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Similar to Topic: Immunology: antigen: antibody reactions
Topic: Immunology: antigen: antibody reactions
- 2. Introduction Immunity Antibody Antigen Antigen antibody reactionand their applications Diagnostic testsofperiodontal disease activity Conclusion References
- 3. ▻ The termimmunityis derived fromimmunitas (Latinfor exemption fromcivic duties or paying taxes) ▻ The term‘immunity’is defined as resistance exhibited by thehost against any foreignantigen including microorganisms. ▻The ability of an organism to resist a particular infection or toxin by the action of specific antibodies or sensitized white blood cells is called immunity.
- 4. An antigen isa substance which when introduced intoa body evokes immune response toproduce a specific antibody withwhich itreacts in an observable manner. Itcan be classified as- A. Complete Antigen B. Incomplete Antigen (Haptens) ‣ Complete antigens are substances which can induce antibody formationby themselves and can react specifically withthese antibodies.
- 5. Haptens are substancesunable toinduce antibody formation on itsown butcan become immunogenic (capable of inducing antibodies) when covalently linked to proteins,called carrierproteins.They can be simple or complex. Proantigens are low molecular weightsubstances which do not induce antibody formationbutcan cause delayed hypersensitivity reaction. Epitope is thesmallest unitofantigenicity. The combining siteon theantibody molecule, corresponding totheepitope is called Paratope.
- 7. Foreignness Chemical nature Size Organ specificity Heterophilespecificity Auto specificity Antigenic specificity Species specificity Susceptibility totissue enzymes
- 8. These are moleculesthatcan interactwithantigen presenting cells and T lymphocytes in a non specific manner. These antigens do notinvolve theendocytic processing as required in typicalantigen presentation. Viral proteins and staphylococcal enterotoxins are examples ofsuperantigens.
- 9. These are substanceswhich are formed in theserum and tissue fluidsin response toan antigen and react withthatantigen specifically and in some observable manner. Secreted by plasma cells, occur in twophysical forms, a soluble form thatis secreted fromthecell, and a membrane-bound form thatis attached tothesurface ofa B cell and is referred toas the B cell receptor (BCR). The BCR is found only on thesurface ofB cells and facilitates the activation ofthese cells and theirsubsequent differentiation intoeither antibody factoriescalled plasma cells or memory B cells thatwill survive in thebody and remember thatsame antigen so theB cells can respond fasterupon futureexposure
- 11. Its uses are 1.In vivo Forms basis ofimmunity against infectious diseases May lead totissue injuryin hypersensitivity reactions and autoimmune diseases 2. In vitro For diagnosis ofinfections Helpful in epidemiological studies For identificationofenzymes Detection and quantitationofantigens or antibodies
- 12. Reaction is specific,an antigen combines only withits homologous antibody and vice versa. However cross reactions may occur due toantigenic similarity. Entire molecules ofantigen and antibody react and not the fragments. Only thesurface antigens participate in theantigen antibody reaction. The reaction is firmbutreversible. The firmness of combination depends on theaffinityand avidity.
- 13. Precipitation reactions Agglutination Complement fixationtest Neutralisation test Opsonisation Immunofluorescence Radioimmunoassay Enzyme linked immunosorbent assay Chemiluminescence assay Immunoelectronmicroscopic tests Immunoblotting
- 14. When a solubleantigenreacts withitsantibody in the presence ofelectrolytes atan optimal temperature and pH, antigen antibody complex formsan insoluble precipitate that usually sediments atthebottomofthe tube and itis called precipitation. Precipitation may occur in liquid media or in gels such as agar, agarose or polyacrylamide gels When instead ofsedimenting, theprecipitate is suspended as floccules thereaction is called flocculation
- 16. Identificationofbacteria.eg Lancefield groupingof streptococcus Detection ofantibody fordiagnostic purposes.eg VDRL in syphilis Forensic application in identificationofhuman blood and seminal stains Testing forfood adulterants To standardise toxinsand antitoxins
- 18. Itis an antigenantibody reaction in which a particulate antigen combines withitsantibody in presence of electrolytesatan optimal temperature and pH resultingin visible clumping of particles. Latticeformationhypothesis holds good foragglutination also. Occasionally incomplete antibodies (eg antiRh and anti Brucella) are formedthatcombine withtheantigen butdo not cause agglutination.They actas “blocking antibodies” inhibiting theagglutinationby thecomplete antibody added subsequently.
- 20. Principle The antigen antibodycomplexes have abilityto‘fix’ complement. This reaction has no visible effect. To detectfixationofa complement, an indicator system consisting ofsheep erythrocytes coated withamboceptor ( rabbitantibody tosheep erythrocytes) isused.
