Download Antibodies in plantscience

Acta Bot.
Need.
40(1), March 1991,
p.
1-27
Review
Antibodies
in
plant science
M.
D.
Dewey
D.
Horsley
Evans
and
C.
Coleman
J.
R.
Priestley
R.
Hull
Hawes
;*
Department ofPlant Sciences, University of Oxford, South Parks Road, Oxford 0X1
*
School
of Biological
Oxford 0X30BP.
3RA
UK and
and Molecular Sciences, Oxford Polytechnic, Gipsy Lane, Headington,
UK
CONTENTS
Introduction
Antibodies in
1
plant biochemistry
Preparation
of
and molecular
antigens
2
Native protein, synthetic peptides
expressed
in bacterial
Monoclonal
versus
Purification of
2
biology
plant proteins
or
3
systems?
polyclonal
antigens,
3
criteria of
purity
and
hapten
3
conjugation
Screening
antibodies
6
Purification of antibodies
7
Uses of antibodies in molecular
Immunodiagnostic
assays for
Immunodetection of plant
biology and biochemistry
8
plant pathogens
8
viruses
9
Immunodetectionof phytopathogenic
bacteria and
9
phloem-restricted prokaryotes
Immunodetectionof
Commercial
plant-invading fungi
10
potential
15
Immunocytochemistry
15
17
Light microscope immunocytochemistry
Whole cell techniques
17
Sectioning techniques
18
Visualizing
Future
the
19
antigen
developments
21
Immuno-electron microscopy
Post-embedding
Other
21
labelling
21
23
techniques
Conclusions
23
Key-words: antibodies, immunochemistry, immunocytochemistry,
immunodiagnostics.
INTRODUCTION
Antibodies are useful for the
extracellular constituents of
detection,
plants
measurement
and of their
and
purification
pathogens.
1
Their
of intracellularand
high sensitivity coupled
2
M. DEWEY
with
potential for high specificity,
a
in whole
given antigen
Antibodies
useful in
are
cellular constituents
other
changes
growing
disease
where
niques
be
cell
Antibodies
of
or
is also
plant pathology
and
to
tech-
immunological
outline
to
We do
problems.
and
and
techniques
seek
not
to
an
reviews
general texts,
be useful in research in
to
and
provide
plant
pathology.
AND
method for
specific
very
They
also be used
can
not
binding
the
identification and
specific inhibitors,
as
surprising
also
on
and
consideration of the
antibodies
in
basis for
a
are
and molecular biology.
biochemistry
a
provides
that antibodies
in Table 1 and include work
deal with
use
problems likely
of
use
and
have found
we
biology
Antibody-antigen
presented
will
of
variety
hydrates, growth regulators, signal receptors
produce
localize key
because
frequently results in steric effects which inhibit the activity of the
tool in both
powerful
scientists
BIOCHEMISTRY
constituents.
It is therefore
antigens.
discussion
a
tool for identificationof
specific
where the
areas
plant
a
techniques
molecular
sensitive
a
antigen
to an
those
to
with
PLANT
plant
of interest.
cation are
and
to
of
material.
BIOLOGY
provide
measurement
and
biology
amount
plant
quantify developmental
to
Their usage in
powerful
a
of
extracts
of the literaturebut instead have included those
IN
MOLECULAR
cation of
identify
dealing
biology,
ANTIBODIES
antigen
to
invaluable aid
which illustrate the
biochemistry,
binding
seek
we
an
coverage
papers
proteins, etc.),
gene products.
detection provides
available for
approaches
exhaustive
identify specific
antigen
review,
can
structural
purified
cell and molecular
plant biochemistry,
(enzymes,
to
indicate the presence and
to
and in crude and
early stage.
at an
In this
and
and
be used
can
cells, tissues,
ETAL.
plant
to
be
a
useful
of their appli-
proteins,
carbo-
other cell constituents. Further
strategies
biochemistry
be encountered in their
Examples
enzymes, structural
many
affinity purifi-
proving
and
which
can
molecular
be
employed
to
and
the
biology
will
biology,
use.
Preparation of antigens
In
general,
be
a
the
cation of
yields)
a
antigen
for
point
for
permit
mixed
An
(although
will be
for workers in
antibody
possible
and
(providing
alternative
preparation
Where the
from
approach
an
this does
homology
the
problems
less
likely
to
possible
of
antigens
of contaminant
be present in
use
of
purification
antigens
antibodiesraised
between
antigens,
plant
from alternative
the
such that a
alternative source,
perhaps
species
as
fractions
possible
so
of
that
a
purified
to
prohibitively
also
a
purify
higher
possible
are
small
starting
starting
available
antibodies from
a
to
a
plant tissue is its
from another
possible,
kingdom.
it eliminates many of
mammalian contaminants
much
higher yields
litre antibodies
may
mixed
from
tissue
makes it
species
or
are
antigen
from
(for instance)
from another
against
result in
monospecific antibody is obtained.
cells. In many instances,
sources,
not
screening procedures
it is also
interest;
careful
to
and molecular
number of different criteria) is the best
production. Antigen-enriched
polyclonal antibody preparation
purifying
a
monoclonal antibodies if subsequent
selection of antibodies of
Similarly,
plant biochemistry
necessarily completely pure) antigen. Frequently, purifi-
not
antigen (determined using
pure
point
are
starting point
well defined
can
well be useful in
of
are
antigen
be obtained.
screening
plant antigen preparation.
and
ANTIBODIES IN PLANT SCIENCE
Native
The
protein, synthetic peptides
techniques
of
plant
biology
immunogen
in addition to
protein!)
available, peptide sequencing
tion of
are
These will
antigen.
which
produced
be
a
but
proteins.
the
of
plant proteins expressed
ability
expression libraries, generally
and
Sambrook
identifying
al.
et
1989)
is
expressed protein
are
available;
the
and the
plant
it will
as
tertiary
will
antigen
use
of the
required
Monoclonal versus
A
good
other
of native
to
methods,
forms and
ease
production
as
of
amount
production
and
a
new
which do
are
not
be obtained,
pure
as
a
to
several:
serum
animals
vary in
permitting study
selective
of
and/or
be
to
are
the
be
to
may prove
advantages
over
extracted/purified
polyclonal antibody
and
quite large (20-50 ml)
litre. The
goats)
yet
of further
production
well result in
amounts
the
available can
but few laboratories have the
and molecular
of
of
of antibodies is
antibody
may
to
just
biology require
polyclonal
possible
be obtained
one
epitope
screening protocol
to cost
one
if that
ofpurity
techniques
Affinity chromatography
time in
epitope
epitope
It may also lead
be used.
can
and
to a
may
is
over
Their
many years
of the
antigen
Monoclonal antibodies have
production,
the
lead to lack of
fact that
a
given
apparent specificity
altered, for instance by fixation
a
can
need not be
litre apparently lower than for
recognize
and
signifi-
a
antisera.
structure/function relationships; immunogen
in addition
criteria
equivalent
antibodies directed
antigen recognition
variety
many
ideal for
are
Affinity purification
than
resources
properties;
Purification ofantigens,
wide
likely
expressed protein.
two
in many
available is limited and
plant biochemistry
in
antiserum which contains antibodies that
nature.
the last
antigen
Rabbits
(e.g. sheep,
large quantities
eletrophoretic procedures.
A
plant protein
but this is
specific high-titre preparation.
however,
loss of
(e.g.
bacterially
small. However, unlike monoclonal antibodies, the
antibody preparation only recognizes
or
of
of native
series into another rabbit and
may
injection
investment
highly
drawbacks
of
available is
serum
handle such animals.
to
very
greater
advantages
handling.
of
relatively
are
Monoclonal antibodies for
cantly
and
quantity
by using larger
a
use
may differ from that in the
affords
frequently
litre, recognition
of antibody-containing
needed
yield
can
of
of antibodies with differentproperties
be increased
resources
terms
preparation
the total
antibody requires
The
elsewhere
impossible.
proves
antiserum
polyclonal
least in
immunization doses
total
plant protein
of such
preparation
and authenticate the
using;
a
permits
Drawbacks of the method
expressed protein
produce
of interest
polyclonal
monospecific
at
for
amounts
as
are
obtained from cDNA
are
require further purification
excess.
used
which is present in
presented
are
produc-
synthetic peptides
antigen
These
small
only
sufficient
then be
can
not one
the
protein
Given sufficient resources, all three methods are worth
successful ifthe
and
encoding
the
be present in
structure
and effort
cost
that
ensure
be further considered here.
not
useful alternative when
a
that the
possibilities
will
which
coli; methodologies
clones that express
and
to
in bacterial systems.
in Escherichia
protein produced
straightforward
more
of the
obtaining
(and
sufficient informationfor the
yield
cDNA
identify
to
for
routes
cells. If facilities
plant
synthetic peptides
unique region
of
libraries
will
alternative
provided
from
is needed
care
variety
use
of
amounts
pure,
represent
The
have
purification directly
comparatively large
in bacterial systems?
plant proteins expressed
or
molecular
3
number of sites in the
or
in
polyclonal
antigen.
hapten conjugation
are
available
is
particularly
to
purify
useful
as
antigens,
a
depending
method for
rapid
on
their
and efficient
4
M,
Table
1. Some
of uses of antibodies in
examples
Antigen
plant biochemistry/molecular biology
Antibody
Plant growth substances (indole-
Reference
type
Monoclonaland
3-acetic acid, abscisic acid,
DEWEY ETAL.
See Weiler et al. 1986 for
references
Polyclonal
cytokinins, gibberellins)
Cell wall
polysaccharides
Isocitrate
Polyclonal
Northcote et al. 1989
lyase
Polyclonal
Martin & Northcote 1982
Endopolygalacturonase
Polyclonal
Grierson
Auxin-bindingprotein
Monoclonaland
Napier et
et
al. 1985
al.
1988
Polyclonal
Phytochrome
Polyclonal
Ldbler & Klambt 1985
Monoclonal and
See
refs in: Thomas
et
al. 1986
Polyclonal
Plasma membrane H
+
Plasma
Monoclonal
with other
purification (possibly coupled
yield
relatively
preparative step
antigen
acts to
antigen
pure
for the
from polacrylamide
present
gels
are
which
and
are
keyhole
the
experience
prior
on
the
nature
permit
the
to
to
of the
stances.
to
that
may
Weiler
have been added
antigen
procedures
are
such
as
of the
hapten;
to
be
prior
to
Bodanszky
&
el
al.
which
conjugated
bovine
linked
is
a
be used
and
to
a
presented
cut
acrylamide
relatively
pure
techniques
large,
with
to use.
highly
more
known
are
as
strongly antigenic
as
haptens
albumin, ovalbumin,
used for hapten conju—NH
as
2
,
—COOH,
protein (Bauminger
If the
Bodanszky 1984; Tijssen 1985).
may
high
to
introduce
a
and
hapten
functional
have been used in plant
number of
the techniques
plant growth
used
are
sub-
described
(1986).
(PBS)
preferably
be immunized
using antigen
and in the absence of toxins
should be avoided if
weakly immunogenic
are
to a
serum
to
the
and
are not
(e.g.
during purification. Adjuvants (compounds
response)
as
injection; here,
functional groups such
covalently
a
useful final
a
major chromatographic
thyroglobulin. The techniques
hapten
animals should
buffered saline
strong immune
the
5,000),
they
plant growth substances involved
Experimental
also
will indicate the best
raise antisera and monoclonal antibodies
extensively by
phosphate
any of the
permit conjugation. Hapten conjugation techniques
The
usually produces
after initial purification; bands
determine purity
investigator
lacks such groups then derivatization procedures
science
it
as
al. 1989
experimental animal)
purified by
large proteins,
and
Wilchek 1980; Kabakoff 1980;
group and
et
immunization. Such small molecules
to
limpet haemocyanin
—SH
al. 1988;
electrophoresis is
antigens
small molecules (<
to
generally conjugated
or
may be
rapidly degraded, requires
molecule
gation depend
—OH
of peptide
which may also be used
are
preparative steps)
short time. Gel
a
et
frequently strongly antigenic (especially
antigens
Production of antibodies
antigenic
Briars
Evans
stimulate the immunesystem of the
peptide antigens,
or
in
preparation
is present. Other
techniques,
and
Polyclonal
transporter
of
Oleski & Bennett 1987
Polyclonal
transporter
2+
membrane Ca
or
in small
in Harlow & Lane
at
all
possible
quantities.
(1988).
sodium
dialysed
azide),
intended
but may be
to
into
which
provoke
necessary
a
if
Full details of immunization
ANTIBODIES IN
Fig.
