serology part1

7/28/2019 Serology Part1

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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

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