Ireland’s Response to the Looming World Energy Crisis by Colin Campbell ASPO.
Improved surface technology for protein arrays Colin Campbell
Transcript of Improved surface technology for protein arrays Colin Campbell
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
•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