High sensitivity chemiluminescence enzyme immunoassay for detecting staphylococcal enterotoxin A in multi-matrices

Analytica Chimica Acta 796 (2013) 14–19

Contents lists available at ScienceDirect

Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca

High sensitivity chemiluminescence enzyme immunoassay for detecting staphylococcal enterotoxin A in multi-matrices Chunmei Zhang a,1 , Zhijia Liu a,b,1 , Yongming Li a,c , Qi Li a , Chaojun Song a , Zhuwei Xu a , Yun Zhang a , Yusi Zhang a , Ying Ma a , Yuanjie Sun a , Lihua Chen a , Liang Fang a , Angang Yang a , Kun Yang a,∗ , Boquan Jin a,∗∗ a

Department of Immunology, The Fourth Military Medical University, No. 169 Changle West Road, Xi’an 710032, Shaanxi Province, PR China The Organ Transplant Institute, The 309 Hospital of Chinese PLA, Beijing 100091, PR China c The Research Laboratory of Biochemistry, Basic Medical Institute, General Hospital of PLA, Beijing 100853, PR China b

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• This CLEIA for detecting SEA was developed by using a pair of highly specific mAbs. • This CLEIA for the detecting SEA is highly sensitive and specific. • It can be successfully applied to the analysis of SEA in a variety of environmental, biological matrices.

a r t i c l e

i n f o

Article history: Received 23 April 2013 Received in revised form 4 July 2013 Accepted 16 July 2013 Available online 25 July 2013 Keywords: Staphylococcal enterotoxin A Chemiluminescence enzyme immunoassay Detection Sensitivity Diagnosis

a b s t r a c t In this study, detection of staphylococcal enterotoxin A (SEA) in multi-matrices using a highly sensitive and specific microplate chemiluminescence enzyme immunoassay (CLEIA) has been established. A pair of monoclonal antibodies (mAbs) was selected from 37 anti-SEA mAbs by pairwise analysis, and the experimental conditions of the CLEIA were optimized. This CLEIA exhibited high performance with a wide dynamic range from 6.4 pg mL−1 to 1600 pg mL−1 , and the measured low limit of detection (LOD) was 3.2 pg mL−1 . No cross-reactivity was observed when this method was applied to test SEB, SEC1, and SED. It has also been successfully applied for analyzing SEA in a variety of environmental, biological, and clinical matrices, such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, human urine, and serum. Thus, the highly sensitive and SEA-specific CLEIA should make it attractive for quantifying SEA in public health and diagnosis in near future. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: CLEIA, chemiluminescence enzyme immunoassay; mAbs, monoclonal antibodies; SEA, detection of staphylococcal enterotoxin A; LOD, low limit of detection; SEs, staphylococcal enterotoxins; SFP, staphylococcal food poisoning. ∗ Corresponding author. Tel.: +86 29 84774598; fax: +86 29 83253816. ∗∗ Corresponding author. E-mail addresses: [email protected] (C. Zhang), [email protected] (Z. Liu), ym [email protected] (Y. Li), [email protected] (Q. Li), [email protected] (C. Song), [email protected] (Z. Xu), [email protected] (Y. Zhang), zhangyusi [email protected] (Y. Zhang), merry [email protected] (Y. Ma), syjfl[email protected] (Y. Sun), [email protected] (L. Chen), [email protected] (L. Fang), [email protected] (A. Yang), [email protected] (K. Yang), immu [email protected] (B. Jin). 1 These authors contributed equally to this work.

Staphylococcal enterotoxins (SEs) belong to a broad family of pyrogenic toxins produced by Staphylococcus aureus. It is found in nostril and on the skin of warm-blood animals, and the hands of food handlers. This is considered to be the primary source of food contaminations [1]. The S. aureus could grow and produce SEs in a wide variety of foods over an extensive range of temperature, pH, and water activity [2,3]. SEs are the most causative agents of foodborne diseases worldwide. Since SEs are heat stable, they present also in food causing staphylococcal food poisoning (SFP) while the Staphylococcus aureus that produces SEs are destroyed through heat treatment of the food [4]. Therefore, the diagnosis of a conclusive staphylococcal food poisoning is mainly based on the detection of

