Generic Hapten Synthesis, Broad-Specificity Monoclonal Antibodies Preparation, and Ultrasensitive ELISA for Five Antibacterial Synergists in Chicken and Milk

Article abstract of DOI:10.1021/acs.jafc.8b03834

An antibody with broad specificity and principally depending on hapten structure and size is a key reagent for developing a class-selective immunoassay. In the present study, three new generic haptens of antibacterial synergists (ASGs) were proposed using

Full text of DOI:10.1021/acs.jafc.8b03834

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New Analytical Methods
Generic haptens synthesis, broad-specificity monoclonal
antibodies preparation and ultrasensitive ELISA
for five antibacterial synergists in chicken and milk
Hongfang Li, Shaoqin Ma, Xiya Zhang, Chenglong Li, Baolei Dong, Mari Ghulam Mujtaba,
Yujie Wei, Xiao Liang, Xuezhi Yu, Kai Wen, Wenbo Yu, Jianzhong Shen, and Zhanhui Wang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03834 • Publication Date (Web): 25 Sep 2018
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Generic hapten synthesis, broad-specificity monoclonal
antibodies preparation and ultrasensitive ELISA for five
antibacterial synergists in chicken and milk
a
b
a
a
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Hongfang Li , Shaoqin Ma a, Xiya Zhang , Chenglong Li , Baolei Dong , Mari
Ghulam Mujtaba , Yujie Wei a, Xiao Liang , Xuezhi Yu , Kai Wen , Wenbo Yu ,
a
c
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Jianzhong Shen a, Zhanhui Wang a,*
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a Beijing Advanced Innovation Center for Food Nutrition and Human Health, College
of Veterinary Medicine, China Agricultural University, Beijing Key Laboratory of
Detection Technology for Animal-Derived Food Safety Beijing Laboratory for Food
Quality and Safety, 100193 Beijing, People’s Republic of China
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College of Food Science and Technology, Henan Agricultural University, 450002
Zhengzhou, People’s Republic of China
c College of Veterinary Medicine, Qingdao Agricultural University, 266109 Qingdao,
People’s Republic of China
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* Author to whom correspondence should be addressed
Tel: +86-10-6273 4565
Fax: +86-10-6273 1032
E-mail: wangzhanhui@cau.edu.cn
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ABSTRACT： An antibody with broadꢀspecificity and principally depending on
hapten structure and size is a key reagent for developing a classꢀselective
immunoassay. In the present study, three new generic haptens of antibacterial
synergists (ASGs) were proposed using trimethoprim as the start molecule. These
haptens contained carboxyl groups on the meta position of trimethoxybenzene for
conjugating to protein, while, the common moiety of ASGs, i.e., diaminopyrimidine,
was intentionally and maximally exposed to the immune system in animals in order to
induce antibodies with broadꢀspecificity against ASGs. Five monoclonal antibodies
(mAbs) were finally obtained, and 5C4 from the hapten with a short spacer arm,
named Hapten A, showed not only uniform broad specificity but also high affinity to
all five ASGs. We further determined the possible recognition mechanism of mAbs in
terms of conformational and electronic aspects. An indirect competitive ELISA
(icELISA) basedꢀ5C4 was established and exhibited IC50 values of 0.067–0.139 ꢁg
L−1 with crossꢀreactivity (CR) of 48.2%–418.7% for the five ASGs in buffer under
optimal conditions. The calculated limits of detection of the icELISA for chicken and
milk were 0.06 ꢁg kg−1–0.8 ꢁg kg−1 and 0.05 ꢁg L−1–0.6 ꢁg L−1, respectively. The
recoveries in spiked chicken and milk samples were 75.2%–101.4% with a coefficient
of variation less than 14.3%. In conclusion, we developed, for the first time, a rapid
and reliable icELISA for ASGs with significantly improved sensitivity and
classꢀselectivity.
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KEY WORDS: antibacterial synergists, generic haptens, broad specificity antibodies,
high sensitivity, food samples
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43 ꢀ INTRODUCTION
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Antibacterial synergists (ASGs, shown in Figure 1a), also known as sulfonamide
potentiators, are a group of dihydrofolateꢀreductase inhibitors that function by
blocking folic acid metabolism in bacteria.1 ASGs are commonly used in combination
with sulfonamides or other antimicrobials, such as fluoroquinolones, βꢀlactams and
aminoglycosides, and are efficient in inhibiting Gramꢀpositive and Gramꢀnegative
bacteria.2ꢀ4 To date, the ASG family consists of 9 drugs, including trimethoprim
(TMP), diaveridine (DVD), brodimoprim (BOP), ormetoprim (OMP), baquiloprim
(BQP), tetroxoprim (TXP), metioprim (MTP), aditoprim (ADP) and iclaprim (ICL).
