S 24 Immunology Lecture 6 Introducton to Immunoglobulin - Antibody.pdf

Immunology BIOL 4201 Lecture 6 Introduction to the Immunoglobulin / Antibody Immunoglobulins (Ig) / Antibody (Ab) Soluble proteins of the beta and gamma globulin fraction of the serum that contain antibody activity; they are induced by and react with specific Immunogen Serum electrophoresis – separa...

Immunology BIOL 4201 Lecture 6 Introduction to the Immunoglobulin / Antibody Immunoglobulins (Ig) / Antibody (Ab) Soluble proteins of the beta and gamma globulin fraction of the serum that contain antibody activity; they are induced by and react with specific Immunogen Serum electrophoresis – separates serum proteins by charge to demonstrate Igs in serum: + Serum sample Separated by electric charge - Immunoglobulins – Heterogeneous group of proteins IgG IgM IgA IgD IgE Ig = Immunoglobulin Basic Structure and Terminology: To determine structure you need a protein Source of pure Ig proteins = Myeloma patients – Multiple myeloma = Cancer of Ig producing cells. Plasma cell tumor. Tumor cell multiples producing large numbers of identical protein molecules. Myeloma protein = homogeneous immunoglobulins produced by the cells resulting from a single malignant plasma cell – Antibody producing cell in the bone marrow Normal serum Multiple myeloma – neoplastic plasma cells in BM Edelman and Porter determined basic Ig structure 1959 Edelman and Porter succeeded in predicting the structure of Ig using gel filtration and by reducing Ig molecules to their primary structure by breaking protein disulfide bonds with enzymes Heavy and Light Chains Digestion with papain Edelman reduced globular Ig proteins into polypeptide chains and used Gel Filtration to separate the chains by molecular size What did Edelman Find: Edelman’s Results: High molecular weight molecule – Heavy (H) chains = MW 50,000 – Came out of the column first Low molecular weight molecule - Light (L) chains = MW 25,000 – Came out of the column second Ratio of L to H – 1/1 Total MW for native molecule = MW 150,000 ___________________________________________ Thus, 2 H chains + 2 L chains = MW 150,000 50,000 Edelman proposed this molecular structure 25,000 25,000 --------------------------------------------------------------------150,000 Further dissection of Ig molecule using pepsin (Edelman) and papain (Porter) that break disulfide bonds – Basic structural regions Experiments using Papain and Pepsin Digestion helped define immunoglobulin regions and functional units Papain Digestion: 3 segments 2 Fab – binds Ag 1 Fc – has biological properties Pepsin Digestion: 2 segments 1 F(ab’)2 – binds Ag 1 Fc fragments – has biological properties Fab = Fragment antigen binding Fc = Fragment crystallizable Simple Ab molecular structure: H-chain L-chain Ag binding site Bisulfide bonds Further dissection of Ig molecule – Light and Heavy Chains Bence Jones Proteins – Proteins produced by myeloma patients that, instead of whole Ig molecules, are L-chains. Since they are small molecular weight they are passed in urine. They used L-chains from many patients to study structure. Heavy Chain Disease - Proteins produced by myeloma Patients that, instead of whole Ig molecules, are H-chains. Since they are large molecular weight they are in serum. Henry Bence Jones They used H and L-chains from many patients to study structure. L-Chains: Amino acid sequencing demonstrated two regions: Constant Region – 110 aa sequences in length. Amino acid sequence relatively the same for different light chains – high degree of homology 110aa 110aa Variable Region – 110 aa sequences in length. Great variability in amino acid sequence for different light chains. There are two classes of L-chains defined by variability in the C-regions Kappa chains – 60% Lambda chains – 40% Myeloma Patients with L-Chain Disease 110 aa Amino acid sequence different Thus, Variable Region 110 aa Amino acid sequence identical Thus, Constant region L-chain from different myeloma patients Why do you think this is? 110aa H-Chains: 110aa Amino acid sequencing demonstrated two regions: Variable Region – 110 aa sequences in length. Great variability in amino acid sequence for different light chains Constant Region – Three 110 aa sequences each equal in length. Amino acid sequence relatively the same for different heavy chains – high degree of homology 110aa 110aa V-regions of L and H chains make up antigen binding site. aa heterogenicity allows for variety of physical shapes needed to bind different antigenic determinants Hypervariable regions in the L-V and H-V regions are were the action is with respect to the antigenic determinant Thus, most of the specificity for Ag binding is found in the HVR or CDR Hypervariable Regions Globular Protein Structure Isotypes – 5 isotypes defined by differences in the H-chain H-chains: Found there were 5 distinct C-regions which gave rise to the 5 Ig classes or Isotypes: Gamma – IgG – 4 subclasses Mu – IgM – 2 subclasses Alpha – IgA – 2 subclasses Decca – IgD Epsilon – IgE Among individuals within a species all the IgG C-region aa sequences are the Same (Iso), but they are different from the aa sequence for IgM Example of Ig Subclasses 95% homology Minor differences in the H-chain Terms in addition to Isotype Allotypes - Variations between individuals of the same species in the C-region of an Ig isotype due to minor aa sequence differences in the H-chain C-region. These variations are inherited and are based on genetic differences (different allelic forms give rise different forms of the same gene at a given locus). Idiotypes – Describe the unique antigen binding region of and Ab molecule formed by the L- and H-chain V-regions. They vary between Ig molecules depending on Ag specificity. Major differences in H-chain aa sequence defines Different Ig classes within a species Very minor differences in H chain aa sequence, aa substitution, of the same Ig within a species Can different isotypes have the same idiotype? Allotypes were important in forensic medicine, why? Differences in Fab V regions , defines the Ag binding site Immunoglobulin Classes IgG: MW = 150,000 70-75% of serum Ig Diffuses into vascular spaces Can fix complement Fc region binds WBC 4 subclasses ½ life = 23 days Rate of synthesis = 2.3g/d/70kg Can cross placenta Transport of IgG across placenta from mother to fetus – Passive immune transfer Syncytiotrophoblastepithelial covering of the highly vascular embryonic placental villi, which invades the wall of the uterus to establish nutrient circulation between the embryo and the mother. IgM: Structure is pentameric, but also monomeric MW = 950,000 First Ig produced in IR Confined to vascular space Effective agglutinater Can fix complement 2 subclasses ½ life = 5 days Rate of synthesis =.4g/d/70kg IgA / SIgA (Secretory IgA): Serum IgA IgA MW = 155,000 7S 15% of serum Ig 2 subclasses; monomeric SIgA Found in body fluids Structure is a dimer MW = 390,000. Made up of: Dimer = 300,000; J-chain = 15,000; Secretory component = 75,000 Secretory Component = stabilizes the molecule and renders it resistant to digestion by proteolytic enzymes present in body fluids. Synthesized by epithelial cell of lymph nodule J-chain joins the two monomers into a dimer J-chain = Holds dimer together. Synthesized by plasma cell Produced in lymph nodules. Fix complement Present on colostrum and breast milk Plasma cell makes Ig molecule and J-chain that binds 2 monomers forming a dimer Epithelial cell makes the secretory component that serves as a receptor for the dimer Polymetric Ig Receptor (PIGr) Lymph Nodule Passive Transfer of SIgA from mother to newborn IgD: Found on the surface of B-Cells and serves as the Ag receptor MW = 180,000 0.2% of serum Ig IgE: Associated with hypersensitivity – allergic reactions MW = 190,000 0.0004% of serum Ig Associates with Mast Cells Called “gate keeper” because of interaction with mast cells resulting in release of histamine and histamine’s effect on the vasculature. FcE Recepter Associated with allergic reactions What can Immunoglobulin do alone: Neutralize toxins Agglutination / Aggregation Opsonization Viral Neutralization Bacterial Neutralization Miracle Molecule Opsonization Opsonization – Means prepare to eat. Ab can coat Ags such as bacteria, viruses, etc. Phagocytic cells bind to the Ab Fc region using Fc receptors. Thus, Ab provides a holdfast for WBCs and tags Ag as foreign. Agglutination / Aggregation Agglutination / aggregation – Ab can bind Ags together facilitating phagocytosis or increasing phagocytic efficiency. Bacterial Neutralization Bacterial neutralization – Ab can block bacterial binding structures; thus, they cannot colonize to cause infection. Viral Neutralization Viral neutralization – Ab can inactivate the infectivity of viruses by blocking their binding site, thus they cannot bind to cells to cause infection. This is mainly SIgA blocking virus outside the body. Neutralize toxins Neutralize soluble toxins – Ab can bind to antigenic determinants on or near the toxin active or attachment sites blocking the toxin from binding to a target cell. Primary and Secondary Antibody Immune Response Primary Immune Response is the reaction of the immune system when it contacts an antigen for the first time. Secondary Immune Response is the reaction of the immune system when it contacts an antigen for the second and subsequent times. Exponential Phase Lag Phase 7 -10 days Steady State Decline Phase 1-2 days Lag Phase: Differentiation Clonal Expansion Clonal Selection Clonal Expansion Differentiation Protein Synthesis Memory / Anamnestic Response Memory Cells Clonal Selection Protein Synthesis Ab class switch From IgM to IgG Immunization / vaccination based on production of long-lived plasma cells in bone marrow and memory cells If we don’t get our act together this might happen, ;->