Immunoassay of SARS-CoV-2 in dogs and cats V.1

Collect the serum, plasma or whole blood samples from dogs, cats, mink, ferrets, cattle, sheep, goats, horses and any other susceptible species.

Allow the reagents to reach room temperature (21 °C ± 5 °C) before use.

Homogenize all reagents and serum samples by immersion or vortexing.

Add 25 µL of Negative control to wells A1 and A2 and 25 µL of Positive control to wells A3 and A4.

Add 25 µL of Diluent 13 to the remaining wells.

Add 25 µL of the serum samples, which previously had been homogenized by vortexing, to the remaining wells (Figure 1).

Cover the plate and incubate for 45 minutes ± 5 minutes at 37 °C ( ± 2°C).

Empty the wells and wash each well 5 times with at least 300 µL of wash solution, avoid drying the wells between washes.

Prepare the 1X conjugate by diluting the 10X conjugate 1:10 with diluent 13.

Add 100 µL of the 1X conjugate to each well.

Cover the plate and incubate for 30 minutes at 21°C (± 5°C).

Empty the wells.

Add at least 300 µL of wash solution so that this substrate reacts with the secondary antibody enzyme and provides a visible signal that can be quantified.

In the presence of antibodies, a blue coloration should appear (Figure 2), avoid drying of the wells between washes.

Add 100 µL of the developing solution into each well.

Cover the plate and incubate 20 min ± 2 min at 21°C (± 5°C) in the dark.

Add 100 µL of stop solution to each well in order to stop the substrate reaction (Figure 3).

Finally, the optical density (OD) is read in a Cytation 3 multimodal microplate reader (BioTek Instruments, Inc. Winooski, VT, USA) using a wavelength of 450 nm.

The assay is validated if:

• The mean value of the Positive Control O.D. (DOcp) is greater than 0.350.

DOcp > 0.350

• The ratio of the mean of the optical density values of the positive and negative controls (DOcp and DOcn) is greater than 3.

DOcp/DOcn > 3

After the laboratory reading, the sample/positive control (S/P) ratio was calculated with the data obtained, which was expressed as a percentage, using the following formula:

Samples presenting a S/P%:

• Less than or equal to 50% are considered negative.

• Between 50% and 60% are considered doubtful.

• Greater than or equal to 60% are considered positive.

Results Status

S/P % ≤ 50 % Negative

50 % < S/P % < 60% Doubtful

S/P % ≥ 60 % Positive