United States Patent: Mcconnell Et Al. (10) Patent N0.: (45) Date of Patent

US007192722B2
(12) United States Patent
(10) Patent N0.:

(45) Date of Patent:
McConnell et al.
(54)
PHENETHANOLAMINE-DERIVED
(56)
U.S. PATENT DOCUMENTS
AND CONJUGATES
6,274,334 B1
(75) Inventors: Robert Ivan McConnell, Ballymena
(Continued)
(73) Assignee: RandoX Laboratories, Crumlin (GB)
Primary ExamineriLong V. Le
Subject to any disclaimer, the term of this

patent is extended or adjusted under 35
Assistant ExamineriSha?qul Haq

(74) Attorney, Agent, or FirmiWoodcock Washburn LLP
U.S.C. 154(1)) by 49 days.
(57)
(21) Appl. No.: 11/271,282

(22)
(30)
(52)
reacting a phenylethanolamine derivative of formula D With

a phenylalkylcarbonyl derivative of formula E:
Oct. 5, 2006
Foreign Application Priority Data
Nov. 10, 2004
(51)
petitive immunoassays for the detection of ractopamine,

isoxsuprine and ritodrine. The haptens are prepared by
Prior Publication Data
US 2006/0223132 A1
ABSTRACT
The present invention relates to a method for preparing

haptens of formulae 1 and 11 that are useful in the preparation
of immunogens, antibodies and conjugates, for use in com
Nov. 10, 2005
(65)
Shelver et a1. ........... .. 435/792
Elliot, C.T. et al., Screening and Con?rmatory Determination of

Ractopamine Residues in Calves Treated with Growth Promoting
Doses of the fi-Agonist, Analyst, May 1998, 123, 1103-1107.
Antrim (GB); Andrew Philip LoWry,

Belfast (GB)
Filed:
8/2001
OTHER PUBLICATIONS
(GB); Stephen Peter Fitzgerald,

Crumlin (GB); El Ouard Benchikh,
Notice:
Mar. 20, 2007
References Cited
HAPTENS, IMMUNOGENS, ANTIBODIES
(*)
US 7,192,722 B2
(EP)
Int. Cl.
G01N 33/53
G01N 33/532
G01N 33/533
C0 7K 1/10
C0 7K 1/13
C0 7K 1/04
C0 7K 16/00
................................ .. 04078100
Z 1/
(2006.01)
(2006.01)
(2006.01)
(2006.01)
(2006.01)
(2006.01)
(2006.01)
US. Cl. ................... ..
435/7.1; 435/7.92; 435/793;
435/975; 436/544; 436/546; 436/56; 530/402;

530/403; 530/388.9; 530/389.8
(58)
Field of Classi?cation Search ............... ..
in Which, in formula 1, Z1 is a crosslinker and Z2 is H and,

in formula 11, Z1 is H and Z2 is a crosslinker.
435/7.1,
435/7.92, 7.93, 975; 436/544, 546, 56; 530/402,

530/403, 388.9, 389.8
See application ?le for complete search history.
27 Claims, 7 Drawing Sheets
Chemical structures nl ?clognmine Ritodrige gig Qmuugrine
OH
1
N
w
HO
Rmogamine
Ritorlrine
on
US 7,192,722 B2
Page 2
Smith, D]. et al., issue Residues of Ractopamine and Urinary
OTHER PUBLICATIONS
Excretion of Ractopamine and Metabolites in Animals Treated for
Haasnoot, W. et al., Determination of Fenoterol and Ractopamine
in Urine by Enzyme Immunoassay, Analyst, 1994, 119, 2675-2680.

Lommen, A. et al., Nuclear Magnetic Resonance Controlled
Method for Coupling of Fenoterol to a Carrier and Enzyme, Food
& Agricultural Immunology, 1995, 7, 123-129.

Shelver, W.L. et al., Production and Characterization of
Monoclonal Antibody
against
the
fi-Adrenergic
Agonist
Ractopamine, J Agric. Food Chem, 2000, 48, 4020-4026.

Shelver, W.L. et al., Development of an Immunoassay for the
fi-Adrenergic Agonist Ractopamine, Journal of Immunoassay,

2000, 21(1), 1-23.
Shelver, W.L. et al., Determination of Ractopamine in cattle and
Sheep Urine Samples Using an Optical Biosensor Analysis: Com

parative Study With HPLC and ELISA, J Agric. Food Chem, 2003,
51, 3715-3721.
Siegel, M.G. et al., The Use of High-Throughput Synthesis and
Puri?cation in the Preparation of a Directed Library of Adrenergic
Agents, Molecular Diversity, 1998, 3, 113-116.
7 Days With Dietary Ractopamine, J Anim. Sci., 2002, 80, 1240

1249.
Haasnoot, W. et al., Immuno?ltration as Sample Cleanup for the
Immunochemical Detection of [5-Agonists in Urine, Analyst, The
Royal Society of Chemistry, 2002, 127, 87-92.

Madding, G.D., Synthesis of Tritium Labeled IsoXsuprine Hydro
chloride, Journal of Labeled Compounds, 1971, 7(4), 393-397.
Ren, J .Z. et al., Synthesis of Ritodrine, Zhongguo I?yao Gongye
Zazhi, 2000, 31(6), 241-242.
Shelver, W.L. et al., Application of a Monoclonal Antibody-Based
Enzyme-Linked Immunosorbent Assay for the Determination of
Ractopamine in Incurred Samples from Food Animals, J Aric.
Food Chem., 2002, 50, 2742-2747.
Terando, N.H., Synthesis of 14C-Radiolabeled Ractopamine

Hydrochloride, Journal of Labelled Compounds and
Radiopharmaceuticals, 1992, 31(9), 651-658.
U.S. Patent
Mar. 20, 2007
Sheet 1 0f 7
US 7,192,722 B2
Chemical structures of RactojlamineLRitodrine and Isoxsuprine
OH
10
H
N
10'
OH
HO
Ractogamine
OH
H
N
HO
VK3OH
Ritodrine
OH
HO
Isoxsugrine
Figure - 1
U.S. Patent
Mar. 20, 2007
Sheet 2 0f 7
US 7,192,722 B2
Chemical Structure of Haptens A, B, C, D, and E

COZH
OH
HO/UK/ W
H
N
COZH
Hagten A
OH
H
N
0
HO
Hagten B
CO2H
OH
ZI
OH
HaQten C
OH
HO
Hagten D
OH
COZH
ZI
HO
Hagten E
Figure - 2
U.S. Patent
Mar. 20, 2007
Sheet 3 0f 7
US 7,192,722 B2
l)NaH/DMF
>
HO
2) Br M 0025:
IN
ID)
002151
OH
NH2
0
OH
HO
H
0025:
Methanol, TEA, NaBHJCN

HO
_5.
NaOH (2N)
Methanol
0
OH
COZH
HO
Hagten A
Conjugation
O
OH
CONHBSA
HO
Immunogen A
Figure - 3
U.S. Patent
Mar. 20, 2007
Sheet 4 0f 7
US 7,192,722 B2
Chemical reactions for the re aration 0f Ha ten B and Immuno en B
O
O
l) NaH IDMF
___>
MeO2C
Br
2
'
COzMe
0H
NH;
0
OH
HO
Methanol, TEA, NaBHJCN

HO
COZMe
NaOH (2N)
Methanol
HO
/@i/
COZH
CONHBSA
Hagten B
Conjugation
OH
Ho/Uk/
Immunogen B
Figure - 4
U.S. Patent
Mar. 20, 2007
Sheet 5 0f 7
US 7,192,722 B2
Chemical reactions for the greparation of Hagten C and lmmunogen C

OH
OH
NHBOC
NH;
(BOC)2O- c?zclz
"_
TEA
HO
HO
Br/\/\CO;El
OH
OH
NH;
NHBOC
CF3CO2H / CHZCIZ
4
11
.2.
00251
NaBHJCN
co2E:
MeOH, TEA
OH
OH
NaOH (2N) I Methanol
-->
2
OH
COZER
()H
Conjugation
O
OH
Hagten C
CO2H
Immunogen C
CONHBSA
Figure - 5
OH
U.S. Patent
Mar. 20, 2007
Sheet 6 0f 7
US 7,192,722 B2
Chemical reactions for the re aration 0f Ha ten D and Immune en D
OH
NH2
O\)l\ MeOH
+
NaCNBH3
>
TEA
HO
13
H
OH
H
N
\r\O/ tert-butyl bromoacetate
-----_>
HO
K2CO3 / Acetonitile
E
OH
H
CO2tBu
\I/\O
CF3COZH
_>
CH2Cl2
H
OH
H
COQH
|\
W/\O /< >
.__>
m
OH
H
N
BSAHN
v0
0
W212
Figure - 6
O/
U.S. Patent
Mar. 20, 2007
Sheet 7 0f 7
US 7,192,722 B2
Chemical reactions for the gregaration of Hagten E and Immunogen E
OH
OH
ter-butyl bromoacetate
Ho
K2CO3 /CH3CN
17
E
COZtBU
OH
NH2
PCC / CH2Cl2
CH0 +
0
HO
12
COZtBU
OH
COZtBu
MeOH/ NaCNBH3
>
TEA
HO
Q
OH
HCl (4N) / Dioxane
CO2H
_->
0
HO
Hagten E
OH
H
N
CONHBSA
___>HO
Immunogen E
Fi
re-7
US 7,192,722 B2
1
PHENETHANOLAMINE-DERIVED
samples Were enzymatically hydrolysed prior to analysis,
HAPTENS, IMMUNOGENS, ANTIBODIES
correlated Well With LC-MS-MS but, as With Haasnoot et al,

the antibodies Were raised in a non-targeted fashion, against
AND CONJUGATES
a fentoterol derivative.
FIELD OF THE INVENTION
Shelver and Smith (Journal of Immunoassay (2000),