0003-2670/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2013.07.044

C. Zhang et al. / Analytica Chimica Acta 796 (2013) 14–19

SEs in food. Among SEs, staphylococcal enterotoxin A (SEA) drives special attention as it is the most common cause of SFP and one of the leading causes of gastroenteritis resulting from consumption of contaminated food [5]. In addition, it is evident that the presence of SEA or sea gene in patients with S. aureus blood stream infection is associated with septic shock and positively associated with the severity of sepsis caused by the proinflammative [6,7]. The SEs are extremely potent and people who ingest as little as 20–100 ng of enterotoxins could cause poisoning featured with symptoms including copious vomiting, diarrhea, abdominal pain or nausea, and so on [8]. When SEs are present in the blood stream, they could activate more than 20% of all peripheral T cells at the concentration of about 1 fm (10−15 mL−1 ) [8]. Since the extreme potency of the SEA, it is needed to develop rapid and sensitive methods to detect it at very low level in foods and serum. Many sensitivity methods have been successfully introduced to detect SEA in food and beverages. The T cell proliferation assay, which involves the super antigen potency evaluation also, is very sensitive (at pg mL−1 level), but there are some pros and cons and limitations when applied to detect the SEA in food [9]. It could not distinguish the SEA from other staphylococcal enterotoxins that have super antigen activity. Therefore, when the assay is applied to detect SEA in food samples, it must be identified subsequently by other immunoassays, such as enzyme-linked immunosorbent assay (ELISA), Western immunoblot, or a biosensor. In addition, T cell proliferation assay is not suitable for rapid detection and diagnosis, which takes 3 days to complete it. Various immunological methods, such as ELISA and antigen/antibody-based biosensor, have been used for detecting SEA in food and beverages samples [10–14]. The ELISA for detecting SEA was rapid and easy to perform, but it suffered from low sensitivity, and limit of detection (LOD) was about ng mL−1 level. Therefore, this method was restricted in clinical diagnosis of SEA-associated diseases. Mass spectrometry coupled with liquid chromatography has recently applied for the analysis of proteins, which could rapidly detect SEA without cross-reaction with other types of SEs. However, the sensitivity of this assay could not approach to the performance level of ELISAs [15]. Several biosensors had been exploited for the detection of SEA, such as amperometric immunosensor and piezoelectric immunosensor [16–18]. Some of these biosensors for the detection of SEA are highly sensitive. For example, a flow injection capacitive immunosensor could detect the SEA at picogram level [19]. However, most of these immunosensors are expensive and only exploited in scientific researches of special professional department, therefore they are not suitable for high-throughput detection. Since enzyme immunoassays such as ELISA have advantages, such as easy, specific, swift, high throughput, and so on. The sensitivity of ELISA can be enhanced, when the chemiluminescent substrates are applied in the enzyme immunoassay. Recently, the CLEIA, especially the enhanced chemiluminescent immunoassay has been widely used due to its high sensitivity and wide dynamic range [20,21]. It has been reported that the sensitivity of chemiluminescent assay often exceeds that of radioactivity assays. We have recently reported the development of sensitive sandwich CLEIA for the detection of SEB, with a detection limit of 10 pg mL−1 , which was the most sensitive immunoassay to detect SEB by then [22]. With two newly raised monoclonal antibodies against botulinum neurotoxin serotype A, we also applied the CLEIA in the detection of BoNT/A successively [23]. In this study, by using two specific monoclonal antibodies, we developed a very highly sensitive CLEIA for detecting SEA spiked in many food and beverages samples, as well as in clinical samples such as serum and urine. In the microplate CLEIA system, the captured antibody was directly coated onto the microplate well

