Technical note: Development of an enzyme-linked immunosorbent assay for the

determination of florfenicol and thiamphenicol in swine feed

P. J. Luo, W. X. Jiang, X. Chen, J. Z. Shen and Y. N. Wu

J ANIM SCI 2011, 89:3612-3616.

doi: 10.2527/jas.2010-3403 originally published online July 1, 2011

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 Technical note: Development of an enzyme-linked
immunosorbent assay for the determination of florfenicol
and thiamphenicol in swine feed1
P. J. Luo,*2 W. X. Jiang,†2 X. Chen,‡3 J. Z. Shen,† and Y. N. Wu*3

*Key Laboratory of Chemical Safety and Health, National Institute for Nutrition and Food Safety,
Chinese Center for Disease Control and Prevention, Beijing 100050, People’s Republic of China;
†Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine,
China Agricultural University, Beijing 100193, People’s Republic of China; and ‡State Key Laboratory
for Infectious Disease Prevention and Control, National Institute for Infectious Disease Control and Prevention,
Chinese Center for Disease Control and Prevention, Beijing 102206, People’s Republic of China

ABSTRACT: One polyclonal antibody against flo- phenicol ranged from 86.4 to 118.6%, whereas intraas-
rfenicol and thiamphenicol was produced and a com- say recoveries of both drug ranged from 90.1 to 126.5%
petitive ELISA was developed for the detection of flo- with less than 15% CV. Results obtained from HPLC-
rfenicol and thiamphenicol in swine feed. The ELISA tandem mass spectrometry indicated this ELISA pro-
gave a 50% inhibiting concentration of 1.02 ng/mL for cedure could be used as a convenient method for rapid
florfenicol. For swine feed fortified with 0.05 to 3.0 mg/ screening of florfenicol and thiamphenicol in swine feed.
kg, the interassay recoveries of florfenicol and thiam-
Key words: enzyme-linked immunosorbent assay, florfenicol, polyclonal antibody, swine feed, thiamphenicol

©2011 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2011. 89:3612–3616
doi:10.2527/jas.2010-3403

INTRODUCTION ing HPLC (Nagata and Sakei, 1991; Hormazabal et al.,

Florfenicol (FF), a fluorinated analog of thiam-
phenicol (TAP), or chloramphenicol (CAP) is a broad
spectrum, bacteriostatic antibiotic that inhibits bacte-
rial protein synthesis by binding to the 50S ribosomal
subunit and inhibiting peptidyl transferase activity
(Lundén et al., 1999). The FF was widely used in vet-
erinary clinical practice to control disease and improve
the growth rate of animals. However, drug residues in
food-animal tissues and the potential for the emergence
of drug-resistant bacteria may lead to adverse effects
on human health. For consumer protection, the Euro-
pean Union has banned all antibiotics, including FF
and TAP, for growth promotion.
To monitor FF and TAP in biological matrices, sev-
eral analytical methods have been developed, includ-
1 a competitive ELISA for the simultaneous detection of
This work is supported by the State Key Program of National
Natural Science of China (20837003; Beijing, China), and the Nation-
al Science & Technology Pillar Program in the Eleventh Five-Year
Plan of the Ministry of Science and Technology (2007BAC27B02;
Beijing, China).
2
These authors contributed equally to this work.
3
Corresponding authors: china_cdc@yahoo.cn and chenx-
ia602@163.com
Received August 5, 2010.
Accepted June 21, 2011.
1993, 1996; Hayes, 2005; Dai et al., 2006; Li et al.,
2006a), HPLC-tandem mass spectrometry (HPLC/
MS; van de Riet et al., 2003; Chen et al., 2005; Peng et
al., 2005; Zhang et al., 2008; Luo et al., 2010), gas chro-
matography (Pfenning et al., 2000; Zhang et al., 2006),
and gas chromatography/mass spectrometry (Nagata
and Oka, 1996; Li et al., 2006b). However, these meth-
ods require expensive instruments and time-consuming
sample preparation steps.
Immunoassay is one of the most suitable methods
to detect drug residues in the veterinary field because
of its rapidity, mobility, and high sensitivity. Several
immunoassays for FF or TAP have been reported (Du-
mont et al., 2006; Hao et al., 2006; Fodey et al., 2007;
Wu et al., 2008; Luo et al., 2009a,b). However, there
has been no report on the ELISA method for the simul-
taneous screening of FF and TAP in feed. In this study,
FF and TAP in swine feed was developed and validated
by the HPLC/MS method.
MATERIALS AND METHODS
This study was approved by the Institutional Animal
Care and Use Committee of China Agricultural Uni-
versity.

