Serological Techniques. Antigen-Antibody Reactions
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Transcript of Serological Techniques. Antigen-Antibody Reactions
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I. Agglutination Reactions:
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Agglutinins
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1. Slide agglutination:i. Identification and typing of isolated
bacteria (Serotyping):ii. Blood grouping
2. Tube agglutination
3. Haemagglutination (Passive & Active)i. Passive haemagglutinationii. Active haemagglutination
4. Latex agglutination
5. Coagglutination test6. Coomb' s test:
i. Direct Coomb' s testii. Indirect Coomb' s test 5
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1. Slide Agglutination:
i. Identification and typing ofisolated bacteria (Serotyping)
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ii. Blood Grouping
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This is an example of active haemagglutination.
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2. Tube Agglutination
It is a semiquantitative test
Used mainly to detect Abs in patients' sera
Widaltest for the diagnosis of entericfever (typhoid and paratyphoid)
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Widal test
Serial dilution of pt serum + Ags
1/20 4080 160 320 640
THAH
BH
CH
O
M
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3. Haemagglutination (Passive &Active)
i. Passive Haemagglutination
Red cells sensitized with the Ag can be used todetect antibodies
Example TPHA for detection of treponemalantibodies.
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ii. Active Haemagglutination:
This character can be used in ahaemagglutination inhibition test to detectviral antibodies.
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Influenza virus
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Haemagglutination inhibition test
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4. latex agglutination:
latex beads could be conjugated to antibodies and used
for agglutination of antigens in solutions.
Rapid identification of pathogenic bacteria and fungisuch as N.meningitidisand Cryptococcus neoformans.
It is also possible to conjugate latex particles with the
Ags and use it for detecting antibodies in patients' seraor other body fluids.
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5. COAGGLUTINATION TEST:
This test takes advantage of this protein A
Uses Staph aureus as an antibody-carrying
particleIf we add the appropriate bacteria or Ag to
be tested, it well attach to the specific Fabsite and the reaction will then be visualizedby clumping of staphylococci
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6. Coomb' S Test
Incomplete or blocking antibodies are able to bindto specific antigen without producing visible effect
They can be looked for by Coombs test
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i. Direct Coomb' S Test: It detects Abs already present on red cells.
In erythroblastosis foetalis (E.F.), baby' s redcells are coated with anti- Rh Abs.
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II. Precipitation Reactions:
Requirements:
1. The Ag has to be soluble (in solution).
2. The Ag and the Ab have to be in optimalconcentration.
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PRECIPITATION IN SOLUTION
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i. Slide precipitation:as RPR and VDRL fordiagnosis of syphilis.
ii. Tube precipitation :as Lancefield test forgrouping streptococci ( Ring test).
iii.Agar ( Gel) Diffusion tests:
A. Double immunodiffusion
B. Single (Radial) immunodiffusion
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i. Slide Precipitation
RPR and VDRL for diagnosis of syphilis.
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VDRL
Heat inactivated patient serum +Cardiolipinlecithin cholesterol antigen---->
Examined microscopically .
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RPR
Carbon containing cardiolipinantigen+unheated patient serum ---}rotate
Aggregates of carbon particles asblackclumps ,read by the naked eye.
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ii. Tube Precipitation
Lancefield test for grouping streptococci ( Ringtest).
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b. Toxin-antitoxin Reaction As In Elek's Test
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B. Single (Radial) Immunodiffusion:
The Ab is incorporated in the agarose
gel plate.The Ag is placed in a well punched in
the agarose.
This is a quantitative method tomeasure the concentration of theunknown Ag.
This technique is used for estimatingthe amount of different Ig classes inthe serum for example IgG
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SINGLERADIALIMMUNODIFFUSION
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III.Complement Fixation Test (C.F.T):
This is an antigen antibody reaction thatoccurs in the presence of a third componentknown as the complement.
The antigen reacts with its specific antibodyand the resulting complex fixes thecomplement.
The C.F.T. is used in the diagnosis of manydiseases by detecting C.F. antibodies in theserum of patients, as in syphilis, whoopingcough, chronic gonorrhoea, typhus and otherdiseases as well as many viral infections.
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Principle Of C.F.T:
The heated serum of a patient
(unknown antibody) is added to theknown antigen, then thestandardized complement is added
no visible reactionA second step should be done in
which anindicator systemis
added made ofsheep red cellscoated with their antibody.If the complement is free (as with
negative sera), it will causehaemolysis of red cells.