The
I. Flow chart
of
amounts
and the
antigen
PLANT SCIENCE
of
a
amounts
induce
an
production
to
be raised. Our
intervals of 0-1-1 0 mg
monthly
membrane calcium
total
in
amounts
pump
at
into mice
primary
as
a
to
screening (see
as
guide
and will need
antigen
evoke
an
further
a
contaminant antibodies.
1% of the
has
into rabbits
2-week intervals of 6-60 pg
gave
are,
the
on
experience
plant protein
a
weak
figures
and
be amended
possible
depending
own
however,
from
immunization
depending
seldom
effects of any
antigen
response
the
on
required.
contaminants;
would be
injected
in
a
immune response. Immunization schedules should be
pre-immune
below and
injection
as
to
injection
per
contaminant present
carefully, by taking
obtained
a
of 1 mg
of antigen will also increase the
sufficient
boost,
monitored
excess
of antibodies.
immune response vary
are
while about three injections
be taken
only
for instance 10 pg of
is
to
antibodies
in the
viable monoclonal antibodies could be raised. These
antigen;
1 mg
at
good antibody litre,
schedules should
Larger
which
injections
purified plant plasma
which
outlining the major steps
antigen required
animal in
indicated that three
produced
5
is
Fig.
1).
likely
The
to
bleed and trial bleeds
injection
increase the
and
carrying
should stop when
a
high
out
litre
likelihood of the presence
of
6
M. DEWEY
Table 2. Uses
of antibodies in
plant biochemistry
Method
Label
Immunosorbent assays
and molecular
biology
second antibody
on
Suitable for measurement?
ELISA (enzyme conjugates)
RIA
(radiolabelled antibody
Yes by comparison
Yes
or
preferably antigen)
blotting/Western
(competition
assay very
specific)
conjugate
Fluorescent
Dot
ETAL.
Yes
Enzyme conjugate
Yes
Gold conjugate/silver
Yes
Biotin/avidin/streptavidin
Yes
by comparison
blotting
Chemiluminescence
Yes
Radio-isotope
Yes
Radiolabelledantigen
Immunoprecipitation
Yes
or
secondary antibody
Gel
Table
3.
Yes
electrophoresis
Applications of antibody/antigen detection systems in plant biochemistry/molecular
biology
Technique
Application
ELISA
Primary screening of antibodies. Measurement of antigens; usual to
perform
ELISA against dilution series of antigen;
comparison between
RIA
Western
particularly useful where
Measurement
of dilutionseries with standard
comparison
between
Measurement of
blotting
assay
comparison
Screening expression
lift/blotting
that
available
Primary screening of antibodies.
blotting
Filter
antigen
note
always valid
Measurementof antigens; competition
radiolabelledpure
Dot
assays not
assays
not
always
of antigens by
set.
Note that
valid
libraries
Measurement difficult
antigens.
(requires scanning
densitometer; values obtained not absolute and
vary
between
experiments). Gives absolute identification of antigen
Measurementof
Immunoprecipitation
or second
antigens. Preferably requires
antibody.
Use
either labelled
of gel electrophoresis
to
antigen
identify antigen
advisable
Screening
antibodies
Techniques
for
Johnstone and
techniques
screening
antibodies
Thorpe 1982;
available
immuno-sorbent
are
assay)
is
are
detailed in
Harlow and Lane
summarized
probably
in Tables 2
most
a
number of
1988)
useful
as
and will
and
a
3.
laboratory
not
The
be
manuals
presented
ELISA
primary screening
(e.g.
here. The
(enzyme-linked
method
as
most
ANTIBODIES IN PLANT SCIENCE
antigens
be
can
supernatants
one
coated
however,
assay
so
results. Plates
verify
the
onto
be screened
can
wells
rapidly.
absolute
immunize the animals and the
in which
blotting’,
nitrocellulose (or
After
does
test
used in
commonly
and the
(1979),
electric
given
are
incubated with
mary
antibody
a
bound
antibody specific
the
to
phosphatase
reaction
product,
or
because
Western
a
for
preparation
the
of
diffusion
double
into
use
agar
an
this
assay
antibody-antigen
followed
by analysis of
for which
antigen prior
to
is
the
the
is
is
onto
supernatants
‘Western’
which
be
(or similar) by appliTowbin
sites
some
to
antigens
antibody
fails
antibody
quantity
are
used,
technique
1968), antigen
and
has
is
affinity
silver intensifi-
of
secondary
the
can
is
Other
to
the
In
into
placed
site.
give
or
In the
anti-
required;
are
any
small
too
antigenic
suggested.
is
recognize
to
antigen
but if this fails
antibody.
a
clear
Ouchterlony
circular
techniques
wells
that
cut
yield
followed by SDS-PAGE of the pre-
preparation
which bind
insoluble coloured
using
studying antibody specificity
containing
pri-
secondary
(commonly
enzyme
an
identified
has masked
immunoprecipitation
complex,
an
a
indicate the location of
the
cases,
non-denaturing gels
the
of radio-iodinationof antibodiesand
use
be because the
for
al.
et
on
the presence of
the paper in
produce
subsequently
is
albuminand the paper
serum
to
blotting
SDS PAGE
separated by
paper
either
conjugated
labelling,
In
may
central well
cipitated
bovine
or
also available
(Ouchterlony
a
antibody
unbound antibody,
peroxidase),
can
are
procedures
additional information induce
material(s)
powder
immunoprecipitation
surrounding
to
screen,
dotted
antigen
technique, developed by
electrophoresis/transfer
may be retained if
genicity
to
used
as
rapid primary
to
antibody.
nitrocellulose
to
washing
which
systems.
Blot;
latter case, alternative
result
after
which
which
techniques,
of the
antigen is indicated by incubating
primary
chemi-luminescent assay
on
be included
must
Thorpe 1982). Non-specific antibody binding
solution of milk
gold,
to
a
monoclonal
or
details of this
horse-radish
or
as
binding
procedure; antigen
transferred
(full
include biotin/streptavidin
antibodies,
material
are
the
to
by its
specificity
for this
primary antibody;
alkaline
cation. Other
gel
current
using
or
within
only reproducible
antigen preparation
which is also used
titre antisera
high
in Johnstone &
blocked
are
paper
an
same
of antisera
variety
a
is difficultand standards
is assayed
demonstrate
to
laboratory
of the
proteins
cation of
our
are
paper.
screening,
be screened further
must
and
differentiateantibodies to contaminantsfrom
not
ELISA,
the antibody
similar)
primary
plates
coated with the
those of interest. An alternative to
‘dot
of ELISA
The results obtained
comparison
generally
are
7
of
an
antibody
affinity
both demonstrate the
without
involving
column
nature
of the
denaturation of the
antibody-antigen binding.
Purification of antibodies
Both monoclonal supernatants and
use.
the
polyclonal
Purification methods may be divided into
serum/medium,
HPLC, gel
body
class
(see
of the
Harlow & Lane
1988 for
details);
and
are
purification
given
in Johnstone &
immunoprecipitation (see above)
and the
A
of
A,
Protein
depending
one
antibody
(1981)
antibody is immunoblotted against antigen and the portion of nitrocellulose
ing
the
buffers which
disrupt
the
cut
out.
Antibody
antibody-antigen
is then washed
from
in which
contain-
off the nitrocellulose
interaction. While
G,
anti-
Alternative methods
of Olmsted
the
antibody/antigen
on
thorough consideration
Thorpe (1982).
technique
of antibodies from
Protein
all used
serum/medium by affinity chromatography.
before further
purified
purification
precipitation,
ion-exchange chromatography
methods available is
include
areas;
for which ammonium sulphate
filtration and
others within the
antisera may be
two
only yielding
very
using
small
M. DEWEY
8
Fig.
2. Flow chart for the steps taken in the purification of antibodies.
See section
on
amounts
of
Purification
Uses
The
in molecular
basis: the need to
same
and
is coated
centre
where
antigen
of
antibody;
antibody
on
and
and effective and has been used
rapid
biology
is
successfully
biochemistry
and molecular
quantify antigens.
The
around three basic
methods; ELISA,
or
identify
ELISA
applied
and
to
wells and
identified or
3
used
essentially the
broadly
are
the
similar techniques, where antigen
subsequently quantified; immunoblotting,
where
quantified.
governed by
indicate the
have
biology
techniques
similar
and its
indicated
paper
presence
or
immunoprecipitation
circumstances and will be
and
plate
nitrocellulose
subsequently
used. Tables 2
and
biochemistry
and
plastic
onto
is
procedure
of antibodies in both
same
an
this
laboratory (S. Montgomery, personal communication).
of antibodies
uses
technique of Olmsted (1981).
of Antibodies p. 7.
antibody,
in the authors’
‘Purification
ETAL.
the
the
antigen
is
by
use
precipitated using
an
The selection of
success
of the initial
the basic methodologies
techniques
depends
screening procedures
applicable
to
variety
a
of
problems.
A number of these
preparation
techniques
of antibodies to
ion pumps and
enzymes.
calcium
to
pump,
a
In several
plant actin, and
antigenic homology between
enced. This
was
have been
number of
used
to
a
were
Raising
antibodies
cation
required
protocols
to
advantage
cases
plant-coated
to
actin
for actin did
in the authors’ laboratories in the
proteins, including
(antibodies
when
in
an
antibody
general
our
for immunization and
plant
cell
the
to
vesicles)
plant
structural
plasma
problems
proved
not
exist.
to
a
be
to a
mammalianprotein
experience
low litre
Use of
a
very
were
was
from
experi-
used for
indicated that larger doses of
antibody production
particularly
proteins,
membrane
resulting
plant protein and native mammalian proteins
subsequent experiments. However,
antigen
employed
plant
difficult because
resulted.
good purifi-
simple procedure resulting
in
an
ANTIBODIES IN PLANT SCIENCE
followed
preparation,
impure
polyacrylamide
gel
detection of the enzyme serine:
aration was
by
Olmsted
bound
problem.
Talian
et
This gave
al.
1986).
This
excellent
an
to
permit
an
(1983)
for
preparation
of
the
desired
was
clathrin, they
column
affinity
prepared against
clathrin as it
case
Immunogdiagnostic
The
also useful
required
was
diagnostics
field.
provide
Immunoassays
detection of disease.
an
important
role in
needed for the
methods of
same as
which
early
detect
are
unique
detection and
the
large
for
in
to
the
‘visualizing
pathogen,
as
samples
review of
to
where
screening
plant
in
pathogens
easy
correct
fungal
visual
diseases.
be either
upon
actin and
plant
In the
protein.
horticulture and
from the
adapted
relatively inexpensive.
symptoms and permit early
pathogens
Immunoassays
on
have
These
on or
the
detector
or
known
plates. Such
assayed
ELISAs,
assays
are
see
to cover
are
easy
are
upon
antigen,
organism,
or
the
are
antibody
enzyme
simultaneously. They
all of the
or
as
an
are
assays
techniques
within the target
played
based
are
page 18 of this review. The
tagging
usually
assays,
will be made in this section of the review
highlight
been
and
complexes.
can
recent
plant
of each
agriculture,
have
identificationof
antigen’
depend
be
pure
PATHOGENS
replicate
to
confirming
plant immunoassay techniques
Instead this review will
PLANT
the
some
not
are
used for immunizationand
was
fluorescein (immunofluorescence),
conjugate
numbers of
mass
FOR
specific antigen-antibody
to
1986).
method of choice
a
of viral and bacterial diseases. Similar
spread
Current methods
extract.
general
antibody
preparation of monospecific antibodies.
sensitive,
detection of many
Enzyme-antibody
sively
in which
Monoclonal antibodies
key epitopes
to
impure antigen
as
for
micro-titre wells linked together in strips
enable
prepof the
purification
antigen preparations
membrane calcium pump,
fast method of
fluorescent molecule such
ticle.
They
limiting
visualizing
be
to
a
but
increasingly important
used
Early
those referred
must
soluble
the
a
for
the methods described
and is
prepared successfully.
ASSAYS
are
assays
techniques
be
to
raise antibodies specific
IMMUNODIAGNOSTIC
medical
by
(1984),
immunocytochemistry
plant plasma
to
were
selection after screening
forestry.
achieved
was
and
from
prepared
were
here, although
produced
and Smith & Fisher
when antiserum of sufficient titre is available,
were
Blue stained band
antibodies
Polyclonal
nitrocellulose after Western Blotting is eluted and used (Montgomery
to
enough
Coomassie
a
glyoxylate aminotransferase;
required (Montgomery
(1981),
of
pure, contaminantantibodies were
relatively
antiserum was
excision
by
this
overcame
9
or a
with
gold
performed
now
all the aspects
advances and review
of
(1986).
in
handle and
to
used
exten-
steps have been automated. For
Barbara & Clark
a
par-
a
No attempt
plant immunoassays.
plant-fungal diagnostics
in
greater depth.
