Chemiluminescence Reaction Kinetics-Resolved Multianalyte

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Chemiluminescence Reaction Kinetics-Resolved Multianalyte Immunoassay Strategy Using a Bispecific Monoclonal Antibody as the Unique Recognition Reagent Hui Ouyang,†,§ Limin Wang,‡,§ Shijia Yang,† Wenwen Wang,† Lin Wang,† Fengquan Liu,*,‡,∥ and Zhifeng Fu*,† †

Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China ‡ College of Plant Protection, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China ∥ Institute of Plant Protection, Jiangsu Academy of Agricultural Science, Nanjing, Jiangsu 210014, China S Supporting Information *

ABSTRACT: The multianalyte immunoassay (MIA) has attracted increasing attention due to its high sample throughput, short assay time, low sample consumption, and reduced overall cost. However, up to now, the reported MIA methods commonly require multiple antibodies since each antibody can recognize only one antigen. Herein, a novel bispecific monoclonal antibody (BsMcAb) that could bind methyl parathion and imidacloprid simultaneously was produced by a hybrid hybridomas strategy. A chemiluminescence (CL) reaction kinetics-resolved strategy was designed for MIA of methyl parathion and imidacloprid using the BsMcAb as the unique recognition reagent. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were adopted as the signal probes to tag the haptens of the two pesticides due to their very different CL kinetic characteristics. After competitive immunoreactions, the HRP-tagged methyl parathion hapten and the ALP-tagged imidacloprid hapten were simultaneously bound to the BsMcAb since there were two different antigen-binding sites in it. Then, two CL reactions were simultaneously triggered by adding the CL coreactants, and the signals for methyl parathion and imidacloprid detections were collected at 0.6 and 1000 s, respectively. The linear ranges for methyl parathion and imidacloprid were both 1.0−500 ng/mL, with detection limits of 0.33 ng/mL (S/N = 3). The proposed method was successfully used to detect pesticides spiked in ginseng and American ginseng with acceptable recoveries of 80−118%. This proof-of-principle work demonstrated the feasibility of MIA using only one antibody.

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monoclonal antibody (BsMcAb) bearing two different antigenbinding sites can bind with two completely different antigens and, therefore, should be more efficient in MIA. BsMcAb can be obtained by chemical cross-linking,11 recombinant DNA technique,12 or cell fusion.13 It has been widely used for the immunotherapy of different diseases such as acute myeloid leukemia,14 breast cancer,15,16 colon cancer,16 etc. Recently, BsMcAb has also been applied as a novel recognition reagent to conduct a single-analyte immunoassay.17−19 For example, a BsMcAb, which could recognize green fluorescent protein and zinc oxide simultaneously, was bound to a zinc oxide-coated plasmonic chip for sensitive detection of green fluorescent protein.17 Kreutz and Suresh18 developed a BsMcAb that could bind prostate-specific antigen

he multianalyte immunoassay (MIA) has attracted increasing attention in disease diagnosis, food safety, and environmental monitoring due to its high sample throughput, short assay time, low sample consumption, and reduced overall cost.1−10 Most of the reported MIA approaches are based on array and multitag modes. In the array mode utilizing one universal probe, array detectors such as the chargecoupled device camera or multichannel electrochemical workstation are adopted to collect multiple optical1,2,4,5 or electrochemical signals.6,7 Differing from the array mode, the multitag mode demands multiple probes to tag different analytes, and the signals from different probes can be easily distinguished by such parameters as potential,8 wavelength,9 and decay time.10 No matter whether array or multitag mode is adopted, the reported MIA strategies always demand multiple antibodies since each antibody can recognize only one antigen. Differing from the widely used regular monoclonal antibodies, bispecific © XXXX American Chemical Society

