The AGID test is based on the formation of lines of precipitation at the meeting points of specific antibody and soluble antigen where the reactants are in optimal concentrations. The test is performed by letting antibody (CBPP antiserum) and antigen (mycoplasma vaccine suspension) to diffuse against each other from wells cut in solidified agar gel.
I. Equipment
II. Materials
III. Test Procedure
Loosen the cap on a bottle of stored M. mycoides subsp. mycoides identity agar gel and stand the bottle in a boiling water bath until the agar is completely molten.
Pipette 3 ml of molten agar into each of 60 × 15 mm petri dish rested on a horizontal surface.
Allow the gel to set, wrap in aluminium foil an dkeep at 4°C overnight.
Using a template to outline the well pattern and a 4 mm diameter (internal) gel punch cut out a central and six peripheral wells. Distances between wells should be approximately 3.5 mm.
Using an 18 gauge needle or a suction pump with Pasteur pipette, the cut out gel pieces are removed. Identify each well by the numbers 1 to 6.
Reconstitute one vial per batch of CBPP vaccine using a volume of cold, sterile distilled water equivalent to the filling volume of the vial. Reconstitute a vial of standard mycoplasma antigen in a similar manner.
Add 30 μl of anti-PG1 serum into the central well.
Add 30 μl of PG1 suspension into each of wells 1 and 4 (Positive Antigen Controls).
Add 30 μl of mycoplasma broth medium into well 3 (Negative Control).
Add 30 μl of vaccine suspension under test into wells 2, 5 and 6.
Place the agar test plates in a humidified plastic box and incubate at either room temperature or at 37°C for 18 to 24 hours.
Examine the plates and note any lines of precipitation between the antiserum and vaccine sample wells. Note also the relationship of these lines with those formed between the antiserum and positive antigen control wells. Record the observations.
IV. Interpretation of the result
A line of precipitation should be clearly visible between the antiserum and standard (PG1) antigen wells. Similar lines of precipitation between the antiserum and test sample wells which merge with the reference antigen versus antiserum precipitin lines indicates a positive identification of M. mycoides subsp. mycoides and the sample passes the test for identity in the agar gel immunodiffusion test. The test is valid if no line of precipitation appears between the antiserum and mycoplasma broth medium wells. A negative result indicates either absence or very low titre of M. mycoides subsp. mycoides in the vaccine.
