This article was downloaded by: [Michigan State University] On: 10 February 2015, At: 02:22 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Immunoassay Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii19
A Competitive Enzyme Immunoassay for Albuterol: Its Application for the Drug Screening in Urine a
a
b
Albert Adam , Huy Ong , Andre Gravel , Jacques c
a
a
Messier , Monique Bellemare , Francine Lantin , d
Gilles Sauvé & Peeter Tyssen
d
a
Faculté de pharmacie , Université de Montréal , P. O. Box 6128, Station A, Montréal (Québec), H3C 3J7, CANADA b
Agriculture Canada, Division de l'hygiène de la viande et des produits de la volaille Agriculture , Canada c
Santé et Bien-Etre social Canada, Bureau des médicaments vétérinaires , Ottawa (Ontario), K1A 1B7, CANADA d
Institut Armand Frappier , Laval, Québec, H7V 1B7, CANADA Published online: 23 Oct 2006.
To cite this article: Albert Adam , Huy Ong , Andre Gravel , Jacques Messier , Monique Bellemare , Francine Lantin , Gilles Sauvé & Peeter Tyssen (1991) A Competitive Enzyme Immunoassay for Albuterol: Its Application for the Drug Screening in Urine, Journal of Immunoassay, 12:2, 207-223, DOI: 10.1080/01971529108055067 To link to this article: http://dx.doi.org/10.1080/01971529108055067
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JOURNAL OF IMMUNOASSAY, 1 2 ( 2 ) , 207-223 ( 1 9 9 1 )
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A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL: ITS APPLICATIOM FOR THE DRUG SCREENING I N URINE.
A l b e r t Adam(l), Huy Ong(1) , Andre G r a v e l ( 2 ) , Jacques Messier(3). Monique Belleniare(1) , Francine L a n t i n ( l ) , G i l l e s Sauv8(4), P e e t e r T y s s e n ( 4 ) . (1) F a c u l t 6 de pharmiacie, Uriiversith d e Montrbal P.O. Box 6128, S t a t i o n A , Montrbal (Quhbec) H3C 3 5 7 , CANADA. ( 2 ) A g r i c u l t u r e Canada, D i v i s i o n de l ' h y g i h n e d e l a viande e t d e s p r o d u i t s d e l a v o l a i l l e , A g r i c u l t u r e Canada ( 3 ) S a n t e e t Bien- t t r c s o c i a l Canada, BUPXU de s mOd i c anietit s vB t &r i na i r e s , Ottawa ( O n t a r i o ) K 1 A 1R7, CANADA arid ( 4 ) I n s t i t u t Armand F r a p p i e r , Lava1 (Qubbec) H7V 1 B 7 , CANADA
ABSTRACT A c o m p e t i t i v e enzyme inununoassay using p u r i f i e d monoclonal IgGl arid an a l k a l.ine pho spha t ase - a l b u t e r o 1 d e r i v a t i v e has been The developed f o r t h e q u a n t i f i c a t i o r i o f a l b u t e r o l i n u r i n e . c a l i b r a t i o n curve obtained i n optimal i n c u b a t i o n c o n d i t i o n s is c h a r a c t e r i z e d by a minimum d e t e c t a b l e l e v e l of 26 fniol/well arid a working range from 52 fniol t o 4.2 pmol/well. T h i s method a l l o w s the p r e c i s e and a c c u r a t e q u a n t i f i c a t . i o n of a l b u t e r o l i n h o r s e Moreover t h e u r i n e without any c l e a n up o r e x t r a c t i o n s t e p . d e f i n i t i o n of i t s s p e c i f i c i t y shows a c r o s s - r e a c t i v i t y of the a n t i b o d y w i t h the glucurono-/sulfa-conjugates of a l b u t e r o l . T h i s p r o p e r t y is p a r t i c u l a r l y i n t e r e s t i n g for t h e s c r e e n i n g of u r i n a r y a l b u t e r o l r e s i d u e s i n meat producing animals. (KEYWORDS: a l b u t e r o l , cleributerol , enzyme inununoassay, meat p r o d u c t i o n , drug residues). 207 Copyright 0 1991 by Marcel Dekker, Inc.
ADAM ET AL.
208
INTRODUCTION Albuterol (salbutaiiiol) , a PZ-adrenergic
agent , used f o r t h e
treatment of lung d i s e a s e s a s s o c i a t e d with a i r flow o b s t r u c t i o n
i n humans (1). has a l s o been applied i n v e t e r i n a r y medicine f o r
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illegal
its
purposes:
sniooth nuscle has been
relaxing
capacity
the
on
bronchial
e x p l o i t e d f o r race horse doping
(2).
Mo~-eove~*, a l b u t e r o l , a s o t h e r (32-adrenergic a g e n t s , is known a s a p o t e n t growth f a c t o r . an
increase
content
in
production threat
iri
This p r o p e r t y has been used t o produce
skeletal
sheep,
pig
muscle and
the
cattle
t h e body
Residues
(3).
fat
these
of
i n meat products may r e p r e s e n t
enhancing drugs
to
and t o reduce
consumers
health.
Facing
this
a
potential
important public h e a l t h problem (4) , t h e Veterinary Branches of t h e European Conmiunities reconmiend t e s t i n g of animals f o r t h e detection (5-6).
of
these
drug
residues , i n
The physicochemical methods
the albuterol
deterniination
the
slaughter
houses
a v a i l a b l e now ( 7 - 1 1 )
for
a r e time consuming and cannot be
processed i n a l a r g e s c a l e f o r screening purpose.
However, t h e
d e t e c t i o n of t h e s e drugs i n u r i n e h a s r e c e n t l y been recommended (5).
We d e s c r i b e here t h e development and t h e a n a l y t i c a l v a l i d a t i o n of
a competitive
enzyme
inuiunoassay
for
albuterol
arid
a p p l i c a t i o n f o r t h e screening of t h i s drug in u r i n e samples.
its
209
A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL MATERIAL AND METHODS
Mater i a 1 The f o l l o w i n g m a t e r i a l s were purchased f coni t h e s u p p l i e r s indicated.
A l b u t e r o l , a l b u t e r o l h e m i s u l f a t e , &glucurotiidase
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(Sigma Chemical Co, St-Louis, MO) Scientific,
Muskegori,
P r o t e i n G Sepharose Uppsala,
Succiriic anhydride ( F i s h e r
Intestine
MI).
paranitrophenylphosphate
.
(Boehringer Plannheim,
(Mab t r a p G ) ,
Sweden) *
alkaline
[
14
Orit. )
.
(Pharinacia
1-ethoxycarboiiyl- 2- ethoxy- 1,2- dihydro.
anhydride
Iiimiurioplates
Denmark.
G~L-III~II~)
.
f3H]
albuterol
(Negev Nuclear Research C e n t e r , B e e r Sheva,
succinic
Cl
.
West
Sepharose CNBr
quinolirie (EEDQ) ( A l d r i c h , Wilwaukee, WI) Ci/nunol)
yhosphatase,
Behcing
A/C
(111 niCi/nuuol)
Nuric
(Anisrsham,
were o b t a i n e d
ELISA p r o c e s s o r
(BEP)
frvia
(Behririg
Israel). Oakville, Roskilde, Institute,
Marburg, West Ge~71ia1iy)was used f o r t h e ELISA development. other
reagents
were of
analytical
grade
arid
(17
obtained
All
from
Fisher Scientific.
Methods Production arid P u r i f i c a t i o r i of Morioclorial IRG Against A l b u t e r o l The p r o d u c t i o n of a monoclonal a n t i b o d y a g a i n s t a l b u t e r o l h a s been d e s c r i b e d p r e v i o u s l y
(12).
