1. The Immune Response 1Dr. Nikhat Siddiqi

2. All vertebrates have an immune system capable of distinguishing molecular “self” from “nonself” and then destroying those entities identified as nonself. In this way, the immune system eliminates viruses, bacteria, and other pathogens and molecules that may pose a threat to the organism. 2Dr. Nikhat Siddiqi

3. The immune response consists of two complementary systems, the humoral and cellular immune systems. The humoral immune system (Latin humor, “fluid”) is directed at bacterial infections and extracellular viruses (those found in the body fluids), but can also respond to individual proteins introduced into the organism. The cellular immune system destroys host cells infected by viruses and also destroys some parasites and foreign tissues. 3Dr. Nikhat Siddiqi

4. The proteins at the heart of the humoral immune response are soluble proteins called antibodies or immunoglobulins, often abbreviated Ig. 4Dr. Nikhat Siddiqi

5. Humans are capable of producing more than 108 different antibodies with distinct binding specificities. This extraordinary diversity makes it likely that any chemical structure on the surface of a virus or invading cell will be recognized and bound by one or more antibodies. 5Dr. Nikhat Siddiqi

6. Any molecule or pathogen capable of eliciting an immune response is called an antigen. An antigen may be a virus, a bacterial cell wall, or an individual protein or other macromolecule. A complex antigen may be bound by a number of different antibodies. An individual antibody or T-cell receptor binds only a particular molecular structure within the antigen, called its antigenic determinant or epitope. 6Dr. Nikhat Siddiqi

7. Molecules of Mr 5,000 are generally not antigenic. However, small molecules can be covalently attached to large proteins in the laboratory, and in this form they may elicit an immune response. These small molecules are called haptens. The antibodies produced in response to protein linked haptens will then bind to the same small molecules when they are free. Such antibodies are sometimes used in the development of analytical tests described later in this chapter or as catalytic antibodies. 7Dr. Nikhat Siddiqi

8. Immunoglobulin Immunoglobulin G (IgG) is the major class of antibody molecule and one of the most abundant proteins in the blood serum. IgG has four polypeptide chains: two large ones, called heavy chains, and two light chains, linked by noncovalent and disulfide bonds into a complex of Mr 150,000. The heavy chains of an IgG molecule interact at one end, then branch to interact separately with the light chains, forming a Y- shaped molecule. At the “hinges” separating the base of an IgG molecule from its branches, the immunoglobulin can be cleaved with proteases. Cleavage with the protease papain liberates the basal fragment, called Fc because it usually crystallizes readily, and the two branches, called Fab, the antigen-binding fragments. Each branch has a single antigen-binding site. 8Dr. Nikhat Siddiqi

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10. In many vertebrates, IgG is but one of five classes of immunoglobulins. Each class has a characteristic type of heavy chain, denoted α, ᵟ, ε,ᵞ, and μ for IgA, IgD, IgE, IgG, and IgM, respectively. Two types of light chain, and, occur in all classes of immunoglobulins. 10Dr. Nikhat Siddiqi

11. Binding of IgG to an antigen 11Dr. Nikhat Siddiqi

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13. Affinity chromatography Specific antibody can be isolated from an antiserum by affinity chromatography, which exploits the specific binding of antibody to antigen held on a solid matrix. Antigen is bound covalently to small, chemically reactive beads, which are loaded into a column, and the antiserum is allowed to pass over the beads. The specific antibodies bind, while all the other proteins in the serum, including antibodies to other substances, can be washed away. The specific antibodies are then eluted, typically by lowering the pH to 2.5 or raising it to greater than 11. Antibodies bind stably under physiological conditions of salt concentration, temperature, and pH, but the binding is reversible as the bonds are noncovalent. Affinity chromatography can also be used to purify antigens from complex mixtures by using beads coated with specific antibody. The technique is known as affinity chromatography because it separates molecules on the basis of their affinity for one another. 13Dr. Nikhat Siddiqi

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15. Production of Antibodies Most of the antibodies used in immunochemistry are raised by injection of a solution or suspension of appropriate antigen into rabbit. After a period of time 5-50 cm3 of blood is obtained from immunized rabbit by an incision in the posterior marginal vein of the ear. 15Dr. Nikhat Siddiqi

16. The blood is allowed to clot at 37 C for one hour. The clot is detached from the slides of its container to retract and left at 4 C to contract and exude 2-25 cm3 of serum. The serum is separated by centrifugation. Proteases and complement are inactivated by incubating the serum at 56 C for 45 minutes. Sheep, goats, horses are used for large scale antibody production. 16Dr. Nikhat Siddiqi

17. When weakly antigenic compounds are used, there are two approaches for obtaining antisera of reasonable titre. Firstly the period of time that the immune system is exposed to antigen may be extended either by repeated inoculation or establishing within the rabbit depots of antigen that slowly release the antigen over weeks. 17Dr. Nikhat Siddiqi

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