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Journal of Immunoassay Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii19
Radioimmunoassay for Albuterol Using a Monoclonal Antibody: Application for Direct Quantification in Horse Urine a
a
b
b
A. Adam , H. Ong , D. Sondag , A. Rapaille , a
a
a
S. Marleau , M. Bellemare , Ph. Raymond , D. c
d
Giroux , J. K. Loo & N. Beaulieu
d
a
Faculté de pharmacie , Université de Montréal , P. O. Box 6128, Station A, Montréal (Québec), H3C 3J7, CANADA b
Transfusion sanguine, Université de Liège , B-4000, Sart Tilman, BELGIQUE c
Faculté de médecine vétérinaire , Université de Montréal , St-Hyacinthe (Québec), J2S 7C6, CANADA d
Health Protection Branch, Pharmaceutical Chemistry Division Ottawa , (Ontario), K1A OL2, CANADA Published online: 23 Oct 2006.
To cite this article: A. Adam , H. Ong , D. Sondag , A. Rapaille , S. Marleau , M. Bellemare , Ph. Raymond , D. Giroux , J. K. Loo & N. Beaulieu (1990) Radioimmunoassay for Albuterol Using a Monoclonal Antibody: Application for Direct Quantification in Horse Urine, Journal of Immunoassay, 11:3, 329-345, DOI: 10.1080/01971529008055036 To link to this article: http://dx.doi.org/10.1080/01971529008055036
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JOURNAL OF IMMUNOASSAY, 11(3),
3 2 9 - 3 4 5 (1990)
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RADIOXMMUNOASSAY FOR ALBUTEROL USING A MONOCLONAL ANTIBODY: APPLICATION FOR DIRECT QUANTIFICATION I N HORSE U R I N E .
H a r l e a u ( l ) , M. B e l l e m a r e ( l ) , Ph. Rayniond(l), D. GiroUX(3) J . K . Loo(4). N . B e a u l i e u ( 4 ) (1) F a c u l t e d e yharmacie, U r i i v e r s i t e be Montreal P.O. Box 6128, S t a t i o n A , Montreal (Quebec) H3C 357, CANADA. ( 2 ) Transfusion sanguine, U n i v e r s i t e d e Liege, B-4000 S a r t T i lmari, BELGIQUE. ( 3 ) P a c u l t e de medecine v e t e r i n a i r e , Uriiversite de M o n t r e a l , St-Hyacirithe (Quebec) 525 7 C 6 , CANADA. (4) H e a l t h P r o t e c t i o n Branch, P h a r m a c e u t i c a l Chemistry D i v i s i o n Ottawa ( O n t a r i o ) K 1 A OL2, CANADA. S.
ABSTRACT A monoclonal a n t i b o d y was s y l i t h e s i z e d i n niouse a g a i n s t t h e 0- (3-carboxypropionyl) d e r i v a t i v e of a l b u t e r o l l i n k e d t o b o v i n e serum albumin. Isotypirig of t h i s m a t e r i a l r e v e a l e d t h e IgGl class c h a r a c t e r i z e d by an a f f i n i t y c o n s t a n t of 1 . 0 3 nM-’ arid a d e n s i t y of s i t e s of 0.55 nM. T h i s a n t i b o d y was found i t s cross- r e a c t i v i t y t o s t r u c t u r a l l y r e l a t e d s p e c i f i c as m o l e c u l e s was less t h a n 1%e x c e p t for c l e n b u t e r o l (75%). A radioinmunoassay was set up w i t h c u l t u r e s u p e r n a t a n t (f irial d i l u t i o n 1/1000) arid [3H] a l b u t e r o l . The c a l i b r a t i o n c u r v e was c h a r a c t e r i z e d by a raaxinium b i n d i n g of 28%, an ED50 of 1 . 1 5 pniol p e r t u b e , t h e d e t e c t i o n limit was 2 8 . 8 f m o l l t u b e arid t h e This R I A l i n e a r i t y of t h e r e s p o n s e was up t o 39.8 pmolltube. method has been used f o r d i r e c t y u a n t i t a t i o n of a l b u t e r o l i n horse u r i n e w i t h o u t any c l e a n - u p OK- e x t r a t i o n s t e p . (KEY WORDS : a l b u t e r o l , c l e n t u b e r o l , radioinuiunoassay, niorioclorial antibody).
329 Copyright 0 1990 by Marcel Dekker, Inc.
ADAM ET AL.
3 30 INTRODUCTION Beta-2 treatment
agonists of
have
constrictive
been
extensively
used
for
the
and o b s t r u c t i v e pulmonary d i s e a s e s ,
i n human and v e t e r i n a r y niedicine.
Aniorig t h e s e d r u g s , a l b u t e r o l
has been used f o r the iniprvvenierit of t h e pulmoriary f u n c t i o n i n
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r a c e doping arid f o r t h e i n c r e a s e of the muscular inass of and c a l v e s .
For t h e s e r e a s o n s , t h e monitoring of a l b u t e r o l i s drug r e s i d u e s i n meat as
r e q u i r e d f o r the q u a l i t y c o n t r o l of
we11
as
oxen
for
t h e d e t e c t i o n of
t h i s substance i n anti-doping
purposes. The
methodologies
for
available
the
determination
of
a l b u t e r o l i n b i o l o g i c a l samples i n c l u d e g a s chromatography-niass spectrometry
(11, h i g h
fluoroiuetric
(2) and
pressure
liquid
electrochemical
chromatography
with
detection,
high
(3-4)
p e r f o m a n c e t h i n l a y e r chromatography (5) arid radioiiiwiunoassay using a polyclonal require
laborious
antiserum
Most of
(6).
extraction
steps
and
these
is
this
procedures
a
limiting
f a c t o r for t h e p r o c e s s i n g of a l a r g e number of samples. The p r e s e n t to
quantitate
method
has
s t u d y d e s c r i b e s a s e n s i t i v e radioinmunoassay
albuterol
been
using
validated
for
a
monoclonal the
direct
antibody.
