Monoclonal antibody based enzyme-linked immunosorbent assay for the specic

detection of ciprooxacin and enrooxacin residues in shery products

Kun Hu
a,1
, Xuanyun Huang
b,1
, Yousheng Jiang
a
, Wei Fang
a
, Xianle Yang
a,b,

a
Aquatic Pathogen Collection Centre of Ministry of Agriculture, Shanghai Ocean University, 999 Huchenghuanlu Road, Shanghai 201306, PR China
b
Laboratory of Fishery Environments and Aquatic Products Processing, East China Sea Fisheries Research Institute Chinese Academy of Fishery Sciences, Shanghai 200090, PR China
a b s t r a c t a r t i c l e i n f o
Article history:
Received 23 December 2009
Received in revised form 4 August 2010
Accepted 10 August 2010
Keywords:
Ciprooxacin
Monoclonal antibody
Enzyme-linked immunosorbent assay
Fishery products
Ciprooxacin (CPFX) residues in shery products were dangerous for human health and still remain a big
challenge in China. However, there are a few available assays to detect specically CPFX residues in shery
products. This study aimed at generating CPFX-specic monoclonal antibodies (mAbs) and developing a
competitive indirect enzyme-linked immunosorbent assay (ci-ELISA) for the sensitive and specic detection
of CPFX residues in shery products. Firstly, the CPFX was conjugated with BSA and the conjugates were
immunized to Balb/c mice. After characterizing anti-CPFX sera, we generated hybridoma cell lines and found
that one hybridoma clone, 1C9, produced anti-CPFX mAb. Characterization of IC9 mAb revealed that it
belonged to IgG2a, with an afnity of 3.7510
10
mol/l for CPFX. Furthermore, we developed ic-ELISA using
150 ng/ml of CPFX-OVA for coating antigen and 0.22 g of mAb. We found that this assay detected CPFX at
45.25 ng/ml and had 84.60% of cross-activity to enrooxacin (ENR), but not to ooxacin, levooxacin,
dioxacin, saraoxacin, furacilin, chloramphenicol and nalachite green. Using this assay, we detected the
CPFX in shery products prepared with recovery rates from 80.57% to 94.61% and relative standard deviation
(RSD) varying from 1.78% to 8.02%, similar to that by HPLC analysis. These data, together the protocol for the
preparation of shery products, indicate that the ic-ELISA is able to detect CPFX and ENR residues in shery
products sensitively and specically within 2 h. Therefore, this assay can be used for monitoring CPFX and
ENR in commercial shery products, improving the safety of commercial meats in China.
2010 Published by Elsevier B.V.
1. Introduction
Ciprooxacin (CPFX) is an antibacterial agent and used as a
veterinary medicine in the world, particularly in developing countries,
like China. The CPFX residues in the consumed animal tissues raise
potential risks of the development of drug-resistance and chronic
adverse effects in the public health (Hernandez et al., 2002). The
maximum residues limits (MRLs) of CPFX in the food products of
animal origin have been established in European Union (DOCE, 1990)
and adapted by many other countries. However, CPFX-related shery
product-involved incidents often occur and have been a serious
concern to human health. Therefore, the careful monitoring of CPFX
residues in shery products for human consumption by sensitive and
specic methods is crucial for food safety and human health.
Currently, the available methods to determine the CPFX in
commercial meats are mainly dependent on the complex high
performance liquid chromatography (HPLC) (Garcia Ovando et al.,
1999), capillary electrophoresis (Hernandez et al., 2002) and
microbiological method (Montero et al., 2005). However, these
methods usually require for the professional operators and expensive
equipments. Enzyme-linked immunosorbent assay (ELISA) is a
simple, sensitive, economical, and high throughput procedure for
the specic detection of antigen. Furthermore, monoclonal antibody
(mAb) based ELISA has greater advantage than that of polyclonal
antibodies (pAbs), particularly for its specicity and stability. In
addition, mAb can continue to supply the well-dened specic and
stabilized antibodies with little batch variation. Several mAbs against
uoroquinolones (Holtzapple et al., 1997; Watanabe et al., 2002; Huet
et al., 2006; Wang et al., 2007; Li et al., 2008) have been generated.
However, most commercial kits for the detection of CPFX residues are
made by pAbs (Snitkoff et al., 1998; Duan and Yuan, 2001). The few
available mAbs against CPFX have been predominately applied to
livestock and poultry (Huet et al., 2006; Wang et al., 2007; Li et al.,
2008). Currently, there is still no available of commercial kit for the
detection of CPFX residues in the shery products.
