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Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay Yuan Liu a,b, Aihua Wu a,b, Jing Hu b, Manman Lin b, Mengtang Wen b, Xiao Zhang b, Chongxin Xu b, Xiaodan Hu b, Jianfeng Zhong b, Lingxia Jiao b, Yajing Xie b, Cun-zhen Zhang b, Xiangyang Yu b, Ying Liang b, Xianjin Liu a,b,⇑ a b

College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing 210014, China

a r t i c l e

i n f o

Article history: Received 8 December 2014 Received in revised form 21 April 2015 Accepted 22 April 2015 Available online xxxx Keywords: 3-Phenoxybenzoic acid Lateral flow immunoassay Monoclonal antibodies Colloidal gold

a b s t r a c t 3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA–bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I50 and I10 values of purified mAbs were 0.63 and 0.13 lg/ml, respectively, with a dynamic range between 0.19 and 2.04 lg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 lg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples. Ó 2015 Elsevier Inc. All rights reserved.

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Due to their low mammalian toxicity and efficient control of insects, pyrethroids are one of the most widely used insecticides in agriculture and private households [1,2]. However, increasing use of pyrethroid insecticides has resulted in concerns regarding potential effects on the ecosystem and human health. In addition to their high toxicity to aquatic life [3], some research also indicates that pyrethroids exposure may cause developmental neurotoxicity [4], adverse affects of male reproduction [5], and endocrine system disruption [6]. 3-Phenoxybenzoic acid (3-PBA)1 is a general metabolite of a number of common synthetic pyrethroids (Fig. 1) such as cypermethrin, deltamethrin, permethrin, cyhalothrin, fenvalerate, ⇑ Corresponding author at: Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing 210014, China. Fax: +86 25 84390401. E-mail address: [email protected] (X. Liu). 1 Abbreviations used: 3-PBA, 3-phenoxybenzoic acid; ELISA, enzyme-linked immunosorbent assay; CG, colloidal gold; BSA, bovine serum albumin; OVA, ovalbumin; KLH, keyhole limpet hemocyanin; mAb, monoclonal antibody; NC, nitrocellulose; CBS, carbonate–bicarbonate buffer saline; PBS, phosphate-buffered saline; PBST, PBS containing Tween 20; CPBS, citrate–acetate buffer; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TMB, tetramethylbenzidine; TEM, transmission electron microscope.

fenpropathrin, sumithrin, and possibly other pyrethroid insecticides [7–9]. Thus, 3-PBA is commonly used as a generic biomarker for multiple pyrethroids exposure for humans [8,10]. In the environment, 3-PBA is more mobile and persistent than its parental compounds and has widespread occurrence in surface water, sediment, and soil; the detection of 3-PBA may also reflect the multiple residues of pyrethroid insecticides [2,11,12]. Several chromatographic methods have been reported for the analysis of 3-PBA in different matrices [13–16]. More recently, enzyme-linked immunosorbent assays (ELISAs) and other bioanalytical approaches have been established as alternative methods for exposure monitoring with a detection limit of ppb (parts per billion) levels [17–20]. However, most of them need to be used in laboratories and operated by trained personnel. The colloidal gold (CG)-based lateral flow immunoassay is a popular and user-friendly format in terms of simplification and rapid on-site testing. It can be rapidly completed in one step, and the results can be observed with naked eyes [21]. To our best knowledge, this is the first report for development a CG-based lateral flow immunoassay for 3-PBA and its application to river water samples.

http://dx.doi.org/10.1016/j.ab.2015.04.022 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Y. Liu et al., Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.04.022

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Fig.1. Chemical structures of pyrethroid pesticides and 3-phenoxybenzoic acid (3-PBA).


Materials and methods

Production of mAbs



Chemicals and buffers



Competitive ELISA procedures



Protein–hapten conjugates and immunization


3-PBA was used as the hapten, and PBA–BSA and PBA–OVA were synthesized by using the active ester method and mixed anhydride method, respectively [22]. Four 6-week-old BALB/c female mice each received four 100-ll intraperitoneal injections at 2-week intervals. Injections consisted of a 1:1 emulsion of 100 lg of PBA–BSA in PBS and Freund’s adjuvant (complete for the first dose and incomplete for subsequent ones). The titers and affinity of the antiserum were determined by ELISA. Three days before the fusion, the mouse with the highest serum affinity to PBA was boosted with 200 lg of PBA–BSA in 200 ll of PBS.

