Serology

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Serology

  1. 1. SEROLGYAntigen and Antibody Reactions Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  2. 2. Serology• The branch of laboratory medicine that studies blood serum for evidence of infection and other parameters by evaluating antigen- antibody reactions in vitro Dr.T.V.Rao MD 2
  3. 3. Serology • Serology is the scientific study of blood serum. In practice, the term usually refers to the diagnostic identification of antibodies in the serum We can detect antigens too Dr.T.V.Rao MD 3
  4. 4. Types of Antigen and Antibody reactions1. Agglutination tests2. Double diffusion precipitation tests3. Immunoelectrophoresis4. Western blot tests5. Complement fixation tests6. Immunofluorescence testing7. Immunoassays Dr.T.V.Rao MD 4
  5. 5. What happens in Antigen and Antibody reactions• React with each other in a observable manner.• Uses 1 Helps antibody mediated immunity in infection, and tissue injury 2 Helps diagnosis of Infections. 3 In epidemiological surveys 4 Detections and quantization of antigens and antibodies Dr.T.V.Rao MD 5
  6. 6. Detectable reactions 2nd Stage• Reaction can occur as 1 Precipitation 2 Agglutination 3 Lysis and killing of live antigens 4Neutralizatiobn of toxins 5 Fixation of complement 6 Immobilization of motile microbes 7 Enhancement of Phagocytosis. Dr.T.V.Rao MD 6
  7. 7. General features of Antigen and antibody reactions.• Specific reaction – combines with specific antigen• Entire molecule reacts not fragments• No denaturation of antigen or antibody• Combination occurs as surface antigens to surface of antibodies• Commination is firm but reversible depends on affinity and avidity• Both antigens and antibodies participate• Combine in varying proportions Bivalent and multivalent Dr.T.V.Rao MD 7
  8. 8. Terms used in evaluating test MethodologySensitivity–Analytical Sensitivity – ability of a test to detect very small amounts of a substance–Clinical Sensitivity – ability of test to give positive result if patient has the disease (no false negative results) Dr.T.V.Rao MD 8
  9. 9. Specificity• Analytical Specificity – ability of test to detect substance without interference from cross-reacting substances• Clinical Specificity – ability of test to give negative result if patient does not have disease (no false positive results) Dr.T.V.Rao MD 9
  10. 10. Affinity• Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody.• It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site . Dr.T.V.Rao MD 10
  11. 11. Avidity • Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies • Avidity is influenced by both the valence of the antibody and the valence of the antigen. • Avidity is more than the sum of the individual affinities. Dr.T.V.Rao MD 11
  12. 12. Dilution• Estimating the antibody by determining the greatest degree to which the serum may be diluted without losing the power to given an observable effect in a mixture with specific antigen Dr.T.V.Rao MD 12
  13. 13. Measurement of Antigen and Antibody reactions• Measured as Mass Nitrogen (microgram)• As Units• As titer Dr.T.V.Rao MD 13
  14. 14. Titer • Different dilutions of serum are tested in mixture with a constant amount of antigen and greatest reacting dilution is taken as the measure or Titer Dr.T.V.Rao MD 14
  15. 15. Common methods in creating dilutions Dr.T.V.Rao MD 15
  16. 16. Expression of Titers• Expressed in term of the was in which they are made• Dilution 1 in 8 is a dilution made by mixing one volume of serum with seven volumes of diluents (Normal Saline )• Incorrect to express dilution as 1/8 Dr.T.V.Rao MD 16
  17. 17. Majority Diagnostic tests are Serological tests • There are several serology techniques that can be used depending on the antibodies being studied. These include: ELISA, agglutination, precipitation, complement-fixation, and fluorescent antibodies. Dr.T.V.Rao MD 17
