Antigen And Antibodies

ANTIGEN AND ANTIBODIES Definition "An antigen is a substance which when introduced into a host induces the formation of specific antibodies and T lymphocytes that are reactive against the antigen." Immunogenicity and Antigenicity Antigens have two very important characteristics: immunogenicity, or the ability to stimulate the specific immune response, and antigenicity: the ability to react specifically with antibodies. An antigen with both these characteristics is called a complete antigen. Determinants of Antigenicity · Macromolecular size · Molecular complexity · Biodegradibility · Foreignness · Specificity. Macromolecular Size; proteins of molecular weight exceeding 10,000 daltons are good antigens. Molecular Complexity; The antigenic potency of macromolecule increases with the complexity of structure and accordingly quaternary structures are antigenically most potent. Biodegradability; If a substance is insoluble in body fluids and cannot be converted to soluble forms by tissue enzymes, it may not act as an antigen. Foreignness; To be antigenic, the macromolecule must be foreign to the animal being immunized. Specificity; Antigen attaches specifically to an antibody because of the "fit" between the antigenic determinant on its surface as well as the receptor on antigen binding site on antibody. The effect of antibody is activated only after the "fit" has taken place. Isospecificity; is called when the antigens differentiate members of same species viz HLA and blood group antigens. Antigen Nomenclature The antigens that require T cells in order to generate an immune response are called as T celldependent (TD) antigens. Antigens that stimulate B cells without the intervention of T cells are the T cell-independent (TI) antigens. The booster or memory response is mediated through T cells and hence can be initiated only by TO antigens. There are certain other terms which are in common use regarding some varieties of antigens. Autologous Antigen: It is one's own antigen, which under appropriate circumstances would induce autoantibody formation. Thus, autologous antigen is synonymous with auto or self-antigen. Heterologous Antigen: It is merely an antigen different from that used in immunization; it may or may not react with the antiserum depending on its chemical similarity to homologous antigen. Homologous Antigen: It is the antigen used in the production of antiserum. Isophile Antigen: Isophile antigens or isoantigens are the molecules of one individual of a species that are antigenic in another member of same species. Best example of isoantigens is blood group system. Cross Reactivity of Antigens An antigen can be a complex mixture of many antigenic molecules, e.g. a microbe. Cross reactivity may occur if different complex antigens have similar antigenic molecules. Cross reactivity may also result from the presence of a variety of molecules in the preparation, some of which are shared. Antigenic Determinant Sites (Epitopes) The whole antigen does not induce an immune response. Only a limited part of an antigen molecule is inducer of Band T cell responses. It is also that part of antigen with which the antibody or T cell reacts. This is called an antigenic determinant site or epitope.

Haptens Haptens are too small molecules to be antigenic in their own right. Injection of hapten into an animal does not normally induce an immune response. At the same time hapten is capable of reacting with antibody induced by injection of a hapten-carrier complex. Thus, a hapten is defined as a molecule that is not immunogenic in itself but that can react with preformed antibody of right specificity. Haptens can be covalently coupled to existing established antigens (carrier) to create new antigenic determinants. These hapten-antigen (carrier) complexes, or conjugated or neoantigens, generate antibodies with specificity for haptenic groups. Haptens are of two types: a. Simple haptens combine with specific antibody but do not produce any antigen-antibody product viz precipitation. b. Complex haptens do combine with specific antibody to produce precipitates because of presence of multiple antibody combining sites on its surface.