The 96-well plates were coated with 2 lg/ml PBA–OVA (100 ll/well) in CBS and incubated overnight at 4 °C. The plates were washed three times with PBST by automated microplate washer and were blocked by incubating with 1% OVA in PBS (200 ll/well) for 1 h at 37 °C. After the washing step, 50 ll of PBA standards and 50 ll of cell culture or purified mAbs were added. After incubation for 1 h at 37 °C, the plates were washed. Subsequently, 100 ll/well of diluted horseradish peroxidaselabeled goat anti-mouse IgG was added and incubated for 1 h at 37 °C, and after the washing step 100 ll/well of a tetramethylbenzidine (TMB) solution (120 ll of 10 mg/ml TMB–DMSO [dimethyl sulfoxide] and 30 ll of 0.65% [v/v] H2O2 diluted with 11.85 ml of CPBS) was added. The reaction was stopped after 15 min by adding 50 ll/well of 2 mol/L H2SO4, and absorbance was read by a microplate reader at 450 nm.



3-PBA, bovine serum albumin (BSA), ovalbumin (OVA), keyhole limpet hemocyanin (KLH), Hybri-Max polyethylene glycol solution (average molecular weight = 1450), complete and incomplete Freund’s adjuvants, and mouse monoclonal antibody (mAb) isotyping reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA). Murine myeloma cells (Sp2/0-Ag14) were a gift from Xinxia Xia (Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, China). HiTrap Protein G HP columns were obtained from GE Healthcare (Piscataway, NJ, USA). Peroxidase-labeled goat anti-mouse IgG was obtained from KPL (Gaithersburg, MD, USA). RPMI 1640 medium and HT and HAT supplements were obtained from Life Technologies (Carlsbad, CA, USA). Fetal bovine serum was obtained from Boster (Wuhan, China). Nitrocellulose (NC) membranes (HF135), sample pads, absorbing pads, and CG pads were obtained from Millipore (Bedford, MA, USA). All other chemicals used in the current study were of analytical grade. River water samples were collected from local rivers of Nanjing, China. Carbonate–bicarbonate buffer saline (CBS: 50 mmol/L, pH 9.6), phosphate-buffered saline (PBS, 50 mmol/L, pH 7.4), PBST (PBS containing 0.05% [v/v] Tween 20), and citrate–acetate buffer (CPBS: 25 mmol/L citrate and 62 mmol/L sodium phosphate, pH 5.5) were used for immunoassay. GB-A solution (0.02 mol/L Tris– HCl [pH 8.6], 0.1% PEG 20,000, 4% sucrose, and 0.02% NaN3) was prepared to resuspend the CG–mAb conjugates.

The fusion protocol was based on Köhler and Milstein’s standard method with some modifications. Briefly, 2.25  108 mouse spleen cells from the immunized mouse and 4.5  107 murine myeloma cells (Sp2/0-Ag14) were fused using Hybri-Max polyethylene glycol solution at 37 °C. Fused cells were selected in HAT medium for 10 days, followed by 1 week in HT medium. Hybridomas were then grown in RPMI 1640 medium supplemented with 20% fetal bovine serum. Hybridoma supernatants were double-checked by noncompetitive ELISA and competitive ELISA. The positive clones were subcloned by the limiting dilution method. The immunoglobulin subclass was determined using mouse mAb isotyping reagents. The choosing positive clones were used to produce ascitic fluids and then purified by a protein G column and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).

Preparation of CG and CG–mAb conjugates


All glassware used in the experiment was soaked in aqua regia and rinsed thoroughly in water prior to use. After 100 ml of 0.01% (w/v) chloroauric acid (HAuCl4) solution was heated to the boiling point, 2 ml of freshly made 1% sodium citrate was added under


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constant stirring for 30 min. The size of CG was determined by transmission electron microscope (TEM). The pH of CG was adjusted to 8.5 by 0.2 mol/L potassium carbonate solution. The optimal concentration of the mAbs for conjugation was determined according to Horisberger and Rosset [23]. For conjugation, 50 lg of mAbs in 500 ll of 5 mM NaCl solution (pH 7.0) was added to 10 ml of CG. The mixture was stirred for 30 min at room temperature, and then 200 ll of 10 mg of KLH solution (10%, w/v) was added and stirred for another 30 min and then centrifuged at 11,000g and 4 °C for 60 min. The supernatant was carefully removed, and the segment was resuspended with 1 ml of GB-A solution.

