The immune response antigen antibody reactions
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The Immune Response: Antigen-Antibody Reactions. Introduction. Antibodies are bifunctional - they bind to the target antigen they recognize as foreign, and they enable other defense components to react with it Variable domain (Fab) - binds to target antigen

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  • Antibodies are bifunctional - they bind to the target antigen they recognize as foreign, and they enable other defense components to react with it

    • Variable domain (Fab) - binds to target antigen

    • Constant domain (Fc) - interacts with cells of the immune system and other host defense mechanisms

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  • Binding is due to weak, noncovalent bonds

  • Shapes of epitope and binding site must be complementary for efficient binding

  • The high complementarity provides for the high specificity associated with antigen-antibody binding

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  • The complement system is a series of protein components that must be activated in a cascade fashion (i.e., the activation of one component results in the activation of the next, and so on)

    • Results in lysis of antibody-coated bacteria and eukaryotic cells

    • Mediates inflammation

    • Attracts and activates phagocytic cells

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  • There are three activation pathways

    • Each results in destruction of the target cell, but their triggering mechanisms are different

  • The classical pathway is dependent on antigen-antibody interactions to trigger it; it is fast and efficient

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  • The lectin pathway is activated by mannose-binding lectins (MBLs) that have been secreted by liver; activation leads to opsonization

  • The alternative pathway does not require antigen-antibody binding; it is nonspecific and inefficient, but contributes to innate resistance

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  • The final step in the pathway is the formation of a membrane attack complex that creates a pore in the membrane of the target cell

  •  The pore allows entry of destructive enzymes or leads to osmotic rupture of the target cell

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  • Ag-Ab Interactions – cont.

  • Toxin neutralization - antibody (antitoxin) binding to toxin renders the toxin incapable of attachment or entry into target cells

  • Viral neutralization - binding prevents viral attachment to target cells

  • Adherence inhibiting antibodies - sIgA prevents bacterial adherence to mucosal surfaces

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  • Antibody-dependent cell-mediated cytotoxicity - involves the complement system, NK cells, or release of cytotoxic mediators from effector cells that attach to the target cell by means of Fc receptors

  • IgE and parasitic infection - in the presence of elevated IgE levels, eosinophils bind parasites and release lysosomal enzymes

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  • Opsonization

    • Prepares the microorganism for phagocytosis

      • Phagocytes recognize the Fc portion of IgG or IgM antibodies coating the surface of the foreign microorganism

    • Phagocytosis can also be stimulated by components of the complement system, whether initiated by the classical or alternative pathways

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  • Inflammation can be mediated by IgE attachment to mast cells and basophils, or by the binding of one of the complement components to mast cells and platelets

    • This complement component is also a powerful chemoattractant for macrophages, neutrophils, and basophils

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  • Immune complex formation - two or more antigen-binding sites per antibody molecule lead to cross-linking, forming precipitins (molecular aggregates) or agglutinins (cellular aggregates)

    • Agglutination that specifically involves red blood cells is called hemagglutination

    • In vivo testing involves immediate or delayed skin testing for the presence of antibodies to various antigens

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  • Agglutination - visible clumps or aggregates of cells or of coated latex microspheres

    • If red blood cells are agglutinated, this is called hemagglutination

  • Agglutination inhibition can be used to detect serum antibodies or to detect the presence of specific substances (e.g., illegal drugs) in urine samples by a competition assay

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Box 33.2

Rapid urine testing for drugs, e.g cocaine – Abuscreen method

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Figure 33.10 – Viral Hemagglutination

Some viruses bind to RBC and cause hemagglutination. If serum contains anti-viral Abs, then hemagglutination is inhibition – positive test.

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Q: What is the titer in rows A-H? antibody titer.

Figure 33.11: A microtiter plate illustrating hemagglutination. Ab in wells 1-10. Positive control = row 11, negative control = row 12. RBCs added to each well. If enough Ab is available to agglutinate the cells, they bind as a mat to the bottom of the well. If insufficient Ab is available, they form a pellet at the bottom.

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  • Complement fixation antibody titer.

    • Irreversible alterations to complement components that are initiated by the binding of antibody to antigen;

    • Used to detect the presence of serum antibodies, thereby indicating prior exposure to a pathogen

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  • If antibody titer. immune complexes are formed, then complement is used up and lysis will not occur when sensitive indicator cells are added

  • If immune complexes are not formed, then complement is not used up and lysis will occur when sensitive indicator cells are added

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  • Enzyme-linked immunosorbent assay antibody titer. (ELISA)

    • Indirect immunosorbent assay - detects serum antibody

      • Antigen is coated on test wells and serum is added

      • If antibodies are present, they will bind antigen; if not, they will wash off

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  • Add to the plate an enzyme that is covalently coupled to a second antibody against first immunoglobulin

  • If antigen was present, the second antibody will bind; if not, it will wash off

  • Add colorless substrate (chromogen) for the enzyme and measure colored product formation spectrophotometrically;

  • No colored product will form if everything washed off

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  • Double antibody sandwich assay second antibody against first immunoglobulin - detects antigens in a sample

    • Antibody is coated onto test wells and sample is added

    • If antigen is present in sample it will bind; if not it will wash off

    • React with antibody against the antigen; if antigen was present in the sample, this antibody will bind; if not, it will wash off

    • Continue with steps (c), (d), and (e) as in the indirect assay

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  • Immunodiffusion second antibody against first immunoglobulin - involves the precipitation of immune complexes in an agar gel after diffusion of one or both components

    • Single radial immunodiffusion (RID) assay - quantitative

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Figure 33.15 second antibody against first immunoglobulin

Mancini technique – Single radial immunodiffusion assay

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  • Double diffusion assay second antibody against first immunoglobulin (Ouchterlony technique) - lines of precipitation form where antibodies and antigens have diffused and met; determines whether antigens share identical determinants

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Figure 33.15 second antibody against first immunoglobulin

Ouchterlony technique – Double diffusion agar assay

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  • Immunoelectrophoresis second antibody against first immunoglobulin - antigens are first separated by electrophoresis according to charge, and are then visualized by the precipitation reaction

    • Greater resolution than diffusion assay

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Figure 33.16 second antibody against first immunoglobulin

Classical Immunoelectrophoresis

- Used to separate major blood proteins in serum for diagnostic tests

Precipitin arcs form where Ab and Ag precipitate.

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  • Immunofluorescence second antibody against first immunoglobulin - dyes coupled to antibody molecules will fluoresce (emit visible light) when irradiated with ultraviolet light

    • Direct - used to detect antigen-bearing organisms fixed on a microscope slide

    • Indirect - used to detect the presence of serum antibodies

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Figure 33.17 – Direct and Indirect Immunofluorescence second antibody against first immunoglobulin

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  • Immunoprecipitation second antibody against first immunoglobulin- soluble antigens form insoluble immune complexes that can be detected by formation of a precipitin

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  • Neutralization and precipitation ring test. - an antibody that is mixed with a toxin or a virus will neutralize the effects of the toxin or the infectivity of the virus; this is determined by subsequent assay

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  • Radioimmunoassay and precipitation ring test. (RIA) - purified antigen labeled with a radioisotope competes with unlabeled sample for antibody binding

  • Serotyping - antigen-antibody specificity is used to differentiate among various strains (serovars) of an organism