improved surface technology for protein arrays colin campbell · common abo blood types a 1 b o a 2...
TRANSCRIPT
Impro
ved s
urf
ace t
echno
logy
for
pro
tein
arr
ays
Co
lin C
am
pbe
ll
Intr
od
uction
•R
ation
ale
•B
ackgro
un
d
•S
urf
ace c
hoic
e f
or
pro
tein
mic
roa
rray
•D
ete
ctio
n a
nd d
iscri
min
atio
n o
f div
ers
e s
urf
ace a
ntige
ns
•Lab
el-fr
ee d
ete
ction
•A
na
lysis
tools
Com
mon A
BO
blo
od types
A1
B
OA
2
The b
asic
unit o
f th
e A
BO
syste
m is t
he O
antigen:
fucose-t
erm
inate
d p
enta
sacchari
de
The A
and B
antigens a
re d
eri
vatives
In t
he c
ase o
f A
the s
ugar
is N
-acety
l ga
lacto
se
In t
he c
ase o
f B
it
is g
ala
cto
se.
A1B
Curr
ent m
eth
ods o
f blo
od typin
g
Oly
mp
us p
late
reader
Dia
me
dchro
mato
gra
ph
y “
card
”
•D
efin
e su
rfac
e ch
emis
try:
Gla
ss s
lides
hav
e bee
n p
repar
ed a
nd c
oat
ed w
ith
1. P
oly
ion
2. F
unct
ional
sil
ane
layer
3. F
unct
ional
hyro
gel
layer
Pro
tein
arr
ay s
urf
ace c
hem
istr
y
H
05
10
15
20
BR
AD
3JL
DM
3L
A2
LB
2
An
tib
od
y
S/Ngold
Hydro
gel I
Epoxy S
ilane
Hydro
gel II
Poly
-l-lysin
e
Com
parison o
f arr
ay s
urf
aces
Surf
ace e
nhanced flu
ore
scence
Effe
ct of m
eta
llic s
urf
ace
:F
luo
roph
ore
qu
ench
ing
0-5
nm
Incre
ased
ra
dia
tive
de
ca
y r
ate
/qu
an
tum
yie
ld 5
-20
nm
B
0
50
10
0
15
0
20
0
25
0
30
0
BR
AD
3
NEA
T
DA
M1
NE
AT
ES
15
A(B
)
NE
AT
JLD
M3
NEA
T
LA
2 D
IL
1/8
LB
2 D
IL
1/2
Anti
bo
dy
S/N
A1
05
01
00
15
02
00
25
03
00
35
0
BR
AD
3
NE
AT
DA
M1
NE
AT
ES
15
A(B
)
NE
AT
JL
DM
3
NE
AT
LA
2 D
IL
1/8
LB
2 D
IL
1/2
An
tib
od
y
S/N
O
0
0.51
1.52
2.53
BR
AD
3
NEA
T
DA
M1
NEA
T
ES
15A
(B)
NEA
T
JLD
M3
NEA
T
LA
2 D
IL
1/8
LB
2 D
IL
1/2
Antibody
S/N
A2
0
50
100
150
200
250
300
350
BR
AD
3
NEA
T
DA
M1 N
EA
TES
15A
(B)
NEA
T
JL
DM
3
NEA
T
LA
2 D
