Immunology for 4th bio.students 1
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    Immunology for 4th bio.students 1 Immunology for 4th bio.students 1 Presentation Transcript

    • ‫﴿بسم ا﵀ الرحمن الرحيم﴾‬‫سنريهم آياتنا فى اآلف اق وفى انفسهم حتى يتبين لهم‬ ‫أنه الحق أو لم يكفى بربك أنه على كل شيء قدير‬ ‫صدق ا﵀ العظيم‬
    • Immunity (resistance): It the sum of all naturally occurring defense mechanisms that protect human from infectious disease Non – specific Specific Naturally acquired ( Innate ) ( Acquired )- Mucous membranes- Phagocytic cells - Placental transfer of antibodies( Passive )- Enzymes in secretion - Recovery from disease ( Active )-Interferons ( α,β,γ) - - Administration of antitoxin ( Passive )- NKCs- - Vaccinations ( Active )-Skin-. Macrophages- Artificially acquired
    • Naturally acquired Artificially acquiredactive passive active passiveFirst: Non – specific Immunity ( Innate):- This is a physiologic mechanism which is inherent or innate with the - following properties A single mechanisms Do not depends on specific Protect It does not exhibit specificity recognition of a foreign Against material many paths
    • Natural ( Innate ) Specific ( Adaptive )-Less specific . or- Skin & mucous membrane . (Acquired)-NK cells .- Complement cascade .- Phagocytosis .- C- reactive protein . Active Passive-Induced by contact with foreign antigens .- - Induced by antibody performed in -- Consist of clinical infection , immunization with live or - another host- killed infectious agents or their toxins . - - Ab injected in the incubation period -- Long term. - - Short term .
    • - Humoral immunity - Cellular immunity . Antibody mediated immunity. - Cell mediated immunity . ( B- lymphocyte) - (T- lymphocytes-Mediated) help help CD4 CD8 B- lymphocyte Helper T- LYMPH . Cytotoxic T-lymphocyte(Protection is mediated bythe produced antibodies) TH1 TH2
    • Haematopoietic stem cell Lymphoid stem cell Myeloerythroid progenitor NK cellB-lymphocyte T-lymphocyte monocyte neutrophil eosinophil macrophage basophile RBC platelets
    • * B- Lymphocytes This cell type consists ( 20 – 25%) of the total peripheral lymphocytes n mammals , they mature in bone marrow , then, migrate to secondary lymphoid organs ( e.g. spleen & Lymph nodes ) .* Upon exposure to antigen , B-Lymphocytes are stimulated to proliferate ,(large lymphocytes) differentiate and mature into LARGE PLASMA CELLS * The large mature B-Lymphocytes have short life span ( days to weeks ) . Secreted Immunoglobulins or Humoral antibodies L-CHAIN Antibody H-CHAIN
    • * Some large mature B-Lymphocytes (B- cells ) can be converted into small B- cells which have long life span This type of cells isSecondary involved in the Immune Memory cells And serve asResponseActivation & differentiation of B-Lymphocytes , in certain instances , needs a Helper T- Lymphocytes activity to enhance the above toprocesses in that B-Lymphocytes.
    • T-Lymphocytes :-*- THEY CONSTITUTE 65 -80 10 of total peripheral lymphocytes .*- They have long life span ( months to years ).*- They mature in thymus gland before migrating to lymphoid organs*- Upon exposure to antigen , T -cell proliferate . How ever , their specific effectors molecules are not secreted and remains firmly Attached to their cellular membranes Giving what is called cell-mediated immune response*- They are involved in a variety of cell-mediated immunological responses defense against malignant cells graft rejection hyper sensitivity reactions bacteria & protozoa Fungi viruses
    • T-CELLST-HELPER (TH) T-SUPRESSOR(TS) T-CYTOTOXIC (TCs) T-DELAYEDT- Helper :Their Surface Antigen : is T4 (CD4) . Helper- *They Promote Maturation Of Antigen . *Stimulated B and T cells. Sensitivity And Cells ( TdH) Enhance their responseT – suppressor cells: * Their Surface antigen is T8(CD8). and * they suppress the effect of T – helper cells . T-CELL MEDIATED i.e. *Suppress T &B – response . IMMUNITYT –cytotoxrc: * their Surface antigen T8(CD). (Tcmi ) * they specifically destroy target cells.virus infected cells unacceptable grafted cells tumor cellsT – delayed hypersensitivity & T cell mediated immunity.CD4 (T4) *they are responsible for delayed hypersensitivity reactions to different antigens , particularly those of intra cellular parasites & contact allergen .In general : * some of the stimulated T-cells release soluble substanceslymphokines that modulate the behavior of other cells.
    • *- most antigens which have a small number of epitopes and require carrier needT – cell cooperation with B- cells for antibodies production . * Deficiency of B – cells (andor) T-helper cellsleads to defective synthesis of antibodies.* its over activity lead to Autoimmune disorders the majority of B-lymphocytes express both surface IgM & IgD, very few expresssurface IgG & IgA or IgE in the circulation.*the majority of B-cells also carry class 2 major histocompatibility complex (class ПMHC) products which are functionally important in Regulation of immune response
    • Co -operation of innate & specific Immunity in Host defense against infection *Antibodies promote Phagocytosis or activate complement to kill microbes *T-lymphocytes enhance phogocytic and microbial functions of macrophages INNATE IMMUNITY SPECIFIC IMMUNITY complement In direct lyses by C. + +BACTERIA PHOGOCYTE PHOGOCYTE Opsonization B-Lymph INEFFECTIVE BACTERIA And Phagocytosis Direct lyses + SERM Bacterial lyses + COMPLEMENT BACTERIA B-Lymph a Ab b LYSISBACTERIA s Cell + MediatedBACTERIA PHOGOCYTE bacteria T-Lymph response
    • classification of acquired Immunity:-- passive Acquired Immunity :-Definition: acquired Immunity by given already form antibodies or antitoxic serum or gammaglobulins from normal or convalescent individuals or Trans placental or lactation . Trans placental . NaturalTypes Lactation (Colostrum). Antitoxin serum tetanus. (Anti_ cobra venom) Artificial Gamma globulins.- characters :- * -Rapidly developed . * -Short duration .[ Rapidly eliminated in 2-4 WKS due to the formation of anti – antibodies (a disadvantage )]. *-Heterogeneous antibodies . * -Cellular mechanism not stimulated . (No memory ).* - Side effects:- *- Hyper sensitivity reactions against the foreign serum *-Neurological affection in some cases ( Encephalitis ). *-Superadded in infections e.g. (AIDS & HEPAT) .