3612

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 Florfenicol and thiamphenicol detection
Table 1. Cross-reactivities of antibody 2 with related antibiotics
Antibiotic
Florfenicol
H3C
Thiamphenicol
H 3C
Florfenicol amine
H3C S
Chloramphenicol O 1,048.31
N+
O-
1
IC50, 50% inhibiting concentration.
Reagents
Florfenicol amine and FF were obtained from Scher-
ing-Plough Corp. (Kenilworth, NJ). Succinic anhydride
(SH), CAP, TAP, maleic anhydride (MH), BSA, hu-
man serum albumin (HSA), N-hydroxysuccinamide,
N,N-dicyclohexylcarbodiimide, ovalbumin (OVA),
Freund’s complete and incomplete adjuvants, goat anti-
rabbit IgG-horseradish peroxidase conjugate, and tetra-
methylbenzidine were obtained from Sigma-Aldrich Co.
LLC (St. Louis, MO). Other reagents were analytical
grade and obtained locally (Beijing Chemical Reagent
Co., Beijing, China). In this study, the PBS consisted
of Na2HPO4, NaH2PO4, NaCl, and pure water, and
PBS with different concentrations or pH values could
be made by changing concentrations of Na2HPO4 and
NaH2PO4 in the assay buffer. The assay buffer was PBS
containing 0.02 M phosphate buffer (pH 7.2) with 0.46
M NaCl, and the washing solution was 0.01 M phos-
phate buffer containing 0.05% Tween 20. The blocking
solution consisted of 0.01 M phosphate buffer and 0.1%
casein.
Antibody Preparation
The haptens were synthesized by coupling FF with
SH (Diener et al., 1981; Xu et al., 2005) or FF male-
ate (Krasnova et al., 2001). Briefly, a solution of FF
(3.57 g, 10 mmol) in dry pyridine (20 mL) was added
to SH (3.0 g, 30 mmol) at room temperature. After
stirring for 24 h, the mixture was concentrated with
a rotary evaporator. The residue was dissolved with
ethyl acetate (30 mL) and then was washed 3 times
with hydrochloric acid (0.1 M, 40 mL) followed by
purified water (30 mL) for 2 times. The solvent was
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3613
IC50,1 Cross-
Structure ng/mL reactivity, %
O OH Cl 1.02 100
S N
O Cl
F O
O OH Cl 2.55 40.0
S N
O Cl
HO O
O OH 298.83 0.3
NH2
O
F
0.1
OH Cl
N
Cl
HO O
evaporated and dried in vacuum for 48 h. The FF-SH
was obtained as a pink solid. Analysis of the pink solid
by mass spectroscopy gave similar molecular weight to
that of the title compound [MS m/z (%): 456 (M-1)−,
356 (M-COCH2CH2COOH)]. In the same way, FF-MH
was obtained as a white solid. The molecular weight of
the white solid was consistent with the title compound
[MS m/z (%): 454 (M-1)−, 354 (M-COCHCHCOOH)].
The haptens were coupled to BSA or HSA by the ac-
tive eater method (Xu et al., 2005; Luo et al., 2009a) to
prepare 4 immunogens (FF-SH-BSA, FF-SH-HSA, FF-
MH-BSA, and FF-MH-HSA). Briefly, the hapten (50
mg, 0.11 mmol) dissolved in N,N-dimethylformamide
(4 mL) was mixed with N-hydroxysuccinamide (27 mg,
0.23 mmol) and N,N-dicyclohexylcarbodiimide (49 mg,
0.24 mmol). The mixture was stirred for 2 h at room
temperature; BSA (50 mg, 0.00076 mmol) or HSA (50
mg, 0.00073 mmol) was dissolved in 14 mL of PBS to
which 2 mL of N,N-dimethylformamide was added. The
solution of an active ester was added dropwise to the
stirred protein solution. This solution was stirred over-
night and dialyzed against NaCl (0.9%) for 72 h at 4°C.
The coating antigens, FF-SH-OVA and FF-MH-
OVA, were synthesized by mixed anhydride reaction
(Gendloff et al., 1986). These contents were dialyzed
against NaCl (0.9%) for 72 h at 4°C and then centri-
fuged at 1,000 × g for 5 min at 4°C to discard sediment.
Eight female New Zealand white rabbits (about 2
kg) were immunized with 4 different immunogens (2
rabbits for each immunogen). Each rabbit was pre-bled
a week before immunization. A 1-mL volume of blood
was collected from the marginal ear vein and stored in
a plastic tube without preservative. Two milligrams of
the conjugate dissolved in 1 mL of NaCl (0.9%) was
emulsified with Freund’s complete adjuvant (1:1, vol/
3614 Luo et al.

