Antigen-Antibody reactions

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Antigen – Antibody reactions Dr. Pendru Raghunath Reddy Assistant Professor of Microbiology Dr. VRK Women’s Medical College

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Antigens and antibodies combine with each other specifically and in an observable manner In the body, they form the basis of antibody mediated immunity in infectious diseases, or hypersensitivity and autoimmune diseases Antigen – antibody reactions in vitro are known as serological reactions In laboratory, they help in diagnosis of infections, in epidemiological surveys, in the identification of infectious agents, enzymes

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Stages of Ag – Ab reactions Primary stage Initial interaction between Ag & Ab – invisible Rapid, occurs at low temperatures & obeys the general laws of physical chemistry & thermodynamics Reaction is reversible Ag & Ab is bound to each other by weak Van der Waal’s forces, Ionic bonds & Hydrogen bonding

Ag-Ab interactions:

Ag-Ab interactions Bonds: Hydrogen Ionic Hydrophobic interactions Van der Waals forces Each bond is weak; many are strong To “hold” they must be close  requiring high amts of complementarity!

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Secondary stage Demonstrable events – Precipitation, agglutination, lysis of cells, killing of live antigens, neutralization of toxins, complement fixation, immobilization of motile organisms & enhancement of phagocytosis. Precipitin – Ab participate in precipitation Agglutinin - Ab participate in agglutination Precipitinogen – Ag participate in precipitation Agglutinogen - Ag participate in agglutination

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Tertiary stage Includes neutralization or destruction of injurious agents or tissue damage Also includes humoral immunity against infectious diseases as well as clinical allergy & other immunological diseases

GENERAL FEATURES OF Ag – Ab REACTIONS:

GENERAL FEATURES OF Ag – Ab REACTIONS The reaction is specific Entire molecules react and not the fragments There is no denaturation of the antigen or antibody during the reaction The combination occurs at the surface. So surface antigens are immunologically relevant The combination is firm but reversible. The firmness is influenced by the affinity & avidity of the reaction Antigens & antibodies can combine in varying proportions. Both Ags & Abs are multivalent

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Affinity = ∑ a ttractive and repulsive forces Ab Ag High Affinity Ab Ag Low Affinity Affinity Refers to the intensity of attraction between the antigen & antibody molecules. It is the function of closeness of fit between the epitope & antigen binding region of its Ab

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Avidity Strength of the bond after the formation Ag- Ab complexes The overall strength of binding between an Ag with many determinants and multivalent Abs Y K eq = 10 4 Affinity Y 10 6 Avidity Y Y Y Y Y 10 10 Avidity

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Two important parameters of serological tests are Sensitivity Specificity Sensitivity Refers to the ability of the test to detect even very minute quantities of antigen or antibody When a test is highly sensitive, false negative results will be absent or minimal

Specificity:

Specificity Refers to the ability of the test to detect reactions between homologous antigens and antibodies only, and with no other In a highly specific test, false positive reactions are absent or minimal In general, sensitivity and specificity of a test are in inverse proportion

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Cross Reactivity The ability of an individual Ab combining site to react with more than one antigenic determinant. The ability of a population of Ab molecules to react with more than one Ag Anti-A Ab Ag A Anti-A Ab Ag B Shared epitope Anti-A Ab Ag C Similar epitope Cross reactions

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Types of Antigen – Antibody Reactions Precipitation reaction Agglutination reaction Neutralization reaction Opsonisation Serological tests based on Ag – Ab reactions Complement fixation test Immunofluorescence Radioimmunoassay Enzyme immunoassay

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PRECIPITATION REACTION PRINCIPLE When a soluble Ag combines with its Ab in the presence of electrolytes ( NaCl ) at a suitable temperature & pH, the Ag- Ab complex forms an insoluble precipitate. When instead of sedimenting , the precipitate remains suspended as floccules – Flocculation reaction Precipitation can take place in liquid media or in gels such as agar, agarose or polyacrylamide .

