Highly Sensitive Microplate Chemiluminescence Enzyme

Aug 27, 2010 - (1) SEB is stable and can be prepared easily and spread in the form of aerosol. ... (18-20) It has been reported that the chemiluminesc...
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Anal. Chem. 2010, 82, 7758–7765

Highly Sensitive Microplate Chemiluminescence Enzyme Immunoassay for the Determination of Staphylococcal Enterotoxin B Based on a Pair of Specific Monoclonal Antibodies and Its Application to Various Matrices Fei Liu, Yongming Li, Chaojun Song, Bangquan Dong, Zhijia Liu, Kui Zhang, Haitao Li, Yuanjie Sun, Yuying Wei, Angang Yang, Kun Yang,* and Boquan Jin* Department of Immunology, The Fourth Military Medical University, No. 17 Changle West Road, Xi’an 710032, Shaanxi Province, People’s Republic of China A highly specific and sensitive microplate chemiluminescent enzyme immunoassay (CLEIA) was established and validated for the detection of staphylococcal enterotoxin B (SEB). A pair of monoclonal antibodies (mAbs) that recognizes different epitopes of SEB was selected from 20 SEB-specific mAbs, and the experimental conditions were examined and optimized for the development of the CLEIA. This method exhibited high performance with a dynamic range of 0.01-5 ng/mL, and the measured limit of detection (LOD) was 0.01 ng/mL. Intra- and interassay coefficient variations were all lower than 13% at three concentrations (0.2, 0.4, and 2 ng/mL). For specificity studies, when this method was applied to test staphylococcal enterotoxins A, C1, and D, no cross-reactivity was observed. It has been successfully applied to the analysis of SEB in a variety of environmental, biological and humoral matrices such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, and human urine and serum. The aim of this article is to show that the highly sensitive, specific, and simple microplate CLEIA, based on a pair of highly specific monoclonal antibodies, has potential applications for quantifying SEB in public health and military reconnaissance. Staphylococcal enterotoxins (SEs) represent a group of low molecular mass (26-30 kDa) toxic proteins that can remain biologically active after exposure to both proteolytic enzymes and high temperatures. As one of the major SEs causing food poisoning, staphylococcal enterotoxin B (SEB) is extremely toxic with a half-lethal dose (LD50) of about 20 ng/kg and a halfeffective dose (ED50) of about 0.4 ng/kg.1 SEB is stable and can be prepared easily and spread in the form of aerosol. According to the Centers for Disease Control and Prevention * To whom correspondence should be addressed: (phone) +86-29-84774598; (fax) +86-29-83253816; (e-mail) [email protected] (B.J.) or yangkunkun@ fmmu.edu.cn. (1) Gill, D. M. Microbiol. Rev. 1982, 46, 86–94.

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(CDC) list, it is a category B potential biological weapon.2,3 Highly sensitive and specific methods for detection of SEB in food, environmental water, and even body fluids such as serum and urine of individuals suspected to have been exposed to toxin are of critical importance in food safety and the war against terrorism. Different assay formats have been developed with the aim of detecting SEB in an ultrasensitive, specific, simple, and rapid way. Traditional detection methods, such as reversed passive latex agglutination (RPLA),4 enzyme-linked immunosorbent assay (ELISA),5,6 and immunofluorescence technique7-9 are often of poor detection sensitivity, and among these the best sensitivity reported was 0.02 ng/mL of SEB in assay buffer.10 A new eightchannel lab-on-a-chip (LOC) for SEB detection without a laboratory has been reported.11 Recently, several useful antigen/antibodybased biosensors for SEB assay have been proposed, such as surface plasmon resonance (SPR) biosensors,12 resonant acoustic profiling (RAP),13 biological semiconductors (BSCs),14 fluidic force discrimination (FFD) assay, flow-injection capacitive biosensor, and piezoelectric-excited millimeter-sized cantilever (PEMC) sensors. Some of these biosensors for the detection of SEB are highly sensitive, especially the flow-injection capacitive biosensor with (2) Ferguson, J. R. JAMA, J. Am. Med. Assoc. 1997, 278, 357–360. (3) Rosenbloom, M.; Leikin, J. B.; Vogel, S. N.; Chaudry, Z. A. Am. J. Ther. 2002, 9, 5–14. (4) Fujikawa, H.; Igarashi, H. Appl. Environ. Microbiol. 1988, 54, 2345–2348. (5) Sasaki, T.; Terano, Y.; Shibata, T.; Kawamoto, H.; Kuzuguchi, T.; Kohyama, E.; Watanabe, T.; Ohyama, T.; Gemba, M. Microbiol. Immunol. 2005, 49, 589–597. (6) Morissette, C.; Goulet, J.; Lamoureux, G. Appl. Environ. Microbiol. 1991, 57, 836–842. (7) Khan, A. S.; Cao, C. J.; Thompson, R. G.; Valdes, J. J. Mol. Cell. Probes 2003, 17, 125–126. (8) Alefantis, T.; Grewal, P.; Ashton, J.; Khan, A. S.; Valdes, J. J.; Del Vecchio, V. G. Mol. Cell. Probes 2004, 18, 379–382. (9) Poli, M. A.; Rivera, V. R.; Neal, D. Toxicon 2002, 40, 1723–1726. (10) Khreich, N.; Lamourette, P.; Boutal, H.; Devilliers, K.; Creminon, C.; Volland, H. Anal. Biochem. 2008, 377, 182–188. (11) Yang, M.; Sun, S.; Kostov, Y.; Rasooly, A. Lab Chip 2010, 10, 1011–1017. (12) Soelberg, S. D.; Stevens, R. C.; Limaye, A. P.; Furlong, C. E. Anal. Chem. 2009, 81, 2357–2363. (13) Natesan, M.; Cooper, M. A.; Tran, J. P.; Rivera, V. R.; Poli, M. A. Anal. Chem. 2009, 81, 3896–3902. (14) Yang, M.; Bruck, H. A.; Kostov, Y.; Rasooly, A. Anal. Chem. 2010, 82, 3567–3572. 10.1021/ac101666y  2010 American Chemical Society Published on Web 08/27/2010

