Development of monoclonal antibody-based ultrasensitive enzyme-linked immunosorbent assay and fluorescence-linked immunosorbent assay for 1-aminohydantoin detection in aquatic animals

Article abstract of DOI:10.1016/j.jpba.2017.06.068

Monitoring and rapid evaluation of nitrofurantoin metabolite, 1-aminohydantoin (AHD), are important for food safety and human health. Herein, we established the monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) an

Full text of DOI:10.1016/j.jpba.2017.06.068

Accepted Manuscript
Title: Development of monoclonal antibody-based
ultrasensitive enzyme-linked immunosorbent assay and
fluorescence-linked immunosorbent assay for
1-aminohydantoin detection in aquatic animals
Authors: Qi Sun, JinHua Luo, Lei Zhang, Zhihao Zhang, Tao
Le
PII:
S0731-7085(17)31325-0
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jpba.2017.06.068
PBA 11374
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date:
Revised date:
Accepted date:
23-5-2017
28-6-2017
30-6-2017
Please cite this article as: Qi Sun, JinHua Luo, Lei Zhang, Zhihao Zhang,
Tao Le, Development of monoclonal antibody-based ultrasensitive enzyme-
linked immunosorbent assay and fluorescence-linked immunosorbent assay for
1-aminohydantoin detection in aquatic animals, Journal of Pharmaceutical and
Biomedical Analysishttp://dx.doi.org/10.1016/j.jpba.2017.06.068
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Development of monoclonal antibody-based ultrasensitive
enzyme-linked
immunosorbent
immunosorbent
assay
assay
and
for
fluorescence-linked
1
-aminohydantoin detection in aquatic animals
a
b1
a
a
a *
Qi Sun , JinHua Luo , Lei Zhang , Zhihao Zhang , Tao Le
a
College of Life Science, Chongqing Normal University, Chongqing 401331, P. R. China
Chongqing Institute of Biotechnology, Chongqing 401123, P. R. China
b

Corresponding author. Tel.: +86 23 67301531; fax: +86 23 67301531; E-mail address:
hnxylt@163.com.
1
Co-first author.
Highlights
1
. This is the first report of QDs-FLISA to detect AHD developed based on a specific
monoclonal antibody.
2
3
. The FLISA offers higher sensitivity in comparison with ic-ELISA.
. Excellent correlations of the ic-ELISA/LC-MS/MS and FLISA/LC-MS/MS data were
observed for processed samples.
1

Abstract
Monitoring and rapid evaluation of nitrofurantoin metabolite, 1-aminohydantoin (AHD)，
are important for food safety and human health. Herein, we established the monoclonal
antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and
quantum dots (QDs) -fabricated fluorescence-linked immunosorbent assay (FLISA).
Monoclonal antibody specific to nitrophenyl derivative of AHD was derived from
hybridoma cell lines 3.2.4/5A8. For another, CdTe core QDs with emission wavelength of
6
05 nm were also synthesized. The performances of the proposed ic-ELISA and FLISA
were further examined and the corresponding results were also validated by standard
LC-MS/MS analysis. The obtained results indicated that both ic-ELISA and FLISA
exhibited good dynamic linear detection for NPAHD over the range from 0.1 to 3.0 ng
-1
mL . Meanwhile, proposed immunosorbent assays are characterized by satisfactory
recovery rates of 81.5-113.7%. The experimental data suggested these two immunoassays
could be facile, cost-effective and rapid tools for the prospective quantitative method for
AHD analysis in food matrix.
Keywords: Nitrofurantoin metabolite; 1-aminohydantoin; Enzyme-linked immunosorbent
assay; Fluorescence-linked immunosorbent assay; Aquatic animals
2