  18. 18. How Antigen – Antibody reaction occurs• Primary stage – Interaction between antigen and antibody occurs without any visible effects• The reaction is rapid even at low temperature but the reaction is reversible• Weaker _ intermolecular forces Van der wall’s force and ionic bonds• Can be measured with radioactive isotopes and Florescent dyes. Dr.T.V.Rao MD 18
  19. 19. Ag-Ab interactions Bonds: • Hydrogen • Ionic • Hydrophobic interactions • Van der Waals forces Each bond is weak; many are strong To “hold” they must be close  requiring high amts of complementarity! Dr.T.V.Rao MD 19
  20. 20. What happen further ?• 1 Precipitation• 2 Lysis of cells.• 3 Killing of live antigen• 4 Neutralization of toxins• 5 Complement fixation• 6 Immobilization of motile microbes• 7 Enhancement of Phagocytosis• 8 Entire molecule react no fragmented• 9 No denaturation 10 Combination occurs on surface 11 Firm but reversible Dr.T.V.Rao MD 20
  21. 21. Types of serological tests1. Agglutination tests2. Double diffusion precipitation tests3. Immunoelectrophoresis4. Western blot tests5. Complement fixation tests6. Immunofluorescence testing7. Immunoassays Dr.T.V.Rao MD 21
  22. 22. Immunology/ Serology? Precipitation Reactions• Capillary tube precipitation (Ring Test)• Ouchterlony Double Diffusion (Immunodiffusion)• Radialimmunodiffusion (RID)• Immunoelectrophoresis (IEP)• Rocket Electroimmunodiffusion (EID)• Counterimmunoelectrophoresis (CIEP) Dr.T.V.Rao MD 22
  23. 23. Antigen – Antibodyreactions presenting with precipitation Dr.T.V.Rao MD 23
  24. 24. Precipitation reactions• Precipitation- classic demonstration of antibody-antigen interaction• Antibody and soluble antigen aggregate to form a visible precipitate• Antibody must be bivalent (Fabs won’t work)• Antigen must be multivalent Dr.T.V.Rao MD 24
  25. 25. Precipitation - Reaction• In precipitation antigen combines with its antibody in the presence of electrolytes ( Nacl ) at a suitable temperature and Ph the antigen and antibody complexes form a insoluble precipitate suspended as floccules.• Reaction can take place in liquid medium, gels, agar, agarose, polyacrylamide Dr.T.V.Rao MD 25
  26. 26. Precipitation• Principle – Soluble antigen + antibody (in proper proportions) –> visible precipitate – Lattice formation (antigen binds with Fab sites of 2 antibodies)• Examples – Double diffusion (Ouchterlony) – Single diffusion (radial Immunodiffusion) – Immunoelectrophoresis – Immunofixation Dr.T.V.Rao MD 26
  27. 27. How precipitation occurs• Precipitation occurs with most antigens because the antigen is multivalent (i.e. has several antigenic determinants per molecule to which antibodies can bind). Antibodies have at least two antigen binding sites (and in the case of IgM there is a multimeric complex with up to 10 antigen binding sites), thus large aggregates or gel-like lattices of antigen and antibody are formed• Specific reaction Antigen combines with homologusantibody - vice versa Dr.T.V.Rao MD 27
  28. 28. Mechanism of Precipitation• Lattice hypothesis – Multivalent antigens combine with bivalent antibodies in varying proportions depending on the antigen and antibody ratio in the reacting mixture Dr.T.V.Rao MD 28
  29. 29. ZONE of Equivalence No Soluble AgANTIGEN ANTIBODYEXCESS or Ab EXCESS Ab CONC Dr.T.V.Rao MD 29
  30. 30. Precipitation Reactions ( no precipitate is formed( Lattices or if an Ag contains only alarge aggregates ) single copy of each epitope )FIGURE 6-4Precipitation reactions in fluids yield a precipitin curve. Dr.T.V.Rao MD 30
  31. 31. Applications of precipitation reactions• Qualitative and quantitative• More useful for antigen detection to as least as 1 microgram• Blood, seminal stains, Food adulteration Dr.T.V.Rao MD 31
  32. 32. Slide test -Flocculation test – VDRL test • VDRL Test – a drop of VDRL antigen to a drop of patients serum, • Shake • The reaction observed under microscope • Observe for flocculation reaction • A Khan test is done in a test tube Dr.T.V.Rao MD 32
