Antibodies (aka immunoglobulin or Ab) are produced by B cells and specifically bind to antigens (aka Ag) in solution (as opposed to TCRs, which bind antigens on cell surfaces). An antigen is any substance that binds specifically to a T cell receptor or B cell receptor. This antibody-antigen binding can: cause the antigen to be engulfed by macrophages (opsonization); can neutralize a virus and prevent it from entering cells; and can induce the complement cascade
(causes bacterial lysis). All antibodies exist in both secreted and membrane-bound (mIg) forms, differing in their carboxy terminal sequence. Secreted antibodies have a hydrophilic terminus, while membrane immunoglobulins have a hydrophobic sequence which inserts into the plasma membrane and a short cytoplasmic sequence. Antibodies have various functions, described below:
| Agglutinins | Cause clumping-together and destruction of foreign cells. For example, agglutinin activity will clump transfused blood cells of a foreign blood type. |
| Antitoxins | Neutralize toxins from foreign microbes. |
| Opsonins | Facilitate engulfment of foreign microbes. |
| Precipitins | Form flocculate (cloudy, lumpy) precipitates of cell-free supernatant of foreign microbes. |
Antibodies consist of heavy chains and light chains. The different istotypes of antibody have structurally different heavy chains, leading to functional differences as well. IgG is the most common immunoglobulin isotype, and its relatively simple structure (consisting of two γ heavy chains, and two light chains) is shown to the right. There are five different types of heavy chain to differentiate the five antibody isotypes (IgA, IgD, IgE, IgG and IgM).
Antibody chains contain constant domains (same amongst antibodies) and variable domains (different amongst antibodies). Heavy chains have four or five constant domains at one end, a variable region at the other and also at least one carbohydrate moiety attached. There are many types of heavy chain variable regions. Light chains have a constant region at one end and a variable region at the other end. Light chain variable regions are either kappa (κ) isotype or lambda (λ) isotype. The two light chain isotypes have no known functional differences. Free light chains are known as Bence-Jones proteins.
Variable regions contain relatively conserved regions and hypervariable regions (aka complementarity determining regions). Complementarity determining regions (CDRs) of heavy and light chain variable regions group together to form a complex that directly interacts with antigens. X-ray crystallography has shown that CDRs are loops which stick out for accessibility, and that relatively conserved regions are merely a scaffold to make sure these CDR loops stay in place.
| Next Steps | Study the different immunoglobulin isotypes. |
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Antigens are foreign bio-organic molecules that interact with B cells (via antibodies) and T cells (via T cell receptors). Blood-borne antigens are concentrated in the spleen; lymph-borne antigens are concentrated in nearby lymph nodes and nodules. Upon detection by the acquired immune response, antigens stimulate production of antigen-specific antibodies. Toxins, invading bacteria and viruses, and the cells of transplanted organs can all function as antigens. Bio-organic chemicals are those based on carbon and the atoms which bond to carbon (hydrogen, oxygen, nitrogen, phosphorous and sulfur — aka CHONPS). An antigen’s configuration of CHONPS is called its antigenic determinant. By ignoring any non-CHONPS chemical, the immune response does not recognize sand, mercury, minerals and other potentially hazardous contaminants. By just recognizing bio-organic antigens, the immune response has evolved to detect only antigens encoded or controlled by genes.
| Hapten-Carrier | Hapten | Carrier Protein |
| ARS-OVA | Azophenylarsonate | Ovalbumin |
| DNP-BGG | Dinitrophenol | Bovine gamma globulin |
| LAC-HGG | Phenyllactoside | Human gamma globulin |
| NIP-KLH | S-nitrophenyl acetic acid | Keyhole limpet hemocyanin |
| TNP-BSA | Trinitrophenyl | Bovine serum albumin |
Antigens are any substance that binds specifically to B cell receptors or T cell receptors. There are two types of antigens: immunogens and haptens. An immunogen is a any substance that can elicit an innate or acquired response, and haptens are research-useful small molecules which must be attached to a carrier molecule to elicit a response.
The immune response has two cells that recognize antigens: B cells and T cells. B cells present immunoglobulin (antibody molecule) and T cells present T cell receptor (TCR). The function of these cells is to bind antigens and to remove them from the system. Antibodies and TCRs are specific to the antigenic determinant of a particular antigen. B cells respond to different antigens in different ways (thymus-dependent and thymus-independent) and this is described in the first two paragraphs of B cell activation.
