BCH 422 Lecture Notes 2022 - Antigen-Antibody Reactions PDF

Antigen – Antibody Reactions Objectives: Students should be able to 1. Discuss the forces stabilizing the antigen-antibody reactions 2. Explain the nature of antigen-antibody reactions 3. Discuss the factors affecting antigen-antibody reactions 4. Discuss the characteristics of antigen-antibod...

Antigen – Antibody Reactions Objectives: Students should be able to 1. Discuss the forces stabilizing the antigen-antibody reactions 2. Explain the nature of antigen-antibody reactions 3. Discuss the factors affecting antigen-antibody reactions 4. Discuss the characteristics of antigen-antibody reactions 5. Describe common types of antigen- antibody reactions 6. Explain the applications of antigen-antibody reactions  Introduction Interaction between antigen and antibody combining together (antigen – antibody interaction) Normally shortened as Ag – Ab interaction The basis of humoral or antibody mediated immunity Serology - science that deals with in vitro interaction of antibodies with antigens  Introduction Interaction of Ag – Ab interaction occurs in three stages:  Formation of Ag – Ab complex (primary Ag – Ab reaction – invisible) rapid (occurring in seconds) not dependent on electrolytes (salts or buffered solutions)  Conversation of invisible primary reactions to macroscopically visible ones (secondary Ag – Ab reactions – precipitation or agglutination)  Destruction or neutralization of the antigens (tertiary)  Introduction How is the interaction possible???  Nature of Ag – Ab reactions Antibody interlocks with antigen like a key in a lock. Non-covalent forces hold antigenic determinants within the Ab combining site  Hydrogen bonds, hydrophobic, Van der Waals  NB: distance or closeness between interacting molecules Reaction is reversible k1 Ag + Ab Ag - Ab K = k1/k-1 Affinity k-1  Properties of Ag – Ab reactions Affinity: strength of the reaction Avidity: overall strength of binding between a single antigenic reaction of an Ag with many determinant and a single combining antigenic determinants and site of Ab multivalent Abs NB: sum of repulsive and attractive NB: it is affect by the valence of Ab forces between antigenic determinants More than sum of individual affinities and combining sites  Properties of Ag – Ab reactions Specificity: ability of an Ab Cross Reactivity: ability of an Ab combining site to react with one combining site to react with more antigenic determinant or population than one antigenic determinant or of Ab molecules to react with only population of Ab molecules to react one antigen with more than one antigen NB: A type of Ab destined to bind to a particular Ag  Factors affecting Ag – Ab interactions measurements Directly or indirectly detecting complex formed between Ag and Ab Affinity: higher affinity for antigen the more stable the reaction and ease to detect the interaction Avidity: reactions between multivalent Ags and multivalent Abs are most stable and thus easy to detect Ag:Ab ratio: size of complex formed is related to concentration of Ag and Ab  Factors affecting Ag – Ab interactions measurements Physical form of the antigen: physical form influences how it reacts with Ab Temperature: high temperature affect binding of Ag to Ab pH: ???? Ionic strength: ????  Tests for Ag –Ab interactions Agglutination Precipitation Immunoassays Complement Fixation Western blotting Immunofluorescence  Agglutination (clumping) Tests When a particulate antigen reacts with an Ab Haemagglutination???? Types  Quantitative Qualitative Agglutination inhibition  Qualitative Agglutination Tests Ab is mixed with a particulate antigen and a positive is indicated by agglutination of the particulate Ag Typical example: ABO blood groupings The hCG pregnancy testing  Qualitative Agglutination Tests Coagulase test for Staphylococcus aureus: use human or rabbit plasma  Quantitative Agglutination Tests Make serial dilutions of samples to be tested for Ab Add fixed number of RBCs or bacteria or particulate antigen Determine maximum dilution that gives agglutination Titer: the highest dilution that still causes agglutination beyond which no agglutination occurs Titer is reported as reciprocal of maximum dilution  Prozone Effect Occasionally, at high Ab (lower dilutions) there is no agglutination and as sample is diluted agglutination occurs Lack of agglutination at high Ab concentration - prozone effect Abs are present in excess so Ag/Ab ratio is out of balance Every epitope on one particle may have bound individual Ab molecules thus preventing cross-linking between different particles  Haemagglutination Inhibition Measures ability of antigen to inhibit the agglutination of Ag-coated RBCs by Abs Fixed amount of Abs to Ag in question is mixed with fixed amount of RBCs coated with Ag Add amount of sample to be analyzed for the presence of the Ag If sample contains Ag, the Ag will compete with Ag coated on RBCs for binding to the Ab therefore inhibiting agglutination  Some Applications Pregnancy testing Using illicit drugs (cocaine)  False positive results since some legal drugs have similar structures as illegal ones Individuals have been exposed to some viruses  Precipitation Tests When a soluble antigen reacts with an Ab in the correct proportion Constant Abs in tubes and add increasing concentration of Ags Three zones  Zone of excess Abs Equivalence zone Zone of excess Ags Equivalence Zone: proportion of Ab and Ag is optimal for maximal precipitation At zones of excess Ag or Ab proportion of reactants do not lead to effective cross linking thus preventing precipitation  Radial Immunodiffusion (Mancini Test) Ab is incorporated into agar gel and different dilutions of Ags placed in holes punched in the agar As antigen diffuses into the holes it reacts with Abs At the equivalence point, rings of precipitation are formed Measure diameter of rings and determine unknow  Applications Determination of immunoglobulin levels in patient samples Antimicrobial sensitivity testing  Immunoelectrophoresis Complex mixture of Ags are placed in well on agar gel and Ags electrophoresed so that they are separated based on charges After electrophoresis, Abs are added into a trough cut in the gel Diffusion of Abs into the agar cause formation of precipitin lines at the equivalence point,  Applications Evaluate purity of isolated serum proteins Determine the presence or absence of immunoglobulins in serum of patients  Immunoblotting (Western Blotting) Mixture of Ags are separated in a gel Separated material is blotted onto a nitrocellulose sheet (binds Ag strongly) Ab applied to the sheet to bind to specific Ag Ab may be labeled  Diagnosis of AIDs by applying a patient serum to a nitrocellulose sheet on which HIV antigens are found Binding of specific Ab is strong evidence of infection b the virus