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Rabbit Monoclonal Antibody-BasedLateral Flow Immunoassay Platformfor Sensitive Quantitation of FourSulfonamide Residues in Milk and SwineUrineNa Liu a , Dongxia Nie b , Zheng Han b , Xianli Yang b , Zhiyong Zhao b

, Jiner Shen a , Gang Liu b , Aibo Wu b & Xiaodong Zheng aa School of Biosystems Engineering and Food Science, ZhejiangUniversity , Hangzhou , P. R. Chinab Institute for Agri-food Standards and Testing Technology, ShanghaiAcademy of Agricultural Sciences , Shanghai , P. R. ChinaAccepted author version posted online: 28 Aug 2012.Publishedonline: 02 Jan 2013.

To cite this article: Na Liu , Dongxia Nie , Zheng Han , Xianli Yang , Zhiyong Zhao , Jiner Shen , GangLiu , Aibo Wu & Xiaodong Zheng (2013) Rabbit Monoclonal Antibody-Based Lateral Flow ImmunoassayPlatform for Sensitive Quantitation of Four Sulfonamide Residues in Milk and Swine Urine, AnalyticalLetters, 46:2, 286-298, DOI: 10.1080/00032719.2012.718827

To link to this article: http://dx.doi.org/10.1080/00032719.2012.718827

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Immunoassay

RABBIT MONOCLONAL ANTIBODY-BASED LATERALFLOW IMMUNOASSAY PLATFORM FOR SENSITIVEQUANTITATION OF FOUR SULFONAMIDE RESIDUESIN MILK AND SWINE URINE

Na Liu,1 Dongxia Nie,2 Zheng Han,2 Xianli Yang,2

Zhiyong Zhao,2 Jiner Shen,1 Gang Liu,2 Aibo Wu,2 andXiaodong Zheng11School of Biosystems Engineering and Food Science, Zhejiang University,Hangzhou, P. R. China2Institute for Agri-food Standards and Testing Technology, ShanghaiAcademy of Agricultural Sciences, Shanghai, P. R. China

Based on the available rabbit monoclonal antibody (RabMAb), a rapid and sensitive lateral

flow immunoassay (LFA) platform has been developed for quantitative detection of four sul-

fonamide residues(SRs) of sulfadiazine (SD), sulfathiazole (STZ), sulfapyridine (SP),

and sulfamethoxazole (SMX).Within the designed LFA competitive format assay, which

was based on antigen-antibody properties, the hapten conjugate N1-[4-(carboxymethyl)-

2-thiazolyl] sulfanilamide linked to protein ovalbumin (TS-OVA) and goat anti-rabbit anti-

body were sprayed as capture and control reagents, respectively, and then the antibody was

conjugated to colloidal gold particles as the detection reagent. With quantitative assessment

aided by a colorimetric strip reader, the sensitivities of the established LFA method for SD,

STZ, SP, and SMX were 0.91 ngmL�1, 0.10ngmL�1,0.12ngmL�1, and 2.13ngmL�1, and

the half-maximum inhibition concentrations (IC50) were 5.19 ngmL�1, 1.25 ngmL�1, 0.66

ngmL�1, and 24.14 ngmL�1, respectively. The recoveries at three spiked levels (5, 20,

50 ngmL�1for SD, STZ, and SP; 20, 50, 100 ngmL�1 for SMX) were in the range of

78.02–135.10% and 76.40–137.16% for milk and swine urine, respectively. More impor-

tantly, the detection performance of the established platform was consistent with that of

in-parallel LC-MS/MS analysis. In conclusion, the proposed LFA platform has showed

the potential for fast, sensitive and relatively accurate quantification of four sulfonamide

residues in practical uses.

Keywords: Lateral flow immunoassay; Quantitation; Rabbit monoclonal antibody; Sulfonamides

Received 5 June 2012; accepted 28 July 2012.

This research was supported by the National High Technology Research and Development

Program of China (863 Program) No. 2007AA10Z436.