Of these ASGs, ADP and DVD are exclusively used in animals, while, ICL is used in
humans and the remainder are both human and veterinary medicines.1,5ꢀ8 DVD, OMP,
and especially TMP are the most commonly used medicines in animal husbandry for
the treatment of bacterial and protozoal diseases.9 For example, compounds of
TMP/sulfadiazine are used for urinary, gastrointestinal and respiratory tract infections
and compounds of DVD/sulfaquinoxaline are used for antiprotozoal drugs in food
animals.4
However, the irrational and excessive use of ASGs could result in residues in
the food chain. The residues of ASGs create a potential threat to human health and
ecosystem, including chromosome variations, thrombocytopenia, leukopenia, emesis,
nausea and antibioticꢀresistance.10 Therefore, the maximum residue limits (MRLs) of
ASGs in some food matrices have been established. The MRLs for TMP have been
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set at 50 µg kg−1 in edible tissues including muscle, liver, kidney, fat and milk in
most countries and the MRL for DVD in chicken is 50 ꢁg kg−1 in Japan. The MRL
of BQP has been set at 30 µg kg−1 (in milk) and 50 µg kg−1 (in porcine liver) by
European Union.11ꢀ14 Thus, a sensitive and reliable method is essential to be
established to detect residues of ASGs in animalꢀderived foods. Instrumental
methods are currently the most commonly reported techniques for the quantification
of ASGs, and include gas chromatography and mass spectrometry,15 high
performance liquid chromatography–tandem mass spectrometry1,16 and
highꢀperformance liquid chromatography.17 Most of these instrumental methods are
used for the simultaneous detection of TMP and sulfonamides rather than ASGs
alone. Although instrumental methods are accurate and sensitive, complex extraction
steps are always necessary, which are not appropriate for screening numerous test
samples in a short term. As an effective alternative, immunoassay methods, just like
enzymeꢀlinked immunosorbent assay (ELISA), possess the merit of simplicity and
costꢀeffectiveness, which make it particularly convenient to achieve screening
purposes. Nowadays, simultaneous detection of several analytes using immunoassay
has attracted increasing attention. Kuang’s research group worked on the
development of multiꢀanalyte immunoassays, i.e., the simultaneous detection of 26
sulfonamide, 32 (fluoro) quinolones and so on.18ꢀ21
At present, a few immunoassays have been developed to analyze TMP residues
only.22ꢀ27 For effective surveillance, an icELISA which can detect multiple ASGs at
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the same time would be preferable. To realize this goal, the preparation of antibodies
with broadꢀspecificity for ASGs is essential. To author’s knowledge, there exist no
reports on the production of antibodies with broadꢀspecificity or the development of
a classꢀselective ELISA. Hapten design is a critical procedure for generating
antibodies with the desired affinity and specificity. As shown in Table 1, two haptens
were synthesized to produce antibodies against TMP and showed negligible
crossꢀreactivity (CR) with other ASGs. In the present study, we prepared three
generic haptens of ASG with different spacer arms and broadꢀspecificity mAbs
against ASGs were obtained. An ultraꢀhigh sensitive indirect complete (icELISA)
based on the broadꢀspecificity mAbs was developed for the simultaneous detection
of five ASGs (TMP, DVD, OMP, BOP and BQP) in chicken and milk samples. The
performance of the icELISA was assessed using fortified and recovery studies.
98 ꢀ MATERIALS AND METHODS
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Materials. TMP, DVD and OMP were bought from Dr. Ehrenstorfer Gmbh
(Ausburg, Germany). BOP and 3, 4, 5ꢀtrimethoxycinnamic acid, 5ꢀ(3, 4,
5ꢀtrimethoxybenzoyl)ꢀ2, 4ꢀpyrimidinediamine (TBP), 3, 4, 5ꢀtrimethoxyphenylacetic
acid, and gallic acid trimethyl ether were obtained from J & K Chemical Technology
(Beijing, China). BQP, 2, 4ꢀdiaminopyrimidine, 2, 4ꢀdiaminoꢀ6ꢀ(4ꢀfluorophenyl)
pyrimidine, 5ꢀphenylꢀ4ꢀpyrimidinamine, pyrimethamine, 3ꢀ(3, 4, 5ꢀtrimethoxyphenyl)
propionic acid, sulfaguanidine, sulfamethizole, sulfamerazine, norfloxacin and
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furazolidone were supplied by Sigma–Aldrich (St. Louis, MO, USA). Hemocyanin
from megathura crenulata (KLH), ovalbumin (OVA), bovine serum albumin (BSA),
hypoxanthine aminopterin thymidine (HAT), complete and incomplete Freund’s
adjuvant, poly (ethylene glycol) (PEG) 1500, fetal calf serum were acquired from
Sigma–Aldrich (St. Louis, MO, USA). Goat antiꢀmouse IgG (HRP labeled) and
mouse mAb isotyping kit were obtained from BioꢀRad Laboratories, Inc. (Hercules,
CA, USA). Cell culture medium (DMEM) was supplied by Thermo Fisher Scientific
Inc. (Waltham, MA, USA). TMB substrate, N, Nꢀdicyclohexylcarbodiimide (DCC)
and Nꢀhydroxy succinimide (NHS) and were purchased from Beyotime (Shanghai,
China). The remaining chemical reagents were supplied by Sinopharm Chemical
Reagent, Inc. (Beijing, China). Flatꢀbottomed highꢀbinding polystyrene ELISA plates
were obtained from Corning Life Sciences (New York, NY, USA). The buffer
solutions used are listed in Supporting Information.