- 22. Bacterial exotoxins arecapable ofproducing neutralising antibodies (antitoxins)which play a role in protectionagainst diseases such as diphtheria and tetanus. The t oxicity of bacterial endotoxins is not neautralised by antisera.
- 25. Itis a processby which a particulateantigen becomes more susceptible tophagocytosis. Opsonic index is defined as ratioofphagocytic activityofthe patient’s blood fora given bacterium tothatofa normal individual. Phagocytic index is theaverage number ofphagocytosed bacteria per polymorphonuclear leukocytes fromstained blood films. Phagocytic index denotes thephagocytic activityofblood and thushelps in measuring opsonic index.
- 27. Fluorescence is thepropertyofcertain dyes which absorb rays ofone particularwavelength(ultraviolet light) and emit rays witha differentwavelength(visiblelight). Most commonly used dyes are 1.fluorescin isothiocyanate 2.lissamine rhodamine Two types are Direct and Indirect.
- 28. Specimen (Positive forantigen) + labelledantibodies ▼ Flurescence observed ▼ antigen is present in the specimen
- 29. It is asensitive method to diagnose rabies by detection of rabies virus antigen in brain smears. It is commonly employed for detection of bacteria,viruses or other antigens in blood, CSF,urine,faeces,tissues and other specimens. Note – A separate specific fluorescent labelled antibody has tobe prepared against each antigen tobe tested.
- 30. Unlike directmethod, antigenis known in this method. Letshave a look attheflowchartforclear understanding.
- 32. Berson and Yallow(1959)firstdescribed radioimmunoassay and since then ithas been utilisedfor quantitationofhormones, drugs, hepatitis B surface antigen, IgE and viral antigens. This testcan detectantigens uptopicogramquantities. It is based on competition for a fixed amounts of specific antibody between a known radiolabelled antigen and an unknown unlabelled testantigen.
- 34. The RAST test(radioallergosorbent test)isan example of radioimmunoassay. Itis used to detect thecausative allergen foran allergy.
- 35. Advantages are- Highly sensitive Canbe used fordetection ofsmallquantities Quantification possible Disadvantages are – Expensive Requires isotopes
- 36. Enzyme Linked ImmunosorbentAssay is as sensitive as radioimmunoassay. Requires only microlitre quantitiesoftestreagents The enzyme acts on substratetoproduce a color in a positive test. Itcan be used fordetection ofantigen or antibody Types are Sandwich, Indirect, Competitive orCassette Elisa
- 42. Detection ofHIV Antibodiesin serum Detection ofmycobacterial antibodies in tuberculosis Detection ofRotavirus in faeces Detection ofHepatitisB markers inserum Detection ofenterotoxin ofE.coli in faeces
- 44. Immunoferritin tests Immunoelectron microscopy Immunoenzymetests Immunoblotting
- 45. Ferritin(electron dense substance)conjugated antibody is used toreact withan antigen.This antigen antibody reaction can be visualised under microscope Used in identificationofLegionella pnemophila
- 46. Viral particles aremixed withspecific antisera and are observed under electron microscope. These are seen as clumps. Used in detection ofHepatitisA virus
- 47. Tissue sections aretreatedwithperoxidase labelled antisera todetectcorresponding antigen. The peroxidase bound to antigen is visualised under electron microscope.
- 48. In immunoblots, antibodiescan detectproteins in mixtures.The mixture ofproteins is electrophoretically separated in a gel. The separated proteins are transferredfromgel to nitrocellulose paper. These nitrocellulose paper stripsare reacted withtest sera and subsequently withenzyme conjugated anti human immunoglobulin.
- 50. This testhas beenwidely used toconfirmtheELISA positive HIV antibody cases. The above procedure may also be applied toanalyse DNA or RNA. When DNA is transferred on nitrocellulose strips fromgel, thistestis referredtoas Southern Blot test.Similarily, if RNA is transferreditis named as Northern Blot test.
- 51. Potential biomarkers ofthediseaseactivitywould need tobe involved in thedisease process in some way and therefore need toundergo extensive and careful basic research investigation before undergoing clinical evaluation. Only when thesource,precise nature and therole of potential marker are known and understood can clinical evaluation be meaningful.
