antigen- antibody reactions -...

ANTIGEN- ANTIBODY REACTIONS

Jyothi.ChLecturer

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

1. Temperature

Attachment of antibody to antigen

Clinically significant antibodies react best at 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-ANTIBODYREACTIONS

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-ANTIBODYREACTIONS

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-ANTIBODYREACTIONS

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

DILUTIONEstimating 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

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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

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EXPRESSION OF TITERSDilution 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

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COMMON METHODS INCREATING DILUTIONS

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APPLICATIONS OF SEROLOGICAL TESTS

Serologic tests are cheaper & easier Serological tests are more widely used in clinical

settings

STRENGTH OF ANTIGEN-ANTIBODYINTERACTIONS

affinity Avidity

Ag+ab K1 Ag-AbK2

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

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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 .

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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.

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THE FORCES BINDING ANTIGEN TOANTIBODY

Electrostatic : between attraction 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.

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THE RATIO OF ANTIGEN / ANTIBODY

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form ( (soluble immune complexes)

Zone of equivalence-optimal ratio of Ag/Ab –

insoluble precipitate

Post-zone – excess of Ag (soluble immune

complexes)

Lattice Hypothesis or Prozone Phenomenon:: Constant antibody concentration: increasing addition of antigen

The goalof humoralresponses isto achieve Abexcess toremove thepathogen

PROZONE

Absence of agglutination at higher antibody concentration.

It is due to many factors includingPresence of blocking antibodies at low

titers Inaccessible antigenic determinantsWeak avidityPoor lattice formation.The problem can be avoided by use of

standard serial dilution.

TYPES OF ANTIGEN- ANTIBODYREACTIONS

ANTIGEN-ANTIBODY BASEDIMMUNOLOGICAL TESTS I. Agglutination reactions: Abs + particulate Ags = agglutination Larger aggregates easier to visualize

Direct agglutination tests Indirect agglutination testsTypes 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.Hemagglutinationb. Latex agglutinationc. 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 AGGLUTINATIONREACTIONDIRECT AGGLUTINATION. To test patient’s sera

(contain antibody) against large antigen.

Direct agglutination can be used to determine antibody titer.

INDIRECT AGGLUTINATION- CONVERTING APRECIPITATION REACTION TO ANAGGLUTINATION 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 AGGLUTINATIONMETHODS

ease of performance. speed of performance, usually requiring few

minutes. high degree of sensitivity.

DISADVANTAGES OFAGGLUTINATION 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

autoagglutinableUsed 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 sensitizing antibody.

This completes the antigen-antibody bridge, allowing agglutination to occur.

Y

4. HETEROPHILE AGGLUTINATION TEST:a. Weil Felix reaction:Proteus strains agglutinate with the sera from

Rickettsial patientsb. 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 ( IgMor IgG classes) bind to the Fc portion of other IgG molecules, and form IgG-anti-IgGcomplexes 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.aureusFc portion of the so added ab binds to the protein a

ag, and Fab region remains freeNow 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 BASEDIMMUNOLOGICAL TESTS

II. Precipitation reactions: Abs + soluble Ags = precipitate

Ring test Immunodiffusion tests Flocculation tests Immunoelectrophoresis

PRECIPITATIONPrinciple

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

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PRECIPITATIONTESTS 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.

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PRECIPITATION INGELSBased on different rates of diffusion of Ag

and Ab into the gel, depending on their : concentration physicochemical properties gel structureMost widely used gels – agar or agaroseTests are performed by pouring molten agar

(agarose) onto glass slides

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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 REACTIONSPolyclonal antibodies can form lattices, or large aggregates.

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

( Lattices or large aggregates )

( no precipitate is formed if an Ag contains only a single copy of each epitope )

PRECIPITATION CURVE

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Precipitation Curve

Ab excess Ag excessEquivalence –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

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.

1.RING TEST: USED FOR TESTING C REACTIVEPROTEIN, FOR LANCEFIELD GROUPING OFSTREPTOCOCCI

Test Tube reaction

Capillary Tube reaction

2.FLOCCULATION TESTS: Slide test-VDRL for Syphilis, Tube test- Khan test for Syphilis

VDRL-SCREENING TESTSFOR SYPHILIS

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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.