Thus, haemolysis means negativeserum.
If the complement is fixed (as withpositive sera); no haemolysisoccurs.
No haemolysis means positiveserum. 32
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Complement Fixation Assay
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Complement Fixation Assay
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IV. Antibody (AB) Labeled Assays:
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1. Fluorescent Antibody techniques
A. Direct immunofluorescence (D I F)B. Indirect immunofluorescence ( I I F)
i. For detection of antibodies
ii. For detection of antigens
2. Enzyme Linked Immunosorbent Assay( ELISA)A. Sandwich method ELISA (Double antibody) fordetection and measurement of unknown Ag
B. Indirect ELISA for detection and measurement ofunknown antibody
3. Radioimmuno assay (RIA)A. Liquid phase RIA
B. Solid phase RIA
4. Chemiluminescence
Fluorescent Antibody
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Fluorescent AntibodyTechniques
A.Direct immunofluorescence (D I F)The Ag to be detected is present in a smear
The fluorescein labeled specific Ab is added
The slide is then examined by an ultravioletmicroscope (UV)
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Principal steps in direct immunofluorescence
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B. Indirect immunofluorescence ( I I F)
i. For detection of antibodies
Placing the Ag on a clean slide, the patient' serum is added,
Antispecies (Anti-human gamma globulin) labeledwith fluorescein is added
Examined by the UV microscope.
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B. Indirect Immunofluorescence ( I I F)
ii. For detection of antigens
Specimen suspected to contain theantigen is fixed on a slide,
Specific antibody is added,
Antispecies antibody conjugated withfluorescent dye is added,
Examined by U.V microscope.
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2. Enzyme Linked Immunosorbent Assay( ELISA)
Principle of the test:It is based on two
assumptions that :
Antigen or antibody can
be attached to a solidphase (plastic surface),
Either antigen orantibody can be linked toan enzyme such as
alkaline phosphatase orperoxidases and thecomplex retain bothimmunological andenzymatic activity.
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2. Enzyme Linked Immunosorbent Assay(ELISA)
A. ELISA for detection ofunknown Ag (Sandwichmethod )
1. The wells are coated with specificantibody
2. The test solutions thought tocontain antigen are incubated inthe sensitized wells.
3. The conjugate consisting ofenzyme- labeled specific antibodyis then incubated in each well.
4. The enzyme substrate is added.
5. A color change upon degradationcan be assessed visually ormeasured in a spectrophotometer(ELISA reader).
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2 E Li k d I b t A
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2. Enzyme Linked Immunosorbent Assay(ELISA)
B. Indirect ELISA fordetection of unknownantibody
1. Wells are sensitized bypassive adsorption with therelevant antigen
2. The test samples areincubated in the sensitizedwells
3. Enzyme-labeled anti-humanIg conjugate is incubated in
the wells,4. Enzyme substrate is added
5. The rate of degradation ofthe substrate is indicated bya color change 42
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3. Radioimmuno Assay (RIA)
Principle:
It isbased onlabelling either the
Ag or the Ab withradioactive
substance such asiodine or tritiatedthymidine.
The amount of
radioactivity ismeasured bygamma or betacounter 43
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3. Radioimmuno Assay (RIA)
A.Liquid phase RIA
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3. Radioimmuno Assay (RIA)
B.Solid phase RIAAg is coated
The test Ab isadded.
Radioactive labeledanti-species Ab isadded,
The radioactivity ismeasured.
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4. Chemiluminescence
Principle:It depends on labelling the Ab with
acetylaminofluorene ; a luminescent
substance detected by luminometer
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Intradermal Skin Tests
I. Neutralization tests:
These are in- vivomanifestations ofAg-Ab reactions.
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A. Schick Test
To test the susceptibility of a person to diphtheria.
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B. DICK TEST
Test the susceptibility of a person to get scarletfever.
Using the erythrogenic toxin ofStreptococcus
pyogenes.
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C. Schultz-Charlton Reaction
Used for the diagnosis of scarlet fever.
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II. Hypersensitivity TESTS
a. Immediate type skin test: As testing for the sensitivity to penicillin.
An immediate wheal and flare indicates that
the person is hypersensitive to penicillin.
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b. Delayed Type Skin Test:
The Ag is injected intradermally, and the test isread after 48-72 hours.
Delayed type hypersensitivity tests areinduced by sensitized lymphoid cells(cellularreaction).
Examples: tuberculin, lepromin, brucellin andDucrey tests.
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