This
quaxititation
of
a l b u t e r o l i n h o r s e u r i n e samples.
MATERIAL and METHODS Material
The following m a t e r i a l s were purchased from the s u p p l i e r s indicated:
albuterol
hemisulfate,
terbutaline,
bovine
serum
331
RADIOIMMUNOASSAY FOR ALBUTEROL USING A MONOCLONAL ANTIBODY
albumin arid bovine ganuna g l o b u l i n s Cohn f r a c t i o n 11, I11 (Sigma
,
S t . Louis,
Chemical
Co.
charcoal
Norit,
(Fisher Sci,
,
MO)
tris-hydroxyniethylan~irioet~iaI~e,
polyethylene
Muskegori,
glycol
dextrari T70
MI),
Scintiverse
8000,
(Phamacia,
BD
Uppsala,
Sweden), Freurid coniplete adjuvant (CFA) arid cell c u l t u r e media:
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Hypoxarithine Memorial (Nunc,
amiriopteririe
Institute
Roskilde, Alto,
Palo
(RPMI) ( D i f c o ,
Denmark),
Biochemicals,
Costa Mesa,
CA)
.
Rosewell
DETROIT, M I ) .
U l t r a s p h e r e ODs
Ouchterloriy
CA).
(HAT) ,
thymidine
Microplates (Beckman,
coluniri
inuiunodiffusion Mouse
kit
Inuiunoglobulin
(ICN
Subtype
I d e r i t i f i c a t i o n k i t (Boehringer Mannheim, I n d i a n a p o l i s , I N )
arid
Fenoterol Boehringer custom
(GFR) .
Irigelheim
labelled
were
cleributerol
by
the
Negev
a
3
Nuclear
.
gift
of
albuterol
was
generous [ HI
Tritiated
Park
Research C e n t e r
(Beer
Sheva, Israel) w i t h a s p e c i f i c a c i t v i t y of 17Ci/nrniol.
Methods S y n t h e s i s of
salbutamol s u c c i r i a t e d e r i v a t i v e arid i t s coupling
t o bovine serum albumin (BSA)
The
synthesis
of
the
.
0- (3-carboxypropionyl)
derivative
of
salbutamol has been performed a c c o r d i n g t o Beaulieu e t a1 ( 7 ) . B r i e f l y , 200 nip of salbutaniol b a s e were d i s s o l v e d i n 25 n i l of a b s o l u t e e t h a n o l i r i presence While
stiririg,
both
succiriic
of
lo7
dpm of
anhydride
(90
[
3
HI salbutaniol.
mg)
arid
[
14 Cl
3 32
ADAM ET A L .
labelled
6
succinic anhydride (2.7 x 1 0
dpm) were added.
The
l i a r v e s t e d c r i s t a l s were washed w i t h e t h a n o l u n t i l a cotistarit ratio
14 H/ C was o b t a i n e d .
3
derivative
conf inned
The nielty p o i n t
the
salbutaniol
(142OC)
benzylic
of
the
succinate
d e r i v a t i v e was o b t a i n e d .
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F o r t y mg of t h i s d e r i v a t i v e were d i s s o l v e d i n 25 nil of dioxarie, 3 n i l water arid 300 p 1 t r i e t h a r i o l a n i i n e were coupled t o BSA (86
nig)
previously
reserice of
dissolved
in
25
ml
of
distilled
water
in
The s o l u t i o n was f i r i a l l y
300 p l t r i e t h a n o l a m i n e .
d i a l y z e d a g a i n s t d i s t i l l e d water g i v i n g a y i e l d of 3.87 moles
of hapteri/riiole of BSA.
P r o d u c t i o n of t h e nwnoclonal a n t i b o d y
Inunun iz a t i o n p r o t o co 1 Three Biozei
ruice
intraperitoneally with
(IFFA Credo, 20
pg
of
France)
were
inmiuriized
t h e 0- (3-carboxy propioriyl)
d e r i v a t i v e of a l b u t e r o l c o v a l e n t l y l i n k e d t o BSA, eniulsif i e d i n 0.25
niL
witlidrawi
of
t h e CFA.
from
anti-albuterol
the
Three weeks
tail
antibodies.
was
later,
screened
for
t h e blood the
The mouse e x h i b i t i n g
of
mice
presence
of
the highest
t i t e r w a s b o o s t e d i n t r a p e r i t o n e a l l y w i t h 4 0 pg of
albuterol
c o n j u g a t e e m u l s i f i e d i n 0.25 niL CFA, 3 t o 4 days p r i o r t o the fusion.
333
RADIOIMMUNOASSAY FOR ALBUTEROL USING A MONOCLONAL ANTIBODY FUs i o n 8
Spleen cells ( 1 . 4 x 10 niyelonia c e l l l i n e
c e l l s ) were fused w i t h SP2/0 mouse
( 2 . 8 x 10
7
using
cells)
the
polyetliylene
g l y c o l method d e s c r i b e d by Fazekas d e S a i n t Groth ( 8 , 9 ) . 6
s p l e e n c e l l suspension (10
cells per w e l l )
T?ie
was seeded i n 96
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w e l l m i c r o p l a t e s w i t h mouse p e r i t o n e a l macrophages a s f e e d e r layer.
Fusion h y b r i d s were m i c r o s c o p i c a l l y observed, 10 days
after.
