ANTIGEN- ANTIBODY REACTIONS

Jyothi.Ch Lecturer Dept. of Microbiology INTRODUCTION

 Serology: use of serum Abs to detect & measure Ags & vice versa  Immunoassay: assays, or tests using immunological reagents (Ags & Abs)  Monoclonal Abs are often used in immunoassays Factor Comments

Sensitization Stage Attachment of antibody to antigen

Clinically significant antibodies react best at 1. Temperature 37ºC

2. pH Most antibodies react at pH 5.5- 8.5

3. Ionic strength Reducing the ionic strength of the Medium facilitates interaction of Ab w/ Ag (LISS)

4. Antigen: antibody ratio Too much antibody can cause prozone (false-negative). Optimum serum to cell ratio 80:1. Usually 2 drops serum + 1 drop Ag. QUANTIFYING ANTIGEN-ANTIBODY REACTIONS

 Detectable specific Abs in serum until 7-10 days after infection  Seroconversion: change from negative serum w/out specific Abs to serum positive for specific Abs.  Progression of infection, amount of Abs in the blood called ‘titer’ increases  Rise in titer of Abs is characteristic of an active infection QUANTIFYING ANTIGEN-ANTIBODY REACTIONS

 Blood serum, plasma, urine, CSF, sputum & other body constituents may contain Ags or Abs  Amount of Abs or titer of a serum sample is usually determined in serial dilutions QUANTIFYING ANTIGEN-ANTIBODY REACTIONS

 Reciprocal of the last dilution showing a detectable Ag-Ab rxn is taken as the titer  Thus if positive reaction is observed in the dilution 1:256 but not in 1:512, the titer is 256.  Both positive & negative controls should be included DILUTION  Estimating the antibody by determining the greatest degree to which the serum may be diluted without losing the power to given an observable effect in a mixture with specific antigen

25-08-2018 7 TITER

 Different dilutions of serum are tested in mixture with a constant amount of antigen and greatest reacting dilution is taken as the measure or Titer

8 EXPRESSION OF TITERS  Dilution 1 in 8 is a dilution made by mixing one volume of serum with seven volumes of diluents (Normal Saline )  Incorrect to express dilution as 1/8

9 COMMON METHODS IN CREATING DILUTIONS

10 APPLICATIONS OF SEROLOGICAL TESTS

 Serologic tests are cheaper & easier  Serological tests are more widely used in clinical settings STRENGTH OF ANTIGEN-ANTIBODY INTERACTIONS

 affinity  Avidity

 Ag+ab K1 Ag-Ab K2

 Ka= affinity constant= (Ag-Ab) (Ag) (Ab) AFFINITY  Affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site.  Affinity is the equilibrium constant that describes the antigen-antibody reaction

25-08-2018 13 AFFINITY

 Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody.  It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site .

14 AVIDITY

 Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies  Avidity is influenced by both the valence of the antibody and the valence of the antigen.  Avidity is more than the sum of the individual affinities.

15 THE FORCES BINDING ANTIGEN TO ANTIBODY 25 - 08 -

 Electrostatic : between attraction 2018 - oppositely charged ionic group – (-NH3 ) of lysine and (-COO-) of aspartate.  Hydrogen bonding – relatively weak and reversible hydrogen bridges between hydrophilic group (-OH, -NH2, COOH).  Hydrophobic– non-polar, hydrophobic side chains of Val, Leu, Ile (hydrophobic groups come close together and exclude water molecules between them. The force of attraction increases.  Van der Waals – forces which depend upon interaction between the external „electron clouds“. Non-specific attractive forces. 16 THE RATIO OF ANTIGEN / ANTIBODY 25 - 08 - 2018

Prozone : Ab excess, Zone of equivalence- Post-zone – excess of precipitate does not optimal ratio of Ag/Ab – Ag (soluble immune 17 form ( (soluble immune insoluble precipitate complexes) complexes) Lattice Hypothesis or Prozone Phenomenon:: Constant antibody concentration: increasing addition of antigen

The goal of humoral responses is to achieve Ab excess to remove the pathogen PROZONE

 Absence of agglutination at higher antibody concentration.  It is due to many factors including  Presence of blocking antibodies at low titers  Inaccessible antigenic determinants  Weak avidity  Poor lattice formation.  The problem can be avoided by use of standard serial dilution. TYPES OF ANTIGEN- ANTIBODY REACTIONS ANTIGEN-ANTIBODY BASED IMMUNOLOGICAL TESTS