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ZONE PHENOMENON The amount of precipitate formed is greatly influenced by the relative proportions of Ags & Abs If increasing quantities of Ags are added to the same amount of antiserum in different tubes, precipitation is found to occur most rapidly & abundantly in the middle tubes Preceding tubes – Ab excess ( Prozone ) Middle tubes – Ag & Ab in equivalent proportions ( Zone of equivalence ) Later tubes – Ag excess ( Post zone )

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Mechanism of precipitation Marrack (1934) proposed the lattice hypothesis – mechanism of precipitation The multivalent antigens combine with bivalent Abs in varying proportions, depending on the Ag – Ab ratio on the reacting mixture Precipitation results when a large lattice is formed consisting of alternating Ag & Ab

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Marrack’s hypothesis

Applications of Precipitation reaction:

Applications of Precipitation reaction It can be carried out as either a quantitative or qualitative test Sensitive for the detection of Ags Identification of bacteria eg: Lancefield’s grouping of Streptococcus Detection of antibody for diagnostic purposes eg: VDRL in syphilis

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Types of precipitation reactions Ring test Flocculation test Immunodiffusion Electroimmunodiffusion

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RING TEST Consists of layering Ag solution over a column of antisera in a narrow tube Eg : Ascolis thermoprecipitin test, Grouping of Streptococci by Lancefield technique

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Flocculation test Slide test When a drop of Ag & antiserum is placed on a slide & mixed by shaking, floccules will appear Eg : VDRL test & RPR test for syphilis

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Tube test The Kahn test (tube flocculation) for syphilis This is also employed for the standardization of toxins & toxoids Serial dilutions of toxin/ toxoid are added to the tubes containing a fixed quantity of antitoxin The amount of toxin that flocculates optimally with one unit of the antitoxin – Lf dose

IMMUNODIFFUSION (precipitation in gel):

IMMUNODIFFUSION (precipitation in gel) Advantages of immunodiffusion: Reaction is visible as a distinct band of precipitation Stable, can be stained for preservation Indicates identity, cross reactions, non identity between different Ags

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Various immunodiffusion tests 1. Single diffusion in one dimension ( Oudin procedure) Ab is incorporated in agar gel in a test tube & Ag solution is layered over it Ag diffuses downward through the agar gel – forming a line of precipitation .

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2. Double diffusion in one dimension (Oakley Fulthorpe procedure ) Ab is incorporated in agar gel Above which is placed a column of plain agar The Ag is layered over it The Ag & Ab move towards each other through the intervening column of plain agar & form the precipitate

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3. Single diffusion in two dimensions (Radial immunodiffusion ) Here the antisera is incorporated in a gel & poured on a flat surface Wells are cut on the surface to which Ag is added It diffuses radially from the well & forms ring shaped bands of precipitation concentrically around the well

Radial Immunodiffusion (Mancini):

Radial Immunodiffusion (Mancini) Interpretation Diameter of ring is proportional to the concentration Quantitative Ig levels Method Ab in gel Ag in a well Ag Concentration Diameter 2 Ag Ag Ag Ag Ab in gel

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Uses 1. It has been widely employed for estimation of immunoglobulin classes i.e. IgG , IgM , IgA in sera 2. It has also been used for screening sera for antibodies to influenza viruses

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4 . Double diffusion in two dimensions ( Ouchterlony procedure ) Helps to compare different antisera & antigens directly Agar gel is poured on a slide & wells are cut Antiserum – central well Different Ags in the surrounding wells

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Reaction of identity Partial identity Lack of relatedness

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Elek’s gel precipitation

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5. Immunoelectrophoresis This involves the electrophoretic separation of composite Ag into its constituent proteins, followed by immunodiffusion against its antiserum – separate precipitin lines It is performed on an agarose gel with an Ag well & Ab trough cut on it The test serum is placed in the antigen well & electrophoresed for about 1 hour Ab against human serum is placed in the trough & diffusion allowed for 18 – 24 hrs

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Immunoelectrophoresis

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Uses 1. By this technique, a number of antigens can be identified in human serum 2. It is particularly useful for detection of normal and abnormal serum proteins like myeloma proteins

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ELECTROIMMUNODIFFUSION The development of precipitin lines can be speeded up by electrically driving the Ag & Ab Two types Counterimmunoelectrophoresis (One dimensional double electroimmunodiffusion ) Rocket electrophoresis (One dimensional single electroimmunodiffusion )

