1	CLSC320 Principles of Immunology Fundamental Concepts in Immunology Program for Clinical Laboratory Science Unit - 05 Immunoglobulin Molecules
2	Immunoglobulin Molecules - 1 Unit - 05 Guidelines Reading assignment: Pages 90 - 123 of textbook Learning objectives: Those listed on page 91 of textbook Key terms: Those listed on page 91 - 93 of textbook
3	Definition of Immunoglobulin Immunoglobulins: a generic designation that refers to all forms of immunoglobulins esecreted immunoglobulins : Uantibodies emembrane bound immunoglobulins Uantigen receptors -B-cell surface immunoglobulins (sIgs) -or B-cell receptors (BCRs) Antibodies: the designation that refers to secreted forms of immunoglobulins that are produced by plasma cells
4	Humoral Immune Response - 1 Initiation of response: edepends upon immune systems ability to recognize antigen by T-cell receptors on: UCD4+ cells UCD8+ cells edepends upon major histocompatibility complex (MHC) on antigen-presenting cells (APC). UB-cells Umacrophages Process of response: eantigen-presenting cells (APC) phagocytizes antigen and process it. Antigen fragments then placed on surface in association with MHC class molecules for T-cell to respond. ethis MHC restricted T-cell response insures self-recognition
5	Humoral Immune Response - 2 Summary of response: APC + Antigen Phagocytized Antigen APC + antigen fragment on MHC molecule If MHC molecule is type I = CD8+ is activated If MHC molecule is type II = CD4+ is activated
6	Role of Lymphocytes - 1 Lymphocyte participation in humoral response by: eserving as antigen-presenting cells eby producing two kinds of immunoglobulin molecules: nB-cell surface immunoglobulins (BCR=s) nsecreted immunoglobulins (antibodies) Antigen recognition and binding: erequires T_helper cells (CD4+) interaction erequires cytokines interaction erequires accessory cell interaction
7	Role of lymphocytes - 2 B-Lymphocyte participation in immune response by: ebinding unprocessed antigen to B cell receptors with same specificity (antigen recognition) ethis antigen recognition is not MHC restricted ethis binding of antigen does not cause an immune response eBCR=s cannot differentiate between foreign and self-antigens B-cell and T-cell interaction erequires T_helper cells (CD4+) interaction erequires cytokines interaction erequires B-cell to act as APC
8	Role of Lymphocytes - 3 B-cell and T-cell interaction - continued B-cell antigen on MHC II T_helper (CD4+) TCR = CD3 1.TCR on CD4+ cell recognizes antigen on MHC II of B-cell 2.CD4+ cell secretes cytokines that cause B-cell to proliferate, differentiate, and secrete antigen specific antibodies UIL-2 UIL-4 IL-2 + IL-4 3.the interaction of CD40 on B-cell with gp39 on CD4+ cell causes B- cell proliferation gp39 CD40
9	Role of Lymphocytes - 4 T-cell Dependent Antigen: eB cell acts as antigen-presenting cell eantigen recognition is MHC restricted erequires interaction of T-cells with B-cells T-cell Independent Antigen edoes not requires T cells interaction eB-cells are activated when antigen comes in contact eAntigens that may act in this way include: Ulipopolysaccharides Uproducts of bacteria eMacrophages are stimulated to secrete cytokines UIl-1 UIL-6 UTNF" eresponse is not long lasting eno IgG produced eonly IgM produced
10	Production of Immunoglobulins - 1 Initiation of humoral immune response: eantigen recognition by CD4+ t-cells eactivation of: Umature B-cells Umemory B-cells eculminates in production of plasma cells esynthesis and secretion of antigen-specific antibodies such as: UB-cell receptors -also known as surface immunoglobulins (sIg) -secreted by B-cells and are bound to their surface -bind antigen at the variable region of sIg.
