Ag ab rxns


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over view of common antigen antibody reactions, their applications, sensitivity, advantage and disadvantage with pictorial illustrations for postgraduate and undergraduate reading

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Ag ab rxns

  2. 2.  Antigen antibody reaction is a bimolecular association in which the antigen and antibody combines with each other specifically and in an observable manner.  Non covalent Interaction between epitope of antigen and complementary determining region of antibody.
  3. 3. Purpose :  Antibody mediated immunity in infectious disease or injury, hypersensitivity and autoimmune disease  In laboratory for diagnosis of infectious disease, detecting antigen or antibody.
  4. 4. General features of antigen antibody( Ag-Ab ) reation:  Ag – Ab reactions are specific  Entire molecules and not the fragments react  No denaturation of Ag or Ab  Combination occurs at the surface. Surface Ag which is immunologically active  Both Ag and Ab participates  Ag and Ab combines in varying proportions. Ab are generally bivalent and Ag have valencies up to hundred
  5. 5. Strength of antigen antibody reaction:  The Ag-Ab binding includes non covalent bonds – H bond, Ionic bond, hydrophobic interactions & vanderwals forces.  Close fit between Ag and Ab requiring greater degree of complementarities between Ag and Ab.  Affinity and avidity
  6. 6.  Affinity The combined strength of the noncovalent interactions between a single antigen-binding site on an antibody and a single epitope is the affinity of the antibody for that epitope.  Strength of attraction
  7. 7. Avidity:  Strength of bond after formation of antigen antibody complex  Binding capacity within biological systems  Interaction of an antibody molecule with an antigen molecule at one site will increase the probability of interaction between those two molecules at a second site.  Strength of such multiple interactions determines avidity
  8. 8. Stages of antigen antibody rection:  Primary reaction  Secondary reaction  Tertiary reaction
  9. 9. Primary:  Without any visible effects  Rapid, follows general law of physical chemistry, low temperature  Reversible  Weaker intermolecular forces
  10. 10. Secondary:  Precipitation, agglutination, lysis of cell, killing of live antigen, neutralization of toxins & fixation of complement and immobilisation of motile phagocytosis. organisms, enhancement of
  11. 11. Tertiary reactions:  Chain reactions  Neutralization or destruction of injurious antigens  Humoral immunity, clinical allergy and immunological diseases.
  12. 12. Cross reactivity:  Antibody excited by one antigen can cross react with unrelated antigen  Different antigens sharing similar or identical epitope.  Affinty of cross reacting epitope is less  Most commonly among polysaccharide Ag.  Homologous determinants microbial and host antigenic
  13. 13. Examples:  ABO Blood group antigens Antibodies to missing antigen develops due to exposure to cross reacting microbial antigens.  Streptococcal M antigens Simulating myocardial and skeletal muscle protein  Cross reacting vaccines
  14. 14. Antigen antibody reactions in serology:  Precipitation  Agglutination  Complement fixation  Enzyme immuno assay  Radioimmuno assay  Neutralization  Western blotting  Immunofluresence
  15. 15. Definition :  A soluble antigen combines with its antibody in the presence of electrolyte at a suitable temperature and pH, the Ag-Ab complex forms an insoluble precipitate  If the precipitate remains suspended its known as flocculation  In liquid or gel media (agar, polyacrylamide)  Sensitive for detection of antigens agarose or
  16. 16. Zone phenomenon:  Relative proportions of antigen and antibody  Increasing amount of antigen added to constant amount of antisera in different test tubes.  Maximal precipitation at zone of equivalence
  17. 17. Mechanism of precipitation: Marrach hypothesis:  Multivalent antigen combines with bivalent antibody.  Lattice formation  In antigen or antibody excess lattice does not enlarge.