Instruments. The chemical structures of the haptens were confirmed by liquid
chromatography triple quadrupole mass spectrometry (Agilent Technologies, Santa
Clara, USA) and nuclear magnetic resonance spectrometry (NMR) DRXꢀ600 (Bruker,
Rheinstetten, Germany). The conjugation ratio was determined by matrixꢀassisted
laser desorption ionization time of flight mass spectrometry (MALDI–TOFꢀMS)
(Bruker, Rheinstetten, Germany). Values of optical density (OD) were measured
using Multiskan FC microplate reader (Thermo Scientific).
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Synthesis of Haptens and Conjugates. A schematic representation of the
synthesis of three haptens is shown in Figure 2.
Hapten A, 4-(4-((2, 4-diaminopyrimidin-5-yl) methyl)-2, 6-dimethoxyphenoxy)
butanoic acid (3), was synthesized as follows: Briefly, TMP (20 g, 68.9 mmol) was
firstly added to the HBr (48%, 240 mL) in batches and then stirred at 100°C for 30
min. Then KOH solution (50%, 67 mL) was slowly added to the hot HBr solution to
quench the reaction. Then, mixture was cooled to room temperature followed by
stirring slowly to precipitate a white solid. The precipitate was filtered and then
washed by ice water. The pH of the mixture was then neutralized to 7 using ammonia
(28%) followed by filtering, washing and drying steps. The crude product was
chromatographed and eluted with dichloromethane/methanol (20/1) to afford
compound (1) (2.9 g, yellow powder). Cs2CO3 (6.84 g, 21 mmol) was added to a
solution of compound (1) (2.9 g, 10.5 mmol) in DMF (35 mL) and stirred at room
temperature for 20 min. Ethyl 4ꢀbromobutyrate (3.29 g, 15.8 mmol) was added into
the solution of compound (1) and the resulting mixture was stirred and refluxed at
40°C for 2 h. The mixture was extracted by ethyl acetate four times when it was
cooled to room temperature. The obtained organic phases were combined,
successively washed by water, saturated aqueous NaHCO3 solution, saturated brine
and then dried using anhydrous sodium sulfate. The crude product was
chromatographed and eluted with the mixture of dichloromethane/methanol (25/1) to
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afford compound (2) (3.2 g, yellow powder). Then, NaOH aqueous solution (2 mol
L−1, 24.6 mmol) was added to a methanol solution of compound (2) (3.2 g, 8.2 mmol
in 35 mL) and stirred at 45°C for 2 h. After the mixture was cooled, methanol was
rotary evaporated. The pH of the crude product which was dissolved in 8 mL water
was neutralized to 7 using 1 mol L−1 HCl aqueous solution. The yellow precipitate
which was obtained after stirring for 30 min was filtered and dried to obtain the final
1
product of Hapten A (2.6 g, yellow powder). MS m/z 363.2. [MꢀH]ꢀ; H NMR (400
MHz, DMSO–d6): δ 7.50 (s, 1H), 6.54 (s, 2H), 6.19 (s, 2H), 5.86 (s, 2H), 3.80 (t, J =
6.0Hz, 2H), 3.70 (s, 6H), 3.52 (s, 2H), 2.41 (t, J = 7.2Hz, 2H), 1.75–1.82 (m, 2H).
Hapten B, 6-(4-((2, 4-diaminopyrimidin-5-yl) methyl)-2, 6-dimethoxyphenoxy)
hexanoic acid (5). Hapten B was synthesized similar to the previous procedure.
Compound (1) was reacted with ethyl 6ꢀbromohexanoate to obtain Hapten B (2.5 g,
1
yellow powder). MS m/z 391.2. [MꢀH]ꢀ, H NMR (400 MHz, DMSOꢀd6): δ 7.50 (s,
1H), 6.54 (s, 2H), 6.22 (s, 2H), 5.84 (s, 2H), 3.76 (t, J = 6.4Hz, 2H), 3.70 (s, 6H), 3.52
(s, 2H), 2.21 (t, J = 7.2Hz, 2H), 1.51ꢀ1.60 (m, 4H), 1.38ꢀ1.44 (m, 2H).
Hapten C, 4-((4-((2, 4-diaminopyrimidin-5-yl) methyl)-2, 6-dimethoxyphenoxy)
methyl) benzoic acid (7). Hapten C was prepared with the similar procedure to that of
Hapten A. Compound (1) was reacted with ethyl 6ꢀbromohexanoate to obtain Hapten
1
C (4.0 g, yellow powder). The MS m/z 411.1. [MꢀH]ꢀ; H NMR (400 MHz,
DMSOꢀd6): δ 7.93 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.45–7.49 (m, 3H),
7.15 (s, 2H), 6.64 (s, 2H), 4.93 (s, 2H), 3.75 (s, 6H), 3.59 (s, 2H).