 I. Agglutination reactions:  Abs + particulate Ags = agglutination  Larger aggregates easier to visualize  Direct agglutination tests  Indirect agglutination tests Types of agglutination reactions:  1.Slide agglutination  2. Tube agglutination  3.Coomb’s test or Antiglobulin test  4. Heterophile agglutination test  5. Passive agglutination test: a.Hemagglutination b. Latex agglutination c. Coagglutination AGGLUTINATION TEST

 It is one of important laboratory method to detect antigen antibody reaction.  It provides flexible and useful method for semi quantitating of either antigen or antibody concentration.  The reaction occurs between insoluble (particulate)antigen and appropriate antibody.  The reaction will results in forming aggregate or agglutinate.  Ag= agglutinogen, Ab= agglutinin STAGES OF AGGLUTINATION REACTION

Phase one  Antibody reacts with single antigenic determinants on or close to particle surface.  It is a rapid reaction. SECONDARY PHASE

 A single antibody molecule binds to antigenic determinants on adjacent particles.  The visible reaction occur under appropriate conditions and over time, particles remain connected and interconnected by antibody bridge. TYPES OF AGGLUTINATION REACTION DIRECT AGGLUTINATION.  To test patient’s sera (contain antibody) against large antigen.  Direct agglutination can be used to determine antibody titer. INDIRECT AGGLUTINATION- CONVERTING A PRECIPITATION REACTION TO AN AGGLUTINATION REACTION

 serum is mixed with latex spheres (inert substance) with the soluble antigens attached.  Antibodies will then cause visible agglutination of the latex spheres with the soluble antigens attached. ADVANTAGES OF AGGLUTINATION METHODS

 ease of performance.  speed of performance, usually requiring few minutes.  high degree of sensitivity. DISADVANTAGES OF AGGLUTINATION METHODS

 the reaction are only semiquantitative.  the occurrence of the prozone phenomenon, in which agglutination is inhibited by extreme antibody excess as a result of poor lattic formation. APPLICATION OF AGGLUTINATION TEST

 several antibodies can be detected by this method such as Rheumatoid factor. 1.SLIDE AGGLUTINATION TEST:

 Ag suspension in saline added onto a slide or tile, now a drop of appropriate antisera (ab) is added, mixed  Instant clumping occurs  Clumping after a minute is cosidered false  Control needs to be processed as well- No antisera only ag + saline, to check if the ag is autoagglutinable Used for detecting Typhoid 2.TUBE AGGLUTINATION TESTS:

 A quantitative method  Serum diluted serially and equal amount of particulate ag added to all the tubes.  Antibody titre noted  Used for detecting Typhoid via WIDAL test, Typhus fever, Brucellosis via Weil Felix reaction 3.COOMB’S OR ANTI GLOBULIN TEST:

 To detect incomplete anti Rh abs generated in the Rh – ve mother after the first delivery.  Such a serum is mixed with Rh positive RBCs,the incomplete globulin coats the RBCs but no agglutination results (in vitro)  To the above antiglobulin or Coomb’s serum( rabbit antiserum generated against human gamma globulin-) is added, now cells agglutinate.  Direct Coomb’s, Indirect Coomb’s test  Sensitization of RBCs with the incomplete abs occurs in vivo in direct and in vitro in indirect  Used for detecting any incomplete abs COOMB’S SERUM

 The main reagent used in the antiglobulin test is anti-human globulin (AHG).  Also called Coombs serum.  Anti-human globulin (AHG) is an IgG antibody directed against human immunoglobulins. AHG ACTION

 AHG combines with the Fc portion of a Y sensitizing antibody.  This completes the antigen-antibody bridge, allowing agglutination to occur. 4. HETEROPHILE AGGLUTINATION TEST: a. Weil Felix reaction: Proteus strains agglutinate with the sera from Rickettsial patients b. Paul Bunnel Test: Sheep RBCs are agglutinated by sera of infectious mononucleosis 5. PASSIVE AGGLUTINATION:

 Conversion of precipitation reaction to an agglutination raection by attaching carrier molecules  As agglutination is more sensitive  If ab is adsorbed onto the carrier- reverse passive agglutination A.LATEX AGGLUTINATION:

 Latex particles + abs, such ab coated latex particles now when added with the ag source, agglutination occurs  Used for detecting presence of Hepatitis B ag, N.meningitidis, etc RHEUMATOID FACTOR (RF)

 This test is done to diagnosed Rheumatoid arthritis, which is one of important autoimmune disease.

