Antibody Structure AND Function PDF

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Page | 1II.) ANTIBODY STRUCTURE ANDFUNCTIONANTIBODIES/IMMUNOGLOBULINSWhen B lymphocytes are stimulated by antigen and undergo differentiation Glycoprotein substances synthesized by plasma cells in response to antigenic stimulation.  Structurally all antibodies are immunoglobulin but functionally, n...

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CLINICAL IMMUNOLOGY AND SEROLOGY II.2.) ANTIBODY FUNCTION

STRUCTURE

AND

TETRAPEPTIDE STRUCTURE OF IMMUNOGLOBULINS

ANTIBODIES/IMMUNOGLOBULINS

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When B lymphocytes are stimulated by antigen and undergo differentiation Glycoprotein substances synthesized by plasma cells in response to antigenic stimulation. Structurally all antibodies are immunoglobulin but functionally, not all immunoglobulin are antibodies. Immunoglubulins are considered to be the humoral branch of the immune response. 82 – 96% protein and 2-14% carbohydrate. With high molecular weight. The ability of immunoglobulins to bind antigen is determine by amino acid sequence in variable region Present in serum/plasma, saliva, semen, CSF and other body fluid Five major classes: Ig GAMED Appear primarily in the gamma (y) band with serum electrophoresis at pH 8.6

Basic four-chain polypetides o Two large heavy (H) chains o Two small light (L) chains Held together by noncovalent forces and disulfide interchain bridges The amino-terminal end of each chain is variable region, while the carboxy-terminal end is one or more constant region Single, unique variable region (amino-terminal end) o with varying antibody specificity (variable region) One or more constant regions (carboxy-terminal end) o with constant amino acid sequence (constant region) Gerald Edelman and Rodney Porterelucidated the basic structure of immunoglobulins (IgG)

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ANTIBODY FRAGMENTATION Fc Fragment Has no antigen-binding ability Represents the carboxyl-termibal halves of two H chains Held together by S-S bonding Important in effector functions of immunoglobulins molecules o Opsonization o Complement fixation Fab fragment One L chain One-half of an H chain Held together S-S binding Obtained by papain digestion of an immunoglobulin Pepsin digestion yield F(ab)2 fragment, with all the antibody activity, and an FC1 fragment Papain digestion yield two F(ab) fragments and an Fc portion

CLINICAL IMMUNOLOGY AND SEROLOGY LIGHT CHAINS (BENCE JONES PROTEIN) Discovered in 1854 by Dr. Henry Bence Jonce o Bence Jonce Protein – In cases of malignancy such as Plasma Cell Leukemia L chains secreted by malignant plasma cells Two types o Kappa (K) – found in chromosome #2 o Lambda ( λ)- found in chromosome #22 Contains between 200 and 220 amino acids Constant region o Is the C-terminal and contain similar amino acids for each class of antibody Variable region o Includes 110-130 amino acids of the light chain and is responsible for binding to antigen. This part of the antibody shows variation in amino acids when the specificity of the antibody for antigen is changed.

designated CH1, CH2, and CH3 Unique to each class and give each immunoglobulin type its name o IgG- Gamma heavy chains o IgM- Mu heavy chains o IgA- Alpha heavy chains o IgD- Delta heavy chains o IgE Epsilon heavy chain ANTIGENIC PROPERTIES OF ANTIBODIES Isotype: A unique amino acid sequence that is common to all immunoglobulin molecules of a given class in a given species Allotype: Minor variations of isotype sequences that are present in some individuals but not others o Found in the constant region and are inherited in simple Mendelian Fashion o Occur four in IgG subclasses in one IgA subclass and in the kappa light chain Idiotype: Variations in variable regions that give individual antibody molecules specificity. o Found in the amino-terminal endsof both L and H chains o Essential to the formation of the antigen-binding site o Together they serve as an antigen-recognition unit.

HEAVY CHAINS SEQUENCING Variable domain- first 110 amino acids at the amino-terminal domain Constant regions- Remaining amino acids o Three or more regions with very similar sequences Page | 2 RIGIDOR S. PADAYHAG | MLS 3D |UNIVERSITY OF THE IMMACULATE CONCEPTION

HINGE REGION Flexible amino acid stretch in the central part of the heavy chains of the IgG and IgA immunoglobulin classes, which links these 2 chains by disulfide bonds o High content of proline and hydrophobic residues o Flexibility assists initiation of the complement cascade  Allows two antigenbinding sites to operate independently Rich in cysteine and proline amino acids, extremely variable in amino acid sequence No resemblance to any other immunoglobulin region Gamma, delta and alpha chains have hinge region Mu and epsilon chains do not have hinge region

