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BS in Medical Technology | Emilio Aguinaldo College-Cavite LESSON 4: ANTIBODY STRUCTURE AND FUNCTION ANTIBODIES Isotype: a unique a...

BS in Medical Technology | Emilio Aguinaldo College-Cavite LESSON 4: ANTIBODY STRUCTURE AND FUNCTION ANTIBODIES Isotype: a unique amino acid sequence that is common to all immunoglobulin molecules of a given class in a given species are immunoglobulins Allotypes: minor variations of isotype sequences that are o glycoproteins found in the present in some individuals but not others serum/plasma (carbs+protein) Idiotype: variations in variable regions that give individual o 82% to 96% polypeptide and 2% antibody molecules specificity. (idiosyncrasy: uniqueness) to 14% carbohydrate o Five major classes: IgG, IgM, IgA, Hinge Region IgD, IgE Segment of H chain located between the CH1 and CH2 regions Are the key element of the humoral immune Has a high content of proline, allowing flexibility, and response hydrophobic residues Appear primarily in the gamma (ᵧ) band with serum Flexibility electrophoresis at pH 8.6; proteins: albumin & globulin o Allows 2 antigen-binding sites operate independently Tetrapeptide Structure of Immunoglobulins o Assists initiation of complement cascade Basic 4-chain polypeptide Carbohydrate Portion o 2 large heavy (H) chains Found in all types of immunoglobulins o 2 small light (L) chains Localized between the CH2 domains of the two H chains Held together by noncovalent force & Increases the solubility of immunoglobulin disulfide interchain bridge Provides protection against degradation Single, unique variable region (amino- Enhances functional activity of the Fc domains terminal end) Three-Dimensional Structure of Antibodies One or more constant regions (carboxy-terminal end) or -COOH Folded into compact globular subunits stabilized by intrachain Fragment Crystalline (Fc) Fragment disulfide bonds Fragment Crystalline: Crystallizes upon purification Immunoglobulin fold back and forth forming Beta-pleated sheets Has no antigen-binding ability o Strategic location: Hypervariable Region – CDR1, Represents the carboxy-terminal halves of two H chains CDR2, & CDR3 (Complementary Determining Regions) Held together by S-S bonding o 30 amino acids residues found in variable of both heavy Important in effector functions of immunoglobulin molecules and light chain o Opsonization: process in which the antibodies will help o CDR – between 9 to 12 residues long in variable region attract the phagocytic cells; also important for o Antigen binding site is determined by the apposition of complement fixation the 6 hypervariable loops (3 from each domain) o Complement fixation: ▪ Antigen binds in the middle of the CDR with Fragment for Antigen-Binding (Fab) Fragment at least 4 of the CDRs involved in the binding A monomer One L chain; one-half of an H chain IgG Held together by disulfide bonding Predominant immunoglobulin in humans (75%-80% of the total Obtained by papain digestion of an immunoglobulin serum immunoglobulins); a monomer o After digestion, Ig will be divided into two: 1 Fc & 2 Fab Has the longest half-life of any immunoglobulin class (23 days, F(ab’)₂ predominance in serum) Four Subclasses Obtained by pepsin digestion o IgG1: 66% Two antigen-binding sites together o IgG2: 23% Fc’ portion in pieces o IgG3: 7% Light Chains (Bence Jones Proteins) o IgG4: 4% Discovered in 1845 by Dr. Henry Bence Jones Differ in number & position of disulfide bridges between y chains All subclasses can cross the placenta, except for IgG2 L chains secreted by malignant plasma cells o Majority of HDN cases are caused by IgG Two types: Macrophages, monocytes, and neutrophils have Fc receptors o Kappa (k) o Lambda (λ) specific to the Fc region of IgG, increasing the efficiency of phagocytosis Contain between 200 and 220 amino acids A high diffusion coefficient allows IgG to enter extravascular Constant region spaces more readily than other immunoglobulin types Variable region IgG plays a major role in neutralizing toxins and viruses Heavy Chains IgG3 Variable Region Heavy chains are unique in a Has the largest hinge region First approximately 110 amino acids different class of Has the largest number of interchain disulfide bonds immunoglobulins; dictates the Constant Regions class of immunoglobulin Most efficient at binding complement Remaining amino acids IgG1 and IgG3: Induced in response to protein antigens Three or more regions with very similar sequences designated IgG2 and IgG4 CH1, CH2, and CH3 Have shorter hinge segments Heavy Chains Poor mediators of complement activation Unique to each class & give each immunoglobulin