The c h a r a c t e r i z a t i o n of
t h i s m a t e r i a l r e v e a l e d t h e monoclonal n a t u r e of t h i s a n t i b o d y belonging t o t h e IgG1 i s o t y p e w i t h an a f f i n i t y constarit of 1 . 0 3 nM
-1
.
I t s s p e c i f i t y h a s been assessed by t e s t i n g
i t s CL-oss-
2 10
ADAM ET AL.
r e a c t i v i t y towards t o a l b u t e r o l a n a l o g s .
The a n t i b o d y d i s p l a y s
o n l y s i g i i i f icarit c r o s s - r e a c t i v i t y t o cleributerol (75%) ( 1 2 ) . A s c i t i c f l u i d s have been obtained a f t e r 18 days i r i Balb/c mice
by
intraperitoneal
i n j e c t i o n of
1. 6
6
x 10
cells/mouse.
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These animals w e r e p r e t r e a t e d w i t h p r i s t a r i e arid i r r a d i a t e d a t 200 r a d , the day b e f o r e i n j e c t i o n ( 1 3 ) . I g G l i n the a s c i t i c f l u i d was p u r i f i e d u s i n g the Mab t r a p G
k i t according t h e i n s t r u c t i o n s o f t h e manufacturer.
The p u r i t y
of t h e m a t e r i a l was checked using t h e 278 Iln1/251
absorbance
rim
r a t i o ( > 1 . 5 ) . arid its recovery by absorbance a t 278 rim ( 1 4 ) .
Preparatiori of t h e A l k a l i n e Phosphatase-Albuterol D e r i v a t i v e The a l k a l i n e p h o s p h a t a s e - a l b u t e r o l d e r i v a t i v e was obtained i r i a two s t e p s r e a c t i o n .
Albuterol
hemisucciriate
was
first
synthesized
according
3 [ HI a l b u t e r o l Beaulieu e t a l . (151, w i t h minor m o d i f i c a t i o n s . 14 arid [ C] s u c c i n i c anhydride were added f o r t h e monitoring of
the
derivatizatiori
The s y n t h e s i z e d m a t e r i a l 3 14 p u r i f i e d by d i a l y s i s t o c o n s t a n t 1 HI/[ C ] r a t i o . Albuterol
procedure.
hemisucciriate
was
coupled
to
calf
was
intestine
a l k a l i n e phosphatase u s i n g EEDQ, a s d e s c r i b e d by B e l l e a u arid Malek ( 1 6 ) .
0.075 nuuol of t h e a l b u t e r o l hemisucciriate ( 2 5 mg)
were f i r s t a c t i v a t e d with 0.121 nmol EEDQ (30 mg) ethanol
f o r 1 6 hours a t 20°C.
in
2 niL
The a c t i v a t e d d e r i v a t i v e w a s
t h e n added t o 1 7 . 8 iimol ( 2 . 5 mg/lmL) of
a l k a l i n e phosphatase
A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL
211
The coupling procedure w a s
s o l u t i o n i r i 0 . 1 H NaHC03 a t pH 9 . 5 .
perfornied f o r 1 6 h o u r s a t room temperature.
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then
against
dialysed
containing
ZnS04
procedure
was
buffer
Tris-HC1
(10 mg/L).
estimated
The s o l u t i o n was 100
nW,
the
The e f f i c i e n c y of by
counting
pH
the
7.4,
coupling
radioactivity
i n c o r p o r a t e d and measuring t h e enzynie a c t i v i t y .
Enzyme Tmunoassay Nuric inmunoplates w e r e c o a t e d (1 h r a t 3 7 T ) w i t h a f f i n i t y
p u r i f i e d monoclonal IgG (100 p 1 c o n t a i n i n g 250 n g / w e l l ) d i l u t e d
i n t h e c o a t i n g b u f f e r (Tris-HC1 50 nM, pH 8 , 5 , NaCl 100 mM). The p l a t e s were then waslied 5
thes
with
the washing b u f f e r
(Tris-HC1
50nC4, pH 7 . 4 , N a C l 100 IIW, Tween 20 0 . 5 niL/L)
incubated
€OK*
buffer
1 hour
(Tris-HC1
albumiric 2 g / L ) .
at
50 ruM,
37°C w i t h pH
200 p 1 of NaCl
7.4,
arid
the saturation
100 nW,
bovine
seruni
T?ie p l a t e s were then r i r i s e d 3 times w i t h the
washing s o l u t i o n and i n c u b a t e d w i t h t h e r e a c t i o n medium which c o n s i s t s of
a l k a l i n e phosphatase
labelled albuterol
a l b u t e r o l s t a n d a r d s o l u t i o n s ( 5 0 pL) o r b i o l o g i c a l PI.)
completed
t o 50 p 1 w i t h the i n c u b a t i o n
g i v i n g a f i n a l volume of
buffer
100 pL i n each w e l l .
a l k a l i n e pliosyhatase corijugate was used
1/2000 corresponding t o 89.48 €moles of l i n k e d t o 20.94 pnioles of a l b u t e r o l .
(50 pL)
,
sample (10 (40
pL),
The a l b u t e r o l
a t f i n a l d i l u t i o n of alkalilie phouphatase
ET AL.
ADAM
2 12
The p l a t e s were incubated f o r a p e r i o d of 4 hours a t 37°C. A f t e r 3 washirig s t e p s , a l k a l i n e phosphatase e n z y m a c t i v i t y was developed
using
100
pL
substrate
of
solution
nM
(100
diethanolaniine b u f f e r pH 9,8, parariitrop?ieiiylphosphate 4n1M and
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per- w e l l .
100 nig/L)
MgS04,
The enzynie r e a c t i o n wds stopped
a f t e r h a l f an hour by adding 100 p1 of hJaOH 2 IU.
were then measured with t h e BEP System a t 405
Tim
Absorbances u s i n g 490
iini
a s a r e f e r e n c e wavelength.
S p e c i f i c i t y of t h e Enzmie Tnuiiunoassay To e v a l u a t e t h e s p e c i f i c i t y of detected
by
enzynie
albuterol
and
submitted
to
The
were
inuiurioassay,
urine
horse u r i n e a f t e r a high
chromatograms compared
perforniance obtained
to
the
inmunoreactive s i g n a l
the
samples
spiked
10 mg I V dose
liquid
following
(12)
chromatography fluorinietric
inmunoreactivity p r o f i l e s
e l u t e d f r a c t i o r i s by enzynie inununoassay procedure.
with
were
(HPLC)
.
detection
set
on
the
A l i q u o t s (1
niL) of t h e u r i n e samples were f i r s t submitted t o a clean-up e x t r a c t i o n by inmiunoaf f i r i i t y chromatography followed by an HPLC step
as
previously
detection. adjusted
Eluted at
7.4
described
fractions
with
Tris
(17)
(1 nil)
500 nM
using
were (50
fluorinietric
collected
~ 1 )f o r t h e
and
pH
enzynie
inununoassay d e t e c t i o n . I n o r d e r t o i d e n t i f y glucurono-/
s u l f o - c o n j u g a t e s of a l b u t e r o l ,
u r i n e samples were submitted t o HPLC b e f o r e arid a f t e r enzymatic
A
COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL
213
h y d r o l y s i s according t o the p ~ * o c e d u ~ -of e Koster et a l . Chromatogranis
obtained
by
fluorinietric
detection
(18).
and
inmiunoreactivity p r o f i l e s w e r e compared i n b o t h c o n d i t i o n s .
