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.