type its name Involved in responses to polysaccharide antigens IgG: γ chain Major Functions of IgG IgM: μ chain IgD: δ chain Providing immunity for the newborn (because IgG can cross the IgA: α chain IgE: ε chain placenta): important in natural passive immunity Gubat, Trisha B. (2024) | MMLS 3-2  BS in Medical Technology | Emilio Aguinaldo College-Cavite LESSON 4: ANTIBODY STRUCTURE AND FUNCTION Fixing complement Secretory IgA Coating antigen for enhances phagocytosis (opsonization) Is synthesized in plasma cells found mainly in mucosal- Neutralizing toxins and viruses associated lymphoid tissue (MALT) Participating in agglutination and precipitation reactions Is released in dimeric form “Secondary Response”; among all Ig’s, IgG is the smallest Is captures by secretory component (SC) on epithelial cells IgG: Laboratory Testing Secretory IgA Functions Agglutination and precipitation reactions take place in vitro Patrols mucosal surfaces and acts as a first line of defense IgG is better at precipitation reactions than at agglutination o Neutralizes toxins produced by microorganisms o Precipitation involves small soluble particles, which o Prevents bacterial adherence to mucosal surface are more easily brought together by the relatively Passively transfers immunity to newborn during breastfeeding small IgG molecule Complexes of IgA and antigen are easily trapped in mucus and IgM eliminated by the ciliated epithelial cells of the respiratory and intestinal tracts Known as “Macroglobulin”; a pentamer o Has a molecular weight of about 900,000 d Aggregation of IgA immune complexes may trigger the alternate o Accounts for 5% to 10% of all serum immunoglobulins complement pathway Has half-life of 6 days (much shorter than IgG) Neutrophils, monocytes, and macrophages possess specific receptors for serum and secretory IgA Can exist as: o Monomer: on surface of B cells Binding to these sites triggers a respiratory burst and o Pentamer: found in secretions degranulation of the cells involved Pentamer Form Both form of IgA can act as opsonins, w/c promote phagocytosis o Held together by J chains, which are linkage points for IgD disulfide bonds between two adjacent monomers Extremely scarce in the serum o Has a star-like shape with 10 antigen-binding sites Less than 0.001% of total immunoglobulins Has a high valency; found mainly in intravascular pool Has a molecular weight of approximately 180,000 daltons Cannot cross the placenta, because of its molecular weight The δ H chain Known as the “primary response antibody” o Has a molecular weight of 62,000 daltons o Appears first after antigenic stimulation and in the o Appears to have an extended hinge region consisting maturing infant of 58 amino acids o Synthesized only as long as antigen remains present Is more susceptible to proteolysis than other immunoglobulins No memory cells o Proteolysis: enzyme that can degrade protein Primary versus Secondary Response Has a short half-life (1-3 days) Onset: IgM Found on the surface of immunocompetent but unstimulated B lymphocytes Convalescent: IgG present Appears second (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/macrophages o Does not cross the placenta Functions of IgM IgE Complement fixation 0.0005% of total serum immunoglobulins Opsonization Has an ε H chain composed of around 550 amino acids that are Agglutination distributed over one variable and four constant domains Toxin Neutralization o Rise up to 10 times you when have allergic reaction Produced by plasma cells are located primarily in lungs & skin IgA Does not participate in complement fixation, agglutination, or Accounts for 10% to 15% of all circulating immunoglobulins in opsonization serum Is incapable of crossing the placenta One variable and three constant regions Attaches to basophils, eosinophils, and tissue mast cells through Serum IgA: appears as monomer with a molecular weight of high-affinity Fc ε RI receptors approximately 160,000 Function: Allergic Reactions Subclasses of IgA 2 adjacent IgE molecules on a mast cell bind a specific antigen IgA1 Cascade of cellular events results in degranulation of mast cells Mainly found in serum and release of vasoactive amines (histamine and heparin) Acts as anti-inflammatory agent Type I Immediate Hypersensitivity Results Downregulates IgG-mediated phagocytosis, chemotaxis, o Hay fever, asthma, vomiting, and diarrhea, hives, life- bactericidal activity, and cytokine release threatening anaphylactic shock IgA2 Function: Parasitic Infections Predominantly found in secretions at mucosal surfaces Eosinophils play a major part in the destruction of large antigens, Dimer along the respiratory, urogenital, and