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Characterization of purified mAbs


Antibodies are the key regent for establishing a lateral flow immunoassay. The purified mAbs (Fig. 3) were employed to establish a competitive ELISA. Results of the inhibition curve for 3-PBA standards in 10% methanol–PBS (Fig. 4) showed that the mAb response ranged between 0.19 and 2.04 lg/ml (calculated as the concentration of analyte giving 20 to 80% inhibition), and the detection limit was 0.13 lg/ml (I10). The I50 value of the ELISA was 0.63 lg/ml.


Development of lateral flow immunoassay strips

Determination of size of CG


The quality of CG directly determined the stability of CG–mAb conjugates. The TEM was used to verify the homogeneity of CG, and the average particle size was 15 nm (Fig. 5).


Optimization of working concentration of coating antigen and CG– mAbs


Coating antigen and CG-labeled antibodies are the two decisive factors to establish a lateral flow immunoassay. Excessive coating antigen or CG-labeled antibodies would decrease the sensitivity



Test and control lines were spotted on the membrane by means of a Biodot ZX-1010 platform. The control lines and test lines were coated with 0.5 mg/ml (1 ll/cm) goat anti-mouse IgG and 2 mg/ml (1 ll/cm) PBA–OVA in the NC membrane, respectively. The distance between the two lines was 0.5 cm. After drying at 4 °C overnight, the membranes were blocked with PBS containing 1% BSA at room temperature for 5 min and washed with PBS for 15 s. Then, the NC membranes were dried at 37 °C for 60 min. The CG pad was coated with CG–mAbs (5 ll/cm) and dried at 37 °C. The NC membranes (2.5  0.3 cm), CG pads (0.8  0.3 cm), and sample and adsorbent pads (1.5  0.3 cm) were assembled together and cut into sections (6  0.3 cm). The lateral flow immunoassay strips were stored under dry conditions at 4 °C.


Test procedures


First, 200 ll of 3-PBA standards or river water samples was added into 96-well plates. The test strips were dipped in the wells at the sample pad side. The samples migrated into the sample pads driven by the capillary forces. When the samples arrived at the CG pad, the CG–mAbs (chromogen) were solubilized by redissolving in the sample. If the samples contained 3-PBA, it would form the complex of 3-PBA and CG–mAbs. So, the complex could not react with the PBA–OVA in the test lines. The color intensity of the test lines inversely correlated with the 3-PBA concentration in the samples. Whenever the samples contained 3-PBA, the complex or additional CG–mAbs would move to the control lines and react with goat anti-mouse IgG and then form visible red lines in control lines. The whole test needed 10 min, and the test results were visually evaluated. The negative test resulted in two red lines: test and control lines. The positive lines gave only control lines. If no control lines were present, the tests were considered to be invalid (Fig. 2).

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Fig.2. Test results of the lateral flow immunoassay.

Fig.3. Identification of purified mAbs by SDS–PAGE. Lane 1: marker; lane 2: ascitic fluid fragments from protein G that eluted by washing buffer; lanes 3 to 7: purified mAbs; lane 8: ascitic fluids.

Fig.4. 3-PBA competitive ELISA dose–response curve.

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Fig.5. TEM image of CG.


of assay. But inadequate coating antigen or CG-labeled antibodies also cause low color intensity. Thus, the minimal concentration of coating antigen in test lines and the dispensing amount of CG– mAbs in CG pads were optimized to generate a visible red line in test and control lines. In Fig. 6, the optimal conditions established for the lateral flow immunoassay were 2 mg/ml coating antigen in test lines and 5 ll/cm CG–mAbs in CG pads.


Blocking NC membranes

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Fig.7. Lateral flow immunoassay of 3-PBA standards in 10% methanol–PBS (3-PBA concentrations from left to right: 8, 4, 2, 1, 0.5, 0.25, and 0 lg/ml).

