Methods

METHODS

  • The concentration of the gel used in AGID varies depending upon the serum sample.
  • Prepare 1% agarose by adding 1 gm of agarose, 0.89 g of sodium chloride, to 100 ml of distilled water and heating the mixture slowly to boiling point. ( Use 8 g sodium chloride in 100 ml of distilled water for poultry diseases).
  • A clean, scratch free microscopic slides are precoated by first dipping them in 0.3 to 0.5 % agar or 1% agarose solution and then drying in air.
  • Place precoated slides on a horizontal leveled surface
  • When the agarose cooled to 500C, add 4 to 5ml of agarose onto glass microscopic slides inorder to achieve a gel thickness of 2 to 2.5mm .
  • When the agarose has solidified, cut wells of 4mm diameter in various patterns as per requirement with the help of template and gel cutter . The inter well distance should be 4 to 6 mm. 
  • Remove the agarose gel from wellswith the help of needle.
  • Fill 25 µl of known serum in the central wells (if antigen to be detected in the material) with help of micro pipette .
  • Fill 25 µl of known antigen in the 2nd outer well (positive control) and add 25 µl of test samples on other peripheral wells.
  • While adding, the contents of the well should not overflow.
  • Incubate the loaded slides in humid chamber at room temperature or at 37°C for 24h to allow diffusion of immunoreactants.

Note

  • Humidity should be maintained to prevent drying out of gel during long incubation.
  • Antisera predominately having IgM require longer incubation period (upto 72hr) for precipitates to occur.
  • For labile proteins incubate slides in cold for several days.
  • Over incubation may lead to loss of sharpness of bands.
Last modified: Tuesday, 23 August 2011, 10:52 AM