Address correspondence to Xiaodong Zheng, School of Biosystems Engineering and Food

Science, Zhejiang University, 388 Yuhangtang Road, Hangzhou, 310058, P. R. China. E-mail: xdzheng@

zju.edu.cn or to Aibo Wu, Institute for Agri-food Standards and Testing Technology, Shanghai Academy

of Agricultural Sciences, 1000 Jinqi Road, Shanghai 201403, P. R. China. E-mail: wuaibo@saas.sh.cn

Analytical Letters, 46: 286–298, 2013

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032719.2012.718827

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INTRODUCTION

The sulfonamides (SAs) are a group of antibacterial agents commonly used tofeed animals for prophylactic or therapeutic purposes (Haasnoot, Bienenmann-Ploum, and Kohen 2003). Due to the harmful effects of veterinary drugs, residuesof SAs by incorrect administration of antibiotics impose the greater potential risksin humans. The main risk of antibiotic residues to human health is allergic or toxicreactions and resistance to drugs (Hoff and Kist 2009). Furthermore, some SAs aresuspect carcinogens which have caused considerable debate on food safety issues. Inmost countries including China, the European Union and the United States, themaximum residue limit (MRL) for total sulfonamides was set 100 mg kg�1in edibletissues (European Commission1999; Food and Drug Regulation 1991; Ministry ofAgriculture of the People’s Republic of China 2002).

Due to its widespread and high toxicity, several instrumental analytical meth-ods have been thoroughly developed and proved to be sensitive and reliable forquantitative analysis of sulfonamides (Hela et al. 2003;Sheridan et al. 2008). Chro-matography methods such as high-performance liquid chromatography (HPLC)and liquid chromatography-mass spectrometry require a well-equipped laboratory,trained personnel, high capital expenditure, time consuming sample preparation,and are not suitable for dealing with large amounts of samples (G. Zhang, Wang,et al. 2008). A proper analytical strategy for monitoring residues is comprised ofscreening with a high-throughput method to prevent false negative results and thenconfirmation with accuracy quantitation methods (HPLC, GC-MS, LC-MS) to pre-vent false positive results (Ngom et al. 2010). Enzyme-linked immunosorbent assay(ELISA) as a screening method has been extensively used for detection of variousdrug residues in many fields due to the advantages of rapidity, convenience, low cost,and high throughput. However, the necessary photometric devices made it in appro-priate for on-site detection (J. Wang et al. 2011;Byzova et al. 2011). In recent dec-ades, another assay based on the reaction between an antibody and its antigen isthe lateral flow immunoassay (LFA) on a nitrocellulose member (Anfossi et al.2011). Detection methods based on rapid and low-cost LFA have been developedfor on-site monitoring residues including various veterinary drugs, and the resultscould be directly judged by the naked-eyes or simple colorimetric strip reader. Sev-eral LFA methods for rapid detection of single sulfonamide residues have beenreported. (O’Keeffe et al. 2003; X. L. Wang et al. 2007; Li et al. 2009; Ngom et al.2011; Guillen et al. 2011; G. Zhang, Wang, et al. 2008). Meanwhile, a method todetect multiple drug residues simultaneously would be more efficient in which vari-ous analytes could be simultaneously detected in one single immunoassay instead ofseveral individual assays (X. L. Wang et al. 2007; H. Y. Zhang, Zhang, and Wang2008; Guo et al. 2010). Hitherto, most previous LFA methods of sulfonamide detec-tions were qualitative. Also practically, higher sensitivities are also needed accordingto the requirements of MRLs in practical monitoring.

Based on the aforementioned considerations, the general objective of thepresent study was to develop a simple, fast, and sensitive applicable chromogenicLFA platform based on the previously obtained rabbit monoclonal antibody(RabMAb), which could be applicable for quantitative analysis of SD, STZ, SP,and SMX in milk and swine urine.