  18. 18. Precipitation reactions in liquid:  Ring test  Slide test  Tube flocculation test Precipitation reactions in gel:  Immunodiffusion  Electroimmunodiffusion
  19. 19. Ring test:  Layering of antigen solution over a column of antiserum in a narrow tube  Precipitate at the junction  E.g. Ascoli’s thermoprecipitin test Grouping of streptococci test. by Lancefield
  20. 20. Slide flocculation test  Antigen and antiserum placed on slide and mixed by shaking  Appearance of floccules
  21. 21. Tube flocculation test:  Khan test for syphilis  Standardization of toxins and toxoids  Serial dillutions of toxins or toxoids added to fixed quantity of antitoxin  Amount of toxin/ toxoid that flocculates with fixed quantity of anti toxin is defined as 1 Lf dose.
  22. 22. Precipitation reactions in gel:  Immunodiffussion  Agar matrix (1%)  Antibody incorporated into the agar.  Antigen diffuses into the agar containing matrix  Line of precipitation stained and preserved  Multiple antigen in reacting mixture can be identified  Detects identity, cross reactivity and non identity between antigens
  23. 23. Modifications of immunodiffusion:  Single diffusion in one dimension  Double diffusion in one dimension  Single diffusion in two diffusion  Double diffusion in two dimension  Immunoelectrophoresis  Electroimmunodiffusion
  24. 24. Single diffusion in one dimension: ( Oudin procedure )  Antibody incorporated in agar gel and antigen layered over it.  Line of precipitation at the advancing front of antigen  Number of bands indicates number of antigens
  25. 25. Double diffusion in one dimension ( Oakley fulthorpe procedure)  Antibody incorporated in agar  Intervening column of plain agar  Antigen layered over it  Both antigen and antibody moves towards each other
  26. 26. Single diffusion in two dimension ( Radial immunodiffusion / Maccni’s method)  Antiserum incorporated in agar poured on petridish  Antigen is added to the wells cut on the surface of gel  Concentric circles of precipitation diameter of halo gives estimate of antigen  Estimation of immunoglobulin classes in sera screening sera for antibodies to influenza
  27. 27. Double diffusion in 2 dimension ( Oucherlony procedure):  Most widely employed  Compare different antigen and antibody  Agar gel on slide with multiple wells  Antiserum in central well antigens in surrounding wells and different
  28. 28. Elek’s test for diphtheria bacillus
  29. 29. Immunoelectrophoresis:  Electrophoretic seperation of composite antigen  Immunodiffusion against its antiserum  Separate precipitation lines for individual protein  For detection of normal and abnormal proteins present in the serum
  30. 30. Electroimmunodiffusion:  Electrical driving up of antibody.  a. b. antigen and Methods: One dimensional double electroimmuno diffusion One dimensional single electro immumodiffusion
  31. 31. One dimensional double electro immunodiffusion  Counter immuno electrophoresis  Precipitation lines formed in 30 min  10 times more sensitive than standard double diffusion  Application: alpha fetoprotein in serum, specific antigen of cryptococcus and meningococcus.
  32. 32. Counter immunoelectrophoresis
  33. 33. One Dimensional Single Electroimmunodiffusion ( Rocket Electrophoresis)  Quantitative estimation of antigens  Antiserum is incorporated into the agarose gel on a glass slide  Antigen in increasing concentration is added to wells punched on gel  Pattern of immunoprecipitation resembles rocket
  34. 34. Nephelometry:  Immune complexes are monitored by spectrometry  Adding constant amount of optically clear specific antisera to varying amounts of antigen  Quantitative measurement of immunecomplexes using photoelectric cell as optical density
  35. 35.  When a particulate antigen combines with its antibody in the presence of electrolytes at suitable temperature and pH the complexes are clumped or sedimented  Antibodies involed in agglutination- agglutunins  Sensitive for detection of antibodies
  36. 36. Principle of agglutination:  Antigen and antibody in optimal proportion  Large lattice formation: antibody interaction with multivalent antigen antigenic determinants.  Prozone phenomenon with repeating
  37. 37. Prozone – mechanism:  High antibody concentration, exceeding the number of epitopes. Monovalent binding of antibody with antigen failing to form cross linkge.