    • Definition :- the body forms his OWN IMMUNITY when stimulated (sensitized ) by introduction of immunogenic agent. Natural InfectionTypes *living attenuated vaccine * killed vaccine . Artificial bacterial products *Endotoxins. * Exotoxins. Others .Characters :- * slowly developed . *longer duration(and leave a potential immunity , so there is A rapid response in the future to the Same antigen ) leads to ?? *-Homogenous antibodies *- Cellular defense mechanism play a role Mechanism of Acquired immunity :- Humeral Ab Cellular T_Cells
    • Embryo Liver stem cell In Bone marrow central or primary lymphoid organs (tissues)SecondaryLymphoidOrgansSpleen or +Bone marrow + A9 A9 T_Cells B_Cells Effector PLASMA Killer cells memory cells CELLS B T HUMORAL ANTIBODIES
    • Specific memory and self-limitation of Immune response Ag A Secondary anti A infection response Serum Primary Anti A AB RESPONSE weeks 12 weeks*- Antigen enhance THE production of specific Antibody A.*- the secondary response to Ag A is more rapid and larger then the primary response ( memory cells ) .*- Antibodies Titer decline ( with time ) after each immunization .
    • Specific immune response : It is developed as a result of exposure to a variety of agents capable of inducing an immune response ( i.e. immunogens ) vaccines microbes that colonize Macro molecules in the diet in the bodyA special case Antigen in the form of hapten Hapten is a micomolecule may be conjugate with a carrier protein in the blood to be immunogen (antigen) Specific immune response Humoral cellular B. Cells T-CELLS *- They are two interrelated & interdependent mechanisms .
    • Specific immune response can be further Classified according to its components intoprimary secondaryInitial exposure to a particular on farther orInfectious agent or immunogen repeated exposureInduction phase of lymphocytes to antigen ( same )proliferation T-CELLS B-CELLS PLASMA CELLS increased resistance develops Sensitized T-CELLS Antibodies Cellular Immune response humoral through Humoral Cellular response response
    • Acquired immune responseHas both good ( desirable ) andBad ( undesirable ) consequence undesirable DesirableProtection Allergies (hypersensitivity)From infectionsagents Autoimmune diseases Immune responseControl ofPre-cancerous Graft rejectiongrowths
    • Interactions & functions of the major components of the immune systemANTIBODY – MEDIATED CELL MEDIATED REPONSE IMMUNE RESPONSE Two major componentsMain defense against T-HLPER & MACROPHAGES Cytotoxic* exteracellular, encapsulatedpathogenic bacteria Intracellular bacteria T-CELLSe.g. streptococci & staphylococci * ( mycobacterium &tuberculosis) Viruses*Neutralizations of toxins e.g.( • * Fungi Acts bytetanus) Destroying* viruses ( Hepatitis C,A,B…….)• Virus- infected cells
    • VIRUS infection cell MHC Class I T-Cell receptor virus MHC Killing Class II CD8 IgM CYTOTOXIC T-Cell RECEPTOR B-CELLS CD4 ( T_HELPER ) INTERLEUKIN-4 INTERLEUKIN-5VIRUS ANTIGEN Defense mechanism against viral infectionVIRUS
    • *- Recognition of phases :- antigen recognition ( binding of Ag to specificreceptor on mature lymphocyte ( exist prior to ag exposure )*- activation proliferation & differentiation of lymphocytes is the sequenceof events induced in lymphocytes as result of Ag recognition .*- Effectors phases elimination of antigen [ is the stage of the responseAt which the sensitized cells perform the function that (eliminate of Ag)Some antigen – stimulated lymphocytes die by process called programmed celldeath ( apaptosis ). Elimination OF AgT OR + AgB Phagocytosis NATIVE complement LYMPHOCYTES Recognition ACTIVATION Effector programmed cell phase phase PHASE Death
    • Immunogenicety ability to induce immune responseAntigenicity ability of the substance to react specifically withimmune system must be Antigenic Immunogenic are not necessary to beHappen is incomplete antigen ( di nitro phenol or penicillin)It cannot stimulate humoral or cellular reactions but can react with theseproducts specifically so it is Antigenic not immunogenicIf they reacted with larger carrier protein (e.g., albumin , globulin orsynthetic poly peptide ) . It will be ImmunogenicAnimals injected with this hapten – proteinComplex will make antibodies to this hapten ,Only if it is ( hapten ) covalently linked tothe carrier (chemically bonded)
    • CARRIER HAPTEN ANTIBODYPRTOCAL PROTEIN i NO YES NO II YES NO Anti carrier only III YES YES Anti carrier only ( not chemically linked) IV YES YES Anti carrier (CHEMICALLY LINKED ) & Anti hapten
    • Immune response :- its characterized by the produuction ofproteins ( Igs) and specificially reactive lymphocytes (T-cells ) when ananimal encounters aforeign macromolecules or cells . The inducing substances are called antigens i.e ( antibody generators ) or immunogens*- Immunogenicity & antigenicity : Interchangeable terms used during discussion of the immune reponse. *- Immunogenicity : it the inherent ability of asubstance ( Immunogen ( complete antigen ) to induce a specific immune response .*- Antigenicity : the ability to react with the products of that response .HAPTEN HAS AN ANTIGENICITYHAPTEN PLUS PROTIEN CARRIER IS IMMUNOGENAntigens are the aligands that react with the products of an immuneresponse .
    • Epitope ( - antigenic determinants ) :- are the sites either (on or) within the antigen with which antibodies or T-cells receptor reactsparatope :- the sites on antibodies which react with the antigen .epitope size ( small ) conformational linear conformational site are on antigen surface or internal that expressed only when the antigen has been partially degraded in vivovalency of antigen :- e.g multivalent i.e the antigen molecule carry a number of different epitopes ( some times 2 or>)some of which specify antibody A others specify antibody B .valency = total no . of epitopes the antigen pocesses .
    • The valence of A.g increases proportionally with molecular size .Macro molecules are easily to induce phagocytic ( as example ) and easier to be phagocytosed Quaternary structure are the most ImmunogenicThe more complexity , the more Immunogenicety
    • .factors Affecting ImmunogenicityForeigness chemical complexity molecular sizeA – foreigness :-the immunogenic substance must be forign to prduce immune response . The greater the foreignness, the more will be the reponse *- identical twins smaller or no response *- brothers with the higher immune response same tissues compatibility the same blood groups .etc ………….B.CHEMICAL COMPLEXITY :-*- MOST of organic molecules are immunogenic expert lipids*- proteins are the strongest immunogenic substance .*-Polysaccharides most of them are haptens but they become complete Ag incases of * peneumococcal polysaccharide . * Lip polysaccharides in cell membrane of gram (– ve ) bacteria.