15

without pretreatment. The sensitive HRP chemiluminescent substrate used in our assay had the ability to produce long periods of light emission. The actual sensitivity of our sandwich CLEIA achieved 3.2 pg mL−1 , which was more sensitive than that of ELISA (generally in the 1 ng mL−1 ). We demonstrated that the microplate CLEIA has the ability to detect SEA with high sensitivity and specificity and has great practical value in food safety and clinical diagnosis. 2. Materials and methods 2.1. Animals Female BALB/c mice were provided by the Laboratory Animal Center of The Fourth Military Medical University. All animal experiments were carried out under the approval of the Ethics Committee for Animal Experiments of The Fourth Military Medical University (Xi’an, China), and the experimental protocols strictly conformed to the institutional guidelines and the criteria outlined in the “Guide for Care and Use of Laboratory Animals.” 2.2. Chemicals and materials Staphylococcal enterotoxins (SE) A, B, C1, and D were obtained from the Academy of Military Medical Sciences, Beijing, China. The toxins were dissolved in phosphate-buffered saline (PBS; 80 mM potassium phosphate buffer with 145 mM NaCl, pH 7.6) to make a 1 mg mL−1 stock solution and stored at −70 ◦ C until used. Freund’s complete adjuvant, Freund’s incomplete adjuvant, Tween 20, ABTS (2,2 -azino bis(3-ethylbenzothiozoline)-6-sulfonic acid), and horseradish peroxidase (HRP) were purchased from Sigma (St. Louis, MO). RPMI 1640 was purchased from HyClone, (Logan, UT, USA). PEG from Merck (MW4000, Germany) was used in the studies. Fetal bovine serum (FBS) was purchased from Gibco (Invitrogen Corporation, Gran Island, NY, USA). SuperSignal ELISA Femto Maximum sensitivity substrate (Pierce, Rockford, IL) served as enhanced chemiluminescence substrate. SBA Clonotyping System was from SouthernBiotech (Birmingham, AL, USA). The 0.05 M carbonate/bicarbonate buffer (pH 9.5) was used as coating buffer and the phosphate buffered saline (PBS buffer, 0.10 M sodium phosphate–0.15 M sodium chloride, pH 7.2) consisting of 0.05% Tween 20 (v/v) was used as washing buffer. The PBS containing 10% FBS (v/v) and 0.3% Tween 20 (v/v) was applied as blocking buffer, and 3% FBS (v/v) containing 0.3% Tween 20 (v/v) was used as dilution buffer. ABTS solution containing 5 mg ABTS, 20 ␮L of 3% H2 O2 , and 10 mL substrate buffer (0.1 M citrate phosphate buffer, pH 5.0) was used as substrate for indirect and competitive ELISA, and TMB solution containing 1 mg TMB, 10 ␮L of 3% H2 O2 , and 10 mL substrate buffer was used as a substrate buffer in sandwich ELISA. All chemicals applied in this study were of analytical grade and used as received from the manufacturer. 2.3. Apparatus The ELISA plates were purchased from Corning (Corning-Costar, Corning, New York, USA), and the white opaque 96-well flatbottomed well plates were from Greiner (Greiner, Germany). A microplate colorimetric ELISA reader (Bio-Rad, Hercules, CA, USA), a microplate luminometer (GENios, Tecan, Austria) were also used in this study. 2.4. Establishment of SEA CLELA and its application 2.4.1. Generation and preparation of mAbs to SEA Hybridoma cell lines secreting mAbs against SEA were generated as conventional protocol in our laboratory [23]. The