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Q u a n t i f i c a t i o n of A l b u t e r o l in Urine b y Erizvme Tnmiurioassay Q u a n t i f i c a t i o n of a l b u t e r o l was performed d i r e c t l y , without any e x t r a c t i o n s t e p , the
study
of
011
the
t h e same samples than t h o s e used f o r
specificity.
concentratioris
The
of
inmiuxioreactive a l b u t e r o l were c o r r e l a t e d w i t h those iueasured by direct: radioiriuiiurioassay ( 1 2 ) and by HPLC/f luorescerice a f t e r an inuiurioaffinity clean-up (17).
Data a r i a l y s i s . The results were analyzed u s i n g a f i t t e d c a l i b r a t i o n curve (19).
RESULTS C h a r a c t e r i s t i c s of the ELISA System Density of a l k a l i n e phosphatase l a b e l l i n g w i t h a l b u t e r o l . The
ef f i c i e r i c y
of
conjugation
of
albuterol
to
alkaline
phosphatase was e s t i m a t e d by determining the i n c o r p o r a t i o n of J
[ HI
a l b u t e r o l g i v i n g a y i e l d of 234 moles of hapten p e r mole
of enzyme.
T h i s l a b e l l i n g s t e p d i d n o t r e s u l t i n any loss of
enzyme a c t i v i t y . ELISA C a l i b r a t i o n Curve
F i g u r e 1 shows a t y p i c a l c a l i b r a t i o n curve o b t a i n e d i n the conditions selected
above,
in presence
of
f i x e d amounts of
ADAM ET AL.
2 14
1.6
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1.2
0.8
0.4
50
5000
500
ALBUTEROL ( fmol/well )
Figure 1: C a l i b r a t i o n curve f o r t h e enzynie a l b u t e r o l i n incubation b u f f e r (--O-) of 10 p 1 of h o r s e u r i n e (-0-1.
[albuterol-enzyme] albuterol, detectable
complex
ranging
with
inunurioassay of o r i n presence
increasing
from 50 fino1 t o 5 pniol/well.
concentration
of
albuterol
amounts
of
The mininial
determined
as
the
absorbance of t h e zero mean concentration minus 2 SD equals 26 fmol/well. 4.2
The working concentration range is f r o m 5 2 fmol t o
ymol/well.
The
absorbance
of
this
upper
limit
being
s t a t i s t i c a l l y d i f f e r e n t (mean p l u s 2 SD) t h a t t h i s measured f o r
a blank value.
The non
i n t e r f e r e n c e of horse u r i n e i n t h i s
d i r e c t assay has been demonstrated by s t a t i s t i c a l a n a l y s i s of
215
A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL
tlie c a l i b r a t i o n curves i n b u f f e r w i t h arid without h o r s e u r i n e (10
All
by
pl/well)
Fit
program
No
(20).
d i f f e r e n c e could b e d e t e c t e d (F (4.8) = 0.49, P
--
significant
0.05).
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Analytical Validation I r i t r a arid i n t e r a s s a y p r e c i s i o n was a s s e s s e d on 15 r e p l i cates of pooled u r i n e samples spiked a t 3 d i f f e r e n t a l b u t e r o l 1 6 0 , 80, 4 0 pmol/mL i n f i v e r e p l i c a t e s .
levels:
were
analyzed
011
three
differerits plates.
The samples
The i n t r a - a s s a y
v a r i a t i o n CV were 2.4, 7 . 8 arid 7.9%, r e s p e c t i v e l y . f o r i n t e r - a s s a y CV were 7.4, 10.7 and 7.6%.
The v a l u e s
The accuracy of
t h e a s s a y was t e s t e d u s i n g a b l a n k u r i n e spiked w i t h a l b u t e r o l t o c o n c e n t r a t i o n s of
40,
20,
160 and
80,
320 pmol/mL.
The
l i n e a r r e g r e s s i o n curve f o r t h e c o r r e l a t i o n between a l b u t e r o l c o n c e n t r a t i o n s expected and t h o s e measured i s d e s c r i b e d by ttie 2 following equation: ?’ = 1.099X t 0.45 (r .= 0.983). S i m i l a r l y , a h o r s e u r i n e a f t e r an I V dose of
was
albuterol
immunoassay.
9
.= 0.014X
+
serially
diluted
and
assayed
10 mg
by
of
enzym
The r e s u l t s are shown i n f i g u r e 2 (r2 = 0.964, 3.63).
S p e c i f i c i t y of t h e ELISA Method Typical
chromatograms
set
by
fluorescence
and
inmuno-
enzymatic d e t e c t i o n on e l u t e d f r a c t i o n s a f t e r HPLC of a blank urine
extract,
arid
an
extract
of
an
horse
urine
sample
ADAM
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216
ET AL.
URINE DILUTION
Figure 2:
2
C o r r e l a t i o r i (r- =. 0.964, 0 = 0.014X t 3.63) between s u c c e s s i v e d i l u t i o n s of a horse u r i n e c o n t a i r i i n g a l b u t e r o l and the coricentratioris measured by erizynie inmiurioassay. Each v a l u e KwpreseIitS t h e mati of two coriceritratioris.
c o n t a i n i n g a l b u t e r o l are shown i n f i g u r e 3 .
The i:iuiiunoreactive
signal
that
detected
standard tiine
vf
by
following 10
miri.
ELISA
corresporids
fluorimetric However,
the
to
detection,
with
chromatographic
of
albuterol
a
retention
profiles
of
e x t r a c t s of h o r s e u r i n e samples a f t e r a n IV dose of a l b u t e r o l d i s p l a y 2 p e a k s e l u t e d a t 6 . 4 arid 10.18 m i r i s d e t e c t e d b o t h by f l u o r i n i e t r i c arid inmiunological iiietliods. To c o n f i r m the n a t u r e of the peak e l u t e d a t 6.4 mixis, t h e
u r i n a r y sample has been s u b m i t t e d t o /3--glucuronidase d i g e s t i o n
217
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A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL
lB
t
A
J
E
2
0
RETENTION TIME ( min )
10
!i
v
5
0
I
6 ELUTED FRACTIONS( mL )
Typical chromatogranis (A) arid inunuriograrts (B) of a blank urine (11, ail h o r s e urine extract a f t e r inu-iunochromat o g raphy clean UP (2) arid P-glucuronidase h y d r o l y s i s ( 3 ) .
ADAM ET AL.
218 prior
the
chroniatography
step.
g lucurono-/ s u l f o- d e r i v a t i ves the
disappearance of
the
of
Indeed,
hydrolysis
of
demonstrated
by
the
a1bu t e r o 1 i s
inmiunoreactive peak
eluted
a t 6.4
mins, with a s h i f t of t h e s i g n a l t o t h e e l u t i o n p o s i t i o n of t h e
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albuterol
standard
confirming
therefore
t h e presence
of
the
conjugate d e r i v a t i v e of a l b u t e r o l both i n t h e chroniatogram arid inuiunogram of t h e s e u r i n e e x t r a c t s . The
cross-reactivity
of
the
antibody
used
with
the
m e t a b o l i t e s h a s been estimated by determining t h e cuniulative amount of drug e x c r e t e d i n u r i n e v e r s u s time by b o t h s p e c i f i c
HPLC inethod and by ELISA.
The cuniulative amount of a l b u t e r o l
e x c r e t e d estimated by ELISA is found t o be t h e double of
by HPLC procedure which allows t h e s e p a r a t i o n of
determined
unchanged a l b u t e r o l from i t s m e t a b o l i t e s . therefore
that
a
full
cross- r e a c t i v i t y
We could conclude
of t h e antibody w i t h t h e
m e t a b o l i t e s p a r t i c u l a r l y t h e glurucono-
arid s u l f o -
derivatives
which are found t h e major ones e x c r e t e d i n u r i n e .