    • *- Glycoproteins :-Are immunogenic ex blood group Ags ( A,B,AB,O,RH )*- POLYPEPTIDES & nucleic acids :-Are weak immunogens*- lipids :- are not antigenic or immunogenicC.molecular size :- usually the larger the molecule the stronger the Immunogenicety .M.Wt below 5000 DA ARE NOT IMMUNOGENICMACRO MOLECULES are the most potent immunogens .( e.g. albumin m.wt 40.000 DaGlobulin m.wt 160 kDaMacrocyanin m.wt 1000 kDa
    • EPITOPE (ANTIGENIC DETEREMNANT):- The portion of Ag that binds specifically withthe binding site of Ab (paratope) or a receptor(s) on T_lymphocyteSIZE CONFORMATIONAL STRUCTURE The size and the structure of epitope are complementary to that of paratope.i.e. they must have approximately the same dimensionsWITH RESPECT TO THEIR STRUCTURE ,A g MAY HAVE THE FOLLOWINGCHRACTES :- Ag may have only a single epitope of a given specificity on its surface which iscapable to bind with antibodies , such Ag is called UNIVALENT AND UNIDETRMINANT(one kind of specificity ) for example hapten Ag may have two or more epitopes (which determine the specificity ) the A g calledin the case MULTIVALENT (which determine the number).If the epitopes are of the same type called also UNIDETERMINANT and if they are ofdifferent types called MULTIDETERMINANT (specificity ). UNIVALENT MULTIVALENT MULTIVALENT UNIDETREMNANT UNIDETREMINANT MULTIDETRMENANT
    • Hapten-carrier conjugates have nativeantigenic determinants of the carrier aswell as new determinants of the hapten
    • Antigenic determinants are usually limited to those portions of the antigenthat are accessible to antibodies shownin black for this iron-containing protein
    • In an antigen, the same antigenic determinant repeated many times
    • T-dependent antigens are characterized by a few copies of many different antigenic determinants
    • MULTIVALENT since it has only one kind of determinant but many of such determinant on each molecule Ex. Many poly saccharides & homo polymer (e-g peptide chain of the some .A. Acids .)*- some antigens are multi determinant & valent such molecules have manyepitopes of different kinds (multi specificity ) but only one of each kind ( monovalent ) Ex. Most proteins .*- High M.WT , chemically complexed compounds or polymerized proteins(quaternary structure or heteropolymerized proteins are usually .*-Multi determinant Ag ( multi specific) , multivalent Ag (more than oneepitope of each kind) )What kind &How many of such kind (
    • Antibody binding site ( Paratope ). Binding of Ag & AbAffinity :- the strength of attraction and binding between an epitope( monovalent ) of an Ag and the antigen combining site of Ab molecule ( Paratope).Avidity :- The strength with which ( multivalcnt ) Ag bind to itsantibodies ( Abs). ( chemical complexity ) This depends on the affinities of the individual combining sites of the determinants on the antigen
    • ANTIBODIES and their STRUCTURESElectrophoretic separation of serum proteins
    • ANTIBODY STRUCTUR *Classes of antibodies . IgM , IgG , IgE , IgA & IgD . CH1A = COMPLEMENT BINDING SITE Constant A Constant A CH2 Heavy chainB = NEUTROPHILS & MACRO- PHAGE BINDING SITE Hinge bonds Constant B Constant BVARIBLE = ANTIGEN BINDING SITE . CH3
    • i-Immunization II_ FUSION antigen + MICE + MYELOMA CELLS B-CELLS fusion TUMOR MICE MICEAscetic fluid selection of Desired Clonesupernatant Tissue cultureFluid HYPRID CELLS HybrIdoma technique
    • Immunoglobulins "Humoral antibodies”They are formed of two identical units each of them is formed of :- A) heavy chain B) light chain C) hing regionA) Light chain 2( lambda) but never 1 and 1K K ( Kappa ) 2B) Heavy chains :* M-Wt 53.000 - 75.000 Da*- heavy chains are hold together with (disulphide bonds) .*- Fixed region contain 2k or 2  .*- The variable region contain a mixture of K,.*- both L& H chains contains the following region : Light chain contain variable (VL) and hyper variable (VH) regions Heavy chain contain variable and hyper variable regions.
    • * Amino terminal * carboxyl terminal* The amino acids differ * A. As are similar in different on  to another specificity. * it contain the effectors domain which is responsible for the* The VL & VH are adjacent to initiation of the processeach other forming paratope . by which the body gets-rid of Ag. .* They have sub-regions of the variable region (hypervariable) It is responsible for Designation of Ab class & its These regions have extreme - distribution. variability in their A .As sequence in different antibodies - and they are responsible for binding with Ag(s) -. - (hyper variable ) CDRs}{ comptementary detemining regions
    • C) Hinge region :- * CH 1, CH 2 , CH 3 : occupies ¾ that of Heavy chains the other ¼ is VH . * The Hinge region lies between CH 1 & CH 2 . * It is flexible & allows movement between the two antibody binding sites . * The hinge region is digested by protease (e.g. pa pain ) which splits it into :-( i ) antigen binding fragments (fab) = They are 2 identical fragments containing theantigen binding site .(ii ) crystallization fragment (FC ):-It contains the effectors )
    • Structures and function Of Specific Immunoglabulins*- Ig(s) are glycoproteins in the gamma globulin fraction of serum proteins (albumin ,fibrinogen , globulins ( ,  and  ) .*- they are produced by B- lymphocytes or plasma cells in response on immunogen (or Ag ). General Ig structure :-*- 4 poly peptide chains.*- they are linked covalently by disulphide bonds*- the 4 chains , monomeric Ig structure ,arecomposed of 2 identical heavy poly peptide chains (H) 2 identical light poly peptide chains (L)*- Heavy and light chains :*- H- chain :*- Have a M.wt of 50-75 KD (Twice that of L chain )*- H chains contain  400 A.As (Twice that of L chain )*-A. As differences in the .COOH terminal portion of the heavy chain (CH) identify 5 distinct H-chains isotypes .