intestinal mucosa such as parasitic worms, that cannot be easily phagocytized Keeps antigen from penetrating farther into the body Is more resistant to some bacterial proteinases that are able to cleave IgA1 Gubat, Trisha B. (2024) | MMLS 3-2  BS in Medical Technology | Emilio Aguinaldo College-Cavite LESSON 4: ANTIBODY STRUCTURE AND FUNCTION Antibody Diversity Theories SUMMARY Side-Chain Theory The basic structural unit for all immunoglobulins is a tetrapeptide Established by Paul Ehrlich in the early 1900s composed of two L and two H chains joined together by disulfide Postulated that certain cells had specific surface receptors for bonds antigen that were present before contact with antigen occurred The five classes of antibodies are IgM, IgG, IgA, IgD, and IgE. If antigen introduced, combines with the proper receptors to Kappa and Lambda (L chains) are found in all types of break off and enter the circulation as antibody molecules immunoglobulins, but the H chains differ for each Postulated that process could be repeated with further contact immunoglobulin class. with antigens Each immunoglobulin molecule has constant & variable regions. 1950s: Jerne and Burnet’s Clonal Selection for AB Formation Variable region (Fab fragment) determines the specificity of that molecule for a particular antigen. Lymphocytes are genetically preprogrammed to produce one Constant region (Fc fragment) is responsible for binding to type of immunoglobulin effector cells. A specific antigen finds the particular cells capable of responding The five different types of heavy chains are called isotypes. to it, causing them to proliferate IgG is small and easily penetrates into tissues. Would require a large number of genes IgM is large and excels at complement fixation. 1965: Dryer and Bennett IgA has an SC that protects it from enzymatic digestion while it Constant and variable portions of immunoglobulin chains are patrols mucosal surfaces. coded for by separate genes An extended hinge region gives IgD an advantage as a surface Genes Coding for Immunoglobulins receptor for antigen. IgE binds to mast cells to initiate a local inflammatory reaction. Chromosomes contains building blocks from which genes can The primary response to an antigen takes 5 to 7 days before be assembled antibody can be detected. Human immunoglobulin genes are found in three unlinked The secondary response to antigen occurs more rapidly and clusters: persists for a longer time. o H chain genes on chromosome 14 Ehrlich’s side-chain theory is based on the antigen selecting the o k chain genes on chromosome 2 correctly programmed B lymphocyte. o λ chain genes on chromosome 22 The clonal selection hypothesis postulated that lymphocytes are rearrangement is needed for genes to become functional generally pre-endowed to respond to one antigen or a group of antibody molecules antigens more than one gene controls synthesis of a particular Several genes code for a particular immunoglobulin; through a immunoglobulin random selection process, these individual segments are joined o H chains to make antibody of a single specificity. ▪ Variable-region genes: VH, D, and J Monoclonal antibodies are made when a cancerous cell or ▪ Constant-region genes: set of C genes myeloma is fused with an antibody-producing cell to form a o L chains: lack a D region hybridoma. Through a random selection process, these individual segments are joined to commit that lymphocyte to making antibody of a single specificity Monoclonal Antibodies Mainly used for diagnostic testing and therapeutic purposes o In vitro diagnostic testing o Delivery of therapeutic agents in diseases 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 fusion of an activated B-cell with a laboratory-grown myeloma cell that cannot make its own DNA because of deficiency of HGPRT (Hypoxanthine, Guanine, & Phosphoribosyltransferase? Production involves: o Immunizing a mouse with a certain antigen o Harvesting spleen cells o Combining spleen cells with myeloma cells in the presence of PEG o Selecting fused cells and screening for presence of desired antibody Gubat, Trisha B. (2024) | MMLS 3-2  BS in Medical Technology | Emilio Aguinaldo College-Cavite LESSON 5: ADAPTIVE IMMUNITY ADAPTIVE IMMUNITY Autoimmune Disorders: T-Suppressor Cells o High Antibody because nothing suppresses its 3rd line of defense of our body when it comes to infections. production. (prevent too much production of Ab) Is specific to each pathogen or microbial agent. o Too much antibody can cause autoimmunity. o Antibodies are created for the purpose of dealing with T Cell Receptors: or attaching to antigens. Each antibodies are specific. o CD2: Sheep RBC Receptor Remembers prior exposure. o CD3: Part of T cell antigen-receptor complex o Due to production of memory cells during the o CD4: Receptor of MHC Class II molecule (T Helper sensitization of B cells, adaptive immunity has a Cells) capability in remembering a prior exposure. o CD8: Receptor of MHC Class I molecule (T Increases response to a pathogen upon repeated exposure. Suppressor and T Cytotoxic Cells) Takes longer to become activated but is longer lasting. T-CELL DIFFERENTIATION: DOUBLE-NEGATIVE STAGE o Innate immunity has a faster response, but it is short when it comes to duration of the response. 