DESCRIPTION
The EIA kit is used in the diagnosis of equine infectious anemia, revealing the serum precipitating antibodies specific to the EIA virus, using the agar gel immunodiffusion technique.

COMPOSITION
The EIA antigen: is a concentrated suspension of viral components obtained by cultivating an EIA virus strain on equine dermis cell cultures.
The EIA positive serum: it is a mixture of sera from horses with specific seroconversion to the EIA virus.
Diluent: sterile deionized water with a pH of 6.5-7.5.
The kit for the serological diagnosis of EIA using the immunodiffusion technique consists of:

DIRECTIONS FOR USE
I. The immunodiffusion test is performed in 1% agar gel prepared in borate buffer (NaOH 2 g; H3BO3 9 g; distilled water 1000 ml), pH = 8.6, distributed in 17 ml in Petri dishes with a diameter of 100 mm. Before use, check to make sure that it is not too dry or it is not covered by condensation.
II. In the sufficiently hardened agar, create wells (Fig. 1), using a mold with 7 wells (a central one and 6 peripheral ones), the diameter of the well is 5.3 mm and the distance between wells is 2.4 mm (OIE, Manual of Standards for Diagnostic Tests and Vaccines, rev. 2011, chapter 2.5.6).
Before filling the well, if necessary, remove the condensation fluid.
III. The distribution of reactants is as follows (Fig. 2):
- In the central well: the antigen (each bottle solubilized in 2 ml of diluent for 125/250 tests or in 2.7 ml of diluent for 185/370 tests);
- In wells 1, 3 and 5, the positive serum (each bottle solubilized in 5.4 ml of diluent);
- In wells 2, 4 and 6, the test sera.
Fill the wells all the way and evenly with a quantity of 43 µl/well for each of the reagents used.
Leave the dishes for a few minutes on the work table, then incubate them at 23-25°C in a closed, moist room.

INTERPRETATION OF THE REACTION
The reading is done 24, 48 and 72 hours after the contact of reagents.
The intensity of the reaction varies depending on the concentration of antibodies found in the test sera.
The reaction is interpreted in comparison with the reaction between the positive control serum and the antigen of the diagnostic kit.
The following situations can be encountered:

A. NEGATIVE REACTIONS (Fig. 3)
The control precipitation line joins the test sample well without bending.
The test sera do not give precipitation lines with the EIA antigen in the kit.

B. POSITIVE REACTIONS (Fig. 4)
The control precipitation line joins the line given by the test serum and the antigen in the kit, forming a continuous line

C. WEAK POSITIVE REACTIONS (Fig. 5)
The control precipitation line is slightly bent forming a line which is tangent or nearly tangent to the well with the test sample.
In order to confirm the diagnosis, it is necessary to repeat the reaction.

D. INTENSE POSITIVE REACTIONS (Fig. 6)
The control precipitation line continues close to the central well with a thick and diffuse precipitation line given by the reaction between the antigen and the test sample.
Testing the serum in 1/2 - 1/4 dilutions is recommended, in which case there is a clear and distinct precipitation line.

NECESSARY MATERIALS NOT SUPPLIED IN THE KIT
- Adjustable single channel pipettes (for 43 µl);
- Agar;
- Petri dishes with a diameter of 100 mm;
- Borate buffer with a pH of 8.6;
- The mold for making the wells in the agar gel;
- Laboratory glassware;
- 23-25°C thermostatic chamber,