Immunoenzymatic assays
The screening for C. burnetii antibodies was done using an
indirect C. burnetii ELISA (Q fever C. burnetii antibody
test kit, IDEXX Laboratories, Switzerland), B. abortus , L.
Hardjo , N. caninum , and by BVDV antigen capture ELISA according
to the kit manufacturer’s instructions. After diluting test sera and
positive and negative controls to 1:400 in the kit wash buffer solution,
100 µL/well of each were dispensed into 96 microtiter plate wells
pre-coated with inactivated C. burnetii antigen. After incubating
the plates at 37◦C for 60 min, they were washed three times with the kit
wash buffer, 100 µL/ well of a peroxidase labelled anti-ruminant IgG
conjugate addedm and the plates then incubated for 60 min at 37◦C. The
plates were then washed three times with the kit wash buffer like before
and 100 µL/well of the TMB substrate added. After stopping the reaction,
the ELISA plates were read at rT in a spectrophotometer (Bio Tek
Instruments. Inc. Highland Park, USA) at 450 nm and the results then
expressed as a percentage of the ratio of the test sample
OD450 to the positive control OD450 (S/P
%). The test samples with S/P % ≥ 40% were interpreted to be positive
while those with S/P% < 30% were deemed negative.
For the indirect ELISA for B. abortus antibodies, 100 µL/well of
test sera, as well as the positive and negative controls, were diluted
1:10 in the kit sample diluent were placed in the 96 microtitre wells
and the plates tightly sealed. After incubating at 37◦C for 60 min, the
plates were washed three times using 300 µL/well of the kit wash
solution and 100 µL/well of peroxidase labeled anti-ruminant IgG
conjugate added. After tightly sealing the plates, they were further
incubated at 37◦C for 60 min. Following the incubation step, the plates
were washed three times with 300 µL/well of the kit wash buffer and 100
µL/well of 3,3’,5,5’-tetramethylbenzidine or TMB substrate then added.
After stopping the reaction, the plates read at 450 nm using a
spectrophotometer (Bio Tek Instruments. Inc. Highland Park, USA).
Samples having S/P % < 80 % were deemed negative while those
with S/P % values ≥ 80 % were determined to be positive.
The screening of test sera for L. Hardjo antibodies was done
using indirect ELISA (Leptospira Hardjo Ab bovine ELISA,
Demeditec Diagnostics, Germany) according to manufacturer’s
instructions. After washing the plates five times with 100 µL/well of
the kit wash buffer, 100 µL/well of test sera diluted at 1:100 in the
kit sample diluent as well as the negative and positive controls diluted
at 1:50 were separately dispensed into the microtiter plates coated withLeptospira antigen. The plates were then incubated at 37⁰C for 60
min after which they were washed five times with kit wash buffer, 100
µL/well of horse radish peroxidase labelled anti-bovine IgG conjugate
added, and the plates incubated for another 60 min at 37⁰ C. After five
washes, , 100 µL/well of TMB substrate were added and the plates
incubated at rT in darkness for 15 min. After stopping the reaction, the
plates were read at 450 nm in a spectrophotometer (Bio Tek Instruments,
Inc. USA). Test samples with S/P % ≥ 34% were considered positive forL. Hardjo antibodies while samples with S/P% < 34%
deemed negative.
The screening for N. caninum antibodies was done using an
indirect ELISA (Neospora caninum Antibody Test Kit, IDEXX
Laboratories, USA) following the kit manufacturer’s instructions.
Briefly, 100 µL/well of test samples diluted at 1:100 in the kit sample
diluent as well as undiluted negative and positive controls were
dispensed into microtiter plates coated with N. caninum antigen.
The plates were then incubated at rT for 30 min after which they were
washed four times with kit wash buffer and then 100 µL/well of
peroxidase labelled anti-ruminant IgG conjugate added and the plates
incubated at rT for 30 min. The plates were then washed four times with
kit wash buffer, 100 µL/well of TMB substrate added to each well, and
the plates further incubated at rT in darkness for 15 min. After
stopping the reaction, the plates were read using a spectrophotometer
(Bio Tek Instruments. Inc. Highland park, USA) at 630 nm. Test samples
with S/P % ≥ 50% were determined to be positive while those with S/P%
<50% were deemed negative.
The detection of BVDV antigen was done using an antigen capture ELISA
(Bovine Viral Diarrhoea Virus antigen test kit/serum plus, IDEXX
Laboratories, Switzerland) according to the manufacturer’s kit
instructions. Briefly, 50 µL of the detection antibodies were dispensed
into a microtiter plate wells pre-coated with monoclonal antibodies
specific for BVDV antigen. This was followed by 50 µL of the test sera
alongside the negative and positive controls. The plates were then
tightly sealed and incubated at 37⁰C for 2 h. The plates were then
washed five times with the kit wash buffer. After incubating at rT with
100 µL/well of peroxidase labelled anti-ruminant IgG conjugate for 30
min, the plates were washed five times with the kit wash buffer after
which 100 µL/well of TMB substrate were added to each well and the
plates incubated at rT for 10 min. After stopping the reaction, the
plates were read at 450 nm using spectrophotometer (Bio Tek Instruments.
Inc. Highland park, USA). The results were expressed as corrected OD
values (S-N) for each sample using negative control. Test samples with
(S-N) ≤ 0.300 were considered negative, while test samples with (S-N) ≥
0.300 were considered positive.