  38. 38.  Polyclonal antibodies containing high concentration of antibodies tat can bind to antigen but do not cause agglutination. Known as blocking antibodies ( IgG class)
  39. 39.  Excess protein concentration on the bacteria or antigenic particle causing electrostatic repulsion.
  40. 40. Strength of agglutination: Appearance Report • Small aggregates +1 • Medium size aggregates +2 • Several large aggregates +3 • one solid aggregate +4
  41. 41. Determination of Antibody Titre:  Highest dilution of the biological sample which gives visible agglutination with antigen with no agglutination at higher dillutions  Using serial dilutions
  42. 42. Factors affecting Agglutination:  Buffer pH  Relative concentration of antigen and antibody  Location and concentration of antigenic determinant on the particle  Electrostatic interaction between particles  Electrolyte concentration  Antibody isotype  Temperature
  43. 43. Types of agglutination reactions:  Direct agglutination  Indirect agglutination  Reverse agglutination  Latex particle agglutination inhibition reactions  Quantitative agglutination reactions  Haemagglutination  Viral haemagglutination  Viral haemagglutination inhibition
  44. 44. Direct(active) agglutination:  Antigen is intrinsic component of the particle e.g bacterium, erythrocytes  Detection of antibodies in biological fluids  E.g. antibodies to brucella, weil-felix , widal test, blood grouping
  45. 45. Indirect (passive) agglutination:  Antigen adsorbed to the particle  Erythrocytes, charcoal, clay (bentonite), colloidal gold are particles to which antigen is complexed with  Testing of organisms which are very pathogenic
  46. 46. Latex particle agglutination test:  Uniform size latex particle coated with antigen will agglutinate in the presence of antibodies specific for the antigen  Samples: CSF, serum  E.g. R F, ASLO
  47. 47. Reverse (passive) agglutination:  Antibody is attached to the particle  Biological sample tested for antigen  e.g: C- reactive protein, candida species, Neisseria gonorrhoeae.
  48. 48. Coagglutination:  It’s a type of reverse passive agglutination test  Bacteria is added as inert particle to IgG antibodies, Fab region is free for antigen to bind  Applications: typing Gonococcal and streptococcal
  49. 49. Latex particle agglutination inhibition assay
  50. 50. Haemagglutination  Agglutination of RBC s  Direct- antigen a component of blood cell  In direct- soluble antigens are complexed with red blood cell e.g. thyroglobulin  Reverse haemagglutination- antibody is complexed to the red cell
  51. 51. Antigens binding to red cell:  Spontaneous adsorption  Covalent binding using chemical links  Tannic acid treatment
  52. 52. Viral haemagglutination:  Non immune agglutination  Agglutinate red cells in the absence of antibody  Dengue, rubella, influenza, mumps viruses  Haemagglutinin of influenza with human sheep and chicken red cells  V bottom micro titer plates  Adherence of RBC s to bottom of plates.
  53. 53. Viral Haemagglutination Inhibition:  Detection of antibodies in biological sample  Competetive binding assay done in two steps  Absence of haemagglutination is positive  End point haemagglutination - reciprocal of highest dilution of patient’s serum that completely inhibits haemagglutination
  54. 54. Antiglobulin Tests:  Coomb’s, Mourant and Race in 1945  To detect anti Rh antibodies  Anti Rh antibodies when mixed with Rh possitive red cells, they coat the surface of erythrocytes without agglutination  Incomplete antibodies  Coomb’s reagent – anti human antibody specific for Fc portion of IgG.  Types- direct and indirect.
