefficient for a particular compound is a constant at the specified wavelength." -- R.C. Denney, Dictionary of Spectroscopy, 2nd ed. (Wiley, 1982), p.119-20. Molar absorptivity Synonym: Molar (decadic) absorption coefficient. Decadic absorbance divided by the path-length l and mole concentration c, of the absorbing material. ε = A10/cl. The molar absorptivity is a Beer-Lambert absorption coefficient. SI unit: m2 mol-1. -- Handbook of Vibrational Spectroscopy, v.5 p.3772 (Wiley, 2002) Extinction coefficient A term that has been widely used for the molar absorptivity, unfortunately often with values given in ill-defined units. Use of this term has been discouraged since the 1960s, when international agreement with non-chemical societies reserved the word "extinction" for diffusion of radiation, i.e. the sum of the effects of absorption, scattering, and luminescence.

ELISA Presentation Transcript

  • 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. Enzyme LinkedImmunosurbent Assay ELISA
  • 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. What is ELISA? ELISA
  • 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. 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. 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. Histor yELISA
  • 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. 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. 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. 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. 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. 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. ApplicationsELISA
  • 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. 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. 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. 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. 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. Immunochemistry ELISA Limitations  Results may not be absolute  False positive possible  Falsenegative possible  Antibody must be availableJune 26, 2012 Total slides : 115 21
  • 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. Mechanism & Reagents ELISA
  • 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. 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. 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. 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. Types of ELISA ELISA
  • 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. 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. 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. 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. 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. 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. MethodsELISA
  • 36. Immunochemistry ELISA BASIC FORMAT Solid phase = 96 / 384-well microplateJune 26, 2012 Total slides : 115 36
  • 37. Immunochemistry ELISA 96 Well 350µL Polypropylene PlatesJune 26, 2012 Total slides : 115 37
  • 38. Immunochemistry ELISA 96 Well 500µL Polypropylene PlatesJune 26, 2012 Total slides : 115 38
  • 39. Immunochemistry ELISA 96 Well 600µL Polypropylene PlatesJune 26, 2012 Total slides : 115 39
  • 40. Immunochemistry ELISA 96 Well 1mL Polypropylene PlatesJune 26, 2012 Total slides : 115 40
  • 41. Immunochemistry ELISA 96 Well 2mL Polypropylene PlatesJune 26, 2012 Total slides : 115 41
  • 42. Immunochemistry ELISA 384 Well Polystyrene Assay PlatesJune 26, 2012 Total slides : 115 42
  • 43. Immunochemistry ELISA 384 Well Polypropylene Microtiter PlatesJune 26, 2012 Total slides : 115 43
  • 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. Immunochemistry ELISA 2. Block free binding sites. Incubate. Wash. Analyte = antibody Analyte = antigenJune 26, 2012 Total slides : 115 45
  • 46. Immunochemistry ELISA 3. Add sample. Incubate. Wash Analyte = antibody Analyte = antigenJune 26, 2012 Total slides : 115 46
  • 47. Immunochemistry ELISA 4. Add conjugate. Incubate. Wash. E E E E Analyte = antibody Analyte = antigenJune 26, 2012 Total slides : 115 47
  • 48. Immunochemistry ELISA 5. Add substrate 6. Incubate, stop, measure colour change ENZYME Colourless OD CONCENTRATIONJune 26, 2012 Total slides : 115 48
  • 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. 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. 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. 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. 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. 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. Immunochemistry ELISA AMPLIFICATION E Directly conjugated developing antibody may give weak signalJune 26, 2012 Total slides : 115 55
  • 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. 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. 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. 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. Immunochemistry ELISAJune 26, 2012 Total slides : 115 60
  • 61. Immunochemistry ELISAJune 26, 2012 Total slides : 115 61
  • 62. Immunochemistry ELISAJune 26, 2012 Total slides : 115 62
  • 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. 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. Immunochemistry ELISA INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES 2. Sample (human) antibody 1. AntigenJune 26, 2012 Total slides : 115 65
  • 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. 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. 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. 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. 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. 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. 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. 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. Immunochemistry ELISA ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS eg. hormones drugs tumour antigens cytokines 1. Anti-analyteJune 26, 2012 Total slides : 115 74
  • 75. Immunochemistry ELISA ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS 2. Sample 1. Anti-analyteJune 26, 2012 Total slides : 115 75
  • 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. 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. 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. Immunochemistry ELISA COMPETITION ELISA TO DETECT ANTIGENS (antigen-coated plate) 1. AnalyteJune 26, 2012 Total slides : 115 79
  • 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. 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. 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. Immunochemistry ELISA COMPETITION ELISA TO DETECT ANTIGENS (antibody-coated plate) 1. Anti-analyteJune 26, 2012 Total slides : 115 83
  • 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. 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. 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. 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. 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. 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. 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. HIV TestELISA
  • 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. 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. Immunochemistry ELISA An ELISA plateJune 26, 2012 Total slides : 115 94
  • 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. Immunochemistry ELISA  Negative ELISA  PositiveELISA Test TestJune 26, 2012 Total slides : 115 96
  • 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. Immunochemistry ELISA Sources of Error for HIV EIA TestsJune 26, 2012 Total slides : 115 98
  • 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. 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. 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. 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. 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. Immunochemistry ELISA Equipment : Pipettes  Single and Multi channel Pipettes should be calibrated on a monthly basis.June 26, 2012 Total slides : 115 104
  • 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Thank you Questions
  • 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Immunochemistry ELISAJune 26, 2012 Total slides : 115 133
  • 134. Immunochemistry ELISAJune 26, 2012 Total slides : 115 134
  • 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