16

C. Zhang et al. / Analytica Chimica Acta 796 (2013) 14–19

monoclonal antibody was purified by the ion-exchange chromatography column using the FPLC system. The immunoglobulin class and subclass of each mAb were determined by the use of SBA Clonotyping System following the manufacturer’s recommendations. 2.4.2. Indirect ELISA The ELISA plates were coated with 2 ␮g mL−1 of SEA (unless otherwise stated) in 100 ␮L of coating buffer to each well and incubated overnight at 4 ◦ C. The plates were washed three times. Then SEA mAbs(1 mg mL−1 ) were serially diluted from 1:102 to 1:108 with dilution buffer, added to the wells(100 ␮L/well), and incubated for 1 h at 37 ◦ C. After three times washing, HRP-labeled goat anti mouse IgG diluted 1:2500 were added, and the plates were incubated for 45 min at 37 ◦ C. After a final washing, 100 ␮L ABTS solution was added to each well and incubated for 15 min at 37 ◦ C, then the absorbance at 405 nm was measured with an ELISA plate reader. 2.4.3. Competitive ELISA The ELISA plates were coated with 2 ␮g mL−1 SEA and incubated overnight at 4 ◦ C. After the plates were washed three times, 50 ␮L/well competitive anti-SEA mAbs (100 ␮g mL−1 ) and 50 ␮L/well HRP-labeled anti-SEA mAbs (1 ␮g mL−1 ) were added to each well and incubated at 37 ◦ C for 1 h. After a final washing, 100 ␮L ABTS solution was added to each well, incubated for 15 min at 37 ◦ C, and the absorbance at 405 nm was measured with an ELISA plate reader. 2.4.4. The pairwise interaction analysis of the mAbs to SEA The pairwise interaction analysis ELISA was carried out according to the procedures described previously [23]. Briefly, the ELISA plates were coated with anti-SEA mAbs in coating buffer at a concentration of 10 ␮g mL−1 and incubated overnight at 4 ◦ C. After extensively washing, 100 ␮L SEA (10 ng mL−1 ) in dilution buffer was added to each well and incubated for 1 h at 37 ◦ C. After another washing procedure, the HRP-labeled anti-SEA mAbs were diluted to 10 ␮g mL−1 with dilution buffer and added to the wells (100 ␮L/well), then incubated as before. After a final washing, the plates were incubated with 100 ␮L/well ABTS solution for 15 min at 37 ◦ C, the absorbance of each well was read at 405 nm as earlier. 2.4.5. Sandwich ELISA The ELISA procedures were carried out following the routine method [23]. 2.4.6. SEA CLEIA The white opaque 96-flat-bottomed well plates were coated with anti-SEA mAb of FMU-SEA-2 in coating buffer and incubated at 4 ◦ C overnight. After washing three times, free-binding sites of the wells were blocked with 300 ␮L/well of blocking buffer for 1 h at room temperature. The blocking buffer was discarded before the plates were tapped over adsorbent paper, and washed three times. Then, SEA solutions (100 ␮L/well) with serially diluted were added. After incubation for 1 h at 37 ◦ C and washing thrice, 100 ␮L/well of HRP-labeled FMMU-SEA-33 mAb diluted in dilution buffer (2.5 ␮g mL−1 ) was added and incubated for another 1 h at 37 ◦ C. After washing, 100 ␮L/well of the chemiluminescent substrate (mixture of Super Signal ELISA Femto Luminol Enhancer Solution and Super Signal ELISA Femto Stable Peroxide Solution in a ratio of 1:1) was added to each well. The chemiluminometric signals were measured with a micro-plate luminometer (GENios, Tecan, Austria). The chemiluminometric signal generated from the HRPluminol-H2 O2 system was emitted at a wavelength of 425 nm and measured with a microplate luminometer. After entering the detection system, a glass fiber guides the light from the sample to the detection unit of a photomultiplier tube (PMT), which is designed

for application in luminescence. Reasonable adjustment of the PMT gain can assure a wide dynamic measuring range of sample concentrations. The undesired diffraction orders produced by the optical gratings can be blocked by the filter wheel [22]. The measurement result of luminous intensity is usually represented as relative light unit (RLU). 2.4.7. Intra- and inter-variations of SEA CLEIA The intra- and inter-variations of SEA CLEIA were calculated from three SEA-spiked samples with three concentrations of 3.2 pg mL−1 , 64 pg mL−1 , and 1600 pg mL−1 . Based on the procedures described in SEA CLEIA, the intra-variation of SEA CLEIA was measured by analyzing RLU for each concentration. Ten samples for each SEA concentration were run, and the assay was repeated thrice. The inter-variation was estimated from 10 separate experiments in quadruplicate performed on different days. All the assays were carried out by the same operator. 2.5. Sample preparation For solid matrices, 10 g samples of roast beef, peanut jam, or cured ham chopped into small pieces with 10 mL PBS (pH 7.6) and different concentrations of SEA were mixed by shaking in widemouth glass vials, then incubated for 20 min at room temperature with gentle vortexing [22]. For liquid matrices, 5 mL liquid samples of sewage, tap water, river water, orange juice, skimmed milk, whole milk, or human urine and serum were spiked with different concentrations of SEA in centrifuge tube, and incubated for 20 min at room temperature with gentle vortexing. The matrix–toxin mixtures were centrifuged at 4000 rpm for 30 min at 4 ◦ C to remove solid particles. Subsequently, the supernatant solution was taken and spiked with dilution buffer to produce a serial concentration of SEA from 10 pg mL−1 to 1500 pg mL−1 prior to CLEIA. The food (roast beef, cured ham, nonfat dry milk, milk, and orange juice) used for the analysis was purchased from local grocery stores. 3. Results and discussion 3.1. The screening of antibody pairs for sandwich SEA CLEIA Followed the routine protocol in our laboratory [24], 37 hybridoma clones secreting mAbs to SEA were obtained and designated FMU-SEA-1 to FMU-SEA-37. The screening of optimized antibody pairs for the establishment of sandwich SEA CLEIA was selected by pairwise mapping ELISA. The results of the 37 × 37 mapping ELISA are shown in Fig. 1 in which 6 mAb pairs with the optical density signal higher than 1.5 were selected to form sandwich ELISA. Finally, the pair with most sensitive and wide dynamic range was selected, which was the FMU-SEA-2 mAb and FMU-SEA-33 mAb as the capture antibody and detecting antibody, respectively. 3.1.1. Optimization of CLEIA for detecting SEA Several parameters were optimized as described before [22]. As shown in Fig. 2A, the concentration of HRP-labeled anti-SEA mAbs was optimized by serially diluted from 1:2000 to 1:32000, and the signal-to-noise (S N−1 ) ratios at SEA concentration from 2.5 to 40 pg mL−1 were obtained. As shown in Fig. 2A, the S N−1 ratios varied with the different concentrations of HRP-labeled antiSEA mAbs. When the 1:4000 dilution ratio of HRP-labeled anti-SEA mAb was applied, the range of the S N−1 ratios was from 2.13 to 17.06, which were higher than the S N−1 ratios of other dilution of HRP-labeled anti-SEA mAbs at each SEA concentration. Therefore, the dilution ratio of 1:4000 of HRP-SEA mAb was employed in the subsequent work.