Immunodetection ofplant
Over
the years
viruses.
They
(ISEM),
a
number of immunological
(1984).
There is
assay systems that
For
lose
immunosorbent
enzyme-linked
dot-blot or
can
practical guides
an
dip-stick
increasing
a
assays
the
by
to
immunosorption-electron
(ELISAs)
subject
towards
and
consult
dot-blot
Cooper
development
of
detect
dip-stick
or
& Edwards
quick
‘user
the farmer or food processor, such
plant
microscopy
as
(1986)
friendly’
dip-stick,
assays.
and dot-blot
e.g.
on
move
be used ‘on site’
agglutination
surface,
methods have been used
include immunofluorescence
(IMF),
assays. For reviews and
and Hill
viruses
flexible
assays
plastic.
the
is pre-coated
capture antibody
When sap is
squeezed
onto
onto a
nitrocellu-
the coated surface the viral
10
M.
if present,
particles,
in between. If
colourless.
It is
series. For
the
positive
that
important
of such
example
an
detected
are
the substrate and the
antibody-enzyme conjugate,
washings
and
trapped
are
dip-sticks
and
positive
an
incubation
by
stopping
controls
negative
assay system
if
are
Mitchell et al.
see
with the
successively
with
solution,
coloured,
appear
DEWEY ETAL.
appropriate
remain
negative they
included in each
who
(1988)
test
developed
a
system, initially for the detection of potato viruses in leaves of standing crops.
The
majority of viral diagnostic
antibodies. In viral
antibodies is
able. In
necessarily
of the
viral
same
only recognizes
against missing
one
antisera
useful in
nate
a
such
leafroll disease
the
as
(Gugerli
al.
et
polyclonal
studies,
and
ingenious
to
developed
on/in
Vuurde
(ISDP)
an
have
in which bacterial
as
well
by incubating
form. Colonies
cence.
can
Several
outer
pathogens
extracted and
to
than that
whole
phloem-restricted
using
raise antisera specific
pathogens,
Preparation
impossible.
but also
of the
to
plant
not
easy.
pathogenic
incu-
bacteria. Van
technique
Petri dishes
and those that remain
or
are
LPS
been
strains
of
used
level of
immunogen
(1986)
are
easily
of LPS is
purposes
(Lyons
more
and
&
for
Taylor
against
1989).
can
be grown in culture. Bove
spiroplasmas
is
serogroups. It
very
only present
in the
is
with antisera raised
mycoplasma-like organisms, partially
they
and
spiroplasmas, mycoplasma (MLO)
number of
intovarious
syringae
etal.
spirolplasmas
a
diagnostic
specificity
pathogens:
for
for
(Wyatt
the
organisms
rehydration.
(LPS) present
highly antigenic
Pseudomonas
of bacteria
specific
is
to
immunofluores-
detected and, if desired, the
stained colonies after
against purified preparations
has
high
are
staining by
A
have been
extracts are
dilution-plating
by washing
(BLO), only
because
Lin & Chen
1985).
common.
antibodies,
antibody-coated
onto
endocellular
antisera
detect and differentiate the
to
of
lipopolysaccharide
bacteria.
cells and
cultivar-specific
the
to
Antiserum raised
polysaccharide (EPS)
and others
trapped
fluorescently
raised antisera
and bacteria-like organisms
(1984)
different
grapevine
definitive signals
molecules is
of monoclonal
down the agar and
by drying
Other workers have obtained a
the extracellular
Of the
host
and/or
use
removed
are
proved
discrimi-
cause
cause
& De Boer
give strong
to
immunosorbent
an
are
Gram-negative
purified.
differentiation of
1990).
low
that only viable bacteria
ensures
of
but
that
not
trapped bacteria with selective media and allowing colonies
be re-isolated from the
membranes
specific
the
workers have
monoclonal
phloem-restricted prokaryotes
are too
the
as
developed
detected
are
This method
bacteria
IV)
(Halk
problems. Commonly aliquots
inoculation rods. Unbound bacteria
detected
and
enrichment medium to increase the numbers of
(1987)
the
viruses have
physical identity
same
contaminant bacteria
methods,
circumvent these
al.
et
to
antibody
safeguard
assays
by
desir-
different
other prokaryotes, directly from infected plant tissue is
with
cross-reactivity
number of
bated
or
the numbers of bacteria present
Frequently
monoclonal
antibody
detected
luteoviruses and others
1990),
a
rarely
recognizing
where antisera do
(GLRaV I,II,III
Immunodetectionof phytopathogenic bacteria and
Detection of bacteria,
many antibodies
higher signal than
and taxonomic
closteroviruses
rather than monoclonal
be achieved with monoclonal
monoclonal antibodies
between different strains that have the
symptoms,
can
target epitope
one
However,
aetiological
that
Detection of individual strains is
Furthermore,
strains where the
new
employ antisera
containing
protein give
coat
epitope.
antibodies may have changed.
particularly
assays still
high specificity
advantage.
an
general, monospecific
epitopes
that
not
the
diagnostics
in
have been able
to
difficult, however,
because
they
are
obligate
small numbers in infected
plants.
without contamination from host molecules is almost
and Clark
et
al.
(1989)
have found monoclonal antibodies
ANTIBODIES IN
be
to
PLANT SCIENCE
useful in
particularly
11
Aster Yellows and
differentiating
Primula Yellows
MLOs
respectively.
Immunodetection of
The
development
plants
are
plant-invading fungi
of
When tested
specific.
extracts
from
appear to be
tions of
antigens
when tested
non-specific antigens
material and
fungal
producing
culture
or
by
fungi
diluting
are
may be useful in
non-specific
out
rarely satisfactory,
competition
and others
present in both the insoluble and soluble frac-
(Dewey
al.
et
antibodies
but there is
assays
filtrates,
extracts, but
or
by immunodiffusion.Chard (1985a,b)
are
have shown
we
antisera that
against mycelial fragments,
of solid cultures
washings
in diseased
fungi
1990)
the
that
converse:
present in both the soluble and insoluble fractions. Attempts
are
specificity
related
surface
difficulty
antisera raised
IMF,
or
in
with both related and unrelated fungi and host tissues
species-specific
have shown that
due to the
partly
ELISA
by
lyophilized mycelia,
widely
cross-react
for the detection of
immunodiagnostic techniques
has been slow. This has been
al.
et
with
evidence that fungal antisera
increasing
(Kitagawa
antisera
cross-absorbing
or
specific
improve
to
N. S.
1989;
Lyons
al.
et
personal
communication).
A number of workers have shown that reasonablelevels ofspecificity may be obtained by
of culture filtrates
using protein precipitates
Examples
include antisera raised
in
Phomopsis longicolla
tissues
root
(Gerik
Antisera raised
et
al.
1987)
against
carbohydrates,
generally
raised antisera
to
antisera
are
have
ing
far, relatively
results with
et
al.
aphanidermatum
and
specificity.
not
and
factors.
the
of
Many
antibodies
P. megasperma
species
on
plants
or
fungus
fungus,
immunfluorescence,
al.
1989).
soluble
(< 1987)
have
that such
food stocks
raw
are
the
against fungi. Disappoint-
animal house facilities
monoclonal antibodies
been raised for diagnostic purposes. However,
var.
al.
et
raised
against
Phytophthora
glycinea (Wycoff
1989) clearly
some,
have
et
al.
such
have
raised
as
the
cinnamomi
1987), and Pythium
diagnostic
potential.
It is
aphanidermatum
the basis of zoospores. Specific assays for the detection of
soil would be
more
Wright
et
useful.
al.
(1987) have successfully
(dot-blot)
raised and used
assays for the detection of
Sirococcus strobilinus and sporocarps of the vesicular-arbuscular
Glomus occultum.
wilt
et
or
1988);
cotton
(Mohan
and showed
the monoclonal antibodies for P. cinnamomi and P.
in infected
banana
fungi
determine if
to
& Pitt
toxins
enzymes,
Notermans
need for
monoclonal antibodies in ELISA and membrane
fungus
as
from several
be used
can
costs
Mitchell & Sutherland (1986) and
the seedborne
in strawberries
such
immunogen.
processing.
to
(Estradia-Garcia
differentiate
mycelia
discouraging
1986),
unfortunate that
only
prior
monoclonal
species-specific
of
high degree
fungal antisera,
against fungi have
(Hardham
a
carbohydrates
the
as
(Barker
Verticillium dahliaein
1987);
few monoclonal antibodieshave been raised
been
undoubtedly
al.
et
Phytophthora fragariae
genus-specific
contaminated with moulds
So
and
(Gleason
extracts
anemone corms
specific fungal fractions,
heat-stable
almost
seeds
soybean
mycelial
or
Colletotrichumin
to
Wong
Fusarium
et
al.
(1988)
have raised
oxysporum
the thick-walled
f. sp.
a
monoclonal
cubense
chlamydospores
that
will
to
the
differentiate,
by
antibody
of strain 4 from those of strains
1
and 2.
Our
own
experiences
species
of
causes
Dutch Elm
are
fungi
involved
in
raising
have been varied.
in
monoclonal antibodies
Development
disease, Ophiostoma
post-harvest spoilage
of
ulmi (Dewey
of rice
that
diagnostic
et
grains
al.
and
are
specific
assays for the
1989a)
to
various
pathogen
and three
fungi,
copra, Humicola
that
which
lanuginosa
12
M.
(Dewey
et
al.
Pencillium islandicum
1989b),
(F.M. Dewey unpublished)
have had
for
level,
1988).
difficulty
the
The
sponse. The site and
by
our
but
immunogen
that
the
non-specific,
adjuvant
the
as
antigen
and
IgG,
IgG 2a
ti tre wells coated with
whereas
species-specific
with
readings
example
between
distinguished
raised
we
were
the
tested
healthy
We
extracts
have
and
we
fragments,
to
specific
response.
with
and
by
found
species-
were
IgGs belonging
were
ELISA
to
mostly IgM
micro-
against
correlation
no
was
infected from non-infected
tissue
whereas
tissue
healthy
to
clearly
another
give
higher
Another monoclonal anti-
Ceratocystis
vitro but
in
not
all
mycelial
we
monoclonal antibodies
extracts.
It did
1989a),
and three
were
healthy
produced
plants.
secrete
raise
soluble
a
gave
low
absorwhen
high readings
with
cross-react
washings
or
from
extracts
P. islandicum, H.
This
high
were
and
fungi
lanuginosa
methodof
simple
of the
directly
the
as
the addition of Freund’s adjuvant
only
and A.
flavus
antigen preparation
fusions
one or two
monoclonalantibodies with the
monoclonal
extracts
All these
response
points
freezing
or
to
antisera titres
to
and
specificity
suitable for the
with 1 in 200,000 for H.
antibodies specific
etal.
and
were
were
to
(Dewey
was
very
needed
needed to
to
the
by
carbohydrate
were
detection of the
surface
fractions
or
has been weak and
hyphal fragments
against
lanuginosa. Higher
pathogen
1990).
washings
ELISA
including
to
develop
only
surface
washings
Eyespot
washings, mycelial
(F.M. Dewey
tried with and without Freund’s
these different immunogens
for antisera tested
(Dewey
antibody
protein
immunogens
from mice immunized with surface
used
diseased
have used several differentimmunogens
unpublished).
pared
of
assays.
trying
pathogen
end
and
cross-reacted
cell-free surface
unpublished).
cases
cell lines that
diagnostic
general
source
ulmi using
al.
30w/v),
distinguish
to
et
antibodies
in
(1
species-specific
against antigens
without concentration
effective. In both
In
specific
a
Ophiostoma
tested
were
of both Ophiostoma
used
successfully
1989b/1990
identify
about the best
different workers and the
by
species-specific
diseased
than with
from diseased
to
genus-specific
ability
of the
from
antibody
species
raise monclonal antibodies
al.
hyphae
by gently washing
tissue.
immunogen
et
one
extracts
when tested
against
walls of the
stimulating
genus-specific
diseased tissue
and the
extracts
monoclonal
these
that recognized
body
bance values
from
extracts
fungal specificity
material. For
plant
in
immunogen (Dewey
antibodies. When all the monoclonal antibodies
found between
no consensus
re-
generally
not
antigens recognized
cross
be removed
can
schedules used
about one-third were
The monoclonalantibodies that
sub-class
is
attained.
with Freund’s
most were
specific.
comparison of the various
a
and the presence
proteins
that the
(Fig. 5)
adjuvant
of monoclonalantibodies that
panel
homogenates
same
the effectiveness of Freund’s
specificity
Of the
of this
solid slant culture with PBS. There is
a
or
See Table 4 for
levels of
some
we
the genus
non-T cell stimulated
a
present in the walls and
are
at
herpotrichoides (Dewey
sub-species-specific antigens
of P. islandicum we have shown
case
the spores and that
the surface of
and
species-
monoclonal antibody
specific
not
of
nature
In contrast,
even
immunogenicity of the different
that induce
glycoproteins
or
Aspergillus flavus
quick.
specific,
are
Pseudocerosporella
be related to the
to
and
1990)
forward and
reflects both the levels of soluble
carbohydrates
known but in the
of cereals
pathogen
probably
turn
of non-specific
al.
et
relatively straight
of difficulty appears
degree
(Dewey
monoclonal antibodies that
raising
Eyespot
which in
fungi,
in
has been
DEWEY ETAL.
non-specific.
had low
were
1988,
adjuvant.