IL 1
/8L
B2 D
IL 1
/2
An
tib
od
y
S/N
ab
cd
A1
B
A2
O
An
ti-R
hD
An
ti-A
An
ti-A
(B)
-ve
co
nt
An
ti-A
An
ti-B
An
ti-R
hD
An
ti-A
An
ti-A
/B-v
eco
nt
An
ti-A
An
ti-B
An
ti-R
hD
An
ti-A
An
ti-A
(B)
-ve
co
nt
An
ti-A
An
ti-B
An
ti-R
hD
An
ti-A
An
ti-A
(B)
-ve
co
nt
An
ti-A
An
ti-B
AB
O d
ete
ction
A1B
+
0
50
100
150
200
BR
AD
3
NE
AT
DA
M1
NE
AT
ES
15A
(B)
NEA
T
JLD
M3
NE
AT
LA
2 D
IL
1/8
LB
2 D
IL
1/2
Anti
bo
dy
S/N
A1+
0
50
10
015
0
20
0
25
0
30
0
BR
AD
3
NEA
T
DA
M1
NEA
T
ES
15A
(B)
NEA
T
JLD
M3
NE
AT
LA
2 D
IL
1/8
LB
2 D
IL
1/2
An
tibo
dy
S/N
Ax+
0
50
10
015
0
20
0
25
0
30
0
BR
AD
3
NEA
T
DA
M1
NE
AT
ES
15A
(B)
NEA
T
JLD
M3
NE
AT
LA
2 D
IL
1/8
LB
2 D
IL
1/2
Anti
bo
dy
S/N
B+
0
100
200
300
400
BR
AD
3
NE
AT
DA
M1
NE
AT
ES
15A
(B)
NE
AT
JL
DM
3
NE
AT
LA
2 D
IL
1/8
LB
2 D
IL
1/2
An
tib
od
y
S/N
O+
0
50
100
150
200
BR
AD
3
NE
AT
DA
M1
NE
AT
ES
15A
(B)
NEA
T
JLD
M3
NEA
T
LA
2 D
IL
1/8
LB
2 D
IL
1/2
Anti
bo
dy
S/N
A2B
+
0
50
100
150
200
250
BR
AD
3
NEA
T
DA
M1
NEA
T
ES
15
A(B
)
NEA
T
JLD
M3
NEA
T
LA
2 D
IL
1/8
LB
2 D
IL
1/2
Anti
bo
dy
S/N
ab
cd
ef
A1B
Rh
DA
2B
Rh
D
BR
hD
A1R
hD
AXR
hD
OR
hD
An
ti-R
hD
An
ti-A
An
ti-A
(B)
-ve
co
nt
An
ti-A
An
ti-B
An
ti-R
hD
An
ti-A
An
ti-A
(B)
-ve
co
nt
An
ti-A
An
ti-B
An
ti-R
hD
An
ti-A
An
ti-A
(B)
-ve
co
nt
An
ti-A
An
ti-B
An
ti-R
hD
An
ti-A
An
ti-A
(B)
-ve
co
nt
An
ti-A
An
ti-B
An
ti-R
hD
An
ti-A
An
ti-A
(B)
-ve
co
nt
An
ti-A
An
ti-B
An
ti-R
hD
An
ti-A
An
ti-A
(B)
-ve
co
nt
An
ti-A
An
ti-B
Rhesus a
ntigen
Most
com
mon R
hesus a
ntige
ns a
re D
, C
or
c a
nd E
or
e
Encode
d o
n t
wo s
tructu
rally
hom
olo
gous g
enes o
n t
he R
hlo
cus.
Both
are
417 a
am
ulti-pass inte
gra
l m
em
bra
ne p
rote
ins w
ith 6
expose
d p
ep
tide
loops o
n e
ryth
rocyte
surf
ace p
resentin
g d
iscre
te a
ntige
nic
epitopes.