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Counterimmunoelectrophoresis (CIE) This involves simultaneous electrophoresis of Ag & Ab in gel in opposite directions resulting in precipitation at a point between them Used only when Ag and Ab have opposite charges Produce precipitation lines within 30 mins Clinical application: detecting Ags like alphafetoprotein in serum, Ags of Cryptococcus & Meningococcus in the CSF It is also applied for detecting hepatitis B antigens and antibodies

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Ag Ab - +

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2. Rocket electrophoresis Used for quantitative estimation of Ags The antiserum to the Ag to be quantitated is incorporated in agarose gel on a slide Ag in increasing concentrations, is placed in wells punched in the set gel The Ag is electrophoresed into the Ab containing agarose The pattern of immunoprecipitation resembles a ROCKET The length of these rocket like structures corresponds to the concentration of the antigen

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Rocket electrophoresis

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Laurell’s two dimensional electrophoresis Variant of rocket electrophoresis Used to quantitate each of the several Ags in a mixture In the first stage, the Ag mixture is electrophoretically separated In second stage, electrophoresis is done perpendicular to that of first stage to get rocket like precipitation

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Agglutination When particulate antigen combines with its antibody in the presence of electrolytes at an optimal temperature and pH, resulting in visible clumping of particles More sensitive than precipitation for the detection of antibodies The agglutination reaction takes place better with IgM antibody Lattice formation hypothesis holds good for aggltination too Blocking antibodies inhibit the agglutination by the complete antibody added subsequently

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Types of agglutination reactions Side agglutination test Tube agglutination test The antiglobulin (Coombs) test Heterophile agglutination test Passive agglutination test

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Slide agglutination test A uniform suspension of antigen is made in a drop of saline on a slide and a drop of the appropriate antiserum is added Reaction is facilitated by mixing the antigen and the antiserum with a wire loop or by gently rocking the slide Clumping occurs instantly or within seconds when agglutination test is positive A control consisting of antigen suspension in saline, without adding antiserum must be included on the same slide

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Uses 1. It is a routine procedure to identify the bacterial strains isolated from clinical specimens ( eg : Identification of Salmonella species) 2. It is also used for blood grouping and cross matching

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Tube agglutination test

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What is the titer of Ab ? The titer is customarily reported as the reciprocal of the highest dilution of Ab that causes an obvious agglutination

Tube Agglutination Test:

No agglutination Agglutination 1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl In this case, the titre is 40 Tube Agglutination Test

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Uses Used for serological diagnosis of Enteric fever ( Widal test) Typhus fever (Weil-Felix reaction) Infectious mononucleosis (Paul- Bunnel test) Brucellosis (SAT) Primary atypical pneumonia (Streptococcus MG agglutination test)

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Problems related to tube agglutination Prozone phenomenon Blocking antibodies Blocking or incomplete antibodies may be detected by performing the test in hypertonic (5%) saline or albumin saline Antiglobulin (Coombs) test is more reliable for detecting these antibodies

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The antiglobulin (Coombs test) Originally devised by Coombs, Mourant and Race (1945) for the d etection of incomplete anti- Rh antibodies There are two types of Coombs test Direct Coombs test Indirect Coombs test

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+ ↔ Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Patient’s RBCs Coombs Reagent (Antiglobulin) Direct Coombs test

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Y Y Y Y Y Patient’s Serum Target RBCs + ↔ Step 1 + ↔ Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Coombs Reagent (Antiglobulin) Step 2 Indirect Coombs test The only difference between the two is that the sensitisation of the erythrocytes with incomplete antibodies takes place in vivo in direct type whereas it occurs in vitro in indirect type

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Uses of Coombs test For detection of anti- Rh antibodies For demonstration of any type of incomplete antibody ( eg : Brucellosis)

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Heterophile agglutination test Heterophile antibodies have a property to react with microorganisms or cells of unrelated species due to common antigenic sharing i ) Weil-Felix reaction Some proteus (OX19, OX2, and OXK) strains are agglutinated by sera of patients with rickettsial infections This is due to antigenic sharing between these Proteus strains and Rickettsial species