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EXPERIMENTAL

Reagents

Gold chloride (HAuCl4.3H2O) and sodium citrate (C6H5Na3O7.2H2O) wereused to prepare colloidal gold nanoparticles. Goat anti-rabbit immunoglobulins(IgG), SD, and SMX were purchased from Sigma (St. Louis, Mo, USA). The SPand STZ were purchased from Veterinary Drug Control (Beijing, China). TheRabMAbSAs-80-8 as detector reagent was produced in our previous study. Samplepad (spun bonded polyester, 6613), gold conjugate pad (borosilicate glass fiber withPVA binder, 8964), and Nitrocellulose (NC) membranes (vivid 170) were purchasedfrom PALL Corporation (NY, USA). Absorbing pad was purchased from ShanghaiGoldbio Tech Co., Ltd. (Shanghai, China).

Apparatus

The HGS510 dispenser and sprayer were obtained from Hangzhou AutokunTechnology Co., Ltd (Hangzhou, China). The strips were cut using a HGS201 Cutterfrom Hangzhou Autokun Technology Co., Ltd (Hangzhou, China). The CHR100Chromogenic Reader was purchased fromKAIWOODTechnology Co., Ltd (Taiwan,China). Deionized water was purified byMilli-Q Gradient A10 system (Millipore, Bill-erica, MA, USA). The TSQ QUANTUM ULTRA LC-MS=MS (Thermo Scientific,Brookfield, USA) was used to confirm the LFA results. Oasis HLB (WATERS,USA) was used as solid phase extraction (SPE) cartridges for clean-up of milk samples.

Antigen and Antibody

The sulfathiazole derivative N1[4-(carboxymethyl)-2-thiazolyl)] sulfanilamide(TS) shown in Fig. 1, and TS-OVA conjugate was synthesized according to previouslyreported procedures (Haasnoot et al. 2000). The RabMAbSAs-80-8 was developed byimmunized rabbits using SD-BSA and SMX-BSA synthesized by diazotization(Frank et al. 1999), and splenocytes of chosen rabbit were fused with 240E rabbitmyeloma cells which were supplied by Epitomics, Inc. (Hangzhou, China). The cho-sen clone was subcloned by limited dilution, and the monoclonal antibody was pur-ified by Protein A-Sepharose 4B affinity chromatography. The RabMAbSAs-80-8was previously obtained in our group which recognized four sulfonamides (SD,STZ, SP, and SMX) and were used to conjugate with colloidal gold particles as adetector reagent in this work.

Preparation of Colloidal Gold Nanoparticles

Colloidal gold nanoparticles with a mean diameter of 25 nm were prepared asfollowing procedures. A sample of 2mL of chlorauric acid solution (1%, w=v) wasadded to 200mL of distilled water and brought to a boiling point with vigorousstirring. Then, 5mL of freshly prepared 1% sodium citrate was added with constantstirring. After the color changed to wine-red, the solution continued to be boiled foranother 7–10min; the solution was cooled to room temperature, and deionized waterwas added to meter volume 200mL. The solution was stored at 4�C until use.

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Preparation of Antibody-Gold Nanoparticles Probe

The gold colloid solution was adjusted to pH 8.0 with 1% potassium carbonatesolution. Then, 160mLof purified RabMAbSAs-80-8 (0.62mgmL�1) was added to the10mL of gold colloid solution, and the mixture was vigorously stirred for 40min atroom temperature. An amount of 1mL of BSA solution (5%, w=v) was added to themixture to block the free colloidal gold for 30min at room temperature. The solutionwas centrifuged (10,000 rpm,4�C) for 30min. After the supernatant was discardedand washed twice with PBS (pH 7.4) containing 0.1% BSA and 0.05% sodium azide,the pellets were finally resuspended in 1mL of the same buffer.