1
. Introduction
Nitrofurantoin containing a characteristic 5-nitrofuran ring group is a typical
representative of the family of nitrofuran drugs. As a synthetic broad-spectrum antibiotic,
nitrofurantoin was often used extensively both as active ingredient in the treatment of
gastrointestinal infections and as growth promoter in the feeding of food-producing
animals [1-3]. Most of nitro furan compounds, in particular nitrofurantoin, are thought to
be mutagenic as well as carcinogenic, the residue of which is one of the most critical
health problems. Nevertheless, there is still illegal abuse of nitro furan compounds due to
their low cost and high benefit in some countries and regions [4], which is closely related
to food safety and human health. Thus accordingly, human health agencies around the
world have not only listed nitrofurantoin and other nitro furan compounds as prohibited
drugs in animal husbandry [1, 5], but strictly regulated their legal permissible level in
food-producing animals [4, 6]. For example, Europe March 13, 2003 decisions had set the
minimum requirement performance limit (MRPL) at 1 μg kg-1 for each nitrofuran
metabolite residue in poultry meat and aquaculture products [7]. Hence, the efficiency of
both rapid and precise evaluation of nitrofurantoin is of great importance. However, direct
determination of the above mentioned compound is almost impossible to achieve because
it is highly unstable and rapidly excreted in vivo. No parent drug can be detected in tissues
after withdrawal of medication in animals. As illustrated in Fig. 1, 1-aminohydantoin
(
AHD) is a metabolite of nitrofurantoin which has been recognized as a marker residue
for evaluation of nitrofurantoin in food-producing animals [8]. Under normal
circumstances, AHD is covalently bound to tissue proteins, and must be released from the
3

tissues under acidic conditions and derivatized with o-nitrobenzaldehyde to form the
nitrophenyl-AHD (NPAHD), which is a suitable structure for detection [9].
There have been diverse analytical methods developed for the determination of AHD,
for example, liquid chromatography-electrospray ionization tandem mass spectrometry,
liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) [10-18]. The
above mentioned instrumental approaches are widely accepted by many reference
laboratories owing to their high sensitivity and satisfactory accuracy. However, expensive
instruments, time-consuming preparation steps are usually needed for these assays.
Therefore, the laboratory-based techniques may not meet the requirements for the
practical applications in a wide variety of fields. It is necessary to develop measurements
which are characterized by simple operation, low cost as well as high accuracy.
Immunochemical method, by contrast, is widely drawing considerable attention due to
its low cost, fast analysis, excellent feasibility and high specificity, which make it
particularly useful in routine monitoring of food matrix. Recently, using antibodies
specific to a derivate of AHD, an indirect competitive enzyme-linked immunosorbent
assay (ic-ELISA) has also been employed for the determination of AHD; however,
ELISA method lacks stability because of the use of enzyme as a biomarker [4, 19-21]. On
the other hand, quantum dots (QDs) have attracted interest of the biosensing community
due to their unique luminescent properties. Compared to conventional fluorophores, QDs
are regarded as a promising reporters for immunoassay because of their excellent
fluorescent properties, such as size-tunable fluorescence, high quantum yields, broad
adsorption spectra, narrow and symmetric photoluminescence spectra, strong
4

luminescence, and high resistance to photo-bleaching [22-24]. Since QDs possess
excellent and promising luminescent properties, they have been utilized for the sensitive
screening of small molecule chemicals based on the development of QDs-fabricated
fluorescence-linked immunosorbent assay (FLISA) as shown in our previous works [25,
2
6]. The excellent application of quantum dots could greatly improve the detection ability
of targets. To the best of our knowledge, however, there has been no report yet on the
application of QDs as the fluorescence probe for the determination of AHD. Herein, we
established a hybridoma clone as the source of a monoclonal antibody (mAb) that is
specific to derivate of AHD and then applied it to detect AHD with the development of
immunosorbent assays. The performance of the proposed ic-ELISA and FLISA were
examined in this study and the corresponding determination results were validated by
LC-MS/MS as a reference method.
2
. Materials and methods
2
.1. Chemicals and materials
Nitrofurantoin, nitrofurazone, furazolidone, furaltadone, AHD, nitrophenyl-AHD
NPAHD), 2-nitrobenzaldehyde (2-NBA), 4-carboxybenzaldehyle (4-CBA), Freund’s
(
complete and incomplete adjuvants, bovine serum albumin (BSA) and ovalbumin (OVA)
were obtained from Sigma-Aldrich (St. Louis, MO). Peroxidase-labeled goat anti-mouse
IgG (IgG-HRP) was supplied by Sino-American Biotechnology (Shanghai, Branch,
China). N,N-dicyclohexylcarbodiimide, N,N-dimethylformamide, N-hydroxysuccinimide,
tween 20, methanol, dimethyl sulfoxide and ethyl acetate were obtained from Sinopharm
5

