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Antigen-Antibody Interactions

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Antigen-Antibody Interactions -
Antigen-antibody interactions depend on four types
of noncovalent interactions: hydrogen bonds, ionic
bonds, hydrophobic interactions, and van der Waals
interactions.
 The affinity constant, which can be determined by
Scatchard analysis, provides a quantitative measure of the
strength of the interaction between an epitope of the antigen
and a single binding site of an antibody. The avidity reflects
the overall strength of the interactions between a
multivalent antibody molecule and a multivalent antigen
molecule at multiple sites.
 The interaction of a soluble antigen and precipitating antibody
in a liquid or gel medium forms an Ag-Ab precipitate.
Electrophoresis can be combined with precipitation
in gels in a technique called immunoelectrophoresis.
 The interaction between a particulate antigen and agglutinating
antibody (agglutinin) produces visible clumping, or
agglutination that forms the basis of simple, rapid, and
sensitive immunoassays.
 Radioimmunoassay (RIA) is a highly sensitive and quantitative
procedure that utilizes radioactively labeled antigen
or antibody.
 The enzyme-linked immunosorbent assay (ELISA) depends
on an enzyme-substrate reaction that generates a
colored reaction product. ELISA assays that employ
chemiluminescence instead of a chromogenic reaction are
the most sensitive immunoassays available.
 In Western blotting, a protein mixture is separated by electrophoresis;
then the protein bands are electrophoretically
transferred onto nitrocellulose and identified with labeled
antibody or labeled antigen.
 Fluorescence microscopy using antibodies labeled with
fluorescent molecules can be used to visualize antigen on
or within cells.
 Flow cytometry provides an unusually powerful technology
for the quantitative analysis and sorting of cell populations
labeled with one or more fluorescent antibodies.

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Antigen-Antibody Interactions

  1. 1. MADE BY - JAYSHREE CHATTERJEE MSc 2 nd sem ( BIOTECHNOLOGY ) Place - Jabalpur (MP)
  2. 2. CONTENTS  Antigen And Antibody  Antigen – Antibody Interaction  Stages Of Ag – Ab Reactions  S a l i e n t F e a t u r e s O f An t i g e n – An t i b o d y R e a c t i o n  Chemistry Of Antigen Antibody Binding  Types Of Antigen – Antibody Interaction  Application Of Antigen – Antibody Reaction  Summary
  3. 3. ANTIGEN - Molecules that can be recognized by the immunoglobulin receptor of B cells or by the T-cell receptor when complexed with major histocompatibility complex (MHC) are called antigens. The word antigen is a shortened form of the words “antibody generator.” The antigens that are not immunogenic but can take part in immune reactions are termed as haptens. ANTIBODY - Antibodies also known as Immunoglobulin are antigen- binding proteins present on the B-cell membrane and secreted by plasma cells. Three types of globulins are present in the blood: alpha, beta, and gamma. The antibodies are the gamma globulins. Antibodies are one of the major plasma proteins, and against infection often referred to as “first line of defense”. The most important function of antibodies is to confer protection against microbial pathogens. What Is Antigen & Antibody ?
  4. 4.  The Antigen – Antibody interaction is a bimolecular association similar to an enzyme-substrate interaction, with an important distinction: it does not lead to an irreversible chemical alteration in either the antibody or the antigen. The association between an antibody and an antigen involves various noncovalent interactions between the antigenic determinant, or epitope, of the antigen and the variable-region (VH/VL) domain of the antibody molecule, particularly the hypervariable regions, or complementarity - determining regions (CDRs).  They form the basis for humoral/antibody mediated immunity. They are used for detection of disease causing agents & some non-specific Ag’s like enzymes. When Ag- Ab reaction occurs in-vitro they are known as ‘serological reactions’. The reactions b/w Ag & Ab occurs in 3 stages: 1st = formation of Ag-Ab complex. 2nd = leads to visible events like precipitation, agglutination etc. 3rd = destruction of Ag or its neutralization.
