Downloaded 72 times
More Related Content
Similar to Anigen antiboy reactions
Anigen antiboy reactions
- 1.
- 2. plasma • Albumin • Fibrinogen • Gobulins – Alpha – Beta – Gammaglobulis
- 3. Gammaglobulis • Fivetypes – IgG – IgA – IgM – IgE – IgD
- 9. functions of antibodies • to neutralize – toxins – viruses, • to opsonize microbes – so they are more easily phagocytosed, • to activate complement • to prevent the attachment of microbes to mucosal surfaces. • antibodies have a catalytic (enzymatic) capability
- 10. • Strength ofthe reaction between an antigenic determinant (antigen) and an antigen combining site (antibody) High Affinity Ab Ag Affinity Low Affinity Ab Ag Affinity = attractive and repulsive forces
- 11. Avidity • Theoverall strength of binding between an Ag with many determinants and multivalent Abs Keq = 104 Affinity 106 Avidity 1010 Avidity
- 12. Specificity • Theability of an antibody combining site to react with only an antigenic determinant. • The ability of antibody to combine with an antigen
- 13. Cross Reactivity •The ability of an individual Ab combining site to react with more than one type of antigenic determinant. • The ability of a Ab molecules to react with more than one Ag Anti-A Ab Ag A Cross reaction Anti-A Ab Ag C Similar A.D
- 14. Factors Affecting Happeningof Ag/Ab Reactions • Affinity • Avidity • Ag:Ab ratio • Physical form of Ag Ab excess Ag excess Equivalence – Lattice formation
- 15. Tests Based onAg/Ab Reactions • All tests based on Ag/Ab reactions will have to depend on lattice formation itself or • we will have to utilize ways to detect small immune complexes • All tests based on Ag/Ab reactions can be used to detect either Ag or Ab
- 16. Antigen antibody tests • Used in both directions – Qualitative – Quantitative
- 17. Uses of antigens/antibodyreactions • Diagnosis Infectious diseases • Diagnosis of autoimmune diseases • Determination of blood type and HLA types • Determination of chemical levels
- 18. Diagnosis of infectious diseases • When the organism cannot be cultured-or difficult to culture hep A B C Syphilis • When the organism is too dangerous to culture- rickettsial disease • When the culture technique is not readily available-HIV,EBV • When the organism takes too long to grow e.g Mycoplasma
- 19. Qualitative /quantitative •Qualitative – determines antigen or antibody is present or absent • Quantitative – determines the quantity of the antibody – Titer – The highest dilution of the specimen usually serum which gives a positive reaction in the test
- 21. Antigen and antibodyreactions in the lab • Precipitation tests • Agglutination • Elisa • Radioimmunoassay • Immunofluorescence • Complement Fixation
- 22. Precipitation tests •The antigen and antibody are in, soluble form • Combine to form a visible precipitate • Presence of electrolytes • Positive controls and negative controls
- 23. Precipitation tests •Precipitation techniques – Tube precipitation test – Gel diffusion • Double • Single radial • precipitation in agar with an electric field – Immuno electrophoresis – Countercurrent electrophoresis (CEP),
- 24. Precipitation in acapillary tube • Streptococcus grouping
- 25. Double Diffusion •antigen and antibody are placed in different wells in agar and • allowed to diffuse and form concentration gradients. • Where optimal proportions occur, lines of precipitate form
- 28. Double diffusion method(Ouchterlony) • indicates whether – antigens are identical, – Antigens not identical – Partially identical
- 29. Single immunodiffusion/Radial immunodiffusion • Method – Ab in gel – Ag in a well
- 30. Radial Immunodiffusion (Mancini) • Interpretation – Diameter of ring is proportional to the concentration • Quantitative – Ig levels Ag Concentration Ab in gel Diameter2 Ag Ag Ag Ag
- 31. Precipitation in agarwith an electric field
- 32. Immunoelectrophoresis • Applicationof electric current • Separation of proteins
- 34. Interpertation • serumproteins are characterized in terms of their • presence, • absence • unusual pattern (e.g., human myeloma protein).
- 35. Counter- Immunoelectrophoresis •Method – Ag and Ab migrate toward each other by electrophoresis – Used only when Ag and Ab have opposite charges - + • Qualitative –Rapid Ag Ab
- 36. uses • Themeeting of the antigen and antibody is greatly accelerated • made visible in 30–60 minutes. • detection of bacterial and fungal polysaccharide antigens in cerebrospinal fluid.