 RF is an antibody ( IgM or IgG classes) bind to the Fc portion of other IgG molecules, and form IgG-anti-IgG complexes in the circulation or joint fluid. RHEUMATOID FACTOR (RF)

 RFs are detected in serum in up to 80% of adult patients with RA.  RFs are not specific for RA and occur in other autoimmune disease, in chronic infectious diseases, such as infective endocarditis, tuberculosis, and hepatitis B.  usually at low titer, in up to 20% of overtly normal elderly individuals B.HAEMAGGLUTINATION:

 RBCs sensitized with the ag used for detecting the abs  Rheumatoid arthritis- autoab called RA factor appears in the serum.  RF acts as an ab to gamma globulin.  RF can hence agglutinate RBCs coated with gammaglobulins.  Ag used is the sheep RBCs sensitised with rabbit antisheep abs( amboceptor- a gamma globulin)  Rose Waleer test for detecting arthritis C. COAGGLUTINATION:

Cowan 1 strain of S.aureus (protein A) + (Protein A anti) Abs= ab coated S.aureus Fc portion of the so added ab binds to the protein a ag, and Fab region remains free Now when ag is added to the above, the free Fab region binds to the ag and as a result, agglutination occurs For detecting, N gonorrhoea, Streptococcus pyrogenes, H. influenza ANTIGEN-ANTIBODY BASED IMMUNOLOGICAL TESTS

 II. Precipitation reactions:  Abs + soluble Ags = precipitate  Ring test  Immunodiffusion tests  Flocculation tests  Immunoelectrophoresis RECIPITATION P 25 - 08 - 2018 Principle  Soluble antigen + antibody (in proper proportions) – > visible precipitate  Lattice formation (antigen binds with Fab sites of 2 antibodies)  Examples  Double diffusion (Ouchterlony)  Single diffusion (radial Immunodiffusion)  Immunoelectrophoresis  Immunofixation 43 PRECIPITATION TESTS

 When sufficient antigen and antibody molecules interact, they precipitate out of solution  Too few antigen molecules, little ppt.  Too many, agn-aby cross links not made.  Examples  immunodiffusion: antibody and antigen react in agar to make ppt band or ring.  Immunoelectrophoresis: complex mixture of antigens separated, then reacted with antibody.

44 PRECIPITATION IN GELS 25 - 08 - Based on different rates of diffusion of Ag 2018 and Ab into the gel, depending on their :  concentration  physicochemical properties  gel structure Most widely used gels – agar or agarose Tests are performed by pouring molten agar (agarose) onto glass slides

45 PRECIPITATION SEROLOGICAL TESTS

 One of the easiest of serological tests  Soluble AG & Ab interact and form a lattice that develops into a visible precipitate.  Occur best when antigen and antibody are present in optimal proportions (Equivelance).  Antibodies that aggregate soluble antigens are called precipitins. AG / AB PRECIPITATION REACTIONS Polyclonal antibodies can form lattices, or large aggregates.

However, monoclonal antibody can link only two molecules of antigen and no precipitate is formed.

( no precipitate is formed ( Lattices or if an Ag contains only a large aggregates ) single copy of each epitope ) RECIPITATION URVE

P C 25 - 08 - 2018

48 Precipitation Curve

Ab excess Equivalence – Ag excess Lattice formation PRECIPITATION CURVE Plots of the amount Ag/Ab complexes precipitated when increasing Ag concentrations are added to constant concentration of Ab. It reveals 3 zones:

 1. Zone of antibody excess - precipitation is inhibited and antibody not bound to antigen can be detected in the supernatant;

 2. Zone equivalence - maximal precipitation in which antibody and antigen form large insoluble complexes and neither antibody nor antigen can be detected in the supernatant;