In this study, we aimed at generating mAbs against CPFX and
developing mAb-based ci-ELISA for the detection of CPFX residues in
commercial shery foods. We rst coupled CPFX into carrier proteins
and immunized mice with the hapten-carrier complex. Subsequent-
ly, we generated hybridoma cell lines, which produced high afnity
anti-CPFX mAb. Furthermore, we used the procedures for the
preparation of shery products and mAb-based ci-ELISA, with
Aquaculture 310 (2010) 812
Corresponding author. Aquatic Pathogen Collection Centre of Ministry of
Agriculture, Shanghai Ocean University, 999 Huchenghuanlu Road, Shanghai 201306,
PR China. Tel./fax: +86 21 61900453.
E-mail address: xlyang@shou.edu.cn (X. Yang).
1
These authors contributed equally to this study.
0044-8486/$ see front matter 2010 Published by Elsevier B.V.
doi:10.1016/j.aquaculture.2010.08.008
Contents lists available at ScienceDirect
Aquaculture
j our nal homepage: www. el sevi er. com/ l ocat e/ aqua- onl i ne
which we detected CPFX residues in the shery products sensitively
and specically.
2. Materials and methods
2.1. Materials
Ciprooxacin (Content 98.5%) was purchased from Zhejiang
Guobang Pharmaceutical Co., while bovine serum albumin (BSA),
ovalbumin (OVA), and peroxidase horseradish (HPR) were obtained
from Dingguo biotechnology. The 1-Ethyl-3-(dimethylaminopropyl)-
carbodiimide (EDC, purity99.3%) was from Shanghai Yanchang
Biochemical Technology and 3,3. 5,5-tetramethylbenzidine (TMB),
HRP-conjugated goat anti-rabbit IgG, RPMI 1640, hypoxanthine
aminopterin thymidine (HAT) medium, calf serum claried, incom-
plete Freund's adjuvant (IFA) and complete Freund's adjuvant (CFA)
were purchased from Sigma Chemical (St. Louis, MO, USA). Chemical
reagents, such as sodium chloride, potassium chloride, sulfuric acid,
and dipotassium hydrogen phosphate were from Shanghai Guoyao
Chemical Reagent Co. The mouse monoclonal antibody isotyping
reagents were purchased fromInvitrogen (Carlsbad, CA, USA). The sh
samples of Carassius auratus, Oreochromis niloticus, Ictalurus punctatus,
Scophthalmus maximus, Eriocheir sinensis, Penaeus vannamei, Macro-
brachium rosenbergii, Trionyx sinensis, and Oncorhynchus mykiss were
obtained from a commercial farm in the suburbs of Shanghai, China.
2.2. Generation of mAbs against ciprooxacin
The conjugates of CPFX-BSA and CPFX-OVA were prepared using a
modied Carbodiimide method, as previously described (Huang et al.,
2008), and characterized by spectrophotometer absorption, electro-
phoresis, and mass spectra. Briey, a total of 8 mg BSA, 20 mg CPFX
and 240 mg EDC were mixed in 4 ml PBS (pH 5.0, 0.01 mol/l) at 28 C
for 2 h. The mixture was dialyzed against 200 volumes of PBS (pH 5.0,
0.01 mol/l) for two days. The conjugates were lyophilized and stored
at 80 C. Similarly, the conjugate of CPFX-OVA was generated.
The mAbs were generated by a similar procedure described
previously (Wang et al., 2007). Briey, individual Balb/c mice from
the Second Military Medical University were immunized with 200 g
of CPFX-BSA (dissolved in 0.01 mol/l PBS pH 7.4) in 50% CFA and
boosted with the same amount of antigen in 50% IFA for three times.
Subsequently, blood samples were obtained from individual mice for
measuring the CPFX-specic antibodies. After the antibodies titers
reached 1:60,000, the mice were sacriced and their spleen cells were
fused with SP2/0 myeloma cells, followed by screening the hybrido-
mas using the indirect ELISA. Furthermore, the hybridoma cells were
injected into Balb/c mice for the induction of ascites and mAb in the
ascites was puried using a 5 ml protein G sepharose-4B fast ow
column, according to the manufacturer's instruction (Beijing Search
Biotech, Beijing). In addition, the subclass of the mAb was determined
using the mouse monoclonal antibody isotyping reagents and its
afnity was measured, as described previously (Zhou et al., 2009). The
cross-reactivity of anti-CPFX mAb toward quinolone analogues and
antibiotics were assessed (Holtzapple et al., 1997). The experimental
protocols were approved by the Animal Research Protection Com-
mittee of Shanghai Ocean University.