11	Production of Immunoglobulins -2 Initiation of humoral immune response: esynthesis and secretion of antigen-specific antibodies such as: Uantibodies or secreted immunoglobulins -are free in body fluids -they are bifunctional in that: 1.they are able to bind antigen with variable region as do sIgs. 2.they are able to trigger various class-specific biologic activities and this is a unique function of the constant region
12	Factors Affecting Ab Production -1 eImmune system response Upresence of various cytokines Upresence of accessory cells Uclass-switching Ufunction of CD4+ T-cells eNature of antigen Udose of antigen Usite of entry of antigen into host eNumber of exposures to antigen Uprimary exposure Usecondary exposure
13	Stages in B-lymph Development Pluripotential Stem Cell Lymphoid Stem Cell Pre-B lymphocytes Immature B lymphocytes Mature B lymphocytes Activated B lymphocytes Plasma cells Memory cells
14	B-Lymph Development - 1 Characteristics of Stages in B lymphocyte development: ePluripotential Stem Cell Ua cell found in bone marrow that may differentiate into any cell line (myeloid or lymphoid) eLymphoid Stem cell Ua cell found in bone marrow that may differentiate into any lymphoid cell line) ePre-B lymphocyte U1st cell of B-cell lineage to show : heavy chain Ufound only in bone marrow or fetal liver Udo not express functional IgM on surface Ucannot bind or respond to antigens
15	B-Lymph Development -2 Characteristics of Stages in B lymphocyte development: cont=d eImmature B lymphocytes Uexpress complete IgM on surface Uare not activated by antigens Ucontact with self-antigens may result in: -B-cell death -tolerance eMature B lymphocyte Ufound only in blood and lymphoid tissue Uexpress surface IgD and IgM Uboth surface immunoglobulins express same antigen specificity
16	B-Lymph Development -3 Characteristics of Stages in B lymphocyte development: cont=d eActivated B lymphocytes Umature B lymphocytes that have come in contact with a specific antigen Ucells proliferate, differentiate into plasma cells Uclass switching of heavy chains may occur resulting in production of all isotypes of Ig=s ePlasma cells Usecrete IgM and IgD Uexpress less surface IgD and IgM Uclass switching by activated B lymphs may result in production of all isotypes of Ig=s by plasma cells
17	B-Lymph Development Characteristics of Stages in B lymphocyte development: cont=d eMemory B lymphocytes Uantigen activated B lymphs Umay persist for weeks or months Uexpress surface Ig=s Udo not secrete Ig=s Ucirculate between blood, lymph, and lymph organs Ucontact with specific antigen results in secondary immune response
18	Primary Immune Response Uwhen antibodies (IgM predominantly) are produced on 1st contact with an antigen Uantibody repertoire = antibody specificity exists for a variety of antigens prior to exposure and is due to genetic variability within B-cells Uonce stimulated plasma cells form: antibodies are produced Umemory cells may also be produced
19	Secondary Immune Response Uwhen antibodies are produced on 2nd contact with an antigen Umainly IgG type antibodies produced Usome IgM is produced Uhigher levels of antibody is produced Uantibodies have higher affinity for antigen Uheavy chain switching more common
20	OSF – Immunoglobulin Diversity On pages 100 - 102 AOne Step Further@ presents a more in- depth discussion of the genetic basis for immunoglobulin diversity. This presentation is contained on a separate slide presentation called A One Step Further #7" The student may call up the slide program OSF-7 later or click on the arrow below to view slides now. The student should pay specific attention to the relationship of primary vs secondary immune response to antigenic exposure.