limit of detection (LOD) ) 0.3 pg/mL,15 the PEMC sensors, which can detect SEB in the range of 2.5 fg/mL-50 pg/mL,16 and the FFD assay with LOD ) 1 fg/mL.17 However, these experiments are mostly used in scientific research in special professional departments, and some biosensors are more expensive and not suitable for high-throughput detection. Chemiluminescent enzyme immunoassay (CLEIA), in particular enhanced chemiluminescent immunoassay with a constant and steady glow-type luminescence, has been widely exploited in recent years due to its high sensitivity, wide dynamic range, and suitability for miniaturization.18-20 It has been reported that the chemiluminescent reaction often exceeds the sensitivity of radioactivity assays.21 The basic principle of CLEIA is that the chemiluminescence intensity produced by a chemical reaction is directly proportionate to the amount of analytes present in a sample. One common characteristic of CLEIA is that they all based on antibody-antigen complexes and tracer-labeled reactions, of which the enzyme-linked immunosorbent assays are highly specific and sensitive. The enzymes used for chemiluminescence are peroxidase, alkaline phosphatase, and β-D-galactosidase, the most popular of which is peroxidase. As antibodies and enzyme substrates are often key factors that affect the detection level of the CLEIA, and to address the need for highly specific SEB assays that can be performed in a simple and rapid way and used widely, we developed a microplate CLEIA based on a pair of highly specific monoclonal antibodies (mAbs) and a sensitive horseradish peroxidase (HRP)-luminol-H2O2 chemiluminescence system for detecting SEB. The microplate luminometers are now more readily accessible than the complicated Ru-based electrochemiluminescence (ECL) detectors and multiple types of biosensors in most instances, and they can be relied on for accurate and consistent detection of luminescent output. The analysis speed is rapid, approximately 2 min per 96-sample plate (1 s per sample), which is ideal for clinical practice in hospital where the microplate luminometer is easily available and the high-throughput detection is needed. Also, the enhanced chemiluminescence (ECL) using carbon nanotubes (CNTs) for primary antibody immobilization in advance based on a cooled charge-coupled device (CCD) detector22 and the immunomagnetic-electrochemiluminescent detection where captured antibody is prebound to streptavidin-coated paramagnetic beads23 that adds to the cost and inconvenience of the analysis. But in the microplate CLEIA system, the captured antibody is directly coated onto the microplate wells without pretreatment; this appears to be more simple and user-friendly. The sensitive HRP chemiluminescent substrate used in our assay has the ability to produce long periods of light emission in (15) Labib, M.; Hedstrom, M.; Amin, M.; Mattiasson, B. Anal. Bioanal. Chem. 2009, 393, 1539–1544. (16) Maraldo, D.; Mutharasan, R. Anal. Chem. 2007, 79, 7636–7643. (17) Mulvaney, S. P.; Myers, K. M.; Sheehan, P. E.; Whitman, L. J. Biosens. Bioelectron. 2009, 24, 1109–1115. (18) Xin, T. B.; Wang, X.; Jin, H.; Liang, S. X.; Lin, J. M.; Li, Z. J. Appl. Biochem. Biotechnol. 2009, 158, 582–594. (19) Bi, S.; Zhou, H.; Zhang, S. Biosens. Bioelectron. 2009, 24, 2961–2966. (20) Yang, M.; Kostov, Y.; Bruck, H. A.; Rasooly, A. Int. J. Food Microbiol. 2009, 133, 265–271. (21) Creton, R.; Jaffe, L. F. BioTechniques 2001, 31, 1098–1105. (22) Yang, M.; Kostov, Y.; Bruck, H. A.; Rasooly, A. Anal. Chem. 2008, 80, 8532–8537. (23) Kijek, T. M.; Rossi, C. A.; Moss, D.; Parker, R. W.; Henchal, E. A. J. Immunol. Methods 2000, 236, 9–17.