Tw
o p
rote
ins d
iffe
r b
y 3
6 a
min
o a
cid
s a
nd t
he
antigen
ic r
egio
ns d
iffe
r b
y a
s little
as o
ne a
min
o a
cid
betw
een R
hD
an
d R
hC
E
0.11
10
100
1000
LHM
76/5
8LH
M76
/59
LHM
50/2
BES
D1
LHM
76/5
5LH
M77
/64
LHM
70/4
5LH
M59
/19
LHM
169/
80B
RA
D3 LD
M1
LDM
77/6
4 ESD
1M
H48
DEM
1
An
tib
od
ies
log (S/N)
Rhesus d
iscrim
ination
R1R
1blo
od h
as D
antigen b
ut
neither
c n
or
E
r”r”
has c
and E
but
not
D
Label-fr
ee b
lood typin
g
•M
otivation
–R
em
ove
va
ria
bili
ty a
nd
tim
e b
y r
em
ovin
g
pro
ce
ss s
tep
s lik
e
•P
urificatio
n
•Lab
elli
ng
•C
am
e a
bout fr
om
effort
s to label th
e w
hole
blo
od p
rote
om
e
05
10
15
20
25
DA
M1
ES
15
LA
2LB
2
An
tib
od
y
S/N
488/1
543/1
543/2
543/3
488/1
Repeat
Re
d C
ells
0.7
5
0.8
0.8
5
0.9
0.9
51
1.0
5
350
400
450
500
550
600
nm
Absorbance
An
ti-A
An
ti-A
An
ti-B
Choic
e o
f scanner
settin
gs
585
543
Case 5
43/3
578
543
Case 5
43/2
570
543
Case 5
43/1
530
488
Case 4
88/1
Dete
ction
wa
vele
ngth
(n
m)
Excitation
wa
vele
ngth
(nm
)
An
ti-A
(B)
Gro
up
B
0.1110100
1000
DA
M1
Ne
at
ES
15 N
ea
tL
A2
Ne
at
LB
2 1:
4
An
tib
od
y
log S/N Ratio
Lab
elle
d R
ed C
ells
Wh
ole
Blo
od
- N
eat
Wh
ole
Blo
od
1/5
Dilu
tion
Wh
ole
Blo
od
1/1
0 D
ilutio
n
Gro
up
A1
0.1110100
1000
DA
M1
ES
15
LA
2 L
B2
An
tib
od
y
log S/N Ratio
La
be
lled
Red
Ce
lls
Wh
ole
Blo
od
- N
ea
t
Wh
ole
Blo
od
1/5
Dilu
tio
n
Wh
ole
Blo
od
1/1
0 D
iluti
on
Typ
e O
0123456789
10
DA
M1 N
ea
tE
S15 N
ea
tL
A2 N
ea
tL
B2 1
:2
An
tib
od
y
log S/N Ratio
La
be
lle
d R
ed
Ce
lls
Wh
ole
Blo
od
- N
ea
t
Wh
ole
Blo
od
1/5
Dilu
tio
n
Wh
ole
Blo
od
1/1
0 D
ilu
tio
n
An
ti-A
An
ti-A
(B)
An
ti-A
An
ti-B
An
ti-A
An
ti-A
(B)
An
ti-A
An
ti-B
An
ti-A
An
ti-A
(B)
An
ti-A
An
ti-B
Ch
oic
e o
f sa
mp
le
pre
-tre
atm
en
t
Sho
ws la
bel-fr
ee A
BO
dis
cri
min
atio
n
Conclu
sio
n
•A
rray b
ased m
eth
od c
an d
iscrim
inate
com
mon e
ryth
rocyte
s
•A
uto
fluore
scence
can b
e u
sed a
s a
read-
out m
odalit
y
Evalu
ation o
f a P
rote
in M
icro
chip
Meth
od f
or
Typin
g W
hole
Blo
od
Ste
w –
Lab T
alk
20
thN
ovem
ber
2006
Aim
•T
ake
67
blo
od
sam
ple
s a
nd
eva
lua
te the
arr
ay m
eth
od
of A
BO
p
he
noty
pin
g.