C. Zhang et al. / Analytica Chimica Acta 796 (2013) 14–19

17

Table 1 The comparison of current methods for detecting SEA. Test method

Demonstrated for sample type

Detection limit (pg mL−1 )

ELISA

Food, beverage, urine

Western blot Bioassay Mass spectrometrybased assay CLEIA

Food Assay buffer, food Blood, fruit juice and milk

100 pg mL−1 [10,12], 1000 pg mL−1 [13] 100 pg mL−1 [30] pg molar[31], 20 pg mL−1 [9] 325 pg mL−1 [15], 500 pg mL−1 [32]

A variety of environmental, biological and matrices

3.2 pg mL−1

Table 2 Intra- and inter-variation of SEA CLEIA.

Fig. 1. The screening of antibody pairwise by sandwich ELISA. The “”blankets represent antibody pairs having the absorbance at 405 nm readouts <0.5, “” readouts in the range of 0.5–1.0, “” readouts in 1.0–1.5, the “”readouts >1.50.

3.1.2. Optimization of the concentration of coating anti-SEA mAb To optimize the concentration of coating anti-SEA mAb, the S N−1 ratios of serial coating anti-SEA mAb concentrations were obtained when 1:4000 dilution of the HRP-labeled anti-SEA mAb and 20 pg mL−1 of SEA concentration were applied. As shown in Fig. 2B, with the concentration of coating anti-SEA mAb increased from 2.5 ␮g mL−1 up to 10 ␮g mL−1 , the S N−1 ratios were increased, while the S N−1 ratios went down from 10 ␮g mL−1 to 40 ␮g mL−1 . Therefore, 10 ␮g mL−1 concentration of coating anti-SEA mAb was applied in the subsequent experiments. 3.2. CLEIA for SEA detection Under optimized conditions, a calibration curve was obtained with the SEA concentrations from 6.4 pg mL−1 to 1600 pg mL−1 , which showed a linear correlation (R2 = 0.9952) represented by equation: Y = 23.83X + 192.45,

(1)

Assay type

Conc. of SEA complex (pg mL−1 )

Mean measured concentration (pg mL−1 )

Inter-assay (n = 10)

3.2 64 1600

3.28 ± 0.17 64.21 ± 2.33 1618.64 ± 127.25

5.07 3.62 7.86

Intra-assay (n = 10)

3.2 64 1600

3.31 ± 0.31 65.34 ± 5.24 1627.46 ± 170.40

9.31 8.28 10.47

CV(%)

where Y represented the RLU, X represented the concentration of SEA (pg mL−1 ). The LOD reached 3.2 pg mL−1 , which was defined as RLU signals for background point adding twice the standard deviation of the point (LOD = background mean +2SD, 16 replicates). These results indicated that the microplate CLEIA system for SEA is highly sensitive. Table 1 lists the current methods, which have been used for SEA detection and their detection limits. Obviously, this optimized CLEIA provides the most sensitive way for detecting SEA. The reproducibility of CLEIA was measured by the inter- and intra-assay coefficient of variation (CV), which was calculated in assay buffer spiked with SEA at three concentrations covering from a low concentration to a high concentration within the standard curve. The CV for the intra- and inter-assay precision determinations ranged from 3.62 to 7.86% and from 8.28 to 10.47%, respectively (Table 2), indicating that the method exhibited high reproducibility. The accuracy of the SEA CLEIA was determined by adding increasing amounts of SEA to dilution buffer and measured the

