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Antigen and antibody reaction

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Antigen and antibody reaction

  1. 1. 1. Introduction.2. Salient Features of Antigen – Antibody Reaction.3. Strength of Antigen – Antibody Reaction.4. Properties of Antigen – Antibody Reaction.5. Types of Antigen – Antibody Reaction.6. Application of Antigen – Antibody Reaction.7. Conclusion.
  2. 2. INTRODUCTION:• The antigens and the antibodies combine specificallywith each other. This interaction between them iscalled Antigen-Antibody reaction.• It may be abbreviated as Ag – Ab reaction.• These form the basis for humoral immunity orantibody mediated immunity.• These reactions form the basis for detection ofinfectious disease causing agents and also some non-specific Ag’s like enzymes.
  3. 3. • When Ag – Ab reactions occur invitro, they areknown as serological reactions.• The reactions between Ag and Ab occur in threestages. In first stage the reaction involves formation of Ag-Ab complex. The second stage leads to visible events like precipitation, agglutination etc. The third stage includes destruction of Ag or its neutralization
  4. 4. Salient Features of Antigen – AntibodyReaction:• Specificity of Antigen – Antibody Reaction.• Immune complex.• Binding Site of Antigen – Antibody Reaction.• Binding Force of Antigen – Antibody Reaction.
  5. 5. Specificity of Antigen – Antibody Reaction:•Specificity refers tothe ability of anindividual antibodycombining site toreact with only oneantigenicdeterminant or theability of apopulation ofantibody moleculesto react with onlyone antigen.
  6. 6. •For example, the antibody produced against lensantigen will react only with lens-antigen. Similarly,the antibody produced against kidney antigen willreact with only kidney- antigen. A standard lock canbe opened by its own key only as one antibody canreact with its own antigen.Immune Complex:•An immune complex is formed from the integralbinding of an antibody to a soluble antigen.•The bound antigen acting as a specific epitope, boundto an antibody is referred to as a singular immunecomplex.
  7. 7. •Mechanisms of antigen-antibody interaction leadingto inflammation. Antigen-antibody immune complexformation results in complement activation,opsonization of target cells, assembly of membraneattack complexes and release of complementactivators for chemotaxis.•Fc receptor mediated cell activation triggers cellularresponses, such as phagocytosis, antibody-dependentcellular cytotoxicity (ADCC) and release ofinflammatory mediators. Ag + Ab Ag-Ab complex
  8. 8. Binding Site of Antigen – Antibody Reaction:• In antigen - antibody reaction, the antibody attacheswith the antigen.• The part of antigen which combines with antibody iscalled Epitope.• An epitope, also known as antigenic determinant, isthe part of an antigen that is recognized by theimmune system, specifically by antibodies, B cells, orT cells.•The part of an antibody that recognizes the epitope iscalled a paratope.
  9. 9. Binding Force of Antigen – AntibodyReaction:• The binding between antigen and antibody in ag – abreaction is due to three factors namely:  Closeness between antigen and antibody.  Non – covalent bonds or Intermolecular forces .  Affinity of antibody.
  10. 10.  Closeness between antigen and antibody: When antigen and antibody are closely fit, the strength of binding is great. When they are apart binding strength low. Non – Covalent Bonds: The bonds that hold the antigen to the antibody combining site are all non- covalent in nature. These include hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds. Affinity of antibody: Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody.
  11. 11. Strength of Antigen – Antibody reaction:•The non – covalentinteraction that formthe basis of antigen– antibody bindinginclude hydrogenbond, ionic bond,hydrophobicinteraction and Vander Waalsinteraction.
  12. 12. •A strong antigen – antibody interaction depends on avery close fit between the antigen and antibody whichrequires high degree of specificity.Properties of Antigen – Antibody Reaction:The properties of antigen and antibody can beexplained with the help of three points. They are:• Antibody Affinity.• Antibody Avidity• Cross reaction.
  13. 13. Affinity of Antibody:Interactions between antigen and antibody involvenon-covalent binding of an antigenic determinant(epitope) to the variable region (complementaritydetermining region, CDR) of both the heavy and lightimmunoglobulin chains. These interactions areanalogous to those observed in enzyme-substrateinteractions and they can be defined similarly. Todescribe the strength of the antigen-antibodyinteraction, one can define the affinity constant (K) asshown:
  14. 14. [Ab - Ag] Affinity K = = 104 to 1012 L/mol [Ab] [Ag]Avidity of Antibody:• It is the strength of the bond after the formation ofAg-Ab complexes.• It is used to denote the overall capacity of antibodiesto combine with the multivalent antigen.• A multivalent Ag has many types of antigenicdeterminants.• When injected into the blood, each antigenicdeterminant stimulates the production of a particularantibody.