•A
rra
ys w
ere
pri
nte
d w
ith
mu
ltip
le s
potte
d r
ep
lica
tes o
f a
ntib
od
ies
sp
ecific
for
eith
er
A (
LA
2)
or
B (
LB
2)
blo
od
typ
e a
ntige
ns, a
lon
g w
ith
P
BS
an
d Ig
G o
nly
sp
ots
as n
eg
ative
co
ntr
ols
•In
to
tal 3
2 s
ep
ara
te s
ub
-arr
ays w
ere
prin
ted
per
slid
e, w
ith
each
su
b-a
rra
y c
onta
inin
g 5
re
plic
ate
s o
f L
A2
an
tib
od
y, 7 r
ep
lica
tes o
f L
B2
, 2
re
plic
ate
s o
f Ig
G a
nd
7 r
ep
lica
tes o
f P
BS
•T
he
refo
re e
ach
arr
ay c
onta
ine
d the
fo
llow
ing
num
bers
of re
plic
ate
sp
ots
: L
A2
(n =
16
0),
LB
2 (
n =
224
), IgG
(n
= 6
4)
an
d P
BS
(n
= 2
24)
Exp
ecte
d R
esu
lts
•T
hre
e b
loo
d typ
es w
ere
ana
lyse
d: A
, B
an
d O
•T
wo
an
tib
od
ies w
ere
use
d to
dete
ct th
ese
diffe
ren
t b
loo
d typ
es:
–LA
2 =
anti-A
blo
od t
ype a
ntig
en
–LB
2 =
anti-B
blo
od t
ype a
ntig
en
•T
yp
e A
blo
od s
hou
ld r
eact w
ith
th
e L
A2
an
tib
od
y
•T
yp
e B
blo
od s
hou
ld r
eact w
ith
th
e L
B2
an
tib
od
y
•T
yp
e O
blo
od s
hou
ld n
ot re
act w
ith e
ith
er
an
tib
od
y (
neg
ative
respo
nse)
RO
C C
urv
es
•R
OC
curv
es w
ere
in
itia
lly d
eve
lop
ed
in
th
e 1
94
0s in
ord
er
to m
ake
se
nse
of ra
dio
sig
na
ls c
on
tam
ina
ted
by n
ois
e
•R
OC
’sare
a g
rap
hic
al p
lot o
f se
nsitiv
ity v
s (
1-s
pe
cific
ity)
or
true
p
ositiv
es v
s fa
lse
po
sitiv
es
•T
he
op
tim
um
pre
dic
tive
ou
tco
me
is th
ere
fore
a c
urv
e in
th
e u
ppe
rle
ft c
orn
er
of th
e g
raph
i.e
10
0%
se
nsitiv
ity (
all
true
positiv
es fo
un
d)
an
d 1
00
% s
pe
cific
ity (
no
fals
e p
ositiv
es fo
un
d)
•In
co
ntr
ast a c
om
ple
tely
ran
do
m p
red
icto
r w
ou
ld g
ive a
str
aig
ht lin
e
at 4
5 d
egre
es fro
m th
e h
orizo
nta
l, fro
m b
otto
m le
ft to
to
p r
igh
t
Use
of R
OC
s to
se
t th
resh
old
s
•A
n In
de
x S
core
wa
s o
bta
ine
d for
ea
ch
arr
ay w
here
the
med
ian
sig
na
l-b
ackg
rou
nd
va
lue for
the
LA
2 a
ntib
od
y w
as d
ivid
ed
by th
e
me
dia
n for
the
LB
2, g
ivin
g a
ra
tio
va
lue
fo
r th
e tw
o a
ntib
od
y
resp
on
se
s
•R
OC
curv
es (
se
nsitiv
ity v
s. (1
-sp
ecific
ity))
were
th
en
plo
tte
d to
o
bta
in thre
sho
ld v
alu
es w
ith
in w
hic
h it co
uld
be
con
fid
en
tly s
aid
tha
t a
giv
en
sa
mple
wa
s o
f a
pa
rtic
ula
r b
lood
typ
e
De
fin
ing
a th
resh
old
•R
OC
curv
es w
ere
constr
ucte
d t
o o
bta
in t
hre
shold
va
lues f
or
each o
f th
e b
lood
types,
the R
OC
curv
e f
or
the A
blo
od t
ype is s
ho
wn b
elo
w
•T
he R
OC
curv
e is a
gra
ph
ical plo
t of
sensitiv
ity v
s.
(1-s
pecific
ity).
