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) .