Procedure

PROCEDURE 

Preparation of sensitized RBC suspension

  1. Sheep RBC are commonly used. Sheep RBC is collected in equal volume of Alsever’s solution and washed thrice in Normal saline.
  2. Prepare a 2.5% RBC suspension in PBS . 
  3. In case of protein antigen the erythrocytes are pretreated with tannic acid before coating with the antigen as follows. 
  4. To 1 ml of 2.5% Sheep RBC add 1 ml of 1:20 000 diluted tannic acid in saline and place in cool temperature for 10 minutes. 
  5. The cells are gently washed with Normal saline thrice. 
  6. To 4 ml of Normal saline pH (6.4) add 1 ml of 0.025% protein antigen and then further add 1 ml of tanned RBCs. 
  7. Incubate at 37º C for 45 minutes to 1 hour. 
  8. The erythrocytes suspension is then washed 2-3 times with PBS to remove the uncoated antigen and suspended in PBS to give final concentration of 0.5% coated RBCs.
  9. Take micro titration plate and add 50 µl of PBS in all the wells in the rows.
  10. Add 50 µl of suspected serum in the first well and serially make two fold dilution.
  11. Add 0.5% antigen coated RBCs to each of the well and gently tap the plate so as to mix it properly.
  12. Incubate at 37º C for 45 minutes .
  13. Keep known postive, negative sera and RBC controls.
Last modified: Tuesday, 17 April 2012, 10:50 AM