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Spectrochimica Acta Part A 81 (2011) 544– 547

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

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hemiluminescence enzyme immunoassay for the determination ofulfamethoxydiazine

ongjun Wu ∗, Songcheng Yu, Fei Yu, Nali Yan, Lingbo Qu, Hongquan Zhangollege of Public Health, Zhengzhou University, No. 100 of Science Avenue, Zhengzhou 450001, China

r t i c l e i n f o

rticle history:eceived 2 May 2011ccepted 18 June 2011

a b s t r a c t

Sulfamethoxydiazine (SMD), which is often used for animal disease treatment, is harmful to humanhealth. No SMD residue should be detected in food in some countries, such as USA and Japan. Therefore, itis significant to develop a high-throughput, high-sensitivity and accurate method for the determination

eywords:ulfamethoxydiazinehemiluminescence enzyme immunoassayuantitation

of the content of SMD in food. In this paper, chemiluminescence enzyme immunoassay (CLEIA) wasdeveloped for quantification of SMD. For this method, the limit of detection was 3.2 pg/ml, the linearrange was from 10 to 2000 pg/ml, the within-day and inter-day precision were below 13% and below18%, respectively, and the recovery was from 85% to 105%. Milk and egg were selected as samples to beexamined with this method, and the result indicated that this CLEIA method was suitable for screeningand quality control of food.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Sulfamethoxydiazine (SMD) has effectiveness on urinary andespiratory tract infections and is often used for animal diseasereatment and prevention [1]. It takes a long time to metabolizeMD completely. Therefore, there is SMD residue in meat, dairy andggs if the animal has taken in SMD. Such food is harmful to humanealth. Codex Alimentarius Commission provides that the maxi-um residue limit for sulfonamides in animal tissues is 100 �g/kg

2]. In USA and Japan, there is policy that no SMD residue shouldxit in food. Thus, it is significant to carry out the determination ofMD residual in the meat, dairy, eggs and other kinds of food.

It has been reported that various methods had been appliedor detection of sulfonamides chemical, including thin layer chro-

atography [3], high performance liquid chromatography (HPLC)4], gas chromatography (GC) [5], gas chromatography masspectrometer (GC/MS), high performance liquid chromatographyass spectrometer (HPLC/MS) [6], capillary electrophoresis [7],

tc. These methods had disadvantages, e.g. high-cost and time-onsuming, and were not suitable for fast screening large numberf samples. Another method, radioimmunoassay [8], had advan-

ages of high sensitivity, specificity and good accuracy, but itsollution to the environment hindered its wide application. There-ore, it is significant to develop a high-throughput, high-sensitivity,

∗ Corresponding author.E-mail address: wuyongjun@zzu.edu.cn (Y. Wu).

386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2011.06.047

environment-protection and accurate method for the determina-tion of the content of SMD in food.

The goal of this paper is to establish a method, chemilumines-cence enzyme immunoassay, which is suitable for high-throughputdetermination of the content of SMD in food.

2. Experimental

2.1. Chemicals

All the reagents were of analytical grade. SMD monoclonalantibody was purchased from Guangzhou MeiJin BiotechnologyCo., Ltd. SMD was purchased from SIGMA with optical purities of99.5%. Horseradish peroxidase (HRP) was purchased from ROCHEInc. (Specific Activity > 250 u/mg). Deionized water was purified byMilli Q system (Waters, Milford, MA). In this method, the follow-ing buffers were used. (A) Coating buffer, 0.05 mol/L carbonate:bicarbonate buffer solution, pH 9.6, (B)incubation buffer, 0.01 mol/Lsodium phosphate buffered saline (PBS), pH 7.2–7.4, (C) washingbuffer, buffer B with 0.05% Tween 20, (D) dilution buffer, buffer Bwith 1% BSA.

2.2. Instruments

MP280-based chemiluminescence immunoassay analyzer wasfrom Beijing Tai Geke letter Biological Technology Co., Ltd.Microplate Reader (Tecan Sunrise). HPLC was purchased from the

a Acta Part A 81 (2011) 544– 547 545

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Figs. 1 and 2. The absorption peaks for HRP were 277 nm and406 nm. Those for SMD were 241 nm and 256 nm. And peak at233 nm presented HRP–SMD conjugate. Thus, from Fig. 1, it wasindicated that HRP–SMD conjugate was prepared successfully. And

Y. Wu et al. / Spectrochimic

nited States Diane (P680 HPLC Pump, ASI-100 Automated Samplenjector, Thermostatted Column Compartment Tcc-100, UVD170).

.3. Procedures

.3.1. Synthesis of HRP–SMD conjugatesTwo methods were used to label SMD with HRP. After pre-

iminary experiments, the optimal condition parameters werestablished. The HRP–SMD conjugate was identified by ultravio-et spectroscopy and infrared spectroscopy. Then the binding ratiof them was calculated according to the literature.