On days 1 3 arid 1 9 , t h e s u p e r n a t a n t s of growing h y b r i d s
were screened f o r t h e p r e s e n c e of a n t i a l b u t e r o l IgGs,
using
t h e radioinwiunoassay procedure d e s c r i b e d below.
C l o n in&
A f t e r t h e s c r e e n i n g of tlie c e l l s u p e r n a t a n t s , t h e c l o n i n g
was perfornied t w i c e by l i m i t i n g d i l u t i o n a t 0.5 and 1 c e l l per w e l l i n 96 w e l l s m i c r o p l a t e s u s i n g human u m b i l i c a l cord serum a s feeder.
Screening f o r t h e p r o d u c t i o n of a n t i - a l b u t e r o l IgG a n t i b o d i e s Anti-albuterol mice
and
the
the
IgG a n t i b o d i e s i n
cu t u r e
supernatant
were
sera
of
screened
immunized by
RIA.
B r i e f l y , 100 p L of t h e t e s t sample d i l u t e d t o 1 : 4 w i t h 200 n@ phosphate b u f f e r , pH 7 . 4 , albuterol
in
a final
Polyclonal r a b b i t
were incubated w i t h 100 pL of
volume
antiserum
c u l t u r e niedium were used
of
0.5
niL
for
4 fir
at
[ ' H I 4OC.
( f i n a l d i l u t i o n 112500) (10) and as
positive
and n e g a t i v e c o n t r o l s
3 34
ADAM ET AL.
3
The s e p a r a t i o n of bound from f r e e [ H I a l b u t e r o l
respectively.
was o b t a i n e d by adding 0 . 5 mL c h a r c o a l mixture
consisting
of
c h a r c o a l N o r i t 1 0 g , d e x t r a n T70 1 . 0 g , bovine ganunaglobuliris f r a c t i o n I1 arid I11 2 . 8
i n 200 nM phosphate
g/L
buffer,
The m i x t u r e was incubated f o r 15 m i r i a t 4 O C ,
7.4.
pH
followed by
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c e n t r i f u g i o n a t 3,000 g , f o r 15 m i r i a t 10°C.
The s u p e r n a t a n t s were t h e n decanted arid t h e i r r a d i o a c t i v i t y was determined
i n the external
Branma, Sweden)
s t a n d a r d mode
(Beta c o u n t e r ,
LKB,
.
Radioiriununoassay
for
quarititatiori
of
albuterol
using
the
7.4.
The
moriocloiial a n t i b o d y The
assay
buffer
was
iricubatiori medium (0.5mL) pmol/mL) 10000
o r urine
dpm),
antibody a t
b u f f e r (200 y L ) .
5OnM
of s t a n d a r d s (1.05 t o 266 3
(100 p L ) ,
superriatarit
[ HI
1/1000
a stock solution
non-specific
binding
moriocloiial
(100 pL)
arid a s s a y
was
from 1.05 t o 266 pmol/mL.
def hied
in
the
motioclorial a n t i b o d y arid a q u a l i t y - c o n t r o l blank h o r s e u r i n e s p i k e d w i t h
the s e p a r a t i o n
absence
of
The the
sample which c o n s i s t e d
albuterol
a t 20 pmol/n& was
A f t e r i n c u b a t i o n f o r 2 h r a t room
included i n e a c h a s s a y b a t c h . temperature,
the
(100 p L ,
C a l i b r a t i o n c u r v e s were c o n s t r u c t e d by s e r i a l
d i l u t i o n s of
of
albuterol
containing
f i n a l d i l u t i o n of
pH
TrisIHCl,
consisted
samples
culture the
a
of
bound
froin
free
f r a c t i o n was
335
RADIOIMMUNOASSAY FOR ALBUTEROL USING A MONOCLONAL ANTIBODY
c a r r i e d o u t by adding 40 pL of a r a b b i t anti-mouse IgG antiserum and 0 . 5 niL of PEG 8000 (15% w/w) c e l l u l o s e t o each t u b e .
and 500 pg of m i c r o c r y s t a l l i n e
The t u b e s were then v o r t e x e d , allowed
t o s t a n d f o r 15 min a t 10°C and were c e n t r i f u g e d a t 3,000 g , f o r 15 n i i r i
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arid
a t 4°C.
the
The s u p e r n a t a n t s w e r e c a r e f u l l y a s p i r a t e d o u t
pellet
resuspended
in
corresponding 0.5mL
of
to
the
the
bound
incubation
fraction
buffer,
arid
was the
r a d i o a c t i v i t y was counted i n a b e t a c o u n t e r .
In
to
order
antibody,
the
assess
the
specificity
cross--reactivity
molecules arid t h o s e used cross-reactivity
(4,)
was
of
the
of
the
monocloiial
structurally
related
i n h o r s e doping were examined.
The
defined
50%
as
the
ratio
dose
at
displacement of t h e t r a c e r (ED501 f o r a l b u t e r o l r e l a t i v e t o ED50 for
each
of
isoprotereriol,
the
following
fenoterol,
t e r b u t a 1irie ,
substances:
cleributerol,
dobutaniine,
aniphetamine,
e p h e d r i n e , arid phenylephrine .
Urinary e x c r e t i o n p r o f i l e of a l b u t e r o l followinn an IV dose i n horse One q u a r t e r h o r s e , 6 y e a r s o l d female (465 kg), without any medication d u r i n g t h e week preceding
t h e experience was used.
During t h e experiment, t h e h o r s e w a s r e s t r a i n e d b u t had a c c e s s
t o water weight)
& libitum. i n 100 n&
A
bolus
of
albuterol
(10
pg/kg
body
NaCl 0.9% was i n j e c t e d i n t r a v e n o u s l y v i a a
336
ADAM ET AL.
c a t h e t e r i n s e r t e d i n t h e j u g u l a r vein.
