High-throughput

screening

of

monoclonal

antibodies

against

plant

cell

wall

glycans

by

hierarchical

clustering

of

their

carbohydrate

microarray

binding

profiles

Antibody-producing

hybridoma

cell

lines

were

created

following

immunisation

with

a

crude

extract

of

cell

wall

polymers

from

the

plant

Arabidopsis

thaliana

In

order

to

rapidly

screen

the

specificities

of

individual

monoclonal

antibodies

-LRB-

mAbs

-RRB-

their

binding

to

microarrays

containing

cell

wall

glycans

immobilized

on

nitrocellulose

was

assessed

Hierarchical

clustering

of

microarray

binding

profiles

from

newly

produced

mAbs

together

with

the

profiles

for

mAbs

with

previously

defined

specificities

allowed

the

rapid

assignments

of

mAb

binding

to

antigen

classes

mAb

specificities

were

further

investigated

using

subsequent

immunochemical

and

biochemical

analyses

and

two

novel

mAbs

are

described

in

detail

mAb

binds

to

an

arabinanase-sensitive

pectic

epitope

and

mAb

binds

to

an

epitope

occurring

on

arabinogalactan-proteins

Both

mAbs

display

novel

patterns

of

recognition

of

cell

walls

in

plant

materials

Introduction

Carbohydrate

microarrays

provide

a

means

of

rapidly

screening

the

interactions

between

glycans

and

other

molecules

-LSB-

-RSB-

Applications

for

this

technology

include

the

screening

of

protein

glycan

interactions

characterization

of

carbohydrate-active

enzymes

and

the

analysis

of

the

specificities

of

monoclonal

antibodies

-LRB-

mAbs

-RRB-

and

carbohydrate-binding

modules

-LSB-

-RSB-

mAbs

are

powerful

tools

for

investigating

the

biological

roles

of

glycans

but

producing

them

is

time

consuming

labour-intensive

and

costly

Hybridoma-based

mAb

production

involves

the

fusion

of

spleen

cells

from

an

immunized

animal

with

myeloma

cells

The

resulting

hybridoma

cell

lines

are

both

immortal

and

secrete

antibody

into

the

cell

supernatant

-LSB-

-RSB-

This

procedure

is

conventionally

performed

in

a

one-by-one

fashion

such

that

each

animal

is

immunized

with

a

single

antigen

and

the

resulting

antibodies

are

screened

for

desired

specificities

using

enzyme-linked

immunosorbent

assays

-LRB-

ELISAs

-RRB-

However

ELISA-based

screening

is

low

throughput

because

only

a

limited

amount

-LRB-

μl

-RRB-

of

hybridoma

supernatant

is

available

for

screening

during

the

initial

stages

of

mAb

production

and

this

is

typically

only

sufficient

to

test

each

mAb

against

just

one

or

two

antigens

An

alternative

approach

involves

shotgun

immunisation

with

a

mixture

of

antigens

-LSB-

-RSB-

This

potentially

results

in

the

simultaneous

generation

of

antibodies

with

a

range

of

specificities

but

the

time

limiting

step

then

becomes

the

detailed

retrospective

screening

of

their

specificities

However

microarrays

offer

a

means

of

rapidly

screening

limited

amounts

of

hybridoma

supernatant

against

multiple

antigens

and

therefore

greatly

increasing

throughput

in

the

identification

of

valuable

cell

lines

Here

we

report

the

use

of

shotgun

immunisation

followed

by

microarray-based

screening

of

hybridoma

supernatants

in

order

to

produce

mAbs

against

plant

cell

wall

glycans

Plant

cell

walls

are

fibre

composites

that

contain

some

of

the

most

complex

glycans

known

-LSB-

-RSB-

In

addition

to

their

biological

roles

many

cell

wall

components

have

important

industrial

applications

including

as

functional

food

ingredients

pharmaceuticals

nutriceuticals

fibres

and

increasingly

bio-fuels

-LSB-

-RSB-

Cell

wall

glycans

can

be

broadly

grouped

into

cellulose

hemicelluloses

pectins

and

glycoproteins

-LSB-

-RSB-

Cellulose

microfibrils

are

cross-linked

by

hemicelluloses

such

as

xyloglucans

xylans

and

mixed

linkage

glucans

forming

a

tough

load-bearing

matrix

which

is

embedded

in

pectic

polysaccharides

-LSB-

-RSB-

Pectins

are

the

most

complex

and

heterogeneous

family

of

cell

wall

glycans

and

are

comprised

of

a

series

of

galacturonic

acid-rich

polymers

including

homogalacturonan

-LRB-

HG

-RRB-

rhamnogalacturonan

I

-LRB-

RGI

-RRB-

rhamnogalacturonan-II

-LRB-

RG-II

-RRB-

and

xylogalacturonans

-LRB-

XG

-RRB-

In

addition

structurally

complex

arabinan

galactan

and

arabinogalactan

polymers

may

be

present

as

side

chains

to

the

galacturonan-rich

backbone

domains

-LSB-

-RSB-

The

protein

moieties

of

cell

wall

glycoproteins

are

often

rich

in

hydroxyproline

-LRB-

Hyp

-RRB-

and

these

polymers

which

are

collectively

referred

to

as

Hyp-rich

glycoproteins

-LRB-

HRGPs

-RRB-

include

the

extensins

and

arabinogalactan-proteins

-LRB-

AGPs

-RRB-

-LSB-

-RSB-

The

fine

structures

and

relative

amounts

of

cell

wall

components

vary

greatly

not

only

among

plants

but

also

between

organs

tissues

cells

and

even

between

different

micro-domains

within

a

single

cell

wall

This

complexity

and

heterogeneity

presents

a

major

barrier

to

detailed

analysis

and

our

understanding

of

many

aspects

of

plant

cell

wall

structure

and

function

is

far

from

complete

Several

mAbs

with

specificities

for

diverse

plant

cell

wall

components

have

been

developed

and

these

are

powerful

tools

for

the

analysis

of

cell

walls

-LSB-

-RSB-

However

the

repertoire

of

mAbs

currently

available

covers

only

a

small

proportion

of

the

glycan

structures

that

have

been

identified

and

there

is

a

pressing

need

for

a

wider

range

of

mAbs

to

facilitate

the

further

characterization

of

cell

walls

We

have

developed

a

method

of

rapidly

screening

mAb

specificities

using

microarrays

of

cell

wall

glycans

including

pectins

with

different

degrees

and

patterns

of

methyl-esterification

pectic

side

chains

-LRB-

e.g.

