46 Immunoglobulin's , Complement system, Antigen Antibody reactions

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Immunoglobulin's , Complement system, & Antigen Antibody reactions::

Immunoglobulin's , Complement system, & Antigen Antibody reactions : By Dr. Srinivas rao

Immunoglobulins (Ig) :

Immunoglobulins (Ig) Glycoprotein molecules which are produced by plasma cells in response to an immunogen and responsible for immunity 20% of total proteins in normal individuals.

Basic Immunoglobulin Structure::

Basic Immunoglobulin Structure: Antibody molecules have a common structure of four peptide chains Two identical light (L) chains, 22,000 Da Two identical heavy (H) chains,55,000 Da Each light chain is bound to a heavy chain by a disulfide bond, non covalent interactions as salt linkages, hydrogen bonds, and hydrophobic bonds, to form a heterodimer (H-L). Similar non covalent interactions and disulfide bridges link the two identical heavy and light (H-L) chain combinations to each other to form the basic four-chain (H-L)2 antibody structure.

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Variable Regions: The first 110 or so amino acids of the amino-terminal region of a light (VL ) or heavy chain (VH ) varies greatly among antibodies of different specificity. Constant Regions: exhibits limited variation CL on the light chain (110 aa ) CH on the heavy chain (330-440aa) Hinge Region – p rovide flexibility in the molecule at this point. proline-rich region, mu and epsilon heavy chains lack a hinge region Oligosaccharides - Carbohydrates are attached to the C H2 domain

STRUCTURE OF THE VARIABLE REGION ::

STRUCTURE OF THE VARIABLE REGION : Hyper variable (HVR) or complementarity determining regions (CDR) : constitute the antigen binding site of the antibody molecule. Antibodies with different specificities have different CDR's while antibodies of the exact same specificity have identical CDR's Framework regions: The regions between the CDR's in the variable region are called the framework regions (FR)

Enzymatic Digestion Of Antibodies:

Enzymatic Digestion Of Antibodies Digestion With Papain Yields 3 Fragments 2 identical Fab and 1 Fc Fab :Antigen Binding Fc :Crystallize In Cold Storage Pepsin Digestion F( ab `)2 No Fc Recovery, Digested entirely Mercaptoethanol Reduction And Alkylation irreversibly cleaves disulfide bonds

Light-Chain Sequencing::

Light-Chain Sequencing: The carboxyl-terminal half of the molecule, called the constant (C) region had two basic amino acid sequences. Two light chain types, kappa (κ) and lambda ( λ ). In humans, 60% of the light chains are kappa and 40% are lambda. A single antibody molecule contains only one light chain type, either κ or λ , never both. 4 sub types λ : λ 1, λ 2, λ 3, λ 4. The amino-terminal half of the chain, consisting of 100–110 amino acids, was found to vary: called variable (V) region

Heavy-Chain Sequencing ::

Heavy-Chain Sequencing : The amino-terminal part of the chain showed great sequence variation ,called the variable (V) region. The remaining part of the protein revealed five basic sequence patterns, corresponding to five different heavy-chain constant (C) regions( μ , ε , γ , δ , α ). Each of these five different heavy chains is called an isotype. The heavy chains of a given antibody molecule determine the class of that antibody: Ig M( μ ), Ig G( γ ),Ig A( α ), Ig D( δ ), or Ig E( ε ).

Immunoglobulin Fine Structure : :

Immunoglobulin Fine Structure : Primary structure: the amino acid sequence, accounts for the variable and constant regions of the heavy and light chains. Secondary structure : formed by folding of the extended polypeptide chain back and forth upon itself into an anti parallel β pleated sheet. Tertiary structure: The chains are then folded into compact globular domains, which are connected to neighboring domains by continuations of the polypeptide chain that lie outside the β pleated sheets Quaternary structure: the globular domains of adjacent heavy and light polypeptide chains interact forming functional domains that enable the molecule to specifically bind antigen and, at the same time, perform a number of biological effector functions .

