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

PROCEDURE

• The serum is precipated by ammonium sulphate  precipitation for isolation and concentration of Ig as per the protocol given in exercise-1.
• Coating of beads:
  • 100 µ l of latex beads (10% suspension) is mixed with 900 µ l of carbonate bicarbonate buffer. To this equal volume of immunoglobulin suspension in carbonate bicarbonate buffer is added. Mix the content well and keep at 4 ⁰ C overnight for coating. The coated beads are centrifuged at 10,000 rpm for 15 min and the supernatant discarded and the nonspecific sites of the beads are blocked with 5mg/ml of bovine serum albumin and incubated at 37 ⁰ C for 1 hour. After 1 h. the mixture is centrifuged at 10,000 rpm for 15 min at 4 ⁰ C. The supernatant is discarded and the beads are washed thrice in carbonate bicarbonate buffer.
  • One drop of the coated beads suspension is mixed with one drop of clarified supernatant of the suspected sample on a cavity slide.
  • Positive and negative controls are also used in the test for comparison. Agglutination of coated beads indicate positivity of the sample