The
Antisera
titres;
dilution
1 in 50,000
com-
titres and cell lines secreting monoclonal
obtainedwhen
a
specific protein
fraction
was
ANTIBODIES IN PLANT SCIENCE
13
(1990)1990)
or
species
(
Ref rence
(1983)
al.
(1984)
(1986)
a/.
al.
al.
al.
et
et
et
Sutherland
(1987) (1987)
(1988)
al. al.
etal
.
et
&
&
al.
al.
et
llaanneleicl/ai. Banowetz Hardham Mitchel Wrighhtt WWyycocfoeftu/.
et
et
1986)
(1989b) (1990)
&HaHahhnnee
Estrad-Grcai
Dewey Dewey
&
Wong
al.
al.
(1988) (1989a)
Fox
Fox
al.
al.
et
et
Dewey
et
other
al. al.
etal.
against
et
et et
tested
not
mAb class
NG
NG
— —— — — —
IgM
NG IgM
NG
IgM IgG IgG IgM IgG IgA IgM
ND
ND
=
IgG IgM IgM
IgG IgM
IgM IgG
IgG IgM
IgG
IgM
NSG
&
&
Specifcity
Genus NSG
SpSpecies NSG
NSG NS
NS genus
RaceNSG Race-
blot
Scre ni ng method
Route
ELISA-S ELISA-S ELISA-S IMF-zo sp.ELISA-sol.Ag ELISA-S ELISA-sol.Ag western
Bo ster
i.p.
i.p.
Early
i.p.
F
i.v.
i.v.
i.p.
i.p.
+
against
raised
Injection
+
7
mg
7
7
3mg
10
per
3
10
2xl2x0
mg
1
1
i.p. NG
NG
NG
Intrasplenic
i.p. i.p.
i.p.
—
ELISA-h.f
IMF
i.p.
i.p.
+
±
±
ELISA-sol.Ag ELISA-SW ELISA-SW IMF-zo sp.
i.v.
i.v.
i.p.
i.p.
+
+
7
mg
mg
10
50,50,0000 015015 protein 8xl8x0
pg
40
NG
NG
mg
mg
3
3
Spores Spore extracts Zoosp. Hyphal fragments Spores Culture filtrate
Spores
Conida hyphal fragments Hyphal fragmentsHyphpahall fragments
i.p.
i.p. i.p.
i.p. i.p.
i.p.
—
—
surface
=
fragments.
SW
det rmined,
hyphal
=
h.f.
protein pprrotein
pg pg
SW SW
not
=
ND
zo spores,
=
given,
zo sp.
not
=
NG
antigens,
7
10
10
x
1
1
Zoosp.
2.
with
7
antibodies
soluble
intravenous, cros-eacts
=
=
i.v.
intrapeional,
Ag
sol.
species, genera,
other related
18
against from
i.p.
=
oxysporum
Monclona
4.
Table
washings,
&
i.p. i.p.
i.p. i.v.
150 500
Dose
Nature
i.p. i.p.
i.p. i.p.
i.p.
—
fungi
plant-ivading Im unogen
i.v.
i.v. i.p. i.v.
i.v.
specif c N.S.G. SSppecies ggenusenus Genus* Species Zo spores Cyst;: NSNS Species
Fungus
Fusarium
cinamo i
strobilnus
lyucopersic
sp.
f.
f.
Tiletia
meqasperma oxysporum
lanuginosa islandicum
mel ea
oc ult m
glycinea cubense
Phytophtora Siroc us Glomus Pythophtora Fusarium Armilari Ophiostmoa Humicola Peniclum Pythium
ululmmii
sp.
sp.
f.
f.
adjuvent,
tested species
not
=
Freunds
=
F
NS
against
genera, �Tested
14
M. DEWEY ETAL
Most of the monoclonal
exception
micro-titre wells
infected
of
a
plants
followed
secondary antibody-enzyme
We have found,
as
did Gleason
islandicum in rice
usual. This method has enabled
if
or
only
a
friendly’ dip-stick
detection of
within 5
is
fungus
proved
grains (Dewey
et
few individual
assay that has
of harvest. The
allowed
to coat
grains
re-wetted incubated with
The
gold conjugates.
in
lanuginosa
are
The wells
at
and
lanuginosa
soaked, individually,
then
are
processed
as
low level of infection in all
a
Paddy heaps
developed
a
‘user
soaked
in situ in the Philippines,
membrane Tmmobilon P’
a new
in PBS, in
individually,
dip-sticks overnight.
amplified by
was
Grains
made of
were
The
followed
hybridoma supernatants
signal
antibody.
that these assays
enable detection
to
infected. We have also
are
use
the surface of the infected
near
day.
of
from the
successful under field conditions. It has enabled
dip-sticks
the surface of the
the reporter
as
is sufficient
1989b/1990).
(Millipore, UK). Fungal diffusates from grains
were
on or
next
coating
extracts
invaluable in the detection of H.
al.
proved
of H.
significant growth
days
present in
gold conjugate
present
are, with the
monoclonal antibody and the
determine if there is
to
us
fungi
involve direct
(1987) using polyclonal antisera,
in micro-titre wells and removed the
overnight
grains
or
They
antigens
specific
passive release, by overnight soaking,
low levels. This method has
very
al.
et
well where the
particularly
tissue and where
P.
incubationwith the
by
for
developed
assays
all indirect assays.
membranes with the mixture of
or
commercial
work
antibody diagnostic
of the few commercial assays,
exposure
to
a
dip-sticks
by
Eppendorfs
were
then
commercial
commercial silver
dried,
immuno-
enhancing
solution.
The
P and
specific fungal antigens
to
that
we
have worked with bind
micro-titre wells. We have also found that the
strongly
their respective antibodies. More than 95% of the total monoclonal
the
is bound within 30
antigen
have shown that in the
case
glycoproteins (Dewey
el
Only
few double
a
detection
The
s.
most
sandwich
are
Milleret al.
monoclonal antibodies and
both Immobilon
specific antigens
is
that the
lanuginosa
bind
rapidly
known but
not
to
that binds
antibody
specific antigens
to
we
are
1989b/1990).
antibody
by
of
of P. islandicum and H.
al.
purposes. Most of these
of turf grass diseases
nature
to
specific antigens
assays
(DAS-ELISA)
have been
commercial and have been
(1988, 1990)
polyclonal
for
developed
developed
for
for detection
Agri-Diagnostics. They employ
both
antisera.
Commercialpotential
The
use
of antibodies
poses, is
was
now
estimated
(Klausner
to
to
1987).
viruses but
assays
detect
to
be £3 million in
At the
important
detect
moment
advances
spiroplasmas,
these assays will necessitate the
specificity
needed. Use of
development
plant pathogens,
for both research and commercial
well established. The commercial value of
of
1985 and is
this
are
likely
mainly
to
be made in
to
use
be about £8.5 million in
the
production of
bacteria and
of monoclonalantibodies inorder
growers and
immunoassays by
user
friendly
interpretation of the
assay
results
pur-
assays
1995
concerned with the detection of
mycoplasma-like organisms,
low-cost, on-site,
methods of sampling and
projected
market is
plant immunodiagnostic
importers
kits such
to asses
as
to
commercial
fungi.
Most of
attain the level of
will
depend
dip-stick
on
the
assays. Good
the levels of infection, will be
critical.
IMMUNOCYTOCHEMISTRY
Over the last decade the
tissues
has
emerged
as
use
one
of antibodies
of the
most
as
probes
for macromolecules within cells and
powerful techniques
in cell
biology.
There
are,
ANTIBODIES IN
therefore,
many
PLANT SCIENCE
excellent reviews and
cols that have been
Varndell
1984).
The
be said that the
light
and electron
probes
by
the
has finally
numerous
levels. Most of the
1982,
a
of
problems
of
proto-
However, it
routinely
at
of
inaccessibility
plant tissues,
cells has
plant
to
biology.
be used
can
and
1985, 1989; Polak &
techniques
in animal cell
nature
techniques
1983,
have been
can
both the
antibody
overcome
variety of tissue preparative techniques.
our
intention
to
review
some
of the
the initialfixation and
preparation
of the
that have been
methodologies
of
permit successful immunocytochemical labelling
to
on
the
of age and
come
due to the cell wall and the
development of
emphasis
rapid developments
technique
In this section it is
developed
outlining
application of immunocytochemical
microscope
antigens,
to
texts
Bullock & Petrusz,
developed (See
somewhat lagged behind the
now
15
plant antigens with
cell/tissues prior
to
light
an
or electron
microscope immunocytochemistry.
Light microscope immunocytochemistry
When preparing
tissues
plant
or
be taken into consideration.
cells for
First,
is
the
denatured by fixation and the other steps in the preparative
affect the
adversely
recognizing
in
prepared
probe
a
a manner
the
to
whole
cells,
probe
to
cross-reactivity
single epitope
in
antigen
made
examine
which
permeable,
or
permit
gaining
are
in
of both the
a
has
most
to
are
or
be selected.
tissues have
as
to
whether
the
employed. Finally,
At the
popular. However, the
be
to
and the attached
be taken
to
be
to
often
can
monoclonal antibody
light microscope
use
of colloidal
gold
in combinationwith silver enhancement. At the
popularity
gold
a
antibody
decision has
sectioning techniques
level colloidal
microscope
if
to
be localized is
to
procedures. This
is the fact that the cells
access
In consequence,
still the
antigen
antibody, especially
Second,
primary antibody binding
LM marker is
electron
will
question.
(LM) level fluorescent probes
as an
with the
is used.
three main factors have
immunocytochemistry
the likelihood that
has become the marker of choice
(Hyatt
1990).
However, enzyme-linked secondary antibodies can still be useful with sectioned material.
Wholecell
to
techniques.
The cell wall and the
the entry of antibodies and associated
the
specimen
has
approach
may be
proved
sectionpd
or
plasma
the cell made
three-dimensionalorganization of which
can
permeable
in the
popular, especially
very
membraneform the
probes into the cell. To
staining protocols involving permeabilization (Lloyd
el
al.
1985; Goodbody
pension
culture cells
was
enzymes
technique
used
to
was soon
if washed
or
to
al.
to
et
such
al.
work
1980).
digested
was
with
et
coverslips prior to staining.
al.
1981).
The
1980;
Wick etal.
former,
1981;
the
to
mild
of the wall.
shown that
cellulase and
the
to
of this
steps
use
of
sus-
with
However,
this
roots,
under controlled
poly-L-lysine
technique
in
Roberts
formaldehyde-fixed
pectinase
the
treatment
original morphology,
major disadvantage
on
of the various
restricted
was
In the
These cells adhered
release of cells from the tissue all information
plant cytoskeleton,
rigours
cellulose and the matrix
tissues when it
could be
et al.
release whole cells which retained their
‘square protoplasts’ (Wick
or
1989). Initially
loosen the
extended
adequately,
conditions
et
protoplasts (Lloyd
study
these barriers,
the antibodies. This latter
to
of the
withstand the
barriers
major
two
overcome
the so-called
coated slides
is the fact that
spatial organization
on
of cell types
within the tissue is lost.
Various approaches have been used
cases
fixation with
practice
to
to
make the
paraformaldehyde (3-8%)
further treat the cells with
a
mild
plasma membrane permeable.
will prove sufficient.
detergent
such
a
However,
Triton X-100
or
In many
it is normal
Nonidet P40
16
M.