Preparation of the Conjugate Pad

Colloidal gold nanoparticles prepared in the previous section were diluted10-fold with 0.1M PBS (pH 7.4) containing 10% (w=v) sucrose, 1% (w=v) BSA,and 0.5% (w=v) trehalose. A conjugate pad was produced by dispensing theconjugate solution onto a glass fiber membrane at a speed of 2.0 mLcm�1 usingHGS510 dispenser and sprayer, and then dried for 24 h at 37�C. The glass fiber mem-brane was cut into small pieces as the gold conjugate pad for use.

Figure 1. Chemical structures of the compounds in this study.

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Preparation of Sample Pad and Absorbent Pad

The sample pad was saturated with 0.1M PBS (pH 7.4) containing 1% of BSAand 0.25%of Tween-20, and was then dried and cut in size of 0.5� 1.5 cm. Theabsorbent pad was not treated and cut to 0.5� 2 cm.

Member Blotting

The TS-OVA conjugate (4.8mgmL�1) was used as the capture reagent. Goatanti-rabbit immunoglobulin was used as control reagent. Both the test and controlreagents were diluted with PBS (pH 7.4) to a concentration of 1mgmL�1 and suited0.5 cm apart on the NC membrane by using HGS510 dispenser and sprayer at a jet-ting rate of 1 mL cm�1,then membrane was dried (37�C, 20% of relative humidity).

Preparation of LFA Strips

As shown in the schematic diagram (Fig. 2A), the sample pad, conjugate pad,NC membrane, and absorption pad were assembled on a plastic backing supportboard with a 2-mm overlap. The LFA strips were sealed in a plastic bag and storedunder dry condition at room temperature.

Figure 2. (A) Structure of LFA. The strip is made up of sample pad, conjugate pad, nitrocellulose

membrane (containing a test line spotted with TS-OVA and a control line spotted with goat anti-rabbit

antibody), absorption pad, and plastic backing. Detection results interpretation described for LFA

from B to F; (B) negative result; (C) weakly positive result; (D) positive result; (E) and (F) invalid

result. (Figure available in color online.)

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Calibration Curves for Quantitation

Standard work solutions at various concentrations from 0 to 100 ngmL�1forSD, STZ, SP, and SMX from 0 to 1000 ngmL�1were prepared by dilution of thestock solution, and 50 mL of each solution was loaded onto the strips. The stripswere set aside at 37�C for 15min. The color intensity (CI) of the test line wasrecorded by the CHR-100 Chromogenic Reader, and the CI value of standardsolutions with SAs was denoted as B and that of solutions without SAs(0 ngmL�1) denoted as B0. The standard curve was plotted by the value B=B0

and corresponding concentrations of SAs, and fitted to four parameter logisticequation by SigmaPlot (version 12.0).

Preparation of Spiked Sample for LFA Strip

Before the spiked studies, the swine urine and milk were verified to be negativesamples by LC-MS=MS. Samples were spiked with single SD, STZ, and SP at con-centration levels 5 ngmL�1, 20 ngmL�1, and 50 ngmL�1or SMX at concentrationlevels 20 ngmL�1, 50 ngmL�1, and 100 ngmL�1in triplicate. Spiked milk sampleswere homogenized by vortex oscillation for 3min and kept for a period of 30minfor placement. After the centrifugation 10,000 g for 20min, milk samples werediluted 5-fold with PBS before analysis. Swine urine samples were directly detectedby the test strips after centrifugation.

In-Parallel LC-MS/MS Analysis

Milk samples were prepared according to the China National Standard GB=T22966-2008. The urine samples were centrifuged, and the supernatants were col-lected. After diluted 20 times with mobile phase and passed through a 0.22-mm filter,the filtrate was directly analyzed by LC-MS=MS.