21	Immunoglobulin Molecules - 1 eForm Ueach B-cell has the ability to synthesize and secrete two forms of Igs: -cell surface immunoglobulins (sIgs) -secreted immunoglobulins (antibodies) Uboth forms are structurally similar Uboth forms show the same antigenic specificity eSurface Immunoglobulins (BCR=s or sIgs) Uhas an additional segment of amino acids at the carboxyl end of heavy chain (Fc portion) which anchors it to cell membrane Usince they are only found on B-cells they act as B-cell markers Ualso serve as antigen receptors
22	Secreted Immunoglobulin - 1 eSecreted Immunoglobulins (Abs) Uthose antibodies that specifically react with the infection-causing agent are called serum markers -antigen/antibody complex is when the antigen and antibody bind in vitro -immune complex is when the antigen and antibody bind in vivo UAntigen/antibody reactions serve as a foundation for immunologic procedures used for the detection of specific antibodies or antigens
23	Secreted Immunoglobulin - 2 eSecreted Immunoglobulins (Abs) - continued UMonoclonal antibodies -blood of healthy individuals contains abs produced in response to many different antigens and these are called polyclonal or heterogeneous antibodies -monoclonal antibodies or homogeneous antibodies are produced from a single clone of cells and all have the same antigenic specificity -types of genetically engineered monoclonal antibodies: LAdesigner@ monoclonal abs LAhumanized@ monoclonal abs
24	Secreted Immunoglobulin - 3 eSecreted Immunoglobulins (Abs) - continued UHeterophil antibodies -associated with infectious mononucleosis caused by Epstein-Barr virus (EBV) -may also be present in healthy individuals at low titer -they are non-specific in that they are produced against one antigen but can react with other antigens from other species -Paul-Bunnell test and Davidsohn differential test are based on detection of heterophil abs in 80 - 90% of patients with IM due to EBV
25	Secreted Immunoglobulin - 4 eSecreted Immunoglobulins (Abs) - continued UAnti-Idiotypic antibodies -antibodies produced against specific idiotypes (amino acid sequences) located within the FAB region (variable) of antibody -thus antibodies can act as antigens causing antibodies to be produced against the antibody -an example of this is the anti-human globulins used in blood bank testing
26	Immunoglobulin Structure - 1 eStructural Characteristics Uimmunoglobulins account for 20% of serum proteins Uimmunoglobulins vary depending on: -size -charge -carbohydrate content -amino acid composition Uimmunoglobulins are located primarily within the gamma fraction of serum proteins when seperated by electrophoresis
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28	Immunoglobulin Structure - 2 eStructural Characteristics - continued Uimmunoglobulins all share the same basic four- chain polypeptide structure Uimmunoglobulin differences results in 5 classes (isotypes) and various subclasses 1.IgG 2.IgM 3.IgA 4.IgD 5.IgE IgG₁ IgG₂ IgG₃ IgG₄ IgA monomer IgA secretory
29	Basic Antibody Structure Cytokines Fab portion Fc portion Fab = fragment antigen binding (where antigen binds) Fc = fragment crystallizable (responsible for biological functions) light chains 2 kappa (6) or 2 lambda (8) heavy chains 2 ( or 2 " or 2 : or 2 * or 2 , IgG IgA IgM IgD IgE carbohydrates
30	Immunoglobulin Regions Cytokines v = variable region c = constant region hypervariable regions hinge region amine terminus carboxyl terminus v v v v c c c c c c c c c c
31	Immunoglobulin Chains - 1 ULight chains -has both constant and variable regions -two chains per each immunoglobulin molecule Leither 2 kappa (6) or 2 lambda (8) but not one of each -each light chain has an amino terminus and a carboxyl terminus UHeavy chains -has both constant and variable regions -heavy chains are structurally distinct for each immunoglobulin class -each immunoglobulin class or subclass is associated with characteristic biological functions
32	Immunoglobulin Chains - 2 UHeavy chains - cont=d -classes of immunoglobulins based on type of heavy chain are: LIgG LIgM LIgA LIgD LIgE -( (gamma) type heavy chains -: (Mu) type heavy chains -" (alpha) type heavy chains -2 (delta) type heavy chains -, (epsilon) type heavy chains URegions of immunoglobulin molecule -Variable region -Hypervariable region -Constant region -Hinge region -antigen-binding sites -antigen-binding sites -biological activity -gives molecule flexibility
33	J chains & Secretory Piece UAdditional structures associated with immunoglobulins: -J chains Lpresent in all immunoglobulins with two or more basic molecules joined together (IgA and IgM) Lonly one J chain is found in each molecule regardless of the number of basic units joined together. -Secretory pieces Lpresent in IgA which is in the dimeric form (2 basic units joined by J chain) Lallows IgA to be secreted into body fluids (sweat & saliva)