Received: December 4, 2014 Accepted: January 26, 2015

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was provided by Dr. Ehrenstodfer GmbH (Germany). Methyl parathion was purchased from Shanghai Pesticide Research Institute (China). Bovine serum albumin (BSA), ovalbumin (OVA), and Dulbecco’s modified Eagle’s medium (DMEM) were all obtained from Gibco (USA). Luminol, 3,3′,5,5′tetramethylbenzidine (TMB), HAT medium (hypoxantin, aminopterin, and thymidin), and complete and incomplete Freund’s adjuvants were purchased from Sigma-Aldrich (USA). N-Hydroxysuccinimide (NHS), N,N′-dimethylformamide (DMF), and fenitrothion were obtained from J&K Chemical Ltd. (China). Disodium 3-(4-methoxy-spiro{1,2-dioxetane-3,2′(5′-chloro) tricyclo(3.3.1.13,7) decan}-4-yl) phenyl phosphate (CSPD) and Sapphire-IITM enhancer were supplied by Boson Biotech. Co., Ltd. (China). HRP and ALP were provided by Beijing Biosynthesis Biotechnology Co., Ltd. (China). HRPtagged goat antimouse IgG was purchased from Boster Biotechnology Co., Ltd. (China). The polystyrene high-affinity 96-well microplate was provided by Greiner Bio-One Biochemical Co., Ltd. (Germany). Ginseng and American ginseng were purchased from a local pharmacy and confirmed to be free of methyl parathion and imidacloprid by HPLC. SP2/0 cells were produced and stored in College of Plant Protection of Nanjing Agricultural University. BALB/c mice were provided by Center of Comparative Medicine of Yangzhou University. The mixture of luminol (0.25 mM) and HIOP (3.0 × 10−3 mM) dissolved in 0.10 M Tris-HCl buffer at pH 8.5 was used as Agent 1 of the CL coreactants. Meanwhile, the mixture of H2O2 (10 mM), CSPD (0.25 M), and Sapphire-IITM enhancer (0.5 g/L) prepared in ultrapure water was used as Agent 2 of the CL coreactants. The two mixtures were prepared just prior to use. SuperBlock T20 used as the blocking buffer was purchased from Thermo Fisher Scientific Inc. (USA). The dilution buffer for all immunoreagents was 0.10 M Tris-HCl buffer at pH 7.4. The coating buffer for microplate coating was 0.10 M carbonate buffer saline at pH 9.0. The washing solution was 0.10 M TrisHCl buffer at pH 7.4 containing 0.05% Tween-20. The elution solution for the pesticides was 0.10 M Tris-HCl buffer at pH 7.4 containing 0.05% Tween-20 and 10% methanol. 4-(4-Nitro phenoxy) butyric acid (hapten 1) and 4(methoxy-(4-nitro-phenoxy)-thiophosphorylamino)-butyric acid (hapten 2) were used as the haptens of methyl parathion, and 1-(6-(2-carboxyl ethylthio-3-pyridyl) methyl)-N-nitro-2imidazoline imine (hapten 3) was used as the hapten of imidacloprid. Figure S-1, Supporting Information, shows the structures of hapten 1 (H1), hapten 2 (H2), and hapten 3 (H3). All of the above haptens were home synthesized according to the reported approaches.23,24 H2−BSA and H3−BSA were used as the immunizing antigens for methyl parathion and imidacloprid, respectively. H1−OVA and H3−OVA were used as the coating antigens for methyl parathion and imidacloprid, respectively. H1−HRP and H3−ALP were used as the signal tracers for methyl parathion and imidacloprid, respectively. All of the above-mentioned protein-conjugated haptens were home prepared with the same DCC−NHS method (see the Supporting Information). Preparation of Hybridomas against H2−BSA and Splenocyte Immunized by H3−BSA. Six week-old female BALB/c mice were immunized intraperitoneally with 100 μg of H2−BSA on days 0 and 21 using complete and incomplete Freund’s adjuvant, respectively. Two subsequent injections of 100 μg of H2−BSA using Freund’s incomplete adjuvant were given on days 42 and 63. The polyclonal antibody for H2−BSA