CLINICAL IMMUNOLOGY AND SEROLOGY CARBOHYDRATE PORTION Found in all types of immunoglobulins Localized between the CH2 domains of the two H chains Increases the solubility of Immunoglobulin Provides protection against degradation Enhances functional activity of the Fc domains THREE-DIMENSIONAL STRUCTURE OF ANTIBODIES Basic four-chain structure is folded into compact globular subunits stabilized by intrachain disulfide bonds Beta-pleated sheet o Within regions or domains, polypeptide chain is folded back and forth on itlsef Immunoglobulin fold o Cylindrical structure produced when folded domains of the H lines up with those of the L chains

Hypervariable regions o Strategic locations wherein antigen binds to a small number of amino acids and then captured within barrel o Comparisons of the amino acid sequences of the variable regions of immunoglobulins show the most of the variability resides in three regions called the hypervariable Complementariry-determining Regions (CDRs) o Found in both H and L chains o Occurs as loops in the folds of ariable regions o Antigen binds in the middle of CDRs CLASSESS OF ANTIBODIES IMMUNOGLOBULIN G Predominant immunoglobulin in humans (75% to 80% of the total serum immunoglobulins) Has the longest half-life of any immunoglobulin class (23 days, predominance in serum) Has four subclasses: o IgG1: 66% o IgG2: 23% o IgG3: 7% o IgG4: 4% Differ in number and position of the disulfiede bridges between the y chains

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Equally distributed in the different fluid compartments with detectable amounts in CSF and urine Readily diffusible IgG antibody response appears later than IgM in primary response but they form the major antibody of the secondary immune response. Maternal IgG is actively and selectively transferred across the placenta to the fetus and imparts passive protection to the newborn for 6-9 months Functions: Providing immunity for the newborn because IgG is the only Ab that can cross placenta Fixation of the complement Opsonization (Coating antigen) Neutralizing toxins and virus Agglutination and precipitation reaction SUBCLASSES

Decreasing serum concentration IgG1, IgG3 and IgG4 cross placenta well (EXCEPT IgG2)

CLINICAL IMMUNOLOGY AND SEROLOGY Not all subclasses fix equally: o Ig4 does not fix complement Most effective complement activator (IgG3) o Largest hinge region o Largest number of interchain disulfide IgG1 and IgG3 binds to Fc receptor on phagocytic cells (Opsonization) o Induced in response to protein antigens IgG2 and IgG4- Associated with polysaccharides antigens o Shorter hinge segment o Do not bind to Fc receptors o Poor mediators of complement activation A high diffusion coefficient allows IgG to enter extravascular spaces moer readily than other immunoglobulin types IgG plays a major role in neutralizing toxins and viruses. Agglutination and precipitation reactions take place in vitro IgG is better at precipitation reactions than at agglutination o Precipitation involves small soluble particles, which are more easily brought together by the relatively small IgG molecule o IgM is more efficient in agglutination

IMMUNOGLOBULIN M Known as a macroglobulin o The largest of the Ig accounting for 5-10% of the total immunoglobulin pool. o Has a molecular weight of about 900,00 d Serum half-life (6 days) Star-shaped in the free state; crab-like in Ag-Ab reaction. Known as the primary response antibody o The earliest to appear in the pri immune response but it does not persist for long. o Appear first after antigenic stimulation and on the maturing infant o Synthesized only as long as antigen remains Maternal IgM does not cross the placenta. IgM detection in newborn is a useful indicator of intrauterine infection A powerful agglutinin of a particulate antigen IgM normally exists as a pentamer (19S immunoglobulin) but it can also exist as a monomer. In the pentameric form all heavy chains are identical and all light chains are identical. Thus, the valence is theoretically 10. IgM has an extra domain on the mu chain (CH4) and it has another protein covalently bound via a S- S bond called the

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Joining chain or J chain. This chain functions in polymerization of the molecule into a pentamer. High valency Found mainly in the intravascular pool No memory cells

Functions of IgM: Complement fixation Agglutination Opsonization Neutralization of toxins Surface receptor for antigens (on Bcells) Properties IgM is the third most common serum Ig IgM is the first Ig to be made by the fetus and the first Ig to be made by a virgin B cells when it is stimulated by antigen As a consequence of its pentametric, IgM is a good complement fixing Ig. Thus, IgM antibodies are very efficient in leading to the lysis of microorganism

CLINICAL IMMUNOLOGY AND SEROLOGY As a consequences of its structure, IgM is also good agglutinating Ig. Thus, IgM antibodies are very good in clumping microorganisms for eventual elimination from the body IgM binds to some cell via Fc receptors