arabinan

and

galactan

-RRB-

hemicelluloses

-LRB-

e.g.

xylans

mannans

and

xyloglucans

-RRB-

HRGPs

-LRB-

e.g.

AGP-rich

gums

-RRB-

β-linked

glucans

-LRB-

e.g.

-LRB-

-RRB-

-LRB-

-RRB-

β-glucan

and

-LRB-

-RRB-

β-glucan

-RRB-

and

celluloses

-LRB-

e.g.

hydroxylethyl

cellulose

and

carboxylmethyl

cellulose

-RRB-

A

further

samples

were

sequentially

extracted

from

A.

thaliana

using

CDTA

and

NaOH

which

are

known

to

predominately

solublize

pectic

polymers

and

hemicelluloses

respectively

-LSB-

-RSB-

Experimental

procedures

Immunisation

and

generation

of

monoclonal

hybridomas

Cell

wall

polymers

were

isolated

from

week

old

wild

type

A.

thaliana

plants

ecotype

grown

in

soil

at

°C

with

cycles

of

h

of

h

of

darkness

One

g

dry

weight

of

a

mixture

of

leaves

stems

and

roots

was

homogenized

to

a

fine

powder

in

liquid

nitrogen

The

homogenate

was

incubated

with

ml

mM

acid

-LRB-

CDTA

pH

-RRB-

for

h

at

°C

and

centrifuged

for

min

at

rpm

The

supernatant

was

collected

and

dialyzed

extensively

against

deionized

water

-LRB-

-RRB-

in

dialysis

tubing

-LRB-

kDa

molecular

weight

cut

off

-RRB-

to

remove

low

molecular

weight

molecules

and

freeze

dried

The

material

was

dissolved

in

phosphate-buffered

saline

-LRB-

PBS

-RRB-

to

generate

the

immunogen

Rats

were

used

for

antibody

production

so

as

to

make

subsequent

comparisons

with

existing

mAbs

most

of

which

were

produced

in

rats

as

valid

as

possible

The

immunization

of

rats

hybridoma

preparation

and

cloning

procedures

were

as

described

previously

-LSB-

-RSB-

Briefly

two

male

Wistar

rats

were

each

injected

subcutaneously

with

μl

of

an

emulsion

of

the

isolated

cell

wall

material

at

mg/ml

in

PBS

with

an

equal

volume

Freund

's

complete

adjuvant

on

day

On

days

and

the

injections

were

repeated

using

incomplete

adjuvant

Tail

bleeds

were

taken

days

after

injections

to

assess

the

immune

response

On

day

a

pre-fusion

boost

was

given

to

the

selected

rat

and

days

later

the

spleen

was

removed

and

lymphocytes

were

isolated

and

fused

with

rat

myeloma

cell

line

-LSB-

-RSB-

using

standard

polyethylene

glycol

fusion

of

lymphocytes

and

myeloma

cells

Hybridoma

lines

were

initially

screened

by

ELISA

with

the

immunogen

coated

onto

microtitre

plates

-LRB-

MaxiSorp

Nunc

Roskilde

Denmark

-RRB-

at

μg/ml

Previously

described

monoclonal

antibodies

Arrays

were

probed

with

mAbs

with

previously

described

specificities

and

details

of

these

are

provided

in

Table

All

were

rat

antibodies

except

mAbs

and

which

were

produced

in

mice

and

which

was

produced

by

phage

display

Table

characterized

monoclonal

antibodies

used

to

probe

glycan

arrays

HG

homogalacturonanSpecificityNameRef.Un-esterified

-LSB-

-RSB-

Un-esterified/Calcium

ion

cross-linked

-LSB-

-RSB-

Partially

methyl-esterified

-LSB-

-RSB-

Partially

methyl-esterified

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

Fucosylated

-LSB-

-RSB-

Non-fucosylated

-LRB-

-RRB-

β-mannan/galacto

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LRB-

-RRB-

β-glucanBS

-LSB-

-RSB-

-LRB-

-RRB-

β-glucanBS

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

Glycan

samples

used

on

the

array

Details

of

the

defined

glycans

used

are

provided

in

Table

Most

glycans

were

dissolved

in

Arabinoxylan

and

glucuronoxylan

were

prepared

by

boiling

in

for

min

and

then

standing

for

h

at

°C

before

use

Glucomannan

was

prepared

by

wetting

with

ethanol

followed

by

addition

of

The

mixture

was

heated

to

boiling

point

and

stirred

for

min

until

dissolved

Pachyman

was

prepared

by

dissolution

in

a

minimal

volume

of

-LRB-

w/v

-RRB-

sodium

hydroxide

followed

by

neutralization

with

acetic

acid

samples

on

the

arrays

were

cell

wall

polymers

extracted

from

A.

