MLS Review - Immunology-Serology

MEDICAL LABORATORY SCIENCE REVIEW TWO PROPERTIES OF ANTIGENS IMMUNOLOGY-SEROLOGY IMMUNOGENICITY OUTLINE • • Immunology-Serology Antigens o Two Properties of Antigens o Definition of Terms o Affinity vs. Avidity • • Antibodies o Immunoglobulins Immune Response o Primary Response o Secondary Response o Comparison of Primary & Secondary IR IMMUNOLOGY AND SEROLOGY • • • • Specific antigen-antibody reactions occur and these are used for the detection of specific infectious agents IS lab used in diagnosis of infectious diseases and detection of immune response (ab present in patient) Ag-ab are reactants that exist that may be used as reagent to detect other one Ag-ab reactions are specific reactions ANTIGENS • • • • • • • • • • Any molecular structure that when introduced is capable of antibody production o Agents that induce/stimulate immune response (antibody production) In the body, there are many antigens that exist The immune system is capable of distinguishing self from nonself should only react when a nonself or foreign antigen is present No healthy immune system should not react with a normal ag Only nonself antigens can induce response A healthy immune system should only respond to nonself or foreign agents The capacity of immune system to react to foreign or nonself agents is due to Major Histocompatibility Complex (MHC) Not all antigens are created equal o They have varying sizes and characteristics Typical antigen has carrier & epitope Parts of Antigen o Carrier portion – responsible for molecular weight of the antigen ▪ MW – dictates the mass of the structure • Determines if an antigen can successfully elicit/induce immune response o Epitope or Determinant – responsible for the specificity of the antigen ▪ Interacts with the Fab portion of the antibody • Fab – antigen-binding fragment; specific binding occurs in the Fab • • • • Inherent ability of a substance to induce the specific immune response resulting in the formation of immune lymphocytes or antibodies Antibody-producing capability of antigen when present in circulation or detected by the immune system Immunogenic - antigen capable of inducing ab production Factors that Affect Immunogenicity o Foreigness ▪ The immunogen must be recognized as foreign or nonself to induce immune response ▪ Dictates the level or extent of immune response ▪ More foreign = more immunogenic ▪ Foreigness may depend on the structure of the antigen or structures present in the surface ▪ Antigen Types • Autoantigen - antigens that belongs to host; aka. self-antigens o Do not evoke/elicit immune response under normal circumstances o The immune system would recognize these as self-antigens o If the immune system/body elicits ab production when autoantigen is detected in circulation, this denotes an defective/abnormal immune response o Autoimmune disorder - occur when autoantigens elicit immune response from autoantibodies which can lead to tissue/organ damage ▪ When autoab attack autoag, it can lead to damage causing disease • Alloantigen - antigens that are derived from other members of the host species or other individuals o Capable of eliciting immune response o In BB, alloantibodies produced by alloantigenic stimulation are detected in antibody screening and identification ▪ Alloantibodies - common cause of transfusion-associated reactions o Crossmatching and compatibility testing uses alloantigens of donor cells against serum of patient to determine if serum has alloab that could react with alloags • Heteroantigen - derived form other species (animal, plants, microorganism) o Can induce immune response o During infection state, when a person is infected by a microbial agent, the heteroantigen in the surface of the agent is responsible for production of heteroantibodies (detected in the lab) o Eg. Salmonella, Treponema, SARS-CoV • Heterophile antigen - heteroantigens that exist in unrelated plants or animals o Either identical or closely related in structure so that when they stimulate immune response, the antibodies produced will cause cross reaction with ag of each other 1 ▪ o o Graft Types • Graft - tissue transplant o Can be a rich source of antigens o Tissue typing prior to tissue transplantation is crucial o Differentiated based on origin/source • Autograft - derived from individual’s own body o Ex. severe 3rd degree burns (skin grafts from px’s lower extremities for repair and reconstruction of skin) • Isograft/Syngraft - derived from identical individuals (twins) o Twin’s tissue can be used for transplantation for the other twin • Allograft - derived from other non-identical individuals o Most common graft type transplanted in health institutions o Kidney - most common allografted organ o Other common tissues transplanted through allograft: blood (transfusion) • Heterograft/Xenograft - derived from other species other than human o Tissues from animals that are transplanted for human use Size ▪ The larger the molecule, the more immunogenic the antigen becomes • The size dictates the number of epitopes present on the surface ▪ The