Air
(0-5-001%).
been used
To
(Wick
al.
plasts
proved
1981; Goodbody
popular.
osmotically bursting
PM
to
are
PM
left
adhering
the
Palevitz 1989; Goodbody
Sectioning
techniques.
tissue
integrity
These
can
be
al.
et
Cryosections:
either
2 pm. Sections
by
then stuck
are
a
very
simple
fixation
cryo-stub, Sakaguchi
tions of shoot
tips of
system of the apical
now common
et
Vinca
whole cell
either
for the tissue
In
that
al.
report from
major
infusion of the
roots
was
10
to
up
and
same
pm
incubated for
at
of 5
al.
staining
of pea
Wax sections:
has
not
been
likely
labels.
to
cryo-
be restricted
to
1
slides
gelatin-subbed
or
a
longitudinal,
be able
some
in
specimens
to
the
drop of
microtubule
complete
such
produce
up
to a
water on
6-8 pm cryosec-
supporting matrix into
good
cryo-
the tissue. It
concentrationof 2
to
prior
M
as
to
support
a
roots
al.
of slides
prepared by
Although
a
a
a
strip
of
were
be
cryo-
(Fig. 3).
a
1
-5
m
laboratory
water
a
of slides
placed
face
film and
and heated
to
the adhesion
useful technique for adhering
3 demon-
subsequent processing. Figure
monoclonal
antibody
yeast tubulin in thick
to
the methods described above.
to use
paraffin
in
being
dissolved
1985, Hawes 1988). Sections
histological studies
of animal
wax-embedded material in
molecular weight
the PEG
on
be
sectioning
to
poly-L-lysine coating
markedly strengthened
probably
popular technique
practice
the
root
found
further
the surface of
on
then rinsed in ultra-pure
slides for
of microtubules with
common
et
were
treatment
a
of the tissues
Cleaned multiwell slides
poly-L-lysine
was
sucrose,
the surface of the slide.
the effectiveness of
kD
glass
them up
onto
it
1990)
m
of the
regions
fragmentation
this
al.
et
in 2
markedly improved
by picking
the slide and this will
to
little
very
roots
pea
vacuolated
highly
manner.
mg/ml 390,000
foundthat this
embedding matrix,
Lammeren
to
sucrose
touching
1990)
following
cytochemistry. However, high
an
and
least 2 h. The slides
was
cryosections
cryosection
antibodies and
to
for
because
ultramicrotome with
they labelled
m sucrose
slides
glass
loop
et
problematical plant specimens
strates the
an
treated
the
paraformaldehyde-fixed
a wire
report (Hush
70°C for 2 h. It
of thick
1989),
technique
cryoprotectant giving better freezing and
in thickness with
held in
drops
onto
by
or
freezing
tissue with
a
with 10% gelatine in 1-5
also enhanced in the
down
al.
et
of cryo-sections
of the authors’ laboratories (Hush
one
then transferred to
were
droplet
In the
is accessible
poly-L-lysine
however,
characteristics of the tissue. This enabled
sucrose
again proved
antigens (Cyr &
satisfactory
use
section thickness is
within which
rare,
both
incubation of
following
Sections
the
obtained excellent
(1988)
perfuse
to
acts as
face of the
during sectioning (Hawes 1988).
a recent
sectioned
has
of
‘footprints’
clathrin (Hawes
as
and
1988).
procedure
dome. It is
practice
this then
sectioning,
or
cytoplasmic
technique
most
material is
sections of plant material without the infusion of
is
such
the
Probably
system
onto
immunostaining (Hawes
to
proteins
conventional cryostat
a
with the latter
attachment, although
Using
This
1980).
of burst proto-
1989).
is maintained and the
cut
have also
min)
coated slides
patches
associated with the
al.
et
labelling of sectioned
immunocytochemical
the
Valk
use
poly-L-lysine
to
microtubule-associated proteins and intermediatefilament
microtubules,
prior
protoplasts
for the examination of structural
popular
or
(Van der
membrane (PM) the
distilled water, circular
glass. Any antigens
then be localized
can
or
ETAL.
1989).
plasma
By sticking
them with buffer
10°C for 6-8
(—
treatments
al.
et
associated with the
study antigens
has
and cold methanol
drying
et
DEWEY
as
plant
polyethylene glycol (PEG)
out
thin
of the section
as
prior
0-5 pm, with this
to
can
tissues,
it
immunobe used
as
staining (Van
technique
can
be
ANTIBODIES IN PLANT SCIENCE
Fig.
3. Immunofluorescent
the meristem
cells
1-5
of the root
m
well
sucrose
slides.
Sidney.
dry
sucrose
and
on
=
a
staining of
10%
pm,
glass
solution
after
B bar
(8-10 tun) cryosections of pea
were
were
fixed
in
in
orientation, whilst
3%
stuck onto heat
contained
of Julia
fish
to
vacuolate
infiltrated with
poly-L-lysine
gelatine
roots. Behind
larger
paraformaldehyde,
activated
1%
in
coated
reduce
multi-
background
Hush, Biology Department, University
of
60 pm.
an
ultramicrotome and transferred
to a
slide
on
a
drop of
1988).
advantage
possible
block
tural localization of
etched from the
Roots
oblique.
1990). Micrographs courtesy
=
knife with
Resin sections: The
same
are
in thick
predominantly transverse
primary antibody staining
(Hawes
lies in the fact that it is
and that the
are
(w/v) gelatine. Cryosections
Hush et al.
30
cortical microtubules
microtubules
the microtubules
washes
(see
A bar
cortical
(B)
All
fluorescence
cut
(A)
17
can
using
cut
resin-embedded material for
sections of tissue that are
be used for both thick and thin
antigens.
surface
of
to
In
of the
most
section
cases
to
the resin
expose
the
sectioning
must
be
antigenic
immunochemistry
difficult
to
cryo-section
for the ultrastruc-
chemically
sites
prior
removed
to
or
staining
18
M.
&
(Perrot-Rechenmann
sodium ethoxide
of resins is
family
retained. Gubler
Gadal
suitable
not
(1989)
With epoxy
1986).
methoxide
or
(Hawes
1988).
levels of
satisfactory
as
reported
on
the
resins
In many
successful
this
polymerized by long
with
Resin
acetone.
embedded sections
is
preparation
Visualizing
removal
(Gubler
may
al.
et
however,
cases,
be
not
The
antigen.
each
An
should be taken
preparative techniques
air-dried
protoplasts
However,
primary antibody
if the
is
stained
One
background
In
our
1 % fish
second
antibody conjugated
antibody
and
schedules have
demonstratedin
with
carrots
a
be
to
localization of
to a
an
be
to
a recent
be
can
paper
same
antigen
&
Duniec
by Goodbody
was
only
for
dependent
dipped
could
set
no
developed
optimized (Wick
antigen
concen-
Therefore,
monoclonal antibody
be constructed for each
to
with
be used
can
as
with
fluorescence,
compounds
pre-immune
on
et
al.
achieved in
in methanol.
be observed in
the control
as
the control with
as
which should
well
polyclonal
with the tissue
not cross-react
of tissues which should
staining
as
substitution
experiment. Commonly
is used
serum
antibody,
an
gelatine
greatly
immunofluorescence is
the
latter due
found in many
in addition
laboratories,
reduce
tissue
to
not
antigen.
major problem
fluorescent
a
is
immunostaining
elegantly
antibodies. With monoclonals
contain the
Lowicryl-
approaches
which had been treated with Triton X-100 and
Suitable controls have
being
was
without methanol treatment.
preparations
of the
low-temperature
blocking agents
staining
microtubule-like distribution of the
a
This resin
tips.
root
with different antibodies.
standard and
as
of fibrillar bundles in
(1989). Staining
not
are
LM immunochemical localizations.
most
buffer types,
of how the successful
example
for
between tissues and
particular experiment
1986).
the
greatly
vary
procedure
with
necessary
two-layer technique, using
protocols used, including
exact
trations,
of this
immunofluorescent localization of
A summary of these various
fluorochrome, is still the method of choice
The
in the tissues
antigenicity
with
out
use
of the section
temperatureand dissolved out
room
1987).
the
in Table 5.
given
the
ultraviolet at
be carried
can
microtubules in sections of buty-methyl methacrylate-embedded
wave
DEWEY ETAL.
to
the
to
a
wide
of blocking
all solutions after incubationin the
to
the level of
of
variety
such
plant cells,
use
non-specific staining
as
general
naturally occurring
various
agents
and
such
and
phenols
as
BSA,
primary antibody
auto-
pigments.
the addition of
has been found
to
fluorescence (B. Satiat-Jeunemaitre personnal
background
communication).
It should be remembered that other markers
to
visualize
the second
sectioned material
can
(Van
arise in tissues
with
linked to the second
The
use
binding
and
endosperm
1987).
A
own
as
more
gold
as
With
a
al.
1981;
the
of Scadoxus
to
to
at
gold
as
fluorescent probes
labelling techniques
Bullock & Petrusz
levels of
have
marker
right, whereby
available
be used
on
1982) although problems
peroxidase activity
the LM level is
is
seen as
and other enzymes
a
increasing
dark red stain
in
popularity.
over
It may
sites of antibody
great effect in the study of microtubules in the liquid
(Haemanthus)
1981; Holgate
(De
sp.
is
et
al.
to
Mey
et
amplify
al.
the
1982; Morejohn
signal
from the
this
technique
reaction monitored microscopically and
stopped
et
al.
gold by
1983; Van den Bosch 1986). With this
shell of silver is grown, under controlled conditions, around the
recently developed reagents
are
can
be used.
satisfactory technique, however,
silver enhancement (Danscher
procedure
may
a
such has been used
cells
et
naturally high
antibody
of colloidal
be used in its
Driessche
well
as
Peroxidase
layer antibody.
can
be carried
when the
out
gold particles.
in the
required density
light
of
and the
staining
is
ANTIBODIES IN PLANT SCIENCE
19
1986
1980
al.
Ref rence
1980 1989
al.
et
Valk
etal.
et
der
Lloyd Hawes Van
1985
1989
1989
al.
et
Schindler 1980
&
al.
1988
1986
1981
etal.
Duniec
1989 1990 1990 al.
et
al.
Go dbody Meiners Lloyd Wick Wick Knox
et
&
et
etal. etal.
Stacy Hush
Sak guchi
Gadal
&
al.
et
Lam er n
Perot-Rchnma
Van
1985
1987
al.
et
1989
1989 etal.
Gubler Gubler Roberts
etal.
Wick
antigens
of
localiz ton
imunoflresc
filaments
Antigen
Tubulin
carboxylase
A
r
a
b
i
n
o
g
l
c
t
p
e
s
A
r
a
b
i
n
o
g
l
c
t
p
r
e
i
n
C
a
l
m
o
d
u
i
n
/
t
b
l
Clathrin Tubulin Interm diate Con exin Tubulin Tubulin
Tubulin Tubulin Tubulin a- mylase
PEP
for
prot plast
S
p
e
c
i
m
e
n
techniques
cultures
cultures cultures
cell
cell
cells
cells
Car ot Car ot Tobac o Car ot
bean
cell
tips
tips
root
apices
root
Onion roots
embryos
&
roots
shoot
Soy Car ot Onion Pea Car ot Car ot Pea Vinca
Tubulin Tubulin Tubulin
spp.)
cells
tips strips
pollen leaves aleurone
(various
Epidermal
Leaves
root
Gasteria SorghumBarley Onion
pre ar tive
tissue
cells
various
culture
pm)
(0-5-1
of
Examples
5.
Table
prot plast ghosts
Technique Permeable Prot plast
su pension
Permeable
—conf cal
prot plast cryosections cryosections
embed ing embed ing
strips tis ues
Square
PEG Resin
Tis ue Thick
Thin Thick
20
M. DEWEY ETAL.
Fig.
4.
Immunogold localization
pumpkin cotyledon
1%
paraformaldehyde and
SGAT
polyclonal
(pH 8-2) with
of Sue
0-5
serum
of serine
glyoxylate
amino transferase
in LR White resin and
embedded
1%
glutaraldehydein
followed
by
MNaCl,01% (w/v)
15
gold
nm
01
polymerized at
M
PIPES
labelled
Tween-20 and
1%
(SGAT)
buffer, pH
anti-rabbit
on a
50°C for 24 h.
6-9.
Staining was
IgG. Staining
globulin-freeBSA.
Bar
peroxisome
Specimens
=
buffer
200
nm.
give
the best
signal,
conjugated
with the second
for
of the silver
growth
material
or
naked
deposits.
cells.
small
a
antibody
This
giving
technique
B.
Recently,
size colloidal
particle
thus
the
is
optimum
a
section of
fixed with
rabbit anti-
20 mM Tris-HCl
(Micrograph courtesy
not
permeable
labelling
with
colloidal
gold
with
enzymes
gold
5nm
and
and
probes
immunoglobulin fragments
procedure
available
The main
to a
advantage
of the tissue is
(1989)
a
use
to
recent
fragments
plant
can
be
deposited silver,
the
staining
to
successfully
sectioned
immunothe cell
by making
prior
introduction of
(papain
cleaved
should make this
to
1-nm
45 kD
staining
cell types.
of silver-enhanced
seen
In combinationwith
the
applicable
Fab
should be
communication)
single antigen binding site),
within cells
possible.
to
personal
enhancement. The
gold-conjugated
wide range of
silver-blue fluorescence
and should prove
methylamine
silver
with
of the
the distribution of antigen
staining
(or
(5 pm)
restricted
necessarily
Satiat-Jeunemaitre
sol
gold
number of nucleation sites
localized microtubules in epidermal strips of mung bean hypocotyls
a
with
was
a
Montgomery, Plant Sciences, Oxford.)
achieved. To
walls
in
were
of
gold staining
at
the LM level is that
with transmitted light and further
epipolarized light,
this becomes
plant
cells
as
a
very
well
which will
impart
impressive technique
as
animal tissues
(Ellis
etal. 1989).