Fig. 2. The optimization of SEA CLEIA. Points represent the mean absorbance values of duplicate wells. Where not visible, the error bars are within the symbols. (A) Optimization of concentration of detecting antibody. (B) Optimization of concentration of capture antibody.

18

C. Zhang et al. / Analytica Chimica Acta 796 (2013) 14–19

Table 3 Recovery of CLEIA. Spike levels (pg mL−1 )

Mean measured concentration (pg mL−1 )

Mean recovery (%)

10 500 1500

9.53 ± 0.54 492.89 ± 32.73 1474.16 ± 121.36

95.3 ± 5.7 98.6 ± 6.6 98.2 ± 8.2

percentage of the recovery by linear regression. The results showed that the average percentages of recoveries were 95.3%, 98.6%, and 98.2% at three concentration of 10 pg mL−1 , 500 pg mL−1 and 1500 pg mL−1 , respectively (Table 3), indicating the accuracy of the SEA CLEIA should be acceptable. 3.3. Method specificity Since the primary sequence homology of the SEs varies from 26 to 80%, the cross reactivity with polyclonal antibodies and monoclonal antibodies has been observed [25]. Therefore, it is important to determine the specificity of the SEA CLEIA. SEB, SEC1, and SED were diluted to the range of the standard curve used for the assay with dilution buffer and tested for cross reactivity. No cross reactivity for any of the serotypes of SEs tested was observed in the SEA CLEIA (Fig. 3), indicating that the mAbs-based sandwich CLEIA is highly specific. 3.4. Detecting SEA in food, beverage, and environmental matrices The SEA is the most prevalent cause of staphylococcal food poisoning and one of the leading causes of gastroenteritis [5]. Because the SEA is heat-resistant toxin, even the bacteria have been destroyed, the activity of the SEA still remains in the SEAcontaminated food or other materials. For this reason, the most related foods are those requiring handling during processing, such as ham or cooked meals, milk, and juice [5]. A wide variety of food matrices have been tested in this study to evaluate the performance of the SEA CLEIA. The food matrix samples were spiked with SEA to the final concentration of 10 pg mL−1 , 500 pg mL−1 , and 1500 pg mL−1 , respectively, and then the concentrations of SEA detected from the food matrix samples were compared with the concentrations detected in dilution buffer with SEA CLEIA. The results were shown in terms of recovery listed in Table 4. The recoveries of ham, beef, and peanut jam were 91.2–98%, 87.3–94.2%, and 87.6–94.5%, respectively. At the concentration of 10 pg mL−1 added in food matrix, all the recoveries were over 87.0%. The beverage matrices we tested were orange juice, whole milk,

and skimmed milk. After adjusting the pH to 7.4, the recovery in orange juice was 93.5–99.2%, indicating that the SEA CLEIA could detect the SEA in orange juice successfully with simple treatment. As shown in Table 4, the milk matrices were treated with centrifugation at 4 ◦ C to eliminate the interference of fat, and the recoveries in whole milk and skimmed milk were 91.3–95.5% and 89.3–91.5%, respectively. As to environmental matrices, all the tested samples produced high recovery (Table 4), suggesting the SEA CLEIA applied in environmental samples may be acceptable. 3.5. Detecting SEA in human serum and urine samples Nowadays, the presence of the SEA in blood stream is considered to be related to the septic shock caused by the proinflammative [6]. It was reported that a young female with S. aureus endocarditis suffered from persistent septic shock after optimal antimicrobials and rapid sterilization of the blood, which was considered to be resulted from the high level of SEA presence in the blood [26]. Presently, very limited diagnostic tests are available for detecting the SEA directly in blood samples of patient, because picomolar amounts of the SEA has been shown to induce lymphocyte proliferation in vitro, the amount of SEA presenting in blood stream is likely at very low concentration, and the sensitivity of tradition immunoassays were failed to detect the SEA in blood stream. Although the mass spectrometry-based assay for targeted and quantification of SEA in serum was developed and used for detecting the SEA in serum and the sensitivity was still low (around 352 pg mL−1 ) [15]. By spiked the SEA in serum samples with escalating concentrations from 10 pg mL−1 to 1500 pg mL−1 , the recovery in serum for the SEA CLEIA was 83.3–90.3% (Table 4), indicating the method we presented here is highly sensitive (3.2 pg mL−1 ) and could be used in the detection of SEA in serum samples of patients. Since the SEA was secreted via the urine, the SEA present in human urine samples may indicate the human exposure of SEA [27,28]. Different laboratories reported contradictious results of urine matrix interference in detecting SEA. Some papers reported no matrix inhibition and quenching of chemiluminescence signal while others found great interference from matrix effect [12,29]. The SEA CLEIA presented here display no matrix interference by human urine, and the recovery was 91.2–94.1% in human urine (Table 4) [22]. When human urine samples spiked with SEA as low as at the concentration of 10 pg mL−1 . Since the SEA CLEIA could detect the SEA in human serum and urine at the concentration of pg mL−1 level and good recovery was obtained, this method may have a potential application to evaluate the clinical specimen from patients with suspicious SEA poisoning.