Immunoglobulin domains ::

Immunoglobulin domains :

Immunoglobulin Classes And their Biological Activities:

Immunoglobulin Classes And their Biological Activities

Immunoglobulin G (Ig G) :

Immunoglobulin G (Ig G) Most abundant immunoglobin 80% of serum Ig ~10mg/ml 4 Ig G subclasses IgG1,2,3,4 (decreasing serum concentration) IgG1, IgG3 and IgG4 cross placenta IgG3 Most effective complement activator IgG1 and IgG3 High affinity for FcR on phagocytic cells, good for opsonization

Immunoglobulin M (IgM):

Immunoglobulin M ( IgM ) 5-10% of serum immunoglobulin 1.5mg/mL 3rd highest serum Ig First Ig made by fetus and B cells Membrane bound IgM (also IgD ) expressed on B-cells as BCR Pentameric version is secreted First Ig of primary immune response High valence Ig More efficient than IgG in complement activation

Immunoglobulin A (IgA):

Immunoglobulin A ( IgA ) 10-15% of serum IgG Predominant Ig in secretions Milk, saliva, tears, mucus Serum mainly monomeric , but polymeric forms possible Secretory Ig : consist of a dimer or tetramer+J -chain polyptetide+secretory component (Poly IgR ) Daily production 5 g to 15 g of secretory IgA into mucous secretions.

Immunoglobulin E (Ig E):

Immunoglobulin E (Ig E) Very low serum concentration, 0.3  g/mL Participate in immediate hypersensitivities reactions. Ex. Asthma, anaphylaxis, hives Binds Mast Cells and Blood Basophils through Fc receptors Binding causes degranulation (Histamine Release) Does not fix complement

Immunoglobulin D (IgD):

Immunoglobulin D ( IgD ) Membrane bound, expressed on B-cell Surface No biological effector activity identified Low serum concentrations, ~30  g/mL

Antigenic Determinants on Immunoglobulins:

Antigenic Determinants on Immunoglobulins Antibodies are glycoproteins, they can themselves function as potent immunogens . Antigenic Determinants on Abs Fall in 3 Categories Isotypic Allotypic Idiotypic Iso typic determinants are constant region determinants that distinguish each Ig class and subclass within a species. Eg:Goat anti- mouse IgG Allo typic determinants are subtle amino acid differences encoded by different alleles of isotype genes. Ex. During pregnancy, Blood transfusion , factor viii inhibitor

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Idiotype : individual antigentic determinant of the variable region. The sum of the individual idiotopes is called the idiotype of the antibody. Anti- idiotype antibody is produced by injecting antibodies that have minimal variation in their isotypes and allotypes . Eg : Humanized Monoclonal Antibodies for Cancer eg : Rituxan B-Cell Receptor: BCR Is An Antibody On surface Of cell membrane bound Ig Very Short Cytoplasmic Tail, cannot Transduce Signal Heterodimeric Molecule Ig-  /Ig-  Transduces (long cytoplasmic tail)

Fc Receptors (FcR) Functions:

Fc Receptors ( FcR ) Functions To Transport Abs Across Membranes Secretion of IgA Across Epithelium into lumen Transport of maternal Abs Across Placenta ( IgG ) Recruit elements of immunity. Ex. Mast Cells, Macrophages, Neutrophils, B, T, NK cell. Opsonization, ADCC Poly IgR Transport of IgA across epithelium FcR N Transport of maternal IgG to fetus

GENERAL FUNCTIONS OF IMMUNOGLOBULINS ::

GENERAL FUNCTIONS OF IMMUNOGLOBULINS : 1) Ag binding: Each immunoglobulin actually binds to a specific antigenic determinant. Protection of the host. 2) Effector Functions : - Opsonization. - Activation of complement. - Antibody dependent cell mediated cytotoxicity (ADCC) - Neutralization.