thaliana

organs

listed

in

Table

using

CDTA

and

M

NaOH

Fifty

milligrams

-LRB-

fresh

weight

-RRB-

of

each

organ

collected

from

at

least

four

separate

plants

were

homogenized

to

a

fine

powder

prior

to

adding

μl

of

mM

CDTA

-LRB-

pH

-RRB-

After

incubating

with

rotation

for

h

at

°C

the

extracts

were

centrifuged

at

rpm

for

min

and

the

supernatants

-LRB-

CDTA

extracts

-RRB-

removed

Pellets

were

resuspended

in

μl

of

M

NaOH

and

samples

were

incubated

with

rotation

for

h

at

°C

prior

to

centrifugation

at

rpm

for

min

Supernatants

were

NaOH

extracts

Table

included

on

the

glycan

arraysAlphanumerical

-LRB-

sugar

beet

-RRB-

-LRB-

apple

-RRB-

-LRB-

lupin

-RRB-

-LRB-

sugar

beet

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

lime

-RRB-

-LRB-

soybean

-RRB-

-LRB-

A.

thaliana

-RRB-

-LRB-

pea

-RRB-

I

-LRB-

apple

-RRB-

II

-LRB-

carrot

-RRB-

III

-LRB-

potato

-RRB-

-LRB-

apple

-RRB-

-LRB-

apple

-RRB-

-LRB-

apple

-RRB-

-LRB-

P.

patens

-RRB-

mucilage

-LRB-

A.

thaliana

-RRB-

-LRB-

tomato

-RRB-

-LRB-

konjac

-RRB-

-LRB-

guar

-RRB-

-LRB-

locust

bean

-RRB-

arabic

-LRB-

acacia

-RRB-

-LRB-

karaya

-RRB-

-LRB-

tragacanth

-RRB-

-LRB-

larch

-RRB-

-LRB-

wheat

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

glucan

-LRB-

lichenan

-RRB-

-LRB-

ivory

nut

-RRB-

-LRB-

tamarind

-RRB-

-LRB-

maize

-RRB-

-LRB-

-RRB-

glucan

-LRB-

avicel

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

glucan

-LRB-

laminarin

-RRB-

-LRB-

-RRB-

glucan

-LRB-

pachyman

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

glucan

-LRB-

pullulan

-RRB-

extract

-LRB-

A.

thaliana

flowers

-RRB-

extract

-LRB-

A.

thaliana

siliques

-RRB-

extract

-LRB-

A.

thaliana

stem

top

-RRB-

extract

-LRB-

A.

thaliana

stem

middle

-RRB-

extract

-LRB-

A.

thaliana

stem

base

-RRB-

extract

-LRB-

A.

thaliana

leaves

-RRB-

extract

-LRB-

A.

thaliana

roots

-RRB-

extract

-LRB-

A.

thaliana

flowers

-RRB-

extract

-LRB-

A.

thaliana

siliques

-RRB-

extract

-LRB-

A.

thaliana

stem

top

-RRB-

extract

-LRB-

A.

thaliana

stem

middle

-RRB-

extract

-LRB-

A.

thaliana

stem

base

-RRB-

extract

-LRB-

A.

thaliana

leaves

-RRB-

extract

-LRB-

A.

thaliana

roots

-RRB-

Alphanumerical

codes

refer

to

the

position

of

samples

on

arrays

Source

organisms

are

in

parenthesesRGI

Rhamnogalcturonan

I

RGII

rhamnogalacturonan

II

MHR

modified

hairy

region

AGP

arabinogalactan-protein

Post-printing

modification

of

glycans

Glycan

samples

on

selected

arrays

were

modified

in

situ

after

printing

by

enzymatic

digestion

For

the

data

in

Fig.

selected

arrays

were

digested

with

endo-α

-LRB-

-RRB-

L-arabinanase

or

endo-β

-LRB-

-RRB-

galactanase

-LRB-

both

from

Aspergillus

niger

Megazyme

-LRB-

Bray

Ireland

-RRB-

used

at

U/ml

in

mM

sodium

acetate

pH

Printing

of

arrays

Glycans

were

applied

to

nitrocellulose

membrane

-LRB-

μm

pore

size

Schleicher

and

Schuell

Dassel

Germany

-RRB-

at

two

concentrations

-LRB-

and

mg/ml

-RRB-

and

in

duplicate

such

that

each

sample

was

represented

by

four

spots

CDTA

and

NaOH

extracted

A.

thaliana

material

was

printed

as

extracted

and

as

a

five

fold

dilution

also

in

duplicate

Printing

was

performed

using

a

microarray

robot

-LRB-

Microgrid

II

Genomic

Solutions

Ann

Arbor

MI

USA

-RRB-

equipped

with

split

pins

-LRB-

MicroSpot

Genomic

Solutions

-RRB-

Pins

were

washed

twice

in

after

deposition

of

each

sample

Probing

of

arrays

Arrays

were

blocked

by

incubation

for

h

in

PBS

-LRB-

mM

NaCl

mM

KCl

mM

mM

pH

-RRB-

containing

w/v

low

fat

milk

powder

-LRB-

MPBS

-RRB-

Arrays

were

then

probed

for

h

with

antibodies

diluted

in

MPBS

All

antibodies

were

used

as

dilutions

except

which

was

used

at

and

and

which

were

used

at

After

washing

with

PBS

arrays

were

incubated

for

h

in

either

anti-rat

or

anti-mouse

secondary

antibody

conjugated

to

alkaline

phosphatase

-LRB-

Sigma

Poole

UK

-RRB-

diluted

in

MPBS

After

washing

in

PBS

arrays

were

developed

using

a

substrate

containing

-LRB-

BCIP

-RRB-

and

nitroblue

tetrazolium

-LRB-

NBT

-RRB-

in

BCIP/NBT

buffer

-LRB-

mM

NaCl

mM

mM

diethanolamine

pH

-RRB-

Scanning

and

analysis

Arrays

were

scanned

converted

to

bit

grey-scale

TIFFs

transformed

to

negative

images

and

uploaded

into

ImaGene

microarray

analysis

software

-LRB-

BioDiscovery

El

Segundo

CA

USA

-RRB-

Semi-automatic

gridding

was

used

to

create

an

analysis

area

for

each

spot

and

a

zone

around

each

spot

was

used

for

calculation

of

local

background

signals

Individual

spot

signals

were

defined

as

the

mean

pixel

value

within

each

spot

area

-LRB-

red

zone

in

Fig.