and horseradish peroxidase (HRP) simultaneously. Using this BsMcAb, an ELISA method for prostate-specific antigen detection was established without chemical tagging of HRP. Wagstaffe et al.19 prepared a BsMcAb bearing two binding sites for Staphylococcus aureus Thermonuclease and fluorescent reporter. Because this BsMcAb showed a much higher affinity to Staphylococcus aureus Thermonuclease, steric hindrancemediated release of the prebound fluorescent reporter was observed after Staphylococcus aureus Thermonuclease was captured by the BsMcAb. Thus, Staphylococcus aureus Thermonuclease could be detected on the basis of its quenching to the fluorescence signal from the reporter. All of the above-reported methods were designed for single-analyte immunoassay because the two binding sites of BsMcAb were not sufficiently utilized to bind two different analytes. Herein, we attempt to adopt BsMcAb to achieve chemiluminescent (CL) MIA based on multitag mode because CL detection has shown such advantages as low background, wide linear range, and simple instrumentation. Unfortunately, differing from other optical methods,9,10 signal intensity is the only considered factor in the CL assay, while wavelength is not considered; therefore, it is very difficult to distinguish CL signals from different probes. Up to now, the various reported CL systems can be classified into three categories based on their different reaction kinetics characteristics. The first one is the flash type producing short-lived but strong CL emission, such as the HRP−luminol system;20 the second one is the glow type providing long-lived and continuously increasing CL emission, such as the alkaline phosphatase (ALP)−1,2dioxetane derivant system,21 and the third one is the oscillating type showing periodically growing and decaying CL emission, such as the Belousov−Zhabotinsky CL system.22 The quite different kinetic characteristics of the above-mentioned CL reactions provide a potential pathway to detect signals from different CL probes in different time windows to establish a novel reaction kinetics-resolved MIA approach. In this investigation, a BsMcAb that could bind methyl parathion and imidacloprid simultaneously was produced from hybrid hybridoma, which was prepared by fusing hybridoma and mouse spleen lymphocyte. HRP and ALP were utilized as the CL probes to tag the haptens of methyl parathion and imidacloprid, respectively. Then, the tagged haptens were simultaneously bound to the BsMcAb by two competitive immunoreactions. The two pesticides were sequentially detected in different time windows after adding the CL coreactants. This proof-of-principle work opened a new avenue for MIA using only one antibody.

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EXPERIMENTAL SECTION Instruments. The CL signals were collected using a MPI-A CL analyzer (Xi’an Remax Electronic Science & Technology Co., Ltd., China) equipped with a photomultiplier operated at −800 V. The absorbance measurements in ELISA tests were performed on an Infinite 200 PRO multifunctional microplate reader (Tecan, Austria). The purification of protein-conjugated haptens was accomplished on an AKTAprime protein purification system (GE healthcare Co., Ltd., UK) equipped with a Sephadex G-25 column with a length of 11 cm. Reagents and Materials. Dicyclohexylcarbodiimide (DCC), isocarbophos, fenthion, acetamiprid, thiamethoxam, thiacloprid, 4-hydroxy-4′-iodobiphenyl (HIOP), 4-(1-imidazoly)-phenol (IMP), and 4-hydroxybi-phenyl (BIP) were all purchased from Aladdin Reagent Ltd. (China). Imidacloprid B