 3. Zone of antigen excess - precipitation is inhibited & Ag. not bound to Ab. can be detected in the supernatant AG / AB PRECIPITATION REACTIONS  All antigen /antibody bindings are reversible according to Law of Mass Action  Free reactants are in equilibrium with bound reactants Equivalent zone  Excess antibody Prozone  Excess antigen Postzone  These zones are demonstrated in the precipitation curve 1.RING TEST: USED FOR TESTING C REACTIVE PROTEIN, FOR LANCEFIELD GROUPING OF STREPTOCOCCI

Ring Precipitate (Ring Test) (Tube Precipitation test) • Involve soluble antigens with antibodies in tubes (test or Capillary tubes). • Layer Ag over Ab • Precipitate occurs at the interface of the two reagents, forming a ring. • Simplest test • Qualitative • Can be bone in Agarose. Test Tube reaction

Capillary Tube reaction 2.FLOCCULATION TESTS:

 Slide test-VDRL for Syphilis,  Tube test- Khan test for Syphilis VDRL-SCREENING TESTS FOR SYPHILIS

 Serologic methods are divided into two classes. One class, the nontreponemal tests, detects antibodies to lipoidal antigens present in either the host or T. palladium; examples are the Venereal Disease Research Laboratory and rapid plasma reagin and tests.

55 NON REACTIVE AND REACTIVE VDRL TESTS

56 FLOCCULATION TEST (A PRECIPITATION REACTION)

(1) Non Reactive (2) Weakly Reactive (3,4) Reactive 3.IMMUNODIFFUSION: TYPES OF IMMUNODIFFUSION TESTS:

Ag-Ab interactions can form visible precipitate A.Gel diffusion tests B.Immunoelectrophoresis A.GEL DIFFUSION TECHNIQUES:

 i. Single diffusion in one dimension- Oudin procedure  ii. Double diffusion in one dimension- Oakley- Fulthorpe procedure  iii. Single diffusion in two dimensions-Radial immunodiffusion  iv. Double diffusion in two dimensions- Ouchterlony procedure B. IMMUNOELECTROPHORESIS:

 i. Counter immunoelectrophoresis/Counter current immunoelectrophoresis  ii. Rocket electrophoresis  iii. Laurell’s two dimensional electrophoresis A.GEL DIFFUSION TECHNIQUES I.SINGLE OR SIMPLE DIFFUSION IN ONE DIMENSION- OUDIN PROCEDURE

the process of diffusion of an antigen in an antibody-containing gel - the process of diffusion of an ag in an ab-containing gel. Immunoprecipitin line is formed at the point of equivalence. II.DOUBLE DIFFUSION IN ONE DIMENSION- OAKLEY-FULTHORPE PROCEDURE

The process of diffusion of an antigen from an ag conatining gel layer to a plain gel layered above the ab containing gel and vice versa

Immunoprecipitin line is formed at the point of equivalence. III.SINGLE DIFFUSION IN TWO DIMENSIONS- RADIAL IMMUNODIFFUSION

• Immunodiffusion procedures are carried out in an agar gel medium.

• The precipitate is easily seen in gels visible precipitin lines

• But no visible precipitate forms in regions of Ab or Ag excess  Ab is put into a gel and Ag is put in a well cut into the gel and a precipitin ring formed when Ag diffuses out in all directions. • The technique is quantitative • is based upon the reaction between an Ag, and a specific Ab during a diffusion period. • Ag placed in a well diffuses into an agar containing the Ag (anti-IgG looking for serum IgG). • The Ag-Ab interaction is manifested by a well-defined ring of precipitation around the Ag well. Interpretation : Diameter of ring is proportional to the Antigen concentration.  In radial Immunodiffusion Antibody is incorporated into the agar gel as it is poured  different dilutions of the antigen are placed in holes punched into the agar.  As the antige diffuses into the gel it reacts with the antibody and when the equivalence point is reached a ring of precipitation is formed  The diameter of the ring is proportional to the concentration of antigen since the amount of antibody is constant. In this example, Anti-dog IgG is Mixed in agar so only what is Placed in wells (Ag) diffuses out 25-08-2018 Dr.T.V.Rao MD 68  By running different concentrations of a standard antibody one can generate a standard cure from which one can quantitate the amount of an antibody in an unknown sample.  Thus, this is a quantitative test.  If more than one ring appears in the test, more than one antigen/antibody reaction has occurred. This could be due to a mixture of antigens or antibodies.  This test is commonly used in the clinical laboratory for the determination of immunoglobulin levels in patient samples. IV.DOUBLE DIFFUSION IN TWO DIMENSIONS- OUCHTERLONY PROCEDURE