21(1), Development of an immunoassay for the [3-adren
The present invention relates to a method for preparing
ergic agonist ractopamine, 1423) disclose preparation of

polyclonal antibodies generated from ractopamine-hemiglu
haptens that are useful for the preparation of immunogens,

antibodies and conjugates, for use in competitive immunoas
tarate-KLH. The hapten is prepared by reaction of racto
says for the detection of phenethanolamines such as, but not
pamine With glutaric anhydride, folloWed by conjugation
limited to, ractopamine, isoxsuprine and ritodrine. The
With an antigenicity-conferring carrier material. The method
present invention also relates to a method and kit for
employed to synthesize ractopamine-hemiglutarate is
detecting or determining phenethanolamines such as, but not
uncontrolled and Will result in derivatisation of ractopamine

at any one of positions 1, 10, 10' and N. The Shelver and
limited to, ractopamine, isoxsuprine and ritodrine.
Smith antibody generated shoWs a sensitivity (ICSO) of 4.2

ng/ml toWard ractopamine. The antibody also exhibits 33%
cross-reactivity With dobutamine.
Shelver et al (J. Agric. Food Chem. 48 (2000), Produc
BACKGROUND OF THE RELATED ART
By detecting is meant qualitatively analysing for the

presence or absence of a substance. By determining is
meant quantitatively analysing for the amount of a sub
20
tion and characterisation of a monoclonal antibody against
the [3-adrenergic agonist ractopamine, 40204026 and their
stance.
US. Pat. No. 6,274,334) disclose the generation ofa mono
Ractopamine is a phenethanolamine leanness-enhancing

agent recently approved as a feed additive for sWine in the
clonal antibody, in Which ractopamine-glutarate-keyhole
United States. Hogs administered dietary ractopamine shoW
limpet haemocyanin Was used as the antigen for antibody

generation. The antibody clone selected (5G10) shoWs 5.3%
cross-reactivity With dobutamine and 3.6% cross-reactivity
25
increased groWth rates and feed ef?ciencies and decreased

fat deposition, relative to untreated animals.
Phenethanolamine [3-agonists have a history of being used

for off-label purposes by livestock producers hoping to
improve the economics of livestock production. Improper
With ritodrine.
A polyclonal antibody is generated by repeated immuni

30
use of [3-agonists can cause a serious risk to human health
due to the residues they leave in the meat and other food
stuffs of animal origin. Indeed, in Europe, all [3-agonists are
banned for use in livestock and for improving athletic
performance under EU Directive 96/22/EC.
The presence of drug residues in animal tissues is a
concern for food safety, especially When the compound has
been used illegally or in a manner proscribed by regulatory
of?cials (off-label use). In an effort to combat such illicit use
of [3-agonist compounds, regulatory organisations around
sation of a mammalian host With the target immunogen. The

resulting antiserum is harvested and the antibodies isolated.
To generate a monoclonal antibody, lymphocytes from an
immunized animal are fused With a myeloma cell line to
35
40
produce hybrid cells or hybridomas. These hybridomas can

be cloned for production of the secreted monoclonal anti
body. A monoclonal antibody Will recognize a single
epitope, Whereas a polyclonal antiserum may recognize
several epitbpes on the same antigen. Hence, monoclonal
antibody technology can usually be applied to generate an
antibody With greater speci?city for the target immunogen.
the World test animal tissues and body ?uids for the presence
In the aforementioned Shelver et al, a scattergun
of such illicit drugs.

Speci?c binding actions, such as antibody-antigen inter
actions, have been used extensively in immunoassays to
detect a variety of substances present in biological ?uids.
Thus, for example, radioimmunoassays could be used for the
determination of phenethanolamines such as ractopamine,
isoxsuprine and ritodrine. Radioimmunoassays are very
sensitive but do require radionucleotide tracers. Enzyme
approach has been used to generate polyclonal antibodies to

ractopamine. The uncontrolled method used to prepare the
45
50
linked immunosorbent assays (ELISAs) are a non-radioac
tive alternative Which could be used for the qualitative and
quantitative determination of phenethanolamines such as,

but not limited to, ractopamine, isoxsuprine and ritodrine.
Haasnoot et al (The Analyst (1994), 119, Determination
of fenoterol and ractopamine in urine by enzyme immu
noassay, 267542680) disclose a urinary enzyme immu
noassay for ractopamine. The Haasnoot antibody Was
derived from a fenoterol derivative and shoWed 20% cross
reactivity With ractopamine, but the analysis of ractopamine
ractopamine hapten suggests that the resulting immunogen

is composed of a mixture of ractopamine linked to keyhole
limpet haemocyanin (KLH) at any one of positions 1, 10, 10'
and N. Immunisation With such an immunogen Will result in
the generation of antisera With a diversity of speci?city. This
is borne out in the cross-reactivity data presented in the
Shelver reference. Their polyclonal antibody cross-reacts
33% With dobutamine, While their monoclonal antibody
cross-reacts 5.3% With dobutamine. In contrast, the targeted
introduction of a crosslinker group onto a single phenolic
55
hydroxyl group of the target hapten, according to the present

invention, produces a surprising effect (a heterofunctional
crosslinker as de?ned herein, is a structure incorporating one
or more functional groups containing one or more hetero
60
atoms, that links, for example through covalent bonding,

With a substrate (in this case a hapten), the crosslinker also
being able to link to a peptide, polypeptide or protein or a
did not correlate Well With GC-MS analysis. Elliott et al
(The Analyst (1998), 123, Screening and con?rmatory
detectable labelling agent). Antibodies generated to the
determination of ractopamine residues in calves treated With
present haptens are highly speci?c and do not cross-react

signi?cantly With dobutamine. In the case of ractopamine,
the antibody generated to the position 10 derivative cross
reacts 0.186% With dobutamine, While the antibody gener
groWth promoting doses of the [3-agonist, 110341107)

disclose an immunoassay for ractopamine residues. The
Elliott antibody, When used in an immunoassay Where the
65
US 7,192,722 B2
3
ated to the position 10' derivative exhibits cross-reactivity of
<0.8% With dobutamine. The only explanation for the cross
reactivity of the Shelver antibodies With dobutamine is that
Shelver et al have generated antibodies to ractopamine
derivatised at the position 1 hydroxyl (hydroxyl group on the
aliphatic chain). This hydroxyl group is absent in dob
utamine. The presence of a single hydroxyl group on one of
the aromatic rings of the hapten derivatives used in the

present application results in a high degree of speci?city for
ractopamine, isoxsuprine and ritodrine. The antibodies of
the present application are also considerably more sensitive
than the Shelver polyclonal and monoclonal antibodies. For
ractopamine, the antibody to the position 10 derivative has
an lC5O of 0.082 ng/ml, While the antibody to the position 10'
derivative has an lC5O of 0.202 ng/ml. The present antibod
The phenethanolamine derivative of formula D may be
prepared, for example, by reacting octopamine hydrochlo
ies exhibit improved speci?city and sensitivity. The present
ride With ZlXl, in Which X1 is a halide. The amine group of
application illustrates hoW a superior antibody can be gen
erated using polyclonal antibody technology and the process

of targeted derivatisation.
20
None of the prior art knoWn to the inventors either
octapamine hydrochloride With the ZlXl, the BOC group
discloses or suggests preparing phenethanolamines haptens

such as, but not limited to, ractopamine, isoxsuprine or
the octapamine hydrochloride may be temporarily protected

With BOC, by reacting the octapamine hydrochloride With
di-tert-butyldicarbonate before reacting the BOC-protected
25
being subsequently removed.