Future
developments.
In addition
to
the introduction of ultra-small
nation with silver enhancement and refinements
protocols,
it is
likely
that the
most
to
the various
significant developments
gold probes
embedding
in LM
in combi-
and
staining
immunochemistry
ANTIBODIES IN PLANT SCIENCE
Fig.
10
5.
species specific antigen
in the
Material
gold-conjugated mousemonoclonal antibody.
nm
maldehyde in
will
of a
Immunogoldlocalization
21
be
in
01
the
PIPES buffer
imaging
which
microscope,
processing
m
will
dimensional
can
make
and low
(pH 7-2)
was
hyphal
fixed in
temperatureembedded
apparatus. The
introduction of
be
epifluorescence
used
in
significant
a
organization
of cells
the
contribution
to
wall of Penicillium
1%
in LR White resin.
the
laser
Bar
1%
=
scanning
and
mode,
our
islandicum
glutaraldehyde and
with
400
nm.
confocal
associated
image
of
three-
understanding
the
With the removal of out-of-focus
(Shotton 1989).
a
parafor-
blur,
resolution in immunofluorescenceimages will be improved and three-dimensional reconstructions from serial
use
of this
microscope
optical
sections
can
is that relatively
easily
be made. Another
large strips
of
tissue,
more
be stained and the distribution of antigens below the surface cell
potentially important
than
one
cell
thick,
layer probed (Wick
can
et
al.
1989).
Immuno-electron
Post
the
microscopy
embedding labelling.
variety
of
plant
as
cytoskeletal proteins
cells due
antigens
can
to
only
has
in
level the
on
biology
cells
proved
very
of
sectioning,
popular
due
available in terms of
pre-embedding labelling
locate structural antigens
1986),
cannot
be used in
plasma membrane-bound surface
labelling
has been used
to
to
(Lawson
protoplasts,
the cell surface. The
in resin and thin
not
preparation
options
be limited. The
detergent-treated
be located after
of molecules
re-embedding
technique
appear
to
the presence of the cell wall. Likewise
to
rearrangement
microscope
with such great effect in animal cell
procedures, used
such
At the electron
preparative techniques
the lack of
which
undoubtedly
of thick
(Raikhel
penetration
the thick cryosection and the technical difficulties of resectioning.
causes
cryosections, prior
et
al.
1984),
of gold
but this
probes
into
22
M. DEWEY
Due
these constraints
to
far the
by
post-embedding labelling techniques
These
techniques
are
discussed here but
Hawes
&
Herman
to
resins
embedding
undertaking
an
both
by
which
use
the
are
1985; Polak &
1983,
1982,
Here
(1988).
application
of these
the
perhaps
two
in the tissue is the
antigenicity
of osmium tetroxide is
plant
these
in
cases
Thus,
in the
majority
glutaraldehyde
Herman
nodules
leguminous
osmication
to
be made
to
be achieved when
or
of
vation and reduction in
and
contrast
Shaw
to a
to
on
the
post-sectioning
to
to
microbody
even
in
in the sections.
paraformaldehyde,
1985; Gubler
quality of
staining
most
protocols
1986). However,
antigenicity
& Henwood
reduction in the
denatured
are
it has been used in
fixation will be restricted
(e.g.
lead
inevitably
effect
major
a
investigations,
mixtures of both
This will
1988).
had
prior
if heat is involved. In
den Bosch & Newcomb
(Van
1986;
Wang
cells the reader is
Many antigens
particularly
precluded although
undoubtedly
be
immunocyto-
localize storage proteins in developing seeds (Craig & Goodchild 1984) and
enzymes
not
the choice of fixatives and
to
major goal
microscopy.
reaction of resins
curing
techniques
to
crucial decisions
most
on
study.
for immunoelectron
specimen
employed
will
protocols
Varndell 1984;
will limit the discussion
we
immunoelectronmicroscopy
fixatives and the
the
cases
the
on
retention of
Satisfactory
preparing
Petrusz,
Fora review
1988).
referred
laboratories. General
many
be found in the many books and review articles
can
chemistry (Bullock
of studies have
ultra-thin sections of resin-embedded material.
on
routine in
now
overwhelming majority
ETAL.
al.
et
1987;
structural preserhave
procedures
to
be
modified accordingly.
The
use
of cryofixation
will
antigenicity
1989). Antigens
now
techniques and freeze substitution
which have
been immunostained
1989).
extreme
at
apparatus (Moor
fixation,
to
aid
to
the
popularity during
sensitivity
such
such
freezing technology,
which will
1987),
in
unobtainable depth within tissues
permit good freezing
(Craig
& Staehelin
as
of
1988),
in the retention of
decade
current
(Elder
have
higher plant actin,
as
the EM level in freeze-substituted cells
in
developments
Recent
increase
undoubtedly
the
&
(Lancelle
high-pressure
plant
tissues
to
Hepler
freezing
hitherto
a
will allow the localization of
fixation-sensitive antigens deep within freeze-substituted tissues.
The choice of
Although
the
embedding
epoxy
resins
procedure
cations due
to
loss of
merization required
resins
of
many
reported
to
preservation
of
structural
preservation
and have
generally
which is also
were
applications (Shaw
be difficult
to use
This
acrylic
resin has the
The resin has
an
polymerized by
extremely
a
Catalytic curing
at
low
viscosity,
of methods
variety
room
den Bosch
et
for conventional electron
the
most
to
be
of
needs
no
mixing
(1)
curing
an
Heat
process.
low-temperature poly-
1985;
tissues
1984;
Lowicryl
good
appli-
et
Gubler
(Herman
et
al.
1982)
al.
1987)
1988).
extremely reliable resin for
Van den Bosch etal.
and many other
and is
curing
not
at
60
toxic. It
or
1989).
advantages.
50°C
can
also be
(Fig. 4),(2)
temperature (the reaction is extremely exothermic), (3)
temperature ultraviolet polymerization
(Fig. 5) (Van
plant
& Miller
follows:
as
heat
procedures (Carlemalm
proved
hydrophilic properties
for the
& Henwood
with many
plant immunocytochemical applications (Craig
antigenicity.
immunocytochemical
exaggerated by the
specifically designed
the London resin LR White has
However,
unsuitable for
immunocytochemical
and have been used in various
but have been
are
antigenicity
hydrophilic Lowicryl
be critical in the
generally give superb
sectioning characteristics, they
The
can
al.
1989). Thus,
microscopy
convenient technique
using
for
or
the
Lowicryl catalyst
the resin
can
temperature polymerization is carried
out
at
—
20°C in
methyl
low
ether
be used with osmicated material
with unosmicated
retaining antigenicity.
benzoin
specimens
and
polymerized by
In the authors’ laboratories lowa
cryostat
fitted with
a
suitable
ANTIBODIES IN PLANT SCIENCE
ultraviolet
5 shows the localization of
Figure
source.
23
walls of Penicillium islandicum after
aldehyde
in the cell
species-specific antigen
a
fixation and
in
low-temperature embedding
LR White.
Other
Ultrathin cryosectioning is
techniques.
tissues
(Tokuyasu
scientists. It
by plant
suitable
be difficult
can
infiltrationit is
sucrose
used this technique with great
nin in
bean
developing
by
no means
success
White resin
rather
than in
temperature resin-embedding
can
be carried
cellular
thus
membranes,
cryosections
of
plant cells,
animal
ignored
but with
Chrispeels (1985)
cellulose
of the
in
alcohol.
polyvinyl
or
preservation
and
major shortcomings
thin
a
Unlike
osmication of the
level of
acceptable
one
Tokyasu technique
the labelled cryosection
techniques, post labelling
overcoming
on
been
immunolocalize phaseolin and phytohemaggluti-
methyl
an
success
largely
Greenwood &
impossible.
embedding of
This will result in
out.
that has
One modification of the standard
cotyledons.
that shouldprove useful is the final
LR
to
technique
ultra-thin
to cut
used with great
frequently
this is another
1986). However,
the low
cryosections
contrast
of
of
layer
in the
aldehyde-only
fixation.
of unembeddedcells
Labelling
a
modification of the
cortical
cells
root
dehydration
and
broken
were
immunolabelled prior
to
antigens
by Goodbody
al.
One
fixing
et
on
area
open
with
under buffer and
to
be
applied
Various
and
to
tissues it is
of plant
cytoskeleton
recently
plasma
by
of inter-
labelling
been described
for the localization of
membrane and the cortical
extensively
tissues is that of freeze-fracture
plant
likely
cells
labelling
on
fractured before
are
has
with various
techniques
(Severs
1989).
labelling.
As
even
techniques improve
techniques
will be
and
freeze-fracturing
These may prove useful for
the surface of membranes and
that these
animal cells
on
cytochemistry.
immunogold labelling
methodologies have been developed for labelling
the distribution of antigens
plant
Similar
should prove useful
face of the
cytoplasmic
paraformaldehyde-fixed
and osmium tetroxide followed
Kengen 1986).
In
protoplasts.
microtubule
of immunochemicaltechnology that has been used
freeze-fracturing
if cells
the
protoplast ‘footprints’
technique
This is the combination of freeze-fracture
or
&
of
mounts
of cells.
but which has yet
protocols.
broken open cells and
on
glutaraldehyde
cleaved
This
(1989).
associated with the
cytoskeleton
be achieved
critical-point drying (Traas
mediate filament
antigens
can
dry cleave technique, whole
developed
within the
for the
studying
lipid bilayer
cryopreservation
and modified for the
of
study
antigens.
CONCLUSIONS
Antibodies
such
are
now
essential tools in
the skills and
limited,
areas.
The
resources
authors’
experiences
number of years need
not
for instance
antibodies
success
and
in
by using
using
be
a
antibodies in
litre;
this involves
screening
of antibodies
a
science
and,
indicate that
daunting task,
purchased
plant
paying
by
plant
unless research is
involved should be available
of
to
given by
or
such
can
be
severely
engaging
facilities
quickly
be
of high
over
in
a
obtained,
other laboratories. The
obtaining preparations
every
to
laboratories
establishing
and useful results
science is in
attention
variety
to
key
to
specificity
step, including careful and thorough
techniques.
ACKNOWLEDGEMENTS
DE and
during
CH
the
were
period
supported by Royal
that
some
Society
1983
of the work discussed here
University
was
Research
carried
out.
Fellowships
24
M. DEWEY ETAL.
REFERENCES
Banowetz, G.M., Trione,
Krygier,
E.J. &
of
Immunological comparisons
wheat bunt
fungi,
bodies and
antisera.
J.D. &
Barbara,
M.F,
In:
Plant Science.
(1984):
of two
using monoclonal
sp
anti-
T.L.
197-219.
in
a
I, &
D.
Pitt,
of
pathogen
Cambridge University
immunosorbent
in
(ELISA).
assay
by
corms
Plant
In;
to
and
E.J.J.
S.
and
&
Pathol.
37;
A,
in
Virus
M.
Wilchek,
M. &
the
old.
(eds) Develop179-193.
Testing.
In:
Langone,
in
Methods
(eds)
Techniques.
A.
(1984):
87-150.
70:
Part
J.M.
Wall-less
(1984):
The Practice
Springer-Verlag,
Briars, S.A., Kessler,
F.
a
plants.
361-396.
& Evans
calmodulin-stimulated
of
prokaryotes
Phytopalhol. 22:
The
(1988):
ATPase ofmaize
140,000 M polypeptide. Ptanta,
coleoptiles
176:283-285.
G.R. &
Techniques
Academic
P.
Petrusz,
Immunocylochemistry.
in
Press,
Resin
Microsc.
Chard,
126:
R.M. &
Villiger,
electron
microscopy
at
low
W,
(1982):
and
temperature.
an
Ini.
J.
132-143.
of
Mycena galopus.
&
antigen
an
Trans.
J.C.
Frankland,
Br.
characteristic
Mycol.
84:
Soc.
235-241.
—,
—
&
—
Mycol.
Use
of
an
anti -Mycena
immunofluorescent
Trans. Br.
ation ofmycoplasma
comparison
of
bodies made
against
organisms.
&
Buss,
L.