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Analytical Chemistry was measured in mouse serum using indirect ELISA. The mouse with the highest titer was selected as the donor of spleen cells for hybridoma production. On day 3 after the last antigen injection, spleenocytes of the mouse were fused with SP2/0 myeloma cells in a ratio of 5:1. Then, the hybridoma (3H9A3) that secreted antimethyl parathion monoclonal antibody was selected through indirect noncompetitive and competitive ELISA tests as described by Liu et al.23 The immune procedure of H3−BSA was similar to the procedure mentioned above, and the mouse with the highest titer was selected as the donor of splenocyte which was used for the experiment as below. Treatment of Hybridoma. The mouse hybridoma cell line (3H9A3) was used for construction of a hypoxanthineguanine phosphoribosyltransferase (HGPRT) deficient mutant, which was selected by cultivating the hybridomas in DMEM culture medium with 20% fetal calf serum containing various concentrations of 8-azaguanine for at least 15 days. As a result, the 8-azaguanine resistant cells were cloned by limiting dilution, and the complete cell death in HAT medium was taken as an indication of HGPRT deficiency (HAT sensitive hybridoma). Preparation of Triomas and BsMcAb. The trioma cell line was prepared by fusing HAT sensitive hybridomas, which secreted antimethyl parathion monoclonal antibodies, and mouse spleen lymphocytes which were immunized by H3− BSA. The fused cells were distributed in 96-well culture microplates and cultivated in HAT medium. After 10 days, culture supernatants were screened by noncompetitive and competitive ELISAs for the presence of antibodies recognizing both methyl parathion and imidacloprid. The selected hybridomas were cloned using a limiting dilution technique. Stable antibody producing clones were expanded and inoculated into BALB/C mice which was previously primed with 0.50 mL of incomplete Freund’s adjuvant to produce ascites. The collected ascites were used as the BsMcAb for the following investigation. Competitive MIA of Methyl Parathion and Imidacloprid. The microplate was coated with 2000-fold diluted BsMcAb (100 μL/well) overnight at 4 °C and then washed thrice with the washing buffer (300 μL/well). It was blocked with the blocking buffer (150 μL/well) for 90 min at 37 °C and then washed thrice with the washing buffer. Subsequently, 80 μL of sample composed of methyl parathion and imidacloprid was mixed with H1−HRP and H3−ALP (both 10 μL) and added into the well. After being incubated at 37 °C for 90 min for the competition immunoreactions, the microplate was washed thrice to remove the unbound immunoreagents. For CL signal triggering, 80 μL of Agent 1 of the CL coreactants was first added into the well. Then, 40 μL of Agent 2 of the CL coreactants was injected rapidly into the well. The concentrations of methyl parathion and imidacloprid were quantified by the CL signals collected at 0.6 and 1000 s, respectively, after Agent 2 was injected.

Figure 1. Absorbance values of the indirect ELISA tests: the microplates were first coated with antigen (H1−OVA or H3−OVA) and then sequentially incubated with BsMcAb and HRP-tagged goat antimouse IgG. (a) Absorbance values obtained from control tests in which coating buffer instead of antigens was coated on the microplates. (b) Absorbance values obtained from blank tests in which the first incubation was performed with BsMcAb solution without methyl parathion or imidacloprid. (c) Absorbance values obtained from tests in which the first incubation was performed with BsMcAb solution spiked with 5.0 μg/mL methyl parathion. (d) Absorbance values obtained from tests in which the first incubation was performed with BsMcAb solution spiked with 5.0 μg/mL imidacloprid.

nonspecific binding of the tracer conjugates. Meanwhile, the coated H1−OVA and H3−OVA showed much stronger binding to the BsMcAb, thus leading to much higher absorbance values of 1.26 and 0.98, respectively (Figure 1b). Absorbance ratios of signal-to-control were 21.0 and 16.3 for H1−OVA and H3− OVA, respectively, demonstrating that the prepared BsMcAb possessed strong affinity to both methyl parathion and imidacloprid. Functional Analysis of BsMcAb: Cross-Talk of Antigen-Binding Sites. Cross-talk of the two antigen-binding sites in BsMcAb was investigated to evaluate the mutual interference between methyl parathion and imidacloprid detections. The detailed investigation process is described in the Supporting Information. To investigate the possible binding of imidacloprid to the methyl parathion binding site, two microplates were coated with H1−OVA. After blocking, the two microplates were incubated with a solution composed of BsMcAb and methyl parathion (solution 1) and a solution composed of BsMcAb and imidacloprid (solution 2), respectively. After the second incubation with the HRP-tagged goat antimouse IgG and the TMB color reaction, the absorbance values were measured to evaluate the interference of imidacloprid to methyl parathion detection. The absorbance values (green) in Figure 1b were used as the blank value, which was obtained from the same process except that the first incubation was performed with a BsMcAb solution without methyl parathion or imidacloprid. As seen in Figure 1c, for the microplate incubated with solution 1, the absorbance value (green) decreased 89.2% in comparison with the blank value, since methyl parathion in solution 1 competes with the coated H1−OVA to bind with BsMcAb. Meanwhile, for the microplate incubated with solution 2, the absorbance value (green) in Figure 1d remained unchanged, implying that imidacloprid in solution 2 did not compete with the coated H1−OVA to bind with BsMcAb. The same investigation was also conducted on two microplates