  33. 33. Non reactive and Reactive VDRL Tests Dr.T.V.Rao MD 33
  34. 34. Agglutination+: ve RPR Agglutination : Dr.T.V.Rao MD 34
  35. 35. Tube test - Precipitation• Kahn test for Syphilis – a tube flocculation test.• Quantitative tube flocculation test used in standardarisation of toxin/toxoid• Serum dilution of toxin or toxoid is added to tubes containing a fixed quantity of antitoxin• The amount of toxin that flocculates optimally with one unit of antitoxin is defined as Lf dose Dr.T.V.Rao MD 35
  36. 36. Single diffusion in one Direction O Oudin Procedure Dr.T.V.Rao MD 36
  37. 37. Immuno Diffusion Tests• Can demonstrate the immunological identity (or not) of two antigen samples; radial Immunodiffusion Dr.T.V.Rao MD 37
  38. 38. Ring Test• The reaction is demonstrated by layering antigen solution over the column of antigen in a narrow tube, The precipitate forms at the junction of two liquids.• Eg Ascoli’s thermo precipitate test• Grouping of streptococci• C-reactive protein Dr.T.V.Rao MD 38
  39. 39. Oudin Procedure• Antibodies in agar gel• Above antigen is layered• Single diffusion in one direction• Called Qudin procedure• Antibody is incorporated in agar• Antigen diffuses down• Precipitation concentration of antigen at the site increases due to diffusion Dr.T.V.Rao MD 39
  40. 40. Dr.T.V.Rao MD 40
  41. 41. Double diffusion in one dimension Oakley Fulthrope procedure• Antibodies in gel• Agar layer• Antigen layered• Antigens and antibodies moves towards each other• Forms precipitate Dr.T.V.Rao MD 41
  42. 42. Single Diffusion In two Dimensions Radial Immunodiffusion• Radial Immunodiffusion is an Immunodiffusion technique used in immunology to detect quantity of antigen by measuring the radius surrounding samples of the antigen, marking the boundary between it and antibody Dr.T.V.Rao MD 42
  43. 43. DOUBLE DIFFUSIONAntigen Antibody Immune Complex Dr.T.V.Rao MD 43
  44. 44. AntibodyAntigen Antigen Immune Complexes Zone of Equivalence Dr.T.V.Rao MD 44
  45. 45. OUCHTERLONY ANALYSISDiffusion of Antigens and Polyclonal Antibodies Non-Identity Antigen 1 Antigen 2 (Molecule #1) (Molecule #2) Antibodies to both antigens The same Animal was injected with antigen 1 and with antigen 2 Dr.T.V.Rao MD 45
  46. 46. OUCHTERLONY ANALYSIS This animal was only injected Partial - Identity with Antigen #4 Antigen 3 Antigen 4 is a part of antigen 4 Remember that Protein Also remember that Antigens have this antibody is amulti-clonal antibody Antibody different such as an anti- antigenicserum to an antigenic Dr.T.V.Rao MD 46 determinants
  47. 47. Single Diffusion In two Dimensions Radial Immunodiffusion • Antigen is added to the wells cut on the surface of the gel • It diffuses radially from well and forms a ring shaped band of precipitation • The halo of precipitation diameter gives the estimate of concentration of antigen • Used in estimation of Immunoglobulins Dr.T.V.Rao MD 47
  48. 48. Precipitation in Agar• These tests are done in agar• Tested for passive immuno diffusion in Agarose• There are several methods in testing this procedure Dr.T.V.Rao MD 48
  49. 49. Elek’s Immunodiffusion Test• Elek Immunodiffusion test. Sterile filter paper impregnated with diphtheria antitoxin is imbedded in agar culture medium. Isolates of C diphtheria are then streaked across the plate at an angle of 90° to the antitoxin strip. Toxigenic C diphtheria is detected because secreted toxin diffuses from the area of growth and reacts with antitoxin to form lines of precipitin Dr.T.V.Rao MD 49
  50. 50. Radial Immunodiffusion• Radial Immunodiffusion, a variation of the agar precipitation technique, is used in clinical immunology for the detection and quantitation of all classes of Immunoglobulins, complement, ceroplastic, transferring, and other serum components Dr.T.V.Rao MD 50
