The document describes the agar gel immunodiffusion (AGID) test, which detects antibodies in a sample by observing the diffusion and precipitation of antigens and antibodies in an agar gel. Key steps include preparing an agarose gel in PBS with sodium chloride, cutting wells in the gel, placing antigen and antibody samples in the wells, incubating for 24 hours, and observing for visible precipitin lines between samples, which indicate a positive result. The test is useful for detecting influenza A antibodies and determining antigenic relationships but requires careful technique and interpretation.

1. Agar Gel Immunodiffusion
(AGID) Test
Principles and Techniques
Dennis A. Senne
dennis.a.senne@aphis.usda.gov
USDA, APHIS, VS,
National Veterinary Services Laboratories,
Ames, IA 50010

2. Agar Gel Immunodiffusion Test
Definition:
• The passive diffusion of soluble
antigens and/or antibodies
toward each other leading to
their precipitation in a gel matrix
Synonyms:
• Ouchterlony test (1949)
• Double immunodiffusion test
• Agar gel precipitin (AGP) USDA-APHIS

3. Agar Gel Immunodiffusion
Test (AGID)
Advantages:
• Group-specific test (detects
antibodies to all subtypes of flu-A,
determine antigenic relationships)
• Easy, requires few reagents/equip
Disadvantages:
• Semi quantitative
• Moderate sensitivity
• Subjective interpretation
• Requires 24 hr USDA-APHIS

4. 100 mm petri dishes – 15-17 ml agar
60 mm petri dishes – 5-6 ml agar

5. Agar Gel Immunodiffusion Test
Gel Preparation:
• 0.9% agarose in PBS (0.01M, pH 7.2)
with 8% sodium chloride
• Dissolve, autoclave 10 min, cool to
about 60-80C and dispense
Pour plates on same day used
15 – 17 ml (100 x 15 mm petri dishes)
5 – 6 ml (60 x 15 mm petri dishes)
USDA-APHIS

7. Template Dimensions
Wells 2.4 mm
apart
Wells 5.3 mm
in diameter
Agar 2.8 mm
thick

8. Source of Template
Cutters
Veterinary Diagnostic Technology, Inc.
4890 Van Gordon St., Suite 101
Wheat Ridge, Co. 80033
Phone: (303) 467-2741
Fax: (303) 467-2799
USDA-APHIS

10. VACUUM ASSEMBLY
Single hole stopper
Side-arm flask
Glass or plasic tubing
VACUUM PUMP
IN
OUT
Vacuum hose Vacuum set at
12 to 20 psi
12 to 14 gauge cannula

11. Placement of Reagents/Samples
Sample #3 Sample #1
As
Ag
As As
Sample #2
As = Positive enhancement serum
Ag = Antigen

12. Numbering Patterns
1 2
3 4 5
6 7
100 x 15 mm
petri dish

14. Agar Gel Immunodiffusion Test
Reagent Sources (AIV):
• NVSL, Ames, IA:
Domestic AIV surveillance – antigen and
enhancement serum are available, free of
charge
Export testing, international – available for
sale
• Charles River (SPAFAS), Norwich, CT:
AIV antigen and enhancement serum
available for sale
USDA-APHIS

15. Filling Wells
50-60 µl

16. Cross section of wells
showing reagent levels
Over Under
Correct
filled filled

17. Hold Tip Vertical When
Filling Wells

18. Method Not Recommended

19. Fill All Wells
PBS Sample #1
As
Ag
As As
PBS

20. Diffusion of Reagents
Ag As
Seen as a precipitin line
when concentrations are
optimal

21. Diffusion of Reagents
Ag As
At 24 hours a
precipitin line is visible

23. Precipitin Lines After 24 Hours
Incubation (Negative samples)
As
#3 #1
Ag
As As
#2

24. Examples of Positive Reactions
Negative
+3 +1 +4
+2 +5
May be seen as light haze

25. Examples of Nonspecific Lines
Negative
Negative
Line of partial
identity
Line of non identity Negative

26. Incomplete Line Formation
+ +
-
Before 24 hours At 24 - 48 hours

27. Potential Positive Sample

28. Additional Problems
“Halos”
+
+ -
+
+

29. AS
#3 #1
AG
AS AS
#2

30. #1
AS AS
AG
#3 #2
AS

31. #1
AS AS
AG
#3 #2
AS

32. #1
AS AS
AG
#3 #2
AS

33. #1
AS AS
AG
#3 #2
AS

34. AS
#4 #1
AG
#3 #2
AS

35. Unbalanced Reagent
Concentrations
As As
Ag Ag
As As As As
As too strong Ag too strong

36. Cutting Errors
May miss the weak
positives