34	IgG & IgM Immunoglobulin eIgG Uthe predominant class (75% of total Igs) Uonly found in the monomeric form (one basic unit) Uhas four subclasses: LIgG₁ LIgG₂ LIgG₃ LIgG₄ -60 - 70% of IgG -14 - 20% of IgG -04 - 08% of IgG -02 - 06% of IgG eIgM Umakes up 10% of total total Igs Ufound in the monomeric form (as sIgs) Ufound in the pentomeric form (5 basic units joined by a J chain)
35	IgA Immunoglobulin eIgA UTwo forms: Lsecretory IgA Lmonomeric IgA -found in secretory fluids -dimeric form (2 basic units held by a J chain) -secretory piece protects Ig from degradation by proteolytic enzymes -found in serum -both forms (monomeric & dimeric) are found in serum Uaccounts for 15% of total immunoglobulins
36	IgD & IgE Immunoglobulins eIgD UTwo classes: LIgD antibodies: LIgD surface imunoglobulins -monomeric form (one basic unit) -0.2% of total immunoglobulins -act as B-cell receptors (BCR=s) -the major receptors for antigen eIgE Umonomeric form (one basic unit) Uaccounts for only 0.004% of total immunoglobulins U50% of the IgE is in circulatory system Uhas an affinity for binding to Fc receptors of mast cells Uplays major role in hypersensitivity reactions
37	Immunoglobulin Functions ePrimary function: eliminate or inactivate an antigen that has passed the host=s natural immune system and has induced a specific humoral immune response. eTwo ways primary function may be achieved: Uthe immunoglobulin binds to the antigen Uthe antibody-antigen complex may induce various class-specific biologic activities
38	Antigen binding Ucurrent view of mechanism: binding of antigen occurs at 3 hypervariable segments called Acomplementarity-determining regions@ (CDR1, CDR2, and CDR3) these protruding CDR surfaces create an antigen- binding site (paratope) which is complementary to the 3-dimensional configuration of the antigen (epitope). antigen epitope light chain of Ig heavy chain of Ig paratope
39	Specificity & Bonds eAntibody specificity the immunoglobulin recognizes the general configuration of the particular epitope that is complementary to it=s binding site (paratope) eBond formation: Ubonds form between amino acides of paratope and the molecules of epitope and may be of the following types: 1.ionic bonds: 2.hydrogen bonds: 3.hydrophobic bonds: 4.van der Waals forces: opposite charges attract polar molecules with COO- join with H⁺ nonpolar molecules exclude H₂O interaction of external electron clouds
40	Affinity – Avidity – Cross reactivity Affinity of antibody: strength of a non-covalent bond that forms between single paratope on an Ig molecule and a single epitope on an antigen Avidity of an antibody: strength of the bonds that forms between a multivalent antibody and a multivalent antigen eCross-reactivity in the cases where the same epitopes are shared between antigens the resulting antibody may bind with the same epitopes regardless of which antigen they are on.
41	Immunoglobulin - IgM eIgM class functions: Uable to form antibody-antigen complexes Umost effective complement binder Ufirst Ig to appear when an immune response is seen Uas IgG is formed IgM levels drop Ufirst class of Igs formed by newborns Ufirst class to appear on B-cell surfaces Uincreased IgM causes increased blood viscosity UWaldenstrom=s macroglobulinemia is increased IgM pentomeric IgM
42	Immunoglobulin - IgG eIgG class functions: Uable to bind complement (except IgG₄) Uthe only Ig that can cross placental barrier Uacts as a defense mechanism for fetuses and newborns Uinfant=s begin producing own IgG at 6 months Uadult levels of IgG reached at age 6 years Udecreased levels result in recurrent infections Uincreased IgG seen in autoimmune diseases Ubind to Fc receptors of macrophages (opsonization) Uonly Ig with antitoxin activity (destroys toxins) UIg produced in secondary immune response monomeric IgG
43	Immunoglobulin - IgA esIgA class functions: Uprovide primary protection against local infections Ufound in colostrum and breast milk - protection of infants monomeric IgA dimeric secretory IgA Uthe function of monomeric IgA is unknown
44	Immunoglobulin - IgD eIgD class function Userum Ig function unknown UsIg function is as antigen receptor on B-cells monomeric IgD
45	Immunoglobulin - IgE eIgE class function Userum Ig function is mediation of immediate hypersensitivity Uthis type of reaction is allergic reaction or allergy monomeric IgE
46	Antibodies in Blood Bank Size of molecule pentamer monomer Heavy chain type Mu (:) Gamma (() Reaction Temperature Room temp 37⁰C Reaction medium saline high protein Sedimentation constant 19S 7S Type of occurance naturally Characteristic IgM IgG immune activation of Complement yes all except IgG₄ placental transfer no yes blood group antibodies ABO Lewis (Le) Ii MN Rh Kell Duffy (Fy) Kidd (Jk) type of antibody Acomplete@ Aincomplete@
47	OSF – Fragmentation of Ig On pages 108 - 109 AOne Step Further@ presents a more in- depth discussion of the fragmentation of immunoglobulin molecules. This presentation is contained on a separate slide presentation called A One Step Further #8" The student may call up the slide program OSF-8 later or click the button below to view slides now. The student should pay specific attention to the fragments that remain when IgG is treated with papain and pepsin.
