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
Blood grouping (hemagglutination reactions) Hemagglutination reactions are used in typing of blood According to the presence of 2 surface antigens (A, B) on red blood cells, there are 4 combinations of these antigens giving 4 different blood groups: A, B, AB,O Another surface antigen exist on human RBCS : Rh factor or D antigen. Accordingly, individuals are either Rh-positive or Rh- negative
Procedure A person’ s blood group is determined by mixing 3 separate drops of blood on a slide with anti A serum, antiB serum, antiD serum in each of 3 squares of the marked slide After mixing each square with separate toothpicks, look for agglutination
Type of serological reaction: hemagglutination reaction Blood group is ……..
Complement fixation test (CF tests) Can be used for both antigen or antibody detection. Example for screening of syphilis Complement proteins are usually found in an inactive form. Once activated, they become involved in a chemical cascade It is the cell- lysing ability of activated complement components that is important in CF tests
Principle The complement fixation tests depend upon 2 distinct systems: 1- test system: involves Ag and Ab (one of which is known, the other is unknown)+ complement: If Ag and Ab are specific for one another, they will combine and fix the added complement 2- Indicator system: sensitized sheep RBCs is used as an indicator system to test for the presence of free complement.
Principle If the complement has been fixed by Ag-Ab complex, none will be available for lysis of sensitized sheep RBCs. If Ag and Ab are not specific for each other (or no antibody present), the complement remains free to attach to sensitized sheep RBCs and lyse them Therefore: a positive complement fixation test gives no hemolysis, a negative test gives hemolysis
Procedure 1- The patient serum is heated in a water bath at 56 C for 30 min to inactivate any complement present in serum 2- the patient serum is then serially diluted in wasserman tubes 2- A known amount of antigen is added to each serum dilution 3- Fixed amount of complement is added to each tube Mixture is incubated for 1 hr at 37 C The indicator system (sensitized RBCs) is then added, incubated at 37C for 30 min Read for hemolysis Control tube is included in the experiment with no antibody : control tube must show hemolysis
Titer: reciprocal of the highest dilution (or least concentration) of antibody which gives a positive result DilutionUndil1/21/41/8Control result+++-- OpaqueopaqueOpaqueClearclear Titer = 4
Results 1.Positive complement fixation: all tubes show no hemolysis, except control tube
2.Positive complement fixation with titer: DilutionUndil1/21/41/8Control result+++-- OpaqueopaqueOpaqueClearclear
3.Negative complement fixation: all tubes show hemolysis including control tube DilutionUndil1/21/41/8Control result----- clearClearclearClearclear
4.Anti complementary reaction: control tube shows no hemolysis, indicating that something is wrong in the test or indicator system, preventing lysis of RBCs DilutionUndil1/21/41/8Control result+++++ opaque