2.3. Immunodiffusion
The glass-plates were poured with 3 ml 1% agar and punched with
holes. Subsequently, the central holes were loaded with antiserum
(1:20) while the peripheral holes were added with single antigen of
CPFX-OVA or OVA, respectively, followed by incubating at 37 C for
24 h. The precipitated antigen/antibody complex was considered as
positive for immunodiffusion assay.
2.4. Sample preparation and extraction technique
The commercial C. auratus, O. niloticus, I. punctatus, S. maximus, E.
sinensis, P. vannamei, M. rosenbergii, T. sinensis and O. mykiss
(5.0 g/sample) were sampled in triplicate and placed into polypro-
pylene centrifuge tubes. The sh samples were mixed with the
extraction solution (30 ml) of 1 M HClacetonitrile (4/500, v/v). After
being vortexed for 5 min and centrifuged at 495 g and 4 C for 15 min,
the supernatants were collected and the residue was re-extracted
with the same procedures again, followed by centrifuging. Subse-
quently, the supernatants were harvested, loaded on a separatory
funnel, and mixed with n-hexane (25 ml). After shaking vigorously for
5 min and then static settlement for 5 min, the aqueous layer was
removed and the organic layer was evaporated for the dryness of the
residues using a vortex evaporator. The resulting residues were
dissolved in 1.0 ml of extraction solution and ltered through 0.45 m
disposable syringe lter equipped with cellulose acetate membranes.
2.5. Indirect competitive ELISA
The CPFX residues in the sh samples were detected using the
mAb-based ic-ELISA, as described previously (Watanabe et al., 2002).
Briey, 150 ng of CPFX-OVA in 100 l of PBS (0.01 mol/l, pH 7.4 were
added into each well of the ELISA plates (Dingguo Biotechnology,
Shanghai, China) and incubated at 37 C for 1 h. After washing for
three times with 250 l washing solution (0.01 mol/l PBS, pH 7.4,
containing 0.5% Tween 20, PBST), the wells were blocked with 1% BSA
in PBST overnight at 4 C. After washing, 50 l of standard CPFX at
different concentrations was rst mixed with 50 l of anti-CPFX mAb
(1:15,000) and the mixture was then added in triplicate to each well,
followed by incubation at 37 C for 1 h. Individual wells with PBST
alone or with anti-CPFX mAb alone were used as negative controls for
the background reading in ic-ELISA. Subsequently, the plates were
washed and the bound mAb was probed with HRP-goat anti-mouse
sera (1/6000 in PBST) at 37 C for 0.5 h. The bound HRP-second
antibodies were detected with the substrate of 100 l of TMB liquid
substrate (1 ml TMB(1.5 mg):5 ml of H
2
O
2
(0.03%):4 ml of 0.2 M NaAc
pH 5.3) for 10 min, and the enzymatic reactions were terminated by
the addition of 2 mol/l sulfuric acid (50 l per well), followed by
reading absorbance at 450 nmin a microplate reader. Standard curves
were obtained by plotting absorbance against the logarithm of CPFX
concentrations, which were tted to a four-parameter logistic
equation: y=(DA)/[1+(x/C)
B
] +A, where A is the minimum
absorbance at innite concentration, B is the curve slope at the
inection point, C is the concentration of IC50, and D is the maximum
absorbance in the absence of CPFX. The limit of detection (LOD) was
calculated as the method, as described by Wang et al. (2007).
The CPFX residues in the prepared samples were also determined
by HPLC using the procedure, similar to that of Tang et al. (2006).
2.6. Recovery rate and relative standard deviation of the assay
The CPFX standard solution was prepared by mixing CPFX with the
metrices of individual sh, as described previously (Duan and Yuan,
2001). Briey, mixing CPFX with the matrices of sh at different ratios
(50 ng/g, 600 ng/g and 1250 ng/g) respectively, the CPFX residues in
the mixture were extracted, as described above. The amount of CPFX
residues in the ltrates was assayed by ic-ELISA and HPLC. The
recovery rate and relative standard deviation (RSD) were calculated
according to the following equations:
Re cov ery % =
Measured value
The oretical value
100%;
RSD =
S
x
100%; S =

xx
2
n1
s
9 K. Hu et al. / Aquaculture 310 (2010) 812
RSD: Relative Standard Deviation; S: Standard Deviation; x : The
average values of determined sample.