The analytes were separated on a Thermo Hypersil Gold columnC18(100mm� 2.1mm, 3.0 mm) at 35�C, with a mobile phase flow rate of 0.30mLmin�1. The mobile phase consisted of (A) water containing 0.05% formic acidand (B) methanol was used for the gradient elution program, giving a total runtime of 7min. The injection volume was 5.0 mL (full loop). The following settingswere used for MS=MS conditions: spray voltage, 3.5 kV; vaporizer temperature,250�C; sheath gas pressure, 30 psi; and capillary temperature, 350�C. The transitionused for STZ quantitation was 256=107.974 with a collision energy of 24 eV, andthe other transition used for identification was 256=92 with the collision energy28 eV; The transition used for SD quantitation was 251=155.974 with a collisionenergy of 16 eV, and the other transition used for identification was 251=92with the collision energy 29 eV; The transition used for SP quantitation was250=155.95 with a collision energy of 17 eV, and the other transition used foridentification was 250=92 with the collision energy 28 eV; The transition used forSMX quantitation was 254=155.944 with a collision energy of 16 eV, and the othertransition used for identification was 254=92 with the collision energy 29 eV.Data acquisition and processing were performed using Xcalibur software (ThermoScientific, USA).

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RESULTS AND DISCUSSION

Principle of the LFA Platform

When the sample loaded onto the LFA strip, the colloidal gold-labeled anti-body was in the sample fluid and moved to the membrane by capillary action(Fig. 2A). If sulfonamide was absent from sample, the colloidal gold-labeled anti-body was trapped by TS-OVA capture reagent and anti-rabbit IgG to form two vis-ible red color lines on both test line and control line which indicated a negative assay(Fig. 2B).When sulfonamide was present in the sample, it competed with the immo-bilized capture reagent TS-OVA capture at the test line for limited amount of detec-tor reagent. If a weak red band color appeared on the test line, it was indicated aweakly positive result (Fig. 2C). With the increase of sulfonamide concentration inthe sample, the weaker red color of the test line appeared. If sufficient sulfonamidewas present in the sample, the limited detector reagent was completed with the freesulfonamide in the sample and no visible color appeared on the test line which indi-cated a positive result (Fig. 2D). If the red control line was invisible, the test resultwas considered to be an invalid result (Fig. 2E and Fig. 2F).

Optimization of Assay Time

Considering the influence of the assay time, several different assay times (5, 10,15, 20, 25min) were selected for comparison, and sample wells were added to 50 mLof SD standard solution with a concentration of 5 ngmL�1. The results showed thatB=B0 were 49.04%, 26.76%, 20.18%, 25.16%, and 29.11%, respectively. Therefore,the assay time of 15min was chosen for the lowest B=B0 value with best inhibition.

Sensitivity and Cross-Reactivity of the LFA Strip

For the quantitative analysis, 50mL of the standard solutions of different concen-trations for SD, STZ, SP, and SMX were loaded onto the strip and evaluated by thestrip reader. As shown in Fig. 3, dose-response curves were obtained for SD, STZ,SP, and SMX. The lower detection limit (LDL) was measured as the amounts in thestandard solution that caused a 20% decrease of the B=B0 response compared with thatproduced by the 0 ngmL�1, and the LDL of SD, STZ, SP, and SMXwere 0.91 ngmL�1

for SD, 0.10 ngmL�1 for STZ, 0.12 ngmL�1 for SP, and 2.13 ngmL�1 for SMX,respectively. The IC50 values obtained from the standard curves for SD, STZ, SP,and SMXwere 5.19ngmL�1, 1.25ngmL�1, 0.66ngmL�1, and 24.14 ngmL�1, respect-ively. The quantitation analysis could be performed for single sulfonamide or a mixtureof SAs including SD, STZ, and SP which have similar LDL and IC50 values.

By naked eye, the visual detection limit (VDL) with unaided visual assessmentwere defined as the concentrations which caused visible differences in color of the testlines comparing with negative sample. The color intensity on the strip graduallydecreased with increasing concentrations of SAs, and the cut-off value was definedas the concentration that caused colorless of the test strips. The VDL for SD, STZ,SP, and SMX were 2.5 ngmL�1, 0.8 ngmL�1, 0.8 ngmL�1, and 25 ngmL�1, andthe cut-off values for SD, STZ, SP, and SMX were 50 ng.mL�1, 15 ngmL�1,10 ngmL�1, and 250 ngmL�1, respectively. Therefore, the developed LFA platform

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was sensitive and suitable for qualitative and quantitative analysis of SD, STZ, SP,and SMX residues.