Characteristics of Blood Group Antibodies::

Characteristics of Blood Group Antibodies: Naturally occurring and immune antibodies: Allo antibodies and Auto antibodies. Polyclonal and monoclonal antibodies

Naturally occurring Vs Immune antibodies:

Naturally occurring Vs Immune antibodies

Allo antibodies Vs Auto antibodies:

Allo antibodies Vs Auto antibodies

Polyclonal Vs Monoclonal antibodies :

Polyclonal Vs Monoclonal antibodies

The Complement system ::

The Complement system : Paul Ehrlich in Berlin coined the term complement, defining it as “the activity of blood serum that completes the action of antibody.” Plays key role in innate and adaptive immunity. Complex group of over 20 circulating and cell membrane proteins. Synthesized mainly by liver. Also produced by blood monocytes , tissue macrophages and epithelial cells of gastrointestinal and genitourinary tracts . Constitute 5% of the serum globulin fraction.

Components of Complement::

Components of Complement: Pro enzymes, or Zymogens , . Complement components are designated by numerals(C1–C9) Four unique serum proteins : factor B, factor D, factor P (Properdin) , IF (initiating factor). Co factors : Ca2+, Mg2+. The smaller fragment is designated “a” ( eg . C3a) The larger fragment designated “b” (eg.C3b) bind to the target near the site of activation The complement fragments that have enzymatic activity designated by a bar over the number or symbol

Complement Activation:

Complement Activation The complement proteins are activated in a cascade of events through three main pathways.

The Classical Pathway:

The Classical Pathway C1 Macromolecular complex Complement activation

Alternative Pathway:

Alternative Pathway The alternative pathway is a component of the innate immune system, because no antibody is required. This major pathway of complement activation involves four serum proteins: C3, factor B, factor D, and properdin. Activated by cell-surface constituents that are foreign to the host. Serum C3, which contains an unstable thio -ester bond, is subject to slow spontaneous hydrolysis to yield C3a and C3b. The C3b component can bind to foreign surface antigens or even to the host’s own cells. Rapid inactivation of bound C3b molecules on host cells(Factor H and I)

C5 convertase:

C5 convertase

The Lectin Pathway:

The Lectin Pathway Does not depend on antibody for its activation. The lectin pathway is activated by the binding of mannose- binding lectin (MBL) to mannose residues on glycoproteins or carbohydrates on the surface of microorganisms. MBL is an acute phase protein produced in inflammatory responses. Its function in the complement pathway is similar to that of C1q. After initiation, mechanism is similar to that of the classical pathway

Membrane-Attack Complex:

Membrane-Attack Complex The terminal sequence of complement activation involves C5b, C6, C7, C8, and C9, which interact sequentially to form a macromolecular structure called the membrane-attack complex (MAC). This complex forms a large channel through the membrane of the target cell, enabling ions and small molecules to diffuse freely across the membrane.

Proteins that regulate the complement system:

Proteins that regulate the complement system

Biological effects mediated by complement products:

Biological effects mediated by complement products

Complement-binding receptors:

Complement-binding receptors

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Functions of Complement :

Binding of complement by RBC antibodies:

Binding of complement by RBC antibodies IgM and subclasses of IgG (human IgG1, IgG2, and IgG3) can activate the classical complement pathway. Antigen-antibody complex induces conformational changes in the Fc portion of the IgM molecule that expose a binding site for the C1 component. Each C1 molecule must bind by its C1q globular heads to at least two Fc sites for a stable C1-antibody interaction to occur When pentameric IgM is bound to antigen on a target surface it assumes the so-called “staple” configuration, in which at least three binding sites for C1q are exposed. Circulating IgM , however, exists as a planar configuration in which the C1q-binding sites are not exposed and therefore cannot activate the complement cascade.

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An Ig G molecule, on the other hand, contains only a single C1q-binding site in the CH2 domain of the Fc, so that firm C1q binding is achieved only when two Ig G molecules are within 30–40 nm of each other on a target surface or in a complex, providing two attachment sites for C1q. This difference accounts for the observation that a single molecule of Ig M bound to a red blood cell can activate the classical complement pathway and lyse the red blood cell while some 1000 molecules of Ig G are required to assure that two Ig G molecules are close enough to each other on the cell surface to initiate C1q binding.

Antigen Antibody Reactions:

Antigen Antibody Reactions

Antigens ::

Antigens : Antigens are generally proteins or polysaccharides, but other substances such as nucleic acids, lipids can also be antigens. Immunogenicity is the ability to induce a Humoral and/or cell mediated immune response. Antigenicity : ability to bind to the subsequently produced antibodies. In blood group serology , the interaction between antigen on cells & the corresponding antibody is normally detected by observing specific agglutination of the cells concerned.