The invention also provides a method of preparing a
ritodrine haptens by the method disclosed herein. The
hapten of formula II:
present method alloWs for the controlled coupling of a
crosslinking group to a single phenolic hydroxyl group of
the hapten. The present invention, by alloWing the prepara
30
Hapten H
tion of haptens and, therefore, immunogens derivatised at a
single phenolic hydroxyl group, facilitates preparation of

antibodies to immunogens derivatised at a single phenolic
hydroxyl group. None of the prior art discloses or suggests
targeted derivatisation of phenethanolamines such as, but
not limited to, ractopamine, isoxsuprine and ritodrine ana
21/
OH
40
SUMMARY OF THE INVENTION
45
Hapten I
Z 1/
Z 1/
2 n
Which is suitable for use in raising an antibody capable of

binding With at least one structural epitope of a phenetha
nolamine. The method comprises the step of reacting a
phenethanolamine derivative of formula D, in Which Z 1 is H,
With a phenylalkylcarbonyl derivative of formula E, in
Which Z2 is a crosslinker; Y is independently H or CH3; W
is independently H or CH3; n is independently l or 2; and m
is independently 0 or 1.
50
N)\(CH/) (0),
OH
35
logues at a single phenolic hydroxy group. Such haptens also

form part of the present invention.
The invention provides a method of preparing a hapten of

formula I:
/ Z2
(0),
55
Which is suitable for use in raising an antibody capable of

binding With at least one structural epitope of a phenetha
nolamine. The method comprises the step of reacting a
phenethanolamine derivative of formula D, in Which Z1 is a
crosslinker, With a phenylalkylcarbonyl derivative of for
mula E, in Which Z2 is H; Y is independently H or CH3; W
is independently H or CH3; n is independently l or 2; and m
is independently 0 or 1.
60
65
The phenylalkylcarbonyl derivative of the formula E may
be prepared, for example, by reacting 4-(4-hydroxyphenyl)

2-butanone With Z2Xl, in Which X1 is a halide.
US 7,192,722 B2
6
5
The invention further provides a hapten derivatised With
a crosslinker at Z1, in Which the hapten has the structural
The invention still further provides a method for detecting

or determining a phenethanolamine such as, but not limited
to, ractopamine, isoxsuprine or ritodrine in a sample, the

method comprising contacting the sample With at least one
conjugate of the hapten of formula II conjugated to a
detectable labelling agent, and With at least one antibody
formula I:
Z 1/
raised against the immunogen comprising the hapten of
T (CH2)"
/<o>m
g
structural formula I coupled to an antigenicity-conferring
carrier material; detecting or determining bound conjugate;

and deducing from the calibration curve the presence of, or
on
(0),
the amount of, the phenethanolamine in the sample.

in Which Z1 is a crosslinker and Z2 is H; Y is independently

H or CH3; W is independently H or CH3; n is independently

conjugate of the hapten of formula II conjugated to a
detectable labelling agent, and With at least one antibody
l or 2; and m is independently 0 or 1.
The invention still further provides a hapten derivatised

With a crosslinker at Z2, in Which the hapten has the
structural formula II:
raised against the immunogen comprising the hapten of

structural formula II coupled to an antigenicity-conferring
20
carrier material; detecting or determining bound conjugate;

and deducing from the calibration curve the presence of, or
Z 1/
the amount of, the phenethanolamine in the sample.
The invention also provides a kit for detecting or deter

mining a phenethanolamine such as, but not limited to,
I (CH2)"
/W
E
on
/ Z2
25
antibody raised against a hapten of structural formula I

coupled to an antigenicity-conferring carrier material.
in Which Z1 is H and Z2 is a crosslinker; Y is independently

30
The invention also provides an immunogen comprising a

hapten of structural formula I, coupled to an antigenicity
conferring carrier material. The invention further provides
an antibody raised against the immunogen Wherein the
antibody is capable of binding at least one structural epitope
of a phenethanolamine. The invention also provides a hapten
conjugated to a detectable labelling agent.
The invention also provides an immunogen comprising a
hapten of structural formula II, coupled to an antigenicity
conferring carrier material. The invention further provides
an antibody raised against the immunogen Wherein the
antibody is capable of binding at least one structural epitope
of a phenethanolamine. The invention also provides a hapten
conjugated to a detectable labelling agent.

ractopamine, isoxsuprine or ritodrine, the kit including at

least one conjugate of the hapten of structural formula I
conjugated to a detectable labelling agent; and at least one
35
antibody raised against a hapten of structural formula II


40
least one conjugate of the hapten of structural formula II

antibody raised against a hapten of structural formula I

45


conjugate of the hapten of formula I conjugated to a detect
able labelling agent, and With at least one antibody raised

least one conjugate of the hapten of structural formula I
(0),
least one conjugate of the hapten of structural formula II

50
against the immunogen comprising the hapten of structural
antibody raised against a hapten of structural formula II

BRIEF DESCRIPTION OF DRAWINGS
formula I coupled to an antigenicity-conferring carrier mate
rial; detecting or determining bound conjugate; and deduc

ing from the calibration curve the presence of, or the amount
55
of, the phenethanolamine in the sample.
FIG. 2 shoWs the structures of Haptens A, B, C, D and E.

FIG. 3 shoWs the reaction scheme for the preparation of


conjugate of the hapten of formula I conjugated to a detect
able labelling agent, and With at least one antibody raised
Hapten/Immunogen A.
60

formula II coupled to an antigenicity-conferring carrier

deducing from the calibration curve the presence of, or the
amount of, the phenethanolamine in the sample.
Hapten/Immunogen B.
Hapten/Immunogen C.
against the immunogen comprising the hapten of structural

material; detecting or determining bound conjugate; and
FIG. 1 shoWs the structures of ractopamine, ritodrine and

rsoxsuprrne.
65
Hapten/Immunogen D.
Hapten/Immunogen E.
US 7,192,722 B2
7
DETAILED DESCRIPTION OF THE
ILLUSTRATIVE EMBODIMENTS
The present invention describes biaromatic haptens deri

vatised With crosslinkers at a single phenolic hydroxyl
group. The structures of ractopamine, isoxsuprine and rito
drine are illustrated in FIG. 1.
The invention also provides a hapten derivatised With a
crosslinker at Z1, in Which the hapten has the structural

formula I
Z 1/
I (CH2)"
/W
Preferably, the phenethanolamine derivative of the for
mula D is prepared by reacting octopamine hydrochloride

With ZlXl, in Which X1 is a halide, preferably a bromide.
OH
20
More preferably, the amine group of the octapamine hydro
chloride is temporarily protected With BOC, by reacting the
octapamine hydrochloride With di-tert-butyldicarbonate

before reacting the BOC-protected octapamine hydrochlo
in Which Z1 is a crosslinker and Z2 is H; Y is independently

l or 2; and m is independently 0 or 1. Additionally, the
invention provides a hapten derivatised With a crosslinker at
ride With the ZlXl, the BOC group being subsequently

25
Z2, in Which the hapten has the structural formula II
removed.
In a further aspect, the invention provides a method of
preparing a hapten of formula II
Hapten II
30
Z 1/
21/
k / (0),
(CH2)"
OH
35
OH
in Which Z1 is H and Z2 is a crosslinker; Y is independently

suitable for use in raising an antibody capable of binding

40
More preferably, the haptens have structural formulae A,
ritodrine, the method comprising the step of reacting a
B, C, D or E as illustrated in FIG. 2.
In a further aspect, the invention provides a method of

45
preparing a hapten of formula I
With at least one structural epitope of a phenethanolamine

phenethanolamine derivative of the formula D, in Which Z 1

is H, With a phenylalkylcarbonyl derivative of the formula E,
in Which Z2 is a crosslinker; Y is independently H or CH3;
W is independently H or CH3; n is independently l or 2; and
m is independently 0 or 1
Hapten I
50
Z 1/
Z 1/
NAM/for"
2 n
OH
Z2
(0);
NH2
55
(0),
suitable for use in raising an antibody capable of binding

With at least one structural epitope of a phenethanolamine
Z2
60
ritodrine, the method comprising the step of reacting a
phenethanolamine derivative of the formula D, in Which Z 1

is a crosslinker, With a phenylalkylcarbonyl derivative of the
formula E, in Which Z2 is H; Y is independently H or CH3;

W is independently H or CH3; n is independently l or 2; and
m is independently 0 or 1
Preferably, the phenylalkylcarbonyl derivative of the for

65
mula E is prepared by reacting 4-(4-hydroxyphenyl)-2

butanone With Z2Xl, in Which X1 is a halide, preferably a
bromide.
US 7,192,722 B2
10
Preferably, the crosslinker is iRiXZ. Preferably, R is a
bivalent link and X2 is a heterofunctional group. By het
pared to 100% for isoxsuprine, of less than 0.75%, prefer

ably less than 0.6%, more preferably less than 0.25%, for
erofunctional is meant a functional group containing at

least one hetero-atom that is able to link the R of the
each of ritodrine and ractopamine. Separately or addition
crosslinker With either antigenicity-conferring carrier mate

rials, to form immunogens or, alternatively, detectable label
When compared to 100% for ritodrine, of less than 0.75%,
ally, the antibodies raised to ritodrine shoW a cross reactivity,
preferably less than 0.6%, for ractopamine.
ling agents, to form conjugates. More preferably, the X2
In a still further aspect, the present invention comprises
heterofunctional group is capable of reacting With either

antigenicity-conferring carrier materials, to form immuno
gens or, alternatively, detectable labelling agents, to form
conjugates comprising the haptens prepared in accordance

With the method of the present invention covalently bonded
to a detectable labelling agent. Preferably, the labelling
conjugates.
agent is selected from an enZyme, a luminescent substance,

a radioactive substance, or a mixture thereof. More prefer
Most preferably, R is selected from the group comprising

a substituted or unsubstituted, straight or branched chain,
ably, the labelling agent is an enZyme, preferably a peroxi
saturated or unsaturated alkylene group and a substituted or
dase, most preferably horseradish peroxidase (HRP). Alter
unsubstituted arylene group such as a benZylene group and
natively, or additionally, the luminescent substance may be
X2 is selected from the group comprising a carboxylic acid
a bioluminescent, chemiluminescent or ?uorescent material.
or an ester thereof; an amine; a maleimide, a halocarboxylic

acid or an ester thereof, an aldehyde, a dithiopyridyl group,
a vinylsulphone group, and a thiocarboxylic acid or an ester
antibodies, the process comprising the steps of immunising
thereof. More preferably, R is a C1_5, most preferably a C1_3,
The invention further provides a process of preparing the

an animal, preferably a vertebrate animal, most preferably a
20
mammalian animal, by repeated administration of an immu

nogen prepared in accordance With the present invention,
and collecting the resulting serum from the immunised
animal. Preferably, the process further comprises ?xing said
25
support, most preferably a polystyrene solid support. Pref

erably, the antibodies are polyclonal. Alternatively, the anti
substituted or unsubstituted, straight chain, saturated alky

lene group. Advantageously, X2 is selected from a carboxy
lic acid or an ester thereof, a thiocarboxylic acid or an ester
thereof, a dithiopyridyl, a maleimide, or an aldehyde group.