-RRB-

minus

the

median

pixel

value

in

the

surrounding

local

background

area

-LRB-

green

zone

in

Fig.

-RRB-

The

mean

of

the

four

individual

spot

signals

for

each

sample

was

defined

as

the

mean

sample

value

-LRB-

MSV

-RRB-

and

total

mean

sample

values

-LRB-

TMSVs

-RRB-

were

the

means

of

MSVs

from

three

separate

experiments

Selected

data

sets

-LRB-

TMSVs

-RRB-

were

used

to

generate

heatmaps

-LRB-

Figs.

and

-RRB-

using

online

heatmapper

software

-LRB-

-RRB-

For

each

antibody

in

the

heatmap

the

maximal

TMSV

was

set

to

and

all

other

values

within

that

data

set

adjusted

accordingly

A

cut

off

of

of

the

maximal

TMSV

was

imposed

Hierarchical

cluster

analysis

was

performed

on

TMSVs

obtained

for

previously

characterized

antibodies

and

newly

produced

ones

using

Epclust

software

-LRB-

-RRB-

Clustering

was

based

on

correlation

measure

based

distance

and

average

linkage

Fig.

of

plant

cell

wall

glycans

a

b

A

library

of

glycan

polymers

were

spotted

onto

nitrocellulose

membranes

in

an

area

mm

mm

Arrays

included

pectins

HRGPs

hemicelluloses

and

glucans

-LRB-

samples

-RRB-

and

the

position

of

these

classes

of

polymer

on

the

arrays

are

indicated

by

colour

coding

in

-LRB-

b

-RRB-

Fourteen

samples

extracted

from

Arabidopsis

thaliana

using

either

CDTA

-LRB-

samples

-RRB-

or

NaOH

-LRB-

samples

-RRB-

were

also

included

Details

of

the

samples

are

provided

in

Table

c

Each

sample

was

printed

at

the

two

concentrations

indicated

and

in

duplicate

d

Microarray

analysis

software

-LRB-

ImaGene

-RRB-

was

used

to

quantify

spot

signals

and

individual

spot

signals

were

defined

as

the

mean

pixel

value

within

each

spot

area

-LRB-

red

zone

-RRB-

minus

the

median

pixel

value

in

the

surrounding

local

background

area

-LRB-

green

zone

-RRB-

e

g

Two

pairs

of

three

arrays

were

printed

and

probed

with

the

anti-homogalacturonan

mAb

on

three

separate

occasions

The

mean

sample

values

-LRB-

MSVs

-RRB-

for

each

pair

were

quantified

and

plotted

against

MSVs

of

an

array

produced

on

a

different

occasion

These

are

presented

as

scatter

plots

with

the

for

the

plots

indicatedFig

of

cell

wall

glycan

arrays

with

mAbs

with

known

specificities

a

Glycan

arrays

were

probed

with

mAbs

with

previously

defined

specificities

and

representative

examples

are

shown

-LRB-

anti-homogalacturonan

-RRB-

-LRB-

anti-

-LRB-

-RRB-

β-glucan

-LRB-

anti-

-LRB-

-RRB-

β-galactan

-RRB-

-LRB-

anti-arabinogalactan-protein

-RRB-

-LRB-

anti-homogalacturonan

-RRB-

A

control

array

was

probed

with

secondary

antibody

but

no

primary

mAb

Arrays

were

scanned

and

converted

into

bit

greyscale

TIFFs

b

Heatmap

showing

the

total

mean

sample

values

-LRB-

TMSVs

-RRB-

for

the

binding

of

each

mAb

to

each

sample

The

maximal

TMSV

for

each

mAb

was

set

to

and

all

other

values

were

adjusted

accordingly

A

cut

off

of

of

the

maximal

TMSV

was

imposed

and

all

values

at

or

below

this

are

represented

as

black

boxes

Indirect

immunofluorescence

labeling

of

plant

materials

Resin

embedded

and

fresh

plant

material

was

sectioned

and

labelled

with

antibodies

as

described

previously

-LSB-

-RSB-

and

hybridoma

supernatants

were

used

as

dilutions

in

MPBS

Sections

were

counterstained

for

cellulose

with

Calcofluor

white

-LRB-

fluorescent

brightener

Sigma

Poole

UK

-RRB-

used

as

a

aqueous

solution

Competitive-inhibition

ELISA

assays

The

ability

of

arabinose

galactose

rhamnose

and

oligoarabinosides

to

inhibit

the

binding

of

and

to

the

immuogen

in

ELISAs

was

assessed

as

described

previously

-LSB-

-RSB-

Sugar

composition

analysis

of

modified

pectic

hairy

regions

-LRB-

MHRs

-RRB-

Sugar

composition

was

determined

using

methanolysis

as

described

previously

-LSB-

-RSB-

MHRs

were

treated

with

N

HCl

in

dry

methanol

for

h

at

°C

followed

by

h

of

M

-LRB-

TFA

-RRB-

at

°C

The

released

sugars

were

analysed

using

high-performance

anion

exchange

chromatography

-LRB-

HPAEC

-RRB-

with

pulsed

amperometric

detection

-LRB-

PAD

-RRB-

as

described

previously

-LSB-

-RSB-

Results

Production

of

microarrays

of

plant

cell

wall

polymers

The

layout

of

the

arrays

is

shown

in

Fig.

and

colour

coding

indicates

the

distribution

on

the

arrays

of

different

classes

of

cell

wall

polymer

-LRB-

Fig.

b

-RRB-

A

detailed

list

of

the

samples

used

is

provided

in

Table

Each

sample

was

represented

on

the

arrays

by

four

spots

-LRB-

two

concentrations

printed

in

duplicate

-RRB-

-LRB-

Fig.