number of epitopes increases proportionally with the size of the molecule ▪ ↑↑ Size → ↑↑ epitope → Enhance immune response ▪ Antigen becomes immunogenic if it has a MW of >10,000 Daltons (Da) ▪ Hapten - antigens with low MW which do not cause immune response • <10,000 Daltons will not cause antigen to elicit immune response ▪ Ex: albumin, hemocyanin • Albumin - good antigen; 30,000 - 60,000 Da o Vaccines has added albumin due to its stability and high MW • Hemocyanin - excellent & highly complex antigen; 100,000 - 1,000,000 Da o More complex = ↑↑ size Chemical Complexity ▪ Overall chemical structure of antigen ▪ Proteins - best and strongest antigen due to their stability (highly stable peptide linkages) • High stability due to covalent bonds • Do not dissociate easily in the blood • Have longer circulation life compared with other ag types • Longer circulation = higher chance of eliciting immune response ▪ Polysaccharides - eg. endotoxin, pneumococcal capsule ▪ Glycoproteins - eg. ABO, Rh antigens ▪ Polypeptide - eg.. insulin ▪ Nucleic Acids, Amino Acids, Lipids – least immunogenic; can easily dissociate in circulation • Have shorter circulation life ANTIGENICITY/SPECIFICITY • • • Ability to react specifically with the antibody or cell that caused it to be produced Epitope/determinant - determines specificity of antigen For specific antibodies, specific antigens are used DEFINITION OF TERMS • • • • Hapten - an incomplete antigen o Not immunogenic by itself o When coupled with a carrier protein, hapten can elicit immune response o Albumin - most common hapten carrier in circulation Immunogen - any substance that can induce immune response o More specific in its action; elicits ab production o Antigens can be self or nonself o Not all antigens are immunogenic, but all immunogens are antigenic/immunogenic Allergen - special class of immunogen that induces hypersensitivity reactions o Only specific groups of individuals develop hypersensitivity reactions to certain allergens ▪ Characteristic can be inherited or acquired due to exposure to high concentrations o May be in the form of perfume, dust, pollen, food o Hypersensitivity - immune response that is exaggerated which becomes damaging/harmful o Exposure to allergen may induce Type 1 hypersensitivity (most fatal type) ▪ Systemic/severe anaphylaxis - fatal Adjuvants - substances added to an immunogen to enhance immune response o May cause immunogen to have greater immune reactivity o Actions ▪ Prolongs retention time of the immunogen in body • Increased chance of antigenic recognition pf immune cells ▪ Increases the effective size of immunogen ▪ Stimulates the influx of macrophage and/or lymphocytes (most immunocompetent WBC) o Examples: ▪ CFA (Complete Freunds Adjuvants): water in oil emulsion of Mycobacterium butyricum or Bordetella pertussis culture ▪ LPS (lipopolysaccharide) ▪ Aluminum adjuvants - most commonly used in the clinical setting AFFINITY VS. AVIDITY • • • • Affinity - The strength of the attraction between an epitope and the antigen combining site of the antibody Avidity- Refers to the strength of interaction between complex antigens and antibodies Similarities o Both pertain to strength of interaction between ag-ab Differences o Affinity - simple interaction between single epitope and Fab portion of antibody o Avidity - complex interactions; sum total of strength of interactions between multiple ag-ab ▪ Involves complex antigens (multiple epitope interaction with antibodies) 2 ANTIBODIES • • • Substances, spec. glycoproteins, produced by B lymphocytes or plasma cells in response to antigenic stimulation Aka: immunoglobulins Associated with humoral immune response (antibody production is end result) DEFINITION OF TERMS • • • • • • Isotype - same with antibody classes o Lies in the heavy chain structures o IgG (Gamma), IgA (Alpha), IgM (Mu), IgE (Epsilon), Allotype - represents genetically determined differences in antibodies specifically in the amino acid sequence in the constant region Idiotype - pertains to the variable region of the antibody o Recognizes different epitopes present J chain - aka Joining chain o Glycoprotein that is responsible for linking or bridging monomers together o Found in abs consisting of two or more monomers o Only found in IgM (pentamer), secretory IgA (dimer) Different antibody structures are released with addition of enzymes and can be studied based on biologic activity Treatment with papain enzyme will produce 3 fragments o One Fc fragment: fragment crystalline; involved in the Ig biologic function ▪ Ig biologic functions: involved in complement fixation, placental transfer of ab/Ig, and serve as the binding site for cell (immunocompetent cells) o Two Fab fragments: capable of antigen binding even without the Fc but cannot agglutinate of precipitate ▪ Can only bind antigens ▪ Divided further into two which become monovalent IMMUNOGLOBULINS STRUCTURE OF IMMUNOGLOBULINS • • • • • A monomeric immunoglobulin is the simplest antibody with Y-shaped structure Composed of four (4) polypeptide chains: 2 identical light chains