Antibodies (Recommended reading: Abbas et al., 4th edition, Chapter 3; Chapter 4; Janeway et al., 5th edition, Chapter 3) Antibodies protect us from a vast variety of pathogens. Indeed the antibody repertoire is immense - the binding or combining sites of antibodies may be able to recognize somewhere in the range of 10 million different shapes. Antibodies share with agglutinins all the features that contribute to the elimination of pathogens, and can also contribute to host defense in some ways that the innate immune system cannot. Antibodies are also called immunoglobulins. A very crude electrophoretic fractionation (separation on the basis of charge) of serum proteins separates albumin from other serum proteins collectively called globulins. The globulins are also further characterized on the basis of charge as α, β and γ -globulins, γ-globulins being the most positively charged. This latter fraction was discovered to be made up largely of antibody molecules. The existence of antibody secreting tumors known an myelomas or plasmacytomas greatly facilitated the study of immunoglobulins and their structure. Tumors are in general derived from a single cell. A single cell and its progeny are generally referred to as a clone and tumors are therefore clonal (or monoclonal) outgrowths. Myelomas and plasmacytomas are derived from differentiated antibody secreting B lymphocytes known as plasma cells These plasma cell tumors each produce large amounts of a single or monoclonal antibody. The portions of the heavy chain and light chain that are involved in antigen recognition are at the N-terminal ends and are referred to as V (or variable) domains. The remaining portions of the heavy and light chains are referred to as constant (or C) regions. Each light chain constant region contains a single constant domain (CL). A domain refers to a portion of a protein which can be separated from the rest of the molecule and still fold into its correct shape. The domains in immunoglobulin molecules have a typical three dimensional structure which is now referred to as an immunoglobulin domain and is found in a variety of proteins (many of which existed before the evolution of immunoglobulins). Each heavy chain constant region is made up of three or four domains (CH). The portion of each heavy chain that is associated with the light chain is the VH domain and the CH1 domain. A cysteine residue in the CH1 domain forms a covalent disulfide bridge with a cysteine residue towards the C-terminal end of the CL domain. When one class of immunoglobulin molecules known as IgG molecules are cleaved with a proteolytic enzyme called papain, these antibodies are cleaved into two identical fragments known as Fab (Fraction antigen binding) fragments and a single Fc (Fraction crystallizable) fragment. Cleavage with pepsin yields an F(ab)2 fragment and an Fc fragment. Each Fab fragment corresponds to an arm of the antibody Y and is made up of a light chain covalently associated (via a disulfide bridge) with a portion of the heavy chain containing the VH and CH1 domains. The Fc portion corresponds to the stem of the Y and is a dimer of the Harvard-MIT Division of Health Sciences and TechnologyHST.176: Cellular and Molecular ImmunologyCourse Director: Dr. Shiv PillaiCH2 and CH3 domains of IgG covalently united via disulfide bridges between the two heavy chains. The variable domains of both heavy and light chains contain a large number of residues that are identical or highly conserved between different antibodies. These are referred to as framework regions. The variable nature of these domains is contributed to by stretches of amino acids that differ from one antibody variable domain to another. These residues make up three hypervariable regions or complementarity determining regions or CDRs. In both the heavy chain and the light chain CDR3 is the most variable of the hypervariable regions. Every constant or variable region domain forms an immunoglobulin fold which looks a lot like two very similar slabs slapped together at a slight angle. Each slab is made up of strands of a polypeptide strung up and down in succession to make a structure known as a β-sheet. The immunoglobulin fold itself is made up of these two slabs of β-sheets compressed against each other (known as an antiparallel β-barrel). At the top and bottom of this sandwich are loops that maintain continuity between individual strands of these β-sheets. When one examines the structure of a variable domain the three CDRs form loops of highly variable configuration that protrude from the "top" of a very conserved immunoglobulin fold structure. It is these very variable CDR loops of a VH and a VL domain that combine to form a binding site for antigen. If an antibody is directed against a small antigenic determinant or epitope the binding site formed by the three CDR loops may resemble a relatively tight pocket into which the epitope might fit. If the epitope is somewhat larger such as the rough terrain forming an antigenic "patch" on the surface of a protein, the combining site of the antibody may be formed by an outward splaying of the six CDR loops. The six hypervariable regions from the heavy and light chains combine to form a complementary surface to that of the protein antigenic determinant with some of the little hills on one surface dipping into the valleys of the other and vice versa Antibodies exist as a number of classes or isotypes (defined on the basis of their heavy chains). The antibody isotype that is made early in an immune response is of the IgM class. Later in an immune response a given B lymphocyte may "switch" from producing IgM antibodies against a specific antigen, to producing antibodies of other isotypes, such as IgG, IgA or IgE, also directed against the original antigen. Class switching of antibodies typically occurs in responses to protein antigens that are driven in part by signals from T lymphocytes. Another important phenomenon that tends to occur in immune responses driven by T cell derived signals is a process by which mutations are introduced into the genes encoding the variable portion of the antibody heavy and light chains. This process is referred to as somatic mutation. As we will discuss in later lectures, the selection by antigen of B lymphocytes whose mutated receptors "fit" the antigen most tightly results in the further differentiation of only those B cells which