-RRB-

and

microarray

analysis

software

was

used

to

quantify

background-corrected

spot

signals

-LRB-

Fig.

-RRB-

As

shown

by

the

arrays

in

Figs.

and

the

consistency

of

the

printing

and

probing

replicates

within

arrays

was

high

However

reproducibility

between

arrays

was

also

tested

A

total

of

six

arrays

-LRB-

pairs

-RRB-

were

printed

and

probed

with

the

anti-HG

mAb

on

three

separate

occasions

The

MSVs

were

quantified

and

the

values

for

each

array

were

plotted

against

those

from

an

array

produced

on

a

different

occasion

The

obtained

for

these

plots

were

all

indicating

a

high

degree

of

reproducibility

between

arrays

-LRB-

Fig.

g

-RRB-

Fig.

of

cell

wall

glycan

arrays

with

newly

produced

mAbs

Glycan

arrays

were

probed

with

mono

or

multiclonal

antibodies

generated

following

shotgun

immunisation

with

plant

cell

wall

polymers

Four

representative

examples

are

shown

Arrays

were

scanned

and

converted

into

bit

greyscale

TIFFs

Probing

of

plant

cell

wall

glycan

arrays

Arrays

were

probed

with

a

range

of

mAbs

with

previously

defined

specificities

for

epitopes

occurring

on

the

major

classes

of

cell

wall

polymers

Details

of

the

mAbs

used

are

provided

in

Table

A

total

of

mAbs

were

tested

and

representative

examples

are

shown

in

Fig.

The

TMSVs

for

the

binding

of

these

mAbs

to

each

sample

-LRB-

derived

from

three

independent

experiments

-RRB-

are

shown

as

a

heatmap

in

Fig.

where

spot

signals

are

correlated

to

colour

intensity

mAb

binding

profiles

were

in

good

agreement

with

the

published

results

for

the

antibodies

tested

For

example

mAb

has

specificity

for

HG

with

a

low

degree

of

methyl-esterification

-LRB-

DE

-RRB-

-LSB-

-RSB-

and

consistent

with

this

bound

to

lime

pectin

samples

with

DEs

of

-LRB-

samples

and

-RRB-

did

not

bind

above

background

to

other

pectin

samples

with

higher

DEs

or

to

any

other

cell

wall

glycans

mAb

is

specific

for

-LRB-

-RRB-

β-glucan

-LSB-

-RSB-

and

of

the

defined

samples

only

bound

to

this

polymer

-LRB-

sample

-RRB-

on

the

arrays

-LRB-

-RRB-

β-glucan

is

known

to

be

extractable

from

plant

cell

walls

using

NaOH

and

consistent

with

this

also

bound

to

weakly

to

NaOH-solubilized

extracts

from

A.

thaliana

-LRB-

samples

-RRB-

As

expected

the

epitopes

recognized

by

some

mAbs

were

detected

on

multiple

samples

and

this

was

the

case

for

mAbs

-LRB-

anti-

-LRB-

-RRB-

β-galactan

-RRB-

-LRB-

anti-AGP

-RRB-

and

However

the

binding

profiles

of

these

mAbs

were

also

consistent

with

their

known

specificities

For

example

bound

with

greatest

avidity

to

pectin

samples

that

contained

abundant

HG

domains

-LRB-

such

as

the

lime

pectins

-RRB-

but

not

to

pectic

fragments

lacking

HG

-LRB-

such

as

the

arabinan

or

galactan

samples

and

-RRB-

To

generate

a

new

series

of

cell

wall-directed

mAbs

rats

were

immunized

with

an

immunogen

consisting

of

a

crude

extract

of

cell

wall

material

solubilized

from

A.

thaliana

using

CDTA

cell

lines

were

selected

by

an

initial

ELISA

analysis

against

the

immunogen

and

these

were

subsequently

probed

against

the

cell

wall

polymer

arrays

and

four

representative

examples

are

shown

in

Fig.

Antibodies

that

bound

with

very

low

avidity

or

appeared

to

have

identical

binding

profiles

to

previously

generated

antibodies

were

not

selected

for

further

analysis

whilst

the

data

for

the

remaining

seven

were

subjected

to

hierarchical

cluster

analysis

Hierarchical

clustering

of

antibody

binding

profiles

Cluster

analysis

was

used

to

rapidly

obtain

information

about

the

specificities

of

new

antibodies

based

on

the

similarly

of

their

binding

profiles

on

the

arrays

to

previously

characterized

mAbs

-LRB-

Fig.