Table 1 Determination of 3-PBA in spiked samples by the lateral flow immunoassay. Spiked concentration of 3-PBA (lg/ml) 0

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Membrane blocking is another factor that usually affects the performance of a lateral flow immunoassay. Some previous studies reported that blocking membranes will slow the flow rate, which leads to increased signals [24]. However, in this study the test lines got more intense color in nonblocked HF135 membranes than the membranes blocked by 1% BSA. Still, the migration of CG–mAbs had severe flow obstruction in nonblocked membranes, it usually needed more than 30 min to get color development in test and control lines, and not all of the CG–mAbs could cross the test and control lines. On the contrary, the speed of signal development in blocking membranes was approximately 10 min, and all of the CG–mAbs could get across the membranes. The sensitivity of blocking membranes was better than that of the nonblocking ones.


Determining the cutoff value of the lateral flow immunoassay


The aim of this study was to establish a rapid screen method for determination of 3-PBA with a visual detection system. Considering the unreliable quantification of visual test by different persons, this assay is more suitable as a semi-quantification test with a cutoff value, which is the minimal concentration of 3-PBA causing the total inhibition of the test lines. As shown in Fig. 7, serials of dilutions of 3-PBA standards in 10% methanol–PBS of 8, 4, 2, 1, 0.5, 0.25, and 0 lg/ml were detected by the test strips, with each concentration being determined in

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Fig.6. (A) Concentrations of coating antigen in test lines (from left to right): 0, 0.5, 1, and 2 mg/ml. (B) Volumes of CG–mAbs in CG pads (from left to right): 0, 0.625, 1.25, 2.5, 5, and 10 ll/cm.

Test result Note. +, positive result;

0.2 (10)


1 +(9),

5 (1)


, negative result.

triplicate. From 1 to 8 lg/ml test lines were completely inhibited, and at 0.5 lg/ml slight pink lines formed in test lines. Thus, the cutoff value for the test strips was 1 lg/ml 3-PBA, and the visual dynamic range was between 0 and 1 lg/ml.


Application in spiked river samples


The test strips were also used to detect 40 spiked river samples with different levels. There were no noticeable matrix effects for the river samples without any sample pretreatment. Assay performance was the same as for the tests using 3-PBA standards in 10% methanol–PBS. For the 40 spiked samples, there was one false negative sample at 1 lg/ml (cutoff value). The false negative rate at this level was 2.5%, and false positive results were not observed (Table 1).


Discussion and conclusions


This study has developed mAbs and established a CG-based lateral flow immunoassay for 3-PBA. The cutoff value of this assay was 1 lg/ml for 3-PBA, and the detection time was within 10 min. In comparison with the chromatographic methods, ELISA, or other bioanalytical approaches that were established before, the greatest advantage of this assay was its simplicity and rapid detection time. The assay could be used without any specific laboratory equipment or training personnel. Test results could be observed by naked eyes within 10 min and did not need any sample pretreatment for river water samples. In addition to simplicity, sensitivity is another important character for a detection method. Although the lateral flow immunoassay also has a visual dynamic range, considering the unreliable color judgment by different persons, this assay was designed to be a semi-quantitative method with a cutoff value. Thus, the sensitivity of the assay was compromised. It was more suitable to be used as a screening method and to detect 3-PBA at high levels. The selectivity of the assay was directly determined by the specificity of mAbs. Because 3-PBA is the generic structure for some pyrethroids, the mAbs produced in this study recognized


Please cite this article in press as: Y. Liu et al., Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.04.022

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b-cypermethrin, flucythrinate, fenvalerate, cypermethrin, and fenpropathrin with cross-reactivity ranging from 5.9 to 39.6% (data not shown). The lateral flow immunoassay could also detect these five pyrethroids at high levels. The application of the assay was demonstrated by 40 spiked river samples. The assay showed accurate and reproducible results. However, a 2.5% negative false rate was also observed. Considering the cross-reactivity of test strips to some pyrethroids (b-cypermethrin, flucythrinate, fenvalerate, cypermethrin, and fenpropathrin), the positive results must be confirmed by highly selective methods.





This project was supported by the National Natural Science Foundation of China (31201535), the 948 Project of the Ministry of Agriculture of China (2011-G5), the Science and Technology Support Program of Jiangsu Province (BE2012750), the Special Public Welfare Industry (Agriculture) Research Fund (201303 088), the Natural Science Foundation in Jiangsu Province (BK20130701), and the Agricultural Independent Innovation Fund of Jiangsu Province (CX(12)5042).



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Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay.

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids expos...
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