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RESULTS AND DISCUSSION Functional Analysis of BsMcAb: Affinity of BsMcAb. The affinity of BsMcAb to the pesticides was estimated by an indirect ELISA method illustrated in the Supporting Information. In brief, three microplates were coated with H 1 −OVA, H 3 −OVA, and coating buffer (as control), respectively. BsMcAb was captured by the coated antigens and then bound with HRP-tagged goat antimouse IgG. After a TMB color reaction, the absorbance was measured to evaluate the affinity. As seen in Figure 1a, the control microplate only showed very low absorbance values around 0.06 due to C

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Figure 2. (A) CL kinetics curves of the HRP−luminol reaction alone for methyl parathion (10 ng/mL) detection and the ALP−CSPD reaction alone for imidacloprid (10 ng/mL) detection. (B) CL kinetics curve of the mixed CL reactions for methyl parathion (10 ng/mL) and imidacloprid (10 ng/mL) detections.

Scheme 1. Schematic Illustration of the CL Reaction Kinetics-Resolved MIA Strategy for Methyl Parathion and Imidacloprid Detections

coated with H3−OVA, and the results are shown as red in Figure 1c,d. For the microplates incubated with solutions 1 and 2, the absorbance values decreased 6.7% and 63.5%, respectively, which indicated that methyl parathion in solution 1 did not compete with the coated H3−OVA to bind with BsMcAb. From the above results, it can be concluded that methyl parathion and imidacloprid bind with BsMcAb at two different antigen-binding sites and the two antigen-binding sites possess good specificity to their corresponding antigens. Kinetics Characteristics of CL Reactions. The HRP− luminol system and ALP−CSPD system were adopted for the CL immunoassay of methyl parathion and imidacloprid, respectively. The reaction kinetics characteristics of the two systems were investigated in detail to demonstrate the signal distinguishability. Figure 2A shows the kinetics curves of the two CL reactions. Obviously, CL emission from the flash type HRP−luminol system increased sharply to the maximum at about 0.6 s after reaction triggering and then decayed quickly within 10 s (curve a). Meanwhile, the ALP−CSPD system was a typical glow type CL reaction, whose emission showed a slow and steady increase in a long period (curve b). Figure 2B shows the CL kinetics curve of the mixed reaction system of HRP− luminol and ALP−CSPD. As seen in this figure, the flash type reaction was not obviously affected by the glow type one;

especially at 0.6 s, the CL response still reached the maximum. Therefore, 0.6 s was an appropriate time window for recording the signal of this flash type system. However, for the glow type reaction, its CL signal was found to be obviously affected by the flash type CL reaction at the beginning of reaction triggering. After 800 s, the CL kinetics curve of the ALP−CSPD reaction in the mixed system (curve c) almost overlapped with that in the individual system (curve b), which indicated that the mutual influence was avoided effectively at this time window. In addition, its detection sensitivity could be greatly improved after longer reaction time. However, too long of a reaction time increased the assay time. Considering the optimal analytical performance and the further development of this method to the high assay speed, 1000 s was chosen as the time window for collecting the CL signal from the glow type system in the following investigation. Principle of CL Reaction Kinetics-Resolved MIA Using BsMcAb. As seen in Scheme 1, the tracer conjugates (H1− HRP and H3−ALP) competed with the free methyl parathion and imidacloprid in the sample solution to simultaneously bind with the BsMcAb possessing two antigen-binding sites. Then, the substrates for HRP and ALP were added to trigger the two CL reactions. Due to the remarkable difference between the reaction kinetics characteristics of the HRP−luminol and ALP− D

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Figure 3. (A) Enlarged view of the CL responses for methyl parathion. (B) The CL responses for methyl parathion and imidacloprid at the concentrations of (a) 1.0, (b) 10.0, (c) 100, (d) 250, and (e) 500 ng/mL. All the tests are conducted under the optimal conditions. (C) The standard curve for methyl parathion. (D) The standard curve for imidacloprid.