    • * Each H chain has 4 or 5 domains :1 domain in the variable and 3 or 4 in the constant3 IgG (  ), IgA( )&IgD( ) Or 4 Igm ( )&IgE(  )Total = 1 Variable + 3 constant or = 4constant +1 Variable Notes (1) -Each L- chain has 2 domains 1 VL 1 CL (2)- Folding of the polypeptides chains brings the hyper variable regions of the VH and VL domains into close proximity . (3)- this folding creates a 3-dimensional structure that is complementary to the epitope (last figure ) The Hinge region*- It is the portion of the H-chain between CH 1& CH 2.*- there is no homology between it and the other H- chain domains, thus .itssequence is unique (sole) for each Ig type and subclass
    • IgM & IgE do not possess a hinge region but have one more CH domain.These structure explain why both IgM & IgE have 4 domains on the CH chains butnot like the other types (which have only 3 domains on CH)*-In this region (hinge), inter chain disulphide bonds forms between the arms of the Fab fragments preventing them from folding and therefore , rendering this portion of the molecule highly susceptible to fragmentation by enzymatic attach .* -The hinge region is highly flexible and allows for movement of the Fab arms in relation to each other .This motility explain why native antibody molecule do not activate complement , whereas those in an immune complex do .This is because , the native Ab is not in the appropriate configuration t1/2 or half life of( Abs) .*- These heavy chain isotypes form the basis of the 5 Class of Immunoglabulins molecule IgG () ,IgA ( ) ,IgM ( ),IgD ( )and IgE ( ).*- H chains Classes  and  are subdivided into subclasses of molecules  1 , 2 , 3 , and 4 And   1 and  2
    • The subdivision is based on the greater similarity of A.As sequence shown bysubclasses of the same classi.e. 1 , 2 , 3 etc,. Than is shown by different classes (i.e.  ,  , or )*- The heavy chain subclasses determine immunoglobulin subclassese.g. 1 = IgG1 2 = IgG2 , 3 = IgG3 etc,.*-L-chains :- * Are composed of  200 A. As . * They are of 2 types ( K= Kappa or  = lambda ) . { based on their structural (antigenic) differences } * All Igs classes have 2K or 2 chains but not k or  k . ex. * The proportion of K/ = 3/2 (human Ig) .- chain Isotypes :-*- There is no isotypic variations in K chains*- There are 4 distinct  chains 4 different isotypes .*- All the 4 subclasses are present in each of the Ig classes i.e. in IgM , IgE , IgD etc.*- Disulphide bonds Hold together the 4 polypeptide chains in Ig molecules . -*There are 2 types of disulphide bonds-:
    • i.Interchain disulphide bonds : occurs between H – H chains H – L chains L – L chains Single L-L only in Hinge But also in Ig A2M (1) region COOH-terminal such bond can of the H chain occurs in all Ig(s)except occur under path- Ig A2M (1) which ogenic conditions. Lacks an inter chain (e.g. Bence JonesThey can be 1:15 depending disulphide bond protein ) seen inOn the class & subclass types urine of some patients with multiple myeloma
    • INTRA CHAIN DISULPHIDE BONDS :*- occurs within an individual chain .*- they are stronger than inter chain bonds .*- they no. of intra-chain disulphide bonds varies depending only on the number ofdomains in the molecular .Light chain have 2 intra-chain bonds .*- human IgG, IgA, IgD heavy chains have 4 intra_chain bonds*- human IgM , IgE heavy chains have 5 intra-chain bonds .*- Each H& L-Chain has a variable (v) and constant (c) region*- V region lies in the – NH2 terminal portion of the molecule .*- The V region has a wide variation in it’s A.A composition .*- The C region lies in the - COOH terminal end of the molecule .*- The C region has a much more constant A.A Sequence except for minor inheritedchanges
    • *- The variable regions associate with appropiate constant regions .so that a variable H – Chain regions (VH) does not occur in an variable L – Chain(VL) and Vise versa .*- However , a particular VH chain sequence may occur in more than oneH – Chain class ( i.e IgG, IgM , IgD ,IgA and IgE ) .*- Thus during class switching in an immune response e.g when B – cells changetheir production from IgM to IgG heavy chainonly the constant regions of the H (CH) changes and the antibody specificityremains the same .HYPER variable regions*- they are particular areas within the variable regionsThat are highly variable in A. As sequence .*- THESE hyper variable regions often called complentary determining regions*- THESE regions occurs at simillar A.A positions in an relatively invariantmolecules .
    • CDRs :- they are short polypeptide segments lining near A. As positions 30,50 CDR1 CDR2 CDR3 AND 90 in the variable regions of both L and H chains . FR1 FR2 FR3 FR4 variabilityFRs CDRS 89-97variable region 24-34 50-56 NO OF AgS Note :- the variability range ( index ) used is an arbitrary scales of the no. of different A.AS found in each position if 100 different Light chain were analyzed . *- the hyper variable regions are important in the structure of the Ag binding site ( paratope ) . *- L – chain have 3 hyper variable regions ( the last figure ) *- H – chain have 4 hyper variable regions although, ONLY 3 OF THE 4 have been associated with epitope recognition *- each Ig chain consists of a series of globular regions or domains enclosed by disulphide bonds ( intra or inter ) ?? Chain disulphide bond . *- The A.AS sequence of the domains show a high degree of homology ( i.e the sequences are very similar ) .
    • Properties of Ig :- IgG IgA IgM IgD IgEH – CHAIN     H – CHAIN SUBCLASS 1, 2 1 2 - - -M.Wt 150 160-400 900 180 190Carbohydrate (%) 3 7 12 13 11Serum conc(mg %) 1200 200 120 3 0.05Seru t ½ ( days) 21 6 10 3 2Functions :-Complement activation ++ - ++++ - -Opsonization ++++ + - - -Antiviral activation ++ +++ + - ?Mast cell sensitization - - - - +
    • Immunoglobulin are glycoproteins :- (3-13 % of their M.WT ) OLIGOSACCHARIGES + PROTEIN*- THESE oligosaccharides are present in CH2 or CH3 .*- N -glycosidic bonds usually link N- acetylglucosamine in the carbohydrate moietyto asparagine residue in the peptide c-chain of Ab[ linkage with the enzyme N -acetylglucosamine .- Asparagine transglycosylase ] transferase*- t ½ of Abs in the circulation depends on the status of oligosaccharide side chain*- the oligosaccharide side chain of Ab terminate with galactose to which sialic acidis bind .*- when Abs have the sialic acid removed by the enzyme neuraminidase , theybecome susceptible to degradation in the liver .*- in this case the terminal galactase bind to a receptor on hepatocytes and theentire molecule is , then , interenalized to the cell for degradation viaProteolytic enzymes in lysosomes of the cells .
    • Restriction enzymes digestion of Abs :1) Papain : digest above hinge region so it leaves 2 Fab fragments each is monovalent S-SAnd crystalline fragment (FC) papain Fab FC Monovalent 2)Pepsin: digest away most of FCFragments below the Interchain disulphide bond(below the hinge region) it give one large fragmentsF(AB)2 which is consist of two Fabfragments joined by the disulphide bondThus , it is bivalent ,possessing the ability to bind and form agglutination S-S F(ab)2 FC Ab
    • Figure 4 Immunoglobulin fragments: Structure/function relationships
    • Classes of antibodiesThey are 5 isotypesThe class of Ab depends on the A.A: sequenceof the constant regions of the heavy chain . IgM*- Immunoglobulin M (IgM) :- * it is a pentamer ( 5 molecules ) . * they are linked together by disulphide bridges at the COOH terminal end of the heavy chains as well as an additional poly peptide chain ( joining chain) * this type of Ab account for 8-10% of the total PLASMA ANTIBODIES . * it is the most abundant Ab produced by the faetus . * it binds with viruses and bacteria*- Immunoglobulin g ( IgG ) :- * it is a monomer * it accounts of ~ 75 % of the total antibodies . * it is important for elicit ting the immune response to Ags * it is only antibody which pass through the placenta to protect the faetus.
