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Agar Gel Immunodiffusion(AGID) Test Principles and Techniques Prof.Dr. Salah M. HassanCollege of Veterinary MedicineUniversity of Mosul

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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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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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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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Unbalanced Reagent
Concentrations
As too strong
Ag too strong
As
As
As
As
As
As
Ag
Ag

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Ag Ab precipitation curve
Increasing antigen concentration
Immune precipitate formed
Typical precipitation curve resulting from titration of increasing Ag concentration against constant amount of Ab
Zone of Ab excess
Zone of equivalence
Zone of Ag excess

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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 .

#2
AS
AS
AS
AG
#1
#3

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Immunodiffusion
Types Immunodiffusion
single Immunodiffusion
double Immunodiffusion
passive
active
J.Oudin 1946

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Agar Gel Immunodiffusion TestDefinition:
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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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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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 24-48 hr

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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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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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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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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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Agar Gel Immunodiffusion TestReagent Sources (AIV):
NVSL, Ames, IA:Domestic AIV surveillance – antigen and enhancement serum are available, free of chargeExport testing – available for saleSPAFAS, Norwich, CT:AIV antigen and enhancement serum available for saleGD Netherlands:AIV antigen and enhancement serum available for saleAny reference lab:

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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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Agar Gel Immunodiffusion TestGel Preparation:
Dissolve the mixture by bringing to boiling, autoclave 10 min, cool to about 70-80C and dispensePour plates on same day used15 – 17 ml (100 x 15 mm petri dishes)5 – 6 ml (60 x 15 mm petri dishes)

100 mm petri dishes – 15-17 ml agar60 mm petri dishes – 5-6 ml agar

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Agar Gel Immunodiffusion TestGel 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.

Agar 2.8 mm thick

Wells 5.3 mm in diameter
Wells 2.4 mm apart
Template Dimensions

Placement of Reagents/Samples

As
As
As
Ag
Sample #1
Sample #3
Sample #2
As = Positive reference serum
Ag = Antigen

Numbering Patterns

1
2
3
4
5
6
7
100 x 15 mm petri dish

Filling Wells

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

Hold Tip Vertical When Filling Wells

Method Not Recommended

Fill All Wells

As
As
As
Ag
Sample #1
PBS
PBS

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Agar Gel Immunodiffusion Testincubation
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 24-48 hr.

Diffusion of Reagents

Ag
As
Seen as a precipitin line when concentrations are optimal

Diffusion of Reagents

Ag
As
At 24 hours a
precipitin line is visible

Reading and interpretation

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

Examples of Positive Reactions

May be seen as light haze
+4
+5
Negative
+1
+2
+3

Examples of Nonspecific Lines

Negative
Negative
Negative
Line of non identity
Line of partial identity

Incomplete Line Formation

Before 24 hours
At 24 - 48 hours
+
+
-

Potential Positive Sample

Additional Problems
“Halos” +
+
+
+
-


#1
#2
#3
AG
AS
AS
AS



#1
#2
#3
AG
AS
AS
AS


#1
#2
#3
AS
AG
AS
AS


#1
#2
#3
AS
AG
AS
AS

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#2
AS
AS
AS
AG
#1
#3


AG
#1
#2
#3
#4
AS
AS

Unbalanced Reagent

Concentrations
As too strong
Ag too strong
As
As
As
As
As
As
Ag
Ag

Cutting Errors

May miss the weak positives

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sm.hassan04@gmail.com