an intense and stable state, which makes it compatible with microplate assay formats. Moreover, the use of mAbs has resulted in a more specific and stronger binding to the target antigen of SEB and precise measurement of SEB. In this study, 20 mAbs were obtained first for SEB, and then the ascites titer and epitope specificities of these mAbs were evaluated by direct and competitive ELISA, respectively. Two mAbs with high titer, high specificity, and different epitope specificities were chosen to further establish a CLEIA for detecting SEB in a sandwich format. The reaction parameters of SEB CLEIA were studied and optimized. In addition, methodology parameters were evaluated, and the matrix effects of various matrices on the SEB CLEIA were investigated. We demonstrate that the microplate CLEIA has the ability to detect SEB with high sensitivity and specificity and has great practical value in food safety, public health, and military security. MATERIALS AND METHODS Reagents. Staphylococcal enterotoxins (SE) A, B, C1, and D were obtained from the Academy of Military Medical Sciences, Beijing. The toxins were dissolved in phosphate-buffered saline (PBS; 80 mM potassium phosphate buffer with 145 mM NaCl, pH 7.6) to prepare a 1 mg/mL stock solution and stored frozen at -20 °C until used. SEs are hazardous and should be handled with care in the experiments. Fetal bovine serum (FBS) was purchased from Gibco (Invitrogen Corp., Grand Island, NY). Freund’s complete adjuvant, Freund’s incomplete adjuvant, Tween 20, ABTS [2,2′-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid], and horseradish peroxidase (HRP) were purchased from Sigma (St. Louis, MO). RPMI1640 (HyClone, Logan, UT) and poly(ethylene glycol) (PEG) (MW4000, Merck, Germany) were also used in the studies. SuperSignal ELISA Femto Maximum sensitivity substrate (Pierce, Rockford, IL) and Lumigen PS-atto substrate (Lumigen, Inc.) served as enhanced chemiluminescence substrates. SBA Clonotyping System was from Southern Biotech (Birmingham, AL). The coating buffer was 0.05 M carbonate/ bicarbonate buffer (pH 9.5) and the washing buffer consisted of 0.05% Tween 20 (v/v) in PBS. PBS containing 10% FBS (v/v) and 0.3% Tween 20 (v/v) were applied as dilution buffer. ABTS solution containing 5 mg ABTS, 20 µL of 3% H2O2, and 10 mL substrate buffer (0.1 M citrate phosphate buffer, pH 5.0) was used as substrate for indirect and competitive ELISA. All chemicals used were of analytical grade and used as received from the manufacturer. The food used for the analysis (roast beef, peanut butter, cured ham, nonfat dry milk, milk, and orange juice) was purchased from local grocery stores. Apparatus. The ELISA plate was purchased from Corning (Corning-Costar, Corning, NY), and the white opaque 96-flatbottomed well plates were from Greiner (Greiner, Germany). A microplate colorimetric ELISA reader (Bio-Rad, Hercules, CA) and a microplate luminometer (GENios, Tecan, Austria) were used in this study. Preparation of Anti-SEB MAbs. Hybridoma cell lines secreting mAbs to SEB were raised by conventional protocol in our laboratory.24 Briefly, female BALB/c mice (8 weeks old) were immunized with 20 µg/mouse SEB immunogen in complete (24) Ouyang, W.; Xue, J.; Liu, J.; Jia, W.; Li, Z.; Xie, X.; Liu, X.; Jian, J.; Li, Q.; Zhu, Y.; Yang, A.; Jin, B. J. Immunol. Methods 2004, 292, 109–117.

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Figure 1. Schematic illustration of sandwich CLEIA for SEB detection.