(1987):
(1989): Prepar-
and
monoclonal
Primula Yellows
Appl.
Biol.
Coleman, J., Evans, D., Hawes,
ation of
S.L.
immunogensfrom plants and
polyclonal
Ann.
Structure
and
higher plant coated
a
anti-
mycoplasma-
114: 111-124.
J.I. &
limitations
In:
tissues.
plant
R.J, &
Palevitz,
from
and microtubule
Danscher,
G.
cal tissue.
Jones,
Edwards,
of
C., Horsley,
molecular
D.
&
organis-
vesicles. J. Cell. Sci. 88:
M.L.
(1986): Variations
enzyme-amplified
R.A.C. and
vicilin
in
treat-
immunogold
ER
and
Golgi.
freezing
pressure
Evaluation
and
of
characteriz-
endoplasmicreticulum
Eur. J. Cell Biol.
systems.
De
B.A.
I. Initial
Localization
111-. 245-260,
of
gold
method
photochemical
bind-
characterization
bundling, Planta
(1981);
A
Microtubule
(1989):
carrot.
microscopy. Histochemistry
in
for
biologi-
light
and
8: 1081-1083.
Mey, J., Lambert, A.M., Bajer, A.S., Moeremans,
M.
& De
Brabander,
Haemanthus
M.
and mitotic
with
endosperm
Proc.
Visualization
(1982):
interphase
method.
Natl.
the
of
cells of
plant
immuno-gold
Acad.
Sci.
USA
19:
1898-1902.
Dewey,
F.M.
of
(1988): Development
immunodiag-
fungal plant pathogens.
Protection
Crop
Brighton
Disease. 777-786.
British
Conference
Crop
In: Proc.
Pests
Protection
MacDonald, M.M. & Philips,
S.L
and
Council.
Devel-
(1989b):
opment ofmonoclonal
antibody—ELISA, —DOT-
Blot and —DIP-Stick
immunoassays
lanuginosa in
—,
—
&
rice. J. Gen. Micro.
J.
Gen.
for
for Humicola
135:361-374.
(1990): Development
antibody —ELISA
immunoassays
grains,
R.A.
Priestley,
monoclonal
Penicillium
of
and —DIP-STICK
islandicum
in
rice
Micro. 136: 753-760,
C.J.
&
clonal
antibodies
Dutch
Elm
Torrance,
and
immunoassays.
L.
Elder,
and
H.Y.
Brasier,
C.M.
specific
to
Disease
(1989a):
Mono-
components
of
pathogen Ophiostoma
(eds) Develop-
(1989): Cryofixation.
P.
Petrusz,
chemistry.
J. &
light
1-28. Academic
Bancroft,
the
ulmi.
M.T.,
White,
G. &
Callow,
bodies
to cell
surface
cysts of the
J.D.
Bullock,
in
G.R.
Immunocyto-
Press,
(1989):
London.
Polarized
enhancement
of
immunogoldsilver preparations:
immunohistology,
Estrada-Garcia,
In:
Techniques
microscopical
immunogold and
Its role in
(eds)
Vol. iv:
Ellis, I.O., Bell,
incident
35-45,
Cooper,
8:
Reps.
Plant Path. 38:9-20.
Soc. 84:243-249.
Clark, M.F., Morton, A.
Cole,
reagent.
galopus
on-grid
Staehelin, L.A. (1988): High
—, Munday,
(1985b):
serum as an
like
Cyr,
—,
T.R.G.
Gray,
Purification
(1985a):
for
embedding
J.M.,
I-IV.
Ini.
and
vicilin,
46: 80-93.
—,
developmentfor
of
Vols
seed
and associated membrane
London.
Carlemalm, E., Garavito,
analysis
(1982, 1983, 1985, 1989):
of
122: 35-44.
nostic assays for
r
Bullock.
&
—
staining
D.E.
resin
Periodate-acid
permits
of pea
microtubules in
Ann. Rev.
is
localization
electron
Berlin.
Bove,
White
Cell Biol.
(1984):
D.J.
sections
ing proteins
New York.
Press,
Bodanszky,
L.
(1980):
Immunochemical
Peptide Synthesis.
with
UK.
H.
Vunakis,
151-159. Academic
Bodanszky,
of
of
ation of novel features of the
test pro-
new
new
Torrance,
Ltd, Suffolk,
Van
Enzymology:
the
Merging
Applications
Lavenham Press
Bauminger,
of
(1986): Application
surveys:
R.A.C, and
Jones,
ments
Goodchild,
intact
O.W.
cedures
139-155.
Testing.
UK.
LR
(1984):
storage protein.
Proloplasma
enzyme-
417-422.
Barnett,
&
—
Detection of the leaf curl
(1988):
C,
Miller,
seed
pea
ment
anemones
&
Ltd, Suffolk,
879-886.
(ed.) Immunology
Press, Cambridge.
Barker,
S.
Craig,
Virus
Applications of
Lavenham Press
improved on-grid immunogold detection
(1986): Immunoassays
Wang,
menl and
76: 51-52.
Mycologia
Clark,
plant pathology.
in
Tilletia
B.B.
teliospores
J. Path.
Green,
J.A.
159:13-16.
J.R.,
Booth,
J.M.,
(1990): Monoclonal
components
of
zoospores
antiand
fungus Pylhium aphanidermatumreveal
species-specific antigens. Exp. Mycol.
13; 348-356.
ANTIBODIES IN PLANT SCIENCE
F.M. &
Evans, D.E., Dewey,
calcium
brane.
and
pumping ATPase
In;
De
Dainty, J.,
(1989):
Plant
(eds)
The
Membrane
231-236.
The Current Position.
Transport,
S.A.
plant plasma mem-
Michelis, M.I., Marre, E.,
F,
Rasi-Caldogno,
Briars,
of the
25
Elsevier,
Amsterdam.
Fox,
of
ELISA
in
of
Rots
Forest
the
field.
monoclonal
by
In:
Conference
Proceedings of
on
458-468.
Trees.
for the
(1988): Prospects
Armillaria
7th International
the
K.
Hahne,
diagnosis
antibody
Root
and Butt
British
Victoria,
Columbia,
&
Huisman, O.C. (1987): A
specific serological staining procedure
dahliae
in
tissue.
cotton root
for Verticil-
Phytopathology,
77:261-266.
detection
Serological
seeds.
of
Ferriss,
On the
R.S.
(1987):
Phomopsis longicolla
Phytopathology 77:
distribution
ated intermediate filament
in
371-375.
A.J. &
Goodbody, K.C., Hargreaves,
(1989):
J.S, &
C.W.
Lloyd,
of microtubule-associ-
antigens
in
hemagglutinin
M.J.
Chrispeels,
localization
cytochemical
the
of
Golgi
complex
Planta
164:295-302.
of
in
plant
The
mRNA
gene
suspen-
endoplasmic
developing
Planta
of
in
Immuno-
(1985):
and
phyto-
reticulum
bean
and
cotyledons.
J. &
of
appearance
tomatoes:
of
one
Tucker,
G.A.
polygalacturonase
series
a
of changes in
ripening.
and
163:263-271.
F.
in
microtubules
plant
tips
root
localization
embedded
Cell Biol. Int.
in
13:
Reps.
137-145.
a-amylase
through
aleurone cell walls.
studies
and
M.E. &
Pudoc,
Halk,
&
antibodies
in
F.
of
(1990): Etiological
grapevine
leafroll disease
antibodies. In:
in
Schots,
Agriculture.
A.
37-47.
Monoclonal
plant
cinnamomi.
and
Suzaki,
cysts
Monoclonal
(1985):
research.
Ann.
Rev.
E. & Lane
(1988):
E.
&
to
of the
Perkin,
J.L.
(1986):
isolate-, species-,
on
the
fungus
surface
and
of
Phytophthora
J. Bot. 64:311-321.
D.
Cold
tory Manual.
Ann. Rev.
London.
Press,
with
the
Physiol.
Plant
electron
localizmicro-
Plant Mol. Biol. 39:
139-155.
S.A.
Methods in Plant
(1984):
Blackwells Scientific
P.N. &
(1983): Immunogold-silverstaining:
immunostaining
Histochem.
Hush,
new cell
with
Cytochem.
J.M., Hawes,
Bird,
a new
enhanced
C.C.
method
sensitivity.
J.
31:938-944.
C.R.
microtubule
polarity
91-129.
Virology.
Publications, Oxford.
Holgate, C.S., Jackson, P., Cowen,
R.L.
Overall,
&
re-orientation
in wounded
(1990):
predicts
a
roots. J. Cell. Sci.
pea
96:47-61.
lanelli, D.J., Papparelli, R., Mariziaro, F,, Scala,
Noviello,
C.
Production
(1983):
secreting
monoclonal
antibodies
Fusarium.
Mycotaxon
17: 523-532.
Johnstone, A.
in
&
R.
Thorpe,
Practice.
Kabakoff,
Blackwell
D.S.
of
F.
hybridoma
the
to
genus
(1982): Immmochemistry
Scientific
(1980); In: Maggio,
Publications,
(1988):
Antibodies.
A Labora-
Subcellular
localization
of
macro-
E.T.
(ed.) Enzyme
CRC Press.
Kitagawa, T., Sakamoto, Y., Furumi, K.
Novel
(1989):
of
&
various
Fusarium
Ogwra,
for
immunoassays
enzyme
H.
specific
species.
Phyto-
flourish
in
pathology79:162-165.
Klausner,
A.
markets.
type,
(1987); Immunoassays
(1989): A
an
early
in the root
labelling
plant
of cell
of cell
P.K,
Hepler,
on
but not cell
(1989): Immunogold
sections
in different
of freeze-substituted
150: 72-74.
(1986): Myosin
D.
glycoproteins
position,
meristem ofDaucus carota L.
Protoplasma
organization
surface
106:47-56.
of actin
cells.
Lawson,
apical
S.A. &
Lancelle,
set
marker
new
5: 551-556.
Biotechnology
J.P.
forms
distribution
areas
fibroblasts.
J.
of
and
actin
antibody-labelled
Cell
Sci.
(Suppl. 5)
45-54.
Lin,
C.P. &
Chen,
and
pathology
T.A.
(1986); Comparison
polyclonal
Aster Yellows
antibodies
in
of mono-
detection
of
mycoplasma-like organism. Phyto-
76:45-50.
Lloyd, C.W., Slabas,A.R., Powell,
A.J. &
(1980): Microtubules, protoplasts
An immunofluorescent
shape:
Spring Harbor Laboratory.
71-105.
Immunoassay.
clonal
components
Can.
Cell
Methods and
gold: Principles.
of macromolecules
scope.
Recent
(1988); Immunocytochemical
quick-frozen
S.H.
disease
antibodies
genus-specific
zoospores
Boer,
23: 321-350.
Hardham, A.R.,
C.
ation
(1989):
L,
coated vesicles.
plant
Vols. I & II, Academic
Development
Brugger, J.J., Bonnard,
Antibodies
De
Phytopathol.
Hawes,
release
The Netherlands.
E.L.
Harlow,
B.,
monoclonal
Monoclonal
(ed.)
(1987): The
gibberellin-treated barley
Tremea,
diagnostic
improved by
J.V.
Planta 172:155-161,
Gugerli, P., Roseiglione,
S., Ramel,
E.M.
Herman,
Knox,
Ashford, A.E. & Jacobsen,
of
Colloidal
Applications.
of
study
Oxford.
13:119-128.
Reps.
detection
Immunofluorescence
(1989):
butyl-methyl-methacrylate.
—,
Biol. Int.
the
Cole,
D. &
Coleman, J., Evans,
advances in
Plant
(ed.)
Oxford.
expression during development
Gubler,
—,
&
phaseolin
Grierson, D., Slater, A., Spiers,
(1985):
Press,
Interphase
sion cells. J. Cell Sci. 93:427-438.
Greenwood,
103-130. IRL
of
S.A. &
Gleason, M.L., Ghabrial,
Soybean
C.H.
Biology.
Hill,
Gerik, J.S., Lommel, S.A.
lium
by microscopy. Shaw,
Molecular
Hayat (1989):
R.T.V. &
rapid
molecules
Lowe,
and
study.
plant
Planta
S.B.
cell
147:
500-506.
Lobler,
M.
protein
and
from
Klambt,
coleoptile
D.
(1985); Auxin-binding
membranes
of
corn
(Zea
M. DEWEY
26
methods
Purification
1:
L.)
mays
and
by
characterisation.
immunological
Biol. Chem.
J.
260:
9848-9853.
tion
the
and
Taylor,
J.D.
identification
detec-
(1990): Serological
of bacteria
conjugate Staphyllococus
from
aureus
plants by
slide
agglutin-
ation test. Plant Path. 39: 584-590.
