Immunology and Serology - 02 Antigen and Antibodies

• IMMUNOLOGY AND SEROLOGY ANTIGEN AND ANTIBODIES OUTLINE • • • • Introduction Antigen o Antigens o Factors affecting Immunogenicity o Relationship of Antigens to the Host Major Histocompatibility Complex o MHC/HLA o MHC o HLA Testing Antibodies o Antibody o Immunoglobulins o Enzyme Hydrolysis • Kinds of Antigen (based on reaction with antibody) o Agglutinogen - antigen in agglutination reactions ▪ Made up of cellular particulate insoluble antigen ▪ Blood typing is based on serologic action o Precipitinogen - antigen in precipitation reactions ▪ Made up of soluble antigen o Virus o Rickettsiae o Toxin o Allergens INTRODUCTION • Immunogenicity o Ability of a molecule or substance to provoke an immune response FACTORS AFFECTING IMMUNOGENECITY • ANTIGEN ANTIGENS • • • • Antigens o Substances recognized as foreign by body o Substance that can stimulate immune response ▪ Immune response - antibody formation, cytokine production o Immunogen - term used in immunology ▪ Can be used interchangeably with antigen ▪ Substance eliciting immune response, specifically antibody formation ▪ Antigen - substance that reacts to antibody produced o Proteins – strongest and most common antigens o Antigens are foreign particles that may elicit an immune response o Immunogens are any foreign particle that elicits an immune response o All immunogens are antigens but not all antigens are immunogens o Lipids and nucleic acids are only antigenic when combined with proteins or polysaccharides Parts of Antigen o Carrier – molecular weight over 10,000 dalton; high MW; almost always protein in nature major bulk of ag o Antigenic determinant or epitope or haptene – portion recognized as foreign; reacts specifically with an antibody receptor ▪ Smaller bulk with lower MW ▪ Part that specifically reacts/binds with ab receptor Functions of Antigen o Major – stimulates production of antibodies or immune response o Minor – shows capability of reacting with antibody Classification o Complete antigen – with 2 parts carrier and haptene; perform two functions o Incomplete antigen – either carrier or haptene; one function is present • • • Foreignness o The more distant the source of antigen, the more it will function as an immunogen o Duck serum protein is more immunogenic o Substances unnatural to the human body are more immunogenic Molecular Size - at least 10,000 daltons to be recognized by the immune system o The larger the size, the more it will act as immunogen Chemical Composition and Complexity o Proteins are powerful immunogens ▪ Most common composition o Carbohydrates are less immunogenic than protein ▪ When couple with protein, become good antigen o Lipids and nucleic acids are weak/poor antigens o Structural complexity directly affects immunogenicity ▪ More complexity = more immunogenic Route, Dosage, and Timing of Parenteral Administration o Administration affects immunogenicity o Route - manner of administration HAPTENS • • • • partial antigens not antigenic must be coupled to a carrier molecule to be antigenic can react with specific antibody alone, but cannot produce a visible reaction such as precipitation or agglutination RELATIONSHIP OF ANTIGENS TO THE HOST • • • Autoantigens o Self-antigens; antigens that belong to the host o do not evoke an immune response Alloantigens o are from other species capable of eliciting an immune response o important to consider in tissue transplantation and in blood transfusions. Heteroantigens o are from other species, such as other animals, plants, or microorganisms. • Heterophile antigens o are heteroantigens that exist in unrelated plants or animals but are either identical or closely related in structure so that antibody to one will cross-react with antigen of the other. CLASS II MHC MOLECULES • • ACTIVATORS OF LYMPHOCYTES • • • • There are three major categories of molecules that can trigger the activation of lymphocytes Monoclonal activators – substances that stimulate cells expressing antigen receptors specific for an epitope. Oligoclonal activators – molecules that are typically derived from bacteria. Polyclonal activators – typically plant proteins that bind to molecules present on all T cells. • • • CLASS III MHC GENES • • MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) • • Cluster of genes found in mammals