Production and Characterisation of specific Antibodies to Bovine Interleukin 5 and 6 and their use in the Development of Sensitive Elisa for their Measurement
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Date
2013-08-12
Authors
Arodi, Washingtone Ouma
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Lack of food especially animal proteins is a major problem in the developing world.
Livestock provide the bulk of these proteins hence the need to increase the supply
through improved livestock health
.
Enzyme Linked Immunos
orbent Assays (ELISA) for
bovine interleukin 5 and interleukin 6 (boIL
-
5 and boIL
-
6) could be applied
for
analyses
of immune responses and advance understanding of a variety of diseases of cattle.
Biological and biochemical characteristics of monoclonal an
tibodies (mAbs) and
polyclonal antibodies (pAbs) against rboIL
-
5 and rboIL
-
6 are described as well as their
use in developing specific and sensitive ELISA for the detection and measurement of
these cytokines.
The ob
j
ective of the study was to produce and
characterize
an
tibodies
(Abs)
to boIL
-
5 and 6 and to use the
se
Abs
in developing sensitive ELISA
to measure
the
respective
cytokine
s
.
Four murine anti boIL
-
5, four murine anti boIL
-
6 mAbs, two
rabbit anti boIL
-
5 and two rabbit anti boIL
-
6 pAbs were
prod
uced
and their binding
specificities
determined
by
in
direct ELISA, Western blotting
, Ouchterlony‘s double
immunod
iffusion
technique
and flow cytometry. Sandwich ELISA for boIL
-
5 was
developed using mAb
25
.1 as the capture antibody and the pAb R
1
as the det
ector
antibody. Sandwich ELISA for boIL
-
6 was also developed using mAb
56.2
as the
capture antibody and pAb R
6
as the detector antibody. The developed ELISA was used
to detect both recombinant and natural boIL
-
5 and boIL
-
6 present in supernatant of
periph
eral blood mononuclear cells (
PBMC
)
stimulated with
Concanavalin A (
Con A
)
and other
mitogens. The
ELISA developed was able to detect as little as 48pg/ml of
boIL
-
5 and 24pg/ml of boIL
-
6
. Using this method it was possible to detect native boIL
-
5 and boIL
-
6 in supernatants of PBMC
from
cattle
as well as
in
cell lines
infected
with
Theileria parva,
indicating the applicability of the
se
assay
s
to a number of
in vitro
culture systems.
Some of the antibodies also showed promising results in intra cellular
cytok
ine staining and flow cytometry.
It is therefore recommended that these
immunoassays be further optimized with a view to making them available for
commercial use.
Description
SF 961 .A72