Urinary Coriceritrations of Albuterol Q u a n t i f i e d by ELISA Table 1 r e p r e s e n t s t h e values of direct
ELISA
fluorescence between
arid after
albuterol
RIA
(12) methods
HPLC
a l b u t e r o l q u a n t i f i e d by arid
those
chromatography.
The
concentrations
measured
by
measured
by
correlation
RLAIELISA
arid
HPLWELISA methods is r e s p e c t i v e l y d e s c r i b e d by t h e following h
equation:
h
Y = 1.168X - 51 (r2 = 0.9981, Y
= 0.690X
-
2
176 ( r
=
219
A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL TABLE: 1
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URINARY LEVELS OF ALBUTEROL (pniol/mL) AFTER AN IV DOSE OF ALBUTEROL QUANTIPIED COMPARATIVELY BY IMMUNOLOGICAL AND INSTRUMENTAL METHODS.
Sample
ELISA
RIA
HPLC
1 2 3 4 5
0 5,333 1,220 792 525 458 346 225 217 187
0
6,208 1,237 862 562 4 71 358 196 187 233
0 3,562 365 370 178 150 92 44 36 36
6 7
8 9 10
These lower l e v e l s obtained by HPLC confirm t h e c r o s s -
0.989).
r e a c t i v i t y of t h e antibody used with t h e a l b u t e r o l m e t a b o l i t e s which
are
separated
from
the
parent
compound
in
the
chroniatographic c o n d i t i o n s used.
DISCUSSION In t h e r e c e n t y e a r s , incentive
for
the
particularly
iioxi
quaiitif icatiori
of
misuse of
development
of
drugs has analytical
radioisotopic these
xeriobiotics.
been
a major
methodologies,
iiimiunoassays
for
Among t h e s e drugs,
the
132
adreriergic a g e n t s have been suspected t o be widely used for
220
ADAM ET AL.
illegal
purposes
clenbuterol,
21).
(5-6,
using
a
an ELISA method
Although
rabbit:
polyclorial
antiserum
has
for been
,
proposed f o r t h e s c r e e n i n g of t h i s a d r e n e r g i c agent (22-23) inmunoassay
with
Downloaded by [Michigan State University] at 02:22 10 February 2015
s t i l l lacking. enzyliie
sensitivity
suf f icierit
We r e p o r t h e r e t h e set up of
inuiuiioassay
a
using
The
a v a i l a b i l i t y of
albuterol
such monoclonal
would a l l o w t h e s t a r i d a r d i z a t i o n o f t h e a s s a y of large
scale
studies.
The
specificity
inmunoassay
is
a competitive
antibody
iiionoclonal
a p p l i c a t i o n f o r t h e d e t e r m i n a t i o n of fluids.
arid
an
its
and
i n biological
(12)
antibody
this drug f u r
reported
here
is
convenient s i n c e it: does not r e q u i r e a c l e a n up e x t r a c t i o n i.n t h e procedure.
tlie p r e s e n t a s s a y i n
The h i g h s e n s i t i v i t y of
t h e fmole range is s u f f i c i e n t f o r t h e s c r e e n i n g of for
which ppb- l e v e l s
(5-6).
Moreover,
albuterol
limits were f i x e d by o f f i c i a l a g e n c i e s
the cross-reactivity
g lucu rono- / s u lfo--conjug a t es
of
of
the
antibody with
a lbu t e r o 1 r e n d e r s
this
a s say
p a r t i c u l a r l y a t t r a c t i v e f o r t h e d e t e c t i o n of t h i s drug arid i t s n i u t a b o l i t e s found a t h i g h l e v e l s
iti
urine.
The a p p l i c a t i o n of
t h e p r e s e n t enzyme iiiuiunoassay i s n o t l i m i t e d f o r t h e s c r e e n i n g of the
t h i s drug i n v e t e r i n a r y medicine b u t would be s u i t a b l e f o r clinical
monitoring
of
this
82
adreriergic
agonist
in
t h e r a p e u t i c a d j u s tnients i n human medicine.
ACKNOWLEDGEMENTS We wish t o thank Dean J . Gagn6, l e "Fonds d e d6veloppement de
l ' U n i v e r s i t 6 de Montr8al" f o r s u p p o r t i n g t h i s work.
A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL Reprints
requests
F a c u l t y of
s h o u l d be
Pharmacy,
addressed
University
of
to:
221 Dr.
Eloritreal,
Albert
P.O.
Adam,
Box
6128,
and
the
S t a t i o n A , Montreal (Quebec) H3C 357 CANADA. REFERENCES
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Weiner,
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Mc Millari P u b l i s h i n g Co, 1985; 145-80.
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Tobin, T., Watt, D . S . , Kwiatkowski S . , T a i , H - H . , Blake, J.W. Nun-isotopic jnuiiurioassay drug tests in racing fiocses: a r e v i e w of t h e i r a p p l i c a t i o n t o p r e and post- race t e s t i n g , drug q u a i i t i t a t i o n , and hiuiiiati drug testing. R e s . Conmiuri. Chern. P a t h o l Pharniacol 1988 ; 62 :371-95.
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Yarig, Y.T., McElligott, M.A. b e t a - a d r e n e r g i c a g o r i i s t s on s k e l e t a l t i s s u e . Biocheni. J . 1989; 261:l-10.
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Weisberger, M . ,
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Hiller, G.L. arid Greenblatt, D.L. Determination of a l b u t e r o l i r i huniari p l a s m by h i g h - performance liquid ctiromatograpliy w i t h f luorescerice d e t e c t i o n . J . Chroinatogr. 1986; 381: 205-8.
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Enuti, T., Lesko, L . J . and L e s l i e , J . Detenniriatiori of a l b u t e r o l i n human serum by r e v e r s e - p h a s e h i g h p e r f orniaiice 1i q u i d chromatography w i t h e 1ec t rochemica 1 d e t e c t i o n . J chromatogr. 1988; 427 : 188--94.
Dresser, W . R . , Wilcke, J . R . Drug animals. JAVMA. 1989; 194: 1700-10.
residues
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P a t r i c k , J . E . , P o w e l l , M.L. Quantitative a n a l y s i s of a l b u t e t w l i n huindn p h s m a by combined gas chromatography and chemical i o n i z a t i o n mass s p e c t r o m e t r y . Biomed I l V d S S SpectlWIlletry 1983; 10~556-8.
.
10
C o l t h u p , P.V., D a l l a s , F . A . , Saynor, D . A . , Carey, P . F . , Skidmore, L.F. and Martin, L.E. Deter-niiriation of salbutaniol i n liumari plasma arid u r i n e by ~ i i g h - p e r f o n n a n c e t h i n - l a y e r chromatography. J . Chronratogr. 1985 ; 345 : 111-8.
ADAM ET AL.
2 22
11. Loo, J . C . K . , B e a u l i e u , N.. J o r d a n , N. arid B r i e r ) R . A spec i f i c r a d i o inunurioassay ( R I A ) f o r s a l bu tamo 1 ( a 1bu t e co 1) In human plasma. R e s . Conun. Cheni. P a t h o l . Pharmacol. 1987 ; 55: 283-6.
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12. Adam, A . , Ong, H . , Soridag. D . e t a l . Radioinunurioassay f o r a l b u t e r o l u s i n g a morioclorial a n t i b o d y : application for d i r e c t quaritif i c a t i o r i i n h o r s e u r i n e . J. Inmiunoassay ( i n press). A method t o induce iiiuriurie 13. Lacy, M . J. arid Voss, E.W. polyclorial ascites f l u i d i r i RALB/c inice u s i n g Sp210-Ag14 cells. J . Inmiuriol . Methods. 1986 ; 87 :169- 7 7. A l a b o r a t o r y niariual. Ed. A L - ~ o w , D . L a m , eds. 14. Antibodies: Cold S p r i n g Harbor L a b o r a t o r y . 1988; 290-1.