 Double diffusion is utilized as a rough estimation of antigen or antibody purity.  Double diffusion in agar can be used for semi quantitative analysis in human serological system.  Both Ab and Ag diffuse from wells into a gel medium

Double diffusion in two dimension

Similar Precipitin lines Precipitin lines precipitin lines do not form a completely cross complete cross 72 Identification

73 • Both antigen and antibody can diffuse independently • It is based upon the simultaneous application of Ag and Ab in separate but adjacent wells of an agar plate. • As the materials diffuse toward one another, ppt. lines form resulting from the Ag-Ab interactions (i.e. it is Qualitative). • If multiple wells of Ag are positioned around an Ab well on the same plate, several patterns of reactivity may be observed. OUCHTERLONY DOUBLE DIFFUSION (IMMUNODIFFUSION) QUALITATIVE TEST

 It is based upon the simultaneous application of Ag and Ab in separate but adjacent wells of an agar plate.  As the materials diffuse toward one another, ppt. lines form resulting from the Ag-Ab interactions.  If multiple wells of Ag are positioned around an Ab well on the same plate, several patterns of reactivity may be observed. OUCHTERLONY DOUBLE DIFFUSION (IMMUNODIFFUSION)

 If the Ag A (patient) is the same as the Ag A (control), the reaction with the Ab will be the same and the result is a solid, continuous, smooth line of identity between the Ag wells and the Ab well. OUCHTERLONY DOUBLE DIFFUSION (IMMUNODIFFUSION)

 If Ag A (patient) is different from Ag B (control), and both react with the Abs to A & B, the precipitin lines cross and a double spur is formed; this is a line of nonidentity. OUCHTERLONY DOUBLE DIFFUSION (IMMUNODIFFUSION)

 If Ag A (patient) and Ag A1 (control) share a common element but are not exactly the same (Abs to A), a single spur is formed. This is the line of partial identity. Simple Immunodiffusion Reactions

The region of equivalence

Diagrammatic representation of radial & double immunodiffusion. (Precipitation reactions in gels yield visible precipitin lines; no visible precipitate forms in regions of Ab or Ag excess.) B.IMMUNOELECTROPHORESIS Immunoelectrophoresis – Antigen is 1st put into wells, charge is applied to separate components of antigen mixture, then troughs are cut and antibody is allowed to diffuse through gel PRECIPITATION AND IMMUNODIFFUSION 25 -

IN GELS 08 - 2018

Plasma Immunoelectropho (mixture of antigens) resis combines Electrophoresis electrophoresis

Antiserum (mixture of separation, antibodies) diffusion and Imunodiffusion precipitation of proteins. 82  In electro-immunodiffusion, diffusion is combined with electrophoresis  Electrophoresis separates antigen molecules according to differences in their electrical charges and molecular weight then specific antibodies diffuse and react with separated antigen forming precipitin bands. A) IMMUNOELECTROPHORESIS  Method  Ags are separated by electrophoresis – Ab is placed in trough cut in the agar + - Ag Ag

Ab

Ag

Ab

• Interpretation- Precipitin arc represent individual antigens • Qualitative - Relative concentration

IMMUNOELECTROPHORESIS

86 87 I. COUNTER IMMUNOELECTROPHORESIS/COUNTER CURRENT IMMUNOELECTROPHORESIS • Reactions occurr between migrating Ag’s and Ab’s during electrophoresis (Ag and Ab migrate toward each other by electrophoresis) • Used only when Ag and Ab have opposite charges  Pairs of wells are punched in agarose plates in which Ag is placed in one well of each pair and Ab in the other.  Following electrophoresis, precipitin lines will be visible between the wells of a pair of wells of matching specificity. • Qualitative – Its major advantage is its speed. - + Ag Ab II. ROCKET IMMUNOELECTROPHORESIS  Antigen is electrophoresed into gel containing antibody.  The precipitin reaction results in a rocket-shaped precipitin formation.  The distance from the starting well to the front of the rocket shaped arc is related to antigen concentration. III.LAUREL’S TWO DIMENSIONAL ELECTROPHORESIS

 A variant of rocket immunoelectrophoresis  Several ags in a mixture can be quantitated  The ab is immobilised in the gel  First stage- ag mixture is electrophoretically separated  Second stage- a perpendicular electrophoresis to the first stage is done, resulting in a rocket MEASUREMENT OF PRECIPITATION BY LIGHT

 Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance.  Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution.  Nephelometry indirect measurement, measures amount of light scattered by the antigen-antibody complexes.