Most advantageously, X2 is a carboxyl group (COOH) or a
serum antibodies to a backing substrate, preferably a solid
thioacetyl (SCOCH3) group.

The resulting haptens are deliberately derivatised at a
bodies are monoclonal.
single phenolic hydroxyl group of phenethanolamines such
In a still further aspect, the present invention comprises a
as, but not limited to, ractopamine, isoxsuprine and rito

drine.
The resulting haptens are employed in the preparation of
immunogens by coupling them to modi?ed or non-modi?ed
antigenicity-conferring carrier materials to provide immu
method for detecting or determining phenethanolamines

such as, but not limited to, ractopamine, isoxsuprine and
ritodrine in a sample, the method comprising contacting the
sample With at least one conjugate of the present invention,
30
and With at least one antibody of the present invention;
detecting or determining bound conjugate; and deducing
nogens for antibody production and conjugates (tracers) that

have excellent sensitivity and speci?city for the detection or
determination of phenethanolamines such as, but not limited
35
to, ractopamine, isoxsuprine and ritodrine.

The invention therefore provides an immunogen compris
ing a hapten prepared in accordance With the present inven
tion, coupled to an antigenicity-conferring carrier material.
Preferably, the carrier material is a protein, a protein frag
ment, a synthetic polypeptide or a semi-synthetic polypep
tide.
In a further aspect, the invention includes a kit for
40
The immunogens obtained are then administered to mam
malian hosts to elicit production of speci?c antibodies,

preferably polyclonal antibodies, Which are then used to
from a calibration curve the presence of, or the amount of,
the phenethanolamine in the sample.
45
detecting or determining phenethanolamines such as, but not

limited to, ractopamine, isoxsuprine or ritodrine, the kit
including at least one conjugate prepared in accordance With
the present invention; and at least one antibody prepared in
accordance With the present invention. The kit may option
ally include instructions for the use of said conjugates and
said antibodies for detecting or determining phenethanola
mines such as, but not limited to, ractopamine, isoxsuprine
or ritodrine in a sample.
develop competitive immunoassays for phenethanolamines
The methods and kits using antibodies and conjugates
such as, but not limited to, ractopamine, isoxsuprine and
raised to ractopamine of the present invention shoW an IC5O

of less than 2.5, preferably less than 1.5, more preferably
ritodrine, employing haptens conjugated to labelling agents

as conjugates or detection reagents.
In a still further aspect, the present invention concerns
50
antibodies raised against the immunogens of the present

invention, the antibodies being capable of binding With at
less than 0.75, most preferably less than 0.25, ng/ml for
ractopamine. The methods and kits, using antibodies and
Preferably, the antibodies are ?xed on a backing substrate.

More preferably, the antibodies shoW a cross reactivity,
conjugates raised to isoxsuprine, shoW an IC5O of less than

2.5, preferably less than 1.5, more preferably less than 0.75,
more preferably less than 0.25, ng/ml for isoxsuprine. The
methods and kits, using antibodies and conjugates raised to
ritodrine, shoW an IC5O of less than 250, preferably less than
150, ng/ml for ritodrine
When compared to 100% for ractopamine, isoxsuprine and

ritodrine, of less than 5% for dobutamine. More preferably,
backing substrate, the backing substrate preferably being a
least one structural epitope of phenethanolamines such as,

the antibodies shoW a cross reactivity, When compared to
55
Preferably, the antibodies are provided, ?xed onto a

60
solid support Which is, for example, a polystyrene solid
100% for ractopamine and isoxsuprine of less than 2.5%,

more preferably less than 1.5%, for dobutamine. Separately
alternatively, a Biochip (Trade Mark) such as is disclosed in
or additionally, the antibodies raised to ractopamine shoW a
US. Pat. No. 6,498,010. Phenethanolamines such as, but not
cross reactivity, When compared to 100% for ractopamine,

of less than 0.75%, preferably less than 0.6%, for ritodrine
and isoxsuprine. Separately or additionally, the antibodies
raised to isoxsuprine shoW a cross reactivity, When com
support. This can take the form of a microtitre plate or,
65
limited to, ractopamine, isoxsuprine or ritodrine, if present

in the standard and sample, compete With conjugates com
prising haptens covalently bonded to a detectable labelling
agent such as, but not restricted to, horseradish peroxidase,
US 7,192,722 B2
11
12
for the limited number of antibody sites on the backing
chloride, 4 in methanol in the presence of triethylamine

(TEA) and sodium cyanoborohydride gives the ester 8. The
hapten B Was obtained after saponi?cation of the ester 8 With
substrate. After incubation at room temperature to alloW a
competition reaction to take place, the backing substrate is
sodium hydroxide (2N) in methanol.

The hapten 10'-O-(3-carboxypropyl)ractopamine ether
(Hapten C4derivatised at position 10') Was prepared in six
steps, according to FIG. 5 of the accompanying draWings,
from octopamine hydrochloride 4. The N-BOC protected
then Washed to remove excess reagents. If the labelling
agent is horseradish peroxidase, its substrate is then added

and an incubation period folloWs to alloW maximum signal
development. In an ELISA format, the colour development
is then stopped by the addition of acid and this produces a
colour change from blue to yelloW. The absorbance is read
octopamine, 9 Was obtained by reaction of 4 With di-tert

butyldicarbonate (BOCZO) in methanol in the presence of
TEA. The reaction of N-BOC octopamine, 9 With ethyl-4
bromobutyrate in acetonitrile in the presence of potassium
carbonate (K2CO3) gives the ester 10 in good yield. The
at 450 nm. A standard curve is then constructed to determine
the concentration of the phenethanolamine such as, but not

limited to, ractopamine, isoxsuprine or ritodrine in the
sample and the standard. Alternatively, in the Biochip for

mat, the signal generated is typically chemiluminescent and
the light level generated is quanti?ed by a charge coupled
treatment of 10 With tri?uoroacetic acid in dichloromethane
gives ethyl 4-[4-(2-amino-1-hydroxyethyl)phenoxy]bu
device (CCD) camera to determine the concentration of the
tanoate, 11. The condensation of octopamine derivative 11

With 4-(4-hydroxyphenyl)-2-butanone 2 in methanol in the
presence of TEA and sodium cyanoborohydride
phenethanolamines such as, but not limited to, ractopamine,

isoxsuprine and ritodrine in the sample or standard.
Preferably, the sample is a solution, such as a biological
?uid. More preferably, the sample is serum or urine.
(NaBH3CN) gives 10'-O-[3-(ethoxycarbonyl)propyl]racto

20
Urine samples may be analysed folloWing a dilution

method. HoWever, if the urine appears dirty or a loWer
detection limit is required, an appropriate extraction method

should be used. Simple dilution involves centrifuging the
urine sample at, for example, 13000 rpm for 10 minutes and
then diluting the centrifuged urine ten fold in diluted diluent/
Wash bulfer, folloWing Which the sample can be applied to
The hapten p-(carboxymethyl)isoxsuprine (Hapten D)

Was prepared in four steps according to FIG. 6 of the
25
accompanying draWings, from ot-(l-aminoethyl)-4-hy

droxybenZyl alcohol hydrochloride 13. The isoxsuprine 15
Was obtained by reaction of 13 With phenoxy-1-propanone
14 in methanol in the presence of sodium cyanoborohydride
and triethyl amine. The ester 16 Was obtained by selective
the backing substrate.

Intra-assay precision of the ELISA kit Was determined
employing negative urine samples spiked With 1 ng/ml and
pamine ether 12. The hapten 10'-O-(3-carboxypropyl)racto

pamine ether, Hapten C, Was obtained after saponi?cation of
the ester 12 by using sodium hydroxide (2N) in methanol.
30
alkylation of isoxsuprine 15 With t-butyl bromoacetate in
5 ng/ml. The results Were 5.95 and 6.62% CV, respectively.

Inter-assay precision of the ELISA kit for the same spiked
acetonitrile in the presence of potassium carbonate. The

treatment of the ester 16 With tri?uoroacetic acid in dichlo
urine samples Was also determined and the results Were
romethane gave the p-(carboxymethyl)isoxsuprine, Hapten
11.83 and 5.90% CV, respectively.

In the method and kit of the present invention, it is
D.
35
The hapten p-(carboxymethyl)ritodrine (Hapten E) Was
preferred that the respective crosslinkers (of the immunogen
prepared in four steps according to FIG. 7 of the accompa
and the conjugate) are different.