Fig. 3. The specificity of the SEA CLEIA. No cross-reaction to different SEs was observed, either (A) concentrations of SEA, SEB, SEC, or SED 10–100 pg mL−1 or (B) concentrations of SEA, SEB, SEC or SED 10–2000 pg mL−1 were added in the CLEIA.

C. Zhang et al. / Analytica Chimica Acta 796 (2013) 14–19

19

Table 4 Recovery in food, beverage, and environmental matrices of SEA CLEIA. Spike levels (pg mL−1 ) 10

The recovery of SEA in different concentrations (%)

Cured ham Roast beef Peanut jam Orange juice Whole milk Skimmed milk River Tap water Sewage Human serum Human urine

4. Conclusions In conclusion, a highly sensitive micro-plate CLEIA for detecting SEA was developed by using a pair of highly specific mAbs, which displayed high sensitivity with a detection limit of 3.2 pg mL−1 and a linear range of 6.4–1600 pg mL−1 and specificity. Thus, the SEA CLEIA we developed may be able to detect SEA at subtoxic concentrations in a wide variety of matrices without apparent matrix interference, which may be useful in food hygiene supervision, environment management, and detection of SEA in clinical samples.

91.2 87.3 87.6 99.2 91.3 89.3 94.8 89.8 97.5 83.3 91.2

[8] [9] [10] [11] [12] [13] [14] [15]

[16] [17]

Acknowledgments [18]

We thank the National Key Technology R&D program of China (2012BAK25B01) and National Key project of China (AWS11C001).

[19] [20]

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2013.07.044. References [1] T. Asao, Y. Kumeda, T. Kawai, T. Shibata, H. Oda, K. Haruki, H. Nakazawa, S. Kozaki, Epidemiol. Infect. 130 (2003) 33. [2] J. Schelin, N. Wallin-Carlquist, M.T. Cohn, R. Lindqvist, G.C. Barker, P. Radstrom, Virulence 2 (2011) 580. [3] J.D. Fraser, T. Proft, Immunol. Rev. 225 (2008) 226. [4] M.A. Argudin, M.C. Mendoza, M.R. Rodicio, Toxins 2 (2010) 1751. [5] N. Balaban, A. Rasooly, Int. J. Food Microbiol. 61 (2000) 1. [6] T. Ferry, D. Thomas, A.L. Genestier, M. Bes, G. Lina, F. Vandenesch, J. Etienne, Clin. Infect. Dis. 41 (2005) 771. [7] O. Dauwalder, D. Thomas, T. Ferry, A.L. Debard, C. Badiou, F. Vandenesch, J. Etienne, G. Lina, G. Monneret, J. Leukoc. Biol. 80 (2006) 753.