  55. 55. Direct coomb’s test:  To detect maternal IgG antibodies bound to Rh antigen on fetal red cells  Erythroblastosis fetalis  Rh negative mother carrying Rh positive baby  Production of both IgM and IgG antibodies  IgG antibodies only crosses placenta  Sensitization of RBCs takes place in vivo
  56. 56. Indirect coomb’s test:  To detect presence of anti Rh antibodies in the serum of Rh- ve mother carrying or given birth to Rh + ve baby  Senstization takes place in vitro  Rh +ve RBCs are incubated with mother’s serum and coomb’s test. test is proceeded as direct
  57. 57. Heterophile Agglutination Test:  Sharing of common antigens by different biological species e.g Forssman antigen  Weil felix test in typhus fever Paul bunnel test in infectious mononucleosis Cold agglutinin in Mycoplasma pneumoniae
  58. 58.  Paul bunnel test is a mono spot test detects sheep agglutinins in patients with infectious mononucleosis  Weil-felix: common antigen between typhus rickettsiae and proteus bacilli  Coldagglutinins ( Ig M) – agglutination at 4°C reversed at 37°C - one week following Mycoplasmal dissapears by 6 weeks - also in Lymphomas & leukemias -complement mediated lysis of red cells infection,
  59. 59. Quantitative Agglutination Tests:  Soluble particle immunoassay ( SPIA)  Disperse dye immunoassay (DIA)  Immunoassay by particle counting (IMPACT)
  60. 60. Immunoassay SPIA Technique Inert Particle Detection Indirect or reverse agglutination Gold- inorganic -Qualitative, colloidal particle color change -Quantitative ,colorimetric analysis DIA Indirect or reverse agglutination Dye- organic -Qualitative, colloidal particle color visualized -quantitative, color measured optically photometric analysis IMPACT Indirect or reverse agglutination Latex particle -quantitative automated particle counter
  61. 61. 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.
  62. 62. Principle: Monitoring the formation of immune complexes by its ability to fix and consume complement proteins. Sensitivity: - 0.04 mg of antibody nitrogen - 0.1 mg of antigen
  63. 63. Components of complement fixation test: Test system  Antigen  Patient’s serum containing antibodies  Complement – fresh guinea pig serum Indicator system  Complement fixation diluents rich in Ca and Mg ions  Sheep erythrocytes  Amboceptors - Antibody to sheep erythrocytes
  64. 64. Preparation of complement:  Serum obtained by cardiac puncture from healthy adult guinea pig (250 to 300 gm) pooled for 18 to 24 hrs  Chlamydial or paramyxoviral infections can give rise to artefactual rections
  65. 65. Preservation of complement:  Freeze drying at – 70° C, reconstituted solution stored at 4° C used with in one week  Richardson’s method: addition of hypertonic sodium chloride or any salt
  66. 66. Titration of guinea pig serum:  One Minimum Hemolytic Dose ( MHD) of complement is defined as the highest dilution of the serum that lyses one unit volume of washed sheep erythrocytes in the presence of excess hemolysin within a fixed time, at a fixed temperature (37°C)
  67. 67.  The MHD of amboceptor is defined as least amount of the inactivated amboceptor that lyses one unit volume of washed sheep erythrocyte in the presence of excess complement within a fixed time and at a fixed temperature
  68. 68. Patient’s serum:  5 ml of blood by venepuncture  Coagulated in a sterile stopper test tube  Serum is pipetted off and diluted to starting dilutions  Heated to 56°C for 30 mins  Reduces nonspecific fixation and inactivates its complement
  69. 69. Procedure: stage 1  Serial dilution of heat inactivated test sera ( paired sera) in buffered CF diluent done in a microtitre plate  1 volume of complement 3 HD50 (50% hemolytic dose) is added to each well  I volume of antigen ( 2 – 4 units) at optimal peak dilution is added to each well  Stack and cover plates and stand overnight at 4 °C
  70. 70. Stage 2  Sensitization of sheep RBCs by slowly adding an equal volume of hemolysin and incubated at 37°C for 10 min or at room temperature for 30 min  Plates warmed for 30 min at room temperature and 0.025 ml of 2% sensitized sheep cells to each well
  71. 71.  Re-incubated for 30 min to allow lysis , shaking at 10, 20 and 30 min to resuspend the cells  Allow deposition of cells
  72. 72. 1 2 3 4 A1, B1, B2, B3- NO HEMOLYSIS – POSITIVE A2, A3, A4, B4 – HEMOLYSIS- NEGATIVE
  73. 73. Controls :  Serum control  Antigen control  Cell control  Positive serum control
  74. 74. Result :  Highest dilution of serum giving a fixation value is taken as the titre of antibody  End point titre – dilution fixing 70 to 100 % of complement  For antigen detection, test is done by diluting the antigen
  75. 75. Indirect complement fixation test:  For seras not able to fix complement  Test is set up in duplicate  After first step as in routine complement fixation test, the standard antiserum known to fix complement is added to one set.  If test serum contained antibody,the antigen being utilized in the first step.  Standard antiserum thus unable to fix the complement.  Haemolysis in indirect test indicate Positive result.