Freund’s adjuvant via multiple subcutaneous (sc) injections. Two booster injections (20 µg/mouse) of the immunogen mixed with Freund’s incomplete adjuvant were given at 1-month intervals. When the titers of sera from immunized mice exceeded 1:64 000, the immunized mice were given another boost (20 µg/mouse immunogen) by intraperitoneally (ip). Three days later, splenocytes from immunized mice and SP2/0 myeloma cells that were cultured in RPMI 1640 containing 20% FBS were fused in the presence of PEG. The positive hybrids were selected by indirect ELISA and subcloned four times through the limiting dilution method. mAbs were produced from supernatants of the hybridoma culture or from ascites of BALB/c mice which had been injected with hybridoma cells (106/mouse) and purified by ion-exchange chromatography column. Coupling of the SEB mAbs with HRP was performed following a previously described method.25 The ascites titers were tested by indirect ELISA, and the immunoglobulin class and subclass of each mAb were determined by use of the SBA clonotyping system following the manufacturer’s recommendations. Indirect ELISA. The wells of the ELISA plate were coated with 2 µg/mL SEB in coating buffer and incubated overnight at 4 °C and then were used immediately. Plates were washed three times with washing buffer, and then ascites we established were serially diluted from 1:102 to 1:109 with dilution buffer, added to the wells (100 µL/well), and incubated for 1 h at 37 °C. After washing, HRP-conjugated goat anti-mouse IgG diluted 1:2500 was added and the plates were incubated for 45 min at 37 °C. After a final washing, the plates were coated with 100 µL/well ABTS solution and incubated for 15 min at 37 °C, and the absorbance was read at 405 nm on an ELISA plate reader. Competitive ELISA. A 96-well microplate coated with 2 µg/ mL SEB overnight was incubated at 4 °C and then was used immediately. The plate was washed three times with washing buffer. Then, 50 µL/well competitive anti-SEB mAbs (100 µg/ mL) and 50 µL/well HRP-labeled anti-SEB mAbs (1 µg/mL) were (25) Nakane, P. K.; Kawaoi, A. J. Histochem. Cytochem. 1974, 22, 1084–1091.

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added to the coated wells and the plate was incubated at 37 °C for 1 h. The plate was washed as before, 100 µL/well substrate solution containing ABTS was added, and the plate was incubated for 15 min at 37 °C. Finally, the absorbance at 405 nm was measured with a microplate reader. SEB CLEIA. The white opaque 96-flat-bottomed well plates were coated with anti-SEB mAbs of FMU-SEB-2 in coating buffer and incubated at 4 °C overnight and then were used immediately. After the plate was washed three times with washing buffer, free binding sites of the wells were blocked with 200 µL/well dilution buffer for 1 h at room temperature. Afterward, 100 µL/well SEB serially diluted with dilution buffer was added. Plates were incubated at 37 °C for 1 h and washed three times, and then the wells were reacted with a detecting antibody (100 µL/well), antiSEB mAbs of FMU-SEB-1 that had been conjugated with HRP and diluted in dilution buffer. After incubation at 37 °C for 1 h and three more extensive washes, the chemiluminescent substrates (mixture of SuperSignal ELISA femto luminol enhancer solution and SuperSignal ELISA femto stable peroxide solution, 100 µL/well) were added to the plates in a ratio of 1:1. The chemiluminometric signal generated from the HRP-luminolH2O2 system was emitted at a wavelength of 425 nm and measured with a microplate luminometer (Figure 1A). 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 ensure a wide dynamic measuring range of sample concentrations. The undesired diffraction orders produced by the optical gratings can be blocked by the filter wheel (Figure 1B). The measurement result of luminous intensity is usually represented as relative light units (RLU). Sample Preparation. For solid matrices, 10-g samples of roast beef, peanut butter, and cured ham were chopped into small pieces thoroughly and then transferred to a mortar and ground to homogeneous matrices. The matrices were spiked with a certain amount of pure SEB and mixed with 10 mL of PBS (pH 7.6) by

Figure 2. Pairwise antigenic epitope mapping of SEB in dilution buffer in a competitive format. (b) HRP-labeled anti-SEB monoclonal antibody was allowed to bind to the antigen-binding site of SEB molecule in the presence of 100-fold concentration of unlabeled SEB mAbs; (O) binding was blocked.

