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32 The Immune Response: Antigen-Antibody Reactions

 

CHAPTER OVERVIEW

This chapter continues the discussion of the immune system with a description of the reactions between antigens and antibodies and the ways in which these reactions protect higher animals against infectious agents, their products, and certain macromolecules. The chapter concludes with a discussion of antigen-antibody reactions in vitro and their uses in disease diagnosis, microorganism identification, and immunological monitoring.

 

CHAPTER OBJECTIVES

After reading this chapter you should be able to:

! discuss the various ways in which antigen-antibody binding initiates the participation of other defense mechanisms, such as the complement system, phagocytosis, chemotaxis, and inflammation

! discuss antibody-dependent, cell-mediated cytotoxicity; opsonization; neutralization; and immune complex formation

! describe various in vitro diagnostic/identification procedures that employ antigen-antibody reactions

 

CHAPTER OUTLINE

I. Introduction

A. Antibodies are bifunctionalCthey bind to the target antigen they recognize as foreign, and they enable other defense components to react with it

B. Variable domain (Fab)Cbinds to target antigen

C. Constant domain (Fc)Cinteracts with cells of the immune system and other host defense mechanisms

II. Antigen-Antibody BindingCoccurs within the pocket formed by folding the VH and VL regions of the Fab domain

A. Binding is due to weak, noncovalent bonds

B. Shapes of epitope and binding site must be complementary for efficient binding

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

III. Antigen-Antibody Reactions in the Animal Body (In Vivo)

A. 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)

1. Results in lysis of antibody-coated bacteria and eucaryotic cells

2. Mediates inflammation

3. Attracts and activates phagocytic cells

4. There are three activation pathways; each results in destruction of the target cell, but their triggering mechanisms are different

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

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

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

5. 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

6. The pore allows entry of destructive enzymes or leads to osmotic rupture of the target cell

B. Toxin neutralizationCantibody (antitoxin) binding to toxin renders the toxin incapable of attachment or entry into target cells

C. Viral neutralizationCbinding prevents viral attachment to target cells

D. Adherence inhibiting antibodiesCsIgA prevents bacterial adherence to mucosal surfaces

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

F. IgE and parasitic infectionCin the presence of elevated IgE levels, eosinophils bind parasites and release lysosomal enzymes

G. Opsonization

1. Prepares the microorganism for phagocytosis; phagocytes recognize the Fc portion of IgG or IgM antibodies coating the surface of the foreign microorganism

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

H. 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

I. Immune complex formationCtwo 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

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

IV. Antigen-Antibody Reactions In Vitro (serology)

A. AgglutinationCvisible clumps or aggregates of cells or of coated latex microspheres; if red blood cells are agglutinated, this is called hemagglutination

B. 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

C. Complement fixationCirreversible 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

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

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

D. Enzyme-linked immunosorbent assay (ELISA)

1. Indirect immunosorbent assayCdetects serum antibody

a. Antigen is coated on test wells and serum is added

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

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

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

e. Add colorless substrate (chromogen) for the enzyme and measure colored product formation spectrophotometrically; no colored product will form if everything washed off

2. Double antibody sandwich assayCdetects antigens in a sample

a. Antibody is coated onto test wells and sample is added

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

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

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

E. ImmunodiffusionCinvolves the precipitation of immune complexes in an agar gel after diffusion of one or both components

1. Single radial immunodiffusion (RID) assayCquantitative

2. Double diffusion assay (Ouchterlony technique)Clines of precipitation form where antibodies and antigens have diffused and met; determines whether antigens share identical determinants

F. ImmunoelectrophoresisCantigens are first separated by electrophoresis according to charge, and are then visualized by the precipitation reaction; greater resolution than diffusion assay

G. ImmunofluorescenceCdyes coupled to antibody molecules will fluoresce (emit visible light) when irradiated with ultraviolet light

1. DirectCused to detect antigen-bearing organisms fixed on a microscope slide

2. IndirectCused to detect the presence of serum antibodies

H. ImmunoprecipitationCsoluble antigens form insoluble immune complexes that can be detected by formation of a precipitin

I. NeutralizationCan 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

J. Radioimmunoassay (RIA)Cpurified antigen labeled with a radioisotope competes with unlabeled sample for antibody binding

K. SerotypingCantigen-antibody specificity is used to differentiate among various strains (serovars) of an organism

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