Chemical Reagent Co. Ltd (Beijing, China).
2
.2. Synthesis of haptens
As demonstrated in Fig.2, carboxyphenyl derivative of AHD (CPAHD) is obtained by the
reaction between AHD and 4-CBA. 1.5 mL of DMF was added to 0.65 g of 4-CBA in 6
mL of methanol with stirring. Then AHD (0.45 g) was added to the reaction mixture and
refluxed for overnight at 65 °C under stirring. Formation of the target products were
monitored by thin-layer chromatography. After the reaction was over according to
thin-layer chromatography, the reaction mixture was filtered and washed with ethanol
several times to remove any unreacted 4-CBA. The CPAHD conjugate was stored at 4 °C
for the following experiment.
2
.3. Preparation of immunogen and coating antigens
Synthetic routes to the derivatives and conjugates are shown in Fig.2. Briefly, 12.4 mg of
CPAHD, 11.6 mg of N-hydroxysuccinimide and 20.6 mg of
N,N-dicyclohexylcarbodiimide were dissolved in 500 µL of N,N-dimethylformamide.
Then the reaction mixture was stirred gently overnight at 4 °C and centrifuged at 2500
rpm for 10 min. Subsequently, the obtained supernatant was added dropwise to 90 mg of
BSA or 60 mg of OVA dissolved in 9.5 mL of phosphate buffered solution (PBS, pH 7.4)
under stirring. The conjugates were stirred at 4 °C for 12 h and purified on Sephadex
G-25. The eluted conjugates were then dialyzed against PBS (0.01 M, pH 7.4) at 4 °C for
3
days and then stored at 4 °C. The formed CPAMOZ-BSA and CPAMOZ-OVA were
used as immunogens and coating antigens, respectively.
6

2
.4. Production of mAb
All animal experiments in this study adhered to the Chongqing Normal University animal
experiment center guidelines and were approved by the Animal Ethics Committee
(
CQNU-2015-02-003). Eight BALB/c female mice (6-8 weeks old, supplied by the
Chongqing Center for Disease Control and Prevention) were immunised with
CPAHD-BSA conjugates. The first dose consisted of 50 μg of CPAHD-BSA that was
intraperitoneally injected in an emulsion of PBS and complete Freund’s adjuvant (1:1,
v/v). It was then injected subcutaneously into multiple sites on the back of each mouse.
After three booster doses of CPAHD-BSA (50 μg) in incomplete Freund’s adjuvant at
2
-week intervals, the antiserum titer was determined by a non-competitive indirect ELISA.
Three days before cell fusion, the mouse exhibiting the highest titer and specificity was
immunized with a final dose of CPAHD-BSA (100 g) in distilled PBS.
Cell fusion procedures were performed as previously described with some
modifications [26]. Hybridoma cell lines were produced through the fusion of myeloma
cells (Sp2/0) at a ratio of 5-10:1, and spleen cells obtained from immunized mice.
Hybridoma from wells having a positive response in the ELISA described below were
cloned three by limiting dilution method. Stable antibody-producing clones were
expanded until monoclones were obtained. After cell culture, the hybridoma cell was
intraperitoneally injected into paraffin-primed mice to produce ascitic fluids. Ascitic
fluids were collected purified by the caprylic acid and ammonium sulfate precipitation
method followed using a protein G affinity column (Amersham Biosciences, Uppsala,
o
Sweden) according to the manufacturer’s protocol. The purified mAb was stored at -20 C
7

