Biosensors and Bioelectronics 54 (2014) 262–265
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Advantages of fluorescent microspheres compared with colloidal gold as a label in immunochromatographic lateral flow assays Quan-Yuan Xie a,1, Yan-Hua Wu a,b,1, Qi-Rong Xiong a,c, Heng-Yi Xu a, Yong-Hua Xiong a, Kun Liu a, Yong Jin c, Wei-Hua Lai a,n a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China Jiangxi Zodolabs Bioengineering Co, Ltd., Nanchang 330047, China c Institute of Food Safety, Chinese Academy of Inspection and Quarantine, Beijing 100123, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 16 August 2013 Received in revised form 31 October 2013 Accepted 1 November 2013 Available online 12 November 2013
Label selection is of vital importance for immunochromatographic assays. In this study, the fluorescent microsphere test strip and colloidal gold immunochromatographic test strip (FM-ICTS and CG-ICTS) were developed for the detection of Escherichia coli O157:H7 on the basis of the sandwich format. Two types of labels, namely, colloidal gold particles (CG) and carboxyl-modified fluorescent microspheres (FMs), were compared while coupling with anti-E. coli O157:H7 monoclonal antibody (mAb). The FM-ICTS and CGICTS were also compared. Results show that the coupling rate between FMs and mAb was higher than that between CG and mAb. Under optimum conditions, the sensitivity of FM-ICTS was eight times higher than that of CG-ICTS. Approximately 0.1 μg of mAb was used in every FM-ICTS, whereas 0.4 μg of mAb was used in every CG-ICTS. The coefficient of variation of FM-ICTS and CG-ICTS was 4.8% and 16.7%, respectively. The FM-ICTS and CG-ICTS can be stored at room temperature for 12 months and specific to five E. coli O157:H7 strains. Milk sample inoculated with E. coli O157:H7 were tested by the FM-ICTS and CG-ICTS. The FM-ICTS sensitivity was 104 CFU/ml while the CG-ICTS sensitivity was 105 CFU/ml. The sensitivity, consumption of antibodies, and coefficient of variation of FM-ICTS were better than those of CG-ICTS for the detection of E. coli O157:H7. & 2013 Elsevier B.V. All rights reserved.
Keywords: Immunochromatographic assay Colloidal gold Fluorescent microsphere Escherichia coli O157:H7
1. Introduction Colloidal gold immunochromatographic test strip (CG-ICTS) has recently seen widespread applications in the fields of medical diagnosis (Chou, 2013; Tripathi et al., 2012), food safety (Chalinan et al., 2013; Chen et al., 2012; Lai et al., 2007; Le et al., 2013), animal health (Kilic et al., 2012; Sheng et al., 2012), agriculture (Hua et al., 2012; Lee et al., 2013; Wang et al., 2013a), and the environment (Liu et al., 2012; Na et al., 2012). This technique is based on an immunochromatographic procedure that utilizes antigen–antibody properties and provides rapid detection of analytes. Colloidal gold (CG) was introduced into immunochromatographic test strip to increase stability. Colloidal gold is essentially inert and forms almost perfectly spherical particles when properly manufactured. Antibodies bind to the surfaces of these gold particles with high strength when correctly coupled, thus providing a high degree of long-term stability in both liquid and solid forms (Chandler et al., 2000). CG-ICTS possesses four
n
Corresponding author. Tel.: þ 86 791 88105487; fax: þ86 791 88333708. E-mail address: [email protected] (W.-H. Lai). 1 These authors contributed equally to this work.
