Antibody Structure & Function PDF

antibody An antibody is a Y-shaped structure produced by plasma cells. It is released in response to antigenic stimulation and is capable of specific interacti...

antibody An antibody is a Y-shaped structure produced by plasma cells. It is released in response to antigenic stimulation and is capable of specific interaction with a provoking immunogen. Antibodies are components of humoral immune response. They are secreted by plasma cells after B-cells differentiate into 5 antibody plasma and memory Bcells general functions Neutralization - antibodies neutralize toxic substances. Phagocytosis - facilitate phagocytosis and kill microbes. Combine with antigens extravascularly – outside of blood vessels within the mononuclear phagocyte system intravascularly – within the blood vessels through the action of complement types of antibodies theories of antibody diversity ALLOANTIBODIES SIDE-CHAIN THEORY Paul Ehrlich formulated the side-chain theory in the early 1900s. produced in response to antigens from He postulated that certain cells had specific receptors for antigen that were present before contact with individuals of the same species. antigen occurred. Once antigen is introduced it would select the cell with the proper receptors, combination would take place, AUTOANTIBODIES and then receptors would break off and enter the circulation as antibody molecules. produced in response to the body’s own antigen or self-antigens. HETEROANTIBODIES Xenoantibodies aka produced in response to antigens from another species TEMPLATE THEORY Felix Haurowitz put forth a second major theory the instructive or template theory in the early 1930s. CLONAL SELECTION THEORY Antibody-producing cells are capable of synthesizing a generalized type of antibody When contact with an antigen occurs, the antigen serves as a mold or template and alters protein synthesis so Niels Jerne and Macfarlene Burnet that antibody with a specific fit is made. This now specific antibody enters the circulation while the antigen independently supported the idea of Clonal remains behind to direct further synthesis. Selection process for antibody formation. The key premise is that individual lymphocytes are genetically pre- programmed to produce one type of immunoglobulin and that a specific antigen finds or selects those particular cells capable of responding to it, causing it to proliferate. How do B cells produce antibodies? properties of antibodies Protein in nature B cells develop from stem cells in the bone marrow of adults. After maturation, B cells migrate to lymphoid With high molecular weight organs (such as lymph nodes or spleen). Present in serum/plasma, CSF, saliva, seminal fluid When a B cell encounters an antigenic recognizes, it is stimulated and it divides into many clones called the Possess high order of specificity plasma cells, which actively secrete antibodies. Each B cell produces antibodies that will recognize only one antigenic determinant. PROPERTIES OF ANTIBODIES Protein in nature With high molecular weight Present in serum/plasma, CSF, saliva, seminal fluid Possess high order of specificity  AGGLUTINATION Antibodies cause antigens to clump together. IgM is more effective than IgG when it comes to agglutination. Example: Hemagglutination – this is the agglutination of red blood cell and is used to determine the ABO blood types, and for the detection of influenza and measles viruses. OPSONIZATION An antigen is covered with antibodies that enhance its ingestion and lysis by phagocytic cells. NEUTRALIZATION IgG inactivates viruses by binding to their surface and toxins are neutralized by blocking their active sites. ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY Used to destroy large organisms such as worms. The target organism is coated with antibodies and bombarded with chemicals from non-specific immune cells. INFLAMMATION essential for fighting infections and healing tissues, but they can also contribute to chronic inflammatory conditions. ACTIVATION OF COMPLEMENT Both IgG and IgM trigger the complement system which results in cell lysis and inflammation. ANTIBODY STRUCTURE / IMMUNOGLOBULINS LIGHT CHAINS Light chains are small chains common to all immunoglobulin Gerald Edelman and Rodney Porter – they worked with IgG. Edelman worked using analytic centrifugation classes. Porter worked using the enzyme papain Molecular Weight of 25,000 Daltons 200-220 amino acids The basic structural unit of immunoglobulin molecules consists Common to all immunoglobulin classes of four polypeptide chains (2 heavy chains & 2 light chains) Two subtypes: kappa and lambda o Kappa and Lambda have linked together by disulfide bonds. different amino acid sequences and are antigenically 1 virus or microbe may have a several antigenic determinant different. sites or epitopes to which different antibody may bind. In human, about 65% of immunoglobulin molecule have Kappa chains, whereas 35% have Lambda chains. If an antigen has an epitope, an antibody has a paratope. Remember: antibodies have only 1 type of light chain. Paratope – region on the antibody reacting to the Never both in one antibody. epitope of an antigen. 2:1 – Kappa to Lambda ration in normal serum Each antibody has at least 2 (two) identical sites (paratopes) that bind an antigen. For the valence of an antibody, this is referred to as the number of antigen binding sites. HEAVY CHAINS LIGHT CHAINS HEAVY CHAINS Molecular Weight of 50,000 – 77,000 Daltons About 440 amino acids M.W. 25,000 Daltons M.W. 50,000 – 77,000 Daltons There are five antigenically distinct isotypes of heavy Approximately 220 amino acids Approximately 440 amino acids chains: gamma, alpha, mu, delta, and epsilon With an amino-terminal variable domain With an amino-terminal variable domain The antibody classes are defined by their unique heavy chain. With a carboxy-terminal constant domain With three to four constant domains Has structurally distinct classes: kappa and lambda chains Structurally different for each of the defined classes of IgG gamma (Y) 4 subclasses: IgG1, IgG2, IgG3, and IgG4 antibody. IgA alpha (𝛼) 2 subclasses: IgA1, and IgA2 Determines the biologic properties of the antibody Variable sequences confer specificity molecule IgM mu (μ) 2 subclasses: IgM1, and IgM2 IgD delta (𝛅) NO SUBCLASSES IgE epsilon (𝛆) NO SUBCLASSES  DISULFIDE BONDS Bonds that hold the four polypeptide chains in a normal immunoglobulin molecule. Interchain bonds Occur primarily in the hinge region H-H can vary in number from 1 – 15 bonds depending on the class and subclass in an immunoglobulin molecule. Occur in most immunoglobulin molecules H-L except IgA2 which does not contain this particular bond. L-L Occur in pathologic conditions such as Bence Jones proteinuria REGIONS Each heavy and light chains consists of two segments: Variable region (V) Intrachain bonds Constant region (C) VARIABLE REGION Individual chain type The number of bonds vary depending on the type It is unique to a specific antibody molecule, it constitutes Light chains have two the idiotype of the molecule. Human gamma, alpha, and delta chains have four Found in the amino-terminal end of each chain contain Human mu, and epsilon have five these regions which are essential to the formation of The distribution of intrachain disulfide bonds form the antigen binding site. the basis for division for each immunoglobulin into Together, they serve as the antigen recognition unit. domains. CONSTANT REGION Shows an unvarying amino acid sequence in the carboxy- terminal portion of the molecule. Found in the carboxy-terminal end of each chain. CH1, CH2, and CH3  VARIABLE REGION CONSTANT REGION DOMAINS Each immunoglobulin chain consists of a series of globular homology regions or domains. Amino acid sequence is subject to change Amino acid sequence is fixed & unchanged Domain consists of about 110 AA residues. Peptide loops of 60 – 70 AA residues enclosed by intrachain disulfide Amino terminals (NH2) Carboxyl terminal (COOH) bonds represent the central portion of each domain. The amino sequences of the loops show a high degree of Concerned with binding to host tissues & complement homology.. Concerned with binding to antigen fixation Domains on the heavy chain VH, CH1, CH2, CH2, CH4 (IgM and IgE) Domains on