Methods
500 µL capillary blood was collected by fingerprick from each participant into an EDTA-coated microtainer tube. Blood samples were transported to the Kenya Medical Research Institute laboratories for same-day testing using the rapid test; blood was separated and plasma samples stored at –80 °C for ELISA testing. The rapid test has three lines on the nitrocellulose membrane: “C” control line, and “G” and “M” test lines. Monoclonal anti-chicken IgY antibody is coated onto the control line region, and monoclonal anti-human IgG antibody and monoclonal anti-human IgM antibody onto the “G” and “M” test line regions. Recombinant COVID-19 nucleocapsid protein conjugated with colloidal gold particles are used as detectors for the “M” and “G” test lines. SARS-CoV-2 antibodies in the specimen combine with recombinant COVID-19 nucleocapsid protein conjugated with colloidal gold particles, producing an antibody–antigen gold particle complex which migrates to the “M” and “G” test lines, and is captured by monoclonal anti-human IgG or IgM antibodies. A violet test line appears in the results window if SARS-CoV-2 antibodies are present in the specimen. 20 µL capillary blood was applied to the specimen well of the test device using the capillary tube provided. Three drops (90 µL) of buffer were added to the specimen well and the test result read at 10–15 minutes. For valid tests, a coloured band was observed at the C test line; the test was considered positive if coloured bands appeared at the M test line (IgM), G test line (IgG), or both.
The Wantai SARS-CoV-2 Ab ELISA is a two-step incubation antigen “sandwich” enzyme immunoassay for qualitative detection of antibodies to the SARS-CoV-2 spike protein receptor binding domain. Polystyrene microwell strips are pre-coated with recombinant SARS-CoV-2 antigen; during the first incubation, SARS-CoV-2 antibodies present in the sample are captured in the wells. The microwells are washed to remove unbound serum proteins, and recombinant SARS-CoV-2 antigen conjugated to horseradish peroxidase enzyme (HRP conjugate) added; conjugated antigen binds to captured antibody inside the wells during a second incubation. The microwells are washed to remove unbound conjugate, and chromogen solution added; in wells containing the antigen–antibody–antigen “sandwich” immunocomplex, colourless chromogens are hydrolysed by bound HRP conjugate to a blue coloured product which turns yellow after stopping the reaction with sulphuric acid. 100 μL of plasma sample was added to each well of the microwell plate and incubated at 37 °C for 30 minutes. Each well was washed to remove unbound antibody before adding 100 µL HRP-conjugated recombinant SARS-CoV-2 antigen, and re-incubated. Following a second cycle of washing, 50 μL each of chromogen solution A and B were added to each well and further incubated. The colour intensity of positive samples was measured using a wavelength of 450 nm with the cut-off at 1 nm.
The Platelia SARS-CoV-2 Total Ab Assay is a one-step antigen capture format assay using wells pre-coated with recombinant SARS nucleocapsid protein. Samples are pre-diluted and mixed with recombinant SARS nucleocapsid protein coupled with peroxidase (conjugate), and incubated in the wells. IgM, IgG and/or IgA antibodies present in the specimen form a complex between recombinant SARS-nucleocapsid protein and recombinant SARS-nucleocapsid protein coupled with peroxidase. After washing, the presence of immune complex is demonstrated after adding a chromogenic solution for colour development. 15 µL of plasma from each sample was pre-diluted and added to each well with conjugate, and incubated for one hour at 37° C. After a washing step, the colour development solution was added and incubated for 30 minutes at room temperature. The optical density (OD) was read at 450 nm within 30 minutes of adding stopping solution, with the cut-off calculated at 1 nm.