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antigen-antibody reactions
This document discusses antigen-antibody reactions, including: 1. Antigen-antibody reactions involve the specific interaction between antigens and the antibodies produced against those antigens. 2. Common antigen-antibody reaction types include precipitation, agglutination, complement-dependent reactions, neutralization tests, opsonization, and various immunoassays. 3. Antigen-antibody reactions have many diagnostic and research applications, including disease diagnosis, epidemiological studies, and quantification of antigens or antibodies.
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antigen-antibody reactions
- 1. ANTIGEN – ANTIBODY REACTIONS Dr.Ajit Kumar Singh MD Laboratory Medicine Chittaranjan national cancer institute Kolkata 700160
- 2. ⚫Definition The interactionsb / w antigens and antibodies. Highly specific . Antigens react only with antibody produced by itself or with closely related antigens .
- 3. ⚫Purposes ⚫The basis ofantibody mediated immunity in infectious diseases . ⚫Help in diagnosis of infections in lab. ⚫In epidemiological surveys . ⚫Detection & quantitation of either Ags or Abs ⚫Invitro – serological reactions
- 4. ⚫ Ag –Ab reactions Occur in 3 stages 1 . Primary stage 2 . Secondary stage 3 . Tertiary stage
- 5. Primary Stage • Initialinteraction • Without visible effect • Low temperature • Reaction reversible • Vander Waal’s forces , ionic bond and hydrogen bonding • Detection - markers – radio isotopes fluorescent dyes ferritin
- 6. Secondary Stage • Demonstrableevents • Precipitation , Agglutination • Lysis of cells , killing of live antigens • Neutralisation of toxin • Complement fixation • Immobilisation of motile organisms • Enhancement of phagocytosis • Agglitinin , Precipitin , • Agglutinogen , precipitoinogen
- 7. Tertiary Reaction Ag-Abreaction in vivo - chain reaction – neutralisation , destruction of injurious Ag, tissue damage
- 8. Types 1 . Precipitation 2. Agglutination 3 . Complement dependent serological tests 4. Neutralization tests 5 . Opsonization 6 . Immunofluorescence 7 . Radioimmunoassay 8 . Enzyme immunoassay 9 .Western blotting 10.Chemiluminescence assay 11.Immunoelectron microscopic tests
- 9. Precipitation When a solubleantigen combines with its antibody in the presence of electrolytes at a suitable • Temp • PH The Ag – Ab complex forms an insoluble precipitate . A lattice is formed between the Ags and Abs . Flocculation When instead of sedimenting . The precipitate remains suspended as floccules .
- 10. Zone phenomenon :lattice hypothesis
- 11. Mechanism of precipitation Latticehypothesis
- 12. Applications Qualitative or quantitativetest sensitive in detection of antigen , 1 µg of protein - can be detected 1. Forensic application - Detection of blood/serum stains 2. Testing - Food adulterants 3. Grouping of Streptococci – Lancefield technique 4. VDRL test for syphilis 5. To standardize toxins and toxoids 6. To test toxigenicity in diphtheria bacillus
- 13. Types 1. Precipitation insolution 2. Precipitation in agar gel 3. In agar with electric field
- 14. Precipitation in solution: 1 . Ring test 2 . Flocculation tests Ring test : Ex 1 . C – reactive protein 2 . Streptococcal grouping by Lancefield
- 15. Flocculation tests 1. Slideflocculation test : Ex : VDRL test 2 . Tube flocculation test Ex : 1 . Kahn test for Syphilis 2 . For standardization of toxins and toxoids
- 16. Precipitation in agergel : Is termed as Immunodiffusion Types : Based on the 1. Number of reactants diffusing 2 . Direction of diffusion Single diffusion in one dimension
- 17. 1 . Singlediffusion in one dimension Oudin procedure
- 18. 2 . Singlediffusion in two dimensions Radial immunodiffusion
- 19. Uses : 1. Forquantitative estimation of Abs & Ags in the serum 2. To measure IgG , IgM , IgA , and Complement 3. To measure Abs to Influenza virus in sera 4. To estimate serum transferrin & Alfa fetoprtein
- 20. 3 . Doublediffusion in one dimension Oakley – Fulthrope procedure
- 21. 4 . Doublediffusion in two dimensions Ouchterlony procedure
- 22. Uses : 1 .Small pox serodiagnosis 2. Identification of fungal antigens 3 . Antibodies to extractable nuclear antigens 4 . Eleks gel precipitation test
- 23. 3 . Precipitationin agar with electric field 1 . Immnunoelectrophoresis 2 . Counter current immunoelectrophoresis 3 . Rocket electrophoresis 4 . Two dimensional immunoelectrophoresis
- 24.