the light chain VL, CL VL and VH for antigen binding CH1 binds complement C4b fragment site of complement system activation CH2 that is complement component C1q fixation CH3 binds Fc receptor on macrophages and monocyte; attachment site for phagocytes, B cells, HINGE REGION cytotoxic killer cells, platelet, and nitrogenous mas cells. Interchange disulfide bonds form between the arms of the antigen binding fragments, preventing them from folding and thus, rendering this portion of the molecule highly susceptible to enzymatic attacks. Portion of the heavy chain between the CH1 and CH2 domains. CH2 and CH3 It is considered as a separate domain because it is not bind to Staph. protein A and also binds to Fc receptor homologous to any of the other known domains. on: placental synctiotrophoblast, neutrophil, killer cell. Highly flexible (high in proline) The amino acid sequences of these domains It lets the two antigen binding site operate independently. determine the biological properties of an antibody Effector functions – initiation of the complement cascade molecule. The gamma, delta, and alpha chains, all have a hinge region, For example, the CH3 of an IgG molecule, gives it the but the mu and epsilon do not. However, the CH2 domains of ability to cross the placenta this latter two chains are paired in such a way to confer CH2 gives the antibody molecule the capacity to flexibility to the antigen binding fragment arms of the initiate complement activation antibody molecule. VL and VH give the antibody molecule the ability to neutralize toxins CH3 or CH4 give the antibody molecule the capacity to attach to cell surfaces  ANTIBODY STRUCTURE important terminologies Ig domain 110 amino acids Polymer Variable domains Present on N terminal an Ig composed of more than a single basic monomeric unit. Because some of immunoglobulin classes are dimers and Constant domains Present on C terminal there’s also pentamer Hinge Region Provides flexibility to Fab arms of the antibody molecule J Chain Fab fragment antigen binding The J stands for “Joining”. So, the J Chain or joining chain is found in IgM and IgA. Fc fragment crystallizable It is a glycoprotein with several cysteine residues. It serves as a linkage point for disulfide bonds between two adjacent monomers. Polypeptide chain which normally holds polymeric Ig; cysteine residue If the antigen has an epitope, the Secretory Component antibody has a paratope. The secretory component is a substance that is attached to polymeric Ig found in secretions The paratope is the region on the antibody reacting to the epitope of an antigen. IMMUNOGLOBULIN VARIANTS FRAGMENTATION OF ANTIBODIES An antigenic determinant is the specific chemical determinant group or molecular configuration against which the Proteolytic enzymes immune response is directed. Because they are proteins, immunoglobulins themselves can function as effective Also referred to as “Peptide bond splitting enzyme”, are used to degrade immunoglobulin molecules into definable antigens when used to immunize mammals of a different species. When the resulting anti immunoglobulins or fragments to facilitate the study of their structure. antiglobulin are analyzed, 3 principle categories of antigenic determinants can be recognized. E.g. papain and pepsin Isotypic Fab Fragment This refers to the differences in heavy chain structure. It refers to the heavy chain that is unique to each immunoglobulin class. Fab fragment has antigen binding capacity. Each Fab fragment is consisting of 1 light chain and ½ of 1 heavy chain held together by ALLOTYPIC disulfide bonding. Again, the Fab fragment has all the antigen This refers to the differences in the constant region. This refers to binding capacity the genetic variation and constant regions of the immunoglobulin molecule. Fc Fragment Idiotypic The Fc fragment is also referred to as fragment crystallizable. It has no antigen binding ability and its spontaneously This refers to the differences in the variable region. This refers to crystallizes at 4 degree Celsius. It represents the carboxy terminal halves of the two H chains. It also plays a role in the the