Previously, there were several reports for individual SD and STZ detection byLFA (X. L. Wang et al. 2007; Guillen et al. 2011). Multi-residual LFA based oncolloidal gold has been developed for the detection of sulfamonomethoxine, sulfa-methoxydiazine, sulfadimethoxine, and sulfadiazine which has a detection thresholdof 10 ngmL�1 with an optical density scanner (X. L. Wang et al. 2007).

To our knowledge, based on a survey of recent studies, no LFA strip based oncolloidal gold for rapid screening of the four SAs (SD, STZ, SP, and SMX) has beendeveloped. In our previous work, the RabMAb SAs-80-8 on which the LFA wasbased was obtained with good recognition of four SAs (SD, STZ, SP, and SMX).The proposed LFA platform aided by the colorimetric strip reader in this currentresearch was capable of quantitation of SD, STZ, SP, and SMX with a detectionthreshold of 10 ngmL�1.

The cross-reactivities against other sulfonamides were also evaluated by ana-logue compounds including sulfachloropyrazine, sulfamerazine, sulfaquinoxaline,sulfamonomethoxine, sulfamethizole, sulfadimethoxine, sulfamethoxypyridazine,sulfisoxazole, sulfachloropyridazine sodium, and sulfamethazine. Standard solutionconcentrations of each sulfonamide were yielded ranging from 100 ngmL�1 to105 ngmL�1. As expected, most sulfonamide drugs had no obvious inhibitionobserved by naked eye with two clear red bands in the test and control lines ofthe strip even though yielding concentrations at high levels, except for sulfamethi-zole and sulfamerazine tested by strip reader with IC50 of 103 ngmL�1 and

Figure 3. Calibration curves for using test strips for detection of SD, STZ, SP, and SMX. The X axle is

expressed on the logarithm of various concentrations of standard solutions. B=B0 represents the represents

the percentage of the CI values according to the standards with sulfonamide compared with the CI value at

0 ngmL�1, and the IC50 was calculated as 5.19 ngmL�1, 1.25 ngmL�1, 0.66 ngmL�1, and 24.14 ngmL�1

for SD, STZ, SP, and SMX, respectively. (Figure available in color online.)

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104 ngmL�1, respectively. Whereas, the IC50 values for the other compounds wereabove 105 ngmL�1. Thus, the strip could be applicable for screening duringpractical monitoring of the presence of SRs of SD, STZ, SP, and SMX belowthe MRLs.

Recovery Studies

Based on the sensitivity of the LFA platform, the samples were spiked withlow, medium, and high concentration levels of the standard solutions (5, 20, and50 ngmL�1 for SD, STZ, and SP; 20, 50, and 100 ngmL�1 for SMX). Urine as fluidbiological sample was easier to handle for clean-up procedures, and the residues inurine had a good parallel relationship with edible tissue as a marker and predictorfor residue presence analysis (G. Zhang, Wang, et al. 2008). Owing to the wideuse of antimicrobial agents in dairy cattle management, the improper administrationwithout observing the withdrawal time for antibiotic could result in antimicrobialresidues in milk and milk products (Bilandzic et al. 2011). Base on the afore men-tioned considerations, swine urine and milk were selected in the study to providean indication of SAs contaminant.

Swine urine samples could be directly analyzed by the established LFA, with-out any dilution steps for the utmost simplicity. The pH value and the ingredient inswine urine could influence the combinations of the detector reagent with free SAsand capture reagent. The concentrations of 20 ngmL�1 for SD, STZ, or SP and100 ngmL�1 for SMX caused visible color decrease on the test line of the stripby visual assessment and the corresponding B=B0 values were 55.4%, 29.4%,39.6%, and 63.5%, respectively. The demand of MRL 100 ngmL�1 for most coun-tries could be satisfied by the LFA strip judged by naked eye in swine urine.Spiked swine urine samples were quantified by the developed chromogenic platforms as shown in Table 1; recoveries were from 76.40 to 112.10% for SD, from79.32 to 122.90% for STZ, from 76.76 to 137.16% for SP, and from 86.77 to136.60% for SMX. The coefficients of variation (CV) ranged from 0.24 to 3.38%for SD, from 0.75 to 5.20% for STZ, from 0.37 to 1.12% for SP, and from 2.01to 11.32% for SMX.