Antigens are difficult for the immune response to detect due to the size of the antigenic universe and because the antigenic universe is constantly changing. The size of the antigenic universe is due to the billions of foreign microbes around. With each microbe containing a few foreign molecules, the number of antigens is inconceivably large. Also, genetic change causes this antigenic universe to constantly change. Bacteria replicate at a fast rate, meaning antigenic drift (change) is too fast for standard mechanisms that deal with diversity.
| Term | Overview |
| Antigen | |
| Immunogen | |
| Epitope | |
| Hapten |
| Next Steps | Study antibodies, their isotypes and the humoral response. |
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The various F(c) Receptors are organized by the type of antibody they bind — F(c)γ Receptors bind IgG (aka γ isotype antibodies) and F(c)ε Receptors bind IgE (aka ε isotype antibodies.
One way antibodies stimulate inflammation and clearing of pathogens is by binding of their F(c) regions to F(c)γ receptors (FcγRs) on effector cells. There are three families of FcγRs: FcγRI, aka CD64; FcγRII, aka CD32; and FcγRIII, aka CD16. FcγRs also stimulate binding and uptake of antigens in human complexes, thus playing an important role in antigen presentation by macrophages and dendritic cells. There is a balance between activator FcγRs and inhibitor FcγRs, with many cells displaying both.
| F(c)γ Receptor | Overview |
| FcγRI (CD64) | FcγRI is expressed only on macrophages and neutrophils. The FcγRI complex includes a γ or ζ (zeta) chain. Mice with their γ chain genes knocked out do not express FcγRI on macrophages and neutrophils. Once FcγRI is bound, the signal is propagated into the cell via the FcγRI immunoreceptor tyrosene-based activation motif (ITAM), located on the γ (or ζ) chain. In response to binding, the ITAM induces antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis. FcγRI is the only receptor which binds antibodies with high affinity. |
| FcγRIIA (CD32) | Not present in mice, FcγRIIA is expressed on macrophages, neutrophils and eosinophils and has signaling motifs in its cytoplasmic tail. FcγRIIA has an immunoreceptor tyrosene-based activation motif (ITAM) and ligation leads to phagocytosis and, in eosinophils, degranulation. |
| FcγRIIB (CD32) | Although present only on B cells, FcγRIIB is similar to FcγRIIA in that both CD32′s have signaling motifs in their cytoplasmic tails. However, FcγRIIB is an inhibitory receptor and contains an immunoreceptor tyrosene-based inhibitor motif (ITIM). Although FcγRs bind the F(c) region antibodies, FcγRIIB only provides an inhibitory response when crosslinked to an entire intact antibody (including the F(ab) fragments). In absence of FcγRIIB — for example, in FcγRIIB knock-out mice– there is a significantly higher production of antibodies after antigen exposure. However, FcγRIIB is not the only antibody production regulatory mechanism. A second inhibitory role for FcγRIIB is inhibition of FcεRI-induced mast cell degranulation. |
| FcγRIIIA (CD16) | FcγRIIIA is a transmembrane receptor with a cytoplasmic tail, and is found on monocytes, macrophages, natural killer and T cells. Like FcγRI (aka CD64), the FcγRIIIA complex includes a γ or ζ (zeta) chain. FcγRIIIA is the only FcγR found on natural killer cells. |
| FcγRIIIB (CD16) | FcγRIIIB is bound to neutrophil membranes by a glycosyl phosphatidyl inositol (GPI) anchor. Mice lack FcγRIIIB. FcγRIIB plays an important role in antibody-mediated (humoral) tumor protection. Inhibitory FcγRIIB receptors somehow stimulate production of antibodies specific to tumor antigens. |
High-affinity F(c)&epsilonRI is found on most mast cells and basophils. It is only activated by cross-linked antibodies, meaning only those antibodies which have already bound their complementary antigen. Once this cross-linking occurs, a Ca2+ flux occurs which triggers granules to swell, move to the membrane and burst out (degranulation). This leads to release of leukotrines and prostaglandins.
One problem with the study of antibodies was that they are heterogenous. For example, an electrophoresis pattern of an animal immunized against albumin (a homogenous protein) would show a spike of albumin and then several much smaller spikes of antibodies (meaning the albumin antibodies are polyclonal, or consisting of different subsets binding different sites on the same antigen). In multiple myeloma, tumorous plasma cells all secrete the tame type of immunoglobulin. This leads to a huge monoclonal spike of antibodies, since there will be huge amounts of a single antibody. The experiments below were all performed to determine the structure of antibodies. After all these experiments, the antibody structure shown above was determined.