Performance of MIA. As seen in Figure 3, the signals from both CL systems decreased linearly with the increasing concentrations of methyl parathion and imidacloprid since a competitive immunoassay format was adopted. The linear ranges for methyl parathion and imidacloprid were both 1.0− 500 ng/mL, with the correlation coefficients of 0.9919 and 0.9974, respectively. At a signal-to-noise ratio of 3, the limits of detection (LODs) for methyl parathion and imidacloprid were both 0.33 ng/mL, which were lower than those of many reported approaches, such as ELISA, electrochemical sensor, and LC-MS/MS (Table S-1 in the Supporting Information). The regression equations for methyl parathion and imidacloprid were I (a. u.) = 7770 − 6.0C (ng/mL) and I (a. u.) = 7430 − 7.7C (ng/mL), respectively, where I was the CL intensity and C was the analyte concentration. The reproducibility was assessed by relative standard deviation (RSD) tests for methyl parathion and imidacloprid at low (10.0 ng/mL), medium (100 ng/mL), and high (250 ng/mL) concentrations. The obtained RSD values for methyl parathion and imidacloprid detections were not higher than 4.3% and 2.5%, respectively (n = 3). In comparison with other detection methods such as fluorescence and electrochemistry, the CL detection utilized in this work showed some obvious advantages including wide linear range, simple instrumentation, and easy manipulation. Specificities of MIA Using BsMcAb. The specificities of the proposed method for methyl parathion and imidacloprid detections were examined by detecting its response to some other pesticides with similar chemical structures (Figure S-1 in the Supporting Information). Fenitrothion, fenthion, and isocarbophos were tested as the interfering agents for methyl parathion, while acetamiprid, thiamethoxam, and thiacloprid were tested as the interfering agents for imidacloprid. All specificity tests were conducted using interfering agents at 250 ng/mL. The degree of interference of these compounds was calculated according to the following equation:

CSPD systems, the signals for methyl parathion and imidacloprid detections could be easily distinguished in different time windows. Methyl parathion and imidacloprid were quantified by the CL signals collected at 0.6 and 1000 s, respectively, after the coreactants injection. Optimization of Assay Conditions. The performance of CL competitive immunoassay usually relies on some assay conditions, such as the amount of the tracer conjugates, the incubation time, the CL enhancer, and the buffer saline for CL reactions. Thus, the effects of these assay conditions on the CL responses were studied using methyl parathion and imidacloprid both at 100 ng/mL. To determine the optimal amount of the tracer conjugates, CL signals resulting from different dilution times of the tracer conjugates were obtained for the analytes and Tris-HCl buffer (as a blank) in parallel. The signalto-blank ratios reached minimal values of 58.3% and 65.9% when the dilution times for H1−HRP and H3−ALP were 1:50 and 1:25, respectively, indicating that the competition capability of the analytes against the tracer conjugates was the strongest under this condition. The effect of the incubation time on the CL responses was also investigated in detail. Both CL responses reached the maximum at 90 min, suggesting that both immunobinding reactions attained the saturation at this incubation time. HRP−luminol and ALP−CSPD systems both showed strong CL emission in weak basic buffered medium. Tris-HCl buffer (0.10 M) at pH 8.5 was chosen as the compromised buffer saline for the CL coreactants since it led to acceptable emission intensities for the both systems. For the HRP−luminol system, some p-phenol derivatives such as HIOP, BIP, and IMP were found to significantly enhance the CL response. After careful comparison, HIOP at 3.0 × 10−3 mM was adopted as the CL enhancer for the HRP−luminol system since it provided the strongest signal intensity. For the same reason, the optimal concentrations of luminol and H2O2 were chosen to be 0.25 and 10 mM, respectively. E

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Table 1. Recovery Tests of (A) Methyl Parathion and (B) Imidacloprid Spiked in the American ginseng and ginseng Samples (n = 3) American ginseng samples added (ng/mL) found (ng/mL) RSD (%) recovery (%)

ginseng samples

A

B

A

B

A

B

A

B

A

B

A

B

250 230 8.1 92

250 276 5.2 110

100 115 2.8 115

100 112 3.8 112

10 8.4 2.7 84

10 8.0 3.4 80

250 214 7.1 86

250 295 8.2 118

100 96 3.3 96

100 100 0.7 100

10 8.3 1.7 83

10 9.3 1.4 93

degree of interference =

C−A × 100% C−B

much wider time windows. Therefore, it possessed many attractive advantages such as low cost, easy manipulation, simple instrumentation, and high efficiency. The recovery tests demonstrated its reliability and application potential in real sample assays. In the future work, it can be expanded to various application areas such as food safety, environmental monitoring, and disease diagnosis, by preparing more BsMcAbs with different antigen-binding sites.

