- Proteins and Peptides
- Lysates and Cell Lines
Antibody Interaction with Antigen
What is an antibody? An antibody is an immunoglobulin protein, secreted by B lymphocytes, that is present in serum or body fluid and combines specifically with an antigen. Antigens are classically defined as any foreign substance that elicits an immune response. An antigen that produces an adaptive immune response after injection into an animal is an immunogen. Immunogens can be designed so that antibodies are generated against specific proteins. Because antibodies can be made against specific proteins, they are very useful tools in science and can be used to investigate specific protein function and location in a dynamic biological system.
The specific region on an antigen that an antibody recognizes and binds to is called the epitope, or antigenic determinant. An epitope is usually 5-8 amino acids long on the surface of the protein. Proteins are three dimensionally folded structures, and an epitope may only be recognized in its form as it exists in solution, or its native form. When an epitope is made up of amino acids that are brought together by the three-dimensional structure, the epitope is conformational, or discontinuous. If the epitope exists on a single polypeptide chain, it is a continuous, or linear epitope. Depending on the epitope an antibody recognizes, it may bind only fragments or denatured segments of a protein, or it may also be able to bind the native protein.
Antibodies can be generated against specific peptide sequences or proteins by being conjugated to a carrier protein that is known to be strongly immunogenic. Common carrier proteins are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). When the immunizing peptide sequence or protein is conjugated to the carrier protein, the antibodies generated by the immunized animal will be specific to epitopes across the surface of the whole carrier complex.
When an animal is immunized, typically a mouse, rabbit, or goat, an immune response results; the antibodies are predominantly found in the serum fraction of the blood. Serum containing antigen-specific antibodies is called antiserum. Five main types of antibodies have been identified, and these types are separated into five classes based upon their unique properties. These five classes of immunoglobulins are: IgM, IgG, IgA, IgD, and IgE.
All antibodies share the same basic structure which consists of four polypeptide chains held together by disulfide bonds. These four polypeptide chains form a symmetrical molecular structure comprised of two identical halves with the antigen binding sites formed between the ends of the heavy and light chains on both sides. The antibody has a hinged region in its center between the heavy chains that allows flexibility to articulate antigen binding. The two light (L) chains are identical to each other and are about 220 amino acids long while the two heavy (H) chains are about 440 amino acids in length and are also identical to each other. These chains are bound together by covalent and non-covalent disulfide bonds, and the number and type of disulfide bonds vary between classes of immunoglobulin.
There are two types of light chain among all classes of immunoglobulin, a lambda chain and a kappa chain; and there are no functional differences known between the two. However, there are five main heavy chain classes, or isotypes, which are unique to each class of immunoglobulin, and some isotypes have several different subtypes. These isotypes determine the biological properties of the antibody in an immune response.
An antibody’s function in the immune system is to specifically bind foreign particles and to signal other cells to eliminate the foreign matter. An antibody’s ability to bind specifically is due to how strongly it can interact with the antigen. An antibody is able to bind with its antigen binding sites that are at the amino-terminal end of each branch of the molecule. The strength of the interaction between antibody and antigen is determined by the strength of interaction between the antibody and a single binding site and by the number of binding sites on the antigen. The strength of binding between the antibody and a single binding site is known as the antibody’s affinity for the antigen, and the binding is reversible. The affinity between the antibody and the antigen binding site is determined by the type of bond formed. Because an antigen can have multiple different epitopes, a number of antibodies can bind to the protein. When two or more antigen binding sites are identical, an antibody can form a stronger bond with the antigen than if only one of the antibody’s sites is bound. Antigens with multiple identical binding sites are called multivalent, and antibodies are able to bind it more strongly. This total binding strength is known as avidity, and is a measure of the strength of the interaction between the antibody and antigen.
The antigen binding portion of an antibody varies extensively among secreted antibodies, and this length of sequence is known as the variable region. The variable region construction during antibody production in the B cell is what enables antibodies to be generated against an infinite variety of antigens. The other biological properties of the antibody and its role in signaling to other immune cells are determined by the constant regions of the heavy chains.
Antibody molecules break into fragments when digested with proteolytic enzymes. The fragments are two Fab fragments (fragment of antigen binding), and one Fc fragment (fragment crystallizable). The Fab fragment contains the entire light chain and the amino-terminal portion of the heavy chain with the antigen binding site between the chains. The Fc fragment contains the carboxy-terminal portion of both heavy chains, and it has no antigenic binding sites. In technical applications, it is the Fc portion that allows the antibody to be bound to matrices for plate based assays such as sandwich ELISAs, or allows the antibody to be used in immunoprecipitations and be recognized by secondary antibodies, or to be affinity purified with protein A or G.
In an animal’s primary immune response, the first antibodies made are the IgM class. IgM molecules form large pentameric polymers in the serum. When the animal’s immune system is challenged with the same antigen again, a secondary immune response occurs. The secondary immune response is largely made up of IgG molecules. IgG molecules are the most common circulating antibodies in the immune system, and these are the most common type of antibody used in cell biology. Because of the nature of an immune response, the immunoglobulins specific to a particular antigenic determinant are only part of a large pool of antibodies. Each immunoglobulin produced from each B cell is the result of a proliferated response from that antigen stimulation, and the serum contains the whole mix of clones from many B cells, and is called polyclonal antiserum.
It is possible to have antibodies generated from one single B cell that results in a homogenous population of antibody, or monoclonal antibody. Monoclonal antibodies can be produced by a hybridoma technique which is the fusion of the B cell with a myeloma tumor cell to form a hybrid cell. In practice, after a mouse has been immunized and has an active immune response, the spleen tissue is removed so that the B cells can be isolated and fused with myeloma cells. The tumor cell lends the ability to grow in culture to the antibody-producing B cell, so that in culture, clones of the hybridoma cells survive and single clones are selected to produce a supply the monoclonal antibody. The antibody can be grown in culture or can also be perpetuated by injecting the hybridoma back into a mouse where monoclonal antibodies are removed in the ascites fluid.
Sometimes an immune response can produce antiserum or monoclonal hybridomas that may work well as unpurified antisera, ascites fluid, or culture supernatant. Commonly, affinity chromatography is used to clean up antibodies. Affinity chromatography directly uses the antibody’s ability to bind in a matrix format to enable the less tightly bound and nonspecific immunoglobulins to be washed away.
Commonly, antibodies are purified using protein A or protein G that is conjugated to a sepharose bead. Protein A and G are isolated from bacteria, and these proteins are used due to their intrinsic antibody-binding properties. These proteins bind the Fc regions of the antibodies. Different species of animal produce antibodies with better binding affinities to either protein A or G.
Antibodies may also be immunoaffinity purified. Immunoaffinity purification entails purifying the antibody against the immunogen, either the original peptide or protein, which is linked to a sepharose bead.