    • *- immunoglobulin D ( IgD):- * It is a monomer ACCOUNTING FOR < 1% & TOTAL ANTIBODIS . * Its function is controversial .*- immunoglobulin E ( IgE):-* It is a monomer ( below 0.004 % & the total Abs)* It is present in spleen , tonsils , mucus membrane of lungs GI* On binding with ag it releases histamine from mast cells leading tohypersensitivity .* It provides immunity to intestinal parasites .*- immunoglobulin A ( IgA):-* MONOMER , DIMER or TRIMER( mostly dimer )* Like IgM the units are linked by disulphide and j chain * it is found in tears , saliva , intestinal treat secretions * it binds with Ag preventing them from tissue adherence , colonization ,andmaking them more phagocytosed .
    • Properties of Ig :- IgG IgA IgM IgD IgEH – CHAIN     H – CHAIN SUBCLASS 1, 2 1 2 - - -M.Wt 150 160-400 900 180 190Carbohydrate (%) 3 7 12 13 11Serum conc(mg %) 1200 200 120 3 0.05Seru t ½ ( days) 21 6 10 3 2Functions :-Complement activation ++ - ++++ - -Opsonization ++++ + - - -Antiviral activation ++ +++ + - ?Mast cell sensitization - - - - +
    • Laboratory Methods SerologyIn vitro Ag & Ab reactions called serologyIt provide methods for i) Identification (Diagnosing) ( ii ) quantization of titre of Ab (and or) AgTitre : or the level of Ab (s) in the serum can be measured by using known AgThe titre may have diagnostic or prognosticEx. A rise in Ab titre between acute &convalescent serum can be used as adiagnostic tool for a specific diseaseThe titre is defined as the greatest dilution of serum (which contain the Ab underconsideration ) that reacts which the antigen ( i.e. gives +ve result ) .
    • - the forces involved in Ag-Ab reactions are greatly affected by various environmental factors :-*- The Ag- Ab complex is not bound firmly together .*_This complex may even dissociate spontaneously .* physiologic ph & salt concentration promote optimal union of them .*- the force of attraction tend to be weaker ina) very acidic .e.g. 0.01Mb) very alkaline medium i.e pH 4 and alkaline ( i.e. above pH 10 )- temperature :- it plays an important role : * the higher the temp ( up to 50 – 55 0 c ) , the more rapid is the rate of reaction between Ag & Ab . * the reason is the increase in kinetic motions of the reactants ( Ag & Ab )
    • various forces act to hold the Ag-Ab complex together :- * The maximum attractive forces stabilizing Ag-Ab complexesAre van der weal forces Ionic bonds 1- van der weal forces :- * occurs because of spatial fit ( the below fig ) * these forces of attraction hold Ag to Ab onlyWhen the two molecules have complementary shapes (a) puratope 2 puratope 2 Epitope 2 (a) (i) significant changes Epitope 2a In the shape of epitope 2 (b) Into 2a
    • these change precludes its ( 2a ) interactions with the matching binding site of theoriginal Ab .* When the molecules have less similar shapes ( b) , these forces are less effective(b)2-Ionic bonds :-* They are patterns of complementary electric charges on the molecule .* The electrostatic interactions tend to hold the molecules together . COO NH3 COO NH3+ COO +NH3Affinity :- the strength of attraction between a single epitope and its matchingparatope is the referred to as the affinity of the reaction between the two reactants . Ag-Ab complex of low affinity dissociate readilyAvidity :-* It is a related term to affinity* It refers to the strength of the interactions between multivalent antigens and thepopulation of Abs that they have included .
    • *- Avidity is influenced by the affinity of individual Abs for their (A) epitope (B) the valency of Ag and (C) the valency of Ab tertiary structure of protein : *- the ability of Ab to bind with Ag can be affected by altering the tertiary structure of any of themex. insulin which is composed of A&B chains Ab to either one of these chains can be produced by(a) splitting the chains(b) purifying tem(b) injecting ( .e.g. a pig)them into foreign host the pig will produces Ab to the particular chain that was injected *- if the host (pig) Abs are injected back into the animal species that supplied the original insulin (man) , the abs will not react with intact insulin molecules . *- This is because the tertiary structure of native insulin is such that the on the A & B chains are not accessible .. epitopes Now , it is generally accepted that in a given poly peptide the A .As that are spatially accessible because of Tertiary structure of this protein are only immune reactive
    • *- The physical state of the antigen is responsible for the identification of Ag –Abreactions and the naming of Abs .*- The same Ab molecule could , in fact , be described by each of the following terms :(1) Agglutinins are Abs that aggregate cellular Ags.(2) Lysins are abs that cause dissolution of cell membrane .(3) Precipitins are abs that form precipitate with soluble Ags .(4) Antitoxins are abs that neutralize toxins . procedures must be involving direct demonstration and observation of reaction .. The relative sensitivities of the tests for Ags and Abs are Presented in table 8.1 page 156 [ immunology , 3rd edn ].A- Agglutination Reactions :- a b Serve to detect and quantities Agglutinins and identify cellular Ags Bacterial cell white blood cells red blood cells .**-- when the cells intact with the appropriate Ab , they clump together and eventually Large enough to be visible form masses with naked eye
    • *- When Ab agglutinates bacteria in the body opsonization occur .*- Agglutination occurs because Abs and at least bivalent .*- Two sites on the Ab and multiple sites on the Ag Ag – Ab lattice formation that can build up into increasingly larges coupled lattice structure Example widal test :- (diagnostic test of typhoid )*-Ab of patient serum is measured by adding a constant mount Ag (e.g. salmonella typhi ) to serially diluted serum .*- After incubation , the test tubes are examined for visible agglutination .*- the last tube (i.e the highest dilation of serum ) showing agglutination is referred as the titre.
    • B- lyses Reactions :- In the presence of a complement an Ag – Ab reaction , on a cell membrane , may result in membrane damage that leads to cell lyses This phenomenon is important in the hosts defense against condition such as microbial infection or cancer ( graft cell , virus infected cells , etc…………….)*i)- Haemolysis :- In which the Hemoglobin is released from R.B.C, is a requisite phenomenon for the complement fixation test .*ii)-- bacteriolysis :- cells of gram (– ve) bacteria are undergoes immune lyses under certaincondition .*iii)-- cytolysis :- involves the destruction of other cells types (e.g. lymphocytes ). C- precipitation:- * occurs when the Ag is soluble instead of cellular *therefore a large number of molecules are required for lattice formation and alarge no .of lattice must be formed for an aggregate to be formed and visibly seen.