before use.
2
.5. The procedures of ic-ELISA
The protocol of ic-ELISA was carried out using the methodology described previously
with a slight modification [26]. Maxisorp 96-well microtiter plates were coated with
-1
-1
CPAHD-OVA (200 µg L ) in 100 µl of coating buffer (0.05 mol L carbonate buffers, pH
9
0
5
.6) overnight at 4 °C. After the plates were washed three times with PBS containing
.1% Tween-20 (PBST), and the excess binding sites were blocked with 5% glycine and
% sucrose in PBS (250 µL per well) for 3 h at 37 °C. Then, the plates were washed with
PBST, and then NPAHD (as the competitor) was serially diluted in PBS at different
concentrations or sample extracts were added at 50 µL per well, and 50 µL per well of the
ant-NPAHD antibody was added to a microtiter plate. After incubation at 37 °C for 30
min, the plates were washed with PBST, and then 100 µL of HRP-IgG (1:5000) were
added and incubated at 37 °C for 30 min. After washing the plate with PBST, 100 µL of
the TMB substrate solution were added to each well and incubated for 15 min at 37°C in
the dark. Then, the reaction was stopped with 50 μL of 2 M H
measured at 450 nm using a Tecan Sunrise 2.5 Microplate Reader (SUNRISE, Austria). A
calibration curve was generated in the form (B/B ) × 100% vs. log C, where B and B
2
SO
4
. The absorbance was
0
0
were the absorbance of the analyte at the standard point and at zero concentration of the
analyte, respectively. The 50% inhibition concentration (IC50) was determined as a
measure of the sensitivity of the ic-ELISA [3]. The limit of detection (LOD) was based on
2
0 blank samples accepting no false positive rates.
2
.6. Determination of cross-reactivity
8

The extent of cross-reactivity (CR) was evaluated by determining IC50 values using the
ic-ELISA described above. Several nitrofuran analogues (nitrofurantoin, furazolidone,
furaltadone, and nitrofurazone) and their metabolites (1-aminohydantoin (AHD),
3
-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethy
l-2-oxazolidinone
(
AMOZ), and semicarbazide (SEM), nitrophenyl derivates (NPAHD, NPAOZ, NPAMOZ,
and NPSEM), carboxyphenyl derivative (CPAHD, CPAOZ, CPAMOZ, and CPSEM), and
derivatizing reagents were selected for testing CR (Table 1). The concentrations of the
-1
above compounds covered the range from 0.01 to 1,000 ng mL . CR values were
calculated as follows: % CR = [(IC50 value of NPAHD) / (IC50 value of other analytes)] ×
1
00%. The mAb with the lowest IC50 for NPAHD was selected for the remainder of this
study.
2
.7. Synthesis of CdTe core QDs
CdTe/CdSe core/shell nanostructure QDs with emission at 605 nm were synthesized
according to a previously reported method [27]. Briefly, 182 mg of CdCl ·2.5H O was
dissolved in 40 mL of toluene, ultrapure water in a round bottom flask, and 240 mg
glutathione, 10 mg trisodium citrate dehydrate, 2 mL of Na TeO and 20 mg of NaBH
2
2
2
3
4
were added into cadmium sol with constant stirring. The pH was adjusted to 10.5 with 1
M of NaOH. The mixtures were kept in ultra-sonicator for vigorous stirring at room
temperature for 30 min. All reactions were carried out under ambient atmospheric
conditions. The as-obtained mixture was prepared at 600 W under microwave irradiation.
Mixture reaction time increased with the time interval of 20 s. Series of high-quality
CdTe QDs was cooled down to 50 °C. The CdTe QDs were precipitated using 2-propanol
9