shaking in wide-mouth glass vials. The matrix-toxin mixtures were pipetted up and down 10 times to mix and incubated on a magnetic stirring apparatus for 20 min at room temperature. For liquid matrices, 5 mL liquid samples of sewage, tap water, river water, orange juice, 10% nonfat dry milk, milk, orange juice, and human urine and serum were spiked with a certain amount of pure SEB in a centrifuge tube; the matrix-toxin mixtures were pipetted up and down 10 times to mix and incubated on a magnetic stirring apparatus for 20 min at room temperature. The solution was centrifuged at 4000 rpm for 30 min at 4 °C to remove solid particles. Subsequently, the supernatant solution was removed and diluted with dilution buffer to produce a serial final concentration of SEB from 0.01 to 5 ng/mL prior to CLEIA. RESULTS AND DISCUSSION Twenty positive hybridoma clones secreting mAbs to SEB were obtained and designated FMU-SEB-1 to FMU-SEB-20. Most of the isotypes of them were IgG1 (κ) except samples 3, 4, 10, and 16, which were IgG 2a (κ). The ascites antibody titer of samples 1, 2, and 4 reached 10-7, while the others were 10-6. These results indicate that all of the mAbs against SEB were of a relatively high titer. Pairwise antigenic epitope mapping of SEB using a competitive format (Figure 2) revealed that epitopes recognized by these 20 mAbs can be divided into six epitope groups: (1) samples 1, 5, and 6; (2) samples 2, 10, 14, 16, and 18-20; (3) samples 3 and 4; (4) samples 7 and 13; (5) samples 8, 11, 12, 15, and 17; and (6) sample 9. Each group represented the same or largely overlapped epitope of SEB. To confirm the relationship, we have performed a sandwich ELISA pairwise interaction analysis. The 20 mAbs were labeled with HRP and each was then utilized as either the capture or detection antibody to determine

the optimum combination. The result indicated that the highest absorbance value was obtained when mAb FMU-SEB-2 was used as the capture antibody and HRP-labeled mAb FMU-SEB-1 was used as the detection antibody. Significantly lower absorbance was observed when any of the other combinations of capture and detection antibody were employed. Optimization of CLEIA for Detecting SEB. According to the results obtained from pairwise antigenic epitope mapping and pairwise interaction analyses, two mAbs of FMU-SEB-1 and FMUSEB-2 with high titer from epitope groups 1 and 2 were used to establish a sandwich CLEIA. With mAb FMU-SEB-2 as capture antibody and HRP-conjugated mAb FMU-SEB-1 as detection antibody, a sandwich SEB CLEIA was established. Several parameters were optimized to develop the CLEIA, including the chemiluminescent kinetic profile, the concentrations of HRP-labeled anti-SEB mAbs, and the concentration of coating anti-SEB mAbs. Standard curves were obtained by plotting relative light units (RLU) against the analyte concentration of SEB. Optimization of Chemiluminescent Kinetic Profile. A comparison of the SuperSignal ELISA femto maximum sensitivity substrate and the Lumigen PS-atto substrate showed that the former produced a much stronger signal and was employed in our experiment. The chemiluminescent kinetic profile of the CLEIA for a sandwich immunoassay with the SEB calibrator of 1 ng/mL was shown in Figure 3A. After adding chemiluminescent substrate to the plate, the signal-to-noise ratio (S/N ) average RLU reading/average RLU reading of negative values) of the SEB CLEIA increased with increasing time up to 20 min and exhibited a plateau lasting for about 40 min, indicating a sufficient interaction between enzyme and substrate. The results showed that the Analytical Chemistry, Vol. 82, No. 18, September 15, 2010

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Figure 3. Optimization of CLEIA for detecting SEB. (A) Chemiluminescent kinetic profile of CLEIA with the SEB calibrator of 1 ng/mL. Each point represents the mean value ( SD of triplicate determinations. (B) Optimization of concentrations of HRP-labeled anti-SEB monoclonal antibody. Each point represents the mean value ( SD of triplicate determinations. (C) Optimized dose-response curve for concentration of coating anti-SEB monoclonal antibody. The dilution ratio of the HRP-labeled anti-SEB monoclonal antibody was 1:16 000. The CLEIA was based on a SEB concentration of 10 pg/mL. Each point represents the mean value ( SD of triplicate determinations. (D) Calibration curve for the determination of SEB. Seven concentrations of SEB (0, 0.01, 0.05, 0.1, 0.5, 1.0, and 5.0 ng/mL) were employed, and the inset is the amplification of 0-0.5 ng/mL. Points are means of triplicate determinations. Error bars within the symbols are not visible.

chemiluminescence reaction time from 20 to 60 min was suitable for the immunoreactions, and this was employed in the subsequent work. Optimization of the Concentration of HRP-Labeled AntiSEB mAb. Optimization of the appropriate dilution of HRP conjugate is one of the key factors determining the sensitivity and working range of CLEIA. Excess HRP in the system depleted the substrate quickly, resulting in an overall increase in background and thereby a decrease in detection sensitivity. The necessary dilution will require optimization for each experimental system. To reduce background noise and to obtain maximized sensitivity in SEB CLEIA, the concentrations of HRP-labeled anti-SEB mAbs were optimized. As shown in Figure 3B, serial dilutions of SEB toxin were prepared in dilution buffer to determine the optimal HRP-labeled anti-SEB mAb concentration providing the best signal-to-noise ratio (S/N). The serial calibrator concentrations were 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 ng/ mL, designated as S0 through S11, correspondingly. The concentration of stock HRP-labeled anti-SEB mAb is 5 mg/mL and the five curves correspond to a series of dilution ratios (1:4000, 7762