Epitopes:

Epitopes Immunologically active regions of an immunogen. Complementary to the combining site of the antibody. These may be sequential as in polysaccharides or polypeptides or may be conformational & involve structures brought into proximity by folding as in proteins. Has an area between one and seven sugar residues for the carbohydrate moiety and four or five amino acids for proteins.

Hapten::

Hapten: The pioneering work of Karl Landsteiner. Small foreign molecule that is antigenic but not immunogenic. Molecular weight is less than 10,000 daltons Hapten cannot function as an immunogenic epitope.But when multiple molecules of a single hapten are coupled to a carrier protein, the hapten becomes accessible to the immune system and can function as an immunogen.

Characteristics of antigens::

Characteristics of antigens:

Blood group antigens: :

Blood group antigens: There are over 300 known blood group antigens Over 1,000,000 different antigen sites on each red blood cell.  These antigens are attached to proteins or lipids on the red cell membrane and are usually complex sugar groups. Some stick out far on the red cell membrane and some are buried within crypts on the membrane surface.

Strength of Antigen Antibody reactions: :

Strength of Antigen Antibody reactions: Non covalent interactions are individually weak. For strong An- Ab reaction interaction: - Large no. of interactions - A very close fit - High degree of complementarity.

Antigen-Antibody Properties:

Antigen-Antibody Properties

Affinity : :

Affinity : Defined as ‘strength of the reaction between a single antigenic determinant and a single combining site on the antibody produced by the summation of attractive and repulsive forces. Affinity is the equilibrium constant that describes the Ag- Ab reaction Ab Ag High Affinity Ab Ag Low Affinity

Equilibrium constant ::

Equilibrium constant : The association between a binding site on an antibody ( Ab ) with a monovalent antigen (Ag) can be described by the equation. Ka is the equilibrium constant for the reaction of binding site of the antibody with the antigen and can be calculated by The equilibrium constant for the dissociation reaction, Kd , the dissociation constant , is equal to the reciprocal of Ka Very stable complexes have very low values Kd , less stable ones have higher values.

AVIDITY::

AVIDITY: The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity. The avidity of an antibody is a better measure of its binding capacity than the affinity of its individual binding sites. High avidity can compensate for low affinity. Y K eq = 10 4 Affinity Y 10 6 Avidity Y Y Y Y Y 10 10 Avidity

Specificity::

Specificity: Refers to the ability of an individual antibody combining site to react with only one antigenic determinant or The ability of a population of antibody molecules to react with only one antigen. Cross reactivity : Arise because antigen shares an epitope in common with the immunizing antigen or because it has an epitope which is structurally similar to one on the immunizing antigen Anti-A Ab Ag A Anti-A Ab Ag C Similar epitope Cross reactions Anti-A Ab Ag B Shared epitope

Detection of RBC Antigen Antibody reactions ::

Detection of RBC Antigen Antibody reactions :

Agglutination::

Agglutination: There are two stages for agglutination to develop Stage 1: known as sensitization , Ab binding occurs. Antigenic determinants on the red cell membrane combine with the antigen combining site ( Fab region) on the variable regions of the immunoglobulin heavy and light chains. IgG molecules fail to bridge the distance between red cells and cause sensitization without lattice formation Stage 2: a lattice structure composed of multiple Ag- Ab bridges between Abs and red cell Ags is formed, visible agglutination is present during this stage. For larger multivalent Ig M molecules, however the direct agglutination occurs easily

Grading Reactions ::

Grading Reactions :

Rouleaux Formation or Pseudo agglutination::

Rouleaux Formation or Pseudo agglutination : Serum from patients with altered albumin-to-globulin ratios (e.g., patients with multiple myeloma) or who have received high-molecular-weight plasma expanders (e.g.. dextran ) may cause nonspecific aggregation of RBCs, known as rouleaux . Differentiation: Cells have a "stacked coin" appearance when viewed microscopically . Rouleaux is observed in all tests containing the patient's serum, including the autologous control and the reverse ABO typing. Rouleaux does not interfere with the AHG phase of testing because the patient's serum is washed away prior to the addition of the AHG reagent. Saline replacement technique.