In a further aspect, the present invention involves the use
of the conjugates of the present invention, or a mixture
thereof, With the antibodies of the present invention, or a
mixture thereof, to detect or determine phenethanolamines
ritodrine in samples such as tissues or biological ?uids.
40
reaction of 17 With t-butyl bromoacetate in acetonitrile in the

presence of potassium carbonate at 600 C. gives 2-[4-(t
butoxycarbonylmethoxy)phenyl]ethanol 18. The oxidation
of the alcohol 18 With pyridinium chlorochromate (pcc) in
Preparation of Haptens
The hapten [10-O-(carboxypropyl)]ractopamine ether
45
(Hapten Aiderivatised at position 10) Was prepared in three

steps according to FIG. 3 of the accompanying draWings,
from 4-(4-hydroxyphenyl)-2-butanone, 2. The reaction of
4-(4-hydroxyphenyl)-2-butanone, 2 With ethyl 4-bromobu
tyrate in dimethylformamide (DMF) in the presence of
nying draWings, from 2-(4-hydroxyphenyl)ethanol 17. The
dichloromethane gives the 2-[4-(t-butoxycarbonylmethoxy)

phenyl]ethanal 19. The ester 20, obtained by condensation
of the aldehyde 19 With ot-(l-aminoethyl)-4-hydroxybenZyl
alcohol hydrochloride 13 in methanol in the presence of
sodium cyanoborohydride and triethylamine. Deprotection

of the t-butyl protecting group of the ester 20 With 4 M HCl
in dioxane at room temperature gave Hapten E in moderate
yield.
50
sodium hydride (NaH), gives ethyl 4-[4-(3-oxobutyl)phe
Preparation of Immunogens and Conjugates

Although the haptens of the present invention provide
noxy]butanoate 3. The ketoester, 3, obtained Was reacted

With octopamine hydrochloride, 4, in methanol in the pres
ence of triethylamine (TEA) and sodium cyanoborohydride
de?ned structural epitopes, they are not in themselves

immunogenic and therefore need to be conjugated to carrier
materials, Which Will elicit an immunogenic response When
55
(Na BH3CN) to give 10-O-[3-(ethoxycarbonyl)propyl]rac
administered to a host animal. Appropriate carrier materials
topamine ether 5, in moderate yields. The hapten A Was

obtained after saponi?cation of the ester, 5, With sodium
commonly contain poly(amino acid) segments and include

polypeptides, proteins and glycoproteins. Illustrative
hydroxide (NaOH) (2N) in methanol.

The hapten 10-O-(4-carboxybenZyl)ractopamine ether
examples of useful carricr materials are bovine serum albu

60
min (BSA), egg ovalbumin, bovine gamma globulin, thy
roxine binding globulin, keyhole limpet haemocyanin

(KLH) etc. Alternatively, synthetic poly(amino acids) hav
(hapten Biderivatised at position 10) Was prepared by the

same method as hapten A in three steps according to FIG. 4
of the accompanying draWings, from 4-(4-hydroxyphenyl)
ing a suf?cient number of available amino groups, such as
2-butanone, 2. The reaction of 2 With methyl 4-(bromom

ethyl)benZoate, 6, in DMF in the presence of NaH, gives
lysine, may be employed, as may other synthetic or natural
methyl 4-[4-(3-oxobutyl)phenoxymethyl]benZoate, 7. The

condensation of the keto ester, 7 With octopamine hydro
65
polymeric materials bearing reactive functional groups. In

particular, carbohydrates, yeasts or polysaccharides may be
conjugated to the hapten to produce an immunogen.
US 7,192,722 B2
14
13
Each hapten prepared in accordance With the present
The residue obtained Was puri?ed by ?ash chromatography
invention can also be coupled to a detectable labelling agent

such as an enzyme (for example, horseradish peroxidase), a
substance having luminescent, chemiluminescent or ?uo
rescent properties or a radioactive label for the preparation
on silica gel using 20% ethyl acetate in hexane as eluant to

give 3 as a colourless oil (16.95 g, 50%).
of conjugates (or detection reagents) for use in the immu

noassays. The ?uorescent substance may be, for example, a
Example 2
monovalent residue of ?uorescein or a derivative thereof.
Preparation of
10-O- [3 -(Ethoxycarbonyl)propyl]ractopamine ether
IR (Film): 1732; 1720; 1612.5; 1512.9; 1245.1 and 1178.
In order to con?rm that adequate conjugation of hapten to

carrier material has been achieved, prior to immunisation,
each immunogen is evaluated using matrix-assisted UV
laser desorption/ionisation time-of-?ight mass spectroscopy
To a solution of 3 (16.95 g, 0.061 mole) and octopam
ine.HCl (11.57 g, 0.061 mole) in methanol (300 ml) Was

added TEA (12.35 g, 0.122 mole) and sodium cyanoboro
hydride (3.83 g, 0.061 mole). The mixture Was stirred at 500
(MALDl-TOF MS). Each of the immunogens of the present

invention is suitable for immunisation, in order to produce
antibodies for the detection of phenethanolamines such as,
C. overnight. Solvents Were removed in vacuo. To the
residue Were added HCl (1N) (150 ml) and Water (150 ml)
and Washed With ether (1><300 ml). The aqueous phase Was
General Procedure for MALDl-TOF Analysis of Immuno

gens
MALDI-TOF mass spectrometry Was performed using a
Voyager STR Biospectrometry Research Station laser-des

orption mass spectrometer coupled With delayed extraction.
20
Were combined, dried over magnesium sulfate, ?ltered and
evaporated to dryness. The crude product Was puri?ed by

?ash chromatography on silica gel using 5*10% methanol in
An aliquot of each sample to be analysed Was diluted in

0.1% aqueous tri?uoroacetic acid (TFA) to create 1 mg/ml
chloroform to give the title compound 5 (7 g, 28%) as a

White solid.
sample solutions. Aliquots (1 pl) Were analysed using a

matrix of Sinapinic acid and bovine serum albumin (Fluka)
IR (KBr): 3506; 3301.2; 1735.8; 1613.2; 1512.6;
Was used as an external calibrant.
Preparation of Antisera
In order to generate polyclonal antisera, each immunogen
of the present invention is mixed With Freunds Adjuvant
adjusted to pH12*13 using sodium hydroxide (6N) and

extracted With ethyl acetate (3><250 ml). The organic layers
1374.03; 1244.1; 1176.9 and 1046.5.
Example 3
30
Preparation of 10-O-(3-Carboxypropyl)ractopamine
ether (Hapten A)
and the mixture is injected into a host animal, such as rabbit,
sheep, mouse, guinea pig or horse. Further injections

(boosts) are made and serum is sampled for evaluation of the
antibody titre. When the optimal titre has been attained, the
host animal is bled to yield a suitable volume of speci?c
To a solution of the ester 5 (7 g, 0.169 mole) in methanol

35
antiserum. The degree of antibody puri?cation required
mixture Was stirred at room temperature for 16 hours. The

solvents Were removed in vacuo and Water (50 ml) Was
depends on the intended application. For many purposes,

there is no requirement for puri?cation, hoWever, in other
added. The pH of the solution Was adjusted to 344 by

addition of HCl (1N) and the resultant precipitate obtained
cases, such as Where the antibody is to be immobilised on a
solid support, puri?cation steps can be taken to remove
(90 ml) Was added sodium hydroxide (2N) (30 ml). The
40
undesired material and eliminate non-speci?c binding.
Was collected by ?ltration and dried in a desiccator over
P205. The hapten A Was obtained as a White solid (5.64 g,
The speci?c antibodies prepared in this invention are
86%).
useful as reagents in immunoassays for the detection or

determination of phenethanolamines such as, but not limited
to, ractopamine, isoxsuprine and ritodrine in biological
Example 4
45
Preparation of Methyl
?uids.
4-[4-(3-oxobutyl)phenoxymethyl]benZoate 7
EXAMPLES
The ester 7 Was obtained by using the same method as
Example 1
Preparation of Ethyl
described in Example 1 using 4-(4-hydroxyphenyl)-2-bu

tanone, 2 (20 g, 0.122 mole), sodium hydride (7.3 g, 0.183
mole) and methyl 4-(bromomethyl) benZoate (33.44 g, 0.146
4-[4-(3-oxobutyl)phenoxy]butanoate 3
mole) 6. The title compound 7 Was obtained as a clear oil
50
(23.2 g, 61%).
To a suspension of sodium hydride (60% dispersion in
55
Example 5
mineral oil) (7.3 g, 0.183 mole) in anhydrous DMF (100 ml)

under nitrogen atmosphere Was added drop-Wise 4-(4-hy
droxyphenyl)-2-butanone 2 (20.0 g, 0.122 mole) in anhy
Preparation of
drous DMF (100 ml) and the mixture Was then stirred at
10-O-[4-(Methoxycarbonyl)benZyl]ractopamine
room temperature for one hour. To this mixture Was added 60
ether 8
drop-Wise ethyl 4-bromobutyrate (28.5 g, 0.146 mole) in

anhydrous DMF (50 ml), and the mixture Was then stirred at
The ester 8 Was obtained by using the same method as
600 C. overnight. The DMF Was removed under reduced

pressure and Water (200 ml) Was then added to the residue
described in Example 2 for the preparation of 5, using the

ester 7 (10.3 g, 0.33 mole), octopamine hydrochloride 4
(6.26 g, 0.33 mole), triethylamine (6.68 g, 0.066 mole) and
sodium cyanoborohydride (2.07 g, 0.33 mole). The ester 8
Was obtained in moderate yield (7 g, 50%).
and extracted With ethyl acetate (3><200 ml). The combined

organic phases Were Washed With brine (1><200 ml), dried
over magnesium sulfate, ?ltered and evaporated to dryness.
US 7,192,722 B2
16
15
Example 10
IR (KBr ); 3171.4; 3025.6; 2942.3; 2850.69; 1717.08;