Table 2. Recoveries of florfenicol (FF) and thiamphenicol (TAP) from swine complete
feed
Interassay (n = 5) Intraassay (n = 3)
Fortified,
Item mg/kg Recovery, % CV, % Recovery, % CV, %

FF 0.05 102.1 9.6 105.5 9.3

0.10 108.8 10.7 112.7 9.8
0.30 118.6 12.5 126.5 14.2
TAP 0.05 98.6 11.3 97.9 10.4
0.10 103.1 10.8 105.3 9.5
0.30 116.5 13.4 121.5 13.9

vol) and injected intradermally at multiple sites on the
back. After 2 wk, each rabbit was administered boost
immunizations with an additional dose of the same im-
munogen emulsified with Freund’s incomplete adjuvant.
After the third immunization, the rabbits were bled
from the marginal ear vein 1 wk after each booster. A
1-mL volume of blood was collected from the marginal
ear vein and stored in a plastic tube without preserva-
tive. Serum was isolated by centrifugation at 3,000 × g
for 15 min at 4°C and stored at −20°C.
Sample Preparation
Swine complete feed or feed supplement sample (2 g)
known be free of FF and TAP was transferred into a
50-mL polystyrene centrifuge tube. Then, FF or TAP
standard solution was added to the sample at 0.05, 0.1,
or 0.30 mg/kg in the complete feed and 0.5, 1.0, or 3.0
mg/kg in the supplement, and air-dried for 10 min.
After adding 20 mL of ethyl acetate, the sample was
mixed briefly and centrifuged at 3,000 × g for 10 min
at 4°C. Then, 1.0 mL of the organic phase was pipet-
ted into a 10-mL test tube, the test tube was placed
in a water bath at 40°C, and the organic phase was
evaporated to dryness under a stream of nitrogen. The
residue was dissolved in 1 mL of hexane by stirring for
1 min, after which 1 mL of assay buffer was added and
the mixture was stirred for 30 s. The mixture was then
centrifuged at 3,000 × g for 10 min at room tempera-
ture. The supernatant was removed and 500 μL of the
remaining aqueous phase was diluted 5 times in assay
buffer for complete feed, and 50 times for the feed sup-
plement sample. This diluted aqueous phase was used
for the assay.
Indirect Competitive ELISA
To select the suitable concentrations of immunore-
agents, competition experiments were conducted by the
checkerboard method (Crowther, 2008). A microtiter
plate was coated with FF-MH-OVA (26 ng/well) and
incubated for 2 h at 37°C. The plate was blocked with
0.1% casein. After the blocking solution was removed,
50 μL/well of the optimal antibody dilution (1:8,000)
and 50 μL/well of analytes or sample extract were add-
ed and incubated for 30 min at 25°C. After a washing
step, 100 μL/well of goat-anti-rabbit IgG-horseradish
peroxidase (1:3,000 in the washing solution) was added
and incubated for 30 min as 25°C. The plate was washed
3 times and 100 μL/well of tetramethylbenzidine sub-
strate system was added. The plate was incubated for
another 10 min at 25°C, and 50 μL/well of 2 M H2SO4
was added to stop the reaction. The absorbance was
read at 450 nm by an ELISA plate reader (Sunrise;
Tecan US Inc., Durham, NC).
RESULTS AND DISCUSSION
Preparation of Anti-FF Polyclonal Antibody
From the titer values and inhibition level of anti-
bodies, antibody 2 against FF-SH-HSA was chosen to
develop ELISA. Because a decreased 50% inhibiting
concentration (IC50) value was obtained based on an-