- 52. Potential sources fromwhichmarkers can be obtained are- 1.blood or serum 2.saliva 3.subgingival plaque sample 4.gingival crevicular fluid
- 53. 1) Bacteria andtheirproduct 2) Inflammatory and immune products 3) Hydrolytic enzymes oftissue origin and those released from dead cells 4) Connective tissue degradation products 5) Products ofbone resorption
- 54. Despite thecomplex interactionthatexists between bacteria and host, a number ofpossible pathogens have been selected on thebasis oftheirassociation with disease progression, animal pathogenicity and their possesion of virulence factorswhich could damage the tissues(Genco et al 1988,Listgarten1992,Socransky and Haffajee 1992).
- 55. Porphyromonas gingivalis Prevotella intermedia Bacteroidesforsythus Actinobacillus actinomycetamcomitans Capnocytophaga ochracea Eikenella corrodens Campylobacter recta Fusobacterium nucleatum Treponema denticola
- 56. There is noevidence forany one specific pathogen in chronic periodontitisand therefore itmay be considered as a non specific bacterial disease (Theilade 1986). The bacteria listedhere tend tobe present in higher numbers atactive disease sites (Socransky and Haffajee 1992). Bacterial species numbers may be determined in variety of ways (Listgarten 1992)and these include thefollowing –
- 57. 1.Dark ground orPhasecontrast microscopy 2.Culture techniques 3.Immunological assays 4. DNA probes 5. BANA assays
- 58. They use eitherpaperpoints or curettebacterial sampling frompocket and include the following- Evalusite (Kodak) This utilizes ELISA’s using antibody against P.gingivalis, P intermedia,A. actinomycetemcomitans. This reaction is carried outin simple chairside kit. Subgingival plaque samples are reacted withthe antibodies and detection substratein a multiwellreaction dishes.
- 59. Omnigeneand BTD(Bio technicaldiagnostics) These are DNA probe systems fora number of subgingival bacteria. A paper pointsample ofsubgingival plaque is placed in a container and sent tocompany for assay. Probes are available forA. actinomycetemcomitans, P.gingivalis, P intermedia, E.corrodens, F.nucleatum,C. Recta and T.denticola
- 60. Perioscan (Oral BLaboratories) This is a chairside testkitsystem which utilises BANA Test forbacterial trypsinlike proteases. They are mainly produced by P.gingivalis and in lesser amounts by B.forsythusand T.denticola. A subgingival plaque sample is reacted in thekitwiththesubstratelinked toa color detection system.
- 61. DiamondProbe/Perio 2000system Combines featureofperiodontalprobe withdetection of volatile sulphur compounds in periodontal pocket.
- 62. Prototek Given by Coxetal in1990, thiscan be used todetect bacterial proteases arg-gingipain/gingivain and DPP in GCF. This is notcommercially available yet.
- 63. Predictive ofdisease activity Simple Resultsavailable in short time Visual result
- 64. Polymicrobial nature ofthedisease Most are notpredictiveofthedisease activity Site Can only detectbacteria we look for Special laboratory required Cost
- 65. Those ofpossible relevancetoperiodontal pathology are- Immune response Antibody TotalIgG and IgG subgroups Complement Inflammatory mediators Arachidonic acid derivatives Cytokines
- 66. Porphyromonas gingivalis hasbeen implicated as major periodontal pathogen and ithas been reported thata positive co relationexists between IgG levels toP.gingivalis and severity of periodontal disease(Gmur etal 1986;Lamster etal 1998;Blackburn 1992) Furthermore elevation in P.gingivalis specific IgG2, IgG1 and IgG4 in rapidly progressing periodontitisand adult periodontitis have been reported.(Kinane etal 1999) Complement proteins are present in GCF fromsiteswith inflammation and thesplitfragments C3 and Factor B have been detected during gingivitis(Patters etal 1989). However, none of these factorshave been associated withdisease activity.
- 67. Periotemp Ithas been developedtomeasure small changes in sublingual and subgingival temperatures and positive cross sectional comparision withclinical parameters have been found. Increased subgingival temperature has been positively corelated withincreased pocket depths, decreased attachment levels, clinical parameters ofgingival inflammation,higher proportions ofputativeperiodontal pathogens and gingival crevicular fluidenzymes( Dinsdale etal 1997, Haffajee etal 1992, Wolffetal 1997)
- 68. Although GCF PGE2has considerable potential as a screening test for periodontal activity, no commercial efforts are currently underway todevelop one. Cytokines are also assayed using ELISA techniques which could be developed intochairside kits.However at present the predictive abilityofthese markers is stillin doubt.
- 69. Only GCF PGE2has been shown tobe predictive of disease activityin longitudinal studies. ELISA techniques can be used todetectcytokines and PGE2 which could be developed intochairside kitswhich are simple touse. ELISA can be read aftershort time. They can be shown tothepatientand related tothe tooth site.