Its products play a role in discriminating self/non-self Participate in both humoral and cell-mediated immunity MHC act as antigen presenting structure. The major histocompatibility complex is found on human chromosome 6, referred to as HLA complex. In mice, MHC is found on chromosome 17, referred to as H-2 complex. Terms HLA and MHC are used interchangeably. o Found in short arm of chromosome 6 at 6p21 between uro pepsinogen 5 and lioxilase Clinically, they are relevant, because they may be involved in transfusion reactions, graft rejection, and autoimmune diseases. Genes controlling expression of these molecules are actually a system of genes known as the major histocompatibility complex (MHC). There were three kinds of molecules encoded by MHC: Class I, Class II, Class III GENETIC MAP • • In humans, there are three MHC Class I alpha chain genes: o HLA-A o HLA-B o HLA-C There are also three pairs of MHC Class II alpha and beta chain genes: o HLA-DP o HLA-DQ o HLA-DR • • • • • • • • • • • Present on all nucleated cells in the body Recognition by CD8+ T cells o CD8+ - cytotoxic suppressor cells highest on lymphocytes and low or undetected on liver hepatocytes, neural cells, muscle cells, and sperm. involved in the recognition and rejection of grafted kidneys and other forms of organ transplant. For protection against viruses and parasites These MHC class I-deficient diseased cells trigger and activate NK cells Chain structure – alpha 2, beta 2 macroglobulin Two groups of MHC genes - structurally and functionally distinct o Class I - recognition by CD8+ T cells o Class II - recognition by CD4+ T cells HLA molecules are responsible for the compatibility of the tissues of genetically different individuals and for the rejection of transplant MHC genes are codominantly expressed in each individual Monozygotic twins have the same histocompatibility molecules on their cells MHC genes are the most polymorphic genes present in the genome (up to 250 alleles identified for some loci) MHC expression o Class I - on all nucleotide cells (no MHC on red blood cells, weak expression on cells in brain) o Class II - found on antigen presenting cells BIOLOGICAL FUNCTIONS • • Induce the differentiation and maturation of T cell to dorm functional T cell repertoire Present antigen to initiate immune response with a phenomena known as MHC restriction o Endogenous Ag is presented to CD8+ T cell by MHC class I molecules o Exogenous Ag is presented to CD4+ T cell by MHC class II molecule GENETIC REGIONS • CLASS I MHC MOLECULES • • Minor MHC antigens Activate complement factor 3 by either classical (C2, C4a, C4b) or alternative pathway (Factor B), TNF MAJOR HISTOCOMPATIBILITY COMPLEX (MHC; PPT) MAJOR HISTOCOMPATIBILITY COMPLEX / HLA • • • • • Found primarily on antigen-presenting cells, which include B lymphocytes, monocytes, macrophages, and dendritic cells. Recognition by CD4+ T cells. o CD4+ - helper T cells Important for antigen presentation and interactions between immunocompetent cells. For protection against bacteria, viruses and other exogenous antigens Chain structure – parallel alpha, beta invariant chain • • Class I gene region o Classical HLA class I genes - HLA-A,B,C o Non-classical HLA class I genes - HLA-E,F,G o MHC class I chain related genes Class II gene region o Classical HLA class II genes - HLA-DP,DQ,DR o Genes associated with antigen processing Class III gene region o Complement genes - C4, C2, Bf o Inflammation-associated genes - TNF, HSP70 MHC CLASS I MOLECULE • • • Heavy chain o α1, α2 domain polymorphic sites o α3 domain - binding of CD8 β-2 microglobulin peptide • • MHC CLASS II MOLECULE • • • α chain o α1 - polymorphic sites o α2 - binding of CD4 β chain o β1 - polymorphic sites o β - binding of CD4 Peptide HLA complex - the MHC of human, a cluster of the genes which encode for HLA and relate to allograft rejection, immune response, immune regulation and cell-regulation HLA VS Transplantation o HLA matching between the donor and the recipient - a predictor of graft survival in the kidney allograft patient o HLA mismatches may cause chronic pathologies in the allograft o It is clear that a negative HLA-A2 recipient receiving an allograft expressing HLA-A2 will have a much higher risk of humoral rejection of the vascular endothelium MHC EXPRESSION • • • • Expression is increased by