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1:8000, 1:16 000, 1:32 000, and 1:64 000). The S/N ratios varied with the dilution ratios of HRP-labeled anti-SEB mAb. When SEB concentrations from S4 to S11 were applied, all the S/N ratios for the dilution ratios of 1:8000, 1:16 000, and 1:32 000 were more than 2, from 2.14 to 1047.29, and the S/N ratios for dilution ratios of 1:4000 and 1:64 000 were less than 2 at S4. When SEB concentrations of S1 and S2 were applied, all S/N ratios were less than 2. The S/N ratio of RLUS3/RLUS0 was studied and the dilution ratio of 1:16 000 corresponds to the highest S/N ratio of 2.79. Therefore, the dilution ratio of 1:16 000 was selected, since it corresponded to the highest sensitivity compared with the other dilution ratios. We also found that the S/N ratio decreased at SEB concentrations more than S10 and a Prozone effect occurred, indicating an antigen excess. Optimization of the Concentration of Coating Anti-SEB mAb. It is most important to determine the optimal antibody concentration for coating in each CLEIA system by suitable titrations, since the actual antibody density may affect results. When 1:16 000 dilution of the HRP-labeled anti-SEB mAb and 10

Table 1. Intra- and Interassay of SEB CLEIA concn of SEB (ng/mL)

mean measd concn (ng/mL)

CV (%)

0.2 0.4 2.0

intraassay (n ) 10) 0.19 ± 0.01 0.39 ± 0.03 1.81 ± 0.16

5.3 7.7 8.8

0.2 0.4 2.0

interassay (n ) 8) 0.19 ± 0.02 0.41 ± 0.04 2.09 ± 0.26

10.5 9.8 12.4

pg/mL SEB concentration were employed, the S/N ratio increased with the concentration of coating anti-SEB mAb from 4 µg/mL up to 12 µg/mL where lower concentrations produced lower S/N ratio, indicating that not all the available antigen of SEB was being captured. Then the S/N ratio went down from between 12 and 20 µg/mL; this means that with greater than 12 µg/mL concentration of coating, anti-SEB mAb gets saturated and this affects the detection sensitivity (Figure 3C). The trapped antibody molecules are packed too close together at these higher concentrations and therefore are not able to capture the antigen effectively because of steric hindrance. The saturated antibodies may also increase the stacking of antibodies, which leads to multilayered binding and allows a less stable interaction of antigen and antibody.26 Hence, the concentration of coating anti-SEB mAb of 12 µg/mL was chosen in our subsequent experiments. Calibration Curve for the Determination of SEB. Under optimized conditions, a calibration curve (Figure 3D) obtained at SEB concentrations from 0 to 5.0 ng/mL showed a linear correlation (R ) 0.9989) represented by Y ) 12 616X + 44.17, where Y represents the relative light units (RLU) and X represents the concentration of SEB (nanograms/milliliter). A linear range of 0.01-5 ng/mL was obtained and the theoretical limit of detection (LOD) reached 0.0043 ng/mL, which was defined as RLU signals for background point with triple the standard deviation of the point added (mean LOD ) background +3SD, 23 replicates). The actual measured LOD is 0.01 ng/mL in this system. These results indicate that the microplate CLEIA system for SEB is very sensitive. Precision. Precision was determined by intra- and interassay, which was calculated from three SEB-spiked samples with a final concentration of 0.2 ng/mL (low concentration), 0.4 ng/mL (medium concentration), and 2.0 ng/mL (high concentration). The intraassay precision of the analytical method was calculated by analyzing each concentration 10 times per run within one time, and the interassay was calculated by analyzing each concentration included in eight assay times. The coefficients of variation (CV) for the intra- and interassay precision determinations ranged from 5.3% to 8.8% and from 9.8% to 12.4%, respectively (Table 1), thus indicating the proposed method exhibited high reproducibility. Accuracy. Accuracy was evaluated by adding increasing amounts of SEB to dilution buffer, measuring the percentage recovery by use of linear regression, and fitting the plotted measured SEB concentration with the spiked levels. Generally, recoveries should be within 20% of target for a CLEIA to be considered accurate. The results showed that the average of (26) Cantarero, L. A.; Butler, J. E.; Osborne, J. W. Anal. Biochem. 1980, 105, 375–382.