Urine was sampled v i a a
Foley
in
catheter
aseptically
installed
the
bladder,
before
a d m i n i s t r a t i o n of t h e drug arid a t each hour f o r a 12 hr p e r i o d . The volume of each sample was measured arid t h e sample s t o r e d a t -30°C.
The pH of u r i n e samples was a d j u s t e d t o pH 7 . 0 - 7 , . 5 arid
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t h e samples wefe t h e n f i l t e r e d o r c e n t r i f u g e d (2 500 g, 10 min) t o e l i m i n a t e p r e c i p i t a t e d phosphate o r u r a t e b e f o r e a l b u t e r o l analysis.
Inmunorram of u r i n a r y a l b u t e r o l To
evaluate
the
specificity
of
the
radioinmunoassay
procedure, a l i q u o t s of u r i n a r y samples from a h o r s e r e c e i v i n g a 10 pg/kg
body weight
s t e p using
PRP-1
were submitted t o a clean-up e x t r a c t i o n
cartridge
(Chromatographic
spec.
,
Milford,
Mass) p r e a c t i v a t e d with 5 mL of methanol and 5 n& of d i s t i l l e d
water.
Following t h e a p p l i c a t i o n of
t h e u r i n e samples ( 1 m L ) ,
the c a r t r i d g e w a s washed w i t h 5 n\L d i s t i l l e d water; was f i n a l l y e l u t e d w i t h 2 mL methanol. evaporated Fa"lningdale,
to
dryness
NJ).
in
a
speed
albuterol
The e l u a t e was then
vac
concentrator
(Savant,
The r e s i d u e w a s r e c o n s t i t u t e d i n 150 pL of
mobile phase arid 100 pL were i n j e c t e d i n t h e HPLC system. The chromatographic system c o n s i s t e d of a pump (Model 510, Waters ASS, M i l f o r d , Mass) an i n j e c t i o n v a l v e (Rheodyne, Model 7125, Berkeley, CA) w i t h a 100 p L loop. colunui used
was U l t r a s p h e r e ODS,
5
The r e v e r s e phase HPLC pin,
25
cm x 4.6 nun I D
337
RADIOIMMUNOASSAY FOR ALBUTEROL USING A MONOCLONAL ANTIBODY (Bechian, P a l o mixture
SO
of
ALto,
ntM
anunonium
a c e t o n i t r i l e (80:8:12 v / v ) . Fractions
of
The
CAI.
1 niL
were
Pliarmacia, Uppsala, Sweden).
mobile
acetate
phase
buffer
consisted pH
6,
in
a
methanol,
The flow rate was a t 0 . 8 nil/min. collected
(FRAC 100
C
collector,
The f r a c t i o n s were then evaporated
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t o d r y n e s s as d e s c r i b e d e a r l i e r t o detetniine a l b u t e r o l by R I A .
Data a n a l y s i s The c u r v e s were arialysed by non l i n e a r r e g r e s s i o n u s i n g a four
pacanieter
logistic
equation
providing
estimates
for
asymptotic maximal arid miriimal b i n d i n g , t h e s l o p e f a c t o r , and ED50 of t h e c u r v e (11).
RESULTS Morioclorial a n t i b o d y c h a r a c t e r i s t i c s Isotypirig of t h e monoclonal antibody I s o t y p i n g of
t h e nionoclorial a n t i b o d y was c a r r i e d o u t
by
Ouchterlony inmiunodif f u s i o n u s i n g t h e conmiercial k i t from ICN. A p r e c i p i t a t i o n band was observed o n l y when the a n t i b o d y r e a c t e d
w i t h r a b b i t a n t i IgG1.
No r e a c t i o n was d e t e c t e d w i t h a n t i s e r u m
a g a i n s t o t h e r mouse IgG s u b c l a s s e s ,
IgA
atid
IgM
(figure
1).
I d e n t i c a l r e s u l t s were confirmed by an ELISA method u s i n g t h e Mouse Inmunoglobulin Subtype I d e n t i f i c a t i o r i k i t and by inuiiunodot (12-13).
ADAM
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338
F i g u r e 1:
ET AL,
Isotypirig of monoclonal a n t i b o d y by Ouchterlony C u l t u r e superria t a n t : A. Rabbit inmiunodi f f u s i o n . a n t i s e r u m a g a i n s t mouse iiimiunog lobu 1 i n : 1( IgGl) , 2(IgG2a), 3(IgG3), 4(IgG4), 5 ( I g A ) , 6(IgM).
S p e c i f i c i t y of t h e monoclonal a n t i b o d y The coniparative c r o s s - r e a c t i v i t y
of
a l b u t e r o l a n a l o g s and
o t h e r d r u g s used i n h o r s e dopixig is shown i n Table 1. interesting
to
CL-OSS. reactivity
note
that
(75%)
only
clenbuterol
followed
by
displays
terbutalirke
It
a
is
high (7%).
Cross- r e a c t i v i t y less than 1%could be d e t e c t e d f o r o t h e r t e s t e d molecules.