- 70. The choice ofmostappropriate biomarker may stillbe difficultatpresent stateofknowledge. There is difficultyin determining thesites tosample and when tosample them. Cost
- 71. 1.Proteolytic enzymes Collagenases Elastase Cathepsin G,B and D Dipeptidylpeptidases Tryptase 2.Hydrolytic enzymes Aryl sulphatase Beta glucuronidase
- 72.
- 73. Collagenase 2(MMP 8),collagenase 1(MMP 1)and collagenase 3(MMP 13)activityare present in gingival tissue,saliva and gcfand can be assayed biochemically withcollagen substrates(Sorsa etal 1990)or withELISA technique (Ingman etal 1996,Matsuki etal 1996). Elastase in gingival tissues is produced by PMN’s and is held in an inactive formprobably bound withan inhibitor.Itis inhibited by secretory leukocyte protease inhibitor(SLPI) and skin antileukoproteinase (SKALP) (Cox etal 2001)
- 74. In humans, GCFtryptaseactivitycorelates withclinical parameters ofdisease severity including probing attachment and bone lossand significantlyreduces followingperiodontal treatment(Eley and Cox 1992). Both tissue DPP 2AND 4corelate withclinical parameters of disease severity and significantlyreduce following periodontal treatment(Cox and Eley 1992)
- 75. Periocheck These system detectspresence ofneutral proteinases such as collagenases in GCF. A paper stripused toobtain GCF sample is placed in contactwithcollagen gel to which a blue dye has been covalently bonded. It is then incubated at43 degrees. Intensityαamount ofenzyme present in the sample
- 76. Prognostik (Dentsply) This systemdetects presence ofserine proteases, elastases in GCF samples Intensity of fluorescence α amount ofenzyme in the sample
- 77. βglucuronidase Itis a diagnostickitthatuses a histochemical substrate forthe enzymes coupled toa color detection system which is releasedif theenzyme attacks the substrate( Lamster etal 1988,1991, 1994) Cysteine andserineproteinases Developed in conjugation in researchers fromPrototek has followingadvantages over it-
- 78. Can be modifiedtodetectnumber ofdifferent proteinases including DPP 2and 4, tryptase etc. GCF sample is collected on a normal paper stripand thereforeleaving group are notintroduced intothe gingival crevice unlike Dentsply Color detection system forthismethod is more convenient foetheuse in dental practice as itrequires no special apparatus.
- 79. Simple Can be readaftershort time Can be shown topatientand related totooth site
- 80. Cost Selection ofsite When tosample Choice ofmost appropriate biomarker may stillbe difficultatpresent stateofknowledge
- 81. Since cell deathis an essential component of periodontal tissue destruction theyshould be released during this process and should pass withthe inflammatory exudate intoGCF. Aspartate amino transferase(AST) Lactate dehydrogenase(LDH)
- 82. Periogard AST inGCF test kit
- 83. Both ofthese potentialmarkers associate withdisease activity butdo notpredict it Simple Can be shown to patient Can be read aftershort period Can be related totoothsite affected
- 84. Cost Selection ofsite When tosample Choice ofmost appropriate biomarker may stillbe difficultatpresent stateofknowledge
- 85. The detection ofbreakdownproducts ofnormal components ofextracellular matrixcould be indicative of tissue breakdown.
- 86. Most complex andexpensive Long collection times Notsuitable forchairside
- 87. Possible markers ofboneresorption and hence periodontal disease activityare- Osteonectin Bone phosphoprotein (N-propeptide) Osteocalcin Telopeptides oftype1collagen Collagen 1 Proteoglycans
- 88. Most ofthepotential markersin thisgroup could be easily adapted intotestkitsas theirdetection involves use of specific monoclonal or polyclonal antibodies. Osteocalcin- ELISA(Kunimatsu etal 1993)or RIA(Giannobile 1995) CTP- RIA(Giannobile 1995) Osteonectin, N Propeptide- ELISA(Bowers etal 1989)
- 89. Some ofthese potentialbiomarkersassociate with the disease butdo notpredict it Can be read afterrelativelyshortperiods Can be shown tothe patient
- 90. Choice ofmost appropriatebiomarker is difficultat the present stateofknowledge Difficultyin determining sitestosample and when to sample them Cost
- 91. Therefore we seetheapplication ofantigen antibody reactions in development ofvarietyof diagnostic tests. Only markers withcredentials should be used in clinical practice forthereasons listed below- 1. To prevent destructive disease 2. To prevent progression ofthe disease 3. To identifyhigh risk patients 4. To targettreatmenttospecificsites 5. To monitortheeffectsofperiodontaltreatment
- 92.