Table 3. Recoveries of florfenicol (FF) and thiamphenicol (TAP) from swine feed
supplement
Interassay (n = 5) Intraassay (n = 3)
Fortified,
Item mg/kg Recovery, % CV, % Recovery, % CV, %

FF 0.5 86.4 7.9 90.1 8.4

1.0 93.6 10.3 93.2 10.8
3.0 106.5 11.5 97.6 11.3
TAP 0.5 87.8 9.3 92.6 9.7
1.0 93.4 9.8 100.1 10.5
3.0 103.6 13.2 109.4 14.1

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 Florfenicol and thiamphenicol detection
Table 4. Florfenicol (FF) or thiamphenicol (TAP) concentration in swine feed samples
from 2 swine operations
FF, mg/kg (mean ± SD)
Sample ELISA HPLC/MS1
1 35.2 ± 3.2 31.9 ± 2.4
2 32.8 ± 4.1 30.2 ± 3.6
3 23.7 ± 2.7 21.9 ± 2.2
4 31.5 ± 3.8 30.3 ± 3.5
5 ND ND
6 ND ND
1
HPLC/MS, HPLC-tandem mass spectrometry.
2
ND, not detected.
tibody 2, the optimized ELISA showed an IC50 of 1.02
ng/mL using FF-MH-OVA, and the range of measure-
ment of the assay was 0.5 to 40.5 ng/mL. Antibody 2
showed 40.0% cross-reactivity with TAP, whereas cross
reactivity was 0.1 and 0.3% with CAP and florfenicol
amine, respectively (Table 1). The IC50 of 2.55 ng/mL
for TAP indicated that the methylsulfonylphenyl and
fluorine atom were important antigenic determinant.
The limit of detection (LOD) was defined as the mean
value of 15 blank samples plus 3 times SD of the mean,
and the LOD was found to be 0.02 mg/kg for both FF
and TAP in the swine feed.
Recoveries and Variations
When the complete feed samples were fortified with
0.05 to 0.3 mg/kg, interassay recoveries of FF ranged
from 102.1 to 118.6% with CV of 9.6 to 12.5%, whereas
recoveries of TAP ranged from 98.6 to 116.5% with CV
of 10.8 to 13.4% (Table 2). Intraassay recoveries of FF
ranged from 105.5 to 126.5% with CV of 9.3 to 14.2%,
whereas recoveries of TAP ranged from 97.9 to 121.5%
with CV of 9.5 to 13.9%. Supplement samples were for-
tified with 0.5 to 3.0 mg/kg, and interassay recoveries
of FF ranged from 86.4 to 106.5% with CV of 7.9 to
11.5%, whereas recoveries of TAP ranged from 87.8 to
103.6% with CV of 9.3 to 13.2% (Table 3). Intraassay
recoveries of FF ranged from 90.1 to 97.6% with CV of
8.4 to 11.3%, and recoveries of TAP ranged from 92.6
to 109.4% with CV of 9.7 to 14.1%.
Validation
To demonstrate reliability of the ELISA method in
this study, 6 field samples from 2 swine operations were
analyzed using the developed ELISA method and the
HPLC/MS method (Zhang et al., 2008). The concen-
tration values of the drugs (Table 4) determined by the
ELISA method were slightly greater than those deter-
mined by HPLC/MS method. This may be attributed
to the special matrix interference in the samples.
In this study, we developed a sensitive ELISA for the
detection of FF and TAP in swine feed based on an
anti-FF polyclonal antibody. The LOD of the method,
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3615
TAP, mg/kg (mean ± SD)
ELISA HPLC/MS
2
ND ND
ND ND
ND ND
ND ND
25.8 ± 3.7 25.1 ± 2.3
50.3 ± 7.1 49.2 ± 3.9
recoveries, and deviations of FF and TAP from fortified
samples were all within acceptable range. The valida-
tion of the ELISA method indicated good reliability
and accuracy. Thus, the ELISA developed herein can
be used as a convenient method for the rapid screening
of FF and TAP in swine feed before confirmation and
quantification by instrumental analysis.
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