  76. 76. Conglutinating complement adsorption assay:  Uses horse complement  Indicator system: sheep erythrocytes mixed with bovine serum.  Conglutinin – beta globulin, antibody to horse complement  Conglutunin causes agglutination of sheep erythrocytes if combined with complement  If the horse complement utilised by the Ag – Ab interaction during first step No agglutination of sensitised cells
  77. 77. Applications of complement fixation test:  Wasserman reaction for syphillis  Immune Adherence – The Ag – Ab complexes of V.cholerae, T.pallidum adhere to RBCs, platelets.  Treponema pallidum immobilisation test  Cytolytic or cytocidal test – vibriocidal antibody test
  78. 78.  First devised to quantify polypeptide hormones by Parker in 1976  Used to measure antigen or antibody  Sensitivity: Permits measurement of analytes up to picograms quantities
  79. 79. Methods: a. Solutions :- Radioimmunoprecipitation - Competition radioprecipitation a. Solidphase: microtitre plate, glass, polystyerene bead, cellulose.
  80. 80. Radioimmunoprecipitation test:  Detection of antibody in test serum  Radiolabelled antigen is reacted with test serum  Antigen antibody complex formed is precipitated and separated from unbound labelled antigen  Comparison of radioactivity between precipitate and supernatant  Ratio gives estimate of amount of antibody in test serum
  81. 81. Separation of bound and unbound antigen:  Antiglobulin  Ammonium sulphate precipitation  Staphylococcal protein A Followed by centrifugation or chromatography
  82. 82. Competition radioimmunoassay:  For antigen detection in test sample  Labelled antigen competes with unlabelled antigen in the test sample for limited amount of antibody reagent  Free and bound labelled antigen separated and measured
  83. 83.  Concentration of test antigen calculated from ratio of bound and total labelled antigen  Compared to standard dose response curve or calibration curve
  84. 84. Solid-phase radioimmunoassay:  Advantage – separation of antigen and antibody easily achieved  Principle is similar to enzyme assay  Radiolabelled immunoglobulins are used  Gama or scintillation counting of bound antiglobulin
  85. 85. Uses of RIA:  Quantification of hormones, drugs, tumour markers, IgE and viral antigen like Hbs Ag Advantages: Specific, precise and highly sensitive Disadvantages:  I125 labelled reagents have shorter half-life  Radioactive hazard  Expensive equipments
  86. 86.  Fluorescence is a property of absorbing light rays of one wave length (excitation) and emitting rays with different wavelength (emission)  Albert coons in 1944 found antibodies could be tagged with molecules with property of fluorescence
  87. 87.  Fluorescent tagged antibodies locates formed immune complexes, detected by colored light emission when excited by light of the appropriate wavelength  Emitted light viewed by fluorescent microscope with a UV light source
  88. 88. Fluorochromes used : Dye Absorbs Fluoresceine isothiocyann ate (organic dye) Rhodamine (organic dye) Blue light (490 nm) Phycoerythri n (from algae) efficient absorber, 30 fold greater than fluoresceine Emits Yellow green fluorescence (517 nm) Yellow green Deep red range ( 515 fluorescence nm) (546 nm) red fluorescence
  89. 89. Types:  Direct immunofluorescence  Indirect immunofluorescence
  90. 90. Direct immunofluorescence:  For detecting antigens in tissues using specific antibody labeled with a fluorescent dye  For identification of viruses, bacteria, membrane bound antigens  Sensitive method for identification of rabies viral antigen in brain smears
  91. 91. Indirect immunofluorescence:  Two antibodies are used  Primary antibody and a secondary antibody which is a fluorescent labeled reagent  Reagent: Fluorescent labeled - secondary antibody, complement, protein A  E.g Fluorescent Treponemal antibody test
  92. 92. Advantages of indirect over direct:  Separate fluorescent conjugate for each antigen is not required, in turn fluorescent tagged antiglobulin to detect human antibodies to many antigen is employed  Sensitivity of staining increased - multiple molecules of the fluorochrome reagent bind to each primary antibody molecule
  93. 93. Advantages:  localization of antigens in tissue sections or in subcellular compartments  To map the actual location of target antigens, fluorescence microscopy is a powerful tool for relating the molecular architecture of tissues Disadvantages:  Non-specific fluorescence
  94. 94. Applications:  Identify subpopulations of lymphocytes, notably the CD4 and CD8 T-cell subpopulations.  For identifying bacterial species  Detecting Ag-Ab complexes in autoimmune disease  Detecting complement components in tissues  Localizing hormones and other cellular products stained in situ.