  51. 51. Dr.T.V.Rao MD 51
  52. 52. Double Diffusion in one Dimension Oakley – Fulthrope procedure • In Double diffusion in one direction antibody in agar gel moves through the layer of plain agar to the antigen above • They react to each other • Form a band of precipitation • Virus antigen is placed in the central well and diffuses outwards. Wells A and C contain positive sera, well B contains a negative sample. The black areas show where antibody in the positive sera have bound to virus antigen and formed a precipitate Dr.T.V.Rao MD 52
  53. 53. Double diffusion in two dimensions Ouchterlony procedure• Both antigen and antibody diffuses independently through agar gel in two dimensions horizontally and vertically• Done in a slide or petridish• In 12 – 48 hours line of precipitates are formed• Useful in serological identification Dr.T.V.Rao MD 53
  54. 54. Immunoelectrophoresis:In Immunoelectrophoresis, a complex mixtureof antigens is placed in a well punched out ofan agar gel and the antigens areelectrophoresed so that the antigen areseparated according to their charge. Afterelectrophoresis, a trough is cut in the gel andantibodies are added. As the antibodiesdiffuse into the agar, precipitin lines areproduced in the equivalence zone when anantigen/antibody reaction occurs. Dr.T.V.Rao MD 54
  55. 55. Immuno-electrophoresis• Mixtures containing multiple antigen species which cross react with the same antiserum may be analysed by running them first on an analytical gel, then cutting a strip from that gel and laying it in a slit cut into the immuno- electrophoresis gel to form a large well. The result is a pattern which shows the positions of strongly reacting antigen species. Dr.T.V.Rao MD 55
  56. 56. Immunoelectrophoresis• Immunoelectrophoresis --migration of molecules due to electric charge Positive particles travel to cathode Negative particles travel anode Precipitin specificity provides a critical indicator of identity Dr.T.V.Rao MD 56
  57. 57. Immuno Electrophoresis• In this procedure the electrophoretic separation of compatible antigen into constituent protein followed by Immunodiffusion against its antiserum resulting in separate precipitation line, indicating relation between each individual protein in the antibody Dr.T.V.Rao MD 57
  58. 58. Electrical gel Immunoelectrophoresis• The reaction is electrically driven, different antigens are separated according to their charges under electrical fields• Number of antigens can be identified Dr.T.V.Rao MD 58
  59. 59. Countercurrent electrophoresis• Method – Ag and Ab migrate toward each other by electrophoresis – Used only when Ag and Ab have opposite charges - + Ag Ab• Qualitative –Rapid Dr.T.V.Rao MD 59
  60. 60. Counter currentImmunoelectrophoresis • In this procedure movement of antigens towards the anode and antibody towards the cathode through agar under electric field • Useful studies on CSF • Hepatitis B surface antigen detection • Detection of fetoproteins Dr.T.V.Rao MD 60
  61. 61. Imunoelectrphoresis (IEP) Qualitative• A serum sample is electrophoresed through an agar medium.• A trough is cut in the agar and filled with Ab.• A precipitin arc is then formed.• Because Ag diffuses radially and Ab from a trough diffuses, the reactants meet in optimal proportions for precipitation. Dr.T.V.Rao MD 61
  62. 62. Rocket electrophoresis Laurel• The reactions appear as rocket• Driven by electric current• Rocket” Immuno- Electrophoresis is used as a rapid way to quantitate antigen in complex samples. Dr.T.V.Rao MD 62
  63. 63. Immuno-electrophoresis gel• Mixtures containing multiple antigen species which cross react with the same antiserum may be analyzed by running them first on an analytical gel, then cutting a strip from that gel and laying it in a slit cut into the immuno- electrophoresis gel to form a large well. The result is a pattern which shows the positions of strongly reacting antigen species. Dr.T.V.Rao MD 63