    • *when soluble Ag (s) come intact with specific Ab. They aggregate (i.e precipitate ) Three conditions are presentA- where the (Ag) is very low with excess Ab (zone of Ab excess ), Formation of complex occurs But Residual Ab remains in the supernatantB- As more Ag is added , large aggregate is formed In the (zone of equivalence) ,maximal Ag-Ab complex are formed and precipitatedC- Instead of reaching a plateau , this curve comes back down to zero with increasing the mount of Ag (zone of Ag excess ) * this is because the lattice size becomes too small to precipitate . * In extreme Ag excess . the complex will be trimmer i.e one Ab +2AgNote:- the soluble Immune complex are not processed efficiently by the reticuloendothelial system ,and ,this cause damage (how??)
    • Amount of precipitate Zone Of equivalence Effects of increasing amounts of Ag on the total immune precipitate obtained from a mixture of soluble Ag and its homologous AbINDIRECT HCG :Examples 1 :- determination( and or) detection of HCG by using indirect methods .(i) an Ag will be added ( HCG ) .(from the kit)(ii) Urine will be added ( excess Ag ) from a female may be pregnant .(iii) Ab to HCG will be added In case of positive In case of negative pregnancyA state of Ag excess a state of equivalence will be reachedTherefore, no precipitation therefore, precipitation occurDirect HCG assay :* (i) Ab to HCG will be added (from the kit)* (ii) Ag ( HCG of the test sample will be added) . If precipitation occur ( positive) if, no precipitation occur ( negative )
    • Hyaluronic acid (HA) assay using excessHA binding protein (HABP) :-* HABP will be added in excess ( known excess ) (ACT AS Ab)* Sample will be added ( containing HA) (ACT AS Ag)* [ A state of Ab excess no ppt ]* An radiolabelled HA will be added ( Ag )Thus, precipitation occur ( IF +ve sample) and immune complex will be separatedand quantities by radio- immune assay technique ,in case of no precipitation, thesample is negativeImmune diffusion* It used for quantization of Ag (s)* Thus, precipitate will also be demonstrated .* If an Ag – Ab reaction takes place in semisolid medium (e.g. agar ) , band ofprecipitate will be formed .* The reason of precipitation , is the diffusion of the components (Ag & Ab ) towardseach other .* A useful example is a double immune diffusion technique :-
    • Procedure :-* Ag& Ab preparations are placed in separate wells that are cut into a thin layer ofagar in a Petri dish .* The reactants diffuse towards each other through the agar until they meet anoptimal proportions [ zone of equivalence ] and forms ( ppt ) bands Solid Chevron Fig (a) Ag PPT Ag Zone of Ab equivalenceThe advantages of the procedure is that antigenic relationshipcan be detected by the precipitation pattern (s)
    • 3 basic patterns are given :(a)- in reaction of identity , the 2 Ags are similar , they will diffuse at the same rateand the two precipitations bonds merges into a solid chevron ( fig b) Aga Aga Fig b Ab2- in reactions of non-identity , the two Ags are completely different and the linesof the precipitate cross (fig c) Ayab Agac Aga Agb Fig c Aba Abb Aba Abb3- reaction of partial identity :-* It is indicated by spur formation indicating that one of theAg(s) is cross-reactive ( but not identical ) to the other one .* The spur occurs because one the Abs (b) does not react with the cross-reactingAg (Ag ac) but migrate past that Ag (Ag ac ) until it reaches an Ag (Ag ab ) that
    • Has an epitope for which it has specificity . B- quantitative radial immune diffusion* It is used routinely to quantities Ab in serum .* For this purpose , an agar coated slide is used .* The agar being impregnated by anti sera ( antibody to human IgG )* SERUM samples are placed in wells in the sugar .* As it diffuse through the agar and encounters the Ab, the IgG in the sample form aconcentric ring or halo precipitate .* The diameter of the halo of precipitate directly correlate with the [ IgG] in thesample .Thus , the levels of IgG in the sample can be determined by referring a standardcurve based on halo diameter (s) of known concentration (s) of IgG C-immune electrophoresis :-* It was developed because the double immune diffusion technique.(i) Could not resolve high complex mixtures of Ags .(ii) and, a more sophisticated technique was needed .
    • In this procedure : (a) Ag is placed in wells in agar on a glass slide and then , subjected toelectrophoresis through application of an electric current . (b) Under these conditions , the individual Ags or antigenic components ( inthe same sample ) migrate through the agar at variable rates . (c) If Ab is placed in a well that runs the length of the slide parallel to the pathof migration , the reactants will diffuse towards one another and form separate arcsof precipitate for each antigenic determinantD- counter-immune ectrophoresis (CIE)This technique Is the double diffusion method + an electric currentWhich plays as the migratory force which:(i) – amplify the speed of reaction ( 24 hrs to 30 min )(ii) Intensifies the precipitation bonds .(iii) Increasing the sensitivity of the assay about 10 fold .
    • Procedure :- (i) Ag & Ab are placed in wells and the current is applied . (ii) in suitable buffer ( eg ph 8.6 ) the negativity charged Ags migratetowards the anode , whereas the Ab [ which has no sufficient net charge ] migrate inthe opposite or counter-direction , as a result of endosmosis .Precipitation occurs where the reactants melt .•D- Antitoxin :-* If a serum contain an antitoxin ( i.e. antibody to a toxin ) , the Ab . Will neutralizethe toxin examples :-- Suppose serum containing antitoxin is mixed with toxin ( in vitro ).- Then , after a few minutes , a small amount of the mixture is injected into anexperimental animal ( in vivo ) .- The animal will be protected against the introduced toxin , and thus , its deleteriousaffects disappear because of antitoxin is present .
    • Clinical example :- ( the virus haemagglutinate R.BCs)* To examine the serum of a patient suspected of having influenza ,*1) The patient serum is mixed with known influenza2)add red blood cellsi- if Ab is present haemagglutination will be prevented .i.e the sample is positive this is due to the ability of Ab to bind with the virus and block its ability tohaemagglutinate the R.B.Csii- if no Ab is present , haemagglutinate will occur . virus + R.B.Cs haemagglutination occur .i.e the sample is negativeE-Flacculation :- it is another form of Ag –Ab reaction that occurs if the Ag is neither cellular nor soluble but it is an insoluble particulate
    • VDRL TEST FOR SyphilisThe venereal disease research laboratory (VDRL) test is a slide flocculation testused for the diagnosis of syphilis .The VDRL make use of heterogenetic ( heterophillic ) antigen shared between the •Spirochete of syphilis & normal beef heart .* The Ag used is a water insoluble cardiolipin that had coated the surface ofcholesterol particles that were added to the system .* These form visible aggregate indicate to the presence of Ab ( reagin ) in the serumof patient for syphilis[ reagin is Ab type which flocculate (or ppt) an Ag that is neither cellular nor solublebut it is insoluble ]* The test can be performed on a glass slide .