48	Immunoglobulin Molecules Press the ESC key to end program
49	One Step Further Genetic Basis for Immunoglobulin Diversity OSF - 7 Pages 100 - 102 7click arrow to return to main program
50	Immunoglobulin Diversity - 2 Immunoglobulin genes: eChromosomes do not contain genes for immunoglobulin synthesis eChromosomes contain multiple coding segments (building blocks) from which developing B lymphocytes assemble the immunoglobulin genes eThese multiple coding segments are called exons when incoporated into the B lymphocyte=s DNA molecule eThese exons are seperated by non-coding DNA segments called introns eThe recombination of immunoglobulin exons to form functional genes takes place whenever the B lymphocyte (plasma cell) needs to produce more immunoglobulins.
51	Immunoglobulin Diversity - 3 Functional Ig Genes: eGenes that code for the Ig molecule: ŽH gene ŽL gene contains code for heavy chains heavy chain contains code for light chains light chain
52	Immunoglobulin Diversity - 4 Heavy Chain genes (H): eHeavy chains consist of variable regions and constant regions variable region constant region exons coding for this: V D J exons coding for this: C( if IgG class C: if IgM class C* if IgD class C, if IgE class
53	Immunoglobulin Diversity - 5 Light Chain genes (L): eLight chains consist of variable regions and constant regions variable region constant region exons coding for this: V J exons coding for this: C₆ if kappa type C₈ if lambda type
54	Immunoglobulin Diversity - 6 Some Nice-to-Know Information: eOnce a L or H chain gene is assembled and becomes functional further gene arrangements are prevented within the same B cell Žthus antigen specificity is maintained Žthis antigen specificity is passed on to progeny Žthe progeny will also have the same light chain types (6 or 8) eHeavy chains may undergo rearrangement during clonal development resulting in progeny with different classes of immunoglobulins (class switching) eSlight changes in the tail end of constant region of heavy chains also determines whether the Ig is attached to the surface or released into circulation.
55	Immunoglobulin Diversity - 6 7click button to return to main program
56	One Step Further Fragmentation of Immunoglobulin Molecules OSF - 8 Pages 108 & 109 7click arrow to return to main program
57	Immunoglobulin Fragmentation - 2 History: e1962 - Rodney Porter proposed the basic structure of the immunoglobulin molecule eUsed proteolytic enzymes to cleave rabitt IgG eThese proteolytic enzymes were: Žpapain Žpepsin eThe results of treatment with these enzymes was the production of different fragments of the IgG molecule
58	Immunoglobulin Fragmentation - 3 Fragmentation with Papain: cleaves molecule at hinge region 2 FAB fragments 1 Fc fragment
59	Immunoglobulin Fragmentation - 4 Fragmentation with Pepsin: cleaves molecule below hinge region 1 FAB fragments 2 Fc fragments
60	Immunoglobulin Fragmentation - 6 Outcomes from fragmentation experiments: eall basic Ig molecules (monomeric forms) have 2 separate antigen-binding sites (FAB) each containing V_L and V_H pair eeach V_L - V_H pair serves as an independent antigen- binding site ethe secreted form of IgM contains five monomeric units - thus 10 separate antigen-binding sites ethe hinge region of an Ig molecule provides flexibility which determines how many of the ab=s antigen-binding sites will bind with epitopes on antigens ethe location and functions of the Fc portion of the Ig are separate from that of the FAB region
61	Immunoglobulin Fragmentation - 7 Outcomes from fragmentation experiments: continued ebiological functions that are activated by Fc regions during the immune response are not antigenspecific ebiological functions that are activated by Fc regions during the immune response are Ig class-specific
62	Immunoglobulin Fragmentation - 7 7click button to return to main program