  15. 15. The various antibodies produced by a single Agcombine with the different antigenic determinants ofthe Ag. nAb+ mAg ↔ AbnAgmWhere nAb is No. of Ab’s and mAg is No. ofAntigenic determinants.Cross Reaction:An antiserum raised against an Ag, can also react witha similar Ag of another type. This is called crossreaction and the Ag which produces the cross reactionis called Cross reactive Ag. But the strength of Abraised against its own Ag is strong.
  16. 16. • The bonds involved in cross reactions are weak.Example: The serum raised against albumin of hen’segg can cross react with albumin obtained fromduck’s egg.The antiserum raised against human insulin will reactwith the insulin of Pig, Sheep, whale etc.The antiserum raised against Pneumococcalpolysaccharides will react with E.coli, blood group Aand collagen Ag’s.
  17. 17. Types of Antigen – Antibody Reaction:The types of antigen – antibody reactions are:• Precipitation Reaction.• Agglutination Reaction.• Complement Fixation.• ELISA – Enzyme Linked ImmunoSorbent Assay.• Immunofluorescence.
  18. 18. Precipitation Reaction:When a soluble Ag combines with its Ab in thepresence of an electrolyte (NaCl) at a particulartemperature and pH, it forms an insoluble precipitateof Ag-Ab complex. The Ab causing precipitation iscalled Precipitin and the reaction is called asprecipitation reaction.
  19. 19.  Function of precipitation reaction: Precipitationoccurs in two media:Liquid.Gel.Precipitation in Liquid:Antigen – Antibody reaction perform by placing aconstant amount of antibody in a series of tubes andadding increased amount of antigen. Antigen –Antibody reacts together resulting in precipitation.Plotting the amount of precipitate against increasingantigen conc. Yeilds a precipitation curve.
  20. 20. Antigen Added
  21. 21. Precipitation curve shows three zones:1. Zone of Ab axis.2. Zone of equivalence.3. Zone of Ag axis.Precipitation in gel:Radial Immunodiffusion (Mancini) :In these methods agar gel or similar gels are used onplates or petriplates. Both Ag and Ab diffuse freely inthe gel system in all directions. At a certain pointdepending on the rate of diffusion and concentrationof the reactants, a zone of equivalence will be formed,which is seen as a visible precipitation.
  22. 22. If Ag or Abpreparationsare complex,multiplebands form.These areagain of 2types- Singlediffusionmethods anddoublediffusionmethods.
  23. 23. Agglutination Reaction:• When a particular Ag is mixed with its Ab’s in thepresence of electrolytes at a suitable temperature andpH, the particles are clumped or agglutinated.• The Ab of the serum causes the cellular Ag’s to formclumps and these are called Agglutinins.• The particulate antigens that are aggregated aretermed Agglutinogens. Slide agglutination: This is a rapid method todetermine the presence of agglutinating antibodies.
  24. 24. To a uniform suspension of particulate Ag, a drop ofsaline is added and then a drop of antiserum is added.The slide is gently rocked or a fine loop is used tomix the contents. If granulation occurs the test isPositive.It takes a minute for the test to complete and is visibleto the naked eye. Some times confirmation may bedone by observing slide under microscope. This test is used for blood grouping(Haemagglutination) and cross matching.
  25. 25. Tube agglutination: This is a standard method forquantitative estimation of Ab. The serum containingAb is diluted serially with saline in several small testtubes, to which a constant volume of Ag suspension isadded.A control tube is kept which has no antiserum. Thetubes are incubated until visible agglutination isobserved. The tube showing highest agglutination isreferred to as the titre.Tube agglutination is employed for the serologicaldiagnosis of typhoid, brucellosis and typhus fever.Widal test is used for the estimation of typhoid fever.
  26. 26. In this test Ab content of the patient’s serum, ismeasured by adding a constant amount of antigen(Solmonella typhi) to the serially diluted serum.