  5. 5. STAGES OF Ag – Ab REACTIONS Primary stage: a) Initial interaction between Ag & Ab – INVISIBLE b) Rapid, occurs at LOW TEMPERATURES. c) Reaction is REVERSIBLE. d) Ag & Ab is bound to each other by WEAK VAN-DER WAAL’S FORCES, IONIC BONDS & HYDROGEN BONDING.
  6. 6. Secondary stage IT IS AN IRREVERSIBLE INTERA- -CTION WITH VISIBLE EFFECTS. Demonstrable events – a) Precipitation b) Agglutination c) Lysis of cells d) Killing of live antigens e) Neutralization of toxins f) Complement fixation g) Immobilization of motile organisms. h) Enhancement of phagocytosis.
  7. 7.  Specificity of Antigen – Antibody Reaction.  Immune complex.  Binding Site of Antigen – Antibody Reaction.  Binding Force of Antigen – Antibody Reaction. SALIENT FEATURES OF ANTIGEN – ANTIBODY REACTION
  8. 8. Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. Each antibody binds to a specific antigen; an interaction similar to a lock and key. 1). SPECIFICITY
  9. 9. An immune complex is formed from the integral binding of an antibody to a soluble antigen. The bound antigen acting as a specific epitope, bound to an antibody is referred to as a singular immune complex. Ag + Ab  Ag-Ab complex 2). IMMUNE COMPLEX
  10. 10. 3). Binding Site of Antigen – Antibody Reaction In antigen - antibody reaction, the antibody attaches with the antigen. The part of antigen which combines with antibody is called Epitope.  An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a paratope.
  11. 11. 4). Binding Force of Antigen – Antibody Reaction  The noncovalent interactions that form the basis of Ag-Ab binding include hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions. Because these interactions are individually weak, a large number of such interactions are required to form a strong Ag-Ab interaction.  Furthermore, each of these noncovalent interactions operates over a very short distance, generally about 1 x 10¯ mm (1 angstrom, Å); consequently, a strong Ag- Ab interaction depends on a very close fit between the antigen and antibody. 7
  12. 12. Chemistry Of Antigen Antibody Binding :- The interaction of an antigen antibody is a reversible binding process that requires several non-covalent interactions like hydrogen bonds, electrostatic forces and hydrophobic interactions. Affinity and Avidity between the antigen antibodies also play a major role in their interaction. Normally antigen-antibody binding site on antibodies are more or less flat and hence spacious so that they can attach large complexes or structures.
  13. 13. AFFINITY - Affinity denotes the intensity of attraction between antigen and antibody. Low-affinity antibodies bind antigen weakly and tend to dissociate readily, whereas high-affinity antibodies bind antigen more tightly and remain bound longer. [Ab-Ag] Affinity K = [Ab] × [Ag] AVIDITY - Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. The various antibodies produced by a single Ag combine with the different antigenic determinants of the Ag. nAb+ mAg ↔ AbnAgm Where nAb is no. of Ab’s and mAg is no. of Antigenic determinants.
  14. 14. Agglutination reaction PRECIPITATION REACTION Radio-immuno assay Immobilization test Neutralization reaction ImmunofluorescenceOpsonisation Complement fixation test Enzyme Immunoassay Types Of Antigen – Antibody Interaction
  15. 15. PRECIPITATION REACTION  Precipitation refers to an antigen-antibody reaction between a soluble antigen & its antibody resulting in the formation of insoluble precipitate. The antibody causing precipitation is called PRECIPITIN.  Precipitation occurs in two media: a) Liquid or solution, b) Gel - agar, agarose or polyacrylamide.  Formation of an Ag-Ab lattice depends on the valency of both the antibody and antigen.  The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments. The antigen must be either bivalent or polyvalent; that is, it must have at least two copies of the same epitope, or have different epitopes that react with different antibodies present in polyclonal antisera.