  64. 64. Counterimmunoelectrophoresis Dr.T.V.Rao MD 64
  65. 65. Electro Immunodiffusion Dr.T.V.Rao MD 65
  66. 66. Dr.T.V.Rao MD 66
  67. 67. Rocket electrophoresis• Crossed Immunoelectrophoresis of antigens and antiserum. In the first dimension, proteins are separated by standard electrophoresis. The separated proteins are then run into the second dimension gel at an angle of 90° from the first dimension. Dr.T.V.Rao MD 67
  68. 68. Serology can be done on various specimens• Some serological tests are not limited to blood serum, but can also be performed on other bodily fluids such as semen and saliva, which have (roughly) similar properties to serum.• Serological tests may also be used forensically, generally to link a perpetrator to a piece of evidence (e.g., linking a rapist to a semen sample). Dr.T.V.Rao MD 68
  69. 69. Measurement of Precipitation by Light• Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance.• Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution.• Nephelometry indirect measurement, measures amount of light scattered by the antigen-antibody complexes. Dr.T.V.Rao MD 69
  70. 70. Measurement of Precipitation by Light• Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance.• Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution.• Nephelometry indirect measurement, measures amount of light scattered by the antigen-antibody complexes . Dr.T.V.Rao MD 70
  71. 71. Agglutination Dr.T.V.Rao MD 71
  72. 72. Agglutination• is the aggregation of particulate matter caused by the combination with specific antibody• 1896: First observed by Gruber and Durham when serum antibody was found to react with bacterial cells Dr.T.V.Rao MD 72
  73. 73. Agglutination• Agglutinins – Antibodies that produce such reactions• Involves two-step process: – Sensitization or initial binding – Lattice formation or formation of large aggregates Dr.T.V.Rao MD 73
  74. 74. Agglutination• Types of particles that participate in such reactions: – Erythrocytes – Bacterial cells – Inert carriers such as latex particles Dr.T.V.Rao MD 74
  75. 75. Secondary phenomenon:LATTICE FORMATION – Ab + multivalent Ag  stable network (visible reaction) – conc. of Ag and Ab – Governed by physiochemical factors: • Ionic strength of milieu • pH • temperature Dr.T.V.Rao MD 75
  76. 76. Secondary Phenomenon• Lattice Formation• The Fab portion of the Ig molecule attaches to antigens on 2 adjacent cells-visible results in agglutination• If both antigen and antibody are SOLUBLE reaction will become visible over time, ie, precipitation Dr.T.V.Rao MD 76
  77. 77. Tube Agglutination Test Dr.T.V.Rao MD 77
  78. 78. Tube Agglutination Test Agglutination No agglutination1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl In this case, the titre is 1/40 Dr.T.V.Rao MD 78
  79. 79. DIRECT AGGLUTINATION-Test patient serum against large, cellular antigens to screen for the presence of antibodies.• Antigen is naturally present on the surface of the cells.• In this case, the Ag-Ab reaction forms an agglutination, which is directly visible. Dr.T.V.Rao MD 79
  80. 80. Slide Agglutination Test• Used for serotyping (e.g. Salmonella)• Antigen: isolated Salmonella in suspension• Antibody: specific antisera against Salmonella• Place test Salmonella in a drop of saline on a slide• Add a drop of antiserum, mix and rock slide for approx 1 minute• Examine for agglutination Dr.T.V.Rao MD 80
  81. 81. Slide Agglutination Test Dr.T.V.Rao MD 81
  82. 82. Agglutination Test Dr.T.V.Rao MD 82
  83. 83. OTHER DIRECT AGGLUTINATION TESTS• The particle antigen may be a bacterium.e.g.: Serotyping of E. coli, Salmonella using a specific antiserum• The particle antigen may be a parasite.e.g.: Serodiagnosis of Toxoplasmosis• The particle antigen may be a red blood cell.e.g.: Determination of blood groups Dr.T.V.Rao MD 83