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ii) Paul-Bunnel test Sheep erythrocytes are agglutinated by sera of infectious -mononucleosis’ iii) Streptococcus MG agglutination test It is positive in primary atypical pneumonia

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Passive agglutination test A precipitation reaction can be converted into agglutination test by attaching soluble antigens to the surface of carrier particles such as latex particles, bentonite and red blood cells Such tests are called passive agglutination tests When instead of antigen, the antibody is adsorbed on the carrier particles for estimation of antigens, it is known as reversed passive agglutination

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Latex agglutination test Polystyrene latex particles (0.8 – 1 µm in diameter) are widely employed to adsorb several types of antigens

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This test is convenient, rapid and specific Used for detection of hepatitis B antigen, ASO, CRP, RA factor, HCG and many other antigens Latex agglutination tile is used to perform this test

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Haemagglutination test Erythrocytes sensitised with antigen are used for detection of antibodies Rose-Waaler test for detection of RA factor in patient serum The antigen used for the test is sheep red blood cells sensitised with rabbit antisheep erythrocyte antibody (amboceptor)

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Coagglutination Some strains of Staphylococcus aureus (especially Cowan 1 strain) possess protein A on their surface When specific IgG molecule is coated on these strains, Fc portion of IgG molecule binds to protein A whereas antigen combining Fab terminal reamains free When the corresponding antigen is mixed with these coated cells, Fab terminal binds to antigen resulting in agglutination This test is used for detection of bacterial antigens in blood, urine and CSF (eg: Gonocooci, Streptococcus pyogenes and Haemophilus influenzae )

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Complement fixation test Complement is a protein (globulin) present in normal serum Whole complement system is made up of nine components: C1 to C9 Complement proteins are heat labile and are destroyed by heating at 56°C for 20 – 30 minutes

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Principle The antigen-antibody complexes have ability to fix complement This reaction has no visible effect To detect the fixation of complement, an indicator system consisting of sheep erythrocytes coated with amboceptor is used

Components of CFT:

Components of CFT Test System Antigen: It may be soluble or particulate. Antibody: Human serum (May or may not contain Antibody towards specific Antigen) Complement: It is pooled serum obtained from 4 to 5 guinea pigs. It should be fresh or specially preserved as the complement activity is heat labile (stored at -30 °C in small fractions). The complement activity should be initially standardized before using in the test Indicator System ( Haemolytic system) Erythrocytes: Sheep RBC Amboceptor ( Hemolysin ): Rabbit antibody to sheep red cells prepared by inoculating sheep erythrocytes into rabbit under standard immunization protocol.

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Controls Antigen and serum controls are included in the test Complement control is used to ensure that the desired amount has been added Cell control to make sure that sensitised erythrocytes do not undergo lysis in the absence of complement

Positive Test:

Positive Test Step 1: At 37°C Antigen + Antibody + Complement Complement gets fixed (from serum) 1 Hour Step 2: At 37°C Fixed Complement complex + Haemolytic system No Haemolysis 1 Hour ( Test Positive)

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Negative Test Step 1: At 37°C Antigen + Antibody absent + Complement Complement not fixed 1 Hour Step 2: At 37°C Free Complement + Haemolytic system Haemolysis 1 Hour ( Test Negative)

Results and Interpretations::

Results and Interpretations: No haemolysis is considered as a positive test Haemolysis of erythrocytes indicative of a negative test 1 2 3 4 A B Microtiter plate showing Haemolysis (Well A3, A4 and B4) and No Haemolysis (Well A1, B1, B2, and B3)

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Indirect complement fixation test Certain avian (eg: duck, parrot) and mammalian (eg: horse, cat) sera cannot fix guinea pig complement Test is done in duplicate and after the first step, the standard antiserum known to fix complement is added in one set

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Positive test First step: Antigen + test serum (positive for antibody) + guinea pig complement Second step: Standard antiserum cannot react with antigen because has been used up by antibody in the first step, hence, complement is free Indicator system: Haemolysis occurs because complement is free