Adjuvants Adjuvants are agents used to potentiate the immune response; both humoral and cell-mediated. Adjuvants are customarily administered with the antigen. An adjuvant may not be an antigen itself. Classification of adjuvants Particulate/Insoluble Aluminium Calcium Oil-in-water Water-in-oil ISCOM Liposomes

Non-particulate/Soluble Muramyl dipeptide Lipid A Saponin Cytokines Carbohydrate polymers Bacterial toxins

The aluminium and calcium salts are best known examples of adjuvants. These adjuvants, by increasing the physical size of the antigen, also enhance phagocytosis. Mechanism of Action of Adjuvants Possible modes of action of adjuvants Principle

Mechanism

Immunomodulation

Modify cytokine network and better processing of antigen

Presentation of antigen

Optimal antigenic determinant is presented to effector cells

Induction of cells

CDS cells are induced for cytotoxic response

Targetting of cells for antigen

Better delivery of antigen to immune effector cells

Depot generation

Sustained and long-term release of antigen for boosting the immune response

ANTIBODY Antibodies are blood proteins, all of which are globulins (hence the synonymous immunoglobulins) part of gamma fraction of serum. In addition to those found in blood (humoral antibodies), some types of antibodies are fixed to body cells or tissues or exist in body secretions (cell bound antibodies). Structure Immunoglobulin (Ig) molecules are symmetrical structures. In solution they become 'Y' -shaped after binding to an antigen. Each molecule consists of four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains. These are designated light or heavy based upon their molecular weight which is 50,000 to 70,000 daltons for H chains and 20,000 to 25,000 daltons for light chains. The L-chain is attached to H-chain by a disulphide bond. The two H-chains are joined together by 1 to 5 S-S-bonds depending on the class of immunoglobulins. The H chains are structurally and antigenically distinct for each class of immunoglobulin. The L-chains are similar in all classes of Ig. They occur in two types: Kappa (K) and lambda (A). A molecule of immunoglobulins could have either kappa or lambda but never both. L and H chains are subdivided into variable regions and constant regions. An L-chain consists of one variable domain (Vl and one constant

domain (Cd). Variable regions are responsible for antigen binding while the constant regions are responsible for biological functions. The antibody molecule can be split by papain to yield two identical fragments, each with a single combining site for antigen. This is called as Fab-fragment antigen binding. The third fragment which lacks the ability to bind to antigen is termed as Fc-fragment crystallizable. In an experimental setting, enzymes can be used to cleave the antibody into Fc and Fab fragments. Fe region: The Fc region (fragment, crystallizable), is derived from the stem of the "Y," and is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. The Fc region binds to various cell receptors and complement proteins so it mediates different physiological effects of antibodies such as opsonization, cell lysis, degranulation of mast cells, basophils and eosinophils and other processes. Fab region: Each end of the forked portion of the "Y" on the antibody is called the Fab region

(fragment, antigen binding). It is composed of one constant and one variable domain of each of the

heavy and light chain. These domains shape the paratope, the antigen binding site, at the amino terminal end of the monomer. The two variable domains bind the epitope on their specific antigens. Classes (Isotype of Ig) Based upon the structure of their heavy chain constant region, immunoglobulins are classed into major groups called classes and also termed as isotypes. In human beings there are five classes: IgG, IgA, IgM, IgD and IgE. Within class IgG, based upon different distinctive heavy chains and differing functional properties, there are four subclasses: IgG1, IgG2, IgG3 and IgG4. Similarly there are two subclasses each of IgA and IgM. Characteristics of Immunoglobulins Physical and biological characteristics of the five major immunoglobulin classes in the humans have been summarised in Table 18.1. The following description shall supplement the information given in Table 18.1. Immunoglobulin G Immunoglobulin G (IgG) is the class of immunoglobulin which has maximum concentration in serum and is the major immunoglobulin to be synthesized during the secondary immune response. It is the major line of defence in a newborn because of its capability to pass through placenta as well as to be secreted in colostrum. IgG can be further sub grouped into four isotypic subclasses - IgG1, IgG2, IgG3, and IgG4. Characteristics of human immunoglobulins Class Molecular weight Valency for antigen binding Heavy chains class Subclasses J chain Secretory piece Present in epithelial secretions Percent of total Ig