Recovery rates and RSD values in the given concentrations were
analyzed by Student T-test. The correlation of the ic-ELISA with HPLC
was determined by logistic regression analysis. A value of Pb0.05 was
considered statistically signicant.
3. Results
To generate mAb against CPFX, the CPFX was conjugated with BSA
by a modied Carbodiimide method with a higher coupling efciency.
The conjugation rates of CPFX-BSA and CPFX-OVA reached 33:1 and
12:1, respectively, which were higher than 13:1 and 6:1 by
conventional method (Zhou et al., 2006). The conjugates were
characterized by spectrophotometric absorption, electrophoresis,
and mass spectra (data not shown). The conjugates were immunized
into Balb/c mice. The blood samples were collected from individual
mice after each boosting and the titers of anti-CPFX sera were
determined by ELISA. Following boosted for three times, the anti-CPFX
sera in most mice reached a titer of 1:62500. The antibodies appeared
to be specically against the CPFX. The agglutination test (Table 1)
indicated that the antibodies were only reacted with the CPFX-OVA,
but not OVA. The high titer of anti-CPFX specic sera in the mice
provided the basis for the generation of mAbs against CPFX.
Following fusion of SP2/0 myeloma cells with the splenic cells
from the immunized mice with high titer of anti-CPFX antibodies,
hybridoma cell lines were screened for antibodies against CPFX and
subjected to cloning by the limited dilution. One of the hybridoma
clones, 1C9, produced high concentrations of mAb and grew stably in
vitro for at least 6 months. To generate large amounts of mAb, the 1C9
clone cells were injected into Balb/c mice for producing ascites and
the mAb 1C9 was obtained from ascites, in which the protein
concentration was 3.25 mg/ml. Characterization of 1C9 mAb revealed
that the 1C9 mAb belonged to IgG2a subclass with an afnity of
3.7510
10
mol/l.
Next, we optimized the concentrations of antibodies and coating
antigens for the ic-ELISA by checkerboard titration. We found that the
experiment using 150 ng/ml of CPFX-OVA for coating antigen and
0.22 g of mAb resulted in the best results. With this experimental
condition, different concentrations of CPFX resulted in a standard
curve in Fig. 1. The four-parameter logistic equation was y=(0.92+
0.07)/[1+(x/2.43)
10.18
] 0.07 while the value of R
2
was 0.997. The
LOD detected by ic-ELISA was 45.25 ng/ml and the linear range was
between50 and 1250 ng/ml witha 50%inhibitionat 245.86 ng/ml. The
specicity of ic-ELISAto other similar antibiotics was assayed by the ic-
ELISA(Table 2). The ic-ELISAdetected 84.6% cross-reactivity with ENR,
a compound with high similarity of structure with CPFX (Fig. 2). In
contrast, the ic-ELISA did not detect any of the other quinolone
analogues and antibiotics tested, because there was no more than
0.01% cross-reactivity of the ic-ELISA to any of other antibiotics.
To test the potential application of ic-ELISA, different shery
samples were mixed with different concentrations of CPFX, and the
recovery rates of CPFX from Carassius auratus, Eriocheir sinensis,
Trionyx sinensis, Oreochromis niloticus, Ictalurus punctatus, Scophthal-
mus maximus, Penaeus vannamei, Macrobrachium rosenbergii or
Oncorhynchus mykiss were determined by ic-ELISA in Table 3. The
recovery rates and RSD of CPFX from these sh samples were in the
range of 80.57%94.61% and 1.78%8.02% respectively. Furthermore,
the recovery rates and RSD of CPFX from different sh samples
reached 84.58%96.54% and 2.47%7.01%, respectively, determined by
HPLC. The recovery rates and RDS values of CPFX by ic-ELISA were
similar to those by HPLC (PN0.05), demonstrating the high sensitivity
and accuracy of ic-ELISA for detecting CPFX in shery products.
4. Discussions
Following the optimization of experimental conditions, we found
that 150 ng/ml of CPFX-OVA for coating antigen and 0.22 g of mAb
Table 1
Titers and specicity assaying results of antisera.