15. B e a u l i e u , N., C h a r e t t e , C1. Loo, J . C . K . , Jot-dan, N. arid Mc G i l v e r a y , I . J . Salbutaniol radioinuriunoassay: sy-nthesis arid p r o p e r t i e s of t h e benzyl s u c c i r i a t e of s a l b u t a m o l . J. Ptiarxi. arid Biomed. A n a l y s i s . 1985; 3:575-9. A n e w coriveriierit reagent: f o r 16. B e l l e a u , B . arid Malek G . J . p e p t i d e s y r i t h e s i s . h i . Chem. Soc. 1968; 90: 1651-2.
A n a l y s i s of 17. Ong, H . , Adam, A . , P e r r e a u l t , S . e t a l . albuterol in human plasrna based on inuwrioaf f i n i t y cliromatography clean up conibined t o h i g h performance l i q u i d chromatography w i t h f l u o r i m e t r i c d e t e c t i o n . J . Chromatogr. 1989; 497: 213-21. A., Hofmaii, G . A . , Frankhiuyzeri-Sierevogel, C . , 18. K o s t e r , Noordhoek, J. Presystemic arid systemic intestinal metabolism of f e r i o t e r o l i n t h e c o n s c i o u s r a t . Drug Metah. D i s p o s i t i o n 1985 ; 13 :4 6 4 - 70.
19. T i j s s e r i , P. P r a c t i c e arid t h e o r y of erizynie inmiurioassays. and iwlecular L a b o r a t o r y .techniques in Biochemistry Biology. Amsterdam, E l s e v i e r , 1985; 385-421. L&ari, A . , Muitsun, P.J. arid Rodbard, D . Siinultarieous a n a l y s i s of f a m i l i e s of s i g m o i d a l curves: a p p l i c a t i o n t o b i o a s s a y , r a d i o l i g a n d a s s a y arid p h y s i o l o g i c a l d o s e - r e s p o n s e c u r v e s . Am. J. P h y s i o l . 1978; 235:E97-E102.
2 0 . De
21. Roy, G. U t i l i s a t i o n i l l k g a l e d'uri s t i m u l a n t de c r o i s s a r i c e chez des veaux. LR m6decin v 6 t 8 r i n a i r e du QuBbec, 1490; 20~35-6.
A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL
223
I. and Xwota, K. Enzyme-inununoassay for clenbuterol an 82-adrenergic stimulant. J . Immunoassay.
22. Yamamoto,
1982; 3:155-71.
P., Degand G., Pelzer G., Gaspar P. and naghein-Rogister C. Dhveloppements analytiques pour la dBtection du clenbutBro1 utilise comnie agent de rhpartition de graisse chez les animaux de boucherie. Ann. M6d. VBt.
23. Schmitz
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1989;133: 427-31.
Journal of Immunoassay Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii19
A Competitive Enzyme Immunoassay for Albuterol: Its Application for the Drug Screening in Urine a
a
b
Albert Adam , Huy Ong , Andre Gravel , Jacques c
a
a
Messier , Monique Bellemare , Francine Lantin , d
Gilles Sauvé & Peeter Tyssen
d
a
Faculté de pharmacie , Université de Montréal , P. O. Box 6128, Station A, Montréal (Québec), H3C 3J7, CANADA b
Agriculture Canada, Division de l'hygiène de la viande et des produits de la volaille Agriculture , Canada c
Santé et Bien-Etre social Canada, Bureau des médicaments vétérinaires , Ottawa (Ontario), K1A 1B7, CANADA d
Institut Armand Frappier , Laval, Québec, H7V 1B7, CANADA Published online: 23 Oct 2006.
To cite this article: Albert Adam , Huy Ong , Andre Gravel , Jacques Messier , Monique Bellemare , Francine Lantin , Gilles Sauvé & Peeter Tyssen (1991) A Competitive Enzyme Immunoassay for Albuterol: Its Application for the Drug Screening in Urine, Journal of Immunoassay, 12:2, 207-223, DOI: 10.1080/01971529108055067 To link to this article: http://dx.doi.org/10.1080/01971529108055067
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JOURNAL OF IMMUNOASSAY, 1 2 ( 2 ) , 207-223 ( 1 9 9 1 )
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A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL: ITS APPLICATIOM FOR THE DRUG SCREENING I N URINE.
A l b e r t Adam(l), Huy Ong(1) , Andre G r a v e l ( 2 ) , Jacques Messier(3). Monique Belleniare(1) , Francine L a n t i n ( l ) , G i l l e s Sauv8(4), P e e t e r T y s s e n ( 4 ) . (1) F a c u l t 6 de pharmiacie, Uriiversith d e Montrbal P.O. Box 6128, S t a t i o n A , Montrbal (Quhbec) H3C 3 5 7 , CANADA. ( 2 ) A g r i c u l t u r e Canada, D i v i s i o n de l ' h y g i h n e d e l a viande e t d e s p r o d u i t s d e l a v o l a i l l e , A g r i c u l t u r e Canada ( 3 ) S a n t e e t Bien- t t r c s o c i a l Canada, BUPXU de s mOd i c anietit s vB t &r i na i r e s , Ottawa ( O n t a r i o ) K 1 A 1R7, CANADA arid ( 4 ) I n s t i t u t Armand F r a p p i e r , Lava1 (Qubbec) H7V 1 B 7 , CANADA
ABSTRACT A c o m p e t i t i v e enzyme inununoassay using p u r i f i e d monoclonal IgGl arid an a l k a l.ine pho spha t ase - a l b u t e r o 1 d e r i v a t i v e has been The developed f o r t h e q u a n t i f i c a t i o r i o f a l b u t e r o l i n u r i n e . c a l i b r a t i o n curve obtained i n optimal i n c u b a t i o n c o n d i t i o n s is c h a r a c t e r i z e d by a minimum d e t e c t a b l e l e v e l of 26 fniol/well arid a working range from 52 fniol t o 4.2 pmol/well. T h i s method a l l o w s the p r e c i s e and a c c u r a t e q u a n t i f i c a t . i o n of a l b u t e r o l i n h o r s e Moreover t h e u r i n e without any c l e a n up o r e x t r a c t i o n s t e p . d e f i n i t i o n of i t s s p e c i f i c i t y shows a c r o s s - r e a c t i v i t y of the a n t i b o d y w i t h the glucurono-/sulfa-conjugates of a l b u t e r o l . T h i s p r o p e r t y is p a r t i c u l a r l y i n t e r e s t i n g for t h e s c r e e n i n g of u r i n a r y a l b u t e r o l r e s i d u e s i n meat producing animals. (KEYWORDS: a l b u t e r o l , cleributerol , enzyme inununoassay, meat p r o d u c t i o n , drug residues). 207 Copyright 0 1991 by Marcel Dekker, Inc.
ADAM ET AL.
208
INTRODUCTION Albuterol (salbutaiiiol) , a PZ-adrenergic
agent , used f o r t h e
treatment of lung d i s e a s e s a s s o c i a t e d with a i r flow o b s t r u c t i o n
i n humans (1). has a l s o been applied i n v e t e r i n a r y medicine f o r
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illegal
its
purposes:
sniooth nuscle has been
relaxing
capacity
the
on
bronchial
e x p l o i t e d f o r race horse doping
(2).
