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

PREPARATION OF REAGENTS

• Antigen
  • Any infected material containing virus such as the infected tissue, fluid and cell culture can be used.
• Antibodies
  • It may be known specific antiserum or serum from infected and convalescent animal. The serum or antiserum is heated at 56 ⁰ C for 30 minutes to remove the effect of complement.
• Complement
  • Fresh serum from Guinea pigs is a rich source of complement.
  • The Guinea pigs are bled and the serum is separated and stored until use at 4 ⁰ C. The titre of the complement is determined before use.
• Hemolysin
  • Rabbit antiserum against sheep erythrocytes can be prepared in the laboratory. Freshly collected sheep blood is washed 3 times in normal saline. Group of healthy rabbits are inoculated with 1 ml of sheep erythrocytes suspension in the ear vein or by S/C route. Four more inoculations are made at five days interval and on the ninth day of the last inoculation, rabbits are bled and serum is separated, pooled, inactivated at 56 ⁰ C for 30 minutes and hemolytic titre is determined.
• Sheep RBC
  • The washed erythrocyte sediment is prepared to contain 2.8% RBC /ml and is ready for sensitization with hemolysin.
• Hemolytic system
  • An equal volume of 2,8% standardized erythrocyte suspension is mixed with equal volume of hemolysin containing at least 4 minimal heaemolytic dose in vernol buffer saline. The mixture is shaken well and incubated at 37 ⁰ C for 30 minutes This indicator system is stored at 4⁰C and can be used for a week.