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Negative test First step: Antigen + test serum (negative for antibody) + guinea pig complement Second step: Standard antiserum will react with antigen and fix the free complement Indicator system: No haemolysis because complement is not free

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Conglutination This is an alternative method for systems which do not fix guinea pig complement Uses horse complement which is non-hemolytic The indicator system is sheep erythrocytes sensitised with bovine serum Bovine serum contains a a beta globulin component named conglutinin, which acts as antibody to complement Conglutinin can cause agglutination of sensitised sheep erythrocytes, if these are combined with complement No agglutination – Positive result Agglutination – Negative result

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Complement dependent serological tests Immobilisation test Immune adherence Cytolytic or cytocidal tests Neutralisation test Opsonisation

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Immunofluorescence Fluorescence is the property of certain dyes which absorb rays of one particular wavelength (UV light) and emit rays with a different wavelength (visible light) Coons and his colleagues showed that fluorescent dyes can be conjugated to antibodies and these labelled antibodies can be used to detect antigens in tissues The commonly used fluorescent dyes are Fluorescin isothiocyanate (Blue green fluorescence) Lissamine rhodamine (orange red fluorescence)

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Immunofluorescence test is of two types Drect immunofluorescence test Indirect immunofluorescence test

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Direct immunofluorescence test

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Uses 1. It is commonly employed for detection of bacteria, viruses or other antigens in blood, CSF, urine, faeces , tissues and other specimens 2. It is a sensitive method to diagnose rabies by detection of the rabies virus antigens in brain smears Disadvantage Separate specific fluorescent labelled antibody has to be prepared against each antigen to be tested

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Indirect immunofluorescence test

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Advantages A single antihuman globulin fluorescent conjugate can be employed for detection of antibody to any antigen All antibodies are globulin in nature, therefore, antihuman globulin attaches to all antibodies This has overcome the disadvantage of direct immuno- -fluorescence test

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Radioimmunoassay (RIA) Berson and Yallow (1959) first described Since then it has been utilised for quantitation of hormones, drugs, HBsAg, IgE and viral antigens Radioimmunoassay is widely-used because of its great sensitivity Using antibodies of high affinity, it is possible to detect a few picograms (10 −12 g) of antigen The greater the specificity of the antiserum, the greater the specificity of the assay

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FIGURE 6-9 A solid-phase radioimmunoassay (RIA) to detect hepatitis B virus in blood samples & A standard curve to determine the conc. of HBsAg in unknown serum. The principle involves competitive binding of radiolabeled Ag and unlabeled Ag to the limited supply of a high affinity Ab

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Disadvantages Radiation hazards: Uses radiolabelled reagents Requires specially trained persons Labs require special license to handle radioactive material Requires special arrangements for Requisition, storage of radioactive material radioactive waste disposal

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Enzyme Linked Immunosorbent Assay (ELISA) Is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample Term was coined by Engvall and Pearlmann in 1971 Similar to RIA, except no radiolabel Very sensitive, pg/mL

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Different types of ELISAs Indirect ELISA Sandwich ELISA Competitive ELISA Cassette or cylinder ELISA

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Indirect ELISA Two commonly used enzymes Horseradish peroxidase (HRP) Alkaline phosphatase (AP)

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Sandwich ELISA Two antibodies required; must recognize different epitopes 1 st Antibody is referred to as capture Ab 2 nd antibody as detection Ab

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Competitive ELISA

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The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabelled) The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal).

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Cassette or cylinder ELISA It is a simple modification of ELISA for testing one or few samples of sera at a time The test is rapid (about ten minutes) as compared with the 2 - 4 hours taken for conventional ELISA

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Uses of ELISA Used for detection of antigens and antibodies for various microorganisms Detection of HIV antibodies in serum Detection of mycobacterial antibodies in tuberculosis Detection of rotavirus in faeces Detection of hepatitis B markers in serum Detection of enterotoxin of E. coli in faeces

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Western blotting (Immunoblotting)

Western Blot for detection of HIV antibody:

Western Blot for detection of HIV antibody HIV-1 Western Blot Lane1: Positive Control Lane 2: Negative Control Sample A: Negative Sample B: Indeterminate Sample C: Positive

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