Mo~-eove~*, a l b u t e r o l , a s o t h e r (32-adrenergic a g e n t s , is known a s a p o t e n t growth f a c t o r . an
increase
content
in
production threat
iri
This p r o p e r t y has been used t o produce
skeletal
sheep,
pig
muscle and
the
cattle
t h e body
Residues
(3).
fat
these
of
i n meat products may r e p r e s e n t
enhancing drugs
to
and t o reduce
consumers
health.
Facing
this
a
potential
important public h e a l t h problem (4) , t h e Veterinary Branches of t h e European Conmiunities reconmiend t e s t i n g of animals f o r t h e detection (5-6).
of
these
drug
residues , i n
The physicochemical methods
the albuterol
deterniination
the
slaughter
houses
a v a i l a b l e now ( 7 - 1 1 )
for
a r e time consuming and cannot be
processed i n a l a r g e s c a l e f o r screening purpose.
However, t h e
d e t e c t i o n of t h e s e drugs i n u r i n e h a s r e c e n t l y been recommended (5).
We d e s c r i b e here t h e development and t h e a n a l y t i c a l v a l i d a t i o n of
a competitive
enzyme
inuiunoassay
for
albuterol
arid
a p p l i c a t i o n f o r t h e screening of t h i s drug in u r i n e samples.
its
209
A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL MATERIAL AND METHODS
Mater i a 1 The f o l l o w i n g m a t e r i a l s were purchased f coni t h e s u p p l i e r s indicated.
A l b u t e r o l , a l b u t e r o l h e m i s u l f a t e , &glucurotiidase
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(Sigma Chemical Co, St-Louis, MO) Scientific,
Muskegori,
P r o t e i n G Sepharose Uppsala,
Succiriic anhydride ( F i s h e r
Intestine
MI).
paranitrophenylphosphate
.
(Boehringer Plannheim,
(Mab t r a p G ) ,
Sweden) *
alkaline
[
14
Orit. )
.
(Pharinacia
1-ethoxycarboiiyl- 2- ethoxy- 1,2- dihydro.
anhydride
Iiimiurioplates
Denmark.
G~L-III~II~)
.
f3H]
albuterol
(Negev Nuclear Research C e n t e r , B e e r Sheva,
succinic
Cl
.
West
Sepharose CNBr
quinolirie (EEDQ) ( A l d r i c h , Wilwaukee, WI) Ci/nunol)
yhosphatase,
Behcing
A/C
(111 niCi/nuuol)
Nuric
(Anisrsham,
were o b t a i n e d
ELISA p r o c e s s o r
(BEP)
frvia
(Behririg
Israel). Oakville, Roskilde, Institute,
Marburg, West Ge~71ia1iy)was used f o r t h e ELISA development. other
reagents
were of
analytical
grade
arid
(17
obtained
All
from
Fisher Scientific.
Methods Production arid P u r i f i c a t i o r i of Morioclorial IRG Against A l b u t e r o l The p r o d u c t i o n of a monoclonal a n t i b o d y a g a i n s t a l b u t e r o l h a s been d e s c r i b e d p r e v i o u s l y
(12).
The c h a r a c t e r i z a t i o n of
t h i s m a t e r i a l r e v e a l e d t h e monoclonal n a t u r e of t h i s a n t i b o d y belonging t o t h e IgG1 i s o t y p e w i t h an a f f i n i t y constarit of 1 . 0 3 nM
-1
.
I t s s p e c i f i t y h a s been assessed by t e s t i n g
i t s CL-oss-
2 10
ADAM ET AL.
r e a c t i v i t y towards t o a l b u t e r o l a n a l o g s .
The a n t i b o d y d i s p l a y s
o n l y s i g i i i f icarit c r o s s - r e a c t i v i t y t o cleributerol (75%) ( 1 2 ) . A s c i t i c f l u i d s have been obtained a f t e r 18 days i r i Balb/c mice
by
intraperitoneal
i n j e c t i o n of
1. 6
6
x 10
cells/mouse.
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These animals w e r e p r e t r e a t e d w i t h p r i s t a r i e arid i r r a d i a t e d a t 200 r a d , the day b e f o r e i n j e c t i o n ( 1 3 ) . I g G l i n the a s c i t i c f l u i d was p u r i f i e d u s i n g the Mab t r a p G
k i t according t h e i n s t r u c t i o n s o f t h e manufacturer.
The p u r i t y
of t h e m a t e r i a l was checked using t h e 278 Iln1/251
absorbance
rim
r a t i o ( > 1 . 5 ) . arid its recovery by absorbance a t 278 rim ( 1 4 ) .
Preparatiori of t h e A l k a l i n e Phosphatase-Albuterol D e r i v a t i v e The a l k a l i n e p h o s p h a t a s e - a l b u t e r o l d e r i v a t i v e was obtained i r i a two s t e p s r e a c t i o n .
Albuterol
hemisucciriate
was
first
synthesized
according
3 [ HI a l b u t e r o l Beaulieu e t a l . (151, w i t h minor m o d i f i c a t i o n s . 14 arid [ C] s u c c i n i c anhydride were added f o r t h e monitoring of
the
derivatizatiori
The s y n t h e s i z e d m a t e r i a l 3 14 p u r i f i e d by d i a l y s i s t o c o n s t a n t 1 HI/[ C ] r a t i o . Albuterol
procedure.
hemisucciriate
was
coupled
to
calf
was
intestine
a l k a l i n e phosphatase u s i n g EEDQ, a s d e s c r i b e d by B e l l e a u arid Malek ( 1 6 ) .
0.075 nuuol of t h e a l b u t e r o l hemisucciriate ( 2 5 mg)
were f i r s t a c t i v a t e d with 0.121 nmol EEDQ (30 mg) ethanol
f o r 1 6 hours a t 20°C.
in
2 niL
The a c t i v a t e d d e r i v a t i v e w a s
t h e n added t o 1 7 . 8 iimol ( 2 . 5 mg/lmL) of
a l k a l i n e phosphatase
A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL
211
The coupling procedure w a s
s o l u t i o n i r i 0 . 1 H NaHC03 a t pH 9 . 5 .
perfornied f o r 1 6 h o u r s a t room temperature.
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then
against
dialysed
containing
ZnS04
procedure
was
buffer
Tris-HC1
(10 mg/L).
estimated
The s o l u t i o n was 100
nW,
the
The e f f i c i e n c y of by
counting
pH
the
7.4,
coupling
radioactivity
i n c o r p o r a t e d and measuring t h e enzynie a c t i v i t y .
Enzyme Tmunoassay Nuric inmunoplates w e r e c o a t e d (1 h r a t 3 7 T ) w i t h a f f i n i t y
p u r i f i e d monoclonal IgG (100 p 1 c o n t a i n i n g 250 n g / w e l l ) d i l u t e d
i n t h e c o a t i n g b u f f e r (Tris-HC1 50 nM, pH 8 , 5 , NaCl 100 mM). The p l a t e s were then waslied 5
thes
with
the washing b u f f e r
(Tris-HC1
50nC4, pH 7 . 4 , N a C l 100 IIW, Tween 20 0 . 5 niL/L)
incubated
€OK*
buffer
1 hour
(Tris-HC1
albumiric 2 g / L ) .
at
50 ruM,
37°C w i t h pH
200 p 1 of NaCl
7.4,
arid
the saturation
100 nW,
bovine
seruni
T?ie p l a t e s were then r i r i s e d 3 times w i t h the
washing s o l u t i o n and i n c u b a t e d w i t h t h e r e a c t i o n medium which c o n s i s t s of
a l k a l i n e phosphatase
labelled albuterol
a l b u t e r o l s t a n d a r d s o l u t i o n s ( 5 0 pL) o r b i o l o g i c a l PI.)
completed
t o 50 p 1 w i t h the i n c u b a t i o n
g i v i n g a f i n a l volume of
buffer
100 pL i n each w e l l .
a l k a l i n e pliosyhatase corijugate was used
1/2000 corresponding t o 89.48 €moles of l i n k e d t o 20.94 pnioles of a l b u t e r o l .