-RRB-

Clustering

indicated

that

most

of

the

antibodies

tested

separated

into

broad

clusters

HG-binders

pectic

side

chain-binders

HRGP-binders

and

hemicellulose-binders

Three

antibodies

with

specificity

for

-LRB-

-RRB-

-LRB-

-RRB-

β-glucan

-LRB-

-RRB-

β-glucan

and

-LRB-

-RRB-

β-mannan

did

not

cluster

with

any

other

mAbs

within

the

-LRB-

distance

measurement

units

-RRB-

cut

off

imposed

These

broad

clusters

were

in

agreement

with

the

previously

defined

specificities

of

the

mAbs

tested

and

this

was

also

the

case

within

sub-clusters

For

example

within

the

HG-binders

cluster

mAbs

and

which

both

bind

to

partially

methyl-esterified

HG

-LSB-

-RSB-

clustered

closely

whilst

mAbs

and

which

both

bind

to

non-methyl-esterified

HG

also

clustered

closely

-LSB-

-RSB-

Similarly

within

the

hemicellulose-binders

the

anti-xylan

mAbs

and

formed

a

sub-cluster

as

did

the

two

anti-xyloglucan

mAbs

and

One

of

the

new

mAbs

-LRB-

designated

when

cloned

-RRB-

was

grouped

with

the

pectic

side

chain-binders

-LRB-

anti-

-LRB-

-RRB-

β-galactan

-RRB-

and

-LRB-

anti-

-LRB-

-RRB-

α-arabinan

-RRB-

However

most

of

the

new

antibodies

tested

were

grouped

in

the

HRGP-binders

cluster

and

within

this

clustered

most

closely

with

the

anti-AGP

mAbs

and

One

of

the

new

mAbs

within

this

group

-LRB-

designated

when

cloned

-RRB-

was

together

with

subject

to

more

detailed

epitope

characterization

in

order

to

further

explore

the

evidence

gained

from

the

clustering

analysis

Fig.

analysis

of

antibody

binding

profiles

to

arrays

mAbs

with

previously

defined

specificities

and

newly

produced

antibodies

-LRB-

red

diamonds

in

black

boxes

-RRB-

were

analyzed

using

Epclust

software

Hierarchical

clustering

was

performed

using

clustered

correlation

measure

based

distance

and

average

linkage

The

binding

profiles

of

all

the

mAbs

analyzed

clustered

into

four

broad

groups

homogalacturonan-binders

pectic

side

chain-binders

HRGP-binders

and

hemicellulose-binders

Three

mAbs

were

non-clustering

outliers

-LRB-

NCO

-RRB-

The

specificities

of

the

previously

defined

mAbs

are

listed

in

Table

Detailed

characterization

of

the

epitopes

recognized

by

and

Cluster

analysis

of

and

suggested

that

the

epitopes

recognized

by

these

mAbs

may

contain

neutral

sugars

such

as

arabinose

and

galactose

that

are

abundant

in

the

side

chains

of

both

pectins

and

HRGPs

The

glycan

arrays

contained

five

samples

of

branched

-LRB-

or

hairy

-RRB-

regions

of

pectic

polymers

modified

by

enzymatic

digestion

-LRB-

modified

hairy

regions

or

MHRs

-RRB-

that

were

rich

in

neutral

sugars

and

for

which

monosaccharide

composition

was

determined

-LRB-

Fig.

-RRB-

The

binding

of

and

to

these

samples

was

therefore

of

particular

interest

bound

only

to

an

MHR

from

carrot

-LRB-

sample

-RRB-

whilst

bound

to

MHRs

from

potato

and

apple

-LRB-

samples

and

-RRB-

It

was

noteworthy

that

the

two

MHR

samples

to

which

did

not

bind

-LRB-

samples

and

-RRB-

had

the

lowest

arabinose

content

suggesting

that

the

epitope

recognized

by

this

mAb

may

contain

arabinose

In

order

to

explore

this

possibility

further

the

sensitivity

of

the

epitope

to

arabinanase

digestion

was

investigated

To

do

this

cell

wall

glycan

microarrays

were

digested

with

arabinanase

prior

to

probing

with

mAbs

and

the

TMSVs

obtained

with

and

without

digestion

were

compared

These

data

are

presented

as

scatter

plots

-LRB-

Fig.

d

-RRB-

in

which

signals

from

enzyme

treated

arrays

were

plotted

against

signals

from

untreated

arrays

As

a

positive

control

arabinanase

digested

and

untreated

arrays

were

probed

with

the

anti-

-LRB-

-RRB-

α-arabinan

mAb

-LSB-

-RSB-

As

expected

the

binding

of

to

all

samples

was

reduced

to

some

extent

by

arabinanase

digestion

-LRB-

Fig.

-RRB-

However

the

effect

of

arabinanase

digestion

on

binding

was

even

more

pronounced

than

for

and

binding

to

all

samples

on

the

arrays

was

essentially

abolished

-LRB-

Fig.

-RRB-

This

suggested

that

the

epitope

recognized

by

either

contained

arabinan

and

was

degraded

by

arabinanase

or

that

the

epitope

was

attached

to

arabinan

and

was

released

from

arrays

by

arabinanase

In

contrast

binding

to

all

samples

was

essentially

unaffected

by

arabinanase

digestion

-LRB-

Fig.

-RRB-

The

effect

of

galactanase

digestion

on

mAb

binding

to

arrayed

samples

was

also

assessed

binding

was

reduced

to

some

extent

by

galactanase

digestion

but

the

reduction

was

far

less

for

than

arabinanase

digestion

-LRB-

Fig.

-RRB-

binding

to

all

samples

was

unaffected

by

galactanase

digestion

and

the

ability

of

this

enzyme

to

degrade

galactan

was

indicated

by

the

fact

that

the

binding

of

the

anti-

-LRB-

-RRB-

β-galactan

mAb

was

essentially

abolished

by

galactanase

digestion

-LRB-

data

not

shown

-RRB-

The

epitope

recognized

by

was

further

characterized

using

competitive

inhibition

ELISA

-LRB-

ciELISA

-RRB-

assays

-LRB-

Fig.