- 37. Agglutination • Visibleclumping together of particulate matter by antigen combining with its specific antibody. • The clumps will be called agglutinates • Performed – Slide – Tube – Tile – Micrtitration plates
- 39. Agglutination • Usedin both directions • Antigen part of a particulate matter Salmonella • Particulate matter – Latex – Carbon particles – cells – Bacteria • Stablised staphylococcal cells
- 40. Agglutination • Activeagglutination test • The antigen part of a particulate matter per se • examples • Salmonella, vibrio,
- 41. Agglutination • Passiveagglutination test • Antigen or antibody are not part of particulate matter but are attached (rided on inert particles like latex, carbon,) + Particulat matter
- 42. coagglutination • stabilizedstaphylococcal cells-protein affinity for FC fragment of antibody – protein A
- 43. Prozone phenomenon •Tube agglutination • The lower dilutions do not show agglutination • the tubes (prior/before the optimum zone ) • The tubes in higher dilutions show agglutination • Reasons/factors – Antibody excess:High level of antibody – Non specific inhibitory factors
- 44. Haemagglutination • ActiveHaemagglutination test – (blood group) • Passive haemagglutination (TPHA) – Known antigen coated on to treated RBC,s – Treated To remove their own antigens – Turkey,s RBC,s are used
- 45. Immunofluorescence • Fluorescentdyes illuminated by ultraviolet light are used to show combination of antigen antibody . • The end point antigen antibody complexes are seen fluorescing against a dark background • Direct • Indirect
- 47. Immunofluorescence • Direct – Ab to tissue Ag is labeled with fluorochrome
- 50. Immunofluorescence • Indirect – Ab to tissue Ag is unlabeled – Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. • Qualitative to Semi- Quantitative
- 51. Fluorescence-Activated Cell Sorting(Flow Cytometry) • the patient's cells are labeled with monoclonal antibody to the protein specific to the cell of interest, e.g., CD4 protein • The monoclonal antibody is tagged with a fluorescent dye, such as fluorescein or rhodamine. • Single cells are passed through a laser light beam, • the number of cells that fluoresce is counted by use of a machine called a fluorescence-activated cell sorter (FACS).
- 52. Immunofluorescence • FlowCytometry – Cells in suspension are labeld with fluorescent tag •Cells analyzed on a flow cytometer
- 53. Assays Based onComplement Lattice formation not required
- 54. CFT • TheCFT is used to detect antibodies or antigens • inference is made by the ability of the antibodies to fix the complement • The fixation of the complement is measured by adding the indicator system • Used in diagnosis of viral parasitic and rickettsial diseases
- 61. Complement Fixation •Methodology – Antibody known mixed with test material to be assayed for Ag – Standard amount of complement is added – Erythrocytes coated with Abs is added – Amount of erythrocyte lysis is determined Ag NO Ag Ag Ag
- 62. RadioImmunoassay • Theradioactivity of the specific labeled antibody or antigen is used to quantify the antigen or antibody in patient ,s serum • HbsAg • Hav IGM
- 64. ELISA • Usesan enzyme system to show the specific combination of antigen antibody • An enzyme labeled or linked to a specific antigen • A substrate • A color reader • Double antibody technique to detect and assay antigen • Indirect technique to Assay antibody
- 65. Direct Elisa •Ag detection – Add labeled antibody – Amount of labeled Ab bound is proportional to the amount of Ag in the sample
- 66. Indirect ELISA •Ab detection – Immobilize Ag – Incubate with sample – Add labeled anti-Ig – Amount of labeled Ab bound is proportional to amount of Ab in the sample • Quantitative Immobilized Ag Solid Phase Ab in Patient’s sample Labeled Anti-Ig
- 67. Western blot •HIV proteins are separated electrophoretically in a gel, • discrete bands of viral protein. • These proteins are then transferred from the gel, i.e., blotted, onto filter paper, and • the person's serum is added. • If antibodies are present, they bind to the viral proteins (primarily gp41 and p24) and can be detected by adding antibody to human IgG labeled an enzyme, • produces a visible color change when the enzyme substrate is added.
- 69. Neutralization Tests •These use the ability of • antibodies to block the effect of toxins or the infectivity of viruses. • They can be used in cell culture ( inhibition of cytopathic effect • in host animals ( mouse protection tests).
- 70.