1612.84; 1595.68; 1511.9; 1281.1; 1250.1 and 1107.
Preparation of
10'-O-[3-(Ethoxycarbonyl)propyl]ractopamine ether
Example 6
12
Preparation of 10-O-(4-CarboxybenZyl)ractopamine
ether (hapten B)
The ester 12 Was prepared by condensation of compound
11 (21.89 g; 0.0574 mole) With 4-(4-hydroxyphenyl)-2

The hapten B Was obtained in 77% yield as a White solid
using the same method as described for the preparation of
butanone; 2 (9.43 g; 0.0574 mole) using the same method as

described for the preparation of 5 and 8. The title compound
12 (10 g; 42%) Was obtained as a yelloW oil.
hapten A (cf.Example 3).

IR (KBr): 3118.8; 3034.2; 1700.7; 1613.6; 1515.56;
1. R(Fi1m): 3297.7(broad); 2936.7; 1732.1; 1612.07;

1514.5; 1446.4; 1375.5; 1174.3 and 1029.7
NMR 13C (CD3OD): 169.6; 158.6; 158.5; 144.2; 134.2;
132.9; 131.3(2C); 130.9(2C); 130.5; 128.4(2C); 128.1(2C);

Example 11
116.4(2C); 116.1(2C); 70.3; 70.1; 52.3; 52.25; 35.4; 31.8

and 16.5.
Preparation of 10'-O-(3-Carboxypropyl)ractopamine
Example 7
20
Preparation of N-[2-Hydroxy-2-(4-hydroxyphenyl)
ether Hapten C
The hapten C Was obtained after saponi?cation of the
ethyl]t-butylcarboxamide 9
ester 12; using the same method as described for the
preparation of haptens A and B; as a White solid (6.2 g; 66%).

To a solution of octopamine hydrochloride 4 (18.96 g; 0.1
mole) in methanol (200 ml) Was added TEA (20.24 g; 0.2
25
NMR 13C (CD30D): 177.17; 160.46; 156.9; 134.3;
132.4; 130.4(2C); 128.4(2C); 116.4(2C); 115.7(2C); 70.0;
mole) and di-tert-butyldicarbonate (26.19 g; 0.12 mole). The
68.1; 55.4; 52.2; 35.6; 31.7; 31.5; 25.8 and 16.5
mixture Was stirred at room temperature overnight. Solvents

Were removed in vacuo and the crude residue obtained Was
puri?ed by ?ash chromatography on silica gel using ethyl
30
acetate as eluant to give N-BOC octopamine 9 (23.2 g, 80%)
Example 12
Preparation of lsoxsuprine 15
as a White solid.
Example 8
To a solution of 01-(1-aminoethyl)-4-hydroxybenZyl alco

35
Preparation of Ethyl 4-{4-[2-(t-butylcarboxamido)
(10.5 g; 0.102 mole). The mixture Was stirred at room

temperature overnight. Solvents Were removed in vacuo. To
1 -hydroxyethyl]phenoxy }butanoate 10
To a solution of N-BOC octopamine 9 (23.2 g; 0.08 mole)
in acetonitrile (400 ml) Was added potassium carbonate
40
(44.29 g; 0.32 mole) and ethyl bromobutyrate (23.41 g; 0.12

mole). The mixture Was stirred and heated at re?ux over
night. The solution Was then alloWed to cool to room
temperature; ?ltered and concentrated to dryness. The resi

due obtained Was puri?ed by ?ash chromatography on silica
gel using 50% ethyl acetate in hexane as eluant to give the
title compound 10 (24.53 g; 78%) as a clear oil.
I.R (Film): 3434.2; 2979.7; 2934.5; 1731.3(broad);
hol 13 (10.0 g; 0.049 mole) and phenoxy-2-propanone 14

(7.4 g; 0.049 mole) in methanol (200 ml) Was added TEA
45
the residue Were added HCl (1N) (100 ml) and Water (100
ml) and this Was extracted With ethyl acetate (1x200 ml).
The aqueous phase Was made basic (pH 12413) using
sodium hydroxide (6N) and extracted With ethyl acetate
(3x200 ml). The organic extracts Were combined and
Washed With brine (1x200 ml); dried over sodium sulfate;
?ltered and evaporated to dryness. The crude product Was
puri?ed by ?ash chromatography on silica gel using 10%

methanol in chloroform to give isoxsuprine 15 (8 g; 54%) as
a White solid; mp. 1024104o C.
50
Example 13
1611.9; 1512.5; 1367.5; 1247.2 and 1172.8.
Preparation of 10'-O-(4-tert-Butoxycarbonylmethyl)
Example 9
isoxsuprine ether 16
Preparation of Ethyl
4-[4-(2 -amino-1 -hydroxyethyl)phenoxy]butanoate
55
tri?uoroacetic acid salt 11.
To a solution of ethyl 4-(carboxypropyl)-N-BOC octo
60
pamine 10 (14.26 g; 0.0363 mole) in dichloromethane (80
lsoxsuprine; 15 (3.1 g; 10.3 mMoles) Was dissolved in

acetonitrile (100 ml). To this solution Was added potassium
carbonate (4.48 g; 32.4 mMoles) and tert-butyl bromoac
etate (3.16 g; 16.2 mMoles) and the mixture heated at re?ux
ml) Was added tri?uoroacetic acid (40 ml) and the mixture
overnight. The mixture Was alloWed to cool; ?ltered and the

?ltrate concentrated in vacuo. The crude product so obtained
Was stirred at room temperature for 1 hour. The solvents

Were removed in vacuo and the crude product obtained Was
methanol in chloroform to give 16 (2.27 g; 52%) as a
puri?ed by ?ash chromatography on silica gel using (10%
Was puri?ed by ?ash chromatography on silica gel using 5%

65
colourless oil. NMR l3C (CDCl3): 168.1; 158.7; 157.0;
methanol in chloroform) to give the title compound 11 (12.2
134.5; 128.5; 127.3; 120.9; 114.5; 83.3; 73.7; 71.9; 65.9;
g; 83%) as a viscous oil.
56.0; 49.6; 28.0; 18.3 and 15.2.
US 7,192,722 B2
17
18
Example 14
solution (4M) in dioxane (25 ml) and the mixture Was stirred
overnight at room temperature. Evaporation of the solvents
in vacuo afforded the crude product Which Was puri?ed by
?ash chromatography on silica gel using 10% methanol in
chloroform to give the hapten E HCl salt.(1.2 g, 34.2%) as
Preparation of 10'-O-(4-Carboxymethyl)isoxsuprine
ether, Hapten D
a foamy solid.
To a solution of 10'-O-(4-tert-butoxycarbonylmethyl)
isoxsuprine ether 16 (2.08 g, 4.8 mMoles) in dichlo

romethane (25 ml) Was added tri?uoroacetic acid (10 ml)
NMR13C(CD3OD): 171.4, 159.6, 158.6, 132.6 (2), 131.6

(2), 130.8(2), 128.3(2), 116.6, 116.1, 84.8, 66.8, 59.8, 52.7,
and the mixture Was stirred at room temperature for tWo

hours. The solvent Was removed in vacuo and the crude
32.6 and 19.4.
Example 19
product obtained Was puri?ed by ?ash chromatography on

silica gel using 10% methanol in chloroform to give the
Conjugation of Haptens A, B, C, D and E to BSA
Hapten D as a TFA salt (1.5 g, 66%).
(Preparation of lmmunogens A, B, C, D and E)