91 TURBIDIMETRY Nephelometry

 precipitation in solution • precipitation in solution • measurement of scattered  measurement of light extraction light (proportional to number (precipitate absorption) of insoluble complexes

 standard curve • standard curve III.NEUTRALIZATION REACTION:

 Neutralization tests (virus neutralization test)  Serum + known viral suspension  If Abs to the virus are present, Abs bind to the virus, preventing its attachment to & subsequent infection of cells  When virus is then added to an appropriate cell culture, it is unable to replicate & cause cell death  Also to used to test for toxins  Neutralizing abs (antitoxins) are produced against bacterial exotoxins (Diptheria, Tetanus)  No such abs are can neutralise endotoxins 1. In vivo tests, 2. In vitro tests 1.IN VIVO TESTS:

 A.Toxigenicity test:  Done for detecting the Diptheria toxin  Intradermal test  1.Animal previously immunised with antitoxin of C.diptheria is intradermally injected with the active toxin- Control  2. Animal not previously immunised with antitoxin of C.diptheria is intradermally injected with the active toxin- tets  2 dies no biological effects are seen in the control.  B.Shick test:  Diptheria toxin – injected intradermally into humans, reaction at the site of injection occurs if the person is immune, as the neutralising circulating abs are present in the sera 2. IN VITRO TETS:

 A. Anti Streptolysin O (ASO) test:  Patient’s sera is added to Streptococcal O haemolysin (toxin)  If the patient is positive for the Streptococcal pyogenes infection, neutralization of the toxin with ASO (antitoxin) happens and vice versa  B. Virus neutralization tests:  Done in cell cultures, eggs, animals  Neutralisation of bacteriophages can be demonstrated by plaque inhibition test.  C.Nagler’s reaction:  C.welchii toxin is neutralised by antitoxin, when the bacteria are grown in egg yolk antitoxin containing medium  A rapid detection test IV. OPSONISATION:

 Opsonins facilitate phagocytosis  A heat labile serum substance present/ ab like substance- opsonin  Particulate ag + opsonin= susceptible to opsonization  Opsonic index= ratio of phagocytic activity of the patient’s blood for a given bacterium to that of a normal individual  Phagocytic index= avg number of ohagocytosed bacteria per PMN leukocyte  Phagocytic index denotes the phagocytic activity of the blood an dthus helps in measuring opsonic index PRIMARY ANTIGEN- ANTIBODY REACTIONS TYPES OF PRIMARY AG-AB REACTIONS

 I. ELISA  II.RIA  III.Immunofluorescences I. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

 Principle:  Uses an immune reaction  Detection based on  Enzyme catalysed reaction OR  Fluorescent probe  NOT radioactivity [great advantage!]

ELISA: ADVANTAGES

 Specific & Sensitive- Wide Application  Equipment cheap & available  Reagents “Cheap”, long shelf life  Assays may be rapid  Simultaneous assays; variety of labels  Potential for automation  No radiation hazards ADVANTAGES OF ELISA  Sensitive: nanogram levels or lower  Reproducible  Minimal reagents  Qualitative & Quantitative  Qualitative  Eg HIV testing  quantitative assays  Eg Ther. Drug Monitoring  Greater scope : Wells can be coated with Antigens OR Antibodies  Suitable for automation high speed  NO radiation hazards ELISA:DISADVANTAGES

 Number of separation methods limited  Expertise required to label and purify conjugates  Susceptible to interference from non specific factors ELISA: ENZYME CHOICES