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The three haptens with carboxylic acid groups were activated and coupled to a
protein (KLH, BSA or OVA) through the activeꢀester method. Firstly, 20 mg of each
hapten was dissolved in 0.6 mL DMF with the addition of NHS (10 mg) and DCC (20
mg). The mixture was reacted using a magnetic stirrer at 4°C for 5 h followed by the
removal of the precipitates. The supernatant of each hapten was divided into three
parts and then dropwise added into dissolved KLH solution (20 mg KLH in 9.5 mL
PBS), BSA solution (20 mg BSA in 9.5 mL PBS), and OVA solution (50 mg OVA in
10 mL PBS), respectively. The solutions containing the haptenꢀprotein conjugates
were stirred for 12 h and then dialyzed in PBS for 2 days. HaptenꢀKLH served as an
immunogen and was stored at −70°C. HaptenꢀBSA and haptenꢀOVA acted as coating
antigens and then were stored at −20°C. The conjugation ratios of haptensꢀBSA were
characterized using MALDI–TOFꢀMS and were calculated as follows:
Conjugation ratio = {M (conjugates)－M (BSA)}/M (haptens) (Eq.1).
Production of Monoclonal Antibodies. All animal treatments were in strict
accordance with Chinese laws and guidelines which were approved by the Animal
Ethics Committee of China Agricultural University. Fifteen eightꢀweek old female
BALB/c mice were divided into three groups randomly (five mice per group) and
were immunized with three immunogens (Hapten AꢀKLH, Hapten BꢀKLH and
Hapten CꢀKLH). The mice were immunized three times with an interval of three
weeks between immunizations. First immunization, 100 µg prepared immunogen (1 g
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Lꢀ1 in PBS) was mixed and emulsified with equal complete Freund’s adjuvant. The
prepared mixture was then injected into BALB/c mice (subcutaneously, 0.2 mL per
mouse). For the second immunization, the mice were boosted with the mixture of
immunogen (100 µg) and incomplete Freund’s adjuvant. The procedure for the third
immunization was the same as that for the second. Ten days after the third
immunization, antisera samples (approximately 50 ꢁL) obtained from the eye socket
of mice were collected to detect the sensitivity and specificity based on the icELISA.
The mice with a low IC50 and broadꢀspecificity were selected for subsequent cell
fusion. The cell fusion procedures were carried out as described previously.28, 29 The
selected mice were injected intraperitoneally with three times the immune dose (300
µg immunogen in 500 µL PBS buffer). Three days later, SP2/0 myeloma cells and
splenocytes from the selected mice were mixed together for cell fusion under the
assistance of PEG 1500. The successfully fusion cells were cultured in HAT medium
for 7 days and the cell supernatant was screened using the icELISA. The homologous
coating antigen (with a carrier protein of BSA) was used and the concentration was
0.35 mg Lꢀ1 for icELISA. The positive hybridoma with high sensitivity was then
subcloned using the limiting dilution method. Subcloning was carried out three times
and the optimum single cell mass was collected and cultured. Finally, one part of the
hybridoma was cryopreserved, and another part was collected to produce ascites via
intraperitoneal injection. The ascites collected from mice were extracted and purified
by the saturated ammonium sulfate.30 The isotypes of the purified mAbs were
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characterized with the mice mAb isotyping kit.
Development of ELISA. The ELISA plates were firstly coated with coating
antigen (100 ꢁL well−1) which was diluted with CB buffer and then incubated at 4°C
for 12 h. The coating solution in the ELISA plates was then discarded. Blocking
buffer was then added to the plates (150 ꢁL well−1) which were placed in an incubator
for 2 h at 37°C. The standard solution (50 ꢁL) of TMP (or other competitors) was
added to the wells as well as 50 ꢁL of diluted mAbs solution. After an incubation of
30 min at 37°C, the plates were washed by PBST for two times. Goat antiꢀmouse IgG
(HRP labeled) (1:5000, 100 ꢁL well−1) was then added and incubated for 30 min at
incubator. 100 ꢁL TMB substrate was added into wells and incubated for 15 min at
37°C after washing. Then 2 mol L−1 H2SO4 (50 ꢁL well−1) was used to stop the
enzymatic reaction and the OD values of 450 nm was measured.
The standard curves were fitted using the following equation:
Y = (A–B)/ [1+(X/C) D] +B
(Eq. 2)
where A and B are the responses at high and low asymptotes of the curve,
respectively, C is the concentration of the targets resulting in 50% inhibition. D is the
slope at the inflection point of the sigmoid and X is the calibration concentration.
Nineteen compounds associated with the structure of TMP were diluted to
produce a series of concentrations to estimate the IC50 using the standard curves. The
CR was calculated using the subsequent formula:
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CR = (IC50 of TMP/IC50 of compounds) × 100% (Eq. 3)
Optimization of ELISA. To improve the performance of the ELISA, the
concentrations and pairing combinations of coating antigen and mAb, some
physicochemical parameters such as pH values, ionic strength and content of organic
solvent were optimized. The effects of these parameters were evaluated by the
Amax/IC50 ratio; Amax represents maximal absorbance, and IC50 was fitted using Eq. 2.