  84. 84. Quantitative MicroHemagglutination Test (HA)Haemagglutination Tests (HA) Dr.T.V.Rao MD 84
  85. 85. HEMAGGLUTINATION• Detects antibody to erythrocyte antigens – sufficient concentration of antibody present-> antibody cross-link= agglutination – non-reactive/insufficient antibody present= no agglutination• Binding different antigens on the RBC surface = detect antibodies to antigen other than those present in the cells Dr.T.V.Rao MD 85
  86. 86. HEMAGGLUTINATION• Chromic chloride, tannic acid, and glutaraldehyde= cross- link antigens to the cell• IgG (does not agglutinate directly)-> need enhancement medium-> AHG• AHG binds to the second antibody present on the erythrocyte. Dr.T.V.Rao MD 86
  87. 87. Haemagglutination RBC Dr.T.V.Rao MD 87
  88. 88. Viral Haemagglutination• Some viruses and microbes contain proteins which bind to erythrocytes (red blood cells) causing them to clump together• NDV• Adenovirus III• AIV• IBV• Mycoplasma Dr.T.V.Rao MD 88
  89. 89. Hemagglutination test: method 1:8 1:2 1:2 1:2 1:2 1:2 virusserial dilution 8 16 32 64 128 256mix with redblood cells side view top view Titer = 32 HA units/ml One HA unit :minimum amount of virus that causes complete agglutination of RBCs Dr.T.V.Rao MD 89
  90. 90. HEMAGGLUTINATION INHIBITION TEST (HI)VIRUSE SERUM Dr.T.V.Rao MD 90
  91. 91. In the absence of anti-virus antibodies Erythrocytes Virus Virus agglutination of erythrocytes Dr.T.V.Rao MD 91
  92. 92. In the presence of anti-virus antibodies Erythrocytes Virus Anti-virus antibodies Viruses unable to bind to the erythrocytes Dr.T.V.Rao MD 92
  93. 93. Dr.T.V.Rao MD 93
  94. 94. Reading/Grading Agglutination Reactions• Done by gently shaking the tubes containing the serum and cells, and observing the cell button as it is dispersed• Hard shaking must be avoided because this may yield to false result• Attention should also be given to whether discoloration of the supernatant is present (Haemolysis). Dr.T.V.Rao MD 94
  95. 95. Antibody Titer• Is the lowest concentratio n of antibodies against a particular antigen. Dr.T.V.Rao MD 95 Figure 18.6
  96. 96. Coombs (Antiglobulin)Tests• Applications –Detection of anti-Rh Ab –Autoimmune hemolytic anemia Dr.T.V.Rao MD 96
  97. 97. Dr.T.V.Rao MD 97
  98. 98. Coombs (Antiglobulin)Tests • Incomplete Ab • Direct Coombs Test – Detects antibodies on erythrocytes + ↔Patient’s RBCs Coombs Reagent (Antiglobulin) Dr.T.V.Rao MD 98
  99. 99. Coombs (Antiglobulin)Tests • Indirect Coombs Test – Detects anti-erythrocyte antibodies in serumStep 1 + ↔ Patient’s Target Serum RBCsStep 2 + ↔ Coombs Reagent (Antiglobulin) Dr.T.V.Rao MD 99
  100. 100. Dr.T.V.Rao MD 100
  101. 101. Passive Agglutination• An agglutination reaction that employs particles that are coated with antigens not normally found in the cell surfaces• Particle carriers include: – Red blood cells – Polystyrene latex – Bentonite – charcoal Dr.T.V.Rao MD 101
  102. 102. Passive Agglutination• Passive agglutination has been used in the detection of : –Rheumatoid factor –Antinuclear antibody in LE –Ab to group A streptococcus antigens –Ab to Trichinella spiralis Dr.T.V.Rao MD 102
  103. 103. Reverse Passive Agglutination• Antibody rather than antigen is attached to a carrier particle• For the detection of microbial antigens such as: ▫ Group A and B streptococcus ▫ Staphylococcus aureus ▫ Neisseria meningitidis ▫ Haemophilus influenzae ▫ Rotavirus ▫ Cryptococcus neoformans ▫ Mycoplasma pneumoniae ▫ Candida albicans Dr.T.V.Rao MD 103
  104. 104. Coagglutination• Name given to systems using inert bacteria as the inert particles to which the antibody is attached• S.aureus: most frequently used because it has protein A in its outer surface that naturally adsorbs the Fc portion of the antibody Dr.T.V.Rao MD 104
  105. 105. • Highly specific but not very sensitive in detecting small quantities of antigen Dr.T.V.Rao MD 105
