Figure 1 Sampling sites in Isiolo (camels), Nairobi (cats and
dogs) & Machakos (camels) are indicated by the red dots.
Camels were sampled randomly as follows: 144 fecal specimens, 144 nasal
swabs and 144 blood samples collected into serum vacutainers. The camels
were identified through their owners whose consent was initially sought;
the animals were restrained using trained animal handlers and samples
taken. The blood samples were processed for serum the same day, labeled,
aliquoted into two portions and stored at -200C. The
duplicate samples were then transported to the Machakos University,
Department of Biological sciences laboratory and the other part
transported to the institute of Primate research, Kenya-snake bite
research center, Karen Kenya.
Samples from cats (16 samples) and dogs (36 samples) were collected at
the veterinary clinics on different dates. The consent was sought from
their owners before drawing of the blood. 6 ml of blood was drawn from
each animal onto a serum vacutainer and stored at 40C
until they were collected and transported to the Institute of primate
Research. The blood sera were transported in vacutainer tubes to the
Institute of Primate Research where the sera separation was done. The
sera were aliquoted and stored at -200C till use.
In order to ascertain that the samples were suitable for the ELISA
assay, we tested a selected number of samples with a commercial kit
based on purified SARS-CoV-2 receptor domain binding site specific ELISA
(PISHTAZ Teb) based ELISA. The kit has been optimized for human samples,
in our test we modified the kit to be able to detect camel, cats and
dogs samples by replacing the kit’s anti-Human IgG-HRP with either
anti-Llama IgG-HRP (for camels and dogs or Invitrogen anti-human IgG-HRP
(for cats samples). To ensure that our modified kits was working well we
used the kits confirmed human positive controls to ascertain that the
kit was also working on camel, cat and dog samples. The rest of the
procedure and reagents used was as described in the kits manual. We
eventually compared the results from the commercial kit to those
obtained using our crude lysate ELISA designed as described below.
Development of a robust in-house ELISA for SARS-CoV-2
antibodies in cats, dogs and camels
The SARS CoV-2 virus was isolated from a COVID-19 patient via
nasopharyngeal swab. The swab was then inoculated onto vero cells
cultured in DMEM media with 15%FBS & Pen/Strep and allowed to infect
the and grow in the cells. The presence of SARS-CoV-2 was confirmed with
RT-PCR reactions. The virus infected vero cells were then cultured in
DMEM media with 15%FBS & Pen/Strep. The cells were monitored and
harvested at 80% CPE. The harvested infected cells were broken down by
freeze thawing and under 5% SDS detergent to inactivate them and viral
particles was separated on sucrose gradient sedimentation solution and
resuspended in 100µl of DMEM media. The harvested virus was further
inactivated by three cycles of freeze thawing and then at 55 degree
Celsius for 1 hour and then stored at -200C. The crude
viral lysate was re- suspended in 400 µl ml of PBS and taken as the neat
lysate. The viral antigen titration curve was done by diluting the viral
lysate in the range of 5 x 10-1 to 5 x
10-10 with PBS. The 50 µl of titrated viral lysate was
used to coat the plates overnight at 40C for the
titration and determination of the appropriate dilution assay. Finally a
dilution of of 5 x 10-7 was adopted and used for
subsequent plate coating and further assays. Wells without coating (No
coats) were also included to compensate for nonspecific interactions of
the serum with other factors other than the antigen. Before addition of
the sera the excess antigen flicked out and the plate tapped upside down
on the soft tissue to remove the excess of the antigen.
In developing the assay further ELISA protocol developed byAmanat et
al., 2020 was adopted with a few modifications as described below.
First, in order to identify the positive and negative camel control sera
a aselected pre-COVID-19 and post COVID-19 camel samples were screened
using PISHTAZ Teb commercial kit. The camel sera was selected, titrated
in the range of 1/100, 1/500, 1/1000, 1/5000, 1/10 000 and 1/50 000, and
finally 1/1000 was adopted. Cat & dogs sera was titrated in the 1/5,
1/10, 1/50, 1/100, 1/500 and 1/1000 and finally 1/50 was adopted. Wells
with antigen coating but no sera (No Sera) were included to check on
nonspecific cross-reactivities. Positive controls sera from camel (C0874
& C1191), cats (CAT 2 & 4) and dogs (DOG 1 & 2) were identified and
used to check if the serum is binding on anything other than the
antigens. Serum dilutions were done in PBS /1% skimmed milk/0.1% SDS
and incubation was done for 1hr at 370C. For the
safety of technologist the animal samples/sera were inactivated at
550C and stored at -200C till used
for the ELISA assay.
The secondary step antibody used was either a reacting anti-Human IgG
HRP for Cats sera or anti-Llama IgG –HRP for camel and dogs samples,
the conjugate incubation was done for 1hr at 370C.
Washing of the wells was done five times with 200µl per well of
PBS/0.1% SDS. 100µl of substrate was used to develop the assay and
incubated for 30 minutes at 370C after which the
reaction was stopped with sulphuric acid and the plate read at 450nm.
To test if our crude lysate ELISA assay could be used for surveillance,
we collected a pool of randomly sampled animal sera from three different
animal species (Camel, n=144, cats, n= 16 and dogs, n=36) and three
different locations (Fig.1). The samples were screened with our crude
lysate ELISA assay for the presence of cross reacting antibodies and
compared with a commercial kits based on the purified recombinant spike
RBD antigen. The camel sera samples were used at 1/1000, while the cats
and dogs sera samples were used at 1/50.