Martin
C.
&
exogenous
mRNA
in
Northcote,
D.
gibberellic
acid
germinating
The
(1982):
action
isocitrate
on
bean
castor
S. &
of
lyase
seeds.
Planla
and diverse
Miller,
M.
homologue in
plant
S.A.,
(1989):
organs.
Rittenburg,
Characterization
cultures
Planla
179:
J.H.,
usable
immunoassays
toring
of
Protection
Crop
795-803.
British
&
—
—, —,
Crop
Patent
Mitchell, L
A. &
Sirococcus
in
assay. Can.
J. Forest. Res.
raised
Howell,
No.
strobilinus
(1989);
genus
Schots,
A.
15-21.
P.J.
(1988):
Detection of
monoclonal
immunosorbent
16: 945-948.
detectingantiserum.
S.
of glyoxysome
ment. DPhil.
Moor,
H.
The
(1986):
antiserum
and
its evalu-
Plant Path. 38:
Thesis.
(1987): Theory
in
and
and
of Oxford.
practice
R.A. and
Biological
and ultra-
peroxisome develop-
University
freezing. Steinbrecht,
Cryotechniques
biochemistry
of high
Zierold,
Electron
A.S.
&
pressure
K.
(eds)
Microscopy.
aniline
D.E.
herbicide,
microtube
(1987): Oryzalin,
a
dinitro-
binds to plant tubulin and inhibits
polymerization
in
vitro.
Planta
172:
252-264.
Napier,
Richardson,
and
Venis,
L.I. &
M.A.,
Bolton,
of
monoclonal
and
clonal antibodies to maize membrane auxin
protein.
Planta
Northcote,
D.H.,
tan in
the
cells.
poly-
binding
176: 519-526.
Davey,
antisera to localize
plant
M.A.,
Butcher, G. (1988): Preparation
characterization
J. Biol. Chem.
O.
256:
R. &
Lay,
cell-plate, primary
and
J.
and
+
ATPase
Plant
Progress
C. &
Plant
in
of ImmunodiffuoAnn
Allergy.
in
Arbor
USA.
P.
Gadal,
In:
immunocytochemistry.
Immunology
antibodies
119955-119957.
Handbook
(1968):
Publishers, Michigan,
(1986): Enzyme
T.L.
Wang,
Science.
59-58.
(ed.):
Cambridge
University Press, Cambridge.
Polak,
for
Varndell, l.M. (1984): Immunolabelling
J.M. &
Electron
Microscopy. Elsevier,
Characterization
adult wheat
Roberts,
of
mediation
S.,
Roberts.
Planta
Cloning,
T.
&
a
B.
des cellules
en
K.
N.
Hara,
from
(1985):
(1988):
the shoot
apex
of
175:403^111.
E.F. &
T.
Maniatis,
Manual
Laboratory
Spring Harbor Laboratory
fibrilles, parietales
(1984):
reorientations:
microtubules in
Satiat-Jeunemaitre,
B.A.
agglutinin
Planta 164: 439-447.
arrays.
Sambrook, J., Fritsch,
Molecular
germ
C.W. &
Hogetsu,
major L.
Vinca
Amsterdam.
Palevitz,
microtubule
helical
by
Sakaguchi,
wheat
Lloyd,
I.N.,
&
Planta 162: 55-61.
plants,
(1989):
2nd edn.
Press.
micro-
(1989): Microtubules,
morphogenese vegetale:
et
extension.
Bull. Soc. Bot.
Fr.,
cas
Actual.
136: 87-98.
N.J.
Severs,
Review
Freeze-fracture
(1989):
of methods. J.
Shaw,
&
P.J.
cytochemistry;
Electron Microsc.
Henwood,
localization
complex,
of
J.A.
Technique
(1985): Immuno-gold
cytochrome
ATP
f,
light-harvesting
and
synthase
ribulose
(1989):
Use of
arabinogalac-
secondary
wall of
Planta 178: 353-366.
Notermans,S.,Wieten,G.,Engel, H.W.B.,Rambouts,
1,5-
Planta
165:
333-339.
D.M.
Shotton,
mens.
(1989):
and its
microscopy
&
Confocal
two
P.A,
nuclear
antigenically
immobilized
of
an
of
on
Identification,
(1984):
and response to heat
shock
forms
major
related
envelope protein
application
optical
175-206.
Fisher,
developmentregulation
of
scanning
applications for biological speci-
J. Cell Sci. 94:
D.E.
purification
callose, xylan
H
vulgaris.
blots of heterogenousprotein
paper
electrophoresis:
Smith,
R.M.,
of Beta
bisphosphate carboxylase/oxygenase.
T.E., Mole-Bajer, J., Bajer,
Bureau,
Fosket,
62:157-166.
(1987):
83: 569-572,
samples.
Science
poly-
different
13: 175-203.
Springer-Verlag, Berlin.
Morejohn, L.C.,
A.B.
tissue
storage
from diazotised
Bot.
structure
from
(1987):
of extracellular
Olmsted, J.B. (1981)Affinitypurificationof
Cold
of
Cross-reactivity
J.H.
Boom,
van
Bacterial.
Appl.
Bennett,
Arrangement of
352-363.
Montgomery,
&
Ethylene-induced
Office.
against Phytophthora species
ation ofa
diag-
88300937.5,
with
enzyme-linked
&
Raikhel, N.V., Mishkind, M.
Agriculture.
European Patent
antibodies
S.B.
in
D.G. &
seed-borne
Mohan,
Diseases.
of modern
Sutherland,J.K. (1986):
an
Brighton
Netherlands.
Application
Blot Device.
field-
Protection Council.
Antibodies
Mitchell, D.H., Rose,
&
and moni-
and
and benefit to the farmer. In:
Monoclonal
Squash
rapid,
J.
species.
N.A.
activity
P.
properties
Perrot-Rechenmann,
F.P.
In:
plants.
Conference—Pests
Pudoc, Wageningen, The
European
of
diagnosis
in
(1990): Development
—
nostic tests
(ed.)
for the
fungal pathogens
148-155.
Petersen,
Grothaus, G.D. (1988): Application
cells
soybean
and
(EPS) antigens produced by
Ouchterlony,
Schindler,
of a connexin
mould
Oleski,
Physiol.
154: 174-183.
Meiners,
Purification
saccharide
N.S. &
Lyons,
R.M., Hoogerhout,
ETAL.
in
improved
antibodies
nitrocellulose
of
a
Drosophila embryos:
method
using
for
affinity
polypeptides
blots. J. Cell Biol. 99:
20-28.
Stacy,
N.J.,
Roberts,
K.R.
&
Knox,
P.J.
(1990):
Patterns of expression ofthe JIM 4 arabinogalactan-
protein epitope in
cell
cultures and
during somatic
ANTIBODIES IN PLANT SCIENCE
in
embryogenesis
Daucus
L.
carota
27
Planla
180:
285-292.
Talian, J.C., Olmsted,
A
rapid
labelled
its
specific
in
use
J.B. &
R.D.
Goldman,
for
procedure
(1983):
fluorescein-
preparing
antibodies from whole
antiserum;
architecture.
analysing cytoskeletal
J. Cell
Biol. 97: 1277-1282.
Thomas,
B.,
Partis,
M.D. &
In;
Science.
B.R.
Jordan,
to the
(1986);
of phyto-
study
Wang, T.L. (ed.) Immunology in
171-196.
Cambridge University
Plant
Press,
Cambridge,
Tijssen,
and
P.
Theory
Kippenberg,
of Enzyme
P.H.
(ed.)
G.W.
Warr,
and
Towbin, H.,
Staehelin,
Wiley
transfer
of
cryoultra-
J.
to nitrocellulose
acrylamide gels
some
and
J.
Proc. Nall.
applications.
and
antigens
(eds) Antibody
Tool.
as a
J.J.
21-58.
Sons, Chichester.
R.,
Atzom, R.,
U.
based
antibody
Antisera
(1986):
and
A.
of
immunoassay
&
monoclonal
T.L.
Wang,
hormones.
plant
(ed.) Immunology
in Plant
Science.
Cambridge University Press, Cambridge.
(1979):
from
sheets:
of
Microsc.
In:
Gordon,
proteins
Plant Science.
Marchalonis,
In:
E.W., Eberle, J., Martins,
S.M. &
J.
Duniec,
(1986):
polyfixatives
and
G.W.
Warr,
Wick,
Electrophoretic
Preparation
(1982);
of immunization.
principles
26-58,
&
139-148.
(ed.) (1986): Immunology in
Cambridge University Press, Cambridge.
Weiler,
143: 139-149.
T.
17:
immuno-
assays. Bulletin
Fejerabend, M,, Jourdan, P.S., Arnscheidt,
of
imraunocytochemistry.
combined
by
221-291.
Immunoassay.
(1986): Applications
to
Bulletin
OEPP/EPPO
T.
bacterial
and immunoidentification
Weiczorek,
K.T.
microtomy
isolation
Practice
Amsterdam.
Tokuyasu,
study.
Vuurde.J.W.L. (1987): New approachindetecting
John
In:
(1985):
ultrastructural
105: 27-43.
phytopathogenic
Wang,
Immunological approaches
chrome.
Van
and
microscopic
escence
Protoplasma
procedure
the
on
calmodulin
of
reactivity
in
Effects of various
plant
cell
tubulin and
immunofluorescence
microscopy.
Acad. Sci. USA.
Protoplasma
133: 1-18.
76:4350-4354.
J.A. &
Traas,
Kengen, H.M.P. (1986):
Gold
labelling
S.
—,Cho,
&
within
deployment
of microtubules in cleaved
A.R.
Mundelius,
studies of sheets of intact
root cells.
J. Histochem.
Cytochem.
34:
den
Bosch,
K.A.
of uricase
of sections
gold labelling
bean nodules. J.
and
(1986): Light
visualization
microscopic
&
immuno-
by
of resin-embedded
Microscopy
E.H.
Newcomb,
fluorescence
and
Cell Biol. Int.
epidermal
13:95-106.
Reps.
electron
—, Seagull, R.W., Osborn, M., Weber, K.
S.E.S.
Immunofluorescence
(1981):
organized
143: 187-197.
microtubule
meristematic
localiz-
(1986): Immunogold
&
Gunning,
of
microscopy
soy-
ized
—
mesophyll
1501-1504.
cells.
Van
Microtubule
(1989):
tissues:
plant
whole mounts ofcortical
arrays
in
cells.
plant
structurally
Cell
J.
stabil-
Biol.
89:
685-690.
ation
bean
—,
of
nodule-specific
root nodules.
Brewin,
mental
N.J. &
Kannenberg,
regulation of a
during growth
uricase in
of
developing soyWong,
Planta 167: 425-436.
E.L. (1989):
Rhizobium cell surface
root nodules.
pea
Developantigen
J. Bacterial. 171:
Driessche,
Kanerek,
L.
Van
Smets,
(1981):
The
in
G.,
Dejaegere,
R,
&
immuno-histochemical
pea
seeds
(Pisum
sativum
Lammeren, A.A.M.,
the
Kieft,
H.
microtubular
developmentin
Keijzer, C.J.,
(1985): Structure
cytoskeleton
Gasteria
verrucosa
Willemse,
and function
during pollen
(Mill.) H.
Duval.
Planta 121: 25-33.
Van
der
f.
cubense
sp.
race
(1988):
to Fusarium
4.
Lett.
J.B. &
Sworobuk,
a vesicular-arbuscular
fungus by using monoclonal
sorbent
LG.
Wright,
antibodies
Appl.
6: 39-42,
Identification of
Wyatt,
Appl.
assay.
G.M.,
J.E.
(1987):
mycorrhiza
antibodies in
Environ.
monas
P., Rennie, P.J.,
Fowke,
L.C.
(1980): Distribution
tubules
in
protoplasts.
Connolly,
J.A.
&
of cortical microAn
immunofluor-
and
syringae
methods. Food
K.L.,
Monoclonal
a
f.
J.G.
Turner,
(1989): Rapid
Wycoff,
Valk,
tobacco
&
immuno-
Microbiol.
53:
2222-2225.
Planta 153:287-296.
M.T.M. &
of
E,,
of lectin
localization
L.).
oxysporum.
Microbiol.
M.
White,
of monoclonal
Wright, S.F., Mortorn,
4537-4542.
Van
W.C.,
Production
specific
&
Morgan,
detection
phaseoli by
pv
Agric.
Immunol.
Jellison,
antibodies
J.
to
&
of
M.R.A.
Pseudo-
immunological
1: 53-56.
Ayers,
A.R.
(1987):
glycoprotein antigens
of
fungal plant pathogen, Phytophthoramegasperma
sp.
glycinea.
Plant
Physiol.
85: 508-515.