[21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]

500 ± ± ± ± ± ± ± ± ± ± ±

3.9 8.2 7.3 6.5 7.6 10.1 7.6 4.6 9.7 5.1 7.4

98.0 94.2 94.5 97.6 95.5 93.8 115.1 97.1 101.6 88.9 94.1

1500 ± ± ± ± ± ± ± ± ± ± ±

12.6 8.8 8.5 10.8 8.5 8.3 3.8 3.2 16.8 6.2 7.5

92.3 88.6 89.8 93.5 91.5 91.5 95.4 97.0 98.2 90.3 91.9

± ± ± ± ± ± ± ± ± ± ±

4.7 6.9 4.4 6.1 7.1 7.2 10.1 6.4 7.5 8.6 7.8

J.D. Fraser, PLoS Biol. 9 (2011) e1001145. R. Rasooly, P.M. Do, FEMS Immunol. Med. Microbiol. 56 (2009) 172. H. Fey, H. Pfister, O. Ruegg, J. Clin. Microbiol. 19 (1984) 34. M.T. Lam, Q.H. Wan, C.A. Boulet, X.C. Le, J. Chromatogr. B 853 (1999) 545. M.A. Poli, V.R. Rivera, D. Neal, Toxicon 40 (2002) 1723. N.E. Thompson, M. Razdan, G. Kuntsmann, J.M. Aschenbach, M.L. Evenson, M.S. Bergdoll, Appl. Environ. Microbiol. 51 (1986) 885. T. Sasaki, Y. Terano, T. Shibata, H. Kawamoto, T. Kuzuguchi, E. Kohyama, T. Watanabe, T. Ohyama, M. Gemba, Microbiol. Immunol. 49 (2005) 589. A. Adrait, D. Lebert, M. Trauchessec, A. Dupuis, M. Louwagie, C. Masselon, M. Jaquinod, B. Chevalier, F. Vandenesch, J. Garin, C. Bruley, V. Brun, J. Proteomics 75 (2011) 3041. M. Yang, Y. Kostov, A. Rasooly, Int. J. Food Microbiol. 127 (2008) 78. M. Salmain, M. Ghasemi, S. Boujday, J. Spadavecchia, C. Techer, F. Val, V. Le Moigne, M. Gautier, R. Briandet, C.M. Pradier, Biosens. Bioelectron. 29 (2011) 140. S. Wang, T. Yin, S. Zeng, H. Che, F. Yang, X. Chen, G. Shen, Z. Wu, PloS one 7 (2012) e30779. J. Jantra, P. Kanatharana, P. Asawatreratanakul, B. Wongkittisuksa, C. Limsakul, P. Thavarungkul, J. Environ. Sci. Health 46 (2011) 560. T.B. Xin, X. Wang, H. Jin, S.X. Liang, J.M. Lin, Z.J. Li, Appl. Biochem. Biotechnol. 158 (2009) 582. X.Y. Yang, Y.S. Guo, S. Bi, S.S. Zhang, Biosens. Bioelectron. 24 (2009) 2707. F. Liu, Y. Li, C. Song, B. Dong, Z. Liu, K. Zhang, H. Li, Y. Sun, Y. Wei, A. Yang, K. Yang, B. Jin, Anal. Chem. 82 (2011) 7758. Z. Liu, C. Song, Y. Li, F. Liu, K. Zhang, Y. Sun, H. Li, Y. Wei, Z. Xu, C. Zhang, A. Yang, Z. Xu, K. Yang, B. Jin, Anal. Chim. Acta 735 (2012) 23. K. Zhang, C. Song, Q. Li, Y. Li, Y. Sun, K. Yang, B. Jin, Hum. Vaccin. 6 (2010) 652. M.M. Dinges, P.M. Orwin, P.M. Schlievert, Clin. Microbiol. Rev. 13 (2000) 16. M. Ellis, A. Serreli, P. Colque-Navarro, U. Hedstrom, A. Chacko, E. Siemkowicz, R. Mollby, J. Med. Microbiol. 52 (2003) 109. G.J. Crawley, I. Gray, W.A. Leblang, J.W. Blanchard, J. Infect. Dis. 116 (1966) 48. M. Nagaki, R.D. Hughes, H.M. Keane, J. Goka, R. Williams, J. Med. Microbiol. 38 (1993) 354. T.M. Kijek, C.A. Rossi, D. Moss, R.W. Parker, E.A. Henchal, J. Immunol. Methods 236 (2000) 9. A. Rasooly, R.S. Rasooly, Int. J. Food Microbiol. 41 (1998) 205. T. Hawryluk, I. Hirshfield, J. Food Prot. 65 (2002) 1183. I. Sospedra, C. Soler, J. Manes, J.M. Soriano, J. Chromatogr. A 1238 (2012) 54.