Table 2. Recovery in Dilution Buffer of SEB CLEIA (n ) 10) spike levels (ng/mL)

mean measd concn (ng/mL)

mean recovery (%)

0.2 0.4 2.0

0.19 ± 0.01 0.39 ± 0.03 1.82 ± 0.16

95.0 ± 5.0 97.5 ± 7.5 91.0 ± 8.0

recoveries was 91.0%, 95.0%, and 97.5%, respectively (Table 2), indicating the accuracy of the SEB CLEIA applied in food, environmental, or biological samples may be acceptable. Method Specificity. Although the SEs are serologically distinct, cross-reactivity with polyclonal antibodies27,28 and monoclonal antibodies29 have been observed owing to their primary amino acid sequence homology, so it is important for us to check the specificity of the SEB CLEIA. Staphylococcal enterotoxins A, C1, and D (SEA, SEC1, and SED) were diluted to 1 ng/mL with dilution buffer and tested in a sandwich format. None of the toxins exhibited cross-reactivity in the SEB CLEIA (Figure 4). The result was different from the SEB ECL assay reported by Kijek et al,23 from which reactivity with SED was observed, indicating that the polyclonal goat detection antibody used in the SEB ECL assay recognized epitopes shared by SEB and SED. The difference in specificity is likely to come from the two mAbs used in our SEB CLEIA system with a high affinity and specificity. Interestingly, in the resonant acoustic profiling (RAP) SEB assay,13 significant cross reactivity with SEC1 was also seen when polyclonal antibodies were used, whereas no cross-reactivity was noted when a monoclonal antibody was used in the assay system. Our result was in agreement with this phenomenon. Matrix Effects on the SEB CLEIA. In order to assess the accuracy and applicability of the present method, a wide variety of food, environmental, and biological matrices have been evaluated in this study. Various matrix samples spiked with SEB at different concentrations of 0.01, 0.05, 0.1, 0.5, 1, and 5 ng/mL were compared for SEB detection in dilution buffer over the dynamic range of the SEB CLEIA (Figure 5). The matrix effect was evaluated by comparing the parallelism of the matrix-specific

Figure 4. Specificity of SEB CLEIA. Represented toxins, including SEA, SEC1, and SED, were diluted to 1 ng/mL in dilution buffer and tested in the SEB CLEIA. Each value represents the mean ( SD (n ) 6). Analytical Chemistry, Vol. 82, No. 18, September 15, 2010

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Figure 5. Dose-response curves for SEB CLEIA performed in various matrices. (A) Matrix effect of solid foods on SEB CLEIA. The solid food matrices used were roast beef, peanut butter, and cured ham. (B) Matrix effect of liquid foods on SEB CLEIA. The liquid food matrices used were 10% nonfat dry milk, milk, and orange juice. (C) Matrix effect of environmental matrices on SEB CLEIA. The environmental matrices used were sewage, tap water, and river water. (D) Matrix effect of humoral matrices on SEB CLEIA. The humoral matrices used were human urine and serum. Each point represents the mean value ( SD of triplicate determinations. The toxin extraction procedures are given in Materials and Methods.

dose-response curves with the curve prepared for the dilution buffer. From the similar tendency of the trendline for both dilution buffer and various food matrices depicted in Figure 5A,B, applicability of the method developed after simple treatment procedures was satisfactory. It should be noted, our prior experiments have confirmed that before the pH of orange juice samples (pH 3.8) was adjusted to 7.4, the SEB recovery has been greatly affected (date not shown), most probably resulting from the pH of solutions that affect the biological activity of antibodies and the binding efficiency between antigen and antibody.30 These limitations need to be more widely recognized and pH adjustment may be made prior to the analysis for foods such as orange juice, apple juice, etc., whose pH is often between 3.7 and 4.5 according to (27) Spero, L.; Morlock, B. A.; Metzger, J. F. J. Immunol. 1978, 120, 86–89. (28) Cook, E.; Wang, X.; Robiou, N.; Fries, B. C. Clin. Vaccine Immunol. 2007, 14, 1094–1101. (29) Thompson, N. E.; Ketterhagen, M. J.; Bergdoll, M. S. Infect. Immun. 1984, 45, 281–285. (30) Matikainen, M. T. J. Immunol. Methods 1984, 75, 211–216.

