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Serological reactions
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Serology
In vitro Antigen- Antibody reactions
Antigen- Antibody reactions are classified
according to the physical state of antigen into:
- Agglutination reactions: antigens are cells or
particles- Precipitation reactions: antigens are soluble
- Flocculation reactions: antigens are suspended
- Complement fixation : indicated by positive orcell lysis
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Agglutination reactions
1- Haemagglutination: e.g. Blood groups
2- Passive haemagglutination: e.g.
Detection of Toxoplasma using microtiter
plate
3- Cell agglutination: e.g. Widal test
4- Passive or Latix agglutination: e.g. ASOtest
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2- Passive haemagglutination
Example: Detection of Toxoplasma antibodies usingmicrotiter plate
Microtiter plate is a plastic tray divided into rows andcolumns containing wells
Toxoplasma Ag are coated with RBCs (hence namedpassive) and are put in the wells of the microtiter plate
Toxoplasma antibodies are detected in patient serum bycarrying out serial dilution of the patient serum in the wellsof the microtiter plate
If there is a positive reaction: Agglutination occurs & fills thewell
If there is a negative reaction: no agglutination occurs andRBCs settle in the well (button like appearance)
Titer is calculated (reciprocal of the highest dilution of
antibody giving positive antigen antibody reaction)
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Passive haemagglutination
The first row showsve Ag-Ab reaction
The second row shows +ve Ag-Ab reaction
In the second row: If the antibody dilution is in the first well, in the 2nd well, 1/8 in
the 3rd well, 1/16 in the 4th well, 1/32 in the 5th well, 1/64 in the 6th well ,titer = 64
Questions: Type of serological reaction: Passive haemagglutination reaction in
microtiter plate
Type of antigen: antigen coated with RBCs e.g. Toxoplasma antigen coated with
RBCs
Uses, Result and titer
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3- Cell agglutination: e.g. Widal test
Widal test is is a kit used to detect
antibodies to Salmonella typhi or
Salmonella paratyphi( which are the
causative agents of enteric fever) inpatient serum
Antibodies to Salmonella may be detected
in patient serum from the 2nd week ofonset of infection
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Widal test
Materials supplied:
O: Somatic antigen of Salmonella typhi
H: flagellar antigen of Salmonella typhi
AH: flagellar antigen of Salmonella paratyphi A
BH: flagellar antigen
of Salmonella paratyphi B
Glass slide
+ve andve controls
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Widal Slide agglutination reaction
The 4 types of antigen (colored suspension) are put on theslide (antigen O,H) of Salmonella typhi and antigen (AH,BH(of Salmonella paratyphi
One drop of patient
serum is added on each We look for
agglutination
not for the color
Questions: Name and type
Serological reaction: widal testCell agglutination reaction
Type of antigens: cellular antigen
Uses of the test, result
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Passive or Latix agglutination: e.g.
ASO test
ASO is a kit used to detect AntiStreptolysin O antibodies(ASO) in patient serum in case of Steptococcus infection
When a person is infected with Streptococcus, thebacterium produces an exoenzyme known as StreptolysinO
The body respond to this antigen by producing ASOantibodies
Detection of ASO antibodies in patient serum indicateStreptococcus infection
At a level of ASO antibodies in patient serum equal orhigher than 200 IU/ml agglutination will occur
It allows qualitative and semi-quantitative determination ofthe antistreptolysin-O titer in serum by agglutination oflatex particles
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ASO kit
Materials supplied:
Antigen (too small)
coated on
Polystyrene latex to
Allow visualization of
Agglutination reaction(hence named passive or latex agglutination)
+ve, -ve control
Disposable slides
Qualitative test should be compared to +ve andve contol tests
QualitativeTest is carried out by mixing 1 drop of latex reagent (coated antigen)with 1 drop of patient serum, rotate the slide for 2 min and observe the result
Quantitative test is carried out by mixing a serially diluted patient serum onconsecutive circles of the slide with one drop of latex reagent. rotate theslide for 2 min and observe the result
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ASO kit
In this test, agglutination is seen only in the first circle. The titer is the reciprocalof the dilution of serum antibody carried out in the first circle
Note: the uniform milky suspension in other circles is the color of polystyrene
latex without agglutination
Questions: Name and type of serological reaction: ASO, latex agglutinationreaction
Type of antigen: particles (Streptolysin O antigen coated on polystyrene latex)
Uses, result, titer
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Flocculation reactions
Example: RPR kit Used to detect Syphilis antibodies in patient serum
Syphilis antibodies are called: Reagin
The test is known as RPR: Rapid Plasma Reagin
Syphilis is a sexually transmitted disease caused by Treponemapallidum
In RPR the antigen used is not syphilis antigen, but is cardiolipinantigen from beef heart (heterophile antigen)
Cardiolipin antigen is suspended on microparticulate charcoal(charcoal particles) to enhance the visual difference betweenpositive and negative results
(-ve) result
+ve result is indicated by flocculation of the microparticulatecharcoal particles which appears as black clumps
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PRP kit
Materials supplied are: reagent (cardiolipin containing microparticulate charcoal),
+ve, -ve control, disposable slides
It may be used for qualitative or quantitative determination
+ve or reactive result is indicated by large or small floccules mainly at the periphery
of the test circle
-ve or non reactive result is indicated by even or smooth appearance,with no visiblefloccules
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RPR kit
-ve control +ve contol
Test is positive
Questions: Name and type of serological reactions: RPR, Flocculation reaction
Type and name of antigen used: cardiolipin suspendedon microparticulate charcoal
Result: +ve orve
Uses of the test
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Precipitation reactions
When soluble antigens are bound by
antibody to form a cross-linked lattice
structure, the reaction is called
precipitation.
Example: Ouchterlony test
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Ouchterlony test
The Ouchterlony test is a double diffusion technique.
In Ouchterlony test, a petri dish with a thin layer ofagarose at the bottom is used
Several wells are created in the agarose gel
Anti-sera will be placed in the central well, and antigenswill be added into the wells around the central well.
Antibody and antigen molecules will diffuse through theagarose.
When antibody meets with its specific antigen at their
equivalent zone (at their optimal concentrations), theprecipitation reaction occurs. Antibody-antigenprecipitates in agarose appear as a light white bandbetween the antibody and the antigen wells.
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Ouchterlony test
Using such a technique, the antigenicrelationship between two antigens (homogeneitybetween 2 antigens) can be analyzed. This isidentified by precepitation lines withcharacteristic patterns
The Ouchterlony test also can be used toestimate the relative concentration of antigens.This is also identified by the position of theprecepitation lines ( away or closer to theantigen well)
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Questions: Name and type of serological reaction: Ouchterlony diffusion test,
Precepitation reaction
Type of antigens: soluble antigens
Result: Precipitation lines between antigens in wells G, E and the central antibody inwell A