Veterinary Epidemiology and Zoonosis

EXERCISE-15: AGAR GEL IMMUNODIFFUSION TEST

Agar gel precipitation tests and Immunoelectrophoresis

• When a soluble antigen combines with its antibody in the presence of electrolytes at a suitable temperature and pH, the antigen-anti­body complex forms an insoluble precipitate.
• When instead of sedimenting, the precipitate remains suspended as floccules, the reaction is known as flocculation.
• Precipitation can take place in liquid media or in gels such as agar, agarose or polyacrylamide.
• The amount of precipitate formed depends on the proportion of antigen and antibody present.
• If the amounts of precipitate in the tubes are plotted on a graph, the resulting curve will have three phases; an ascending part (prozone or zone of antibody excess), a peak (zone of equivalence) and a descending part (post zone or zone of antigen excess). This is called the zone phenomenon.
• Zoning occurs in agglutination and some other serological reactions also.
• The prozone is of importance in clinical serology, as sometimes sera rich in antibody may give a false negative precipitation or agglutination result, unless several dilution are made.
• Precipitation test is classified in to two,
  • Qualitative and
  • Quantitative

Mechanism of precipitation

• Multivalent antigen combines with bivalent antibody in varying proportions, depending on the antigen-antibody ratio in the reacting mixture.
• Precipitation results when a large lattice is formed consisting of alternating antigen and antibody molecules. This is possible only in the zone of equivalence.
• In the zones of antigen or antibody excess, the lattice does not enlarge, as the valencies of the antibody and the antigen, respectively, are fully satisfied. ­

Agar gel immunodiffusion test

• When soluble antigen is combined with its homologous antibody in wells cut in agar gels diffuse and form an opaque band of precipitate of the antigen-antibody complex at the zone of optimal proportions.
• AGID can be either used as quantitative or qualitative test.
• Example: Hydatidosis can be diagnosed by AGID.

Materials required	Quantity and purpose
Agarose (low EEO)	1g for 100mL (1%)
Normal saline	100mL for preparation of 1% agarose gel
Conical flask – 250mL	One number
Aluminium foil	To cover mouth of the conical flask
Stainless steel vessel	To keep the conical flask with agarose for boiling
Gas stove or micro oven	For boiling of agarose
Glass slide or glass petriplate with lid	Adequate number
Glass pipette	One number for casting gel on glass slide
Gel cutter – 4 mm diameter	For cutting gel
Template	For cutting wells
Micropipette and tips	For dispensing about 25 to 30µL of materials into wells
Humid box	Water-soaked cotton in a box to avoid drying of gel during incubation
Incubator	Optional (for incubation of contents at 37ºC)
Known positive antigen of zoonotic portance	30µL per test
Known negative antigen	30µL per test
Known hyperimmune serum against zoonotic disease suspected	30µL per test
Test serum from suspected case of zoonosis	30µL per test

Figure: Template for making wells on agarose gel for AGID test

Procedure

• Boil 1g of agarose (1% agarose) in 100mL of normal saline and boil by keeping the conical flask in a stainless steel vessel with adequate quantity of water to provide moist heat.
• Wait for some time to get down the melted agarose to the temperature of approximately 55ºC.
• Cast the melted agar gel on a clean and scratch free glass slide (~4mL); or petriplate.
• After solidification, cut wells with the help of the template and gel cutter. The inter-well distance should be about 4 mm.
• Remove the agar gel from wells with the help of needle.
• Place 25µL of known antigen (in the central well) for which the test serum to be tested.
• Place 25µL of known positive serum specific to the known antigen of zoonotic importance in one of the peripheral wells (positive control) and 25µL known negative serum in another peripheral well (negative control).
• Charge 25µL of test serum samples in rest of the peripheral wells.
• Incubate at 37ºC in a humid box for up to 72 hours. Cover the plate with the lid if petriplate is used.
• Read the result every 24 hours up to 72 hours of incubation.

Test serum sample  5 and 6 are positive

Figure: Schematic representation of AGID test

Interpretation of result

• Precipitation line will be formed in the positive control. Presence of precipitation line between test serum samples and known positive antigen of zoonotic importance is POSITIVE for the zoonotic disease suspected. Readings are facilitated by oblique light.