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Polymeric form stabilized a short polypeptide chain. It is known as the “antiseptic paint” of mucous membranes. It can activate the bacteriolytic activity through the alternate pathway of complement system and only Synthesized in plasma cells found mainly in mucosalassociated lymphoid Captured by secretory component (SC) on epithelial cells Patrol mucosal surfaces and acts as a first line of defense  Neutralizes toxins produced by microorganism  Prevents bacterial adherence to mucosal surfaces Passively transfer immunity to newborn during breastfeeding Complexes of IgA and antigen are easily trapped in mucus and eliminated by the ciliated epithelial cells of the respiratory and intestinal tracts Aggregation of IgA immune complexes may trigger the alternate complement pathway

IMMUNOGLOBULIN A (IgA) Represents 10-15% of all circulating o immunoglobulins One variable region and three constant regions o Major antibody class in secretions Major immune factor in combating pathogens in the respiratory, urinary o and intestinal tract Presence of J chain and a secretory component (resistant to digestion) Second most common serum Ig Monomeric forms do not activate complement but the dimeric forms fix complement Transport mechanism across o endothelial cells (SC) Forms of IgA: Serum IgA - accounts for about 15o 20% of the total normal serum Ig o can agglutinate motile infectious agents thus promoting their phagocytosis but they cannot activate the complement system o Secretory IgA (sIgA)- predominance in body secretions (saliva, tears, colostrum, bronchial, genitourinary and intestinal secretions) Page | 5 RIGIDOR S. PADAYHAG | MLS 3D |UNIVERSITY OF THE IMMACULATE CONCEPTION

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Neutrophil, monocytes, and macrophages possess specific receptors for serum and secretory IgA Binding to these sites triggers a respiratory burst and degranulation of the cells involved Both forms of IgA can, thus, act as opsonins, or promoter of phagocytosis

Subclasses: IgA1 – Alpha 1 heavy chains o Mainly found in Serum o Act as antiinflammtory agent o Downregulates IgGmediated phagocytosis, chemotaxis, bacterial activity and cytokine release o Can be inactivated by an IgA protease produced by gonococci, meningococci, pneumococci and Haemophilus influenzae

CLINICAL IMMUNOLOGY AND SEROLOGY IgA2 – Alpha 2 heavy chains o Important in mucosal immunity o Predominantly found in secretions at mucosal surfaces o A dimer along the respiratory, urogenital and intestinal mucosa o More resistant to some bacterial proteinases that are able to cleave IgA1

The second immunoglobulin to appear on the surface of B cells (After IgM May play a role in B-cell activation Plays a role in regulating B-cell maturation and differentiation Secreted form in serum does not appear to serve a protective function o Does not bind complement o Does not bind to neutrophils or macrophages o Does not cross the placenta Detectable by highly sensitive assay requiring radio-labelled antisera

IMMUNGLOBULIN D ( IgD) Extremely scarce in the serum Has a molecular weight of approximately 180,000 Heat labile immunoglobulin, accounts for less than 1% of the total serum Ig but is known to be present in large quantities on the membrane of many circulating immunocompetent unstimulated B lymphocytes. Delta Heavy Chain o Has a molecular weight of 62,000 o Appears to have an extended hinge region consisting of 58 amino acids Precise biological action is not known but it may play a role in antigen-triggered lymphocyte differentiation More susceptible to proteolysis than other Immunoglobulins Has a short half-life (1-3 days) Page | 6 RIGIDOR S. PADAYHAG | MLS 3D |UNIVERSITY OF THE IMMACULATE CONCEPTION

IMMUNOGLOBULIN E (IgE) Present in lowest concentration in serum/ least abundant in serum accounting for only 0.0005% of the total serum Ig. Most Heat labile immunoglobulin. Plasma cell present in the mucous membrane of the GI and respiratory tracts. It is unable to fix the complement via the classical pathway. It is homocytotropic due to its affinity for cells of the host species, particularly for tissue mast cells and blood basophils. Because of its ability to attach to the human skin, it is associated with immediate hypersensitivity reactions but also, apparently, with immunity to certain helminthic parasites.

CLINICAL IMMUNOLOGY AND SEROLOGY Also known as reaginic antibody/ nuissance antibody Attaches to basophils, eosinophils, and tissue mast cells through highaffinity Fc e RI receptors IgE Function: Allergic Reactions Two adjacent IgE molecules on a mast cell bind a specific antigen Cascade of cellular events results in degranulation of mast cells release of vasoactive amines (such as histamine and heparin) ALLERGIC REACTION

Implicated in Type 1 immediate hypersensitivity results o Hay fever, asthma, vomiting, and diarrhea, hives, lifethreatening anaphylactic shock IgE Function: Parasitic Infection Eosinophils play a major part in the destruction of large antigens, such as parasitic worms, that cannot easily phagocytized.

