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Antigen and Antibody Reactions Agglutination Tests

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Antigen and Antibody Reactions Agglutination Tests

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Antigen and Antibody Reactions Agglutination Tests

  1. 1. Antigen and Antibody Reactions Agglutination Tests Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  2. 2. 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 2
  3. 3. 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 3
  4. 4. Slide Agglutination Test Dr.T.V.Rao MD 4
  5. 5. Agglutination Test Dr.T.V.Rao MD 5
  6. 6. 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 6
  7. 7. Introduction: • The complement fixation test (CFT) was extensively used in syphilis serology after being introduced by Wasserman in 1909. • Complement is a protein (globulin) present in normal serum. • Whole complement system is made up of nine components: C1 to C9 • Complement proteins are heat labile and are destroyed by heating at 56°C for 20 – 30 minutes. • Complement binds to Ag-Ab complex • When the Ag is an RBC it causes lysis of RBC’s.
  8. 8. Principle • Complement takes part in many of the immunological reactions. It gets absorbed during the combination of antigens and antibody. • This property of antigen–antibody complex to fix the complement is used in complement fixation test for the identification of specific antibodies. • The haemolytic system containing sheep erythrocytes (RBC) and its corresponding antibody (amboceptor) is used as an indicator which shows the utilization or availability of the complement. • If the complement is fixed then there will be no lysis of sheep erythrocytes, thus denoting a positive test. • If the complement is available then there will be haemolysis which is a property of complement, denoting a negative test.
  9. 9. Complement fixation (CF) • Antibody and antigen allowed to combine in presence of complement • If complement is fixed by specific antigen-antibody reaction, it will be unable to combine with indicator system • Precautions • Serum must be heat-activated • Stored serum becomes anti-complementary • Extensive QC/standardization required • Only use for IgM antibodies
  10. 10. Components of CFT Test System • Antigen: It may be soluble or particulate. • Antibody: Human serum (May or may not contain Antibody towards specific Antigen) • Complement: It is pooled serum obtained from 4 to 5 guinea pigs. It should be fresh or specially preserved as the complement activity is heat labile (stored at -30 °C in small fractions). The complement activity should be initially standardized before using in the test. Indicator System (Haemolytic system) • Erythrocytes: Sheep RBC • Amboceptor (Hemolysin): Rabbit antibody to sheep red cells prepared by inoculating sheep erythrocytes into rabbit under standard immunization protocol.
  11. 11. Positive Test • Step 1: At 37°C Antigen + Antibody + Complement (from serum) Complement gets fixed 1 Hour • Step 2: Fixed Complement complex + Haemolytic system At 37°C No Haemolysis 1 Hour (Test Positive)
  12. 12. Negative Test  Step 1: At 37 C Antigen + Antibody absent + Complement Complement not fixed 1 Hour  Step 2: At 37 C Free Complement + Haemolytic system 1 Hour Haemolysis (Test Negative)
  13. 13. Results and Interpretations: • • No haemolysis is considered as a positive test. haemolysis of erythrocytes indicative of a negative test. 1 2 3 4 A B • Microtiter plate showing Haemolysis (Well A3, A3 and B4) and No Haemolysis (Well
  14. 14. Quantitative Micro Hemagglutination Test (HA) Haemagglutination Tests (HA) Dr.T.V.Rao MD 14
  15. 15. 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 15
  16. 16. Haemagglutination RBC Dr.T.V.Rao MD 16
  17. 17. 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 17
  18. 18. HEMAGGLUTINATION INHIBITION TEST (HI) VIRUSE SERUM Dr.T.V.Rao MD 18
  19. 19. In the absence of anti-virus antibodies Erythrocytes Virus Virus agglutination of erythrocytes Dr.T.V.Rao MD 19
  20. 20. In the presence of anti-virus antibodies Erythrocytes Virus Anti-virus antibodies Viruses unable to bind to the erythrocytes Dr.T.V.Rao MD 20
  21. 21. Dr.T.V.Rao MD 21
  22. 22. Heterophile Agglutination Tests • Weil – Felix Test or Reaction in Serodiagnosis of typhus fevers is heterophile agglutination test and sharing of common antigen between typhus Rickettsiae and some strains of Proteus bacilli Dr.T.V.Rao MD 22
  23. 23. Other tests • Red cells as antigens • 1 Paul Bunnell test • 2 Cold agglutination test Dr.T.V.Rao MD 23