and 2 identical heavy chains. o All structures must be identical form o Chromosome 14 - site for heavy chain synthesis ▪ Heavy chains: Alpha, Gamma, Mu, Epsilon, Delta o Light chain - can be kappa or lambda ▪ Chromosome 2 - Kappa light chain synthesis ▪ Chromosome 22 - Lambda light chain synthesis Both light and heavy chains are held together by covalent disulfide bonds Heavy chains are interconnected by disulfide linkages Ig has 2 terminal regions: o Carboxyterminal - with constant amino acid sequence (constant region); dictate allotype ▪ Where the Fc portion is located ▪ Fc - crystallizable fragment/fragment crystalline o Aminoterminal - with varying antibody specifity (variable region) ▪ Determines idiotype of antibody ▪ Binds to the epitope ▪ The hypervariable portion of variable region of ab specifically binds with the epitope of the ag • DOMAINS OF IMMUNOGLOBULIN • • • • • • Hinge region - found between CH1 & CH2 o Flexible region of the antibody o Importance for the ability of the antibody to squeeze through structures (placenta) Treatment with pepsin enzyme results in digestion of Fc fragment, leaving 2 Fab fragments. o The two Fab fragments has two antigen combining sites (bivalent/divalently linked), therefore, it’s capable of antigen binding, precipitation and agglutination. Variable region - responsible for specificity of antibody o Contains the Fab region where epitope binds ▪ Antigen-binding site (Fab) CH2 - constant heavy chain 2 o Involved in the classical complement pathway o Binds with complement proteins (C1q) ▪ C1q - recognition site for C1 tri-molecular complex o When reacts with C1q, it initiates the activation of classical complement pathway (IgM or IgG) CH3 - constant heavy chain 3, end portion of Fc/constant portion o Responsible of cytotropic reactions involving WBCs (macrophages, monocytes, mast cells, cytotoxic killer cells, B cells) o Connects/links to receptors on the cell membrane CL - constant light chain; o Responsible for the light chain type o Can only kappa or lambda, never both o Kappa (65%) - predominant light chains o Lambda (35%) Hinge Region - found between CH1 & CH2 o Responsible for the flexibility (flexible region) o Flexibility is attributed to the amino acid abundantly present in the region ▪ Proline - major amino acid found in hinge region responsible for the flexibility of hinge region 3 CLASSES OF IMMUNOGLOBULINS • • IgG (Gamma) o Predominant Ig in healthy humans ▪ Due to its longest retention time in the circulation ▪ Predominant in terms of concentration o Provides immunity to the newborn ▪ Only ab that can cross the placenta due to small size • Only IgG1 & IgG3 subclasses can cross placenta ▪ Acquired natural passive immunity - when IgG crosses the placenta, the maternal antibody will provide immunity to the newborn • Beneficial to the fetus • Acquired - from another person/source • Natural - passed from one person to another • Passive - antibody is transferred ▪ Incompatible pregnancy - when antigen of fetal cells stimulates maternal immune system to produce antibodies against fetal cells, the maternal IgG antibodies crossing the placenta will sensitize fetal cells • Can lead to HDFN (harmful to baby) • Different blood type of mother and baby o Smallest immunoglobulin o Monomer and has no J chain o Predominant ab in secondary or anamnestic response o Fixation of complement ▪ IgG3 → IgG1 → IgG2 • IgG subclasses based on potency in complement activation o Promotes opsonization (Facilitated phagocytosis) ▪ IgG can act as opsonin ▪ Opsonin - enhances phagocytosis by facilitating the linking or cross-bridging of phagocytes and target antigens o Toxin and virus neutralization (neutralizing agent) o Participates in agglutination and precipitation reaction ▪ IgG - best precipitating antibody o 4 subclasses (differentiated on concentration and number of disulfide bonds) ▪ IgG1 - 66% of total IgG in serum; 2 disulfide bonds ▪ IgG2 - 23% of total IgG in serum; 4 disulfide bonds ▪ IgG3 - 7% of total IgG in serum; 15 disulfide bonds ▪ IgG4 - 4% of total IgG in serum; 2 disulfide bonds IgM (Mu) o Primary response antibody ▪ Predominant in primary immune response o First to displayed in the surface of B cells ▪ Mu heavy chain is the first synthesized o First to appear in phylogeny and the last to leave in senescence o First to appear after a primary antigenic stimulus o Largest antibody (pentamer) ▪ Composed of 5 monomers (pentameric antibody) o Best antibody in terms of fixing complement ▪ Most potent o Functions: ▪ Complement fixation ▪ Agglutination • Best agglutinating antibody ▪ Opsonization ▪ Toxin neutralization • • • IgA o Exist as monomer and dimer ▪ In plasma, IgA is monomer ▪ In secretions, IgA is dimer o Predominant ab in secretions/body fluids o Responsible for mucosal immunity o 2 subclasses: IgA1 & IgA2 o Only antibody with secretory component ▪ Secretory component - responsible for maintaining structure of the IgA antibody by preventing enzymatic degradation of structure ▪ Also plays a role in binding of IgA to the epithelial mucosal surface IgD (Delta) o