and the solution was centrifuged at 4,000 rpm for 5 min. The CdTe QDs dispersion was
resuspended in PBS buffer at pH 7.4. The CdTe QDs were characterised by the Unicam
UV500 UV–vis spectrophotometer (Thermo Spectronic, USA) and luminescence
spectrometer LS-55 (Perkin-Elmer, Inc., Waltham,MA).
2
.8. Preparation of mAb-QDs conjugates
The formation of antibody-conjugated QDs was synthesized using the activated ester
approach. Briefly, 0.1 mL of QDs diluted in 1 mL of reaction buffer (10 mM sodium
borate, pH7.4), was pre-activated with 0.05 mL of a freshly prepared aqueous solution of
-1
1
-ethyl-3-(3-dimethylaminopropyl) carbodiimide (10 mg mL ) for 20 min at room
temperature. Then, 0.2 mg of anti-NPAHD mAbs were added to the carboxyl-activated
QDs and incubated for 30 min at room temperature. Then, 200 mL
N-hydroxysulfosuccinimide solution was added and the same mixture was reacted at 4 °C
for a further overnight incubation. The resulting mixture was centrifuged three times at
1
4,000 rpm to remove any unbound QD labeled antibodies. Then the precipitates were
resuspended with 10% Triton X-100. The conjugated bio-complexes were characterized
by UV-vis absorption and luminescence spectrometer, and then stored at 4 °C.
2
.9. Competitive indirect FLISA procedure
The same pair of immunoreagents, CPAHD-OVA, were selected to develop the FLISA for
AHD determination. The protocol used for the competitive indirect FLISA was similar to
our previously described procedures with some modifications [25]. The checkerboard
procedure was used to optimize the coating antigen and the primary antibody
10

concentrations. To each well of 96-well maxisorp microtiter plate was added 100 µl of
-1
2
00 µg L of CPAHD-OVA solution in 0.05 M NaHCO (pH 9.6), and incubation was
3
performed overnight at 4 °C. The plates were washed three times with PBST and then
incubated (37 °C) with 200 μL per well of 0.1% gelatin in PBS for 2 h. After the blocking
solution was removed, NPAHD (as the competitor) at different concentrations, or 50 μL of
test sample extract, were then pipetted into the coated microtiter well and then 50 μL each
of mAb-QDs were added into each well. After incubation (37 °C) for 1 h, the plates were
washed three times with PBST. Then the fluorescence intensity was recorded by
SpectraMax M2e microplate reader (Molecular Devices, USA). Using an excitation
wavelength of 335 nm and emission wavelengths of 605 nm, standard curves were plotted
from which IC50 values and LODs for NPAHD were obtained.
2
.10. Samples preparation
The tissue samples were minced, homogenized and fortified with AHD at the content of
-1
0
.06, 0.15, 0.6 and 1.5 µg kg . The samples were then derivatized and extracted
according to the procedure described in the literature. In brief, every fortified sample
1.0 g each) was weighed into 50 mL polypropylene centrifuge tubes, then 3 mL
(
methanol was added. The mixtures were vortexed for 10 s at room temperature, and
were processed for 10 min in a boiling water bath. 4 mL deionized water, 0.5 mL 1 M
HCl, and 100 μL 2-NBA (50 mM) in dimethyl sulfoxide were added to the mixtures.
Samples were vortexed for 30 s and incubated at 37°C overnight. Then the samples
were extracted by mixing with 5 mL of 0.1 M K
2
HPO , 0.4 mL of 1 M NaOH , and 5
4
mL of ethyl acetate, followed by centrifugation at 5000 rpm at 4 °C for 20 min. The
11

ethyl acetate fraction was collected and dried under a stream of nitrogen. Then the
residue was re-dissolved in 300 μL of PBS. The remaining extracts were
simultaneously analyzed by ic-ELISA and FLISA. It was noticed that for 1 g of the
-1
sample fortified with AHD at the content of 0.06, 0.15, 0.6 and 1.5 µg kg , after
pretreatment and substituting in 300 μL of PBS, the final concentration of AHD in the
-1
extracted solutions was theoretically 0.2, 0.5, 2.0 and 5 ng mL .
2.11. Validation of ic-ELISA and FLISA
Validation of ic-ELISA and FLISA was carried out using 20 different samples including
in catfish, carp and shrimp muscle purchased from local markets. The samples were
previously confirmed using LC-MS/MS analysis, to be free of AHD compounds. LOD
determination was based on 20 blank samples, accepting no false positive rates, with an
average +3 standard deviation (SD). The accuracy and precision of the method were
represented by the recovery and coefficient of variation (CV), respectively. Recoveries (%)
of fortified AHD were determined using five fortified duplicate blanks at 0.2, 0.5, 2.0 and
-1
5
µg kg in four different analyses. The recovery (%) was calculated by the following
equation: (conc. measured/conc. fortified) × 100. CVs were determined from the analysis
of the above samples. Analysis of each concentration level was repeated in quintuplicate
over a time span of two months.
2.12. Comparison of ic-ELISA, FLISA and LC-MS/MS
In order to demonstrate the applicability of the ic-ELISA and FLISA for real sample
analysis, the proposed methods were used for the determination of AHD residue in some
12