  95. 95.  Designed to automate the analysis and separation of cells stained with fluorescent antibody.  Uses a laser beam and light detector  Cells having a fluorescently tagged antibody bound to their cell surface antigens are excited by the laser and emit light that is recorded by a detector system located at a right angle to the laser beam
  96. 96. Informations obtained by flowcytometry:   Number and percentage of target cells The distribution of cells in a sample population according to antigen densities as determined by fluorescence intensity  The size of cells.  Analyzing cell population - with two or even three different fluorescent antibodies
  97. 97. Applications:  Essential tools for the detection and classification of leukemias  Rapid measurement of T cell population in AIDS
  98. 98.  Viral neutralization test  Toxin neutralization test
  99. 99. Viral neutralization:  Certain viral antigens inducing neutralizing antibodies that react with viruses resulting in loss of infectivity  To type viruses  To titrate the level of antibody in serum
  100. 100.  Phage neutralization – plaque inhibition test Bacteriophages seeded on lawn culture of susceptible bacteria, plaques of lysis produced,specific antiphage serum inhibits plaque formation  Neutralisation in animal, egg, or cell culture
  101. 101.  Incubating virus with sera containing neutralizing antibody and detecting residual viral activity by its ability to infect cell culture or other host system
  102. 102. Mechanism of viral neutralization:  Antibody inhibits the attachment of virus to its receptors by steric hindrance- Extrinsic neutralization  Antibody mediated alteration in viral expression- Intrinsic neutralization  Antigen – antibody complex formed between virus and antibody( lattice)pseudoneutralization
  103. 103. Toxin neutralization:  Bacterial exotoxins are immunogenic inducing neutralizing antibodies - antitoxins  Antitoxins – protection and recovery from disease  Toxin neutralization tests- in vivo or in vitro
  104. 104.  Invivo test : schick’s test- ability of circulating antitoxin to neutralize diphtheria toxin given intradermaly  Invitro test: antistreptolysin O test, anti toxin in human serum neutralizes the hemolytic activity of streptolysin O
  105. 105.  In 1966 Enzyme labeled conjugates introduced to identify antigen in tissues  EIA includes all assays based on measurement of enzyme labeled antigen, antibody or hapten  Types: - Homogeneous - Heterogeneous
  106. 106. Homogenous EIA:  No separation of bound and free fractions  Completed in one step  Used for assay of haptens  E.g: Enzyme multiplied immunoassay technique (EMIT) for opiates, cocaine, barbiturates
  107. 107. Heterogeneous EIA:  Separation of free and bound fraction by centrifugation or absorption on solid phase  Multi step procedure  Enzyme Liinked Immunosorbent Assay is a major type
  108. 108.  Immunoabsorbent is used as absorbent specific for one of the component of the reaction  Particulate or solidphase  Particulate -cellulose, agarose,  Solid phase -polysterene, polyvenyl, polycarbonate tubes, microwells or membrane or discs of polyacrylamide  Sensitivity of 0.1 to 0.01ng
  109. 109. Principle:   To use an enzyme to detect the Ag-Ab binding The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag-Ab binding  Enzymes: alkaline phosphatase, horseradish peroxidase, and galactosidase  Substrates: para-nitrophenyl phosphate and hydrogen peroxide
  110. 110. substrat e Colored product Different antigen in sample
  111. 111. Types of ELISA:  Non competitive Sandwich Indirect Capture  Competitive ELISA
