Inmunodiagnostico AGID
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Transcript of Inmunodiagnostico AGID
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Agar Gel Immunodiffusion(AGID) Test
Principles and Techniques
Dennis A. [email protected]
USDA, APHIS, VS,
National Veterinary Services Laboratories,
Ames, IA 50010
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USDA-APHIS
Agar Gel Immunodiffusion TestDefinition:
• 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)
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USDA-APHIS
Agar Gel Immunodiffusion Test (AGID)
Advantages:• Group-specific test (detects
antibodies to all subtypes of flu-A, determine antigenic relationships)
• Easy, requires few reagents/equipDisadvantages:
• Semi quantitative• Moderate sensitivity• Subjective interpretation• Requires 24 hr
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100 mm petri dishes – 15-17 ml agar60 mm petri dishes – 5-6 ml agar
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USDA-APHIS
Agar Gel Immunodiffusion TestGel 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 used15 – 17 ml (100 x 15 mm petri dishes)5 – 6 ml (60 x 15 mm petri dishes)
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Agar 2.8 mm thick
Wells 5.3 mm in diameter
Wells 2.4 mm apart
Template Dimensions
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USDA-APHIS
Source of Template Cutters
Veterinary Diagnostic Technology, Inc.4890 Van Gordon St., Suite 101Wheat Ridge, Co. 80033Phone: (303) 467-2741Fax: (303) 467-2799
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IN
OUT
VACUUM PUMP
VACUUM ASSEMBLYSingle hole stopper
Side-arm flask
Glass or plasic tubing
Vacuum hose
12 to 14 gauge cannula
Vacuum set at12 to 20 psi
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Placement of Reagents/Samples
As
As As
Ag
Sample #1Sample #3
Sample #2
As = Positive enhancement serumAg = Antigen
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Numbering Patterns
1 2
3 4 5
6 7100 x 15 mm petri dish
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USDA-APHIS
Agar Gel Immunodiffusion TestReagent Sources (AIV):
• NVSL, Ames, IA:Domestic AIV surveillance – antigen and enhancement serum are available, free of chargeExport testing, international – available for sale
• Charles River (SPAFAS), Norwich, CT:AIV antigen and enhancement serum available for sale
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Filling Wells
50-60 µl
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Cross section of wellsshowing reagent levels
CorrectOverfilled
Underfilled
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Hold Tip Vertical WhenFilling Wells
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Method Not Recommended
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Fill All Wells
As
As As
Ag
Sample #1PBS
PBS
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Diffusion of Reagents
Ag As
Seen as a precipitin linewhen concentrations areoptimal
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Diffusion of Reagents
Ag As
At 24 hours aprecipitin line is visible
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Precipitin Lines After 24 HoursIncubation (Negative samples)
AsAs
As
Ag
#1
#2
#3
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Examples of Positive Reactions
May be seen as light haze
+4
+5
Negative
+1
+2
+3
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Examples of Nonspecific Lines
Negative
Negative
Negative
Line of non identity
Line of partialidentity
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Incomplete Line Formation
Before 24 hours At 24 - 48 hours
+ +
-
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Potential Positive Sample
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Additional Problems“Halos”
+
++
+
-
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#1
#2
#3
AG
ASAS
AS
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#1
#2#3
AG
AS
AS
AS
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#1
#2#3
AS
AG
AS
AS
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#1
#2#3
AS
AG
AS
AS
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#2
AS
AS AS
AG
#1
#3
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AG
#1
#2#3
#4
AS
AS
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Unbalanced ReagentConcentrations
As too strong Ag too strong
As
As
As As
As
As
Ag Ag
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Cutting Errors
May miss the weakpositives