    • Technique :- cholesterol particles + normal beef heart extract ( inert support ) ( antigen like substance ) Insoluble antigen serum ( A.Bsource ) visible aggregate Which can either seen by The naked eye Using a microscope and green filter COMPLEX SEROLOGICAL PROCEDURESAg-Ab reactions in which the visible manifestation requires Participation of: a) Accessory factors b) Indicators system c) Specialized equipment
    • A- fluorescent dyes :-e.g fluorescein isothiocyanate ( FITC)* FITC can be conjugated to Ab. Molecules to visualize of the molecule under (uv) or (b)a fluorescence microscope .* such labeled Ab. May then be used to identify Ag(s)(i) Direct immunofluorescence assay :-* The method uses Ab. That is specific for a particular Ag or parasite* This Ab is labeled with a fluorescent dye (FITC)* This conjugate is allowed to react with unknown tissue or organism .* IF the Ab reacts ( i.e +VE the result ) , it will visualized as green stain on thespecimen when it examined under the fluorescence microscope by using uv light
    • Examples :- Identification of Trepenoma palladium ( syphilis ) in an extracted from a patientsuspected of having syphilis .- Procedure :-*1) The slide is coated with the Ag .*2) Ab tagged with FITC is added .* 3)Excess Ab is then washed .* 4)Then , the slide is examined with uv fluorescent microscope .* Trepenoma palladium is fluoresce against the black back groundthis methods can be extended for other pathogens .
    • (ii)-immune peroxides technique :If viral antigen in tissues will be detected, horse radish peroxides is conjugated with the Ab .*-(1) After the enzyme – Ab complex has reacted with the tissue (Ag) .*- (2) Excess Ab is washed .*-(3) And , an appropriate enzyme substrate is added to the tissue section .*- the bound Ab. Is detected by the presence of a dark precipitate*- Advantage of immune peroxides technique over the immune fluorescenttechnique:-*- The specimen can be stained with conventional Histochemical dyesSo structural details can be seen ( noted )*- the tissue con be examined by standard light microscope .(iii)- Indirect immune fluoresce technique:-*- The procedure use Ab ( secondary ) ( against ) another Ab ( primary ) of patient .
    • *- the primary Ab is the patient serum detection of Abs.*- the secondary Ab is covalently conjugated whit fluorescent compound (FITC)*-*- ex. Of secondary Ab is rabbit antihuman( INJECTED Ab in host is FROMHUMAN ) gamma globulin anti sera . .i.e Produced against Ab used to immunize rabbit and that Abwhich will be examined latter on (unknown conc.)Technique :- (this procedure allows for detection of Abs,) Example serodiagnosis of syphilis by the fluorescent trepenomal antibody absorption (FTA-Abs) test*-(i) -T. pallidum is fixed to a slide*-(ii)- the slide is flooded with the patient serum (staining Ab )*-(iii)-If Ab to spirochete are present, the Ab will reacts (bind) with the organism on the slide* (iv)-Excess Ab (serum) must be removed with washing , to detect the bound Abonly .*-(v) the Ag-Ab complex formed is them treated with the fluorescein – tagged Ab tohuman gamma globulin, the excess Ab is washed carefully.*-If the patients serum contains Ab (+ v e) against the T. pallidum, fluoresceinorganism will be seen when the slide is examined with fluorescence microscope
    • FITC **-- Ab fluorescence conjugate is Patient’s binded . Slide coated with ag in Serum is Microscope (uv ) is used (excess) added- Indirect Immune fluorescence assay is also used for Detection of Antinuclear Antibodies (Ana) :- ( e.g. DNA , RNA & His tone) ANA are present in systemic lupus erythroMatosis ( SLE ) , some Times in rheumatoid arthritis and other autoimmune collagen – Vascular diseases .Example (SLE ) :- * The procedure is similes to that of T . palladium . * The Ag is ( DNA ) histone in form such as Animal Buffy coat calls Rat kidney section Human Buffy coat calls beef thymus Lymphoid Thymus Lymphoid organs
    • Haem agglutination Inhibition teat :- It involves The agglutination of R.B.cs by Or (haemagglutinin(s)) Certain virus other Ab(s) particles (influenza) substancesIt demonstrates the presence of serum Ab to haemagglutinating viral substance . Technique :- R.B.C s serum sample Ab from the Which contain Ab kit that make Prevent haemagglutination haemagglutination Agglutination occur no agglutination The sample is negative the sample is positive
    • Similar test can be used to detect soluble Ag(s) which able to react with and neutralize a haemagglutinating Ab . R.B.Cs from kit serum Ag which Abs from kit whichPrevent haemagglutination Capable of haemagglutination haemagglutination haemagglutination takesplace Sample is negative Inhibition Sample is positive
    • - passive agglutination:- in the conversion of a reaction system from one thatprecipitate one that agglutinate Thus yields a more sensitive indication of the presence of antibodies .Example :RHEUMATOID ARTHRITIS- The use of latex particles in the diagnosis of rheumatoid arthritis (soluble Antibodies) is an example of passive agglutination .Principle :- In this disease ,the patient produces an Ab (Mainly IgM) to his own IgGTechnique :-*-(i) latex particles were coated with IgG.*-(ii) patient s serum is added (which contains antibodies IgM)*-(iii) Agglutination indicates the presence of Antibodies (Ig?) (i.e The test is positive )The detectable antibody is called rheumatoid factor
    • Bis-diazotized diphenyl :- it is a coupling reagent that can be used to proteinsconjugate : or to R.B.Cs Haptens and thusPassive haemagglutination Occur.Thus :*- Addition of serum containing Antibodies to these substances (proteins orhaptens ) allow the detection of these specific antibodies to these substance by atechnique called Passive Haemagglutinationrose –waaler Test : which detect rheumatoid factor in serum of the patients suspected tohave an anti-IgG auto-antibody .i.e. .(IgM to an accumulated IgGTannic acid –treated a sheep R.B.Cs (S R.B.Cs) are coated with rabbit IgG -Antibodies specific for these S. R.B.Cs
    • fromTannic acid the kit coated by IgG serum from a patient suspected R.B.Cs to have autoantibodies haemagglutination the sample is positive IgM
    • Coombs (antiglobulin) Test :-* In certain people .Abs directed against antigenic determinants (e.g. R.B.Csantigens )are able to form visible aggregates when subjected to : 1- precipitation 2- Agglutination*-To demonstrate the presence of Abs in such cases the coombs (antiglobulin)testmay be used .*- The test involves the addition of Ab direction against gamma globulin : whichprovides a bridge between two antibodies coated call or particleThus ,The major use of the coombs test is to detect the Non agglutinating(haemagglutinating )anti-red blood Cs Abs.(1) – Direct coombs test * It is used to detect call bound antibodiesTechnique :- You must use EDTA BLOOD then centrifugation*-(1) The red blood calls (bound antibodies )are washed free from serum and theunbound antibodies (to be leaving the bound ones ). (2) Antiglobulin serum is added directly to this call suspension -*
    • *- The direct coombs test is of value in the detection of antibodies toR.B.Cs associated with hemolytic disease of new born (e.g.erythroblastosis fetalis ) and auto immune anemia or disease .*- The Abs associated with these diseases have the ability to attach to butnot agglutinate the target R.B.Cs .*- These absorbed Abs can be detected by the use of Ab (i.e. coombs Kitserum ) to This human gamma globulin .