  24. 24. Paul Bunnell test • Based on the principle of presence of sheep agglutinins in the sera of infectious mononucleosis patents who are absorbed by OX red cells but not by guinea pig kidney extract Dr.T.V.Rao MD 24
  25. 25. Cold Agglutination test • Positive in Mycoplasma ( Primary Atypical ) Pneumonia • The patients sera agglutinated human O group erythrocytes at 4 o c the agglutination being reversible at 37 0 c Dr.T.V.Rao MD 25
  26. 26. Coombs (Antiglobulin)Tests • Applications –Detection of anti-Rh Ab –Autoimmune hemolytic anemia Dr.T.V.Rao MD 26
  27. 27. Dr.T.V.Rao MD 27
  28. 28. Coombs (Antiglobulin)Tests • Incomplete Ab • Direct Coombs Test – Detects antibodies on erythrocytes ↔ + Patient’s RBCs Coombs Reagent (Antiglobulin) Dr.T.V.Rao MD 28
  29. 29. Coombs (Antiglobulin)Tests • Indirect Coombs Test – Detects anti-erythrocyte antibodies in serum Step 1 ↔ + Patient’s Serum Target RBCs Step 2 + ↔ Coombs Reagent (Antiglobulin) Dr.T.V.Rao MD 29
  30. 30. Dr.T.V.Rao MD 30
  31. 31. 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 31
  32. 32. 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 32
  33. 33. 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 33
  34. 34. 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 34
  35. 35. • Highly specific but not very sensitive in detecting small quantities of antigen Dr.T.V.Rao MD 35
  36. 36. 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 men False Negative • Testing before reaching detectable levels of hCG. Dr.T.V.Rao MD 36
  37. 37. 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 37
  38. 38. Fluorescence UV Light Dr.T.V.Rao MD 38 Antigens on Cells or on Tissue Sections
  39. 39. Fluorescence Double layer Sandwich UV Light Antigens Dr.T.V.Rao MD 39
  40. 40. 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 40
  41. 41. ELISA Enzyme Linked Immuno-Sorbant Assay Peroxidase Enzyme is permanently attached to Antibody Probe Substrate that turns from clear to green Ag Ag Microtiter ELISA Antigens are immobilized toMD plastic surface of a the Dr.T.V.Rao Microtiter Plate 41
  42. 42. 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 ELISA Antigens are immobilized toMD plastic surface of a the Dr.T.V.Rao Microtiter Plate 42
  43. 43. ELISA • The ELISA (Enzyme-Linked ImmunoSorbant 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 43
  44. 44. 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 44
  45. 45. 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 45
  46. 46. 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 46
  47. 47. ELISA plate Dr.T.V.Rao MD 47
  48. 48. 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 48
  49. 49. 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 49
  50. 50. Sandwich ELISA Dr.T.V.Rao MD 50
  51. 51. Different methods of ELISA • Direct and Indirect ELISA • Sandwich • Non competitive Sandwich Dr.T.V.Rao MD 51
  52. 52. 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 52
  53. 53. 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 53
  54. 54. 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 54
  55. 55. Rosalyn S. Yalow and Sol Berson Dr.T.V.Rao MD 55
  56. 56. 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 56
  57. 57. 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 57
  58. 58. 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 58
  59. 59. Immunofluorescence Dr.T.V.Rao MD 59
  60. 60. Fluorescent Methods Dr.T.V.Rao MD 60
  61. 61. Direct and Indirect Methods Dr.T.V.Rao MD 61
  62. 62. 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 62
  63. 63. 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 63
  64. 64. Appearance of test readings Dr.T.V.Rao MD 64
  65. 65. 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 cellsurface marker from those that are not) Dr.T.V.Rao MD 65
  66. 66. Dr.T.V.Rao MD 66
  67. 67. 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 67
  68. 68. Chemiluminescence's • Chemiluminescence's 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 68
  69. 69. Chemiluminescence's Immuno Assay Dr.T.V.Rao MD 69
  70. 70. 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 conjugate The membrane is exposed at three windows on the cassette Dr.T.V.Rao MD 70
  71. 71. Immunochromatographic Tests Dr.T.V.Rao MD 71
  72. 72. Immunochromatographic Tests • A colored band appears at the second window • Control also can be recorded Dr.T.V.Rao MD 72
  73. 73. 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 73
  74. 74. • Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Students in the Developing world • Email • doctortvrao@gmail.com Dr.T.V.Rao MD 74

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