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the results of our experiment. As to environmental matrices, dilution buffer produced higher chemiluminescence intensity than any other matrices, except at 0.1 ng/mL for the sewage and river samples (Figure 5C), suggesting dirty samples often entail a combination of multiple factors (i.e., dielectric, ionic strength, etc.) that may affect the binding of antigen and antibody, HRP activity, or chemiluminescent kinetic profile, requiring further study. Finally, we evaluated the performance of these assays in human urine and human serum samples. Different assay methods and laboratories have different results on urine matrix interference. Some papers have reported that results are greatly affected by matrix effect23 while others found no obvious signal quenching.9,13,31 Our SEB CLEIA suffered no matrix interference by human urine (Figure 5D). Moreover, even human urine samples spiked with SEB at 0.05 ng/mL had a recovery of 82.7%, indicating that our SEB CLEIA was more sensitive for the evaluation of human urine (31) Peruski, A. H.; Johnson, L. H.; Peruski, L. F., Jr. J. Immunol. Methods 2002, 263, 35–41.

samples. As SEs are excreted mainly via the urine,32 our SEB CLEIA may have a potential application to evaluate clinical specimens from patients with SEB poisoning. In addition, we also found a signal quenching effect with human serum, which was also reported by other methods.23,31 This may be due to the high prevalence of staphylococcal enterotoxin antibodies found in most of the human sera,33,34 which caused significant interference in the SEB CLEIA. Furthermore, the nonspecific blocking action of human serum on anti-SEB mAbs binding sites may result in a signal quenching effect. It is important to note that the matrix effect of samples can be weakened or eliminated by 5-10-fold dilution in dilution buffer while the spiked samples were still within the detection range based on the large dynamic range of the SEB CLEIA. Moreover, a good recovery from 81.2% to 106.1% was obtained at 1 ng/mL for all of these spiked samples, suggesting that our assay is sufficient to easily detect SEB in various matrices studied here, far below the toxic dose (100-200 ng) that can cause symptoms35 or even while highly diluted. CONCLUSIONS Chemiluminescence sandwich immunoassay systems for SEB detection with high sensitivity are of growing importance and continue to experience active use and development. Recent development in the area was achieved by use of polyclonal antibodies based on a gold nanoparticle-based enhanced chemiluminescence immunosensor and the limit of detection was found to be ∼0.01 ng/mL.20 In our study, the microplate CLEIA using a pair of highly specific mAbs and a supersensitive chemiluminescent substrate for the horseradish peroxidase (HRP)-luminolH2O2 system demonstrated a significantly enhanced performance on detection sensitivity and specificity with a theoretical LOD of 0.0043 ng/mL and an actually measured LOD of 0.01 (32) Crawley, G. J.; Gray, I.; Leblang, W. A.; Blanchard, J. W. J Infect Dis 1966, 116, 48–56. (33) Franz, D. R.; Jahrling, P. B.; Friedlander, A. M.; McClain, D. J.; Hoover, D. L.; Bryne, W. R.; Pavlin, J. A.; Christopher, G. W.; Eitzen, E. M., Jr. JAMA, J. Am. Med. Assoc. 1997, 278, 399–411. (34) LeClaire, R. D.; Bavari, S. Antimicrob. Agents Chemother. 2001, 45, 460– 463. (35) Bennett, R. W. J. Food Prot. 2005, 68, 1264–1270.

ng/mL. No cross-reactivity with other relevant toxins was observed. To the best of our knowledge, this is the first demonstration in which a microplate CLEIA system using two mAbs in a sandwich assay format possesses potential application for the detection of SEB. Analysis of a wide variety of matrices indicates that the microplate CLEIA was able to detect SEB at subtoxic concentrations in these samples without apparent matrix effect, and samples required simple preparation. Up to now, among reported results, it is the most sensitive immunoassay reported so far for the detection of SEB in urine, achieving LODs of 0.05 ng/mL.9,23 Unlike other antibody sandwich-based methods for SEB detection, such as colorimetric ELISA, antibody-based biosensors, etc., in which no more than one monoclonal antibody was used, highly specific mAbs recognizing two epitopes on SEB in a sandwich format are potentially useful for the development of sensitive and specific SEB detection methods. In our earlier work, a colorimetric ELISA for quantifying SEB was established, based on the mAb FMU-SEB-2 used as the capture antibody and HRPlabeled mAb FMU-SEB-1 used as the detection antibody. The detection limit of this quantitative assay reaches 0.0165 ng/mL. Our supersensitive microplate CLEIA was strongly grounded on the sensitive ELISA. Thus, highly specific mAbs to SEB together with supersensitive substrate make the CLEIA highly sensitive, specific, and simple for high-throughput detection of SEB. This may be useful in food hygiene supervision, environment management, detection of SEB in clinical samples, and antiterrorism. ACKNOWLEDGMENT The first two authors contributed equally to this work. We thank the National Science and Technology Infrastructure Program of China (2009BAK61B04) and the National High-tech R&D Program (863 Program) of China (2006AA02A237).

Received for review June 24, 2010. Accepted August 17, 2010. AC101666Y

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