cytokines such as IFNα, IFNβ, IFNγ, and TNF Transcription factors like CIITA (Transactivation), RFX (Transactivator) increase MHC gene expression Some viruses (CMV, HBV, Ad12) decrease MHC expression Reduction of MHC may allow for immune system evasion HLA TESTING • • DIFFERENTIAL DISTRIBUTION OF MHC MOLECULE • • • • Cell activation affects the level of MHC expression The pattern of expression reflects the function of MHC molecules o Class I is involved in the regulation of anti-viral immune responses o Class II involved in regulation of the cells of the immune system Anucleate RBCs cannot support virus replication, hence no MHC class I Some pathogens exploit this (eg. Plasmodium spp.) • There are two main approaches to histocompatibility testing: o tissue matching o tissue typing - microcytoroxicity test/lymphocyte cytotoxicity test Clinical Significance of HLA o Organ transplant and bone marrow transplant. o Platelet transfusion – matching between donor and recipient is useful to patients who are retractile to random donor platelets o HLA compatibility exerts the strongest influence on long-term kidney survival after a transplant. o HLA identical donors for bone marrow transplantation to reduce the frequency of graft vs host disease o Paternity testing o Disease association MHC/HLA & Related Diseases o B27 - ankylosing spondylitis (first to discover) o B8 - Myasthenia gravis, Celiac disease o DR2 - Multiple sclerosis o DR2/DR3 - SLE o DR3 - Grave's / Myasthenia gravis o DR3/DR4 - IDDM / Type I Diabetes o DR4 - RA or Pentigus vulgaris o DR4/DR5 - Hashimoto's disease HLA TESTING • IMPORTANT CONCEPTS • • Transplantation antigen - histocompatibility antigen; antigens which cause immune response to the graft and determine the survival of the graft; they are alloantigen which is specific for each individual Human Leucocyte Antigen (HLA) - the MHS of human which is associated with allograft rejection, immune response, immune regulation and cell-cell recognition • • Microlymphocytotoxicity test / Complementdependent cytolysis o Dye – trypan blue or eosin o Classic method using purified T lymphocytes (class I typing) and B lymphocytes o Has typing sera o Positive result - blue color Mixed lymphocyte culture – measurement of the degree of radioactivity by liquid scintillation counting DNA-based typing methods – nucleic acid amplification (PCR) and sequencing ANTIBODIES ANTIBODY • • • • Aka as immunoglobulins Made in response to exposure to the antigen. Each antibody has at least two identical sites that bind antigen Belong to a group of serum proteins called immunoglobulins CLASSIFICATION OF ANTIBODY • • ANTIBODY STRUCTURE • • • • • • Monomer: A flexible Y-shaped molecule with four protein chains: o 2 identical light chains o 2 identical heavy chains o Joined together by disulfide bonds Variable Regions o Two sections at the end of Y’s arms o Contain the antigen binding sites o Region exposed to antigens o Identical on the same antibody, but vary from one antibody to another. Constant Regions o Stem of monomer and lower parts of Y arms. o Remains constant Hinge region o Area that gives molecule flexibility allowing for multiple binding sites o Located between the CH1 and CH2 domains Two Heavy Chains o Heavy Chains ▪ Molecular weight varies from 50,000-90,000 dalton ▪ Designated as mu (IgM), delta (IgD), gamma (IgG), alpha (IgA), and epsilon (IgE) ▪ Each heavy chain is made of 3 constant domains (globular regions): • CH1 • CH2 • CH3 • one variable (V) domain (VH) Two Light Chains o Light Chains - are designated as : ▪ Kappa (κ) or Lambda (λ) ▪ W. 23,000 d ▪ Kappa chain – consist of 214 amino acid residues ▪ Lambda chain – consist of 213 amino acid residues ▪ Each light chain is made of: • one constant domain, CL • one variable domain VL. • • ENZYME HYDROLYSIS • • • PROPERTIES OF AN ANTIBODY • • • Protein in nature High MW more than 150,000 dalton Present in serum/plasma, CSF, saliva and seminal fluid According to Temperature at which they react o Cold antibodies – react at 4C to room temp o Warm antibodies – react at 37C According to Occurrence o Natural antibodies – appear without any apparent stimulus o Immune antibodies - appear following the introduction of an antigen According to Species o Isoantibodies – produced after introduction of the antigen from the same species o Heterophile antibodies – produced after introduction of the antigen from another species According to its Reaction with an antigen o Agglutinins – responsible for immobilization of motile organisms and for cell clumping o Hemagglutinins – agglutination of RBC o Precipitins – demonstrated only against soluble antigens o Lysins – cause dissolution of antigenic cells o Opsonins – act upon bacterial invaders, o Neutralizing antibodies – render antigenic microorganism (usually viruses) harmless o Allergic antibodies – react with allergens o Antitoxins - which neutralize specific toxins o Complement fixing antibodies o Blocking or inhibitory antibodies – combine with antigen but are not grossly detectable 2 Enzymes – papain and pepsin useful in determining the function of specific areas. Papain splits the following fragments: o 2 Fab capable of causing agglutination or precipitation reaction; o 1 Fc carries no antibody activity but only biological activity such as: ▪ Complement fixation, placental permeability/transport, attachment to phagocytic cells, degranulation of mast cell, histamine release Pepsin results in the following fragments: o F (ab)’2 slightly different from Fab type fragments that not only retain the ability to bind with antigen but capable of causing agglutination or precipitation reaction. o F(c’) cleavage produces two heavy chain fragments that are not joined by s-s bond IMMUNOGLOBULINS • Human Ig classes o IgG - (γ) heavy chains o IgM - (µ) heavy chains o IgA - (α) heavy chains o IgD - (δ) heavy chains o IgE - (ε) heavy chains o IgG Subclasses ▪ IgG1 - Gamma 1 (γ1) heavy chains ▪ IgG2 - Gamma 2 (γ2) heavy chains ▪ IgG3 - Gamma 3 (γ3) heavy chains ▪ IgG4 - Gamma 4 (γ4) heavy chains o IgA subclasses ▪ IgA1 - Alpha 1 (α1) heavy chains ▪ IgA2 - Alpha 2 (α2) heavy chains IMMUNOGLOBULIN STRUCTURE • IgG o o o o o o o o o • • • • • **Fab - variable region; for antibody activity, binding with antigen o Composed of amino terminal group Fc - crystallization fragment; constant region o Heavy chains o Composed of carboxyl terminal o For phagocytosis, complement fixation, placental permeability Hinge region - exposed to enzymatic activity Pepsin o One large F(ab)’2 fragment o LMW peptides Papain o Exposure to papain cause hydrolysis of hinge region causing variable region to split o Two (2) Fab fragments o One (1) Fc MAJOR Ig CLASSES • 5 classes/ Isotypes (constant heavy chain) o IgG: gamma heavy chain o IgA: alpha heavy chain o IgM: mu heavy chain o IgE: epsilon heavy chain o IgD: Delta heavy chain Four subclasses: IgG1, IgG2, IgG3 & IgG4 Monomer Highest concentration in plasma Transported across the placenta Activates complement Opsonizes Main Ab in the secondary immune response Mediates Antibody-dependent cellular cytotoxicity (ADCC) React best at body temp • IgA o Two subclasses: IgA1 & IgA2 o Monomer/ Dimer o Main Ig in external secretions such as milk, tears, saliva & respiratory & intestinal mucus o Protects mucosal surfaces o Major protective factor in colostrum o It is present in the secretion as dimer w/ a J (joining) chain & secretory piece. J chain is made by B or plasma cell; Secretory piece made by epithelial cell • IgM o Produced in the primary response o Pentamer: serum (held by J chains) o Monomer: Ag receptor on B-cell surface o 1st antibody that an infant makes o Most frequently found as natural antibodies against such antigens as blood group determinants o Most efficient at activating complement o Highest agglutinating capacity o Can be removed by 2 mercaptoethano; (2-ME) & dithiothreitol (DTI) • IgE o Monomer o Mediates type I hypersensitivity o Main host defense against helminth infxn • IgD o Monomer o Uncertain o Present in the membrane of mature B cells ANTIBODY VARIANTS/HETEROGENECITY/VARIABILITY • • • Isotypes o Antigenic (amino acid) differences in their constant heavy regions o Heavy chain isotypes: 9 o Total isotypes: 18 Allotypes o additional antigenic features of Ig that vary among individuals o Results from the substitution of only one or two amino acids in the constant regions (usually) of heavy or light chains o No biological significance Idiotypes o Antigenic determinants formed by the specific amino acids in the hypervariable region o Individual, unique differences between antibodies of different antigen binding specificities o Individually specific to each Ig molecule