AD Antigen

Manufacturer: United

U.s. Vet. Lic. No.

245

AD Antigen Indications

Aleutian disease (AD) is a virus disease of mink characterized by glomerulonephritis, arteritis, generalized plasmacytosis, hypergammaglobulinemia and infectious immune complexes. This cell culture propagated antigen, when used in the counterelectrophoresis (CEP) test, provides a rapid serological test for detecting the presence of antibody to AD.¹ It contains 8 or 16 “units” per 0.01 mL.

Test Principles

Detection procedure: AD may be manifested at certain stages by exhibiting elevated globulin levels. This observation led to the first detection procedure, the iodine agglutination test. ² A more specific test was developed which detects antibody to AD by CEP, ¹ and this method provides a reliable and rapid diagnostic tool throughout the life span of the mink. ³

Principal: Mink blood, obtained by clipping a toenail and collecting the blood in a capillary tube, is centrifuged to obtain serum. Antigen prepared by extraction from AD infected mink, ¹ or cell culture, ⁵ will combine with specific antibody to form a visible precipitin line in an agarose gel. ^4,6 The CEP test utilizes the relatively rapid electrophoretic mobility of the antigen and the electroendosmotic properties of the antibody in agarose gel. The antigen migrates toward the anode, whereas, the antibody migrates toward the cathode, resulting in the development of the precipitin lines within 60 minutes.

Test Procedure

Equipment: The CEP test for AD may be performed on a variety of commercially available CEP equipment. A power supply compatible with the electrophoresis cell and test plate, chosen on the basis of convenience, reliability, and in keeping with the number of samples to be run per day, constitute the basic tools. A number of manufacturers also provide viewers. These provide oblique illumination against dark background for easier observation of precipitin lines. A bacteria colony counter may also serve this purpose.

Expendable supplies: CEP test plates are prepared from a high quality grade of agarose (not agar) with tris barbital buffer of ionic strength compatible with the balance of the test system. They may require wicks or be of the self-wicking type. Suitable test slides in a variety of sizes may be prepared by the user. Wells, usually 3.0 mm in diameter and a 10 microliter capacity, may be punched with commercially available or custom made punches. Specific information on sources for supplies may be obtained by writing United Vaccines.

Directions for use:

A. Titer (strength): The AD reagent has been prepared to contain eight (8) or 16 “units” of antigen per 10 mcL (0.01 mL) using a control serum which is positive at a 1:160 dilution. One hundred CEP assays may be performed per 1 mL of eight (8) “units” reagent. 200 tests may be performed per 1 mL of concentrated 16 “units” reagent (recommended dilution); for example (16 “units” only), in 10 microliter wells use one part antigen plus one (1) part saline or CEP buffer; maximum recovery of antigen may be made by rinsing the vial with physiological saline or CEP buffer; for convenience, the appropriate diluent may be added directly to the reagent vial. The titer of the antigen can be influenced by buffer, pH, and the quality of the agarose used for test plates.

B. Procedure:

1. CEP plates (slides) should be prepared as follows:

a. Place clean slides on a leveling table.

b. Prepare agarose by dissolving in distilled water to make a 1.4% to 1.8% solution (the grade and concentration of agarose influences the migration rate of the antigen and antibody). Add warm (approximately 60°C) buffer (gelman buffer at 18 g/1,200 mL H₂O) in a volume equal to the agarose. This results in 0.7% to 0.9% agarose in buffer, pH 8.6. Sodium Azide (NaN₃) at 0.03% may be added as a preservative. Hold in water bath (60°-70°C) with occasional stirring.

c. Dispense the maximum amount of agarose per slide which will not spill over the slides, producing a layer approximately 3 mm deep (17.0 mL per slide, 82 x 101 mm). Allow to gel and store in a humidified container.

d. Punch wells in pairs, 3 mm in diameter and 3 mm to 7 mm apart (inside edge to inside edge). Remove plugs by vacuum aspiration or other mechanical means.

2. Centrifuge the blood, which has been collected in a capillary tube, for three (3) to five (5) minutes.

3. To load the sample into the CEP plate, break the capillary tube at the serum/clot interface and touch the serum containing portion to the bottom of the well on the anode (+) side of the plate. If the serum does not leave the tube by capillary attraction, a small rubber bulb may be used to expel it.

4. Add the AD™ Antigen to the test wells on the cathode (-) side of the plate. This may be accomplished manually with a capillary tube or with a semi-automatic dispensing syringe.

5. Positive control serum should be placed in one well on each plate. Users wishing to gain experience in performing the CEP test should use strongly and weakly positive, as well as negative, control sera. These are available upon request from United Vaccines.

6. Fill the reservoirs with an appropriate volume of buffer, place the test plate on the CEP apparatus in accordance with the manufacturer's directions, and position wicks if the system requires this manner of contact. Adjust power to four (4) to six (6) volts per cm to obtain clear precipitin lines in 40 to 60 minutes.

Reading and interpretation of results:

A. When the migration of reactants is complete, a precipitin line will be visible at the positive control position and at other wells which contain AD positive serum.

B. Precipitin lines will be clearly visible if the plate is illuminated bilaterally from below and viewed against a dark background. Staining or rinsing are optional.

C. Precipitin lines will generally appear as sharp, gray lines halfway between the serum and antigen well, or closer to the serum well. The position of the lines (as well as titer of the reagents) is influenced by buffer, pH, and the qualities of the agarose. Lines may be straight or slightly curved.

D. If the results are obscured by hemolysis, the precipitin lines will become more clearly visible by soaking the plates 15 minutes to 18 hours in 2% saline solution.

Precaution(s)

Stability of antigen: The antigen may be stored in an upright position at 2°-7°C. For extended storage after first use, it may be frozen in an upright position. Freezing and thawing should be limited to not more than two cycles.

AD Antigen Caution(s)

A. Incinerate or autoclave final container and all unused contents. Disinfect test plates after each use.

B. AD^® Antigen is potentially infectious to mink of all genotypes and should be used only as an in vitro diagnostic reagent.

C. Use only high quality agarose (Bio-Rad Standard Low-mr, Sea Kem ME, Litex HSA). Pretest in the 0.7 to 0.9% range to determine the concentration for optimum sensitivity.

D. Buffer should be in the range of pH 8.6 to 8.8.

E. Use clean glassware and avoid contamination of the antigen with mink serum or the mink serum with antigen.

F. Excessive voltage across the CEP plate will distort the precipitin lines or melt the agarose. Four (4) to six (6) volts per cm will generally produce precipitin lines from strongly positive sera in 30 minutes and from weakly positive sera in 60 minutes.

References

Available upon request.

Presentation

2 x 1,000 test vials.

Nac No.

11040121