1st chapter detection of ab -ag_3

8/13/2019 1st Chapter Detection of Ab -Ag_3

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DETECTION OF ANTIGENS & ANTIBODIES

MR. AZMIL AZAM.


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Affinity =

attractive and repulsive forces

Ab

Ag

High Affinity

Ab

Ag

Low Affinity

Affinity

Strength of the reaction between a single antigenic

determinant and a single Ab combining site


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Specificity

The ability of an individual antibody combiningsite to react with only one antigenicdeterminant.

The ability of a population of antibodymolecules to react with only one antigen.


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Cross Reactivity

The ability of an individual Ab combining site to react with

more than one antigenic determinant.

The ability of a population of Ab molecules to react with

more than one Ag

Anti-A

Ab

Ag A

Anti-A

Ab

Ag B

Shared epitope

Anti-A

Ab

Ag C

Similar epitope

Cross reactions


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Factors Affecting Measurement of

Ag/Ab Reactions

Affinity

Avidity

Ag:Ab ratio

Physical form of Ag

Ab excess Ag excess

Equivalence Lattice formation


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Tests Based on Ag/Ab Reactions

All tests based on Ag/Ab reactions will have todepend on lattice formation or they will have toutilize ways to detect small immune complexes

All tests based on Ag/Ab reactions can be used todetect either Ag or Ab


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Agglutinantion


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Agglutination Used in all sorts of clinical applications

erythrocyte typing, pregnancy detection(HCG), rheumatoid factor test, syphilis etc.

Can be separated into 2 types Direct agglutination

Passive agglutination

Direct agglutinationantigen + antibody

Passive agglutinationantibody + (soluble

antigen + insoluble particle)


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Direct agglutination


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Passive agglutination


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In passive agglutination, theres a term calledagglutination inhibition.

It means that soluble antigen combines withantibody and the result is not visible to thenaked eye.

Example : {Thyroglobulin antigen(soluble) + latex beads } + antibody

If add soluble antigen to antibody before Thyroglobulin + latexbeads = agglutination inhibition.


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Precipitation reaction Examples :- radial immunodiffusion,

immunoelectrophoresis etc.

Principles : Reaction that occurs between soluble antigen and antibody.

Divalent antibody + multivalent antigen = lattice.

Lattice will loose solubility eventually and precipitate out ofthe solution.

Termed precipitin reaction


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Precipitation reaction ingel

Precipitation can also takes place in gel.

Soluble antigen and antibody placed in wellscut in gel and the reactants will diffuse in thegel, forming a gradients of concentration.

Highest concentrationsclosest to the wells.

Somewhere in between, concentration ofreactants = zone of equivalence =precipitation.


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Interaction between 1

antigen and 1 antibody,

Line is the zone ofequivalence

Interaction between 3antibody and 3 antigens.

Antigen travels at differentrate.

Antibody travels at the samerate.


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Double diffusion method. A form of gel diffusion

Uses the same principles to show antigenicrelationship between antigens.

Result will form 3 patterns of identity forsubstance. Patterns of identity.

Patterns of nonidentity.

Patterns of partial identity.


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Patterns of identity 2 antigens are

immunologicallyidentical

The antibodies in the

antiserum react withboth the antigensresulting in a smoothline of precipitate.

The antibodies

cannot distinguishbetween the twoantigens.


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Patterns of nonidentity None of the

antibodies in theantiserum react withantigenic

determinants thatmay be present inboth the antigens.

The two antigens are

immunologicallyunrelated as far asthat antiserum isconcerned.


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Patterns of partial identity The antibodies in the

antiserum reactmore with one of theantigens than the

other.

The spur is thoughtto result from thedeterminants

present in oneantigen but lackingin the other antigen


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Radial immunodiffusion Variation of double diffusion test.

Used to measure serum protein concentration.

Multiple wells contains antigens at different

concentration. Antibodies are distributed uniformly in the agar

gel.

Precipitin line is replaced by precipitin ring

around the well. Distance of precipitin ring from the center of

the well = directly proportional to antigenconcentration in the well.


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Immunoelectrophoresis Share the same principles as radial

immunodiffusion, but this time, we use electricfield to separate the mixture of protein.

It is a 2 step process, involving separation of amixture of protein added to a polyacrylamidegel using an electric field, and detection usingantibody.

Comparison of the pattern from normal

human serum with result obtained frompatients sera may reveal an absence,overabundance or other abnormality of oneor more serum protein.


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HemagglutinationTests

Lattice Formation


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Hemagglutination

Definition - tests that have as their endpoint

the agglutination of a particulate antigen

Agglutinin/hemagglutinin

+

Qualitative agglutination test

Ag or Ab


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Hemagglutination

Quantitative agglutination test

o Titer

o Prozone

1/2

1/4

1/8

1/16

1/

32

1/

64

1/128

1/

256

1/512

1/1

024

Po

s.

Ne

g.

Titer

64

8

512


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Hemagglutination

Definition

Qualitative test

Quantitative test

ApplicationsBlood typing

Bacterial infections

Practical considerations

Easy

Semi-quantitative

1/2

1/4

1/8

1/16

1/32

1/64

1/128

1/256

1/512


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Hemagglutination

Definition - agglutination test done with a solubleantigen coated onto a particle

+

Applications

Measurement of antibodies to soluble antigens


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Hemagglutination Inhibition

o Definition - test based on the inhibition of agglutination due to

competition with a soluble Ag

+

Prior to Test

+ +

Test

Patients sample


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Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent

Assays (EIA)

Lattice formation not required

Co etiti e RIA/ELISA fo A


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Competitive RIA/ELISA for Ag

Method

o Determine amount ofAb needed to bind toa known amount of

labeled Ag

+

Prior to Test

Labeled

Ag

+

Test

+

Patientssample

LabeledAg

+ Use predetermined

amounts of labeled Ag

and Ab and add asample containing

unlabeled Ag as a

competitor


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Solid Phase Non-Competitive RIA/ELISA

Ab detection

o Immobilize Ag

o Incubate with sample

o Add labeled anti-Igo Amount of labeled Ab

bound is proportionalto amount of Ab in

the sample

Solid

Phase

AgImmobilized

Ab in

Patients

sample

LabeledAnti-Ig


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Solid Phase Non-Competitive RIA/ELISA

Ag detection

o Immobilize Ab

o Incubate with sample

o Add labeled antibody

o Amount of labeled Abbound is proportional tothe amount of Ag in the

sample

Solid

Phase

Ag

Immobilized

Ag in

Patients

sample

Labeled

Ab


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Tests for CellAssociated

Antigens



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Immunofluorescence

DirectAb to tissue Ag is labeled with fluorochrome

Ag

Fluorochrome

Labeled Ab

Tissue Section


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Immunofluorescence

Indirecto Ab to tissue Ag is unlabeled

o Fluorochrome-labeled anti-Ig is used todetect binding of the first Ab.

Ag

Fluorochrome

Labeled Anti-Ig

Tissue Section

Unlabeled

Ab

Qualitative to Semi-Quantitative


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Assays Based onComplement


C l F


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Complement Fixation

Ag mixed with test serum to be assayed for Ab

Standard amount of complement is added

Erythrocytes coated with Abs is added

Amount of erythrocyte lysis is determined

Ag

Patients

serum

Ag No Ag

Ag

1st chapter detection of ab -ag_3

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