Monomer surface antibody o Function is still unknown but believes to play a role in immunoregulation (control of antibody secretion and release in circulation) o Found on the surface of B-lymphocytes ▪ Second to be expressed (second to IgM) o Postulated to be an anti-idiotypic antibody IgE (Epsilon) o Least abundant Ig in the healthy serum o Heat-labile antibody, originally known as reaginic antibody o Binds strongly to mast cell and basophil ▪ Only Ig with receptor for mast cell • Mast cell - site for histamine production ▪ When IgE carries specific allergen and binds to receptor on mast cell and basophil, histamine is released from mast cell and basophile • Histamine - mediator of allergic reaction/type 1 hypersensitivity o Mediates some types of hypersensitivity reaction o Spikes up during hypersensitivity reactions and helminthic parasitic infections IgG Monomer IgA Monomer (serum IgA) Dimer (secretory IgA) 160-400 T IgM Pentamer Molecular 150 T 900 T Weight Daltons Half-life 21-23 days 5-6 days 5 days Subclasses 4 2 2 Domains 4 4 5 Activate Yes, Alternative Yes, most Complement except pathway efficient IgG4 only (classical (classical pathway) pathway) Other name Serum Ig Secretory Ig Pentameric Ig Other notes Can cross Predominant Predominant the Ig in Ig in primary placenta secretion; immune except With J response; IgG2; chain; Has J chain Major Ig in Exist as secondary monomer in immune serum and response dimer in secretion IgD Monomer IgE Monomer 180 T 190 T 2-3 days 4 No. 2-3 days 5 No. - Reaginic Ig Involved in Assoc with B cell immediate activation; hypersensitivity Susceptible Binds to enzyme basophils and degradation; mast cells; Heat and Elevated during acid labile parasitic infections 4 SECONDARY RESPONSE IMMUNE RESPONSE • • Immune response - body’s/immune system’s reaction when a foreign agent enters the body o The immune system is activated and defends the body against harmful agents Affected by the following factors o Overall health status of the individual - health status determines immune cell activity ▪ Good/excellent overall health = fast/responsive immune cells ▪ Weak/poor health = slow/deficient immune response o Age ▪ Neonates, newborns, geriatrics’ immune system are different from adults/children ▪ Neonates - immune system is still developing; process of identifying foreign antigens and yet to be exposed with different types of antigens • Normal delivery has faster development of immune system for neonates compared to CS due to exposure to vaginal canal ags ▪ Geriatrics - their immune system is already weak so their immune response is slower compared to younger individuals o Concentration/route of administration of antigen to the host body ▪ Greater antigen administered = faster and greater immune response o Genetic control of immune response ▪ Pertains to MHC - determines self from non-self ags • • • • • • COMPARISON BETWEEN PRIMARY AND SECONDARY IR Lag Phase Plateau Phase Decline Phase AB Present AB Titer • PRIMARY RESPONSE • • • Occurs in four phases after exposure to an antigen o Lag ▪ No antibody is detectable • Antibody production is not yet present ▪ Processing phase where immune systems process the foreign agents that entered the body ▪ Antigens are yet to be distributed to the tissues ▪ Phase where the pathogen/agent enters the body ▪ Pathogen will be distributed to the tissues ▪ Immunocompetent WBCs would recognize the pathogens to initiate processing o Log (Exponential phase) ▪ Antibody titer increases logarithmically (steady ↑↑) ▪ Antibody production increase o Plateau (Stationary phase) ▪ Antibody titer stabilizes ▪ There is equilibrium between antibody production and antibody catabolism o Decline ▪ Antibody decreases due to catabolism/degradation ▪ Greater antibody catabolism than antibody production Aka. Anamnestic/memory response Elicited by a second or any subsequent exposure to the same antigen/pathogen/agent Since there is memory due to re-exposure, memory response makes it possible for more rapid ab response o Shorter processing period/lag phase o Previous exposure allows memory response Exhibits same four phases as the primary response IgG - predominant Ig o Long retention time Antibody titer is higher o Increased antibody production and retention time • Primary Secondary Longer Shorter (first exposure) (memory response) Shorter (↓ ab, faster Longer (abs stay longer in decline) circulation, ↑ ab titer) Less gradual/ abrupt More gradual IgM IgG (↑↑), IgM Lower (short IgM Higher (longer IgG retention time) retention time) Correlation with vaccination o Two doses ensure that the patient’s immune system would recognize antigen & produce high antibody titer o 1st dose - primary immune response o 2nd dose/subsequent - secondary immune response o Booster shot - sustain antibody titer in levels that protect from certain infectious agents ▪ The doctor presumes that ab titer of patient is decreasing or lower than normal It is important to maintain excellent health to maintain high titer of antibodies IgM - predominant Ig Requires foreign or nonself antigen to induce response 5