  106. 106. False-Positive Result• If injected with hCG to trigger ovulation or to lengthen luteal phase of menstrual cycle.• Chorioepithelioma, hydatidiform mole or ingestion of aspirin• To detect the presence of a testicular tumor in menFalse Negative• Testing before reaching detectable levels of hCG. Dr.T.V.Rao MD 106
  107. 107. Immunofluorescence• Antibodies can be labeled with fluorescent dye Can localize binding sites on cell• Dyes: Fluorescein, rhodamine, phycoerythrin can be conjugated to Fc region of Ab (so antigen binding is unaffected• Absorb at one wavelength and emit at another Dr.T.V.Rao MD 107
  108. 108. Fluorescence UV Light Dr.T.V.Rao MD 108 Antigens on Cells or on Tissue Sections
  109. 109. Fluorescence Double layer Sandwich UV Light Antigens Dr.T.V.Rao MD 109
  110. 110. Enzyme Immunoassay ( EIA )• Introduced in 1966 alternative to fluorescent methods• Versatile, simple economical• Absence of radiation.• EIA means measuring enzymes labelled antigen, hapten, antibody Dr.T.V.Rao MD 110
  111. 111. ELISA Enzyme Linked Immuno-Sorbant Assay Peroxidase Enzyme is permanently attached to Antibody Probe Substrate that turns from clear to green Ag Ag Microtiter ELISAAntigens are immobilized toMD plastic surface of a Dr.T.V.Rao the 111 Microtiter Plate
  112. 112. Fluorescence UV Light Dr.T.V.Rao MD 112 Antigens on Cells or on Tissue Sections
  113. 113. ELISA Enzyme Linked Immuno-Sorbant Assay Peroxidase Enzyme is permanently attached to the Antibody Probe Substrate that turns from clear to green Ag Ag Microtiter ELISAAntigens are immobilized toMD plastic surface of a Dr.T.V.Rao the 113 Microtiter Plate
  114. 114. ELISA• The ELISA (Enzyme-Linked Immuno- Sorbant Assay) can be used both qualitatively and quantitatively to measure antigen-antibody binding. Depending on what variation you use, it will detect antigen (hormones, enzymes, microbial antigens, illicit drugs) or antibody (anti-HIV in the screening test for HIV infection) in body fluids or tissue culture supernatants. Dr.T.V.Rao MD 114
  115. 115. ELISA• Enzyme-Linked Immuno-Sorbant Assay, also called ELISA, Enzyme ImmunoAssay or EIA, 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. Dr.T.V.Rao MD 115
  116. 116. Enzyme Linked Immuno Assay• The Technique involves use of Immuno-Sorbant and absorbing material specific for one of the components of reaction, the antigen or antibody Dr.T.V.Rao MD 116
  117. 117. Components in ELISA testing• Conjugate – Horseradish peroxidase• Substrate - O-phenyle diamine dihydrochloride• The test is conducted in solid phase Polystyrene, Polyvinyl or polycarbonate tubes or in plastics Dr.T.V.Rao MD 117
  118. 118. ELISA plate Dr.T.V.Rao MD 118
  119. 119. ELISA methodology• 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). Dr.T.V.Rao MD 119
  120. 120. ELISA Methodology Dr.T.V.Rao MD 120
  121. 121. ELISA methodology• 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 Dr.T.V.Rao MD 121
  122. 122. Sandwich ELISA Dr.T.V.Rao MD 122
  123. 123. Different methods of ELISA• Direct and Indirect ELISA• Sandwich• Non competitive Sandwich Dr.T.V.Rao MD 123
  124. 124. ELISA – most popularly used method• The ELISA is probably the most commonly used immunological assay because of its versatility, sensitivity (ability to detect small amounts of antigen or antibody), specificity (ability to discriminate between closely related but antigenically different molecules), and ease of automation. Although some of the substrates are carcinogenic, they are generally considered safer than radioisotopes used in RIA (radioimmunoassay). Dr.T.V.Rao MD 124
  125. 125. Uses of ELISA• Helps detection of Antigens, Antibodies, hormones and Enzymes• Eg in Microbiology Antigens – HbS Ag Antibodies HIV, HCV, CMV, several other disease Dr.T.V.Rao MD 125
