Development of a Multispecies Double-Antigen Sandwich ELISA Using N and RBD Proteins to Detect Antibodies against SARS-CoV-2

Abstract

SARS-CoV-2 infects humans and a broad spectrum of animal species, such as pets, zoo animals, and nondomestic animals. Monitoring infection in animals is important in terms of the risk of interspecies transmission and the emergence of new viral variants. Economical, fast, efficient, and sensitive diagnostic tests are needed to analyze animal infection. Double-antigen sandwich ELISA has the advantage of being multispecies and can be used for detecting infections caused by pathogens that infect several animal hosts. This study aimed to develop a double-antigen sandwich ELISA using two SARS-CoV-2 proteins, N and RBD. We compared its performance, when using these proteins separately, with an indirect ELISA and with a surrogate virus neutralization test. Positive and negative controls from a cat population (n = 31) were evaluated to compare all of the tests. After confirming that double-antigen sandwich ELISA with both RBD and N proteins had the best performance (AUC= 88%), the cutoff was adjusted using positive and negative samples from cats, humans (n = 32) and guinea pigs (n = 3). The use of samples from tigers (n = 2) and rats (n = 51) showed good agreement with the results previously obtained using the microneutralization test. Additionally, a cohort of samples from dogs with unknown infection status was evaluated. These results show that using two SARS-CoV-2 proteins in the double-antigen sandwich ELISA increases its performance and turns it into a valuable assay with which to monitor previous infection caused by SARS-CoV-2 in different animal species.

Keywords: COVID-19; ELISA; SARS-CoV-2; cats; microneutralization; multispecies.

Figure 1

Optimization of the double-antigen sandwich ELISA. (a) Several concentrations of capture antigen (0.5 to 5.0 µg/mL) and detection antigen (0.2 (green bars), 0.4 (light blue bars), 0.8 (yellow bars), and 1.6 (purple bars)) were used to find the optimal combination of capture and detection antigen (N protein). Data are visualized to represent the maximal absorbance resulting from subtracting the mean of the positive controls’ absorbance minus the negative controls’ mean absorbance. In this experiment, human serum samples were evaluated. (b) To find the optimal conditions of the double-antigen sandwich ELISA using N protein, additional concentrations of capture (2 and 3 µg/mL) and detection (3 and 5 µg/mL) antigen were evaluated. Blue dots represent the positive controls, and the green dots represent the negative controls. (c) Optimization of the double-antigen sandwich ELISA using RBD with 2 µg/mL capture and detection antigen and sera from negative controls (Neg, light brown dots), individuals vaccinated and infected (Vac + infect, orange bar and dots), and vaccinated individuals red bar and dots. (d) Optimization of the double-antigen sandwich ELISA using N and RBD as capture (2 µg/mL, 1 µg/mL of N, and 1 µg/mL of RBD) and detection (2 µg/mL, 1 µg/mL of N and 1 µg/mL of RBD) antigens. In this case, we used sera from negative (Neg, green dots and bars), vaccinated and infected individuals (Vac + infect, light blue dots and bars), and infected (Infect, pale yellow dots and bars) humans.

Figure 2

Double-antigen sandwich ELISA using RBD and N proteins was used to evaluate samples from tigers, rats, and dogs. Serum samples from tigers (pink dots), rats (purple dots), and dogs (blue dots) were assessed. Each point represents a sample from tigers (n = 2), rats (n = 51), or dogs (n = 45) whereas each line within groups represent the mean. The dotted line indicates the cutoff established at 0.3615.

Figure 3

Correlation coefficient of reproducibility analysis. Laboratory 1 and Laboratory 2 evaluated 20 identical samples, including positive (n = 9) and negative (n = 11) samples, using the double-antigen sandwich ELISA with RBD and N proteins.