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.11.002
benefits: user-friendly format, a short period of time to acquire test results, long-term stability over a wide range of climates, and relatively inexpensive production. These characteristics render CG-ICTS ideally suitable for on-site testing by untrained personnel. However, CG-ICTS shows serious limitations when high sensitivity is needed. Lateral flow immunoassays have been developed with the advancement of labels, and more sensitive assays using fluorescent microspheres (FMs) instead of CG have appeared in recent years (Song et al., 2013; Xu et al., 2013). However, studies comparing the new labels with CG focused only on sensitivity (Etvi et al., 2012). To our knowledge, direct comparative evaluations of FMs versus CG on the basis of their coupling rate, properties of relative test strip from sensitivity, consumption of antibodies, and coefficient of variation (CV) have not been published. Escherichia coli O157:H7 was used as a model target analyte. The CG and carboxyl-modified FMs were conjugated with anti-E. coli O157:H7 monoclonal antibody (mAb). The coupling rate and amount of antibody conjugating to the surface of the two labels were studied. Label-antibody complex was sprayed on the conjugate and prepared immunochromatographic test strips (ICTS). Two types of ICTS detecting E. coli O157:H7 (FM-ICTS and CG-ICTS) were compared for the sensitivity and CV by different readers.
Q.-Y. Xie et al. / Biosensors and Bioelectronics 54 (2014) 262–265
263
2. Materials and methods
2.6. Evaluation of coupling rate of FM probe and CG probe by ELISA
2.1. Bacterial strains and growth condition
A total of 100 μl of E. coli O157:H7 (108 CFU/ml) solution was added to the bottom of each well and incubated for 2 h at 37 1C. After washing, 100 μl of anti-E. coli O157:H7 mAb (0, 0.01, 0.02, 0.04, 0.08, 0.16, and 0.32 mg/ml) was added to separate duplicate wells and incubated for 2 h at 37 1C. After washing, 100 μl of substrate/chromogen solution was added and incubated for 15 min at room temperature in the absence of light. The reaction was stopped by adding 100 μl of 2 M H2SO4. The absorbance was measured at 450 nm by a Labsystem microplate reader (Helsinki, Finland). The calibration curve was made according to the experimental data. The concentration of residual mAb in supernatant was detected and could be read from the calibration curve. The coupling rate of FM-mAb and CG–mAb could be determined by using ELISA.
E. coli O157:H7 strain ATCC 43888 was used in this study. The strain was cultured in Luria–Bertani medium (LB, Oxoid, Basingstoke, UK) at 37 1C for 20 h before use. To determine the number of viable cells, serial dilutions of cultures in phosphate-buffered saline (PBS, Sigma Chemical Company, St. Louis, MO, 0.01 M, pH 7.4) were made and plated onto trypticase soy agar (TSA; Becton, Dickinson and Company, Sparks, MD). The plates were then incubated at 37 1C for 24 h. 2.2. Reagents Fluorescein isothiocyanate fluorescent microspheres (diameter¼ 175 nm; excitation wavelength¼ 470 nm; emission wavelength ¼525 nm; COOH ¼443 meq/g) were obtained from Merck company (Darmstadt, Germany). The CG particles (40 nm) were prepared in our laboratory (Lai et al., 2009). Nitrocellulose membrane, absorbent pad, sample pad, and conjugate pad were purchased from Millipore (Bendford, MA, USA). The antibody pairs, namely, murine anti-E. coli O157:H7 mAb and goat anti-E. coli O157:H7 polyclonal antibody, and Donkey anti-mouse IgG were purchased from Meridian Life Science, Inc (Memphis, TN, USA). Bovine serum albumin (BSA), N-(3-dimethylaminopropyl)-Nethylcarbodiimide hydrochloride (EDC), and N-hydroxy-sulfosuccinmide (sulfo-NHS) were from Sigma (St. Louis, MO, USA). All solvents and other chemicals were analytical reagent grade.
