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.