| Electrophoretic Migration | Electrophoretic migration analysis was performed on serum from rabbits immunized with ovalbumin (resulting in ovalbumin, α, β and γ peaks), and for serum from rabbits immunized with ovalbumin but with ovalbumin antibodies removed (resulting in ovalbumin, α and β peaks). These results indicated that antibodies were some sort of gamma globulin. |
| Molecular Weight | To determine the molecular weight of this gamma globulin, it was migrated in a centrifugal field. Its migration was 7S, corresponding to a 150,000 dalton molecular weight. |
| Valence | Molecular analysis of immune precipitates between bacterial polysaccharide antigens and their specific antibodies showed a valence of 2. |
| Papain Cleavage | Cleavage of an antibody with papain yielded two different fragments that were separated using ion exchange chromatography: F(ab) and F(c). A whole antibody bound two antigen molecules, F(ab) bound a single antigen molecule (but could not precipitate) and F(c) formed crystals. F(ab) and F(c) each had a weight of 50,000 daltons. Thus, an antibody must contain 2 F(ab) fragments and 1 F(c) fragment. |
| Pepsin Cleavage | Cleave of an antibody with pepsin yields one fragment of 100,000 daltons capable of binding two antigen molecules and could precipitate. |
| Disulfide Cleavage | Cleavage of disulfide bonds yielded two products which could be separated based on their size: a heavy chain (50,000 daltons) and a light chain (25,000 daltons). An anti-L antibody reacted with Fab only. An anti-H antibody reacted with F(ab) and F(c). An anti-F(ab) antibody reacted with both H and L. |
| Next Steps | Study the different immunoglobulin isotypes and antigen-antibody interactions. |
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There are several different isotypes of heavy chain constant regions, broken into classes and subclasses. Classes are differentiated by large structural differences correlated to large functional differences. Subclasses have small but significant differences, also corresponding to separate functions. Most functions of antibodies are mediated (determined) by the heavy chain constant region. However, all antibody functions are triggered only by binding of an antigen to the variable region. The two light chain isotypes (κ and λ) associate with all the different heavy chain isotypes. Each isotype is encoded by a separate gene, and all genes are present in normal individuals.
| Isotype | Heavy Chain | Structure & Function | Subclasses (Human) | |
| IgM | μ | The IgM heavy chain has four constant regions and no hinge, represented as (H2L2)6. However, a J chain is frequently produced as well to create a (H2L2)5J antibody. IgM has great valency, allowing it to avidly bind antigens and be the first antibody to get produced after antigen exposure. IgM’s effector functions are: activation of the classical pathway of complement; and as the antigen receptor of naive B lymphocytes. | None | |
| IgG | γ | IgG is the most abundant antibody. IgG’s effector functions are: opsonization of antigens for phagocytosis by macrophages and neutrophils; activation of the classical pathway of complement; antibody-dependent cell-mediated cytotoxicity (ADCC), mediated by natural killer cells and macrophages; and neonatal immunity, the transfer of maternal antibodies through placenta and gut.
γ heavy chains contain four intrachain disulfide bonds and light chains contain two intrachain disulfide bonds; γ heavy chains and light chains are connected by interchain disulfide bonds. An entire IgG antibody is 150,000 daltons; each γ heavy chain is 50,000 daltons; and each light chain is 25,000 daltons. |
IgG1, IgG2, IgG3, IgG4 | |
| IgA | α | IgA is present in secretions and protects the epithelium. IgA frequently polymerizes with IgM’s J chain. In secretions, it also has a fourth chain (secretory component) which is a product of epithelial cells. Since IgA is secreted into the lumens of the gastrointestinal and respiratory tracts, IgA’s effector function is to protect against pathogens which attack at the mucosal surface. | IgA1, IgA2 | |
| IgE | ε | IgE binds the FcR receptor on Mast cells, and allergies are initiated when the IgE-FcR complex binds an antigen. IgE is present in low concentrations. IgE’s effector functions are: Mast cell degranulation, leading to immediate hypersensitivity (allergy); and antibody-dependent cell-mediated cytotoxicity (ADCC) involving eosinophils. | None | |
| IgD | δ | IgD’s effector function is as the membrane receptor of naive B cells, and is expressed by anergic B cells. | None |
| Next Steps | Study immunoglobulin structure and antigen-antibody interaction. |
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Antibodies bind to antigens in a reversable non-covalent manner via hydrogen bonds, ionic bonds, hydrophobic interactions and van der Waal’s interactions. Antibodies only react with antigens in solution — as opposed to TCRs, which react with antigens bound to cell surfaces. These forces operate at short distances, so the antibody CDR (accounting for most of the antigen-antibody interaction) and the antigen epitope (where the CDR binds) must fit together very well. The more precise the fit, the better the interaction.
Antibodies make contact with protein antigens, usually 15-22 amino acids on the antigen contact a similar number on the antibody giving a complementary surface of 650-900 Angstroms. The amino acids comprising the epitope are adjacent in 3D space, but not necessarily in linear sequence.
When exposed to an antigen, B cells producing antibodies reactive to that antigen will begin to produce vast amounts of antibody. Each B cell produces a monoclonal population of antibodies, meaning each antibody binds the same epitope (site) on the same antigen; however, the B cells together produce a polyclonal population of antibodies, consisting of different antibodies binding a different epitope (site) on the same antigen.
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