  112. 112. Sandwich ELISA:  Sensitive for detection of antigen  Antibody is immobilized on a microtitre well  Sample containing antigen is added and allowed to react with the immobilized antibody .  Well is washed  enzyme linked antibody specific for epitope on the antigen is added  Suitable substrate added  Coloured reaction product  E.g detection of rotavirus in stool sample
  113. 113. Indirect ELISA:  Sensitive for detection of antibody  Antigen coated microtitre well  Serum sample containing primary antibody added ( Ab1)  Washed to remove unbound Ab  Enzyme-conjugated secondary anti-isotype antibody (Ab2)  Substrate  Colored reaction read  E.g: anti- HIV antibody
  114. 114. Capture ELISA:  Sensitive for detection of antibody responses  Especially IgM responses in early disease  For diagnosis of infectious diseases like dengue, rubella, measles, toxoplasmosis
  115. 115. Competitive ELISA:  Sensitive for antigen detection  Antibody is first incubated in solution with a sample containing antigen  Antigen-antibody mixture added to an antigen coated microtiter well  The more antigen present in the sample the less free antibody will be available to bind to the antigen-coated well
  116. 116.  Addition of an enzyme-conjugated secondary antibody (Ab2) specific for the isotype of the primary antibody  Higher the concentration of antigen in the original sample, the lower the absorbance  Detection of antigen in a crude mixture
  117. 117.  Modification of the ELISA  Quantitative determination of cells in a population that are producing antibodies specific for a given antigen or an antigen for a specific antibody  Plates are coated with capture antigen or antibody
  118. 118.     Newer technique devised to detect west nile virus antibodies domain –III envelope protein antigen linked with enzyme serum samples mixed with the enzymelabelled antigen immune complexes formed are recognized by rheumatoid factor coated microtiter plates
  119. 119. Combines sensitivity of EIA with more specificity Steps: 1. Separation of ligand antigen component gel by electrophoresis 2. Blotting of electrophorosed ligand fraction on nitrocellulose membrane 3. Enzyme immunoassay or radio immunoassay
  120. 120. EIA or RIA is done to detect:  Antibody against various ligand fraction bands  Probe with known antisera against specific antigen bands
  121. 121. Types of immunoblotting:  Southern blotting : First evolved, invented by Edwin Southern, detects DNA fragments  Western blotting : detecting specific protein in a complex mixture.  Northern blotting : detecting mRNA
  122. 122.   Chemical reaction emitting energy in the form of light Chemiluminescent compounds are used to tag as a label for Ag-Ab reactions  Compounds – luminol, acridium  5 to 10-18 moles (5 attomoles) of target antigen have been detected
  123. 123.  Simple, economical & reliable  Test system- cassette containing membrane impregnated with antibody (to specific Ag) colloidal gold dye conjugate  Cassette having 3 windows: - test serum - test result - control
  124. 124.  Serum sample added to one window  Serum moves by capillary action  Colored band appears at second site if antibody is present in serum which forms antigen-antibody-conjugate complex  Colored band at 3rd window – control  Test invalid if control band is absent
  125. 125.  Ananthanarayan and Paniker’s Text book of Microbiology, eighth edition  Mackie and McCartney’s practical medical microbology.  Kuby’s text book of immunology
  126. 126.     Kuby’s text book of immunology. Stite’s Medical immunology, tenth edition. Ananthanarayan and Paniker’s Text book of Microbiology, eighth edition Mackie and McCartney’s practical medical microbology.