 Horse Radish Peroxidase  Alkaline Phosphatase  Glucose Oxidase  Urease  Indirect ELISA  Direct or sandwich ELISA  Competitive ELISA TYPES OF ELISA 1. Noncompetitive binding assay A. Sandwich or Cirect ELISA: Antigen measuring system [Titrewells coated with antibodies ; Enzyme labelled antibodies] B. Indirect ELISA: Antibody measuring system [Titrewells coated with antigens ; Enzyme labelled antiantibodies] 2. Competitive binding assay [Titrewells coated with antibodies ; Enzyme labelled antigens] 1.INDIRECT ELISA:  Antibody measuring system  Titre wells coated with suitable antigen  Add patient sample (sera) containing the antibody  Incubate: till antigen antibody reaction is complete  Wash remove unbound antibody  Add Antiantibody labelled with Enzyme  Incubate till labelled antiantibodies binds antigen- antibody complex  Wash  remove unbound labelled antiantibody  Add substrate ; incubate  Enzyme + Substrate  Product  measure colour  Colour proportional to antibody in patient sample 1. INDIRECT ELISA

Used for detecting the abs in the sera 2. SANDWICH OR DIRECT ELISA:  Antigen measuring system  Titre wells coated with suitable antibody  Add patient sample (sera) containing the antigen  Incubate: till antigen antibody reaction is complete  Wash remove unbound antigen  Add Antibody labelled with Enzyme  Incubate till antigen binds labelled antibody  Wash  remove unbound labelled antibody  Add substrate ; incubate  Enzyme + Substrate  Product  measure colour  Colour proportional to antigen in patient sample SANDWICH OR DIRECT ELISA

Used for detecting the ags in the sera COMPETITIVE ELISA COMPETITIVE BINDING ASSAY

 Incubate the ag(sera)-ab complex  Titre wells coated with antigens  Add the above ag-ab complex to the wells  Incubate: till antigen antibody reaction is complete  Wash remove unbound ag-ab complex  Add antiab-labelled with enzyme  Wash to remove the unbound antiab-enzyme  Add substrate ; incubate  Enzyme + Substrate  Product  measure colour  Colour is observed only if the in the first step there is no ag in the patient’s sera , as then the primary ab(present in the complex) would be free and would bind to the ag in the well  The more the ag present in the sample, the less free ab will be available to bind to bind to the ag coated well.  The higher the concentration of the ag in the original sample, the lower the absorbance ENZYME LABELS

 Enzyme labels should have high specific reactivity  Should be easily coupled to ligands & the labelled complex must be stable  The reactivity should be retained after linking of the enzyme to the antigen/antibody  The chosen enzymes should not be normally present in the patient samples  Examples of enzyme labels  Horse radish peroxidase, Alkaline phosphatase, Glucose oxidase APPLICATIONS OF IMMUNOASSAYS [RIA & ELISA]

 Analysis of hormones, vitamins, metabolites, diagnostic markers  Eg. ACTH, FSH, T3, T4, Glucagon, Insulin, Testosterone, vitamin B12, prostaglandins, glucocorticoids,  Therapeutic drug monitoring:  Barbiturates, morphine, digoxin,  Diagnostic procedures for detecting infection  HIV, Hepatitis A, B etc II.RIA-RADIO IMMUNOASSAY

Radioimmunoassay (RIA) is a scientific method used to test antigens (for example, hormone levels in the blood) without the need to use a bioassays.

Radioimmunoassay (RIA) is a Radio-analytical technique with remarkable sensitivity and a high degree of specificity that is widely used for the estimation of a variety of molecules present in complex matrices. Also known as Radio tracer technique and best example of invitro diagnosis technique using radio isotopes.

This technique is used over a wide spectra of substances such as hormones, steroids, vitamins, drugs, tumor markers and viral antigens.

Radio Immuno Assay

Use of radio Antigen Detection of active antibody binding compound material theory This isotopic measuring method was developed in 1959 by two Americans,

biophysicist Rosalyn Yalow and physician Solomon A. Berson.

RIA combines the specificity of an antigen-antibody reaction with sensitivity of

radioactivity measurements.

This is a technique used for detection of micro quantities of protein, viral

antigens, antibodies, structural proteins, vitamins and drug and their

metabolites.

It can also be used for detection of pictogram quantities (10−12 g) of biological

constituents present in biological fluid.

RIA is used in place of bioassay in various branches of science like

Biochemistry, Microbiology, and Hematology and Clinical pharmacology. PRINCIPLE AND THEORY :

RIA works on basic principle of biochemistry that competitive binding between antigens for

same antibody binding site.

The competition of an analyte with its radioisotopically labeled counterpart for a limited

amount of antibody, the specific reagent, is the underlying principle of this technique.