339
RADIOIMMUNOASSAY FOR ALBUTEROL U S I N G A MONOCLONAL ANTIBODY
TABLE 1 COMPARATIVE CROSS REACTIVITY O F ALBUTEROL ANALOGS I N THE RADIOIHMUNOASSAY
ED50 (pniol/niL)
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Drug
Albuterol Cleribu t e r o 1 Terbutaline Fenoterol I s o p r o tererio 1 Dobu t anii fie Aniphe t aniine Ephedrine Pheny l e p h r iria
< <
Journal of Immunoassay Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii19
Radioimmunoassay for Albuterol Using a Monoclonal Antibody: Application for Direct Quantification in Horse Urine a
a
b
b
A. Adam , H. Ong , D. Sondag , A. Rapaille , a
a
a
S. Marleau , M. Bellemare , Ph. Raymond , D. c
d
Giroux , J. K. Loo & N. Beaulieu
d
a
Faculté de pharmacie , Université de Montréal , P. O. Box 6128, Station A, Montréal (Québec), H3C 3J7, CANADA b
Transfusion sanguine, Université de Liège , B-4000, Sart Tilman, BELGIQUE c
Faculté de médecine vétérinaire , Université de Montréal , St-Hyacinthe (Québec), J2S 7C6, CANADA d
Health Protection Branch, Pharmaceutical Chemistry Division Ottawa , (Ontario), K1A OL2, CANADA Published online: 23 Oct 2006.
To cite this article: A. Adam , H. Ong , D. Sondag , A. Rapaille , S. Marleau , M. Bellemare , Ph. Raymond , D. Giroux , J. K. Loo & N. Beaulieu (1990) Radioimmunoassay for Albuterol Using a Monoclonal Antibody: Application for Direct Quantification in Horse Urine, Journal of Immunoassay, 11:3, 329-345, DOI: 10.1080/01971529008055036 To link to this article: http://dx.doi.org/10.1080/01971529008055036
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JOURNAL OF IMMUNOASSAY, 11(3),
3 2 9 - 3 4 5 (1990)
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RADIOXMMUNOASSAY FOR ALBUTEROL USING A MONOCLONAL ANTIBODY: APPLICATION FOR DIRECT QUANTIFICATION I N HORSE U R I N E .
H a r l e a u ( l ) , M. B e l l e m a r e ( l ) , Ph. Rayniond(l), D. GiroUX(3) J . K . Loo(4). N . B e a u l i e u ( 4 ) (1) F a c u l t e d e yharmacie, U r i i v e r s i t e be Montreal P.O. Box 6128, S t a t i o n A , Montreal (Quebec) H3C 357, CANADA. ( 2 ) Transfusion sanguine, U n i v e r s i t e d e Liege, B-4000 S a r t T i lmari, BELGIQUE. ( 3 ) P a c u l t e de medecine v e t e r i n a i r e , Uriiversite de M o n t r e a l , St-Hyacirithe (Quebec) 525 7 C 6 , CANADA. (4) H e a l t h P r o t e c t i o n Branch, P h a r m a c e u t i c a l Chemistry D i v i s i o n Ottawa ( O n t a r i o ) K 1 A OL2, CANADA. S.
ABSTRACT A monoclonal a n t i b o d y was s y l i t h e s i z e d i n niouse a g a i n s t t h e 0- (3-carboxypropionyl) d e r i v a t i v e of a l b u t e r o l l i n k e d t o b o v i n e serum albumin. Isotypirig of t h i s m a t e r i a l r e v e a l e d t h e IgGl class c h a r a c t e r i z e d by an a f f i n i t y c o n s t a n t of 1 . 0 3 nM-’ arid a d e n s i t y of s i t e s of 0.55 nM. T h i s a n t i b o d y was found i t s cross- r e a c t i v i t y t o s t r u c t u r a l l y r e l a t e d s p e c i f i c as m o l e c u l e s was less t h a n 1%e x c e p t for c l e n b u t e r o l (75%). A radioinmunoassay was set up w i t h c u l t u r e s u p e r n a t a n t (f irial d i l u t i o n 1/1000) arid [3H] a l b u t e r o l . The c a l i b r a t i o n c u r v e was c h a r a c t e r i z e d by a raaxinium b i n d i n g of 28%, an ED50 of 1 . 1 5 pniol p e r t u b e , t h e d e t e c t i o n limit was 2 8 . 8 f m o l l t u b e arid t h e This R I A l i n e a r i t y of t h e r e s p o n s e was up t o 39.8 pmolltube. method has been used f o r d i r e c t y u a n t i t a t i o n of a l b u t e r o l i n horse u r i n e w i t h o u t any c l e a n - u p OK- e x t r a t i o n s t e p . (KEY WORDS : a l b u t e r o l , c l e n t u b e r o l , radioinuiunoassay, niorioclorial antibody).
329 Copyright 0 1990 by Marcel Dekker, Inc.
ADAM ET AL.
3 30 INTRODUCTION Beta-2 treatment
agonists of
have
constrictive
been
extensively
used
for
the
and o b s t r u c t i v e pulmonary d i s e a s e s ,
i n human and v e t e r i n a r y niedicine.
Aniorig t h e s e d r u g s , a l b u t e r o l
has been used f o r the iniprvvenierit of t h e pulmoriary f u n c t i o n i n
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r a c e doping arid f o r t h e i n c r e a s e of the muscular inass of and c a l v e s .
For t h e s e r e a s o n s , t h e monitoring of a l b u t e r o l i s drug r e s i d u e s i n meat as
r e q u i r e d f o r the q u a l i t y c o n t r o l of
we11
as
oxen
for
t h e d e t e c t i o n of
t h i s substance i n anti-doping
purposes. The
methodologies
for
available
the
determination
of
a l b u t e r o l i n b i o l o g i c a l samples i n c l u d e g a s chromatography-niass spectrometry
(11, h i g h
fluoroiuetric
(2) and
pressure
liquid
electrochemical
chromatography
with
detection,
high
(3-4)
p e r f o m a n c e t h i n l a y e r chromatography (5) arid radioiiiwiunoassay using a polyclonal require
laborious
antiserum
Most of
(6).
extraction
steps
and
these
is
this
procedures
a
limiting
f a c t o r for t h e p r o c e s s i n g of a l a r g e number of samples. The p r e s e n t to
quantitate
method
has
s t u d y d e s c r i b e s a s e n s i t i v e radioinmunoassay
albuterol
been
using
validated
for
a
monoclonal the
direct
antibody.