•F
or
the R
OC
analy
sis
the A
blo
od t
ype s
am
ple
inde
x s
core
s w
ere
use
d t
o
repre
se
nt
the t
rue p
ositiv
e s
am
ple
s,
whils
t th
e ind
ex s
core
s o
f th
e B
blo
od t
ype
sam
ple
s w
ere
used a
s t
he f
als
e p
ositiv
es
Are
a u
nd
er c
urv
e =
0.9
95
De
fin
ing
a th
resh
old
•T
he
RO
C c
urv
e d
em
on
str
ate
d a
go
od
ab
ility
to
se
pa
rate
th
e A
an
d B
blo
od
typ
es a
s t
he
are
a u
nd
er
the
cu
rve
(0
.99
5)
wa
s c
lose
to
th
e m
axim
um
are
a o
f 1
,in
dic
atin
g a
to
tal
se
pa
ratio
n o
f th
e s
am
ple
se
ts.
•T
his
in
dic
ate
s t
ha
t fo
r th
e c
ho
se
n t
hre
sh
old
cu
t-o
ff v
alu
e o
f 2
.71
fo
r th
e A
blo
od
typ
e
va
lues,
a s
en
sitiv
ity o
f 1
00
% a
nd
a s
pe
cific
ity o
f 9
6.2
% w
as o
bta
ine
d
•T
hre
sh
old
va
lue
s f
or
the
oth
er
blo
od
typ
es w
ere
ob
tain
ed
an
d a
rep
rese
nte
d b
elo
w,
ag
ain
th
e R
OC
cu
rve
s d
em
on
str
ate
d a
go
od
ab
ility
to
se
pa
rate
bo
th t
he
A a
nd
O,
an
d
the
B a
nd
O b
loo
d t
yp
es
100 (
at 0
.929
)
97 (
at 2
.7)
87.5
(at
0.9
29
)
92.3
(at
2.7
)
0.9
29 –
2.7
O
87.5
100
�0.6
4B
96.2
100
�2.7
1A
% S
pec
ific
ity a
t T
hres
hold
% S
ensi
tivit
y a
t T
hre
shold
Th
resh
old
Valu
e S
elec
ted
of
Ind
ex
Sco
re
fro
m R
OC
Cu
rve
Blo
od
Typ
e
Assig
nin
g b
lood
typ
es b
ase
d o
n th
e
se
lecte
d th
resh
old
s
Cla
ssif
ica
tion
A
B
O
Tota
l
Sam
ple
s
To
tal
Sam
ple
s 26
8
33
67
Corr
ect
ly P
red
icte
d
25
7
32
64
Inco
rrec
tly P
red
icte
d
1
1
1
3
% S
am
ple
s C
orr
ectl
y P
red
icte
d
96.2
87
.5
96.9
95.5
Carr
ied o
ut
a “
hold
-out”
valid
ation b
y u
sin
g 9
0%
of
the s
am
ple
s a
s “
train
ing
set”
and 1
0%
as a
“te
st
set”
Co
nclu
sio
ns
•S
ho
wn
tha
t w
e c
an
use
an
tib
od
y a
rra
y tech
no
log
y to
accu
rate
ly
ph
en
oty
pe
RB
Cs b
y d
ete
ctin
g c
om
ple
x m
ixtu
res o
f an
tig
ens o
n the
ce
ll surf
ace
•T
he
syste
m is la
be
l fr
ee a
nd
ca
n d
ete
ct b
loo
d typ
e a
ntig
ens in
who
le
blo
od
sa
mp
les thu
s a
ctin
g to
sig
nific
an
tly s
imp
lifyin
g th
e b
loo
dty
pin
g p
roce
du
re
•T
he
use
of R
OC
curv
es a
llow
s th
e d
efin
itio
n o
f cle
ar
thre
sho
lds
with
in w
hic
h a
n u
nkno
wn
sa
mp
le c
an
be
successfu
lly a
ssig
ne
d a
p
art
icu
lar
blo
od
typ
e
Acknow
ledgem
ents
•D
PM
–P
ete
r G
hazal, J
ohn
Beatt
ie,
Ala
n R
oss,
Ste
wart
Burg
ess,
Thors
ten
Fors
ter
•S
NB
TS
–N
ick O
’loo
ney,
Janin
e
Ro
bb,
Marisa C
ho
ng-
Kw
an,
Jura
j P
etr
ik
•S
co
ttis
h E
nte
rpri
se