1. Agar Gel Immunodiffusion (AGID) Test Principles and Techniques Prof.Dr. Salah M. Hassan College of Veterinary Medicine University of Mosul

2. INTRODUCTION
Serological reaction means Ag and Ab reaction to form AgAb complex.
This complex need detector or indicator to see and interpertat the results.
eg; RBCs in HI and HA test
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3. Immunodiffusion
Means development of non soluble AgAb complex in a semisolid agar medium as a result of their diffusion towards each other.
Used for qualitative and quantitative analysis of Ag and Ab.
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4. Immunodiffusion
Although the formation of AgAb complex in the agar is dependant on buffer electrolytes, pH and temp BUT the most important factor is the relative concentration of Ag and Ab.
The precipitin line forms where the concentration of Ag and Ab is optimum, but the line will shift towards the well of the lowest concentration.
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5. Unbalanced Reagent Concentrations As too strong Ag too strong As As Ag
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6. Ag Ab precipitation curve
Immune precipitate formed
Zone of
Ab excess
Zone of
equivalence
Zone of
Ag excess
Increasing antigen concentration
Typical precipitation curve resulting from titration of increasing Ag concentration
against constant amount of Ab
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7. The prozone phenomenon
Means suboptimal precipitation that occur in the region of antibody excess .
This phenomenon may be a cause of miss interpretation .
Can be also a result in false negative serological tests .
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8. #1 AS AS AG #2 #3 AS

9. Immunodiffusion
J.Oudin 1946
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10. Agar Gel Immunodiffusion Test Definition:
The double passive diffusion of soluble antigens and/or antibodies toward each other leading to their precipitation in a gel matrix form visible line.
Synonyms:
Ouchterlony test (1949)
Double Immunodiffusion test
Agar gel precipitin (AGP)
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11. Agar Gel Immunodiffusion Test
The basic screening test for type A influenza viruses and Gumboro viruses
Detect the circulated Ab to AIV group-specific Ag (RNP and M protein).
Detect Abs to all AI viruses.
Also used as a group- specific test to identify isolates as AI viruses
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12. Agar Gel Immunodiffusion Test (AGID)
Advantages:
Group-specific test (detects antibodies to all subtypes of flu-A, determine antigenic relationships)
Easy, requires few reagents/equipment
Disadvantages:
Semi quantitative
Moderate sensitivity
Subjective interpretation
Requires hr
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13. Agar Gel Immunodiffusion Test (AGID)
I-Material
Equipment
Reagent/supplies
-refrigerator
- Freezer
-incubator or closed container
-autoclave, hot plate
-vacuum pump
-microscope illuminator
-template cutter
-micropipettes
-Agarose or pure agar(1%)
-NaCl (80g)
-PBS(0.01M)
-Reference Ag &Ab
-common lab. glassware
-Petri plates
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14. Agar Gel Immunodiffusion Test (AGID)
II-Preparation for the test
A-Personnel qualification /training
B-Preparation of equipment
C-Preparation of reagents /control procedures
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15. Agar Gel Immunodiffusion Test (AGID)
A-Personnel qualification /training
Personnel must be familiar with :
1-preparation and proper handling of reagents and biological materials.
2-clibration, maintenance, and use of instruments.
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16. Agar Gel Immunodiffusion Test (AGID)
B-Preparation of equipment
The equipment must be calibrated and certified according to the respective standard operating procedures (Sops).
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17. Agar Gel Immunodiffusion Test Reagent Sources (AIV):
NVSL, Ames, IA:
Domestic AIV surveillance – antigen and enhancement serum are available, free of charge
Export testing – available for sale
SPAFAS, Norwich, CT:
AIV antigen and enhancement serum available for sale
GD Netherlands:
Any reference lab:
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18. Agar Gel Immunodiffusion Test (AGID)
C-Preparation of reagents
/control procedures
1% agarose in PBS (0.01M, pH 7.2) with 8% sodium chloride
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19. Agar Gel Immunodiffusion Test Gel Preparation:
Dissolve the mixture by bringing to boiling, autoclave 10 min, cool to about 70-80C 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)
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20. 100 mm petri dishes – 15-17 ml agar

21. Agar Gel Immunodiffusion Test Gel Preparation
The agar can be stored in the flask at 45c for several months and milted and dispensed into plates as needed.
The liquid agar can be kept in a 455c water bath for several weeks and used as needed.
Don’t use agar if mold or precipitate is observed.
The plate should be used the same day they are poured.
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23. Template Dimensions Wells 2.4 mm apart Wells 5.3 mm in diameter
Agar 2.8 mm thick

26. Placement of Reagents/SamplesAs Ag As As
Sample #2
As = Positive reference serum
Ag = Antigen

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

29. Filling Wells

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

31. Hold Tip Vertical When
Filling Wells

32. Method Not Recommended

33. Fill All Wells As Ag
Sample #1
PBS

34. Agar Gel Immunodiffusion Test incubation
Cover the plate after filling and allow the plate few minutes before moving to reduce the possibility of spillage.
Incubate the plate in room temperature or in closed champer if needed for 24-48hr.
Try to read the result during hr.
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35. Diffusion of Reagents Ag As Seen as a precipitin line
when concentrations are
optimal

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

37. Reading and interpretation

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

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

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

41. Incomplete Line Formation+ -
Before 24 hours
At hours

42. Potential Positive Sample

43. Additional Problems
“Halos” + -

44. AS #3 #1 AG AS AS #2

45. #1 AS AS AG #2 #3 AS

46. #1 AS AS AG #3 #2 AS

47. #1 AS AS AG #3 #2 AS

48. #1 AS AS AG #2 #3 AS
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49. AS #4 #1 AG #3 #2 AS

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

51. Cutting Errors
May miss the weak
positives

52. THANK YOU
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