  16. 16. Precipitation In Liquid Or Solution  Soluble antigen + antibody (in proper proportions)  visible precipitate  Ring test and flocculation test are examples of precipitation in solution. a) Ring test :- In this test, antigen solution is layered over antiserum in a test tube or capillary tube. Precipitation between antigen & antibodies in antiserum solution is marked by the appearance of a ring of precipitation at the junction of two liquid layers. C-reactive protein (CRP) & streptococcal grouping by the Lancefield methods are the examples of the ring test. Test - tube Capillary - tube
  17. 17. Precipitation curve  Plots of the amount Ag/Ab complexes precipitated when increasing Ag concentrations are added to constant concentration of Ab. It reveals 3 zones: 1. Zone Of Antibody Excess (PROZONE) - precipitation is inhibited and antibody not bound to antigen can be detected in the supernatant. 2. Zone Of Equivalence - maximal precipitation in which antibody and antigen form large insoluble complexes and neither antibody nor antigen can be detected in the supernatant. 3. Zone Of Antigen Excess (POSTZONE) - precipitation is inhibited & Ag. not bound to Ab. can be detected in the supernatant.
  18. 18. Precipitation in Gels  The precipitation test in agar gel is termed as immunodiffusion test. In this test, reactants are added to the gel and antigen – antibody combination occurs by means of diffusion. The rate of diffusion is affected by the size of the particles, temperature, gel viscosity, amount of hydration, and interactions between the matrix and reactants.  Immunodiffusion reactions have the following advantages: ■ In this test, the line of precipitation is visible as a band, which can also be stained for preservation. ■ The test can be used to detect identity, cross-reaction, and nonidentity between different antigens in a reacting mixture.  Immunodiffusion reactions are classified based on the - (a) number of reactants diffusing and (b) direction of diffusion, as follows:
  19. 19.  Single Diffusion In One Dimension (Oudin Procedure) :-  Ab is incorporated in agar gel in a test tube & Ag solution is layered over it.  Ag diffuses downward through the agar gel – forming a line of precipitation.  The number of precipitate bands shows the number of different antigens present in the antigen solution.  Double diffusion in one dimension (Oakley-Fulthorpe procedure) :-  Ab is incorporated in agar gel. Above which is placed a column of plain agar.  The Ag is layered over it. The Ag & Ab move towards each other through the intervening column of plain agar & form the precipitate. Ag (High Concentration) Two different precipitates (two Ag with different molecular weights) Agar with Antiserum which reacts with Two Ag’s Ag – Ab Reaction Visible Precipitation Antibody
  20. 20.  Single Diffusion In Two Dimension (Radial Immunodiffusion) :-  In this method antiserum solution containing antibody is incorporated in a agar gel on a RID slide or petri plate.  Wells are cut and antigen is applied in the gel. Then Ab present in the gel reacts with the Ag which diffuses out of the well. Precipitation rings are formed around the wells. Double Diffusion In Two Dimensions ( O ucht erl ony Procedure) :-  Ab is incorporated in agar gel.Above which is placed a column of plain agar. The Ag is layered over it. The Ag & Ab move towards each other through the intervening column of plain agar & form the precipitate.  Antiserum – central well. Different Ags in the surrounding wells. Reaction of identity Lack of relatedness P a r t i a l i d e n t i t y RID DD
  21. 21. AGGLUTINATION REACTION  The interaction between antibody & a particulate (Insoluble) antigen results in visible clumping called agglutination. The antibodies that cause agglutination are called Agglutinins & the particulate antigens aggregated are called Agglutinogens.  Particulate antigen include: a) bacteria, b) white blood cells, c) red blood cells, d) latex particles . Agglutination Test antige n antibody positive negative
  22. 22. Types of agglutination reactions Direct Agglutination Passive Agglutination Tube Agglutination Antiglobulin (Coombs’) Test Slide Agglutination Heterophile Agglutination Direct Coombs Test Indirect Coombs Test Latex Agglutination Test Coagglutination Test Hemagglutination Test
  23. 23. APPLICATIONS :- - Blood typing. - Bacterial infections. - Viral Infections. LIMITATIONS :- - Time consuming (1 day) - Cannot distinguish IgG from IgM.