    • (2) indirect coombs test :- *- It is used to detect the presence of Circulating Antibodies.*- It is of value in detecting IgG - associated antibodies in the serum ofwoman who is though to be (a) sensitized to Rh antigen And (b) at risk for carrying an erythoblastbotic febus .Technique :-*(1)- Serum sample (containing Ab ) is incubated with donor R.B.Cs( containRh antigen). Faetus like blood*- (2) Then ,the cells R.B.Cs are washed off ( to remove excess Ab ) .*- (3) The anti globulin (coomb reagent) reagent is added ( kit)
    • Serum Ab is absorbed haemagglutination ( + ve ). serum No Ab .(No absorption ) haemagglutination ( - ve ). Anti globulin commb reagent Viral Neutralization :- It is very similar to haemagglutination Inhibition on ( i.e. it is a neutralization event ) .*- Principle :- The assays is based on the ability of specific Abs to interfere with Some biological function of the virus under consideration ( usually The infective property is blacked ) .( 1 )- Cyotpathic effect ( CPE ) :-- certain virus + cells ( in tissue culture ) cell destruction .2 )- the CPE is useful in the search for virus neutralizing Abs in serum sample.
    • *-Technique:- **_ serum suspected of containing Ab is added to a virus suspension . **_ Susceptible cell culture is inoculated with the mixture . CPE developed (killing ) If the culture fail to develop no Neutralizations CPE (no killing ) ( + ve ) ( culture Cell death ) ( - ve ) Abs are interfere with the Ability of the virus to kill Tissue culture cells– Radio immune assay (RIA ):- * - It is and extremely sensitive method for quantization of any substance that is Immunogenic or heptenic and can be labeled with radioactive isotope e .g. I (25I) .Liquid phase RIA :- It depends on the competition between labeled (Known ) and Unlabeled ( unknown )antigen for the same antibody .
    • Labeled Ag (known amount) Unlabled Ag[unknown amount] Ab specific known amount Immune reaction product (Immune complex) ( i ) Separation of this complex by immunological method by secondary Ab or by Precipitating agent . [ (NH4)(SO4)] ii) Separation of this complex by physiological methods (a) centrifugation (b) Decantation . PPT (commonly used)The radioactivity of either . The supernatantIs then determined CPMA Calibration curve based on using serial dilution of known unlabeled standard ( instead of the serum ) is used for calculating of un known samples conc.
    • *- Solid phase RIA :- Liquid phase is modified by :-( i ) adsorption or covalently linkage of Ab to solid matrix ( solid phase RIA) .( ii ) The unlabeled Ag (sample ) is added followed by the labeled one ( Antigen or Antibody ) . Then The bound versus free Ag can be determined by Using reference calibration curve ( as before) [ washing steps]
    • Enzyme linked Immune Sorbet Assays { ELISA } :- *- ELISA is both highly sensitive ( > 99% ) and specific ( > 99% in high – risk populations ) . *- It can be for the assay of either Ag(s) or Ab(s) . *- Ag or Ab can be attached to solid phase support ( plastic surfaces , paper disks ) and still retains its immunologic activity. *- Either Ag or Ab can linked with an enzyme e.g. ( horse reddish peroxidease alkaline phosphates ) . *- substrate is added and the color absorbed by the enzymatic procluct is then quantization and compared with a calibration curve .Example :- detection of Ab(s) to the human HIV :- *- The virus is grown in vitro in a human T-cell culture . *- purified whole virus is disrupted 8 viral proteins are immobilized onto plastic beads or multi well trays . *- Abs to any of these antigens will bind with them & immobilized . *- Excess proteins are removed by washing the beads ( or wells ) and an enzyme linked anti human gamma globulin antibody is added . *- The presence of this second Ab can be detected calorimetrically by adding a substrate for the enzyme that will yield a colored end product .
    • *- The rate of substrate degradation is determined by the amount of enzyme – labeled Ab that is bound which is proportional with the amount of Ag in the solution being tested. *- the color change can be measured quantitatively in a spectrophotometer .* Double Antibody sandwich ELISA :- It is used for the assay of Ag ( e.g. HBSAg ) uses tow Abs as below :-( i ) first Ab ( specific e.g. HBs Ag ) is coated on a plastic surface ( poly styrene), the solution being tested for HBs Ag is then applied to the surface .( ii ) Washing of any un reacted material .( iii ) The second Ab ( ie enzyme linked anti HBs Ag specific Ab is then applied .( iv ) Any excess conjugate is rewove by washing . (v) finally substrate is added to the detest the present of En2 ABO group & Transfusion Reactions *- ALL human erythrocytes contain all antigens ( i.e. Antigens that vary among individual members of a species ) of the ABO group . *- This is important system , which . is the basis for blood typing & transfusions . *- The A & B antigens are carbohydrates that differ by a single sugar .
    • *- Despite this small difference , A & B antigens do not cross – react*- R.B.Cs have 3 terminal sugars ( in common ) on there are surface.. N – acetyl glucose amine Galactose Fucose H antigen**-- Type A cells have an additional N –acetylgalatose .**-- Type B cells have an additional galactose.N.B . Type A & B genes code for transferaes that add the respective Sugar .*-* Type O have only the H antigen : To avoid Ag – Ab reactions that would result in transfusion, all blood for transfusion must be carefully cross matched .*-* So , Ag the corresponding Ab do not coexist in the some persons blood .*-* Transfusion reactions result when incompatible donors R.B.Cs are transfused e . g . group A In to group B .
    • Ag – Ab – Reaction Involving R . B . Cs antigens :-ABO blood group . Structure of the terminal sugars that Determine ABO blood groups .
    • Group Antigen on R.B.Cs Antibody in plasma A A Anti B B B Anti A AB A&B No Anti A NOR Anti B O No A nor B Anti A & Anti B 4 POSSIBILITES OF CONBINATION