variation in the variable regions that give individual antibody opsonization process as well as in complement fixation. molecular specificity. PAPAIN PEPSIN Papain cleaves the molecule on one side of the disulfide bonds, acting directly on the hinge region, forming 3 Pepsin cleaves a monomer just below the hinge region of most of the Fc fragment, leaving one large fragment FRAGMENTS of approximately equal sizes. consisting of 2 Fab fragments joined by univalent bonds Papain cleaves the antibody molecule into 2 FAB FRAGMENTS and 1 FC FRAGMENT. Papain directly acts on the Alfred Nisonoff – first to perform the pepsin digestion of antibodies. hinged region of the antibody molecule. Papain was first used for the fragmentation of antibodies by: Rodney Porter Each fab fragment possesses only 1 combining site. It can combine This proteolytic enzyme was found to cleave IgG at the but cannot precipitate or agglutinate the antigen. For the Fc, the Fc carboxy terminal side of the interchain disulfide bonds fragment contains the carboxy terminal portion of the heavy chain. yielding 1 single fragment with a molecular weight of Some of its function include the following: 100,000 Daltons and all the antigen binding ability Binds compliments known as the: Contains carbohydrate F(ab')2 and Fc' Dictates whether a given immune globulin can cross the placenta It fixes the antibody to the skin CLASSIFICATIONS OF ANTIBODIES ACCORDING TO SEDIMENTATION COEFFICIENT Gerald Edelman, his work centered on using the analytic ultracentrifuge which as a very high-speed centrifuge that According to sedimentation coefficient (Svedberg classification) he used to separate out immunoglobulins on the basis of their molecular weight. According to temperature which they react Svedberg unit – indicates the sedimentation rate in an analytical ultracentrifuge According to occurrence According to species which produce them According to their reaction with an antigen IMMUNOGLOBULIN SEDIMENTATION COEFFICEINT MOLECULAR WEIGHT According to in-vitro behavior IgG 7 S (Svedberg unit) 150,000 IgA 7S 160,000 (IgA1) 350,000 (IgA2) IgM 19 S 900,000 IgD 7S 180,000 IgE 8S 190,000 ACCORDING TO TEMPERATURE AT WHICH THEY REACT ACCORDING TO THEIR REACTION WITH AN ANTIGEN Cold antibodies react at 4 degree Celsius – room temperature immobilization of motile organisms Agglutinins cell clumping antibodies responsible for immobilization of motile organisms and for cell clumping Warm antibodies react at 37 degree Celsius (Rh antibodies) Precipitins antibodies that cause ppt formation by forming complexes with antigen molecules in solution ACCORDING TO occurence agglutinins that are modified by heat in such a manner that they can no longer Agglutinoids bring about agglutination, but which are still capable of combining with specific Natural antibodies appear without any apparent stimulus (anti-A and anti-B antibodies) agglutinogens Hemagglutinins cause agglutination of RBCs Immune antibodies appear following the introduction of an antigen cause dissolution of antigenic cells Lysins Bacteriolysin – acts upon bacterial cells ACCORDING TO SPECIES THAT PRODUCE THEM Hemolysin – acts upon the RBCs Leucocidin – kills leukocytes with or without lysis Isoantibodies antibodies produced after the introduction of the antigen from the same species Heterophile antibodies antibodies produced after the introduction of the antigen from another species ACCORDING TO THEIR REACTION WITH AN ANTIGEN ACCORDING TO THEIR IN-VITRO BEHAVIOR act on bacterial invaders weakening their resistance to the phagocytizing action POINT OF DIFFERENTIATION COMPLETE ANTIBODY INCOMPLETE ANTIBODY Opsonin of leukocytes. Univalent; blocking coagulating; Synonyms Bivalent; saline-acting conglutinating; monovalent Neutralizing antibodies render antigenic microorganisms harmless Responses to temperature Thermolabile Thermostable also referred to as protective antibodies Ability to cross the placenta Cannot cross the placenta Can cross the placenta Allergic antibodies react with allergens in allergic reactions Occurrence Early in immunization Late in immunization Reaction Saline-acting Albumin-acting Antitoxins