- 25. Main advantage : 1. The number of antigens can be identified 2 . To detect normal as well as abnormal proteins - serum & urine
- 26. 2 . Countercurrent immuno eletrophoresis
- 27. Uses : ⚫Commonly forHepatitis B surface antigens ⚫Alfa feto protein ⚫Hydatid and amoebic Ags ⚫Cryptococcal & meningococcal Ags in CSF
- 28. 3 . Rocketelectrophoresis : One dimensional single elctroimmunodiffusion
- 29. Uses : ⚫Mainly forquantitative estimation of Ag in serum
- 30. 4 . Laurell’stwo dimensional immunoelectrophoresis It is a 2 step procedure
- 31. 2 ) AGGLUTINATION: ⚫An Ag – Ab reaction in which a particulate Ag combines with its antibody resulting in formation of visible clumping of particles . TYPES : 1 . Direct agglutination 2 . Passive agglutination
- 32. 1 . Directagglutination : 1 . Slide agglutination 2 . Tube agglutination 3 . Heterophile agglutination 4 . Antiglobulin ( Coombs ) test
- 33. 1 . Slideagglutination : Uses : 1 . As a routine procedure to identify bacterial strains such as Salmonella , Shigella , Vibrio etc 2 . For blood grouping & cross matching
- 34. 2 . Tubeagglutination test : Uses : 1 . It is a standard method for quantitative estimation of antibodies in the serum 2 . Routinely used for serodiagnosis of Enteric fever , Brucellosis , typhus fever Widal Test: H’ - flagellar antigen - large, loose, fluffy clumps ‘O’ - somatic antigen - tight compact deposit
- 35. Complications : • Prozonephenomenon • Incomplete or blocking Abs
- 36. 3 . Heterophileagglutination test : Ex : 1 . Weil – Felix test 2 . Paul – Bunnel test 3 . Streptococcus MG agglutination test 4. Cold agglutination test
- 37. 4 . Antiglobulin( Coombs ) test ⚫ 2 types 1 . Direct coombs test 2 . Indirect coombs test
- 38. 1 . DirectCoombs test :
- 39. Uses : 1 .Detection of anti Rh antibodies 2 . For detection of incomplete antibodies in Brucellosis and other diseases
- 40. 2 . Passiveagglutination : Types : 1 . Latex agglutination tests 2 . Haemagglutination tests 3 . Coagglutination tests
- 41. 1 . Latexagglutination tests : Uses : 1 . For rapid identification of antigens of group B Streptococcus , Staphylococcus Neisseria meningitidis and Cryptococcus neoformans 2 . For detection of soluble microbial antigens in urine , spinal fluid , serum 3 . To detect RA factor , ASO , CRP , HCG
- 42. 2 . Haemagglutinationtests : Rose – waaler test : Rheumatoid arthritis
- 43. 3 ) Coagglutinationtest : Reverse passive agglutination
- 44. USES : ⚫For detectionof Cryptococcal Ags ⚫For diagnosis of Amoebic & Hydatid Ags ⚫For grouping of Streptococci , and Mycobacteria ⚫For typing of N . Gonorrhoea , Legionella
- 45. 3 ) COMPLEMENT– DEPENDENT SEROLOGICAL TESTS 1 . Complement fixation test 2 . Immune adherence test 3 . Immobilization test 4 . Cytolytic or cytocidal reaction
- 46. • Complement -Ag – Ab • Lyses erythrocytes Kills / lyse bacteria, immobilises motile organism • Promotes phagocytosis • Immune adherence • Tissue damage Complement fixation test
- 47. Principle Ag-Ab complex -fix complement Versatile, sensitive CFT - Two steps Five reagents - Ag Ab Complement Sheep RBC Amboceptor
- 49.
- 50. Uses : 1. Wassermann test for syphilis 2 . Test for antibodies to Mycoplasma pneumoniae Bordetella pertusis Many viruses Fungi such as Cryptococcus spp , Histoplasma , Coccidiodes immitis
- 51. Applications of CFT: ⚫Virology: Herpessimplex virus Picorna virus CFT is useful to identify exposure to Poliovirus but not for type-specific diagnosis. Influenza virus-CFT with the RNPAg of influenza virus type A,B & C are very useful as the antibodies are formed during infection only.CFT can also be done using V antigens for demonstration of strain specific Abs.