This
quaxititation
of
a l b u t e r o l i n h o r s e u r i n e samples.
MATERIAL and METHODS Material
The following m a t e r i a l s were purchased from the s u p p l i e r s indicated:
albuterol
hemisulfate,
terbutaline,
bovine
serum
331
RADIOIMMUNOASSAY FOR ALBUTEROL USING A MONOCLONAL ANTIBODY
albumin arid bovine ganuna g l o b u l i n s Cohn f r a c t i o n 11, I11 (Sigma
,
S t . Louis,
Chemical
Co.
charcoal
Norit,
(Fisher Sci,
,
MO)
tris-hydroxyniethylan~irioet~iaI~e,
polyethylene
Muskegori,
glycol
dextrari T70
MI),
Scintiverse
8000,
(Phamacia,
BD
Uppsala,
Sweden), Freurid coniplete adjuvant (CFA) arid cell c u l t u r e media:
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Hypoxarithine Memorial (Nunc,
amiriopteririe
Institute
Roskilde, Alto,
Palo
(RPMI) ( D i f c o ,
Denmark),
Biochemicals,
Costa Mesa,
CA)
.
Rosewell
DETROIT, M I ) .
U l t r a s p h e r e ODs
Ouchterloriy
CA).
(HAT) ,
thymidine
Microplates (Beckman,
coluniri
inuiunodiffusion Mouse
kit
Inuiunoglobulin
(ICN
Subtype
I d e r i t i f i c a t i o n k i t (Boehringer Mannheim, I n d i a n a p o l i s , I N )
arid
Fenoterol Boehringer custom
(GFR) .
Irigelheim
labelled
were
cleributerol
by
the
Negev
a
3
Nuclear
.
gift
of
albuterol
was
generous [ HI
Tritiated
Park
Research C e n t e r
(Beer
Sheva, Israel) w i t h a s p e c i f i c a c i t v i t y of 17Ci/nrniol.
Methods S y n t h e s i s of
salbutamol s u c c i r i a t e d e r i v a t i v e arid i t s coupling
t o bovine serum albumin (BSA)
The
synthesis
of
the
.
0- (3-carboxypropionyl)
derivative
of
salbutamol has been performed a c c o r d i n g t o Beaulieu e t a1 ( 7 ) . B r i e f l y , 200 nip of salbutaniol b a s e were d i s s o l v e d i n 25 n i l of a b s o l u t e e t h a n o l i r i presence While
stiririg,
both
succiriic
of
lo7
dpm of
anhydride
(90
[
3
HI salbutaniol.
mg)
arid
[
14 Cl
3 32
ADAM ET A L .
labelled
6
succinic anhydride (2.7 x 1 0
dpm) were added.
The
l i a r v e s t e d c r i s t a l s were washed w i t h e t h a n o l u n t i l a cotistarit ratio
14 H/ C was o b t a i n e d .
3
derivative
conf inned
The nielty p o i n t
the
salbutaniol
(142OC)
benzylic
of
the
succinate
d e r i v a t i v e was o b t a i n e d .
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F o r t y mg of t h i s d e r i v a t i v e were d i s s o l v e d i n 25 nil of dioxarie, 3 n i l water arid 300 p 1 t r i e t h a r i o l a n i i n e were coupled t o BSA (86
nig)
previously
reserice of
dissolved
in
25
ml
of
distilled
water
in
The s o l u t i o n was f i r i a l l y
300 p l t r i e t h a n o l a m i n e .
d i a l y z e d a g a i n s t d i s t i l l e d water g i v i n g a y i e l d of 3.87 moles
of hapteri/riiole of BSA.
P r o d u c t i o n of t h e nwnoclonal a n t i b o d y
Inunun iz a t i o n p r o t o co 1 Three Biozei
ruice
intraperitoneally with
(IFFA Credo, 20
pg
of
France)
were
inmiuriized
t h e 0- (3-carboxy propioriyl)
d e r i v a t i v e of a l b u t e r o l c o v a l e n t l y l i n k e d t o BSA, eniulsif i e d i n 0.25
niL
witlidrawi
of
t h e CFA.
from
anti-albuterol
the
Three weeks
tail
antibodies.
was
later,
screened
for
t h e blood the
The mouse e x h i b i t i n g
of
mice
presence
of
the highest
t i t e r w a s b o o s t e d i n t r a p e r i t o n e a l l y w i t h 4 0 pg of
albuterol
c o n j u g a t e e m u l s i f i e d i n 0.25 niL CFA, 3 t o 4 days p r i o r t o the fusion.
333
RADIOIMMUNOASSAY FOR ALBUTEROL USING A MONOCLONAL ANTIBODY FUs i o n 8
Spleen cells ( 1 . 4 x 10 niyelonia c e l l l i n e
c e l l s ) were fused w i t h SP2/0 mouse
( 2 . 8 x 10
7
using
cells)
the
polyetliylene
g l y c o l method d e s c r i b e d by Fazekas d e S a i n t Groth ( 8 , 9 ) . 6
s p l e e n c e l l suspension (10
cells per w e l l )
T?ie
was seeded i n 96
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w e l l m i c r o p l a t e s w i t h mouse p e r i t o n e a l macrophages a s f e e d e r layer.
Fusion h y b r i d s were m i c r o s c o p i c a l l y observed, 10 days
after.