Method 1 [9]: SMD was mixed with HRP by a mass ratio of 1:1..5% glutaraldehyde was added in the mixture and stirred for 4 hree from light. Then 100 �l ethanolamine of 1 mol/L was added, andncubation at room temperature for 2 h. After that it was dialysed

ith incubation buffer for 5 days.Method 2 [10]: 1.0 mg HRP was weighed accurately and dis-

olved with 250 �l incubation buffer. NaNO2 was added to aoncentration of 1 mol/L. Then 300 �l 1 mol/L HCl was added andncubated for 20 min at 4 ◦C. This mixed solution was then added toMD solution (7.0 mg SMD was weighed accurately and dissolvedith 1 mol/L of NaOH solution) slowly until the pH value was sta-

le at 9.5. After incubated at 4 ◦C overnight, dialysis with incubationuffer was kept on for 5 days to obtain HRP–SMD conjugates.

Calculation of binding ratio: the binding ratio was calculatedccording to the following formula:

indingratio = εconjugat − εHRP

εSMD= Aconjugate/Cconjugate − AHRP/CHRP

ASMD/CSMD

ccording to literature [11], the binding ratio between 5 and 25ould meet the requirement and the conjugate could be applied.

.3.2. Procedure for measurement of CLEIAIn this CLEIA, the HRP–SMD conjugate, competed by the SMD

olecules in the sample to be analyzed, combined to the SMD anti-ody coated on the microplate. In this case, the relative luminescentnits (RLU) and the concentration of SMD in sample were nega-ive correlated. Therefore, a quantitative mathematical model wasstablished by the relationship between RLU and concentrations of

serial of SMD standard solutions.After optimization, the optimal condition was established.

00 �l SMD monoclonal antibody in dilution buffer (2.5 �g/ml) wasncubated in a polypropylene microplate with 96 wells at roomemperature. After washed with washing buffer, 100 �l sampleolution and HRP–SMD conjugate solution were added, respec-ively, and incubated for 1 h. Then washed with washing buffer,nd the substrates of HRP (luminol and H2O2) were added. Afterhat, chemiluminescent signal was measured by chemilumines-ence immunoassay analyzer. The light between 425 and 430 nmas issued and the relative luminescent unit (RLU) was determined.

.3.3. Method evaluationThe performance of this CLEIA was evaluated, including limit

f detection (LOD), precision, recovery and method comparison.ccording to literature [11], ten blank solutions were analyzed.hen the average value (X̄) and relative standard deviation (RSD)ere calculated. LOD could be calculated by the formula below:

OD = X̄ − 2RSD. The precision was determined by intra- and inter-ay variations. The intra-day precision was determined by threeests in a single day. The inter-day precision was determined byhree tests carried out in three different days. The variations were

xpressed by the relative standard deviations (RSD). The recoveryxperiment was used to evaluate the accuracy of this method. Accu-ate amount of SMD (spiked value) was added to sample (originalalue), and then the content of SMD (observed value) was deter-

Fig. 1. The UV spectra of HRP, SMD and conjugate HRP–SMD.

mined. The average recovery was determined by the followingformula:

recovery (%) = observed value − original valuespiked value

× 100

The method comparison experiment was carried out by comparingCLEIA with ELISA and HLPC. Two kinds of samples, milk and egg,were analyzed.

3. Results and discussion

3.1. Identification of HRP–SMD conjugates

The binding ratio of HRP–SMD conjugates synthesized withmethod 1 and 2 were four and six, respectively. It was shown thatconjugate synthesized with method 2 could be used in CLEIA.

The results of the identification of HRP–SMD conjugate byultraviolet spectroscopy and infrared spectroscopy are shown in

Fig. 2. The infrared spectra of HRP, SMD and conjugate HRP–SMD.

546 Y. Wu et al. / Spectrochimica Acta Part A 81 (2011) 544– 547

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Table 1The precision of CLEIA at different concentrations of SMD.

SMD (pg/ml) Within-run precision (n = 10) Between-run precision (n = 10)

X̄ SD RSD% X̄ SD RSD%

10 11.8 1.5 12.8 11.3 1.9 17.2100 98.0 11.1 11.3 107.4 14.7 13.7

1000 924.2 115.0 12.4 1041.8 168.2 16.2

Table 2Linear range, coefficient and limit of detection of CLEIA, ELISA and HPLC (n = 5).

Method Linear range Coefficient Limit of detection

CLEIA 10–2000 pg/ml 0.9952 3.2 pg/mlELISA 10–300 ng/ml 0.9970 4.5 ng/mlHPLC 0.05–1.25 �g/ml 0.9996 0.01 �g/ml

Table 3The precision of CLEIA, ELISA and HPLC (n = 10).