food samples of seventy unknown samples, including ten carp, six grass carp, nine
crucian carp, five chub, five catfish, six shrimp, eight chicken muscle, seven swine muscle,
nine milk, and five cattle muscle purchased from local markets and supermarkets. The
content of AHD in the above samples was detected by ic-ELISA and FLISA method. At
the same time, the LC-MS/MS was used as a reference method which was offered by EU
[
13].
3
. Results and discussion
3.1. Preparation and identification of immunogen
It is well known that the hapten design of the corresponding immunogen is critical to the
identification of the target analyte by the specific antibody. As small molecule compounds,
both nitrofurantoin and AHD are firstly needed to bind to carrier proteins in order to
render them highly immunogenic. As described above, direct determination of
nitrofurantoin is almost unrealistic owing to its being extremely unstable and rapidly
excreted in vivo. By contrast, AHD, the metabolite of the target molecule, could be bound
to tissue proteins and persist for considerable period of time in tissues. Therefore, AHD is
treated as the target marker for the detection of nitrofurantoin. In order to prevent its
rebinding, AHD is firstly needed to be reacted with some derivative reagents. In general,
the derivative form of AHD is NPAHD, but its structure is not suitable for the preparation
of the immunogen. On the other hand, it is reported that AHD could be reacted with
carboxybenzaldehyle (3-CBA or 4-CBA) in order to form immunizing haptens [4, 19, 20].
Under this circumstance, AHD was derivatized with the attachment of a carboxyphenyl
13

moiety to form CPAHD whose structure is similar to NPAHD. Therefore, AHD was
reacted with 4-carboxybenzaldehyle to form CPAHD so that we could obtain a desirable
hapten mimicking the NPAHD derivative, which was a key step in antibody production
and illustrated in Fig.2. CPAHD could be conjugated with BSA by the carboxylic acid
spacer to form immunogens. It should be noted that both the benzene ring and the
carboxyl group are critical to the preparation of the mAb. As depicted in Fig.3, UV-VIS
spectroscopy results demonstrated the maximum absorption wavelength to be 310 nm,
2
78 nm and 313 nm for CPAHD, BSA, and CPAHD-BSA, respectively. Moreover, the
above results also indicated that carrier protein BSA has been successfully conjugated to
CPAHD and CPAHD-BSA could be used in following animal immunization experiment.
3
.2. Characterization of the monoclonal antibody
After immunization of mice, the value of antiserum titer for mAb against NPAHD was
calculated to be 32,000 by the indirect ELISA assay. Hybridomas cells (hereinafter
referred to as 3.2.4/5A8), which may produce stable and specific mAb against NPAHD,
could be obtained after the operation of cell fusion and cloning. In order to evaluate mAb
sensitivity, the binding and competitive properties of the antibody were examined by
ic-ELISA assay under the optimal conditions. The value of antiserum titers was 2.56 ×104
for these two hybridomas. Meanwhile, the IC50 value of mAb (3.2.4/5A8) was also
-1
evaluated to be as low as 0.57 ng mL for NPAHD. Furthermore, IC50 values and
cross-reactivities of selected compounds tested by ic-ELISA are shown in Table 1. It
could be inferred that immune response of mAb (3.2.4/5A8) presented in this work is
characterized by a high specificity as well as a good sensitivity when compared with those
14

