VMC 221: Veterinary Immunology and Serology (1+1)

METHOD

Titration of complement and haemolysin

• Add 0.5 ml of Vernol buffer diluent (VBD) to the wells
• Various dilutions of the complements are made
• Plate is shaken gently and stored at 37 ⁰ C for 1 h.
• Various dilutions of haemolysin, 2ml of 2.8% RBC are added and the tubes are shaken well and incubated at 37 ⁰ C for 30 minutes
• Add 25 µ l of indicator system (Sensitized RBC) to the plates.
• Plate is shaken gently, covered and sealed and incubated at 37 ⁰ C in a humid chamber for 1 h with 3-4 intermittent shaking.
• The plates are then kept at 4 ⁰ C overnight and the results are read the next day.
• The highest dilution of the complement that gives 50% lysis with the highest dilution of the haemolysin is taken as 1 unit haemolytic complement 50 and 1 unit haemolytic complement 50 respectively. The degree of haemolysis is ascertained by the degree of setting of unlysed RBC in the bottom of the well and scored in the scale of 0 to 4:100, 75,50,25 and 0% haemolysin respectively.
• For the test, 4 units of haemolysin and 3 units of complement are used.