Milk samples were prepared by performing a 5-fold dilution step with PBS tominimize the matrix interference. When the concentrations reached at 5 ngmL�1 forSD, STZ, and SP and 10 ngmL�1 for SMX, the results of LFA from visual evalu-ation of spiked milk samples were considerably weaker in color on the test linealthough still distinguishable. When the concentrations reached 50 ngmL�1 for SDand SP, 20 ngmL�1 for STZ, and 200 ngmL�1 for SMX, the red color almost disap-peared on the test line of the strip. When SD, STZ, SP, and SMX was at a MRL levelof 100 ngmL�1, the test line was absent for three sulfonamides (SD, STZ, and SP),and a weak signal was observed for SMX. This result demonstrated by LFA could besatisfactory for screening of SD, STZ, SP, and SMX at MRL level by naked eyeassessment in milk samples. As shown in Table 2, spiked milk samples were quanti-fied by the developed chromogenic platforms with recoveries ranging from 78.02 to103.23% for SD (CV, 2.58–6.47%), from 88.63 to 111.37% for STZ (CV,2.76–17.68%), from 86.26 to 108.94% for SP (CV, 3.35–10.88%), and from 89.61to 135.10% for SMX (CV, 4.53–17.19%) in milk. The results of swine urine and milk

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samples indicated that the LFA method could be utilized for detecting SD, STZ, SP,and SMX in swine urine and milk with the recoveries between 70 to 140% of thetheoretical values and CV values below 20%.

Table 1. Recoveries of four sulfonamides in spiked swine urine samples, and the results confirmed by

LC-MS=MS

Swine Urine

LFA LC-MS=MS

Analyte

Spiked

level

(ng=mL) Mean� SD (ng=mL) RC (%) CV (%) Mean� SD (ng=mL) RC (%) CV (%)

SD 5 5.61� 0.19 112.10 3.38 4.52� 0.41 90.37 8.97

20 22.30� 0.05 111.50 0.24 19.00� 3.53 95.00 18.57

50 38.20� 0.32 76.40 0.85 53.03� 2.28 106.06 4.29

STZ 5 4.89� 0.09 97.79 1.87 2.73� 0.73 54.61 14.69

20 24.58� 1.28 122.90 5.20 16.42� 1.38 82.09 8.39

50 39.66� 0.30 79.32 0.75 40.88� 4.52 81.77 11.05

SP 5 6.86� 0.03 137.16 0.37 2.63� 0.41 52.66 15.38

20 17.22� 0.10 86.08 0.55 18.63� 2.66 93.16 13.31

50 38.38� 0.43 76.76 1.12 46.65� 4.40 93.30 8.80

SMX 20 27.32� 3.09 136.60 11.32 14.61� 1.58 73.04 10.81

50 56.62� 3.05 113.24 5.39 43.53� 1.22 87.05 2.80

100 86.77� 2.31 86.77 2.01 90.09� 4.78 90.09 5.31

Note: Swine urine were spiked with SD, STZ, and SP at 5, 20, and 50 ngmL�1 while SMX at 20, 50, and

100 ngmL�1, respectively, and each concentration of samples were analyzed in triplicate. RC%, recovery

percentage; SD, standard deviation; CV, coefficient of variation.