(50 pL)
,
sample (10 (40
pL),
The a l b u t e r o l
a t f i n a l d i l u t i o n of alkalilie phouphatase
ET AL.
ADAM
2 12
The p l a t e s were incubated f o r a p e r i o d of 4 hours a t 37°C. A f t e r 3 washirig s t e p s , a l k a l i n e phosphatase e n z y m a c t i v i t y was developed
using
100
pL
substrate
of
solution
nM
(100
diethanolaniine b u f f e r pH 9,8, parariitrop?ieiiylphosphate 4n1M and
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per- w e l l .
100 nig/L)
MgS04,
The enzynie r e a c t i o n wds stopped
a f t e r h a l f an hour by adding 100 p1 of hJaOH 2 IU.
were then measured with t h e BEP System a t 405
Tim
Absorbances u s i n g 490
iini
a s a r e f e r e n c e wavelength.
S p e c i f i c i t y of t h e Enzmie Tnuiiunoassay To e v a l u a t e t h e s p e c i f i c i t y of detected
by
enzynie
albuterol
and
submitted
to
The
were
inuiurioassay,
urine
horse u r i n e a f t e r a high
chromatograms compared
perforniance obtained
to
the
inmunoreactive s i g n a l
the
samples
spiked
10 mg I V dose
liquid
following
(12)
chromatography fluorinietric
inmunoreactivity p r o f i l e s
e l u t e d f r a c t i o r i s by enzynie inununoassay procedure.
with
were
(HPLC)
.
detection
set
on
the
A l i q u o t s (1
niL) of t h e u r i n e samples were f i r s t submitted t o a clean-up e x t r a c t i o n by inmiunoaf f i r i i t y chromatography followed by an HPLC step
as
previously
detection. adjusted
Eluted at
7.4
described
fractions
with
Tris
(17)
(1 nil)
500 nM
using
were (50
fluorinietric
collected
~ 1 )f o r t h e
and
pH
enzynie
inununoassay d e t e c t i o n . I n o r d e r t o i d e n t i f y glucurono-/
s u l f o - c o n j u g a t e s of a l b u t e r o l ,
u r i n e samples were submitted t o HPLC b e f o r e arid a f t e r enzymatic
A
COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL
213
h y d r o l y s i s according t o the p ~ * o c e d u ~ -of e Koster et a l . Chromatogranis
obtained
by
fluorinietric
detection
(18).
and
inmiunoreactivity p r o f i l e s w e r e compared i n b o t h c o n d i t i o n s .
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Q u a n t i f i c a t i o n of A l b u t e r o l in Urine b y Erizvme Tnmiurioassay Q u a n t i f i c a t i o n of a l b u t e r o l was performed d i r e c t l y , without any e x t r a c t i o n s t e p , the
study
of
011
the
t h e same samples than t h o s e used f o r
specificity.
concentratioris
The
of
inmiuxioreactive a l b u t e r o l were c o r r e l a t e d w i t h those iueasured by direct: radioiriuiiurioassay ( 1 2 ) and by HPLC/f luorescerice a f t e r an inuiurioaffinity clean-up (17).
Data a r i a l y s i s . The results were analyzed u s i n g a f i t t e d c a l i b r a t i o n curve (19).
RESULTS C h a r a c t e r i s t i c s of the ELISA System Density of a l k a l i n e phosphatase l a b e l l i n g w i t h a l b u t e r o l . The
ef f i c i e r i c y
of
conjugation
of
albuterol
to
alkaline
phosphatase was e s t i m a t e d by determining the i n c o r p o r a t i o n of J
[ HI
a l b u t e r o l g i v i n g a y i e l d of 234 moles of hapten p e r mole
of enzyme.
T h i s l a b e l l i n g s t e p d i d n o t r e s u l t i n any loss of
enzyme a c t i v i t y . ELISA C a l i b r a t i o n Curve
F i g u r e 1 shows a t y p i c a l c a l i b r a t i o n curve o b t a i n e d i n the conditions selected
above,
in presence
of
f i x e d amounts of
ADAM ET AL.
2 14
1.6
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1.2
0.8
0.4
50
5000
500
ALBUTEROL ( fmol/well )
Figure 1: C a l i b r a t i o n curve f o r t h e enzynie a l b u t e r o l i n incubation b u f f e r (--O-) of 10 p 1 of h o r s e u r i n e (-0-1.
[albuterol-enzyme] albuterol, detectable
complex
ranging
with
inunurioassay of o r i n presence
increasing
from 50 fino1 t o 5 pniol/well.
concentration
of
albuterol
amounts
of
The mininial
determined
as
the
absorbance of t h e zero mean concentration minus 2 SD equals 26 fmol/well. 4.2
The working concentration range is f r o m 5 2 fmol t o
ymol/well.
The
absorbance
of
this
upper
limit
being
s t a t i s t i c a l l y d i f f e r e n t (mean p l u s 2 SD) t h a t t h i s measured f o r
a blank value.
The non
i n t e r f e r e n c e of horse u r i n e i n t h i s
d i r e c t assay has been demonstrated by s t a t i s t i c a l a n a l y s i s of
215
A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL
tlie c a l i b r a t i o n curves i n b u f f e r w i t h arid without h o r s e u r i n e (10
All
by
pl/well)
Fit
program
No
(20).
d i f f e r e n c e could b e d e t e c t e d (F (4.8) = 0.49, P
--
significant
0.05).
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Analytical Validation I r i t r a arid i n t e r a s s a y p r e c i s i o n was a s s e s s e d on 15 r e p l i cates of pooled u r i n e samples spiked a t 3 d i f f e r e n t a l b u t e r o l 1 6 0 , 80, 4 0 pmol/mL i n f i v e r e p l i c a t e s .
levels:
were
analyzed
011
three
differerits plates.
The samples
The i n t r a - a s s a y
v a r i a t i o n CV were 2.4, 7 . 8 arid 7.9%, r e s p e c t i v e l y . f o r i n t e r - a s s a y CV were 7.4, 10.7 and 7.6%.
The v a l u e s
The accuracy of
t h e a s s a y was t e s t e d u s i n g a b l a n k u r i n e spiked w i t h a l b u t e r o l t o c o n c e n t r a t i o n s of
40,
20,
160 and
80,
320 pmol/mL.
The
l i n e a r r e g r e s s i o n curve f o r t h e c o r r e l a t i o n between a l b u t e r o l c o n c e n t r a t i o n s expected and t h o s e measured i s d e s c r i b e d by ttie 2 following equation: ?’ = 1.099X t 0.45 (r .= 0.983). S i m i l a r l y , a h o r s e u r i n e a f t e r an I V dose of
was
albuterol
immunoassay.
9
.= 0.014X
+
serially
diluted
and
assayed
10 mg
by
of
enzym
The r e s u l t s are shown i n f i g u r e 2 (r2 = 0.964, 3.63).
S p e c i f i c i t y of t h e ELISA Method Typical
chromatograms
set
by
fluorescence
and
inmuno-
enzymatic d e t e c t i o n on e l u t e d f r a c t i o n s a f t e r HPLC of a blank urine
extract,
arid
an
extract
of
an
horse
urine
sample
ADAM
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216
ET AL.
