Section D – Antibodies
amounts of Ag and Ab present (Fig. 1). As the amount of Ag added increases, the amount of precipitate and Ab in the precipitate increases, until a maximum is reached, and then decreases with further addition of Ag. When there is both sufficient Ag and sufficient Ab, the combination of Ag and Ab proceeds until large aggregates are formed, which are insoluble and precipitate (equivalence).
However, inAb excessor in Ag excess, less lattice formation occurs and more soluble complexes are formed.
84 Section D – Antibodies
1 2 3 4 5 6 7 8 9
Quantity of antibody in precipitate
Quantity of antigen added Equivalence
B
A C
Antibody excess
Antigen excess
Fig. 1. Immune complex formation and precipitation. The same amount of Ab to a protein was added to each of a series of tubes (1–9), followed by the addition of increasing amounts of the protein Ag to each successive tube. (A) The zone of Ab excess; (B) zone of
equivalence in which all of the Ag and Ab are incorporated into a precipitate; and (C) the zone of Ag excess.
These reactions occur in vivo during an immune response. Initially, there is Ag excess as no Ab to the Ag is present at the time of first contact with the Ag.
Within days however, plasma cells develop, producing Ab to the Ag which complex with it (Ag excess). As more Ab is produced, equivalence is reached resulting in large Ag–Ab complexes which are removed by phagocytic cells through interaction with their Fc and complement receptors. Plasma cells continue to produce Ab during their short life, increasing the Ab concentration in the serum (Ab excess). However, once Ag has been removed, no further restimulation of B cells occurs and no more plasma cells develop (Topic G4).
Thus, the Ab concentration in the serum begins to decrease as a result of normal catabolism.
If the Ag persists (e.g. with some infectious organisms such asStreptococcus) or is self Ag, immune complexes are continually formed and may not readily be removed due to an ‘overwhelmed’ phagocytic system. This can lead to the deposition of immune complexes in tissues resulting in damaging reactions (type III hypersensitivity, Topic K4). The complexes activate complement and induce an acute inflammatory response (Topic B4). Direct interaction of the immune complexes with Fc and complement receptors on the neutrophils causes the release of proteolytic enzymes that damage surrounding tissues.
As previously described, when there is both sufficient Ag and sufficient Ab, the combination of Ag and Ab proceeds until large aggregates are formed which Precipitation
assays Immune complexes and tissue damage Immune complexes in vivo
are insoluble in water and precipitate (equivalence). The extent to which a lattice forms depends on the relative amounts of Ag and Ab present.
Lattice formation and precipitation are the basis for several qualitative and quantitative assays for Ag or Ab. These assays are done in semisolid gels into which holes are cut for Ag and/or for Ab and diffusion occurs until Ag and Ab are at equivalence and precipitate. In radial immunodiffusion, Ab (e.g. horse anti-human IgG) is incorporated into the gel and Ag (e.g. human serum) is placed in a hole cut in the gel. Ag diffuses radially out of the well into the gel and interacts with the Ab forming a ring of precipitation, the diameter of which is related to the concentration of the Ag (Fig. 2). Similar assays have been devel- oped in which a voltage gradient (electrophoresis) is used to speed up move- ment of Ag into the Ab containing gel (rocket immunoelectrophoresis).
D6 – Antigen/antibody complexes (immune complexes) 85
Unknown
Standards
Precipitation ring
Fig. 2. Measurement of Ag by precipitation in gels. Ab-containing gel is placed on a glass or plastic surface. Holes are cut in the gel and filled with Ag which diffuses radially out of the well and interacts with the Ab in the gel. Soluble complexes are initially formed but as more Ag diffuses equivalence is reached resulting in a lattice and precipitation. The diameter of the precipitation ring is related to the concentration of the Ag and, using known standards, can be quantitated and compared with the levels of Ag in other samples.
In immunoelectrophoresis, Ags (e.g. serum) are placed in a well cut in a gel (without Ab) and electrophoresed, after which a trough is cut in the gel into which Abs (e.g. horse anti-human serum) are placed. The Abs diffuse laterally to meet diffusing Ag, and lattice formation and precipitation occur permitting determination of the nature of the Ags (Fig. 3).
Agglutination involves the interaction of surface Ags on insoluble particles (e.g. cells) and specific Ab to these Ags (Fig. 4). Ab thus links together (aggluti- nates) insoluble particles. Much smaller amounts of Ab suffice to produce agglutination than are needed for precipitation. For this reason, agglutination rather than precipitation may be used to determine blood group types or if Ab to bacteria is present in blood as an indication of infection with these bacteria.
Since IgM has ten binding sites, whereas IgG has two, IgM is much more effi- cient at agglutinating particles or cells.
Although Abs are frequently used by themselves to assay for the presence of an Ag, a second Ab is sometimes used in what is known as a Coomb’s test. In some instances, such as when an autoantibody has been produced against a Agglutination
assays
given cell type, the cells will have human Ab bonded to them, and thus can be identified by a second Ab (an Ab to human immunoglobulin) which will cause agglutination of the cells. In an indirect Coomb’s test, the presence of circulat- ing Ab to a cell surface Ag is demonstrated by adding the patient’s serum to test cells (e.g. erythrocytes) followed by addition of Ab to human Ab.
86 Section D – Antibodies
ⴙ ⴚ
Ag
Antisera
Fig. 3. Identification of antigens using gel electrophoresis. Ag (e.g. serum) is placed in a well cut in a gel and subjected to a voltage gradient which causes the various antigens to migrate different distances through the gel dependent on their charge. After electrophoresis, a trough is cut in the gel into which antibodies (e.g. horse anti-human serum) are placed. The anti- bodies diffuse laterally from the trough until they meet Ag diffusing from its location after electrophoresis. Again, lattice formation and precipitation occurs and, based on immuno- electrophoresis of defined standards, the identity of the Ag can be determined.
Cell Agglutination
Antibody
Agglutination: Antigen insoluble before adding antibody Precipitation: Antigen is initially soluble;
antibody binding to it creates a lattice and makes it insoluble
Fig. 4. Agglutination.
Section D – Antibodies