Example 15
To a solution of hapten A, B, C, D or E (58 mg, 0.15
Preparation of tert-Butyl
mmole) in DMF(1 ml) Was added N,N-dicyclohexylcarbo

diimide (DCC) (34 mg, 0.165 mmole) and N-hydroxysuc
4- [2 -hydroxyethyl]phenoxyacetate 1 8
To a solution of 2-(4-hydroxyphenyl)ethanol 17 (27.6 g,
20
Was removed by ?ltration and the ?ltrate added drop-Wise to

a solution of BSA (200 mg) in 0.1M sodium hydrogen
carbonate, pH8.5 (12 ml). The mixture Was stirred at room
0.2 mole) in acetonitrile (300 ml) Was added potassium

carbonate (110.5 g, 0.8 mole) and tert-butyl bromo acetate
(46.8 g, 0.24 mole) and the mixture Was stirred and heated
at re?ux for tWo hours. The mixture Was alloWed to cool,
?ltered and the ?ltrate concentrated in vacuo. The crude
cinimide (19 mg, 0.165 mmole) and the mixture stirred at

room temperature overnight. The dicyclohexylurea formed
25
temperature overnight. The solution Was dialysed against 50

mM phosphate buffer, pH7.2 (3 changes) for 24 hours at 4
C. and then freeZe-dried.
product obtained Was puri?ed by chromatography on silica

gel by using 30% ethyl acetate in hexane to tert-butyl
4-[2-hydroxyethyl]phenoxyacetate 18 (39.8 g, 83%) as a
MALDI results shoWed 17 molecules of hapten A, 5.8

molecules of hapten B, 10.7 molecules of hapten C, 12.4
molecules of Hapten D and 6.2 molecules for hapten E had
clear oil.
30
been conjugated to 1 molecule of BSA.
Example 16
Example 20
Preparation of tert-Butyl
4-[formylmethyl]phenoxyacetate 19
35
To a solution of 18 (20.0 g, 0.083 mole) in anhydrous

dichloromethane (200 ml) Was added pyridinium chloro
chromate (PCC) (44.7 g, 0.207 mole) and the mixture Was
EDC.HCl (10 mg) Was dissolved in Water (0.5 ml) and

immediately added to a solution of Hapten (A or B or C or
D or E) (2 mg) in DMF (0.2 ml). After mixing, this solution
stirred at room temperature for tWo hours. Ether (500 ml)

Was then added to quench the reaction and the mixture Was
General Method for the Conjugation of Haptens A,

B, C, D and E to HRP (Horseradish Peroxidase)
40
Was added drop-Wise to a solution of HRP (20 mg) in Water
then ?ltered through C elite. (Trade Mark) Evaporation of
(1 ml). Sulfo-NHS (5 mg) Was added and the reaction
the solvents in vacuo afforded the crude product Which Was
mixture Was incubated in the dark at room temperature
puri?ed by ?ash chromatography on silica gel using 20%
overnight. Excess hapten Was removed by desalting With

PD-10 columns (Pharmacia) in series, pre-equilibrated With
PBS (phosphate buffered saline) at pH 7.2. The hapten-HRP
conjugate Was then dialysed overnight against 10 L of PBS
ethyl acetate in hexane to give 19 (16.6 g, 80%) as a yelloW

oil.
45
Example 17
pH 7.2 at 40 C.
Preparation of t-Butyl 4-[-2-(beta-hydroxy-alpha
methyl-4-hydroxyphenethylamino)ethyl]phenoxyac
Example 21
50
Preparation of Antibodies to lmmunogens A and C,
etate, 20
Prepared in Example 19
The ester 20 Was prepared by condensation of tert-butyl
4-[formylmethyl]phenoxyacetate (10.0 g, 0.04 mole) With

ot-(l-aminoethyl)-4-hydroxybenZyl alcohol 13 (8.2 g, 0.04
An aqueous solution of each immunogen Was formulated

55
With Freunds Complete Adjuvant (FCA) to form an emul
sion consisting of 2 mg/ml immunogen in 50% (v/v) FCA.
mole) using the same method as described for the prepara

tion of 5, 8, and 16. The ester 20 (8.18 g, 51%) Was obtained
Three sheep Were immunised With this emulsion (1 immu
as a yelloW oil.
nisation), 0.25 ml being subcutaneously injected at each of

four sites in the ?ank of each animal. Subsequent immuni
Example 18
60
Zations (boosts) contained 1 mg/ml immunogen. All boosts

Were emulsi?ed in 50% (v/v) Freunds Incomplete Adjuvant
Preparation of 4-[-2- (beta-hydroxy-alpha-methyl-4
(ETA) and Were administered in the same manner as the 1
hydroxyphenethylamino)ethyl]phenoxyethanoic
immunisation, at monthly intervals for 1 year. Blood sam
acid, Hapten E
pling took place 7 to 14 days after each boost. Each sample

65
To a solution of tert-butyl ester 20 (3.7 g, 0.92 mMole) in

25 ml of anhydrous dioxane Was added hydrochloric acid
Was processed to produce antiserum, Which Was further
puri?ed by caprylic acid and ammonium sulfate precipita

tion to yield an immunoglobulin G (IgG) fraction. The lgG
US 7,192,722 B2
19
20
fraction Was evaluated by competitive ELISA microtiter

plate assay, as described in Example 22 below.
TABLE 1
Data generated from competitive microtiter plate assays for ractopamine,
employing antisera generated to immunogen A (hapten A-BSA)
Example 22
5
Example 19 and immunogen C hapten C-BSA
Development of Competitive ELISAs for
Fxamnle 22 a)
Ractopamine
a) The Wells of an enhanced binding 96 Well polystyrene
microtiter plate Were coated With the IgG fraction of the
Ractopamine
Ractopamine
Concentration
ng/ml
A450
% B/BO
ngml
A450
% B/BO
0
0.05
0.1
0.5
1
5
2.385
1.276
1.181
0.566
0.404
0.151
100
53.5
49.5
23.7
16.9
6.33
0
0.025
0.1
0.25
0.5
1
1.994
1.683
1.302
0.918
0.68
0.469
100
84
65
46
34
24
IC5O (ng/ml)
bu?cered saline containing TWeen 20 (TBST) and tapped dry.
0.082
IC5O (ngml)
0.202
A450 = absorbance at 450 nm

B = absorbance at 450 nm at x ng/ml standard concentration
Standard solutions of ractopamine Were prepared in TBST at
0, 0.05, 0.1, 0.5, 1, and 5 ng/ml, and 50 pl ofeach Was added

to the appropriate Wells. 75 pl of conjugate A (hapten
Fxamnle 22 b)
Concentration
antiserum raised to immunogen A (hapten A-BSA) (Ex

ample 19), diluted in 10 mM Tris, pH8.5 (125 pl/Well). The
appropriate antibody coating dilution Was determined using
standard ELISA checkerboard techniques. The plate Was
incubated for 2 hours at 370 C., Washed 4 times With Tris
Example 19 .
B0 = absorbance at 450 nm at 0 ng/ml standard concentration
20
IC5O = standard concentration Which produces 50% B/BO

% CR = percentage cross-reactivity based on speci?city to ractopamine
A-HRP) (Example 20), diluted in Tris bulTer (pH 7.2)
this is What is meant by the term cross-reactivity in the present speci?
containing EDTA, D-mannitol, sucrose, thimerosal and
cation.
BSA, Was added to each of the Wells. The appropriate

dilution of conjugate Was also determined using standard
ELISA checkerboard techniques. The plate Was incubated at
370 C. for 2 hours. Excess unbound conjugate Was removed
by Washing 6 times over a 10 minute period With TBST. 125
Example 23
25
Cross Reactivity of Competitive ELISAs for
Ractopamine
pl of tetramethylbenZidine (TMB) substrate solution Was

added to each Well of the plate that Was then incubated for
15 to 20 minutes in the dark at room temperature. The
In order to determine the speci?city of the competitive
30
ELISAs for ractopamine, standard solutions of a range of
structurally similar [3-agonists Were prepared in TBST.

Employing each series of standards in the ractopamine
reaction Was terminated by addition of 125 pl 0.2M HZSO4

to each Well. The absorbance Was then measured at 450 nm
using a microtiter plate reader. The data generated in the

assay is presented in Table 1.
35
competitive ELISAs, calibration curves Were generated and

these Were used to determine the cross-reactivity of each
immunoassay With these substances. The results of this
study are presented in Table 2, cross-reactivity being calcu

lated according to the folloWing formula:
b) In a similar manner to that described in Example 15(a),

the Wells of a 96-Well microtiter plate Were coated With the
IgG fraction of the antiserum raised to immunogen C
(hapten C-BSA) (Example 19), standards Were applied at 0,

0.025, 0.1, 0.25, 0.5 and 1 ng/ml and conjugate C (hapten
C-HRP) (Example 20) Was employed as detection reagent.
% CR :Icso, mmopamin/lcso, CRXlOO