Molecule Alignment and Electrostatic Potential Analysis. All structures were
built in Gaussian 09 (Gaussian, Wallingford, CT, USA) according to the ASGs
configurations in the PubChem database. Then, DFT calculations with the B3LYP
functional and 6ꢀ311++ G (d, p) basis set were performed to optimize the molecules
using the Gaussian 09 package.31ꢀ33 The fundamental vibrations were calculated by
the same method to confirm whether they were true minima or not. TMP was chosen
as the template molecule to align TBP. The Molecule Overlay dialog in Discovery
Studio 2016 (Accelrys Software, Inc., San Diego, CA, USA) was used to conduct
molecular alignment. The alignment rootꢀmeanꢀsquare value was introduced to
measure the degree of molecular superposition in each group. The Gaussian 09 and
Gaussian View 5 packages were employed to conduct the molecular electrostatic
potential (ESP) analysis. The minima and maxima of the ESP were used in the
Multiwfn software package and mapped onto the Van der Waal surface.
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Matrix Effect and Recovery. ASGs negative chicken and milk were both
provided by the National Reference Laboratory for Veterinary Drug Residues
(Beijing, China). Fiveꢀgram chicken (in 50 mL tube) were put in an ultrasonic bath
for 5 min followed by the addition of 50% acetonitrile solution (5 mL for each tube).
Next, the mixture was agitated on a vortex mixer for 20 min and then centrifuged for
10 min at 3500 g. The supernatant was separated and then diluted with PBS to
evaluate the matrix effect using icELISA.22 Milk samples were degreased by
centrifugation for 20 min at 8000 g. Defatted milk was diluted with PBS to eliminate
the matrix effect.29
Blank chicken and milk samples were spiked with TMP, DVD, OMP, BOP and
BQP at three different concentrations, respectively. After pretreatment, the samples
were diluted (20ꢀfold for chicken samples and 15ꢀfold for defatted milk) and
submitted to icELISA for recovery analysis. All the recovery data were detected in
triplicate (N = 3) using icELISA.
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Preparation of Haptens and Conjugates. In previous reports, several strategies
for preparing TMP conjugates were proposed, resulting in antibodies which were
highly specific to TMP with moderate affinity (Table 1). Using those strategies, the
amino groups of TMP were direct derivatives of glutaric anhydride (GA) or maleic
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anhydride, and then coupled to the carrier protein.22ꢀ25 However, the amino groups on
diaminopyrimidine are not typical and are difficult to react with other reagents.
Moreover, the derivation of these two amino groups is not selective, and always
results in mixed and unpurified conjugates. These studies did not obtain purified and
identified haptens. The objective of the present study was to generate mAbs with
broadꢀspecificity for ASGs which can be used to establish a classꢀselective ELISA for
determining the total range of ASGs. To produce antibodies which exhibit the desired
broad specificity, the key is the structure of the haptens which should closely mimic
the common part of target analytes, and maximize the steric, electronic and
hydrophobic similarity to the parent molecules. In the case of ASGs, all analogs share
the common structure of diaminopyrimidine (Figure 1a). Thus, it is highly possible to
generate broadꢀspecificity antibodies against all ASGs by introducing active groups
on the trimethoxybenzene moiety. Of these ASGs, TMP is the most frequently used
ASG and has been used as a start molecule to prepare generic haptens of ASGs.
Previous research showed that haptens that possess spacer arms containing four to six
carbons were acceptable and appropriately sustain epitopes to produce high quality
antibodies.34ꢀ36 Here, we synthesized three novel generic haptens by deriving butyric
acid, caproic anhydride and benzoic acid on the metaꢀOCH3 group of
trimethoxybenzene, and introduced different spacer arms ending with a carboxyl
group, which were named Hapten A, Hapten B and Hapten C, respectively (Figure 2).
All haptens were purified and identified by mass spectrometry and nuclear magnetic
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resonance spectroscopy (Figure S1 and Figure S2), and the results indicated that the
three haptens were successfully synthesized.
The haptens possessing active carboxyl groups were attached to the amino
groups of proteins using the active ester method; thus, the common moiety of ASG
was fully exposed to the immune system and could produce antibodies with
broadꢀspecificity. The BSA conjugates were verified using MALDI–TOF–MS as
KLH and OVA were mixed with an indeterminate molecular weight, which could not
be accurately identified. An observable shift in the peak maximum of BSA conjugates
in comparison to the control protein was observed, proving that the haptens had been
successfully conjugated to the carrier protein (Figure S3). The calculated molar ratios
of the hapten: BSA were2.0:1 (Hapten A: BSA), 8.4:1 (Hapten B: BSA) and 18.9:1
(Hapten C: BSA), respectively. The haptensꢀKLH conjugates were then used as the
immunogen, while, haptensꢀBSA and haptensꢀOVA were used as the coating
antigens.
Characterization of Antisera and mAb. The antisera of three groups of mice
were monitored after the third immunization. Table S1 showed that all immunized
mice had a positive immune response with high titers (1/5000–1/30000). Five ASGs,
including TMP, DVD, OMP, BOP and BQP were used to determine the affinity
(expressed by IC50) and specificity (expressed by CR) of antisera using the
homologous coating antigen. The results indicated that all antisera could recognize
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ASGs with varied affinities (IC50 values of antisera were from 0.11 to 21.32 ꢁg L−1).