- 71. Ag-Ab reactions Testsfor Ag-Ab reactions
- 72. Nature of Ag/AbReactions • Lock and Key Concept • Non-covalent Bonds – Hydrogen bonds – Electrostatic bonds – Van der Waal forces – Hydrophobic bonds • Multiple Bonds • Reversible http://www.med.sc.edu:85/chime2/lyso-abfr.htm Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000
- 74. Coombs (Antiglobulin)Tests •Incomplete Ab • Direct Coombs Test – Detects antibodies on erythrocytes + Patient’s RBCs Coombs Reagent (Antiglobulin)
- 75. Coombs (Antiglobulin)Tests •Indirect Coombs Test – Detects anti-erythrocyte antibodies in serum Patient’s Serum + Target RBCs Step 1 + Coombs Reagent (Antiglobulin) Step 2
- 76. Immunofluorescence • FlowCytometry cont. – Data displayed One Parameter Histogram Unstained cells Green Fluorescence Intensity Number of Cells FITC-labeled cells Red Fluorescence Intensity Green Fluorescence Intensity Two Parameter Histogram
- 77. Agglutination Tests LatticeFormation
- 78. Active Agglutination •Definition - tests that have as their endpoint the agglutination of a particulate antigen – Agglutinin
- 79. Agglutination/Hemagglutination • Quantitativeagglutination test – Titer – Prozone 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 Pos. Neg. Titer 64 8 512 <2 32 128 32 4 Patient 1 2 3 4 5 6 7 8
- 80. Agglutination/Hemagglutination • Definition • Qualitative test • Quantitative test • Applications – Blood typing – Bacterial infections –Fourfold rise in titer • Practical considerations – Easy – Semi-quantitative 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512
- 81. Passive Agglutination/Hemagglutination •Definition - agglutination test done with a soluble antigen coated onto a particle • Applications – Measurement of antibodies to soluble antigens
- 82. Coombs (Antiglobulin)Tests •Applications – Detection of anti-Rh Ab – Autoimmune hemolytic anemia
- 83. Precipitation Tests LatticeFormation
- 84. Radioimmuoassays (RIA) Enzyme-LinkedImmunosorbent Assays (ELISA) Lattice formation not required
- 85. Competitive ELISA forantigen • Method – Determine amount of Ab needed to bind to a known amount of labeled Ag + Prior to Test Labeled Ag + Test + Patient’s sample Labeled Ag + – Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor
- 86. Competitive ELISA forAg • Method cont. – Determine amount of labeled Ag bound to Ab NH4SO4 anti-Ig • Immobilize the Ab • Quantitative – Most sensitive test + Test + Patient’s sample Labeled Ag + Solid Phase Solid Phase – Concentration determined from a standard curve using known amounts of unlabeled Ag
- 87.
- 88. Solid Phase RIAfor antigen/direct • Ag detection – Immobilize Ab – Incubate with sample – Add labeled antibody – Amount of labeled Ab bound is proportional to the amount of Ag in the sample • Quantitative Ag Solid Phase Ag in Patient’s sample Immobilized Labeled Ab
- 89. Solid Phase RIAfor antibody/indirect • Ab detection – Immobilize Ag – Incubate with sample – Add labeled anti-Ig – Amount of labeled Ab bound is proportional to amount of Ab in the sample • Quantitative Immobilized Ag Solid Phase Ab in Patient’s sample Labeled Anti-Ig
- 91. Factors Affecting Measurementof Ag/Ab Reactions • Affinity • Avidity • Ag:Ab ratio • Physical form of Ag Ab excess Ag excess Equivalence – Lattice formation
- 92. Cross Reactivity •The ability of an individual Ab combining site to react with more than one type of antigenic determinant. • The ability of a Ab molecules to react with more than one Ag Anti-A Ab Ag A Cross reactions Anti-A Ab Ag B Shared A.D Anti-A Ab Ag C Similar A.D
- 94. Immunoelectrophoresis • Method – Ags are separated by electrophoresis – Ab is placed in trough cut in the agar + - Ag • Interpretation Ag Ab Ag Ab – Precipitin arc represent individual antigens
- 97. Haemagglutination inhibiotion test • Certain viruses measles and influenza arboviruses agglutinate RBC,s • If specific antibodies are included in the system a virus is identified
- 98. Tests for CellAssociated Antigens Lattice formation not required
- 99. Immunofluorescence • FlowCytometry – Cells in suspension are labeld with fluorescent tag •Cells analyzed on a flow cytometer
- 101. Double Antibody ELISA • Ag detection – Immobilize Ab – Incubate with sample – Add labeled antibody – Amount of labeled Ab bound is proportional to the amount of Ag in the sample • Quantitative Ag Solid Phase Ag in Patient’s sample Immobilized Labeled Ab