antigen- antibody reactions -...
TRANSCRIPT
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