NON REACTIVE AND REACTIVEVDRL TESTS

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FLOCCULATION TEST(A PRECIPITATION REACTION)

(1) Non Reactive (2) Weakly Reactive (3,4) Reactive

TYPES OF IMMUNODIFFUSION TESTS:

Ag-Ab interactions can form visible precipitate

A.Gel diffusion testsB.Immunoelectrophoresis

3.IMMUNODIFFUSION:

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 ONEDIMENSION- 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 agconatining 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 Abor 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-definedring 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

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Dr.T.V.Rao M

D

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 TWODIMENSIONS- 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

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Double diffusion in two dimension

Similar precipitin lines

Precipitin lines completely cross

Precipitin lines do not form a complete cross

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Identification

• 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-Abinteractions.

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.

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.)

The region of equivalence

Simple Immunodiffusion Reactions

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

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Immunoelectrophoresis combineselectrophoresis

separation,diffusion and

precipitation of proteins.

Plasma (mixture of antigens)

Electrophoresis

Antiserum (mixture of antibodies)

Imunodiffusion

In electro-immunodiffusion, diffusion is combined with electrophoresis Electrophoresis separates antigen molecules according to differences in

their electrical charges and molecular weight then specific antibodiesdiffuse and react with separated antigen forming precipitin bands.

A) IMMUNOELECTROPHORESIS Method

Ags are separated by electrophoresis

Ag-+

Ag

Ab

Ag

Ab

– Ab is placed in trough cut in the agar

• Interpretation- Precipitin arc represent individual antigens

• Qualitative - Relative concentration

IMMUNOELECTROPHORESIS

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• 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 agaroseplates 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- +

I. COUNTERIMMUNOELECTROPHORESIS/COUNTER CURRENTIMMUNOELECTROPHORESIS

II. ROCKETIMMUNOELECTROPHORESIS 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 DIMENSIONALELECTROPHORESIS

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 OFPRECIPITATION 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.

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TURBIDIMETRY

precipitation in solution

measurement of light extraction

(precipitate absorption)

standard curve

Nephelometry

• precipitation in solution

• measurement of scattered light (proportional to number of insoluble complexes

• 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 endotoxins1. 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 neutralisingcirculating 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 ohagocytosedbacteria per PMN leukocyte

Phagocytic index denotes the phagocytic activity of the blood an dthus helps in measuring opsonicindex

PRIMARY ANTIGEN-ANTIBODY REACTIONS

TYPES OF PRIMARY AG-AB REACTIONS

I. ELISA II.RIA III.Immunofluorescences

Principle: Uses an immune reaction Detection based on

Enzyme catalysed reaction OR Fluorescent probe NOT radioactivity [great advantage!]

I. ENZYME LINKED IMMUNOSORBENTASSAY (ELISA)

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 ELISASensitive: nanogram levels or lowerReproducibleMinimal reagentsQualitative & 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 speedNO 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 ELISA1. Noncompetitive binding assayA. 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 ELISAUsed 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 ELISAUsed 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

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

Radioimmunoassay (RIA) is a Radio-analytical technique with remarkablesensitivity and a high degree of specificity that is widely used for the estimation ofa variety of molecules present in complex matrices. Also known as Radio tracertechnique 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 active material

Antigen antibody binding theory

Detection of compound

II.RIA-RADIO IMMUNOASSAY

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 reactionLabeled 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

B

C

Antibody

Unlabeled antigen

Labeled antigen

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

Here first antibodies bound with labeled antigen are put into the knownconcentration of analyte solution and it is observed how the labeled antigen freefrom 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 ofthe antiserum, the greater the specificity of the assay.

High sensitivity : Immune reactions are sensitive, Using antibodies ofhigh affinity it is possible to detect a few picograms (10−12 g) of antigen inthe tube.

Accuracy and Precision

Disadvantages

Radiation hazards: Uses radio labelled reagentsRequires specially trained personsLabs require special license to handle radioactive materialRequires special arrangements forRequisition, storage of radioactive materialradioactive waste disposal.

REQUIREMENTS FOR RIA

1. Preparation & characterisation of the Antigen [Ligand to be analysed]

2. Radiolabelling of the Antigen3. Preparation of the Specific

Antibody4. 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 PROCEDUREAdd known amounts of the test sample +

labelled antigen into the microtitre wells Incubate allow the reaction to reach completionDecant & wash contents of the well removes all

unbound antigensRadioactivity 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

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

Methods in 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:COMPETITIVEBINDING

Insulin Abs+ serum(insulin ags) + radiolabelledinsulin ags

Lesser the raioactivity , the person is not diabetic and vice versa

FLOWCHART OFTECHNIQUE

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