  126. 126. Radio Immuno Assay Berson and Yallow• Besides fluorescent dyes other labels can be used• Uses with Radio isotopes• Variety of tests are done for detection of antigen or antibody• The term binder ligand assay has been used• The minute amounts of substances can be detected• Used in Biology and Medicine Dr.T.V.Rao MD 126
  127. 127. Rosalyn S. Yalow and Sol Berson Dr.T.V.Rao MD 127
  128. 128. RIA ( Radio Immuno Assay )• The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. It represented the first time that hormone levels in the blood could be detected by an in vitro assay. Dr.T.V.Rao MD 128
  129. 129. Uses of RIA• Used for detection of Hormones, enzymes,tumour markers• IgE and viral antigens• RIA is a competitive binding assay fixed amount of antibody and radiolabelled antigen react in the presence of unlabelled antigen• Detection is done for free and bound fractions, ratios calculated Dr.T.V.Rao MD 129
  130. 130. Immunofluorescence• The purpose of immunofluorescence is to detect the location and relative abundance of any protein for which you have an antibody. Once you have antibodies to your favourite protein, you can use them to indicate where the protein is located. Dr.T.V.Rao MD 130
  131. 131. Immunofluorescence Dr.T.V.Rao MD 131
  132. 132. Fluorescent Methods Dr.T.V.Rao MD 132
  133. 133. Direct and Indirect Methods Dr.T.V.Rao MD 133
  134. 134. Western Blot• Western blot analysis can detect your protein of interest from a mixture of a great number of proteins. Western blotting can give you information about the size of your protein (with comparison to a size marker or ladder in kDa), and also give you information on protein expression (with comparison to a control such as untreated sample or another cell type or tissue). Dr.T.V.Rao MD 134
  135. 135. Western Blot Test• The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting. Dr.T.V.Rao MD 135
  136. 136. Appearance of test readings Dr.T.V.Rao MD 136
  137. 137. Flowcytometry• FACS- fluorescence-activated cell sorter Analyze cell populations• Sort cells with different features into different containers (e.g., T and B cells; cells that are producing a cell- surface marker from those that are not) Dr.T.V.Rao MD 137
  138. 138. Dr.T.V.Rao MD 138
  139. 139. Uses for flow cytometry• Percentage of a total population of cells• Measuring antigen density within a population of cells• Multiple antibodies can be used to assess several cell surface antigens simultaneously• Clinical analysis (tumor characterization) Dr.T.V.Rao MD 139
  140. 140. Chemiluminescences• Chemiluminescences Chemical reaction emitting energy in the form of light• Chemilumiscence - Luminol or acridinium esters causes signal in the process of antigen antibody reaction• Signal can be amplified, measured, and the concentration of the analyses sample• Uses the automated methods.• Increasingly used where the volume of work is large Dr.T.V.Rao MD 140
  141. 141. Chemiluminescences Immuno Assay Dr.T.V.Rao MD 141
  142. 142. Immunochromatographic Tests• One step in diagnosing• Simple Economical.• Reliable• Eg HbsAg A small cassette system containing a membrane impregnated with antiHbsAg antibody colloidal gold dye conjugateThe membrane is exposed at three windows on the cassette Dr.T.V.Rao MD 142
  143. 143. Immunochromatographic Tests Dr.T.V.Rao MD 143
  144. 144. Immunochromatographic Tests• A colored band appears at the second window• Control also can be recorded Dr.T.V.Rao MD 144
  145. 145. Also called as Dot Methods • The tests can be done by paramedical staff, as they are simple to read • Helps in emergency rooms. • The results are available within few minutes • The HIV and HBV infections can be done at the earliest Dr.T.V.Rao MD 145
  146. 146. Results can be read as Positive and Negative at the earliest Dr.T.V.Rao MD 146
  147. 147. • Programme created by Dr.T.V.Rao MD for Basic learning in Immunology • Email • doctortvrao@gmail.com Dr.T.V.Rao MD 147

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