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Hemolysis: Destruction of red cells by the action of complement that are activated by the action of certain Ag – Ab reactions. PRECIPITATION: The antigen and antibody are in soluble form combine to form a visible precipitate Agglutination inhibition: Soluble forms of blood group substances have the effect of neutralizing (or inhibiting) the reaction of the corresponding blood group antibody. In this test ,soluble antigens are added to the serum containing antibody. Agglutination is inhibited when antigen antibody reaction has previously occurred in the test system. Used in the determination of secretor status.

Factors that influence agglutination reactions::

Factors that influence agglutination reactions : Factors acting on the equilibrium constant - Temperature - pH - Ionic strength - Enzyme treatment of red cells -Antigen-Antibody ratio -Duration of incubation -Centrifugation Other factors(second atage of agglutination) -Ig type. - zygosity Factors affecting the antigen-antibody reaction

Effect of Ph ::

Effect of Ph : The ideal pH is between 6.5 and 7.5, exceptions include some Anti-M, and some Abs of the Pr(SP1) group which show stronger reactivity below pH 6.5. Different types of Abs may exhibit optimal reactivity at different temp. IgM Abs usually react optimally at or below 22°C. IgG Abs usually require 37°C temp. Temperature:

Centrifugation::

Centrifugation: Enhance agglutination reactions as it decreases reaction time by increasing the gravitational forces on the reactants as well as bringing them closer together. Allows for an increase in antigen antibody lattice formation and enhanced agglutination. Duration of incubation: For maximum sensitivity, the antigen-antibody reaction should be allowed to reach equilibrium.  Routine : 30-60 min incubation at 37c is adequate Variables: temperature, Ig class, An- Ab interactions , enhancement agents may effect .

Antibody to antigen ratio::

Antibody to antigen ratio: The ratio between the antigen and antibody influences the detection of Ag/ Ab complexes. Zone of equivalence is when antigen and antibody are present in optimal proportions. Any deviation from this decreases the efficiency of the reaction. Prozone phenomenon occurs when excess of unbound antibody is present. Postzone phenomenon occurs when excess antigen is present.

Ionic strength::

Ionic strength: The rate at which the antigen–antibody reaction occurs is considerably increased when the ionic strength of the medium in which the red cells are suspended is decreased. Zeta potential : a zone of negative charge surrounding RBCs in a cationic media is a part of the force that repels RBCs from each other and is due to sialic acid molecules on the surface of RBCs . This zone is protective and keep RBCs from adhering to each other. IgM and Ig G antibodies have differences in how they react to same zeta potential. Reducing the zeta potential allows the more positively charged antibodies to get closer & increases RBC s agglutination by Ig G

Nature of antibody::

Nature of antibody: IgM antibodies have large diameter (35 nm), which allows them to span the distance between two adjacent RBCs in solution IgG antibodies are smaller (14 nm) , unable to induce visible agglutination without the assistance of secondary enhancing reagents The density and accessibility of specific antigens on the RBC membrane are also critical in second-stage latticework formation . Dosage : weak expression of antigen on RBCs occurs as a result of the inheritance of genotypes that give rise to heterozygous expression. Homozygous antigens will react stronger Heterozygous antigens will react weaker Eg : Rh system, kidd , duffy , MM&MN, Lutheran

Potentiators::

Potentiators: Albumin Serum/cell mixture should incubate at least 20 minutes, 30 minutes preferred; doesn’t enhance warm autoantibodies LISS Incubation time of 10 minutes; lowers ionic strength allowing better reaction; sensitive and quick! PEG Polyethylene glycol Enhances warm autoantibodies ; does not react well with insignificant antibodies ( IgM ) Enzymes ( Papain , Ficin Bromelain , Trypsin ) Reduces RBC surface charge Destroys or depresses some RBC antigens and enhances other

Serological tests in blood bank:

Serological tests in blood bank Blood grouping. Compatibility testing. Antibody screening Antibody identification.

Thank you.:

Thank you.

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