Serological reactions

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 or cell lysis

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. ASO test

2- Passive haemagglutination
Example: Detection of Toxoplasma antibodies using microtiter plate Microtiter plate is a plastic tray divided into rows and columns containing wells Toxoplasma Ag are coated with RBCs (hence named passive) and are put in the wells of the microtiter plate Toxoplasma antibodies are detected in patient serum by carrying out serial dilution of the patient serum in the wells of the microtiter plate If there is a positive reaction: Agglutination occurs & fills the well If there is a negative reaction: no agglutination occurs and RBCs settle in the well (button like appearance) Titer is calculated (reciprocal of the highest dilution of antibody giving positive antigen antibody reaction)

Passive haemagglutination

The first row shows ve 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

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) in patient serum Antibodies to Salmonella may be detected in patient serum from the 2nd week of onset of infection

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 and ve controls

Widal Slide agglutination reaction

The 4 types of antigen (colored suspension) are put on the slide (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 test Cell agglutination reaction Type of antigens: cellular antigen Uses of the test, result

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, the bacterium produces an exoenzyme known as Streptolysin O The body respond to this antigen by producing ASO antibodies Detection of ASO antibodies in patient serum indicate Streptococcus infection At a level of ASO antibodies in patient serum equal or higher than 200 IU/ml agglutination will occur It allows qualitative and semi-quantitative determination of the antistreptolysin-O titer in serum by agglutination of latex particles

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 and ve 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 on consecutive circles of the slide with one drop of latex reagent. rotate the slide for 2 min and observe the result

ASO kit

In this test, agglutination is seen only in the first circle. The titer is the reciprocal of 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 agglutination reaction Type of antigen: particles (Streptolysin O antigen coated on polystyrene latex) Uses, result, titer

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 Treponema pallidum In RPR the antigen used is not syphilis antigen, but is cardiolipin antigen from beef heart (heterophile antigen) Cardiolipin antigen is suspended on microparticulate charcoal (charcoal particles) to enhance the visual difference between positive and negative results (-ve) result +ve result is indicated by flocculation of the microparticulate charcoal particles which appears as black clumps

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 visible floccules

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 suspended on microparticulate charcoal Result: +ve or ve Uses of the test

Precipitation reactions
When soluble antigens are bound by antibody to form a cross-linked lattice structure, the reaction is called precipitation. Example: Ouchterlony test

Ouchterlony test
The Ouchterlony test is a double diffusion technique. In Ouchterlony test, a petri dish with a thin layer of agarose at the bottom is used Several wells are created in the agarose gel Anti-sera will be placed in the central well, and antigens will be added into the wells around the central well. Antibody and antigen molecules will diffuse through the agarose. When antibody meets with its specific antigen at their equivalent zone (at their optimal concentrations), the precipitation reaction occurs. Antibody-antigen precipitates in agarose appear as a light white band between the antibody and the antigen wells.

Ouchterlony test
Using such a technique, the antigenic relationship between two antigens (homogeneity between 2 antigens) can be analyzed. This is identified by precepitation lines with characteristic patterns The Ouchterlony test also can be used to estimate the relative concentration of antigens. This is also identified by the position of the precepitation lines ( away or closer to the antigen well)

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 in well A