reported antibodies previously [4, 19-21].
3
.3. Characterizations of CdTe QDs and mAb-QD conjugates
The morphology and optical properties of QDs were characterized by high-resolution
transmission electron microscopy and spectrophotometer approaches, respectively. As
shown in Fig.4 (A), prepared QDs are almost uniformly distributed in spherical shape
with the average size of 62 nm. The results for optical properties of QDs and mAb-QD
were also depicted in Fig.4 (B). On one hand, these QDs in our study were characterized
by broad excitation spectra and narrow emission spectra. On the other hand, CdTe QDs
displayed absorption and emission peaks centered at 555 nm and 605 nm, respectively as
illustrated in Fig.4 (B). The absorption and corresponding emission maximum
wavelengths of QDs are greatly blue shifted which could be attributed to the size effect of
nanoparticles. The full width at half maximum of QD was 35 nm with high fluorescent
quantum yield around 50-65%. Furthermore, the fluorescence intensity and the peak
wavelength of CdTe QD and mAb-QD conjugates were also detected by fluorescence
spectrometer method. The obtained result suggested that the mAb-QD conjugates have the
same emission peaks centered at 605 nm as QDs (Fig.4 (B)). The full width at half
maximum of QD and mAb-QD conjugates did not change. It could be inferred that there
is no reunion phenomenon occurred during the course of QDs conjugating to antibody.
Based on the above spectra results, we may conclude that QD conjugates to the specific
antibody were effective. The fluorescent area of mAb-QD was smaller than QD and the
interference of QDs emission wavelength and fluorescence intensity by mAb-QD
conjugates is almost negligible. So, it is possible to develop a FLISH assay with the above
15

mentioned tracer.
3
.4. Sensitivity of the ic-ELISA and FLISA
In this study, both ic-ELISA and FLISA method were established for the detection of
AHD residue. The AHD residue was derivatized into NPAHD for the detection by the
developed ic-ELISA and FLISA methods. The standard solutions of NPAHD were diluted
-1
in PBS to produce the concentration range from 0.01 to 100 ng mL . The standard curve
was established using NPAHD diluted in PBS, rather than a matrix matched calibration
standard, because NPAHD was water-soluble and stable in PBS. The sensitivities of the
ic-ELISA and FLISA methods were evaluated using the values of IC50 and LOD, which
were obtained from the standard curves referred by NPAHD molecule. The competitive
inhibition between anti-NPAHD mAbs and CPAHD-OVA was demonstrated in Fig.5 (A)
which was evaluated by increasing NPAHD concentrations. It is clearly noted that B/B
0
value decreased linearly with the rise of the logarithm level of NPAHD over the range
-1
from 0.1 to 3.0 ng mL (Fig.5 (B)) with respect to ic-ELISA assay. The corresponding
-1
-1
IC50 and LOD values were calculated to be 0.57 ng mL and 0.13 ng mL . Specific
features of FLISA assay are also demonstrated here. IC50 value for NPAHD is 0.44 ng
-1
-1
mL and the detection limit is 0.09 ng mL . The concentration in the range of 0.1 to 3.0
-1
2
ng mL with acceptable correlation coefficients (R =0.9915) is also obtained here. By
comparing the results obtained by both immunoassays with different detection modes, it
could be revealed that FLISA method possesses a higher sensitivity than ic-ELISA assay.
In addition, the detection limit and the dynamic range of the as-developed immunoassays
can meet the requirement for monitoring AHD residue in foodstuffs.
16

3.5. Validation of ic-ELISA and FLISA
The animal tissue samples (including catfish, carp and shrimp) collected from local retail
stores, were analyzed by ic-ELISA and FLISA. The sample preparation of the ic-ELISA
and FLISA method included the following procedures: acid hydrolysis, derivatization,
extraction, enrichment, clean up. The developed ic-ELISA and FLISA method involved
acid hydrolysis for the release of tissue bound residues into the solution. During the
sample preparation, the free AHD residues were reacted with 2-NBA into NPAHD to
increase the molecular mass prior to detection. After the extraction, enrichment and clean
up procedures, the NPAHD was detected using the ELISA and FLISA method. Finally,
the concentration of NPAHD was determined and converted into AHD concentration
according to the following formula: Concentration of AHD = (molecular weight of
AHD/molecular weight of CPAHD) × concentration of CPAHD. The matrix effects are
one of the most common challenges in performing immunoassays on complex samples. In
this study, to evaluate the influence of the matrix on the ic-ELISA and FLISA, animal
tissue samples were diluted with PBS, and the schemes of dilution are shown in Table 2.
The LODs of the ic-ELISA and FLISA for NPAHD in catfish, carp and shrimp samples
-1
-1
were 0.13-0.22 μg kg and 0.09-0.14 μg kg , respectively, which corresponded to
-1
0
.06-0.10 μg kg and 0.04-0.06 of AHD after calculation using the above formula. The
-1
LODs for for the three above mentioned animal tissues were below 0.10 μg kg , which is
lower than the MRPL for AHD residue set by the EU. Recoveries of AHD for both
ic-ELISA and FLISA assay were determined and the results are presented in Table 2. For
ic-ELISA analysis, the recoveries ranged from 81.5% to 113.7% and the CVs were less
17