The mice, Hapten AꢀKLHꢀ1# and Hapten AꢀKLHꢀ3# exhibited relatively broad
specificity (CR > 42%) against ASGs. Furthermore, the antisera derived from Hapten
AꢀKLHꢀ1# showed generally lower IC50 values than those of Hapten AꢀKLHꢀ3#. In
mice immunized with Hapten BꢀKLH and Hapten CꢀKLH, the antisera showed a
lower CR towards OMP (CR < 36%) and BQP (CR < 19.7%) compared with those
for Hapten AꢀKLH. These results indicated that Hapten A seemed more effective in
producing antibodies with broadꢀspecificity than Hapten B and Hapten C. A possible
reason for this is that caproic anhydride and benzoic acid in the spacer arm of Hapten
A and Hapten B both had large steric hindrance thus resulting in exposure of the
characteristic structure of TMP. Compared with other reports on the production of
TMP,22ꢀ25 all antisera derived from the three immunogens provided lower IC50 values
ranging from 0.22–2.47 ꢁg L−1 for TMP, indicating that the haptens prepared in the
present study had a more potent effect in inducing a strong immune response than the
immunogens previously reported. In addition to the more precise synthesis,
purification and identification of haptens and conjugates in this study, we conclude
that the high potency of these haptens may have been derived from the high polarity
of diaminopyrimidine which was distal to the protein and completely exposed to
immune system. The recognition of antibodies and haptens is mainly dependent upon
hydrogen bonding, electrostatic, and hydrophobic interactions.37 The two amino
groups of diaminopyrimidine could have contributed to the high immune response
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and then induced a high affinity antibody, while haptens reported by other groups
exposed trimethoxybenzene with high hydrophobicity to the immune system,
resulting in relatively lower affinity. However, this will be proved by separately
immunizing diaminopyrimidine and trimethoxybenzene conjugates in our further
research.
For the cell fusion experiments, the best mice were chosen i.e., Hapten
AꢀKLHꢀ1#, Hapten BꢀKLHꢀ4# and Hapten CꢀKLHꢀ3#. After screening and cloning
three times, five hybridomas were obtained. In detail, mAb 5C4 and 9C9 were from
Hapten A, 3B6 and 14G1 were from Hapten B, and 1F1 from Hapten C, respectively.
The isotypes of all mAbs were determined and are shown in Table S2 and the
preliminary characterization of mAbs are shown in Table S3. Although all mAbs
showed highly affinity to TMP with IC50 values below 0.4 ꢁg L−1, only 5C4 showed
broadꢀspecificity towards other ASGs via homologous coating antigens.
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Optimization and Development of icELISA. Aiming to improve the
performance of icELISA, the concentration of mAbs and coating antigen, coating
antigen types and several physicochemical factors related to the buffer were examined.
As to competitive ELISAs, low concentrations of immunoreagents are required to
obtain the best sensitivity.38 Accordingly, the dilutions of immunoreagents were firstly
optimized to get the lowest IC50 and the optimum Amax values in the range of 1.8–2.2
(Amax is the absorbance value without analyte). We use checkerboard assays to study
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the effect of different dilutions for mAbs and coating antigen on the Amax of icELISA.
The optimal dilutions of mAbs and coating antigens are summarized in Table S4,
which shows that any mAbs could recognize any coating antigens and all dilutions of
immunoreagents were above 1/20000. The highest dilutions of mAb and coating
antigen were for mAb 3B6 and homologous coating antigen, Hapten BꢀOVA. It is
well known that heterologous coating antigens usually lead to higher sensitivity and
lower dilution of immunoreagents in a competitive ELISA. As the heterologous
coating antigen could often result in a weak decrease in antibody recognition
compared with the homogenous coating antigen, this allows the analyte to compete
with the heterogeneous coating antigen at lower concentrations and higher sensitivity
can be obtained.39 Thus, the optimal pair of coating antigen types and mAbs were
then tested using a twoꢀpoint (0 and 0.4 ꢁg L−1 TMP) competitive format and were
evaluated by inhibition rate. The higher inhibition rate represents the higher
sensitivity then better pair of coating antigens and mAbs. Figure 3a shows the
inhibition rate of five mAbs and six coating antigens in the presence of 0.4 ꢁg L−1
TMP. The affinities of mAbs (except 1F1) for TMP were improved using
heterologous coating antigens. Interestingly, BSA coating antigens all produced
higher inhibition rates than the OVA counterparts. Thus, Hapten CꢀBSA was selected
for subsequent studies. The detailed parameters of the five mAbs paired with Hapten
CꢀBSA in addition to the IC50 and CRs are listed in Table S1. The results
demonstrated that the mAb 3B6 and 1F1 showed low CRs values for OMP (< 4.4%)
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and BQP (< 3.2%), while, mAb 9C9 and 14G1 showed similar recognition profiles
with TMP (100%), DVD (> 49.6%), OMP (> 52.6%) and BOP (>893%), but not BQP
(CR < 24.6%). The mAb 5C4 recognized all ASGs (TMP, DVD, BOP, OMP and
BQP) with similar high affinity, and was used for further optimization and to develop
a classꢀselective icELISA for the qualitative detection of total ASGs.