Mast cell has Fc e R1 – a receptor site for the Fc portion of the IgE During a first encounter with an allergen, the Fc receptor of the IgE will bind at the Fc e R1 receptor of the mast cell. During second encounter with an allergen, the allergen will bind at the Fab portion of the IgE causing this to crosslink, thus, resulting to mast cell degranulation releasing the preformed mediatiors (Histamine) of an allergy reaction

ANTIBODY DIVERSITY THEORIES EHRLICH’S SIDE-CHAIN THEORY The first theory to account for antibody diversity establish by Paul Ehrlich in the early 1900s Postulated that certain cells had specific surface receptors for antigen that were present before contact with antigen occurred >If antigen introduced, combines with the proper receptor to break off and enter the circulation as antibody molecules

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Based in the antigen selection (B lymphocytes) Characterized by the presence of specific surface receptors for antigen (surface immunoglobulins IgM, IgD) The lock and key concept Capacity to respond to specific antigen Postulated that process could be repeated with further contact with antigens 1950s: JERNE AND BURNET’S CLONAL SELECTION FOR ANTIBODY FORMATION Lymphocyte are genetically preprogrammed to produce one type of immunoglobulin and that specific antigen finds or selects those particular cells capable of responding to it, causing these to proliferate. Repeated contact with the antigen would continually increase a lymphocyte pool. A theory postulated to explain the specificity of antibody formation, based on the premise that each lymphocyte is genetically programmed to produce a specific

CLINICAL IMMUNOLOGY AND SEROLOGY type of antibody and is selected by contact with antigen Require a large number of gene 1965: Dryer and Bennett Constant and variable portions of immunoglobulin chain are coded for by separate genes TEMPLATE THEORY Antibody producing cells are capable of synthesizing a generalized type of antibody, and when contact with an antigen occurs, the antigen serve as a mold or template and alters protein synthesis so that antibody with a specific fit is made. The “molded” antibody then enters the circulation , while the antigen remains behind to direct further synthesis SELECTIVE THEORY Assumes that antibodies are synthesized in a manner similar to that of other proteins. Instructions for their synthesis are provided by genetic elements in the nucleus of the cell rather from the antigen GENES CODING FOR IMMUNOGLOBINS Tonegawa did some pioneering experiments with DNA and discovered that chromosomes contain no intact immunoglobulin genes, only building blocks from which genes can be assembled. o Confirmed the hypothesis of Dryer and Bennett

Chromosomes contain building blocks from which genes can be assembled Human immunoglobin genes are found in three unlinked clusters: o H chain genes on chromosome 14 o K chain genes on chromosome 2 o λ chain genes on chromosome 22 Rearrangement is needed for genes to become functional antibody molecules o If a successful rearrangement of DNA on one chromosome 14 occurs, then the genes on the second chromosome are not rearranged; this phenomenon is known as allelic exclusion. More than one gene control synthesis of a particular immunoglobulin o H chains  Variable-region genes: Vh, D, and J  Constant-region genes: set of C genes o L chains: lack of D region Through a random selection process, these individual segments are joined to commit that lymphocyte to making antibody of a single specificity o Do not react to random antigen

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The variable and constant regions are joined at the ribonucleic acid (RNA) level, thus conserving the DNA of the constant regions and allowing for a later phenomenon called class switching, whereby daughter plasma cells can produce antibody of another type. MONOCLONAL ANTIBODIES Heterogeneity of the antiserumphenomenon where in several B cell clones are involved, each producing a slightly different Ab molecule to the myriad antigenic determinants, encompassing the whole antigen Polyclonal antiserum – antiserum coming from many antibodies producing B cell clones. Monoclonal antibody – product of one to one cell hybridization; antibody produced to a single determinant by a single hybridized B cell Immunoglobulins derived from single plasma cells Produced by HYBRIDOMA TECHNOLOGY Discovered by GEORGES KOHLER & CESAR MILSTEIN who used SENDAI VIRUS (an influenza) that characteristically causes cell fusion Mainly used for diagnostic testing and therapeutic purposes o In vitro diagnostic test o Delivery of therapeutic agents in disease

CLINICAL IMMUNOLOGY AND SEROLOGY Developed based on knowledge that B cells are genetically preprogrammed to synthesize very specific antibody Are derived from a single parent antibody-producing cell that has reproduced many times Hybridoma Cell lines grew continuously in culture because of the cancerous myeloma hybrid, forming a little tumor. Hypoxanthine Aminopterin Thymidine Medium – this medium is used to select for only the hybrid (hybridoma) cells . Fusion of ...