- 52. Contd…. Mycology: ⚫Histoplasmosis ⚫Coccidioidomycosis ⚫Sporotrichosis ⚫Pneumocystis jirovecii Parasitology: ⚫Protozoans- Trypanosomacruzi(Machado- Guerreiro ) Trypanosoma brucei Leishmania donovani(WKK antigen ) Toxoplasma gondii(Sabin
- 53. Contd…. ⚫Platyhelminthes- Schistosoma haematobium Fasciolahepatica Clonorchis sinensis Paragonimus westermanii ⚫Nemathelminthes-Trichinella spiralis Strongyloides stercoralis Loa loa Onchocerca volvulus
- 54. Virus Neutralisation Tests Neutralisationof viruses Neutralisation of bacteriophages Plaque inhibition Toxin Neutralisation Bacterial exotoxins - Tetanus, diphtheria Diphtheria toxin - Schick test
- 55. Anti streptolysin O(ASO) test – Streptococcal O hemolysin Nagler’s reaction – Clostridium perfringens - alpha toxin
- 56. OPSONISATION ‘Opsonin’ – Wright- 1903 ⚫Heat-labile substance - Facilitates phagocytosis (complement) ⚫Heat-stable serum factor – ‘bacteriotrophin’ ⚫‘Opsonic index’- Progress of resistance - ratio of phagocytic activity of patient’s blood for given bacterium to phagocytic activity of blood from normal individual
- 57. Radioimmunoassay (RIA) ⚫Radioisotopes conjugatedto antigen or antibody ⚫Binder - Ligand assay ⚫Analyte or ligand (Ag) – Substance whose concentration is to be determined ⚫Binder (Ab) – Binder protein which binds to the ligand ⚫RIA – Berson and Yallow, 1959
- 58. ⚫Enzyme-labelled conjugates ⚫Homogenous EIA ⚫HeterogenousEIA ENZYME IMMUNOASSAY
- 59. Homogenous EIA : •Does not require bound and free fractions to be separated • EMIT • Assay of haptens • Drugs – opiates , cocaine barbturates ,amphetamine - serum
- 60. Heterogenous EIA •Requires theseparation of free and bound fractions either by centrifugation or absorption on solid surfaces and washing ELISA
- 61. ELISA ⚫Involves the useof immunosorbent ⚫ for one of the components of the reaction: antigen or antibody ⚫96 – well microtitre plate ⚫Principle illustrated by outlining its application for detection of Rotavirus antigen in feces
- 62. ELISA – Types SandwichELISA Indirect ELISA Competetive ELISA Capture ELISA Cylinder or casstte ELISA
- 63. Sandwich ELISA ⚫Microtitre wellscoated with goat antirotavirus antibody ⚫Fecal samples added after washing ⚫Incubated, wells washed ⚫Guinea pig anti rotavirus antiserum labelled with alkaline phosphatase added ⚫Incubated, wells washed ⚫Substrate (Para – nitrophenyl phosphate) added ⚫Positive – colour change ⚫Negative – no colour change
- 64.
- 65. Indirect ELISA Anti HIVAb • Antigen adsorbed onto microassay plate wells ⚫Test serum added, incubated, washed ⚫Goat anti-human immunoglobulin antibody conjugated with horse radish peroxidase enzyme is added , incubate ⚫Wash, substrate (O- phynelene diamine dihydrochloride )adde ⚫Positive – colour change ⚫Negative – no colour change
- 66.
- 67.
- 68. ⚫Competitive ELISA –Similar to RIA, unknown antigen (sample) and known antigen (standard) compete with each other for fixed antibody ⚫Hapten detection ⚫Cylinder or cassette ELISA – Each sample tested in a separate disposable cassette, in-built controls, result read visually ⚫Ex : Tri - dot test
- 69.
- 70. Enzyme linked immunosorbentassay (EL ISA)
- 71. Applications ⚫Detection of infectiousdiseases – HIV, Hepatitis, EBV, CMV , Dengue, TORCH , Influenza ⚫Rota virus , ET of E .coli in feces ⚫Syphilis IgG /IgM , H pylori IgG , Ag ⚫Food toxins – aflatoxins ⚫Food adulterants – E.coli, Campylobacter, Salomonella Ag ⚫Mycobacterial antibody detection ⚫Human allergic specific IgE & IgA ELISA
- 72. ⚫Chemiluminescent compounds areused in CLIA as a label to provide signal during antigen - antibody reaction ⚫The signal (light) can be amplified, measured and concentration of analyte calculated Chemiluminescence immunoassay (CLIA)
- 73. Immunoelectroblot / westernblot techniques ⚫The technique is a combination of three procedures ⚫Separation of ligand and antigen by gel electrophoresis ⚫Blotting of electrophoresed ligand fraction ⚫Enzyme immunoassay to detect antibody – varius ligand fraction bands ⚫Confirmatory test - HIv
- 74.
- 75. IMMUNOFLUORESCENCE ⚫Fluorescence - Propertyof absorbing light rays of one wavelength and emitting rays of a different wavelength ⚫Direct immunofluorescence test - Specific antiserum labelled with a fluorescent dye, used for identification of antigens , bacteria , viruses
- 76. Direct immunofluorescence tests Diagnosisof rabies Ag – Brain smears Disadvantages – separate F conjugate > each Ag
- 77.
- 78. ⚫Immunohistochemical technique –Helps to visualise antigen - antibody reactions in situ ⚫Flow cytometry –Fluorescence technique used to identify and enumerate cells bearing a particular antigen or surface marker ⚫Cells are made to flow in a single stream through an electronic detection apparatus
- 79. Schematic diagram ofa flow cytometer
- 80. Applications Size ,granularity ,DNA or RNA content, Cellular Ags , receptor levels ⚫Differential leucocyte count ⚫T cell subsets - CD4 and CD8 counts in HIV patients ⚫Diagnosis, prognosis and treatment of cancer ⚫To study the cell cycle , apoptosis
- 81.