40
Where % CR is the percentage cross-reactivity,

ICSO, ractopamine is the concentration of ractopamine that
causes 50% displacement of signal and ICSO, CR is the
The data generated are presented in Table 1. The same
concentration of potential cross-reactant that causes 50%
de?nitions apply for A450, B, B0 and ICSO.
displacement of signal.
TABLE 2
Cross reactivity of the competitive ELISAs for ractopamine
Fxamnle 15 a)
Icso CR
Fxamnle 15 b)
Icso CR
Compound
ng/ml
% CR
ng/ml
Ractopamine
Dobutamine
0.082 100
0.202
44.2
0.186 >50
Shelver pAb
Icso CR
% CR
100
<0.8
ng/ml
4.2
12.7
Shelver mAb
Icso CR
% CR
100
33
ng/ml
2.69
50.3
% CR
100
5.3
Isoxsuprine
DL-Isoproterenol
>50
>50
<0.16
<0.16
>50
>50
<0.6
<0.5
6,200
>100,000
0.1
<0.01
1,391
NA
0.2
NA
Ritodrine
Salmeterol Xinafoate
Methyl Clenbuterol
Fenoterol
Pirbuterol
Tulobuterol
Dopamine
Salbutamol
Clenbuterol
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
<0.16
<0.16
<0.16
<0.16
<0. 16
<0. 16
<0.16
<0.16
<0.16
<0.16
<0. 16
<0. 16
>50
>50
>5 0
>50
>50
>50
>50
>50
>50
>50
>5 0
>50
<0.5
<0.5
<0 .05
<0.05
<0.05
<0.05
<0.01
<0.01
<0.01
<0.01
<0 .01
<0.01
577
1,800
NA
310
NA
NA
NA
79,000
>100,000
>100,000
NA
NA
0.8
0.2
NA
1.3
NA
NA
NA
<0.01
<0.01
<0.01
NA
NA
73.8
1519
NA
2,682
NA
NA
NA
NA
NA
NA
NA
NA
3.6
0.2
NA
0.1
NA
NA
NA
<0.1
<0.1
NA
NA
NA
Metaproterenol
Terbutaline
Cimaterol
US 7,192,722 B2
TABLE 2-continued
Cross reactivity of the competitive ELISAs for ractopamine
Example 15 a)
Example 15 b)
ICSO CR
Compound
Bamethane
() Isoproterenol
Mabuterol
ICSO CR
Shelver pAb
Shelver mAb
ICSO CR
ICSO CR
ngml
% CR
ngml
% CR
ng/ml
% CR
ng/ml
% CR
>50
>50
>50
<0.16
<0.16
<0.16
>50
>50
>50
<0.01
<0.01
<0.01
2,900
NA
NA
0.1
NA
831
NA
0.3
NA
NA
NA
NA
IC5OICR = concentration of potential cross-reactant that causes 50% displacement of signal

% CR = percentage cross-reactivity based on speci?city to ractopamine
Shelver pAb = Shelver polyclonal antiserum (Shelver and Smith, Journal of Immunoassay (2000), 21(1))
Shelver mAb = Shelver monoclonal antibody (Shelver et al., J. Agric. Food Chem. 48 (2000))
The data presented in Table 1 indicate that the ractopam
ine immunoassays developed employing antibodies generated to hapten A and hapten B are highly sensitive With IC5O
_
TABLE 3
20
Values of 0082 and 0202 ng/ml: respecnvely These lmmu'

noassays are considerably more sensitive than those presented in the prior art. For example, Shelver et al. (2000)
reported an IC5O of 4.2 ng/ml for their ractopamine EIA. The
, ,
Data generated from competrtrve mrcrotrter plate assays for
isoxsuprine and ritodrine, employing antisera generated to immunogen

D (hapten D-BSA) (Example 19) a~Hdirnrnu110gn E (hapten E-BSA)
(Fmmnle 19)
Example 24
Example 25
speci?city data presented in Table 2 indicate that the anti- 25
b d.
1
d
h. hl
.? f
t
.
Th
I o res Iemp oye are 1g Iy speIcr c or rac Iopamrne.
ere
Isoxsuprrne
Concmmtion
ng/ml
15 neglrgrble cross-reactrvrty W1th dobutamrne (<0.8%) for

both antrbodres 1n the present applrcatron, compared W1th
A450
2345
% B/B0
100
Rrtodrrne
Concentration
ng/ml
A450
L946
% B/B0
100
that publrshed 1n the prror art. For example, Shelver et al 30
()5
0743
31_6
r_79r
92.0
reported 33% cross-reactivity With dobutamine for their
8-???
12-?
1(5)
{22;
2g;
polyclonal antibody and 5.3% cross-reactivity With dob-
10
0:115
49
50
1:260
64:7
utamine for their monoclonal antibody.
50
0.036
1.5
100
1.088
55.9
100
0.032
1.3
500
0.584
30.0
The ractopamrne-denved antrbodres from the present
200
0022
0I9
1000
0440
22I6
rnventron Wrll therefore yreld hrghly specr?c results When 35
IC50 (ng/ml)
screening biological samples for the presence of ractopam
0.230
1C50 (Hg/ml)
115.230
A450 = absorbance at 450 nm

B = absorbance at 450 nm at x ng/ml standard concentration
ine.
B0 = absorbance at 450 nm at 0 ng/ml standard concentration
Example 24
IC5O = standard concentration Which produces 50% B/BO

40
Example 26
Development of a Competitive ELISA for
Isoxsuprine
In a similar manner to that described in Example 22(a),
45
In a similar manner to that described in Example 23, the
IgG fraction of the antiserum raised to immunogen D
(hapten D-BSA) (Example 19), isoxsuprine standards Were

applied at 0, 0.5, 1, 5, 10, 50, 100 and 200 ng/ml and
conjugate D (hapten D-HRP) (Example 20) Was employed
Cross Reactivity of Competitive ELISAs for

Isoxsuprine and Ritodrine
cross reactivity of each immunoassay With isoxsuprine,
50
ritodrine, dobutamine and ractopamine Was determined. The

results of this study are presented in Table 4.
as detection reagent. The data generated are presented in

Table 3.
TABLE 4
Cross reactivity of the competitive ELISAs for isoxsuprine (Example
24) and ritodrine (Example 25)
Example 25
55
Example 24
Development of a Competitive ELISA for Ritodrine
Icso CR
Compound
In a similar manner to that described in Example 22(a),

IgG fraction of the antiserum raised to immunogen E
60
Isoxsuprine
ngml
0.230
Icso CR
% CR
100
ngml
% CR
569.7
20.2
Ritodrine
Dobutamine
>200
>200
<0.1
<0.1
115.2
>1000
100
<5 .0
Ractopamine
>200
<0.1
>1000
(hapten E-BSA) (Example 19), ritodrine standards Were

applied at 0, 1, 5, 10, 50, 100, 500 and 1000 ng/ml and
conjugate E (hapten E-HRP) (Example 20) Was employed as
placement of signal
detection reagent. The data generated are also presented in
(Example 24) or ritodrine (Example 25)
Table 3.
Example 25
<0.5
IC5O CR = Concentration of potential cross-reactant that causes 50% dis
% CR = Percentage cross-reactivity based on speci?city to isoxsuprine

United States Patent: Mcconnell Et Al. (10) Patent N0.: (45) Date of Patent

Uploaded by

Copyright:

Available Formats

United States Patent: Mcconnell Et Al. (10) Patent N0.: (45) Date of Patent

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

United States Patent: Mcconnell Et Al. (10) Patent N0.: (45) Date of Patent

Uploaded by

Copyright:

Available Formats

US007192722B2

(12) United States Patent

(10) Patent N0.:

U.S. PATENT DOCUMENTS

(75) Inventors: Robert Ivan McConnell, Ballymena

(73) Assignee: RandoX Laboratories, Crumlin (GB)

Subject to any disclaimer, the term of this

Assistant ExamineriSha?qul Haq

U.S.C. 154(1)) by 49 days.

(21) Appl. No.: 11/271,282

reacting a phenylethanolamine derivative of formula D With

Foreign Application Priority Data

Nov. 10, 2004

petitive immunoassays for the detection of ractopamine,

Prior Publication Data

The present invention relates to a method for preparing

Nov. 10, 2005

Shelver et a1. ........... .. 435/792

Elliot, C.T. et al., Screening and Con?rmatory Determination of

Antrim (GB); Andrew Philip LoWry,

(GB); Stephen Peter Fitzgerald,

Mar. 20, 2007

HAPTENS, IMMUNOGENS, ANTIBODIES

US. Cl. ................... ..

435/7.1; 435/7.92; 435/793;

435/975; 436/544; 436/546; 436/56; 530/402;

Field of Classi?cation Search ............... ..

in Which, in formula 1, Z1 is a crosslinker and Z2 is H and,

435/7.92, 7.93, 975; 436/544, 546, 56; 530/402,

27 Claims, 7 Drawing Sheets

Chemical structures nl ?clognmine Ritodrige gig Qmuugrine

Excretion of Ractopamine and Metabolites in Animals Treated for

Haasnoot, W. et al., Determination of Fenoterol and Ractopamine

in Urine by Enzyme Immunoassay, Analyst, 1994, 119, 2675-2680.

& Agricultural Immunology, 1995, 7, 123-129.

Ractopamine, J Agric. Food Chem, 2000, 48, 4020-4026.

fi-Adrenergic Agonist Ractopamine, Journal of Immunoassay,

Sheep Urine Samples Using an Optical Biosensor Analysis: Com

Agents, Molecular Diversity, 1998, 3, 113-116.

7 Days With Dietary Ractopamine, J Anim. Sci., 2002, 80, 1240

Haasnoot, W. et al., Immuno?ltration as Sample Cleanup for the

Immunochemical Detection of [5-Agonists in Urine, Analyst, The

Royal Society of Chemistry, 2002, 127, 87-92.

Terando, N.H., Synthesis of 14C-Radiolabeled Ractopamine

Mar. 20, 2007

Chemical structures of RactojlamineLRitodrine and Isoxsuprine

Mar. 20, 2007

Chemical Structure of Haptens A, B, C, D, and E

Mar. 20, 2007

Methanol, TEA, NaBHJCN

Mar. 20, 2007

Chemical reactions for the re aration 0f Ha ten B and Immuno en B

Methanol, TEA, NaBHJCN

Mar. 20, 2007

Chemical reactions for the greparation of Hagten C and lmmunogen C

Mar. 20, 2007

Chemical reactions for the re aration 0f Ha ten D and Immune en D

\r\O/ tert-butyl bromoacetate

W/\O /< >