  27. 27. Passive agglutination test: It is similar tohaemagglutination test but the physical nature of thereaction is altered.The Ag is coated on the surface of a carrier particleand thereby helps to convert a precipitation reactioninto an agglutination reaction making the reactionmore sensitive. The carrier particles used can be RBC,latex particles or bentonite. Some times RBC coatedwith polystyrene (tanned RBC) can be used.When patients serum is mixed with these, it leads toagglutination. This test is used for the diagnosis ofRheumatoid arthritis.
  28. 28. Carrier Soluble Coated Particle Antigen ParticleCoated Particle Antibody Agglutination
  29. 29. Agglutination Inhibition:Provides a highly sensitive assay for small quantitiesof an Antigen.Example: First home pregnancy test
  30. 30. Complement Fixation:• Lysis of RBC or bacteria requires some non-specificunstable components of fresh serum which are calledcomplement.•This complement system comprises of 11 proteinsand are present in ever individual. They bind to Fccomponent of Ab involved in Ag-Ab complex. Thisability of the Ag-Ab complex to fix complement isused in complement Fixation tests.•In the first stage, the test Ag and the antiserum(heated to 56oC to inactivate complement) are mixedin the presence of known amount of complement.This is incubated at 4oC for 18h.
  31. 31. • If Ab specific for the Ag is present in the serum, Ag-Ab complex will be formed that will fix thecomplement.
  32. 32. ELISA – Enzyme Linked ImmunoSorbentAssay:• In 1971, enzyme labeled Ag’s and Ab’s weredeveloped as serological reagents for the assay ofAb’s and Ag’s.• These are very simple, sensitive, economic and lesshazard when compared to RIA.•The ligand used here is a molecule which can detectthe Ab and is covalently coupled to an enzyme suchas peroxidase, betagalactosidase, alkaline phosphataseetc.
  33. 33. ELISA is of 3types. Indirect ELISA: This technique is used for the detection of HIV. The envelop proteins are developed by recombinant technology and coated on the surface of the of microtire plates. Suspects serum is added, and unbound proteins are washed off. Sandwich ELISA: Used to detect the presence of Ag in a sample. The well is coated with Ab specific to the Ag and then suspect serum is added allowed to react. The wells are washed to remove unbound Ag’s.
  34. 34. Then a labeled Ab against a different epitope of theAg is added. Unbound Ab’s are removed by washingand this is followed by addition of colored substrateand development of color. The intensity of color isdirectly proportional to the concentration of the Ag inthe serum. Competitive ELISA: Another variation for measuring amounts of antigen is competitive ELISA. In this technique, antibody is first incubated in solution with a sample containing antigen. The antigen-antibody mixture is then added to an antigen coated micro titer well.
  35. 35. The more antigen present in the sample, the less freeantibody will be available to bind to the antigen-coated well. Addition of an enzyme-conjugatedsecondary antibody (Ab2) specific for the isotype ofthe primary antibody can be used to determine theamount of primary antibody bound to the well as in anindirect ELISA.Immunofluorescence:•Fluorescence is the property of absorbing light raysof one particular wavelength and emitting rays with adifferent wave length.• Fluorescent dyes show up brightly under UV light asthey convert into visible light.
  36. 36. Coons et al (1942) showed that labeled dyes can beconjugated to Ab’s and these labeled antibodies canbe used to detect Ag’s.•Dyes that are commonly used include:Fluorescein, an organic dye that is the most widelyused label for immunofluorescence procedures,absorbs blue light (490 nm) and emits an intenseyellow-green fluorescence (517 nm).Phycoerythrin is an efficient absorber of light (~30-fold greater than fluorescein) and a brilliant emitter ofred fluorescence, stimulating its wide use as a labelfor immunofluorescence.
  37. 37. Application of Antigen – Antibody Reaction:The chief use of antigen-antibody reactions are:• Determination of blood groups for transfusion.• Serological ascertainment of exposure to infectiousagents.• Development of immunoassays for thequantification of various substances.• To detect the presence or absence of protein inserum.• Determining the characteristics of certain immuno-deficiency disease.
  38. 38. Conclusion:Thereby we conclude up this topic with a quickreview about Antigen – Antibody Reaction. Theantigens and the antibodies combine specifically witheach other. This interaction between them is calledAntigen-Antibody reaction. As per the currentrequirement of the syllabus, the topic has beenincluded, right from its features till the types ofreaction . We show our gratitude towards all of you .Hope this project satisfy the required information byyou and any resemblance of mistakes are dulyapologized.

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