1st stage of T cell differentiation Involves T and B lymphocytes All thymocytes (precursors that are committed on becoming T cells) start on this stage. They lack on both CD4 and CD8 and T CELLS B CELLS are therefore known as double-negative (DN) thymocytes. Arm to Arm combat (respond Bows and Missiles (respond to to foreign materials directly) foreign materials indirectly by No CD4 and CD8, can’t be classified yet. creating antibodies) Chemokines drive differentiation process. Cell-Mediated Humoral Immunity Thymocytes undergo rearrangement of genes coding for T-cell Mature in Thymus Mature in Bone Marrow receptors (TCRs) Creates Lymphokines (type of Creates Antibody CD3/TCR complex cytokine) o Alpha and beta chains that recognize antigen. 60-80% in blood 20-35% in blood o Six other chains in three pairs that assist with signaling when antigen binds to T cells. Longer Lifespan Shorter Lifespan (3-5 hours) o It is the T cell receptor for the antigen itself. Once the chemokines drive the differentiation process, and the Has CD2 (+) on its surface Surface Immunoglobulin (+) rearrangement of genes happened, from double-negative, it Attack invaders outside the B Attack invaders inside the T becomes double-positive. Cell Cell T-CELL DIFFERENTIATION: DOUBLE-POSITIVE STAGE LYMPHOCYTES Thymocytes express CD4 and CD8 and rearrange gene coding Arise from Pluripotential stem cells at the Yolk Sac for the alpha chain. o Three stages of hematopoiesis: Yolk sac (start of o Although the thymocytes express both CD4 and CD8 lymphocyte production) → Liver → Bone marrow on its surface, we still cannot classify double positive T LYMPHOCYTES thymocytes because for it to be classify T Responsible for the immune response and are involved in lymphocytes, it must only possess one surface Antibody regulation. antigen, either CD4 or CD8. Maturation: In the Thymus Positive selection, if functional TCR: Grows in the Bone Marrow: where undifferentiated Thymocytes o It is based upon the recognition of MHC antigens, and are made. that allows only functional T cell receptors to survive. Subsets: o However, negative selection will eliminate T cells that o T-Helper Cells (70%, CD4(+)) – most numerous are capable of autoimmune response → undergo o T-Suppressor Cells (30%, CD8(+)) – 2nd most apoptosis. numerous MHC restriction: only cells that react with host MHC survive o T-Cytotoxic Cells (CD8(+)) - With NK cells that kill Negative selection: cells that react with self-antigen are intracellular organisms. destroyed. ▪ T Cytotoxic cells involved in direct killing of T-CELL DIFFERENTIATION: MATURE T CELLS cells infected with intracellular organisms, Survivors of positive & negative selection exhibit either CD4/CD8 like NK cells, and they also release CD4 T cells are T helper cells: Assist in antibody production. granzymes and perforins. Also known as T inducer cells and they will assist in antibody o T-Delayed Hypersensitivity production. o T-Regulatory Cells (Possess CD4 and CD25; Suppress immune response to self-antigens) CD8 T cells are cytotoxic T cells: Kill target cells by producing ▪ Cell that prevents our immune system from perforins and granzymes. attacking self-antigens, prevents T cells acquire their specificity in the thymus, and they are autoimmunity. responsible in the cell mediated immunity. T Helper: T Suppressor cell ratio: 2:1 T Helper Cells Human Immunodeficiency Virus (HIV): A blood borne pathogen Have CD4 receptor; Recognize antigen & class II MHC protein. that attack T Helper Cells (Low CD4 cell count) Account for two-thirds of periph eral T cells o CD4 cell count is important in assessing HIV in a person to know if the immunity of a patient is sufficient Th1 Cells Th2 Cells T Regulatory Cells in combating secondary infections. Produce interferon Produce IL-4, IL-5, Have CD4 and o HIV’s receptor has an affinity to the CD4 of T helper gamma, IL-2, IL-6, IL-9, IL-10, IL- CD25 cells. (attach to CD4 → enter the cell → replicate Tumor Necrosis 13 inside the cell → destroy the CD4 cells/T helper cells) Factor-β (TNF- β) o