Increasing the analyte concentration inhibits the binding of the labeled analyte to the antibody.

Ag + Ag* + Ab  AgAb + Ag*Ab + Ag

 Unbound Ag* and Ag washed out

 Radioactivity of bound residue measured

 Ligand conc. is inversely related to radioactivity

Ag : ligand to be measured ; Ag*: radio labelled ligand  Competing depression reaction Labeled antigen (*Ag) possesses the same properties of unlabeled antigen (Ag). It can also bind to the correlated specific antibody (Ab) with the formation of labeled antigen-antibody complex or called bound antigen (B), leave the unbound one as free labeled antigen (F). The more Ag is present, the less likely is the *Ag bound to the Ab, thus the amount of B formed is inversely proportional to the Ag originally present in serum, this is so called competing depression reaction. A

Antibody B Unlabeled antigen

Labeled antigen

C

Now how the competition occur with increase the concentration of unlabeled antigen in the system of RIA in three different cases A, B and C.

Here first antibodies bound with labeled antigen are put into the known concentration of analyte solution and it is observed how the labeled antigen free from antibody and unlabeled will bind in place of there. Ag+Ab Ag.Ab+Ag + *Ag

* Ag.Ab (B)+*Ag (F) Advantages

Highly specific: Immune reactions are specific,the greater the specificity of the antiserum, the greater the specificity of the assay. High sensitivity : Immune reactions are sensitive, Using antibodies of high affinity it is possible to detect a few picograms (10−12 g) of antigen in the tube. Accuracy and Precision Disadvantages

Radiation hazards: Uses radio labelled 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. REQUIREMENTS FOR RIA

1. Preparation & characterisation of the Antigen [Ligand to be analysed] 2. Radiolabelling of the Antigen 3. Preparation of the Specific Antibody 4. Development of Assay System DEVELOPMENT OF THE ASSAY SYSTEM

 A crucial step is separation of unbound antigens  This is achieved by binding the antibodies to the microtitre well surface [Solid phase RIA]  Antigens bound to the fixed antibodies remain stuck to the inner surface  Decanting & washing the well removes unbound antigens  Other techniques of separation: Centrifugation ASSAY PROCEDURE  Add known amounts of the test sample + labelled antigen into the microtitre wells  Incubate  allow the reaction to reach completion  Decant & wash contents of the well  removes all unbound antigens  Radioactivity remaining in the Microtitre wells measured by a Counter [GM counter , Scintillation counter etc]  Intensity of radioactivity is inversely correlated with the conc of original unlabelled antigens in the test sample  Sensitive to very low conc of antigens Methods in RIA :

1. Radio labelling of the Antigen or radio labelled production

2. Preparation & characterisation of the Antigen [Ligand to be

analysed]

3. Preparation of the Specific Antibody

4. Development of Assay System or separation techniques

5. Two types of RIA:

Solid phase RIA and Liquid phase RIA 1.SOLID PHASE RIA:

 Microtitre wells+ ab, now add serum (ag) and radiolabelled ag  Incubate  Remove supernatent and measure radioactivity  Greater the infection more will be the radioactivity in the supernatent and vice versa 2.LIQUID PHASE RIA:COMPETITIVE BINDING

 Insulin Abs+ serum(insulin ags) + radiolabelled insulin ags  Lesser the raioactivity , the person is not diabetic and vice versa FLOW CHART OF TECHNIQU E III.IMMUNOFLUORESCENCE:

Fluorescent dyes (fluorescein or rhodamine) attached to known specific Abs, used to detect presence of Abs in serum or Ag (microorganisms) in a sample  Direct fluorescent Ab test: used to detect Ag or microorganism  Indirect fluorescent Ab test: used to find a specific Ab in the serum  Quantitative Immunofluorescence-flow cytometry ANTIGEN LOCALIZATION IN SPLEEN 1.DIRECT IMMUNOFLUORESCENCE:

 Sample(ag)+ flourescein tagged ab  Look for fluorescence 2. INDIRECT IMMUNOFLUORESCENCE:

 Step 1:  Sample( ag)+ untagged ab  Step 2:  To the above add fluorescein tagged antiab  Look for fluorescence 3. QUANTITATIVE IMMUNOFLUORESCENCE- FLOW CYTOMETRY