(1)

Here, A, B, and C are the signals from the interfering agents, methyl parathion or imidacloprid, at the same concentration and the blank sample, respectively. The degree of interference for fenitrothion, fenthion, and isocarbophos to methyl parathion was 45.6%, 1.4%, and 2.9%, respectively. Meanwhile, the degree of interference for acetamiprid, thiamethoxam, and thiacloprid to imidacloprid was 6.4%, 6.9%, and 6.9%, respectively. As seen in the above results, most of other pesticides except fenitrothion showed negligible interference to the proposed strategy for methyl parathion and imidacloprid detections using BsMcAb. The obvious interference of fenitrothion to methyl parathion detection resulted from the great similarity between the structures of the two compounds. Application in Real Sample Assay. As very popular herbal medicines, ginseng and American ginseng are being used worldwide. Routine monitoring of pesticide residues is necessary to ensure the safety of the medication because the two herbal medicines are mainly obtained from commercial cultivation. Ginseng and American ginseng samples were spiked with the two pesticides at low, medium, and high concentrations and detected using the proposed CL reaction kinetics-resolved MIA strategy. The ginseng and American ginseng samples spiked with the two pesticides were pretreated according to the method reported by Wang et al.25 The detailed pretreatment process is illustrated in the Supporting Information. For the extraction of the pesticides from the spiked samples, an elution solution containing 10% methanol was adopted because the effect of methanol on the activity of BsMcAb was negligible when the amount of methanol was no more than 10%. The results listed in Table 1 showed satisfactory recoveries of 83−115% and 80−118% for methyl parathion and imidacloprid, respectively, with RSD values all below 8.2%.

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ASSOCIATED CONTENT

* Supporting Information S

Additional information including Figures S-1, preparation of protein-conjugated haptens, indirect ELISA test for the affinity of BsMcAb, indirect ELISA test for the cross-talk of antigenbinding sites, pretreatment of real samples, and Table S-1 as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-25-8439-9609. Fax: +86-25-8439-6109. E-mail : [email protected] (F.Q.L.). *Tel.: +86-23-6825-0184. Fax: +86-23-6825-1048. E-mail: [email protected] (Z.F.F.). Author Contributions §

Hui Ouyang and Limin Wang contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the National Natural Science Foundation of China (21175111, 21475107, and 31401771), the National High Technology Research and Development Program of China (2012AA101401-1), the Natural Science Foundation of Chongqing (CSTC2013jjB0096), the Fundamental Research Funds for the Central Universities (XDJK2013A025), and the Program for Innovative Research Team in University of Chongqing (2013).

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CONCLUSION In summary, a BsMcAb bearing two different antigen-binding sites was produced from hybrid hybridoma. This BsMcAb showed strong affinity and good specificity to methyl parathion and imidacloprid. In addition, a MIA strategy was proposed on the basis of the distinguishable reaction kinetics characteristics of different CL probes. With the designed CL reaction kineticsresolved strategy, this novel BsMcAb was adopted successfully to MIA of methyl parathion and imidacloprid. For a timeresolved fluorescent assay that also collects signals from different probes at different time windows, a sophisticated timing instrumentation is necessary since the fluorescence lifetime of most probes typically ranges from the picosecond to the millisecond level. In contrast, the present method allowed one to perform the CL immunoassay of multiple analytes with the aid of just a regular timer because the CL reactions showed

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REFERENCES

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DOI: 10.1021/ac5045093 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/ac5045093 Anal. Chem. XXXX, XXX, XXX−XXX