IgG

IgM

IgA

IgD

IgE

150,000

900,000

160,000

150,000

200,000

2

10

2or4

2

2

Gamma 4 -

Mu 1 + -

Alpha 2 + +

Delta -

Epsilon -

-

-

+

-

-

70-80

5-10

10-15

1

0.01

Immunoglobulin A The main function of IgA is to defend the exposed external surfaces of the body against attack by microorganisms. It appears selectively in saliva, tears, nasal fluid, sweat, colostrum and secretions of lungs, genitourinary and gastrointestinal tracts. IgA is synthesized locally by plasma cells. Two subclasses of IgA have also been found, IgA1 and IgA2. Immunoglobulin M Because of high molecular weight (900,000) and a polymer of five four peptide units, the IgM is also referred to as the macroglobulin antibody. These antibodies are extremely efficient, appear early in response to infection and are largely confined to the bloodstream. Immunoglobulin D IgD is present on the surface of a proportion of blood lymphocytes where it seems likely that they may operate as mutually interacting antigen receptors for the control of lymphocyte activation and suppression. Immunoglobulin E Very few plasma cells in body synthesize this immunoglobulin and the concentration of IgE in serum is also very low. IgE antibodies remain firmly fixed for an extended period when injected into human skin where they are probably bound to mast cells. Contact with antigen leads to release of vasoactive amines. The main physiological role of IgE seems to be protection of external mucosal surfaces of the body through triggering an acute inflammatory reaction.

Two theories of Immunoglobulin Formation are: Ehrlich's Instructive Theory As per this theory, the antigen enters a cell that is routinely engaged in normal gamma globulin synthesis. The antigen interferes with this process, possibly by complexing with mRNA in the polysome. This results into a change in gamma globulin synthesis which now takes the shape of antibody. The antibody dissociates itself from antigen and is excreted into the blood. Clonal Selection Theory A clone is a population of cells arising from a single parent cell. As per this theory, in a mature animal the lymphocyte is genetically endowed with the capability of synthesizing immunoglobulins. At rest, unstimulated by antigen, only small amounts of immunoglobulins are formed. On contact with the corresponding antigen, lymphocyte capping heralds a change of that lymphocyte to reproduce and differentiate into a clone of immunoglobulin secreting plasma cells. The resulting clone of cells would consist of a large enough population that the antibody produced would become measurable into the blood. Soluble Antibodies and Membrane Bound Antibodies Antibodies occur in two forms: A soluble form that is secreted from cells and released into the blood and tissue fluids, and a membrane-bound form that is attached to the surface of a B cell and is called the B cell receptor (BCR). The BCR on the surface of B cells allows the B cell to detect when a specific antigen is present in the body. Once the B cell binds to an antigen the B cell can be activated - interaction of the B cell with a T helper cell is necessary to produce full activation of the B cell. The activated B cell differentiates into either soluble antibody generating factories called plasma cells, or into memory cells that will survive in the body for years afterwards, allowing an organism to remember that antigen and respond faster upon future exposures. Antimicrobial Actions of Antibodies The major antimicrobial actions of antibodies are · Opsonization for phagocytosis · Complement activation, enhancing phagocytosis and inducing lysis · Prevention of attachment of Ag to host cells · Prevention of penetration by Ag of host cells · Neutralization of toxins · Inhibition of motility of parasites · Agglutination of parasites · Inhibition of microbial growth and metabolism MONOCLONAL ANTIBODY (HYBRIDOMAS) Hybridomas are produced by fusion of high density antibody forming cells and nonsecreting myeloma cells. These cells have several advantages as: a. Pure antibodies are produced from crude antigen preparation. b. Antibodies to those antigens can be synthesized which are not detectable by conventional means. c. Antibody produced is of single Ig class and specific for single epitope. d. The produce is constant. e. The quality is same since large quantity of uniform quality can be produced. f. Can help in antigen mapping. g. Dynamics of mutation in antibody forming cells can be studied. The hybridoma technique provides a novel avenue for investigating the antigenic nature of infectious agents, tumour antigens, HLA antigens, differentiation of antigens and provides reagents of a purity, that were never before available.