2.7. Preparation of test strips The sample pad, which was treated with 50 mM of borate buffer, pH 7.4, containing 1% BSA, 0.5% Tween-20, and 0.05% sodium azide, was dried at 60 1C for 2 h. The goat anti-E. coli O157:H7 polyclonal antibody (1.5 mg/ml) and rabbit anti-mouse antibody (1.0 mg/ml) were applied to the test and control lines on the nitrocellulose membrane, respectively, and dried at 35 1C. The NC membrane was blocked with 5% BSA in pH 7.2 PBS (0.02 M) for avoiding the background in the strip. Absorption pad and glassfiber membrane were used without treatment. The nitrocellulose membrane, absorption pad, glass fiber membrane, and pretreated sample pad were assembled as the strip.
2.3. Instruments The FM-ICTS reader was from Shanghai Huguo Science Instrument Co, Ltd (excitation wavelength ¼470 nm; emission wavelength ¼520 nm), and the SkanFlexi BioAssay Reader Systems for detecting CG-ICTS was from Skannex Biotech Co., Ltd (Oslo, Norway). 2.4. Preparation of FM probe The EDC-mediating method was used to cross-link carboxylmodified FMs with amine-containing mAb (Wang et al., 2013b). Approximately 10 ml of carboxylated FMs was mixed with sulfoNHS and EDC in MES-buffered saline (pH 4.7) at room temperature for 120 min to form an amine-reactive sulfo-NHS ester. After adding 1 ml of anti-E. coli O157:H7 mAb at concentrations of 25, 50, 100, 200, and 300 μg/ml, the solutions were incubated at room temperature for 2 h. Afterward, 10% BSA was added into the mixture for blocking unreacted active sites for 30 min. The reaction mixture was centrifuged at 8000g for 15 min. After centrifugation, the pellet was suspended in 500 μl of neutral PBS containing 1% BSA, 5% sucrose, 3% trehalose, and 0.4% Tween 20. The FM-labeled mAb compound was dried on an enzyme-linked immunosorbent assay (ELISA) plate for 2 h at 30 1C. 2.5. Preparation of colloidal gold probe About 1 ml of the same anti-E. coli O157:H7 mAb at concentrations of 25, 50, 100, 200, and 300 μg/ml were added into 10 ml of pH-adjusted CG solution and was agitated for 30 min. Afterward, 2 ml of 1% (w/v) BSA solution was added and was agitated for 15 min. The mixture was centrifuged at 9000g for 30 min. After centrifugation, the gold pellets were dissolved in 50 mM of Tris/ HCl buffer. The CG-labeled mAb compound was dried on an ELISA plate for 2 h at 30 1C.
2.8. Sensitivity comparison between FM-ICTS and CG-ICTS The E. coli O157:H7 culture was diluted from 2.0 102 to 1.6 106 CFU/ml by sterile PBS. About 100 μl aliquot of sample was pipetted into the ELISA well, which was added with FMlabeled mAb compound and CG-labeled mAb compound. The test was allowed 10 min for the antibody–antigen complex to form. Thereafter, the samples were transmitted into sample pads of FMICTS and CG-ICTS. After 10 min, the FM-ICTS was detected by the FM-ICTS reader, and the CG-ICTS was detected by the SkanFlexi BioAssay Reader. The properties of FM-ICTS were compared with that of CG-ICTS. All experiments were performed in triplicate. 2.9. Stability comparison between FM-ICTS and CG-ICTS The FM-ICTS and CG-ICTS were subjected to accelerated stability study. They were kept at 37 1C and taken out at different time intervals and tested using E. coli O157:H7 (ATCC 43888, approx. 105 CFU/ml). 2.10. Specificity comparison between FM-ICTS and CG-ICTS Five E. coli O157:H7 strains and 23 non-E. coli O157:H7 strains were tested by the FM-ICTS and CG-ICTS for evaluating the specificity of two methods. 2.11. Detection of food sample between FM-ICTS and CG-ICTS Ultra high temperature sterilized milk samples were purchased from a local supermarket in Nanchang. Each milk sample (25 g) transferred to a stomacher bag was inoculated with 0.1 ml of E. coli O157:H7 innocula (106, 105, 104, and 103 CFU per ml of milk). The samples were tested by the FM-ICTS and CG-ICTS, respectively.