On days 1 3 arid 1 9 , t h e s u p e r n a t a n t s of growing h y b r i d s
were screened f o r t h e p r e s e n c e of a n t i a l b u t e r o l IgGs,
using
t h e radioinwiunoassay procedure d e s c r i b e d below.
C l o n in&
A f t e r t h e s c r e e n i n g of tlie c e l l s u p e r n a t a n t s , t h e c l o n i n g
was perfornied t w i c e by l i m i t i n g d i l u t i o n a t 0.5 and 1 c e l l per w e l l i n 96 w e l l s m i c r o p l a t e s u s i n g human u m b i l i c a l cord serum a s feeder.
Screening f o r t h e p r o d u c t i o n of a n t i - a l b u t e r o l IgG a n t i b o d i e s Anti-albuterol mice
and
the
the
IgG a n t i b o d i e s i n
cu t u r e
supernatant
were
sera
of
screened
immunized by
RIA.
B r i e f l y , 100 p L of t h e t e s t sample d i l u t e d t o 1 : 4 w i t h 200 n@ phosphate b u f f e r , pH 7 . 4 , albuterol
in
a final
Polyclonal r a b b i t
were incubated w i t h 100 pL of
volume
antiserum
c u l t u r e niedium were used
of
0.5
niL
for
4 fir
at
[ ' H I 4OC.
( f i n a l d i l u t i o n 112500) (10) and as
positive
and n e g a t i v e c o n t r o l s
3 34
ADAM ET AL.
3
The s e p a r a t i o n of bound from f r e e [ H I a l b u t e r o l
respectively.
was o b t a i n e d by adding 0 . 5 mL c h a r c o a l mixture
consisting
of
c h a r c o a l N o r i t 1 0 g , d e x t r a n T70 1 . 0 g , bovine ganunaglobuliris f r a c t i o n I1 arid I11 2 . 8
i n 200 nM phosphate
g/L
buffer,
The m i x t u r e was incubated f o r 15 m i r i a t 4 O C ,
7.4.
pH
followed by
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c e n t r i f u g i o n a t 3,000 g , f o r 15 m i r i a t 10°C.
The s u p e r n a t a n t s were t h e n decanted arid t h e i r r a d i o a c t i v i t y was determined
i n the external
Branma, Sweden)
s t a n d a r d mode
(Beta c o u n t e r ,
LKB,
.
Radioiriununoassay
for
quarititatiori
of
albuterol
using
the
7.4.
The
moriocloiial a n t i b o d y The
assay
buffer
was
iricubatiori medium (0.5mL) pmol/mL) 10000
o r urine
dpm),
antibody a t
b u f f e r (200 y L ) .
5OnM
of s t a n d a r d s (1.05 t o 266 3
(100 p L ) ,
superriatarit
[ HI
1/1000
a stock solution
non-specific
binding
moriocloiial
(100 pL)
arid a s s a y
was
from 1.05 t o 266 pmol/mL.
def hied
in
the
motioclorial a n t i b o d y arid a q u a l i t y - c o n t r o l blank h o r s e u r i n e s p i k e d w i t h
the s e p a r a t i o n
absence
of
The the
sample which c o n s i s t e d
albuterol
a t 20 pmol/n& was
A f t e r i n c u b a t i o n f o r 2 h r a t room
included i n e a c h a s s a y b a t c h . temperature,
the
(100 p L ,
C a l i b r a t i o n c u r v e s were c o n s t r u c t e d by s e r i a l
d i l u t i o n s of
of
albuterol
containing
f i n a l d i l u t i o n of
pH
TrisIHCl,
consisted
samples
culture the
a
of
bound
froin
free
f r a c t i o n was
335
RADIOIMMUNOASSAY FOR ALBUTEROL USING A MONOCLONAL ANTIBODY
c a r r i e d o u t by adding 40 pL of a r a b b i t anti-mouse IgG antiserum and 0 . 5 niL of PEG 8000 (15% w/w) c e l l u l o s e t o each t u b e .
and 500 pg of m i c r o c r y s t a l l i n e
The t u b e s were then v o r t e x e d , allowed
t o s t a n d f o r 15 min a t 10°C and were c e n t r i f u g e d a t 3,000 g , f o r 15 n i i r i
Downloaded by [Purdue University] at 04:37 12 March 2015
arid
a t 4°C.
the
The s u p e r n a t a n t s w e r e c a r e f u l l y a s p i r a t e d o u t
pellet
resuspended
in
corresponding 0.5mL
of
to
the
the
bound
incubation
fraction
buffer,
arid
was the
r a d i o a c t i v i t y was counted i n a b e t a c o u n t e r .
In
to
order
antibody,
the
assess
the
specificity
cross--reactivity
molecules arid t h o s e used cross-reactivity
(4,)
was
of
the
of
the
monocloiial
structurally
related
i n h o r s e doping were examined.
The
defined
50%
as
the
ratio
dose
at
displacement of t h e t r a c e r (ED501 f o r a l b u t e r o l r e l a t i v e t o ED50 for
each
of
isoprotereriol,
the
following
fenoterol,
t e r b u t a 1irie ,
substances:
cleributerol,
dobutaniine,
aniphetamine,
e p h e d r i n e , arid phenylephrine .
Urinary e x c r e t i o n p r o f i l e of a l b u t e r o l followinn an IV dose i n horse One q u a r t e r h o r s e , 6 y e a r s o l d female (465 kg), without any medication d u r i n g t h e week preceding
t h e experience was used.
During t h e experiment, t h e h o r s e w a s r e s t r a i n e d b u t had a c c e s s
t o water weight)
& libitum. i n 100 n&
A
bolus
of
albuterol
(10
pg/kg
body
NaCl 0.9% was i n j e c t e d i n t r a v e n o u s l y v i a a
336
ADAM ET AL.
c a t h e t e r i n s e r t e d i n t h e j u g u l a r vein.
