Figure 2: Titration of Crude SARS COV-2 Lysate antigen. Shown on the y-axis is the ELISA OD scores, while the X-axis shows the crude lysate titrations. The serum for the o titration gradient was used at 1/10 for Cat, 1/10 for Dogs sera and 1/1000 for the Camel sera. The camel and cats titres were higher than those of the dogs.
b) Screening of positive and negative controls against best performing Secondary HRP conjugated antibodiesTo check if anti-Llama IgG conjugated with HRP could also cross react with dogs and cats sera and also if anti-human IgG conjugated with –HRP could cross react with camel, cats and dogs sera, we coated the ELISA plates with the crude lysate. The wells were then probed with sera from camel, cats and dogs. The sera were then detected with both Anti-Llama IgG-HRP and anti-Human IgG-HRP. The rest of the conditions were as described in Amanat et al., 2020. Anti-Llama IgG Conjugated – HRP detects /cross reacts with dogs antibodies whereas Anti-human IgG Conjugated – HRP with dogs antibodies do not. Using crude lysate ELISA, it’s possible to discriminate positive & negatives on dog samples: D1, D2, D3 and D5 dogs’ samples are positive (figure 3). Crude lysate ELISA can pick positive & negatives camel sera: Camel D8-874 react positively with both anti-human IgG-HRP and anti-Llama IgG- HRP-conjugated antibody while the negative camel sera (D0) 0852 remains negative.
Figure 3: Bar graph showing positive cat, dogs and camel Sera detected either with Secondary second step anti-human IgG HRP or anti-Llama IgG HRP conjugated antibodies. The selected anti-human IgG conjugated with HRP can discriminate between positive sera (CAT1 & 2) and negative cat sera (CAT3, 4 &5) samples, while anti-Llama IgG-conjugated HRP cannot discriminate between positives and negative cats sera. cat sera dogs sera (D1, 2, 3 & 5). Anti-human IgG conjugated HRP does not detect Camel sera therefore doesn’t distinguish between positive and negatives (C0852-Antihuman IgG HRP, C874 anti-Human) while anti-Llama IgG HRP does detect camel sera and can discriminate between a SARS CoV-2 positive sera (C874 –Anti-Llama IgG-HRP from the negative sera(C0852-Anti-Llama IgG HRP)
c) Titration of serum samples (camel, dogs and cats) against Crude SARS-CoV-2 lysate antigen
To determine the right sera dilution to use for assay and to help avoid non-specific antibody-antigen interactions, we titrated the three serum samples (camel, cats and dogs sera). Camel sera was titrated in the range of 1/100, 1/500, 1/1000, 1/5000, 1/10 000 and 1/50 000, cats and dogs sera was titrated in the 1/5, 1/10, 1/50, 1/100, 1/500 and 1/1000. The diluted sera was then used to perform the ELISA assay and detection of the antibodies was achieved by either Anti-Llama IgG –HRP for Camels and dogs samples while the cats antibodies were detected using anti-Human IgG–HRP.
The camel sera C1191 and C0874 had high titers such that even at a higher dilution series they were still positive for SARS-CoV-2 and it titrated out confirming the positive detection of SARS COV-2, while C0852 remained constantly low confirming its negativity (figure 4a).. The positive SARS CoV-2 cats samples (CAT2, & CAT4) also titrated out while the negative cat samples remained constant low suggesting that the test is capable of discriminating between cats SARS CoV-2 positive and negative sera (figure 4b). The same dynamics was seen with the dogs samples where DOG 1 & 2 shown positive titration trend in detection of SARS COV-2 while DOG4 sera remained negative suggesting the capability of the system to positively detect dogs samples (figure 4c) .