Mouse no. Titers of antisera
a
Immunodiffusion assay
CPFX-BSA
b
CIP-OVA
c
OVA
c
1 1:62,500 Positive Negative
2 1:12,500 Positive Negative
3 1:62,500 Positive Negative
4 1:12,500 Positive Negative
5 1:62,500 Positive Negative
6 1:62,500 Positive Negative
a
The titrations of antibodies in mice after 3
rd
boosting were determined by indirect
ELISA.
b
Immunogen.
c
CIP-OVA or OVA were coated on the plates, respectively, and the specicity of anti-
CPFX sera was characterized by agglutination test.
Fig. 1. Standard curve for detecting CPFX by ci-ELISA. The ci-ELISA was used for
detection of different concentrations (01250 ng/ml) of CPFX and the resulting
absorbance values were calculated as the value of individual concentrations of CPFX
(b)/the value of control without CPFX (b
0
), respectively. The standard curve was
established by different concentrations of CPFX versus the values of b/b
0
. Individual
value represents the meanSD of the concentration of CFPX from three independent
experiments.
Table 2
Cross-reactivity of anti-CPFX monoclonal antibody towards other competitors.
Competitor IC
50
(ng/ml)
Cross-
reactivity %
Competitor IC
50
(ng/ml)
Cross-
reactivity %
CPFX 245.86 100 Saraoxacin N/A b0.01
ENR 290.61 84.6 Furacilin N/A b0.01
Ooxacin N/A b0.01 Chloramphenicol N/A b0.01
Levooxacin N/A b0.01 Malachite green N/A b0.01
Dioxacin N/A b0.01 N/A
N/A means not available.
ciprofloxacin enrofloxacin
HN
N N
N
N N
F
F
O
O
O
O
OH
OH
H
2
C
H
3
C
Fig. 2. Molecular structure of CPFX and ENR.
10 K. Hu et al. / Aquaculture 310 (2010) 812
led to a specic detection of 45.25 ng/ml CPFX with a 50% inhibition at
245.86 ng/ml. Interestingly the ic-ELISA detected 84.6% cross-reactiv-
ity with ENR, a compound with highly structural similarity to the
CPFX, similar to that reported by Duan and Yuan (2001) and Wang et
al. (2007). The cross-reactivity of anti-CPFX with ENR suggested that
the minor difference in the 7th position between CPFX and ENR
(Fig. 2) may have little effect on the recognition and reactivity of anti-
CPFX. Indeed, the position 1 in both molecules contributes to the
predominate antigenicity (Holtzapple et al., 1993, 1997). Importantly,
the ic-ELISA did not detect the other seven quinolone analogues and
antibiotics tested. In addition, the ic-ELISA detected the CPFX with
recovery rates and RSD of 80.57%94.61% and 1.78%8.02%, respec-
tively, which were similar to that determined by traditional HPLC.
Moreover, the sensitivity of ic-ELISA for detection of CPFX was similar
to that reported with mAb (Wang et al., 2007) and higher than that
using polyclonal antibodies (Snitkoff et al., 1998; Duan and Yuan,
2001). Apparently, the ic-ELISA is a highly sensitive and specic assay,
and can be used for the detection of CPFX and ENR in shery products
with high reproducibility.
To prepare optimal shery samples for detection of CPFX, we
extracted the samples with the acidulated acetonitrile, which
improved the extracting efciency, making the extract easy to be
concentrated and puried. Furthermore, we treated the extracts with
n-hexane to maximally remove most insoluble components, such as
sh fats, which usually interfere with the sensitivity and specicity of
ic-ELISA in detecting small molecular compounds. These extraction
strategies should improve the sensitivity and specicity of this assay,
leading to high recovery rates from the shery products.
Notably, ELISA has been demonstrated to be highly sensitive,
specic, and suitable for simultaneous analysis of many samples.
Furthermore, ELISA usually is relatively cheaper than other common
methods and easily performed. In this study, we analyzed the CPFX
residues in different shery products, which were mainly exported
from China, and detected small amount of CPFX in those shery
products within 2 h. Conceivably, the developed ic-ELISA, together
with the extraction protocol, can be used for screening and
characterization of CPFX-contained shery products, thereby improv-
ing the safety of commercial shery products.
In summary, we developed a rapid and sensitive ELISA for the
detection of CPFX and ENR in aquaculture products tissues with a
sensitivity of 10 ng/g. The LOD was below current MRL established by
the EU and China. Therefore, the method can be used as a convenient
tool for the rapid detection of CPFX and ENR in aquaculture products.
Acknowledgements
This study was supported by the grants from the Key Project of
Science and Technology of Shanghai Agriculture Committee
(6660106477) and the earmarked fund for the Modern Agro-industry
Technology Research System of China.
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