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

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 USDA-APHIS

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. USDA-APHIS

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. USDA-APHIS

Unbalanced Reagent Concentrations As too strong Ag too strong As As Ag USDA-APHIS

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 USDA-APHIS

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 . USDA-APHIS

#1 AS AS AG #2 #3 AS

Immunodiffusion J.Oudin 1946 USDA-APHIS

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) USDA-APHIS

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 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/equipment Disadvantages: Semi quantitative Moderate sensitivity Subjective interpretation Requires 24-48 hr USDA-APHIS

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 USDA-APHIS

Agar Gel Immunodiffusion Test (AGID) II-Preparation for the test A-Personnel qualification /training B-Preparation of equipment C-Preparation of reagents /control procedures USDA-APHIS

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. USDA-APHIS

Agar Gel Immunodiffusion Test (AGID) B-Preparation of equipment The equipment must be calibrated and certified according to the respective standard operating procedures (Sops). USDA-APHIS

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: USDA-APHIS

Agar Gel Immunodiffusion Test (AGID) C-Preparation of reagents /control procedures 1% agarose in PBS (0.01M, pH 7.2) with 8% sodium chloride USDA-APHIS

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) USDA-APHIS

100 mm petri dishes – 15-17 ml agar

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. USDA-APHIS

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

Placement of Reagents/Samples As Ag As As 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

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

Hold Tip Vertical When Filling Wells

Method Not Recommended

Fill All Wells As Ag Sample #1 PBS

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 24-48 hr. USDA-APHIS

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 Ag #1 #2 #3

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

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

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

Potential Positive Sample

Additional Problems “Halos” + -

AS #3 #1 AG AS AS #2

#1 AS AS AG #2 #3 AS

#1 AS AS AG #3 #2 AS

#1 AS AS AG #3 #2 AS

#1 AS AS AG #2 #3 AS USDA-APHIS

AS #4 #1 AG #3 #2 AS

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

Cutting Errors May miss the weak positives

THANK YOU sm.hassan04@gmail.com USDA-APHIS