specific toxins antibodies that neutralize specific toxins Complement-fixing antibodies detected by the consumption of the complement by antigen-antibody complexes Blocking or inhibitory they combine with the antigen but are not grossly detectable unless they are antibodies shown to block a reaction or unless the protein species of an antibody can be identified Immunoglobulins immunoglobulin g Antibodies belong to the class of proteins known as Globulins because of their globular structure. Initially, because of MW of 150,000 Da and an S value of 7 S their migratory properties in an electrophoretic field, they were called Gamma globulins. Today, they are known IgG is a monomer collectively as Immunoglobulins. Four subclasses By physicochemical and immunochemical techniques, also known as the immune antibody the immunoglobulins consist of five different isotypes: IgG, IgA, IgM, IgD, and IgE. They differ not predominant immunoglobulin in humans comprising only in antigenic reactivity (serologically) and approximately 70% - 75% of the total serum immunoglobulins. SUBCLASSES POPULATION NO. OF DISULFIDE carbohydrate content but also in size and biological BONDS function. IgG has the longest half-life which is approximately IgG1 66% 2 23 days IgG, IgD, and IgE all exist as monomers IgG2 23% 4 IgA may exist as a polymer or as a monomer warm antibody and it is considered as the most IgG3 7% 15 IgM exist as a pentamer efficient precipitin IgG4 2% 2 the secretory component may be found in IgA the J chain or the Joining chain is found in both IgA IgG precipitates and agglutinates in vitro and IgM Functions: immunoglobulin A Major immunoglobulin in normal human serum IgA is present in the serum and in various bodily Major antibody in secondary immune response secretions, and thus, takes two forms: serum IgA and Only lg that can cross the placenta secretory IgA IgG molecules are capable of fixing complement Mucosal infections, atopy, autoimmune diseases IgG is important in phagocytosis Alternative pathway It is capable of changing its shape IgG are aggregated by treatment with alcohol and heating SERUM IgA Properties: In humans, over 80% of serum IgA exists in monomeric form with a 7 S value. Structure Monomer IgA is inactivated by IgA protease: produced by Percent of Total Ig 70 – 75% Neisseria gonorrhoeae Molecular Weight (Daltons) 150,000 Sedimentation Coefficient 7S SECRETORY IgA Serum Half-Life (Days) 23 days Predominant immunoglobulin in various secretions sIgA is responsible for local immunity Complement Fixation yes sIgA molecules protect mucosal surfaces Crosses Placenta yes SECRETORY COMPONENT immunoglobulin A Mucosal immunity Most primitive Prevents attachment of pathogen to mucosal surface Has a pentameric structure Prevents enzymatic degradation of IgA Possesses J chain Value of 19 S Properties: It is easily dissociated by reducing agents such as 2- mercaptoethanol (2-ME) and dithiothreitol (DTT) Structure Monomer/ Dimer Percent of Total Ig 10 – 15% FUNCTIONS: Molecular Weight (Daltons) 160,000/350,000 Predominant antibody in the early (primary) immune Sedimentation Coefficient 7S response to most antigens and the predominant antibody Serum Half-Life (Days) 5 produced by the fetus. IgM is the most efficient immunoglobulin at fixing Complement Fixation YES complement in lytic reactions and enhancing phagocytosis. Crosses Placenta NO Some IgM is synthesized locally in secretory tissue. Wasserman antibodies, heterophile antibodies, Rheumatoid factor, cold agglutinins, and allohemagglutinins Properties: immunoglobulin D FUNCTIONS: Structure Pentamer One unique structural feature is the presence of IgD occurs in large quantities on the membrane of human only a single HH interchain bond, along with H-L B cells and may, as an antigen receptor be involved in B Percent of Total Ig 10% interchain bonds. cell activation Molecular Weight (Daltons) 900,000 Its function remains to be unknown Anti-idiotypic antibody Sedimentation Coefficient 19 S Heat-and acid-labile Immunoregulation Serum Half-Life (Days) 6 Complement Fixation YES Properties: Crosses Placenta NO Structure Monomer Percent of Total Ig