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3. Results and discussion
Table 3 Signal intensity of T line of FM-ICTS and CG-ICTS.
3.1. Influence of different concentrations of mAb on the coupling rate with FMs The calibration curve was set up by the known concentration of mAb and its absorbance value (y¼ 0.790Ln(x)þ 4.022, R² ¼0.997). The concentration of residual mAb in supernatant corresponding to the absorbance value can be read from the calibration curve. The coupling rate of mAb with FMS can be identified according to the experimental data (Table 1). The results show that the coupling rate of mAb with FM decreased from 98.7% to 83.2% as mAb increased. The number of mAb conjugating on the FM surface also increased. 3.2. Influence of different concentrations of mAb on the coupling rate with CG The calibration curve was set up by the known concentration of mAb and its absorbance value (y¼0.664Ln(x) þ3.111, R² ¼0.993). The coupling rate of mAb with CG was irregular and generally low as mAb increased. However, the number of mAb conjugating on the CG surface increased (Table 2). 3.3. Comparison between FM-ICTS and CG-ICTS The sandwich immunoassay test was used. If E. coli O157:H7 is present in a sample, then the strain will be captured by the mAb – conjugated CG or the mAb – conjugated FMs. As the sample moves along the strip, some of the E. coli O157:H7-mAb-CG complexes or E. coli O157:H7-mAb-FMs complexes are captured by the goat antiE. coli O157:H7 polyclonal antibody coated on the test line. If the concentration of E. coli O157:H7 in the sample exceeds the cutoff level, a red or fluorescent T line will form. The T line will be detected by the reader, indicating a positive result. Some of the conjugated particles will continue to flow toward the control line and form a red-colored or fluorescent band regardless of the presence or absence of E. coli O157:H7. The two model assays were optimized with respect to analytical sensitivity with both types of reporter particles. The FM-ICTS had optimum sensitivity when the mAb content was 50 μg (Table S1). Increasing the mAb did not improve the ICTS sensitivity. Therefore,
Table 1 Effect of different concentrations of mAb on the coupling rate with mAb-FMs. Added mAb (μg)
Coupling rate (%)
Labeled mAb (μg)
25 50 100 200 300
98.7 96.2 89.5 86.7 83.2
24.7 48.1 89.5 173.5 249.5
Table 2 Effect of different concentrations of mAb on the coupling rate with mAb-CG. Added mAb (μg)
Coupling rate (%)
Labeled mAb (μg)
25 50 100 200 300
72.1 52.2 57.7 62.9 58.3
18.0 26.1 57.7 125.8 175.0
Concentration Signal intensity of FM-ICTS in (CFU/ml) T line
2
2.0 10 4.0 102 8.0 102 1.6 103 3.1 103 6.3 103 1.25 104 2.5 104 5.0 104 1.0 105 2.0 105 4.0 105 8.0 105 1.6 106
Signal intensity of CG-ICTS in T line
Mean
SD
CV
Mean
SD
CV
No No No 63 145 196 335 693 822 1301 1586 1665 1703 2012
No No No 3 8 9 14 44 34 63 78 75 75 79
No No No 4.8% 5.5% 4.6% 4.2% 6.3% 4.1% 4.8% 4.9% 4.5% 4.4% 3.9%
No No No No No No 126 298 412 503 614 669 713 797
No No No No No No 21 45 59 70 75 77 80 82
No No No No No No 16.7% 15.1% 14.3% 13.9% 12.2% 11.5% 11.2% 10.2%