Table 2. Recoveries of four sulfonamides in spiked milk samples, and the results confirmed by LC-MS=

MS

Milk

LFA LC-MS=MS

Analyte

Spiked level

(ng=mL)Mean�SD

(ng=mL)

RC

(%)

CV

(%)

Mean�SD

(ng=mL)

RC

(%)

CV

(%)

SD 5 4.84� 0.32 96.76 6.47 6.02� 0.15 120.48 2.41

20 20.65� 0.97 103.23 4.83 23.42� 0.58 117.12 2.47

50 39.01� 1.29 78.02 2.58 44.19� 3.03 88.37 6.86

STZ 5 5.57� 0.88 111.37 17.68 4.91� 0.28 98.26 5.77

20 17.73� 0.35 88.63 7.03 15.86� 2.59 79.31 16.32

50 45.70� 1.4 91.41 2.76 63.95� 4.38 127.90 6.84

SP 5 5.24� 0.57 104.79 10.88 4.33� 0.30 86.54 8.76

20 21.79� 0.73 108.94 3.35 25.60� 0.84 127.86 4.19

50 43.13� 1.76 86.26 4.09 46.65� 4.40 93.30 9.43

SMX 20 27.02� 3.34 135.10 11.26 17.47� 0.92 87.37 5.24

50 45.65� 2.07 91.29 4.53 40.70� 0.46 81.39 1.13

100 89.61� 15.47 89.61 17.19 88.64� 5.61 88.64 6.33

Milk were spiked with SD, STZ, and SP at 5, 20, and 50 ngmL�1 while SMX at 20, 50, and 100ngmL�1,

respectively, and each concentration of samples was analyzed in triplicate. RC%, recovery percentage; SD,

standard deviation; CV, coefficient of variation.

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Comparative Analysis with LC-MS/MS

An overview of the comparative results of LFA and LC-MS=MS are shown inTable 1 and Table 2. Recoveries in swine urine by LC-MS=MS ranged from 90.37 to106.06% for SD (CV, 4.29–18.57%), from 54.61 to 82.09% for STZ (CV,8.39–14.69%), from 52.66 to 93.30% for SP (CV, 8.80–15.38%), and from 73.04 to90.09% for SMX (CV, 2.80–10.81%). Recoveries in milk sample by LC-MS=MS werefrom 88.37 to 120.48% for SD (CV, 2.41–6.86%), from 79.31 to 127.90% for STZ(CV, 5.77–16.32%), from 86.54 to 127.86% for SP (CV, 4.19–9.43%), and from81.39 to 88.64% for SMX (CV, 1.13–6.33%). Due to the sensitivity of LC-MS=MSand the different methods of sample preparation for swine urine and milk, relativelyhigh CV values or low recoveries were obtained for low concentration-spiked sam-ples including 5 ngmL�1 and 20 ngmL�1. The results of LC-MS=MS were similarwith the results obtained by the LFA method for SD, STZ, SP, and SMX for detec-tion in swine urine and milk samples, indicating good agreement between thedeveloped LFA methods and in-parallel LC-MS=MS. The consistent results sup-ported that the colloidal gold-based LFA method is sufficiently reliable for SD,STZ, SP, and SMX detection in both swine urine and milk samples with the advan-tages of rapidity, simplicity, and sufficient sensitivity.

CONCLUSIONS

As proven currently, LFA is a one-step assay method for on-site large screen-ing of SRs in real samples. In this study, antibody-gold nanoparticles based on ahigh affinity RabMAb against four sulfonamides were prepared, and then theLFA provided simple operation and was less time-consuming. Also, the establishedLFA platform aided by colorimetric strip reader achieved quantitation detection ofSD, STZ, SP, and SMX within15min, with desirable sensitivity to SD, STZ, SP, andSMX below 10 ngmL�1. As confirmed by LC-MS=MS, the strip was suitable fordetecting SD, STZ, SP, and SMX residues below 100 ngmL�1 in milk and swineurine for quantitation, which met the detection limit requirements in most countries.As concluded, the proposed LFA platform was confirmed to be a fast, simple, andapplicable approach for detecting SD, STZ, SP, and SMX in practical uses related tofood safety monitoring.

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