  24. 24. COMPLEMENT FIXATION TEST
  25. 25. Enzyme – Linked Immunosorbent Assay (ELISA) •In 1971, enzyme labeled Ag’s and Ab’s were developed as serological reagents for the assay of Ab’s and Ag’s. • These are very simple, sensitive, economic and less hazard when compared to RIA. •The ligand used here is a molecule which can detect the Ab and is covalently coupled to an enzyme such as peroxidase, betagalactosidase, alkaline phosphatase etc. ENZYME IMMUNOASSAY Types Of Enzyme-linked Immunosorbent Assay (ELISA)
  26. 26. OPSONIZATION
  27. 27. Application of Antigen – Antibody Reaction The chief use of antigen-antibody reactions are: a. Determination of blood groups for transfusion. b. Serological ascertainment of exposure to infectious agents. c. Development of immunoassays for the quantification of various substances. d. To detect the presence or absence of protein in serum. e. Determining the characteristics of certain immuno- deficiency disease.
  28. 28. SUMMARY  Antigen-antibody interactions depend on four types of noncovalent interactions: hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions.  The affinity constant, which can be determined by Scatchard analysis, provides a quantitative measure of the strength of the interaction between an epitope of the antigen & a single binding site of an antibody. The avidity reflects the overall strength of the interactions between a multivalent antibody molecule and a multivalent antigen molecule at multiple sites.  The interaction of a soluble antigen and precipitating antibody in a liquid or gel medium forms an Ag-Ab precipitate. Antibody and soluble antigen interacting in aqueous solution form a lattice that eventually develops into a visible precipitate. Antibodies that aggregate soluble antigens are called precipitins.  The interaction between a particulate antigen and agglutinating antibody (agglutinin) produces visible clumping, or agglutination that forms the basis of simple, rapid, and sensitive Immuno- assays.  The enzyme-linked immunosorbent assay (ELISA) depends on an enzyme-substrate reaction that generates a colored reaction product. Other than these many types such as complement fixation test, opsonization, neutralization etc. are the reactions which takes part in Ag – Ab interaction.
  29. 29. References :-  Textbook of Microbiology and Immunology (2nd Ed) - Subhash Chandra Parija .  Kuby Immunology (5th Ed) - Judith A. Owen, Jenni Punt, Sharon A. Stranford, Patricia P. Jones .  Immunology & Microbiology – A.Mani, L.M.Narayanan, Dulsy Fatima, A.M.Selvaraj, N.Arunugam (Saras Publication)  https://www.google.co.in/search?q=complement+fixation+test+ procedure&source  www.google.co.in
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Antigen-Antibody Interactions - Antigen-antibody interactions depend on four types of noncovalent interactions: hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions.  The affinity constant, which can be determined by Scatchard analysis, provides a quantitative measure of the strength of the interaction between an epitope of the antigen and a single binding site of an antibody. The avidity reflects the overall strength of the interactions between a multivalent antibody molecule and a multivalent antigen molecule at multiple sites.  The interaction of a soluble antigen and precipitating antibody in a liquid or gel medium forms an Ag-Ab precipitate. Electrophoresis can be combined with precipitation in gels in a technique called immunoelectrophoresis.  The interaction between a particulate antigen and agglutinating antibody (agglutinin) produces visible clumping, or agglutination that forms the basis of simple, rapid, and sensitive immunoassays.  Radioimmunoassay (RIA) is a highly sensitive and quantitative procedure that utilizes radioactively labeled antigen or antibody.  The enzyme-linked immunosorbent assay (ELISA) depends on an enzyme-substrate reaction that generates a colored reaction product. ELISA assays that employ chemiluminescence instead of a chromogenic reaction are the most sensitive immunoassays available.  In Western blotting, a protein mixture is separated by electrophoresis; then the protein bands are electrophoretically transferred onto nitrocellulose and identified with labeled antibody or labeled antigen.  Fluorescence microscopy using antibodies labeled with fluorescent molecules can be used to visualize antigen on or within cells.  Flow cytometry provides an unusually powerful technology for the quantitative analysis and sorting of cell populations labeled with one or more fluorescent antibodies.

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