Rapid Salivary Test suitable for a mass screening program to detect SARS-CoV-2: A diagnostic accuracy study

Lorenzo Azzi, Andreina Baj, Tiziana Alberio, Marta Lualdi, Giovanni Veronesi, Giulio Carcano, Walter Ageno, Cinzia Gambarini, Lorenzo Maffioli, Salomone Di Saverio, Daniela Dalla Gasperina, Angelo Paolo Genoni, Elias Premi, Simone Donati, Claudio Azzolini, Anna Maria Grandi, Francesco Dentali, Flavio Tangianu, Fausto Sessa, Vittorio Maurino, Lucia Tettamanti, Claudia Siracusa, Andrea Vigezzi, Elisa Monti, Valentina Iori, Domenico Iovino, Giuseppe Ietto, ASST dei Sette Laghi Rapid Salivary Test Nurse staff Research Group, Paolo Antonio Grossi, Angelo Tagliabue, Mauro Fasano, Lorenzo Azzi, Andreina Baj, Tiziana Alberio, Marta Lualdi, Giovanni Veronesi, Giulio Carcano, Walter Ageno, Cinzia Gambarini, Lorenzo Maffioli, Salomone Di Saverio, Daniela Dalla Gasperina, Angelo Paolo Genoni, Elias Premi, Simone Donati, Claudio Azzolini, Anna Maria Grandi, Francesco Dentali, Flavio Tangianu, Fausto Sessa, Vittorio Maurino, Lucia Tettamanti, Claudia Siracusa, Andrea Vigezzi, Elisa Monti, Valentina Iori, Domenico Iovino, Giuseppe Ietto, ASST dei Sette Laghi Rapid Salivary Test Nurse staff Research Group, Paolo Antonio Grossi, Angelo Tagliabue, Mauro Fasano

No abstract available

Keywords: COVID-19; Coronavirus; Lateral flow assay; SARS-CoV-2; Saliva.

Conflict of interest statement

Declaration of Competing Interest The authors declare the absence of any conflict of interests. Alberio Tiziana, Azzi Lorenzo, Baj Andreina, Fasano Mauro and Lualdi Marta are the co-inventors of the Rapid Salivary Test described in this paper and of the Italian patent filing number 102,020,000,006,400 registered on 2020, March 26th.

Figures

Fig. 1

Rapid Salivary Test based on Lateral flow technique and its interpretation. The customized sandwich LFA was designed to detect the presence of SARS-CoV-2 in salivary samples using a polyclonal antibody directed against the viral Spike protein. The same anti-Spike antibody (αSpike) was differentially conjugated in order to work as either capture antibody or detection antibody in the sandwich. Universal nitrocellulose LFA strips were used to perform the immunochromatography test. The applied sample was a mixture of diluted saliva and conjugated antibodies, added with an internal validity control (biotin). LFA results were read based on the appearance of a red “Control-line” (C-line) and a red “Test-line” (T-line) on the strip. (a) The C-line on the strip consists of immobilized streptavidin. 40 nm gold-conjugated biotin (GOLD-biotin) is added to the sample. When the flow of the sample reaches the C-line, streptavidin binds biotin with high affinity and the red C-line appears on the strip (valid test). If the flow does not reach the C-line, the test is invalid. (b) The T-line on the strip consists of immobilized anti-Ulfa-tag antibodies (αUlfa). The capture antibody is conjugated with the Ulfa-tag (αSpike-Ulfa), while the detection antibody is conjugated with 40 nm gold particles (αSpike-GOLD). When the Spike protein is present (positive test), the antibody sandwich forms and the red T-line appears on the strip. By contrast, when the Spike protein is absent (negative test), the sandwich does not form and the red T-line is not detectable. (c) The LFA strip consists of a nitrocellulose membrane, containing a “Control-line” (C-line) and a “Test-line” (T-line). The test is “positive” (presence of SARS-CoV-2) when both red lines are visible. The intensity of the T-line can be qualitatively evaluated using a scoring card. The test is “negative” (absence of SARS-CoV-2) when only the red C-line is detectable. The test is “invalid” when the red C-line is not visible, regardless of the presence of the red T-line. (d) Example of a run with a positive result (on the left) and of a run with a negative result (on the right). Both of these runs were valid since the control line appeared. The scoring card of the commercial kit (Abcam cat# ab270537) is shown on the right.