VLD 421: Veterinary Clinical Biochemistry and Laboratory Diagnosis-II (0+1)

Quantitative AGID

PROCEDURE FOR QUANTITATIVE AGID

• Boil 1 g of agarose in 100 ml of normal saline i.e 1% agarose (use 8.9 g of sodium chloride water for poultry diseases) by keeping the conical flask in a stainless stell vessel with adequate quanity of water.
• Wait for some time to get down the melted agarose to the temperature of approximately 550c.
• Cast the melted agarose gel on a clean and scratch free glass slide (4ml).
• After solidification, cut wells with the help of the template and gel cutter. The interwell distance should be about 4 to 6 mm.
• Remove the agarose gel from wells with the help of needle.
• Prepare two-fold dilution of test serum in a microtitre plate.
  • Add 25 μl of normal saline in five well in a microtitre plate,
  • Add 25 μl of test serum in the first well and mix well,
  • Transfer 25 μl from the first well to second well and so on up to fifth well; and then discard 25 μl from the last well.
  • Now the dilution is 1:2, 1:4,1:8, 1:16 and 1:32.
• Place 25μl of known antigen in the central well.
• Place 25μl of two-fold diluted test serum 1:2, 1:4, 1:16 and 1:32 respectively in 3^rd ,4^th,5^th,6^th and 7^th well. 
• Place 25 μl of known serum in 2^nd well (positive control).
• Incubate at 37^ºC (or at room temperature control) in a humid box for 24 to 48 h. Cover the plate with the lid if pertriplate is used.
• Read the result after 24 to 48 h of incubation. Precipitation line will be formed in the positive control. Reciporal of the highest dilution showing precipitation line between test serum and known antigen is the end point. Readings are facilitated by oblique light.