URINE DILUTION
Figure 2:
2
C o r r e l a t i o r i (r- =. 0.964, 0 = 0.014X t 3.63) between s u c c e s s i v e d i l u t i o n s of a horse u r i n e c o n t a i r i i n g a l b u t e r o l and the coricentratioris measured by erizynie inmiurioassay. Each v a l u e KwpreseIitS t h e mati of two coriceritratioris.
c o n t a i n i n g a l b u t e r o l are shown i n f i g u r e 3 .
The i:iuiiunoreactive
signal
that
detected
standard tiine
vf
by
following 10
miri.
ELISA
corresporids
fluorimetric However,
the
to
detection,
with
chromatographic
of
albuterol
a
retention
profiles
of
e x t r a c t s of h o r s e u r i n e samples a f t e r a n IV dose of a l b u t e r o l d i s p l a y 2 p e a k s e l u t e d a t 6 . 4 arid 10.18 m i r i s d e t e c t e d b o t h by f l u o r i n i e t r i c arid inmiunological iiietliods. To c o n f i r m the n a t u r e of the peak e l u t e d a t 6.4 mixis, t h e
u r i n a r y sample has been s u b m i t t e d t o /3--glucuronidase d i g e s t i o n
217
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A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL
lB
t
A
J
E
2
0
RETENTION TIME ( min )
10
!i
v
5
0
I
6 ELUTED FRACTIONS( mL )
Typical chromatogranis (A) arid inunuriograrts (B) of a blank urine (11, ail h o r s e urine extract a f t e r inu-iunochromat o g raphy clean UP (2) arid P-glucuronidase h y d r o l y s i s ( 3 ) .
ADAM ET AL.
218 prior
the
chroniatography
step.
g lucurono-/ s u l f o- d e r i v a t i ves the
disappearance of
the
of
Indeed,
hydrolysis
of
demonstrated
by
the
a1bu t e r o 1 i s
inmiunoreactive peak
eluted
a t 6.4
mins, with a s h i f t of t h e s i g n a l t o t h e e l u t i o n p o s i t i o n of t h e
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albuterol
standard
confirming
therefore
t h e presence
of
the
conjugate d e r i v a t i v e of a l b u t e r o l both i n t h e chroniatogram arid inuiunogram of t h e s e u r i n e e x t r a c t s . The
cross-reactivity
of
the
antibody
used
with
the
m e t a b o l i t e s h a s been estimated by determining t h e cuniulative amount of drug e x c r e t e d i n u r i n e v e r s u s time by b o t h s p e c i f i c
HPLC inethod and by ELISA.
The cuniulative amount of a l b u t e r o l
e x c r e t e d estimated by ELISA is found t o be t h e double of
by HPLC procedure which allows t h e s e p a r a t i o n of
determined
unchanged a l b u t e r o l from i t s m e t a b o l i t e s . therefore
that
a
full
cross- r e a c t i v i t y
We could conclude
of t h e antibody w i t h t h e
m e t a b o l i t e s p a r t i c u l a r l y t h e glurucono-
arid s u l f o -
derivatives
which are found t h e major ones e x c r e t e d i n u r i n e .
Urinary Coriceritrations of Albuterol Q u a n t i f i e d by ELISA Table 1 r e p r e s e n t s t h e values of direct
ELISA
fluorescence between
arid after
albuterol
RIA
(12) methods
HPLC
a l b u t e r o l q u a n t i f i e d by arid
those
chromatography.
The
concentrations
measured
by
measured
by
correlation
RLAIELISA
arid
HPLWELISA methods is r e s p e c t i v e l y d e s c r i b e d by t h e following h
equation:
h
Y = 1.168X - 51 (r2 = 0.9981, Y
= 0.690X
-
2
176 ( r
=
219
A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL TABLE: 1
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URINARY LEVELS OF ALBUTEROL (pniol/mL) AFTER AN IV DOSE OF ALBUTEROL QUANTIPIED COMPARATIVELY BY IMMUNOLOGICAL AND INSTRUMENTAL METHODS.
Sample
ELISA
RIA
HPLC
1 2 3 4 5
0 5,333 1,220 792 525 458 346 225 217 187
0
6,208 1,237 862 562 4 71 358 196 187 233
0 3,562 365 370 178 150 92 44 36 36
6 7
8 9 10
These lower l e v e l s obtained by HPLC confirm t h e c r o s s -
0.989).
r e a c t i v i t y of t h e antibody used with t h e a l b u t e r o l m e t a b o l i t e s which
are
separated
from
the
parent
compound
in
the
chroniatographic c o n d i t i o n s used.
DISCUSSION In t h e r e c e n t y e a r s , incentive
for
the
particularly
iioxi
quaiitif icatiori
of
misuse of
development
of
drugs has analytical
radioisotopic these
xeriobiotics.
been
a major
methodologies,
iiimiunoassays
for
Among t h e s e drugs,
the
132
adreriergic a g e n t s have been suspected t o be widely used for
220
ADAM ET AL.
illegal
purposes
clenbuterol,
21).
(5-6,
using
a
an ELISA method
Although
rabbit:
polyclorial
antiserum
has
for been
,
proposed f o r t h e s c r e e n i n g of t h i s a d r e n e r g i c agent (22-23) inmunoassay
with
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s t i l l lacking. enzyliie
sensitivity
suf f icierit
We r e p o r t h e r e t h e set up of
inuiuiioassay
a
using
The
a v a i l a b i l i t y of
albuterol
such monoclonal
would a l l o w t h e s t a r i d a r d i z a t i o n o f t h e a s s a y of large
scale
studies.
The
specificity
inmunoassay
is
a competitive
antibody
iiionoclonal
a p p l i c a t i o n f o r t h e d e t e r m i n a t i o n of fluids.
arid
an
its
and
i n biological
(12)
antibody
this drug f u r
reported
here
is
convenient s i n c e it: does not r e q u i r e a c l e a n up e x t r a c t i o n i.n t h e procedure.
tlie p r e s e n t a s s a y i n
The h i g h s e n s i t i v i t y of
t h e fmole range is s u f f i c i e n t f o r t h e s c r e e n i n g of for
which ppb- l e v e l s
(5-6).
Moreover,
albuterol
limits were f i x e d by o f f i c i a l a g e n c i e s
the cross-reactivity
g lucu rono- / s u lfo--conjug a t es
of
of
the
antibody with
a lbu t e r o 1 r e n d e r s
this
a s say
p a r t i c u l a r l y a t t r a c t i v e f o r t h e d e t e c t i o n of t h i s drug arid i t s n i u t a b o l i t e s found a t h i g h l e v e l s
iti
urine.
The a p p l i c a t i o n of
t h e p r e s e n t enzyme iiiuiunoassay i s n o t l i m i t e d f o r t h e s c r e e n i n g of the
t h i s drug i n v e t e r i n a r y medicine b u t would be s u i t a b l e f o r clinical
monitoring
of
this
82
adreriergic
agonist
in
t h e r a p e u t i c a d j u s tnients i n human medicine.
ACKNOWLEDGEMENTS We wish t o thank Dean J . Gagn6, l e "Fonds d e d6veloppement de
l ' U n i v e r s i t 6 de Montr8al" f o r s u p p o r t i n g t h i s work.
A COMPETITIVE ENZYME IMMUNOASSAY FOR ALBUTEROL Reprints
requests
F a c u l t y of
s h o u l d be
Pharmacy,
addressed
University
of
to:
221 Dr.
Eloritreal,
Albert
P.O.
Adam,
Box
6128,
and
the
S t a t i o n A , Montreal (Quebec) H3C 357 CANADA. REFERENCES
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