excess mAb bonds on the surface of FM could not improve the sensitivity of the ICTS. With the FM-ICTS reader detection, the test line value increased with the increase in the concentration of E. coli O157: H7. The CV for each concentration was less than 7%. The mean and standard deviation of the blank sample was zero. Therefore, calculating the limit of detection (LOD) of the FM-ICTS according to mean plus threefold standard deviation of the blank sample is unsuitable (Zhu et al., 2011). The FM-ICTS sensitivity of the E. coli O157:H7 assay was 1.6 103 CFU/ml, and the CV was 4.8% (Table 3). The CG-ICTS had optimum sensitivity when the mAb content was 200 μg (Table S2). With the SkanFlexi BioAssay Reader detection, the test line value increased with the increase in the concentration of E. coli O157:H7. The CG-ICTS sensitivity of the E. coli O157:H7 assay was 1.25 104 CFU/ml, and the CV was 16.7%. It was concluded that the FM-ICTS and CG-ICTS can be stored at room temperature for 12 months according to accelerated stability study (Table S3). Five E. coli O157:H7 strains and 23 non-E. coli O157:H7 strains were tested. The result showed that the FM-ICTS and CG-ICTS are specific to the E. coli O157:H7 strains (Table S4). Milk sample inoculated with E. coli O157:H7 were tested by the FM-ICTS and CG-ICTS. The FM-ICTS sensitivity was 104 CFU/ml while the CG-ICTS sensitivity was 105 CFU/ml. When E. coli O157:H7 was used as model analyte, the results indicate that the fluorescent test strips were about eight times more sensitive than the gold-based test strips are. Furthermore, the mAb was used on every FM-ICTS and CG-ICTS with values of 0.1 and 0.4 μg, respectively.
4. Conclusions The development of labels or markers for lateral flow immunoassay has gone hand in hand with the advances in detection methodology and instrumentation. Sensitive assays by using fluorescent labels have appeared in recent years (Raphael and Harley, 2009). In the study, FM-ICTS and CG-ICTS were developed for the detection of E. coli O157:H7. The two test strips were compared. The fluorescent microsphere immunochromatographic lateral flow assay has advantages over CG immunochromatographic lateral flow assay in terms of coupling rate, sensitivity, CV, and antibody needed. Other important comparison between FM-ICTS and CG-ICTS including the steric incumbrance and total detection time should be studied in the future.
Q.-Y. Xie et al. / Biosensors and Bioelectronics 54 (2014) 262–265
Acknowledgements We are grateful to the Research Program of State Key Laboratory of Food Science and Technology, Nanchang University (Project No. SKLF-ZZB-201307), the National Natural Science Foundation of China (No. 30960301), Jiangxi province main science and technology leader project (20113BCB22007), The Jiangxi education bureau technology put into use project (KJLD13009), and the Nanchang Technological Program (2012-CYH-DW-SP-001) for financial support. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.11.002. References Chalinan, P., Parin, C., Siwaporn, L., Paisarn, S., 2013. Biosens. Bioelectron. 42, 229–235. Chandler, J., Gurmin, T., Robinson, N, 2000. IVD Technol. 〈http://www.devicelink. com/ivdt/archive/00/03/004.html〉. Chen, X.L., Xu, H.Y., Lai, W.H., Chen, Y., Yang, X.H., Xiong, Y.H., 2012. Food Addit. Contam. 29, 383–391. Chou, S.F., 2013. Analyst 138, 2620–2623.