-RRB-

The

binding

of

to

immobilized

arabinan

was

inhibited

to

of

maximal

binding

by

approximately

μg/ml

of

both

and

but

oligoarabinosides

of

lower

degrees

of

polymerisation

and

rhamnose

galactose

and

glucuronic

acid

failed

to

inhibit

binding

at

the

highest

concentration

tested

of

mg/ml

The

binding

of

to

immobilized

antigen

was

not

inhibited

by

any

of

the

haptens

tested

at

concentrations

up

to

mg/ml

-LRB-

data

not

shown

-RRB-

Fig.

of

and

to

samples

to

pectic

modified

hairy

regions

-LRB-

MHRs

-RRB-

a

Monosaccharide

composition

of

pectin

modified

hairy

regions

-LRB-

MHRs

-RRB-

from

apple

-LRB-

samples

and

-RRB-

carrot

-LRB-

sample

-RRB-

potato

-LRB-

sample

-RRB-

b

TMSVs

for

the

binding

of

mAbs

and

to

these

samples

The

maximal

TMSV

for

and

-LRB-

to

all

samples

on

the

array

-RRB-

was

set

to

and

all

other

values

adjusted

accordingly

A

cut

off

of

of

the

maximal

TMSV

was

imposed

and

all

values

at

or

below

this

are

represented

as

black

boxesFig

epitope

recognized

by

is

arabinanase

sensitive

a

d

Scatter

plots

showing

the

effect

of

galactanse

and

or

arabinanase

on

the

binding

of

and

-LRB-

anti-

-LRB-

-RRB-

α-arabinan

-RRB-

to

glycan

arrays

Arrays

were

incubated

in

enzymes

after

blocking

but

before

probing

TMSVs

were

determined

and

plotted

against

each

other

Spots

that

lie

on

the

dotted

line

indicate

that

enzyme

digestion

had

no

effect

on

mAb

binding

whereas

spots

that

lie

below

the

dotted

line

indicate

where

enzyme

digestion

reduced

mAb

binding

e

The

binding

of

to

oligoarabinosides

was

tested

by

competitive

inhibition

ELISA

in

which

arabinan

was

the

immobilized

polymer

Oligoarabinosides

with

degrees

of

polymerization

less

than

five

failed

to

inhibit

binding

even

when

used

at

mg/ml

Both

and

inhibited

the

binding

of

by

of

maximal

binding

when

used

at

approximately

μg/ml

Cluster

analysis

suggested

that

was

most

similar

to

that

of

mAbs

with

specificity

for

pectic

side

chains

whilst

the

binding

of

was

most

similar

to

that

of

anti-AGP

mAbs

AGPs

can

be

distinguished

from

pectic

polymers

using

SDS-PAGE

because

whereas

AGPs

resolve

as

characteristic

smears

pectic

polymers

typically

do

not

enter

gels

-LSB-

-RSB-

Western

blots

were

prepared

using

the

same

A.

thaliana

CDTA-extracted

material

that

was

used

for

immunisation

and

probed

with

or

-LRB-

Fig.

-RRB-

did

not

bind

to

material

on

these

blots

whereas

bound

to

a

high

molecular

weight

smear

-LRB-

kDa

-RRB-

that

is

characteristic

of

AGPs

Fig.

of

monoclonal

antibodies

and

The

CDTA-soluble

Arabidopsis

thaliana

fraction

that

was

used

as

the

immunogen

was

separated

by

SDS-PAGE

with

μg

protein

loading

per

lane

A

Western

blot

of

this

material

was

probed

with

and

did

not

bind

to

the

blot

but

produced

a

smear

in

the

region

of

kDa

which

is

characteristic

of

AGPs

Immunolocalization

of

the

and

epitopes

In

order

to

further

investigate

the

epitopes

recognized

by

and

in

planta

a

range

of

plant

materials

were

probed

with

these

mAbs

using

indirect

immunofluorescence

labelling

In

transverse

resin-embedded

sections

of

A.

thaliana

inflorescence

stems

bound

specifically

to

epidermal

cells

and

most

strongly

to

the

tangential

cell

walls

-LRB-

Fig.

-RRB-

Plant

cell

wall

glycans

may

be

altered

during

extraction

and/or

immobilization

such

that

epitopes

are

changed

from

their

native

state

in

planta

In

order

to

test

if

this

was

a

factor

in

determining

the

arabinase

sensitivity

of

the

epitope

sections

of

A.

thaliana

stems

were

also

digested

with

arabinanase

prior

to

probing

with

As

shown

by

comparison

of

Fig.

b

the

binding

of

to

A.

thaliana

stem

epidermal

cell

walls

was

abolished

following

arabinanase

digestion

indicating

that

the

epitope-structure

immobilised

on

arrays

recognized

by

reflected

that

observed

in

planta

In

contrast

to

bound

to

some

extent

to

all

cell

walls

in

an

equivalent

section

through

an

A.

thaliana

stem

-LRB-

Fig.

-RRB-

Labelling

of

the

surface

of

intact

roots

of

A.

thaliana

seedlings

indicated

that

bound

very

weakly

whereas

bound

strongly

to

the

surface

of

root

hairs

-LRB-

Fig.

and

f

-RRB-

In

transverse

sections

through

tobacco

stems

the

epitope

was

associated

with

cell

walls

adjacent

to

metaxylem

cells

of

the

vascular

tissue

a

distinctly

different

labelling

pattern

to

that

observed

in

A.

thaliana

stems

-LRB-

Fig.

-RRB-

In

contrast

did

not

label

any

cell

walls

in

tobacco

stems

-LRB-

Fig.