Urine was sampled v i a a
Foley
in
catheter
aseptically
installed
the
bladder,
before
a d m i n i s t r a t i o n of t h e drug arid a t each hour f o r a 12 hr p e r i o d . The volume of each sample was measured arid t h e sample s t o r e d a t -30°C.
The pH of u r i n e samples was a d j u s t e d t o pH 7 . 0 - 7 , . 5 arid
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t h e samples wefe t h e n f i l t e r e d o r c e n t r i f u g e d (2 500 g, 10 min) t o e l i m i n a t e p r e c i p i t a t e d phosphate o r u r a t e b e f o r e a l b u t e r o l analysis.
Inmunorram of u r i n a r y a l b u t e r o l To
evaluate
the
specificity
of
the
radioinmunoassay
procedure, a l i q u o t s of u r i n a r y samples from a h o r s e r e c e i v i n g a 10 pg/kg
body weight
s t e p using
PRP-1
were submitted t o a clean-up e x t r a c t i o n
cartridge
(Chromatographic
spec.
,
Milford,
Mass) p r e a c t i v a t e d with 5 mL of methanol and 5 n& of d i s t i l l e d
water.
Following t h e a p p l i c a t i o n of
t h e u r i n e samples ( 1 m L ) ,
the c a r t r i d g e w a s washed w i t h 5 n\L d i s t i l l e d water; was f i n a l l y e l u t e d w i t h 2 mL methanol. evaporated Fa"lningdale,
to
dryness
NJ).
in
a
speed
albuterol
The e l u a t e was then
vac
concentrator
(Savant,
The r e s i d u e w a s r e c o n s t i t u t e d i n 150 pL of
mobile phase arid 100 pL were i n j e c t e d i n t h e HPLC system. The chromatographic system c o n s i s t e d of a pump (Model 510, Waters ASS, M i l f o r d , Mass) an i n j e c t i o n v a l v e (Rheodyne, Model 7125, Berkeley, CA) w i t h a 100 p L loop. colunui used
was U l t r a s p h e r e ODS,
5
The r e v e r s e phase HPLC pin,
25
cm x 4.6 nun I D
337
RADIOIMMUNOASSAY FOR ALBUTEROL USING A MONOCLONAL ANTIBODY (Bechian, P a l o mixture
SO
of
ALto,
ntM
anunonium
a c e t o n i t r i l e (80:8:12 v / v ) . Fractions
of
The
CAI.
1 niL
were
Pliarmacia, Uppsala, Sweden).
mobile
acetate
phase
buffer
consisted pH
6,
in
a
methanol,
The flow rate was a t 0 . 8 nil/min. collected
(FRAC 100
C
collector,
The f r a c t i o n s were then evaporated
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t o d r y n e s s as d e s c r i b e d e a r l i e r t o detetniine a l b u t e r o l by R I A .
Data a n a l y s i s The c u r v e s were arialysed by non l i n e a r r e g r e s s i o n u s i n g a four
pacanieter
logistic
equation
providing
estimates
for
asymptotic maximal arid miriimal b i n d i n g , t h e s l o p e f a c t o r , and ED50 of t h e c u r v e (11).
RESULTS Morioclorial a n t i b o d y c h a r a c t e r i s t i c s Isotypirig of t h e monoclonal antibody I s o t y p i n g of
t h e nionoclorial a n t i b o d y was c a r r i e d o u t
by
Ouchterlony inmiunodif f u s i o n u s i n g t h e conmiercial k i t from ICN. A p r e c i p i t a t i o n band was observed o n l y when the a n t i b o d y r e a c t e d
w i t h r a b b i t a n t i IgG1.
No r e a c t i o n was d e t e c t e d w i t h a n t i s e r u m
a g a i n s t o t h e r mouse IgG s u b c l a s s e s ,
IgA
atid
IgM
(figure
1).
I d e n t i c a l r e s u l t s were confirmed by an ELISA method u s i n g t h e Mouse Inmunoglobulin Subtype I d e n t i f i c a t i o r i k i t and by inuiiunodot (12-13).
ADAM
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338
F i g u r e 1:
ET AL,
Isotypirig of monoclonal a n t i b o d y by Ouchterlony C u l t u r e superria t a n t : A. Rabbit inmiunodi f f u s i o n . a n t i s e r u m a g a i n s t mouse iiimiunog lobu 1 i n : 1( IgGl) , 2(IgG2a), 3(IgG3), 4(IgG4), 5 ( I g A ) , 6(IgM).
S p e c i f i c i t y of t h e monoclonal a n t i b o d y The coniparative c r o s s - r e a c t i v i t y
of
a l b u t e r o l a n a l o g s and
o t h e r d r u g s used i n h o r s e dopixig is shown i n Table 1. interesting
to
CL-OSS. reactivity
note
that
(75%)
only
clenbuterol
followed
by
displays
terbutalirke
It
a
is
high (7%).
Cross- r e a c t i v i t y less than 1%could be d e t e c t e d f o r o t h e r t e s t e d molecules.
339
RADIOIMMUNOASSAY FOR ALBUTEROL U S I N G A MONOCLONAL ANTIBODY
TABLE 1 COMPARATIVE CROSS REACTIVITY O F ALBUTEROL ANALOGS I N THE RADIOIHMUNOASSAY
ED50 (pniol/niL)
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Drug
Albuterol Cleribu t e r o 1 Terbutaline Fenoterol I s o p r o tererio 1 Dobu t anii fie Aniphe t aniine Ephedrine Pheny l e p h r iria
< <