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Etvi, J., Tiina, M., Teppo, S., Tero, S., Kim, P., 2012. Anal. Biochem. 428 (1), 31–38. Hua, X., Yang, J., Wang, L., Fang, Q., Zhang, G., Liu, F., 2012. PLoS One 7, e53099. Kilic, S., Celebi., B., Yesilyurt, M., 2012. Diagn. Microbiol. Infect. Dis. 74 (1), 1–5. Lai, W.H., Fung, D.Y.C., Xu, Y., Liu, R.R., Xiong, Y.H., 2009. Food Control 20 (9), 791–795. Lai, W.H., Xu, Y., Fung, D.Y.C., Xiong, Y.H., 2007. Asia Pac. J. Clin. Nutr. 16 (Suppl. 1), 106–110. Le, T., Yan, P.F., Xu, J., Hao, Y.J., 2013. Food Chem. 138 (2–3), 1610–1615. Lee, E.H., Kim, Y.A., Lee, Y.T., Hammock, B.D., Lee, H.S., 2013. Food Agric. Immunol. 24, 129–138. Liu, X., Xiang., J.J, Tang, Y., Zhang, X.L., Fu, Q.Q., Zou, J.H., 2012. Anal. Chim. Acta 745, 99–105. Na, Y., Sheng, W., Yuan, M., Li, L., Liu, B., Zhang, Y., 2012. Microchim. Acta 177 (1–2), 177–184. Raphael, C.W., Harley, Y.T., 2009. Lateral Flow Immunoassay, first ed. Humana Press, NewYork. Sheng, X.Z., Song, J.L., Zhan, W.B., 2012. J. Appl. Microbiol. 113, 737–744. Song, C.M., Zhi, A.M., Liu, Q.T., Yang, J.F., Jia, G.C., Shervin, J., Tang, L., Hu, X.F., Deng, R.G., Xu, C.L., Zhang, G.P., 2013. Biosens. Bioelectron. 50, 62–65. Tripathi, V., Nara, S., Singh, K., Singh, H., Shrivastav, T.G., 2012. Clin. Chim. Acta 413 (1–2), 262–268. Wang, D.B., Tian, B., Zhang, Z.P., Deng, J.Y., Cui, Z.Q., Yang, R.F., Wang, X.Y., Wei, H.P., Zhang, X.E., 2013a. Biosens. Bioelectron. 42, 661–667. Wang, Y.K., Shi, Y.B., Zou, Q., Sun, J.H., Chen, Z.F., Wang, H.A., 2013b. Food Control 31 (1), 180–188. Xu, W., Chen, X.L., Huang, X.L., Yang, W.C., Liu, C.M., Lai, W.H., Xu, H.Y., Xiong., Y.H., 2013. Talanta 114, 160–166. Zhu, P.X., Shelton, D.R., Li, S.H., Adams, D.L., Karns, J.S., Amstutz, P., Tang, C.M., 2011. Biosens. Bioelectron. 30 (1), 337–341.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Advantages of fluorescent microspheres compared with colloidal gold as a label in immunochromatographic lateral flow assays Quan-Yuan Xie a,1, Yan-Hua Wu a,b,1, Qi-Rong Xiong a,c, Heng-Yi Xu a, Yong-Hua Xiong a, Kun Liu a, Yong Jin c, Wei-Hua Lai a,n a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China Jiangxi Zodolabs Bioengineering Co, Ltd., Nanchang 330047, China c Institute of Food Safety, Chinese Academy of Inspection and Quarantine, Beijing 100123, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 16 August 2013 Received in revised form 31 October 2013 Accepted 1 November 2013 Available online 12 November 2013
Label selection is of vital importance for immunochromatographic assays. In this study, the fluorescent microsphere test strip and colloidal gold immunochromatographic test strip (FM-ICTS and CG-ICTS) were developed for the detection of Escherichia coli O157:H7 on the basis of the sandwich format. Two types of labels, namely, colloidal gold particles (CG) and carboxyl-modified fluorescent microspheres (FMs), were compared while coupling with anti-E. coli O157:H7 monoclonal antibody (mAb). The FM-ICTS and CGICTS were also compared. Results show that the coupling rate between FMs and mAb was higher than that between CG and mAb. Under optimum conditions, the sensitivity of FM-ICTS was eight times higher than that of CG-ICTS. Approximately 0.1 μg of mAb was used in every FM-ICTS, whereas 0.4 μg of mAb was used in every CG-ICTS. The coefficient of variation of FM-ICTS and CG-ICTS was 4.8% and 16.7%, respectively. The FM-ICTS and CG-ICTS can be stored at room temperature for 12 months and specific to five E. coli O157:H7 strains. Milk sample inoculated with E. coli O157:H7 were tested by the FM-ICTS and CG-ICTS. The FM-ICTS sensitivity was 104 CFU/ml while the CG-ICTS sensitivity was 105 CFU/ml. The sensitivity, consumption of antibodies, and coefficient of variation of FM-ICTS were better than those of CG-ICTS for the detection of E. coli O157:H7. & 2013 Elsevier B.V. All rights reserved.
