Anthrax (Bacillus anthracis), Molecular Biology of
317
will obviously not test positive in all three
reactions,
and
additional
methods
will
need to be used to confrm their identity.
Some anthrax detection methods look
For actual bacteria rather than For DNA.
One such method is a
fluorescent anti-
body assay
that has also been validated For
rapid identifcation oF
B. anthracis
.Inth
is
method, two fluorescently labeled mono-
clonal antibodies, one against the cell wall
polysaccharide antigen and one against the
capsule antigen, are incubated with bacte-
rial samples. The cells are then washed to
remove unbound antibody and scored un-
der a fluorescent microscope. Although
other bacillus strains express a similar
polysaccharide antigen, these bacteria lack
the poly-D-glutamic acid capsule and will
not score positive with both antibodies.
The combined assay was 99% specifc,
with 227 oF 230
B. anthracis
isolates scoring
positive, and 100% specifc, with none oF
the 56 non-
Bacillus anthracis
strains scor-
ing positive. It also could provide results
w
i
th
in3to6h
.Seve
ra
lc
l
in
ica
lsamp
le
s
that were positive For
B. anthracis
by the
real-time PCR assay were not detected by
this fluorescent assay, so the latter assay
may not be as sensitive. However, the an-
tibody assay requires less lab space and
sophisticated equipment and, thereFore,
may be useFul in an outbreak situation,
especially as a rapid confrmatory assay.
A
method
that
may
be
useFul
For
the detection oF exposure to
B. anthracis
in clinical samples is an enzyme-linked
immunosorbent assay (ELISA) that detects
antibodies to PA. Only individuals who
have been exposed to or vaccinated For
anthrax will show positive results in this
assay. In this ELISA, recombinant PA
is bound to a microtiter plate. Serum
From the test subject is then added to
the plate and incubated to allow anti-PA
antibodies From the serum to bind to
the plate. ±ollowing extensive washes, an
antihuman IgG antibody labeled with a
reporter molecule is used to measure the
amount oF anti-PA antibody bound to the
plate. The specifcity oF this assay can be
enhanced through competitive inhibition,
where
test
sera
are
incubated
in
the
presence or absence oF excess recombinant
PA
in
solution
beFore
analysis
in
the
standard ELISA. IF the antibodies detected
in the ELISA are truly specifc For PA
(and not just sticking to something in
the assay nonspecifcally), they should be
removed From the assay by this competitive
inhibition, and little antibody will bind to
the microtiter plate. This assay will be
used as part oF a panel oF tests For the
confrmation oF clinical human anthrax.
This
test
should
not
be
used
as
the
sole confrmation oF anthrax because it
is
known
that,
at
least
in
nonhuman
primates,
early
antibiotic
treatment
oF
an
anthrax
inFection
can
abrogate
the
development oF anti-PA antibodies.
3.2
How do You Tell One Strain from Another?
Not only is it important to be able to dis-
tinguish
B. anthracis
From related
Bacillus
species, it has also become important to
distinguish between diFFerent strains oF
B. anthracis
.ThisisimportantFortracking
the history oF an environmental outbreak
and in the investigation oF acts oF ter-
rorism that use
B. anthracis
. It has been
diFfcult to develop methods to distinguish
between
B. anthracis
strains because this
is one oF the most monomorphic species
known – which means that it has very little
genetic variation. OF 1221-A±LP Fragments
examined in 79
B. anthracis
isolates in one
study, 97% were monomorphic – no se-
quence variation was Found between the
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