TITLE: Immunoassay of SARS-CoV-2 in dogs and cats V.1
AUTHORS: Dumar A. Jaramillo-Hernández, María C. Chacón, María A. Velásquez, Adolfo Vásquez-Trujillo, Ana P. Sánchez, Luis F. Salazar Garces, Gina L. García, Yohana M. Velasco-Santamaría, Luz N. Pedraza, Lida C. Lesmes-Rodríguez

[DESCRIPTION]
This protocol describes the qualitative determination of IgG antibodies against the nucleocapsid (N) protein of SARS-CoV-2 in serum from domestic dogs and cats, the indirect ELISA (Enzyme-Linked Immunosorbent Assay) kit ID Screen® SARS-CoV-2 Double Antigen Multi-Species (IDvet, Grabels, France). 
The diagnostic kit detects antibodies against the nucleocapsid (N protein) of the SARS-CoV-2 virus. 
This test can be used on samples from dogs, cats, mink, ferrets, cattle, sheep, goats, horses and any other susceptible species requiring serum, plasma or whole blood samples.

[STEPS]
SECTION: Before you start
1. Collect the serum, plasma or whole blood samples from dogs, cats, mink, ferrets, cattle, sheep, goats, horses and any other susceptible species.
SECTION: Procedure
2. Allow the reagents to reach room temperature (21 °C ± 5 °C) before use. 
SECTION: Procedure
3. Homogenize all reagents and serum samples by immersion or vortexing.
SECTION: Procedure
5. Add 25 µL of Diluent 13 to the remaining wells.
SECTION: Procedure
6. Add 25 µL of the serum samples, which previously had been homogenized by vortexing, to the remaining wells (Figure 1).

  

SECTION: Procedure
7. Cover the plate and incubate for 45 minutes ± 5 minutes at 37 °C ( ± 2°C).
SECTION: Procedure
8. Empty the wells and wash each well 5 times with at least 300 µL of wash solution, avoid drying the wells between washes.
SECTION: Procedure
9. Prepare the 1X conjugate by diluting the 10X conjugate 1:10 with diluent 13.
SECTION: Procedure
10. Add 100 µL of the 1X conjugate to each well.
SECTION: Procedure
11. Cover the plate and incubate for 30 minutes at 21°C (± 5°C).
SECTION: Procedure
13. 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.
SECTION: Procedure
14. In the presence of antibodies, a blue coloration should appear (Figure 2), avoid drying of the wells between washes.
  

SECTION: Procedure
16.  Cover the plate and incubate 20 min ± 2 min at 21°C (± 5°C) in the dark.
SECTION: Procedure
17. Add 100 µL of stop solution to each well in order to stop the substrate reaction (Figure 3).
  

SECTION: Procedure
18. 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.
SECTION: Validation
19. 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
SECTION: Interpretation
20. 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
SECTION: Procedure
15. Add 100 µL of the developing solution into each well.
SECTION: Procedure
4. Add 25 µL of Negative control to wells A1 and A2 and 25 µL of Positive control to wells A3 and A4.
SECTION: Procedure
12. Empty the wells.