Keywords: Immunochromatographic assay Colloidal gold Fluorescent microsphere Escherichia coli O157:H7
1. Introduction Colloidal gold immunochromatographic test strip (CG-ICTS) has recently seen widespread applications in the fields of medical diagnosis (Chou, 2013; Tripathi et al., 2012), food safety (Chalinan et al., 2013; Chen et al., 2012; Lai et al., 2007; Le et al., 2013), animal health (Kilic et al., 2012; Sheng et al., 2012), agriculture (Hua et al., 2012; Lee et al., 2013; Wang et al., 2013a), and the environment (Liu et al., 2012; Na et al., 2012). This technique is based on an immunochromatographic procedure that utilizes antigen–antibody properties and provides rapid detection of analytes. Colloidal gold (CG) was introduced into immunochromatographic test strip to increase stability. Colloidal gold is essentially inert and forms almost perfectly spherical particles when properly manufactured. Antibodies bind to the surfaces of these gold particles with high strength when correctly coupled, thus providing a high degree of long-term stability in both liquid and solid forms (Chandler et al., 2000). CG-ICTS possesses four
n
Corresponding author. Tel.: þ 86 791 88105487; fax: þ86 791 88333708. E-mail address: [email protected] (W.-H. Lai). 1 These authors contributed equally to this work.
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.11.002
benefits: user-friendly format, a short period of time to acquire test results, long-term stability over a wide range of climates, and relatively inexpensive production. These characteristics render CG-ICTS ideally suitable for on-site testing by untrained personnel. However, CG-ICTS shows serious limitations when high sensitivity is needed. Lateral flow immunoassays have been developed with the advancement of labels, and more sensitive assays using fluorescent microspheres (FMs) instead of CG have appeared in recent years (Song et al., 2013; Xu et al., 2013). However, studies comparing the new labels with CG focused only on sensitivity (Etvi et al., 2012). To our knowledge, direct comparative evaluations of FMs versus CG on the basis of their coupling rate, properties of relative test strip from sensitivity, consumption of antibodies, and coefficient of variation (CV) have not been published. Escherichia coli O157:H7 was used as a model target analyte. The CG and carboxyl-modified FMs were conjugated with anti-E. coli O157:H7 monoclonal antibody (mAb). The coupling rate and amount of antibody conjugating to the surface of the two labels were studied. Label-antibody complex was sprayed on the conjugate and prepared immunochromatographic test strips (ICTS). Two types of ICTS detecting E. coli O157:H7 (FM-ICTS and CG-ICTS) were compared for the sensitivity and CV by different readers.
Q.-Y. Xie et al. / Biosensors and Bioelectronics 54 (2014) 262–265
263
2. Materials and methods
2.6. Evaluation of coupling rate of FM probe and CG probe by ELISA
2.1. Bacterial strains and growth condition
A total of 100 μl of E. coli O157:H7 (108 CFU/ml) solution was added to the bottom of each well and incubated for 2 h at 37 1C. After washing, 100 μl of anti-E. coli O157:H7 mAb (0, 0.01, 0.02, 0.04, 0.08, 0.16, and 0.32 mg/ml) was added to separate duplicate wells and incubated for 2 h at 37 1C. After washing, 100 μl of substrate/chromogen solution was added and incubated for 15 min at room temperature in the absence of light. The reaction was stopped by adding 100 μl of 2 M H2SO4. The absorbance was measured at 450 nm by a Labsystem microplate reader (Helsinki, Finland). The calibration curve was made according to the experimental data. The concentration of residual mAb in supernatant was detected and could be read from the calibration curve. The coupling rate of FM-mAb and CG–mAb could be determined by using ELISA.