than 11.8% in various biological matrices. Under the same experimental conditions, the
recoveries ranged from 82.4% to 107.6% with the CVs less than 10.6% for FLISA assay.
It could be indicated that the matrix has no significant effect on the sensitivity of the
ic-ELISA and FLISA followed by the extraction method used in this study. In general, the
commonly accepted recovery value for an ic-ELISA is between 80 and 120% [19].
Percentage recovery tests exhibited excellent recoveries of AHD for proposed
immunological methods (Table 2), indicating that the accuracy and precision of both
proposed assays are within an acceptable range.
3.6. Comparison of ELISA, FLISA and LC-MS/MS method
The analytical performances of the ic-ELISA and FLISA were validated by determining
AHD levels in animal tissue samples. Meanwhile, the concentrations of AHD in samples
were detected following the standard LC-MS/MS method. As shown in Table 3, AHD
was not detected in all studied samples obtained from local markets and supermarkets.
These results suggested that the present ic-ELISA and FLISA are reliable for the detection
of AHD in edible tissues of animals.
4
. Conclusions
The present study reports the production and characterisation of a specificity mAb
3.2.4/5A8) against NPAHD, which showed the cross-reactivity to CPAHD (74.0%) and
(
AHD (14.9%). The mAb can be successfully labeled with CdTe QDs by covalent binding
using EDC. The aim of the present study was to develop rapid, simple, accurate and
sensitive analytical method for the determination of AHD. With respect to two different
18

detection modes, recoveries of 81.5-113.7% were obtained by spiking with AHD blank
animal tissue samples with CVs below 15%. Moreover, the ic-ELISA and FLISA were
also validated by LC-MS/MS with good correlation. The results obtained here suggested
these two immunoassays could be considered as feasible quantitative assays for AHD
evaluation in food-originating animals.
Disclosure statement
The authors declare no competing financial interest.
Acknowledgments
This project was supported by the Scientific and Technological Research Project of
Chongqing China (Project No. cstc2016shmszx80069); China Scholarship Council (CSC
No. 201608505051); the National Natural Science Foundation of China (Grant No.
3
1671939) and Chongqing Normal University.
Appendix A. Supplementary materials
Routes of synthesis of CPAHD, NPAHD, CPAHD-BSA, and CPAHD-OVA; Ultraviolet
wavelength scanning of CPAHD, BSA and CPAHD-BSA.
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Figure Captions
Fig. 1. The mojiangcular structures of nitrofurantoin, AHD, CPAHD, NPAHD, 4-CBA
and 2-NBA.
Fig. 2. Routes of synthesis of CPAHD, NPAHD, CPAHD-BSA, and CPAHD-OVA.
CPAHD and NPAHD were respectively derived with 4-CBA and 2-NBA from AHD.
CPAHDZ was used to synthesize the immunogen and coating antigen, while NPAHD was
the target sample derivative.
Fig. 3. Ultraviolet wavelength scanning of BSA, CPAHD and CPAHD-BSA.
Fig. 4. Characterization of the structural and optical properties of QD. (A) the HRTEM of
QDs; (B) UV-vis absorption and fluorescence spectra curve of QD and mAb-QD. The
emission peak is at 605 nm, with full width at half-maximum of 35 nm.
Fig. 5. The standard curve for NPAHD using ic-ELISA and FLISA. B and B
0
are the
absorbances of the sample with/without NPAHD, respectively. Each value shows the
-1
mean (S.D.) of B/B
0
(n =5). The linearity was good from 0.1 to 3 ng mL which has a
linear regression equation and acceptable correlation (R2).
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