Method Within-run precision (%) Between-run precision (%)

CLEIA 11.3–12.8 13.7–17.2ELISA 12.0–14.5 15.6–17.6HPLC 2.4–4.2 4.9–5.9

Table 4Recoveries of SMD by CLEIA in milk and egg (n = 10).

Sample Theoreticalconcentration(pg/ml)

Detectableconcentration(pg/ml)

Average recovery(%)

Milk 50 45.9 91.8200 188 94.0

2000 2078 103.9

Egg 50 42.4 84.7

Fig. 3. Kinetics curves of chemiluminescence reaction.

his was in accord with the result of infrared spectrum in Fig. 2. Thebsorptions in 3500–4000 cm−1 region, 2250–2500 cm−1 regionnd 1250–1750 cm−1 region were for the amino acids in HRP andhe absorption in 850–1200 cm−1 region was for SMD. Throughomparison between the infrared spectra between conjugate andRP, as well as SMD, the result indicated that the conjugate con-

ained HRP and SMD.

.2. Kinetics of chemiluminescence reaction

According to literature [12], an experiment of kinetics of chemi-uminescence reaction was carried out. The result is shown in Fig. 3.rom the result it could be seen that chemiluminescence reactionas fast and longtime-last. And 1.5 min was selected as the optimal

eaction time in CLEIA.

.3. Calibration curve

A series of standard SMD solution, in which the concentrationsf SMD were 10, 50, 100, 200, 500, 1000, 2000 pg/ml, respectively,ere prepared and determined by CLEIA. Calibration curve (Fig. 4)as drawn by the logarithm of light intensity (y) versus the loga-

ithm of the concentration of standard SMD solution (x). Standardegression equation was:

= −0.0064x + 6.6846

The linear range was from 10 to 2000 pg/ml and the correlationoefficient was 0.9952.

Fig. 4. Calibration curve of CLEIA.

200 175.8 87.92000 1824 91.2

3.4. Limit of detection, precision and recovery

The analytical limit of detection was 3.2 pg/ml. The within-run and between-run precisions were below 13% and below 18%,respectively (Table 1). The recoveries were between 91.0% and104.0% in milk and between 84.0% and 92.0% in egg. The perfor-mance of this method showed that it was suitable for carrying outquantitative determination of SMD in food.

3.5. Method comparison

In this part, CLEIA was compared with ELISA and HPLC by deter-mination of SMD in milk and egg. The sample preparation for ELISAand CLEIA was according to references [13–17]. And that for HPLC

was in line to the literatures [18–21]. The results are shown inTables 2–6.

Table 5Recoveries of SMD by ELISA in milk and egg (n = 10).

Sample Theoreticalconcentration(ng/ml)

Detectableconcentration(ng/ml)

Average recovery(%)

Milk 10 9.02 90.2100 91.7 91.7300 321.9 107.3

Eggs 10 8.34 83.4100 88.9 88.9300 240.6 80.2

Y. Wu et al. / Spectrochimica Acta

Table 6Recoveries of SMD by HPLC in milk and egg (n = 10).

Sample Theoreticalconcentration(ng/ml)

Detectableconcentration(ng/ml)

Average recovery(%)

Milk 0.10 0.0891 89.11.25 0.9684 77.5

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Eggs 0.25 0.2246 89.91.25 1.0127 88.2

The results showed that the detection limit of CLEIA was low-st, that its recovery was better than those of HLPC and ELISA, andhat the precision was suitable for quantification determination.lthough the precision of CLEIA was not as well as HPLC, it wasimple-sample-preparing, high throughput, low-cost. Therefore, its a promising method.

.6. Discussion

As a kind of widely used drug in curing urinary and respira-ory tract infection, sulfamethoxydiazine was widespread in foodelated to animal. Due to the low concentration of sulfamethoxy-iazine in food, a high sensitivity of determination method wasequired for quality control. In order to develop a highly sensitive,ell reproducible, and high throughput assay for the determination

f sulfamethoxydiazine, CLEIA, horseradish peroxidase, luminolnd hydrogen peroxide as chemiluminescence reaction system,as established in this paper. This assay decreased the limit ofetection by thousands of times, compared with the reportedethods for analyzing sulfamethoxydiazine. This increased the

eliability of quality control of food.

. Conclusion

A CLEIA method to determine sulfamethoxydiazine in food wasstablished in this paper. Due to its advantages of good precision,imple sample preparation, high-throughput and low cost, it hadhe potential of wide application for quality control of food.

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Part A 81 (2011) 544– 547 547

Acknowledgment

This work is supported by Henan Province Major Public ResearchProject.

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