Several physicochemical factors were further optimized including pH value (6.0,
6.5, 7.0, 7.4 and 8.0 for PBS), ionic strength (concentrations of NaCl were 0.05, 0.1,
0.2, 0.5 and 1.0 mol L−1) and organic solvent (proportion of methanol and acetonitrile
were 0%, 1%, 5%, 10% and 20%). The Amax/IC50 ratio was chose to be the primary
criterion to evaluate the icELISA and the highest ratio value meant the highest
sensitivity. It can be seen that the ratio of Amax/IC50 was highest at pH 7.4 in the range
6.0 to 8.0 evaluated (Figure 3b). These results illustrated that slight pH differences in
the assay buffer obviously affected the icELISA performance. Similar to pH, the
influence of ionic strength on the icELISA showed a similar tendency. The ratio of
Amax/IC50 was better at an ionic strength of 0.1 mol L−1 (Figure 3c). Deviation from
this concentration was detrimental to the icELISA performance. In addition, organic
solvents are often chosen to extract ASGs from food matrices35, 40. Excessive organic
solvent can result in denaturation of antibodies and lower sensitivity of the icELISA.
Therefore, the assay must be tested to determine the tolerance of organic solvents.
Several buffers containing different concentrations of methanol or acetonitrile were
used for testing. As shown in Figure 3d and Figure 3e, a clear decrease in the
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Amax/IC50 ratio was observed when the concentration of methanol was more than 10%
or the concentration of acetonitrile was more than 5%. The ELISA could be
performed with methanol concentrations up to 10% or acetonitrile concentrations up
to 5%. Thus, the optimum conditions (pH 7.4 and 0.1 mol L−1 NaCl in PBS buffer)
were used in subsequent experiments.
Sensitivity and Specificity of the Optimized icELISA. The sensitivity and
specificity of the icELISA was determined by conducting competitive assays for
ASGs and other analogs (structures shown in Figure 1b). The sensitivity of mAb 5C4
was evaluated using the icELISA calibration curve for ASGs, and showed that the
IC50 was 0.067 ꢁg L−1, 0.076 ꢁg L−1, 0.016 ꢁg L−1, 0.112 ꢁg L−1 and 0.139 ꢁg L−1 for
TMP, DVD, BOP, OMP and BQP, respectively (Figure 3f). The IC50 value of the
developed icELISA was 30 to 100ꢀfold better than other reported immunoassays for
TMP. The limit of detection (LOD) of the icELISA we developed was 2.8 to 300ꢀfold
lower than the LOD or visual limit of detection (vLOD) of reported immunoassays. 23,
24, 27
The specificity of the icELISA was demonstrated by counting CRs against
nineteen compounds, including five ASGs, nine structurally related compounds and
five other antibiotics. It is not surprising that the icELISA showed no CRs (< 0.002%)
for other frequently used antibiotics such as sulfaguanidine, sulfamethizole,
sulfamerazine, norfloxacin and furazolidone. The IC50 values and CR of the icELISA
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for ASGs and other structurally related compounds are summarized in Table S5. It can
be seen that mAb 5C4 only recognized the compounds that possessed both
diaminopyrimidine and metoxybenzene, which was supported by observing the
ignorable CR for 3,4,5ꢀtrimethoxycinnamic acid; gallic acid trimethyl ether;
3ꢀ(3,4,5ꢀtrimethoxyphenyl)propionic acid; 3,4,5ꢀtrimethoxyphenylacetic acid; 2,
4ꢀdiaminoꢀ6ꢀ(4ꢀfluorophenyl)pyrimidine, pyrimethamine and 2, 4ꢀdiaminopyrimidine.
These compounds only contained one moiety of TMP, i.e., diaminopyrimidine or
metoxybenzene. It is reasonable that the mAb could not recognize the compounds
only containing metoxybenzene as this moiety was used to conjugate with protein and
then was blocked when exposed to the immune system. We thought that the mAb
could recognize DAP which was fully exposed to the immune system; however, the
unstable immuneꢀcomplex of mAbꢀDAP may lead to a high dissociation rate constant
resulting in very low affinity.
It should also be noted that diaminopyrimidine and metoxybenzene must be
connected by methylene instead of carbonyl. For example, the structure of TBP is
extremely similar to TMP except for the connect group as mentioned above, and this
difference resulted in an almost 800ꢀfold decrease in the IC50 value for TBP compared
to TMP (Table S5). The recognition profiles of mAb for TBP and TMP were not easy
to explain by only viewing the 2Dꢀchemical structure; thus, we further examined the
possible recognition mechanism of mAb in terms of conformational and electronic
aspects. We aligned TMP with TBP based on their lowest conformations and expected
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that these two molecules would not be well superimposed. As shown in Figure 4a, our
expectation basically agreed with the alignment of these molecules. For concise
purposes, only the backbone of hapten molecules are viewed without hydrogen atoms.
The conformations of TMP and TBP were different to the orientation of
metoxybenzene as we superimposed the common part of diaminopyrimidine. The
dihedral angles between the pyrimidine ring and the benzene ring of TMP and TBP
were 88.67° and 50.79°, respectively. The torsion angles of C7ꢀC6ꢀC9ꢀC11 were 118.6°
for TMP and 16.3° for TBP. The introduction of carbonyl around the connection site
of diaminopyrimidine (C9) resulted in the benzene ring bending away with an
approximately 100° difference in TBP compared with TMP, which may be partly
responsible for the almost 800ꢀfold decrease in mAb recognition ability.