-RRB-

Fig.

immunofluorescence

labelling

of

plant

material

with

and

bound

strongly

to

inner

and

outer

radial

walls

of

epidermal

cells

in

transverse

sections

of

A.

thaliana

inflorescence

stem

sections

The

section

is

counterstained

with

calcofluor

-LRB-

blue

-RRB-

that

labels

β-linked

glucans

b

binding

to

A.

thaliana

inflorescence

stem

sections

when

sections

were

treated

with

arabinanase

Arrowheads

in

a

and

b

indicate

the

epidermal

surface

c

Control

in

which

an

equivalent

stem

to

that

in

a

was

labelled

with

the

absence

of

primary

antibody

d

An

equivalent

section

to

that

shown

in

a

labelled

with

which

bound

to

all

cell

walls

e

bound

very

weakly

to

the

intact

surface

of

the

roots

of

A.

thaliana

seedlings

f

bound

strongly

to

the

surface

of

root

hairs

-LRB-

arrowheads

-RRB-

of

Arabidopsis

seedling

roots

g

In

transverse

sections

through

tobacco

stem

bound

strongly

to

cell

walls

adjacent

to

files

of

metaxylem

cells

-LRB-

mx

-RRB-

h

did

not

bind

to

equivalent

sections

through

tobacco

stems

Scale

bars

μm

for

a

d

g

and

h

μm

for

g

inset

μm

for

e

and

f

Discussion

The

work

presented

here

demonstrates

the

potential

of

glycan

microarrays

for

overcoming

a

major

bottleneck

in

anti-glycan

mAb

production

Specifically

the

use

of

microarrays

enabled

μl

of

hybridoma

supernatant

to

be

screened

rapidly

and

simultaneously

against

potential

epitope-bearing

target

molecules

A

novel

aspect

of

this

work

was

the

use

of

cluster

analysis

of

array

data

to

rapidly

predict

antibody

specificities

by

comparison

with

previously

defined

mAbs

Subsequent

detailed

analyses

of

the

specificities

of

the

new

mAbs

and

indicated

they

bound

to

pectic

and

AGP

class

of

polymers

respectively

as

was

predicted

by

the

cluster

analysis

These

results

indicate

that

if

a

relatively

large

set

of

probes

with

defined

specificities

are

available

to

serve

as

references

this

is

an

effective

method

for

high-throughput

initial

mAb

screening

One

potential

limitation

of

shotgun

immunisation

could

be

immuno-dominance

such

that

an

immune

response

is

elicited

against

a

limited

subset

of

the

injected

antigens

In

this

study

two

mAbs

and

were

selected

with

specificity

for

two

different

classes

of

molecule

However

of

the

seven

mAbs

selected

for

cluster

analysis

of

binding

profiles

six

clustered

with

mAbs

with

specificity

for

AGPs

and

one

with

mAbs

to

pectic

side

chains

suggesting

that

AGPs

were

the

immuno-dominant

antigens

in

this

case

It

is

therefore

likely

that

such

a

multi-antigen

approach

towards

cell

wall

polymers

may

be

most

effective

to

obtain

a

panel

of

mAbs

with

a

range

of

specificities

within

a

single

class

of

polymer

The

arrays

we

constructed

were

based

on

the

non-covalent

attachment

of

glycans

to

nitrocellulose

This

approach

has

the

advantage

that

molecules

can

be

immobilized

directly

without

the

need

to

create

functional

groups

and

nitrocellulose

has

been

previously

shown

to

be

a

effective

substrate

for

the

immobilization

of

diverse

glyans

-LSB-

-RSB-

Non-covalent

attachment

has

the

potential

drawback

that

variations

in

the

effectiveness

of

immobilization

may

result

from

differences

in

the

structural

properties

of

the

arrayed

molecules

-LSB-

-RSB-

All

of

the

arrayed

samples

used

in

this

work

were

recognized

to

some

extent

by

at

least

one

of

the

mAbs

used

-LRB-

as

shown

in

Fig.

-RRB-

indicating

that

all

were

immobilized

to

some

degree

Nevertheless

the

spot

signals

obtained

can

only

provide

semi-quantitative

information

about

mAb

binding

Two

new

probes

and

were

produced

against

cell

wall

polymers

and

both

have

distinctly

different

specificities

to

previously

generated

antibodies

Cluster

analysis

ciELISA

data

immunoblotting

and

arabinanase

sensitivity

suggested

that

the

epitope

recognized

by

is

an

arabinan-containing

structure

that

occurs

as

a

side

chain

on

pectic

polymers

Pectic

side

chains

typically

consist

of

either

galactan

arabinan

or

type

I

arabinogalactan

chains

in

which

arabinose

usually

occurs

as

a

terminal

sugar

-LSB-

-RSB-

It

was

of

note

that

binding

to

arrays

was

reduced

to

some

extent

by

galactanase

digestion

However

the

galactanase

used

had

very

low

arabinanase

side

activity

-LRB-

U/ml

specific

activity

with

an

arabinan

substrate

compared

to

U/ml

specific

activity

with

a

galactan

substrate

-RRB-

It

is

possible

therefore

that

galactanase

digestion

resulted

in

the

indirect

loss

of

the

epitope

by

cleavage

of

galactan

to

which

an

arabinan-containing

epitope

is

attached

The

fact

that

bound

to

both

pectin-derived

MHR

samples

and

to

AGP-like

material

on

a

blot

suggests

that

this

mAb

binds

to

an

epitope

of

type

II

arabinogalactan

that

may

occur

on

both

pectins

and

AGPs

The

novel

patterns

of

recognition

on

A.

thaliana

and

other

plant

materials

indicates

that

these

are

useful

new

probes

for

the

analysis

of

cell

wall

glycans

polymers

and

complex

cell

wall

architectures

O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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carbohydrate microarrays
plant cell walls
monoclonal antibodies
hierarchical clustering