Complementary shape is an essential factor for molecules interacting with mAb, but
not sufficient to complete the stable complex of haptenꢀantibody. The electronic
contribution to antibody recognition was thought to be another important factor. As
seen in Figure 4b, the exit of carbonyl on TBP induced an obvious change in atom
partial charges in comparison with those of TMP, for example, the C2, C4 and C7
carbon (Figure 4b), which were maximally exposed to the immune system. To explain
the difference in TMP and TBP in terms of conformation and electron distribution,
the ESP displayed on van der Waal surfaces of global lowest energy conformation for
TMP and TBP are shown in Figure 4c and 4d (in the same orientation as for Figure
4a). The ESP shows the potential energy of a proton placed at a point near the
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molecule. The most positive potential energy is represented with blue areas which are
repulsive to a proton on the molecules. The most negative potential energy is
represented by red areas which are attractive to a proton. It is clear from Figure 4c
that the surface area in different ESP ranges of TMP and TBP represented similar but
obviously varied electrostatic potential characters. Furthermore, TBP has a stronger
area of negative potential (marked by arrow) which is associated with the carbonyl as
observed in Figure 4d. The comparisons in Figure 4 demonstrated that the connect
group between diaminopyrimidine and metoxybenzene markedly changed the whole
molecular conformations and electron distribution, and then resulted in low affinity.
The icELISA exhibited the highest CR with BOP (418.7%) and showed similar
CR with TMP (100.0%), DVD (88.2%), OMP (59.8%) and BQP (48.2%). Although
the other four ASGs are not commercially available, TXP, MTP, ADP and ICL, we
suggest that the mAb should recognize them with similar affinity to TMP. The
satisfactory CRs indicated mAb 5C4 was a groupꢀspecific antibody against the five
ASGs. The icELISA we developed had significant advantages in terms of sensitivity
and broadꢀspecificity compared with the results of other authors.22, 24ꢀ25
Matrix Effect and Recovery. ASGs are widely used in the feeding of poultry
and dairy cows, thus chicken and milk samples were chosen to determine the matrix
effect and recovery. The matrix effect of samples can influence quantification of the
target. The cleanꢀup step is frequently used to eliminate matrix effects. However,
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direct dilution of samples is a simpler way of eliminating matrix effects in rapid
screening methods, such as ELISA.41, 42 Thus, in this study, the matrix effect of the
extract was expected to be removed just by dilution due to the high sensitivity of the
developed icELISA. Therefore, the pretreated chicken sample was diluted 10 times,
20 times and 40 times with PBS and the defatted milk was diluted 2ꢀfold, 5ꢀfold and
15ꢀfold with PBS, respectively. The standard curves of TMP prepared in diluted
samples were then compared with those in PBS to evaluate the matrix interferences
(Figure 3g, 3h). The influence of the matrix on the performance of the icELISA
decreased as the dilution factor increased. Despite a 40ꢀfold dilution of chicken
samples could farthest improve assay performance in term of Amax, the icELISA
standard curve in 20ꢀfold dilution of chicken samples was more consistent with that in
PBS (Figure 3g). Accordingly, 20ꢀfold dilution of chicken samples in PBS buffer was
chosen to establish the calibration curves. The matrix effects of milk were avoided by
15ꢀfold dilution using PBS buffer (Figure 3h). The calculated limits of detection
(from IC10) were observed at 0.06 ꢁg kg–1ꢀ0.8 ꢁg kg–1 in chicken samples and 0.05 ꢁg
L–1ꢀ0.6 ꢁg L–1 in milk samples, which was below the MRL of ASGs. The linearity
range (from IC20 to IC80) is shown in Table 2.
The recovery and coefficient of variation (CV) of spiked samples were used to
evaluate the effectiveness of sample preparation and utility of the icELISA. Negative
samples were spiked with five individual ASGs (TMP, DVD, BOP, OMP and BQP)
at three different concentrations to determine recoveries with the icELISA (Table 2).
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The recoveries ranged from 75.2% to 94.6% with a CV lower than 13.2% in chicken
samples. The recoveries ranged from 75.3% to 101.4% and the CV between analyses
ranged from 5.7% to 14.3% in milk samples. These results demonstrated that the
icELISA based on mAb 5C4 was ultraꢀsensitive, classꢀselective and reliable for the
detection of ASGs in chicken and milk.
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Support Information
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Buffer used in this article; The structures and mass spectra, and H NMR spectra of
three haptens (Figure S1 S2), MALDI–TOFꢀMS result of haptenꢀBSA (Figure S3);
–
Characterization of antiserum and mAbs (Table S1), the isotypes of mAbs (Table S2),
preliminary characterization of all the mAbs (Table S3), optimum dilution of coating
antigens and mAbs (Table S4), crossꢀreactivity of structurally related compounds with
mAb 5C4 in the icELISA (Table S5).
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ACKNOWLEDGEMENTS
This work is supported by the Beijing Municipal Natural Science Foundation (No.
520
521
6182019) and China Agriculture Research System (CARS–36).
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