E. coli O157:H7 strain ATCC 43888 was used in this study. The strain was cultured in Luria–Bertani medium (LB, Oxoid, Basingstoke, UK) at 37 1C for 20 h before use. To determine the number of viable cells, serial dilutions of cultures in phosphate-buffered saline (PBS, Sigma Chemical Company, St. Louis, MO, 0.01 M, pH 7.4) were made and plated onto trypticase soy agar (TSA; Becton, Dickinson and Company, Sparks, MD). The plates were then incubated at 37 1C for 24 h. 2.2. Reagents Fluorescein isothiocyanate fluorescent microspheres (diameter¼ 175 nm; excitation wavelength¼ 470 nm; emission wavelength ¼525 nm; COOH ¼443 meq/g) were obtained from Merck company (Darmstadt, Germany). The CG particles (40 nm) were prepared in our laboratory (Lai et al., 2009). Nitrocellulose membrane, absorbent pad, sample pad, and conjugate pad were purchased from Millipore (Bendford, MA, USA). The antibody pairs, namely, murine anti-E. coli O157:H7 mAb and goat anti-E. coli O157:H7 polyclonal antibody, and Donkey anti-mouse IgG were purchased from Meridian Life Science, Inc (Memphis, TN, USA). Bovine serum albumin (BSA), N-(3-dimethylaminopropyl)-Nethylcarbodiimide hydrochloride (EDC), and N-hydroxy-sulfosuccinmide (sulfo-NHS) were from Sigma (St. Louis, MO, USA). All solvents and other chemicals were analytical reagent grade.
2.7. Preparation of test strips The sample pad, which was treated with 50 mM of borate buffer, pH 7.4, containing 1% BSA, 0.5% Tween-20, and 0.05% sodium azide, was dried at 60 1C for 2 h. The goat anti-E. coli O157:H7 polyclonal antibody (1.5 mg/ml) and rabbit anti-mouse antibody (1.0 mg/ml) were applied to the test and control lines on the nitrocellulose membrane, respectively, and dried at 35 1C. The NC membrane was blocked with 5% BSA in pH 7.2 PBS (0.02 M) for avoiding the background in the strip. Absorption pad and glassfiber membrane were used without treatment. The nitrocellulose membrane, absorption pad, glass fiber membrane, and pretreated sample pad were assembled as the strip.
2.3. Instruments The FM-ICTS reader was from Shanghai Huguo Science Instrument Co, Ltd (excitation wavelength ¼470 nm; emission wavelength ¼520 nm), and the SkanFlexi BioAssay Reader Systems for detecting CG-ICTS was from Skannex Biotech Co., Ltd (Oslo, Norway). 2.4. Preparation of FM probe The EDC-mediating method was used to cross-link carboxylmodified FMs with amine-containing mAb (Wang et al., 2013b). Approximately 10 ml of carboxylated FMs was mixed with sulfoNHS and EDC in MES-buffered saline (pH 4.7) at room temperature for 120 min to form an amine-reactive sulfo-NHS ester. After adding 1 ml of anti-E. coli O157:H7 mAb at concentrations of 25, 50, 100, 200, and 300 μg/ml, the solutions were incubated at room temperature for 2 h. Afterward, 10% BSA was added into the mixture for blocking unreacted active sites for 30 min. The reaction mixture was centrifuged at 8000g for 15 min. After centrifugation, the pellet was suspended in 500 μl of neutral PBS containing 1% BSA, 5% sucrose, 3% trehalose, and 0.4% Tween 20. The FM-labeled mAb compound was dried on an enzyme-linked immunosorbent assay (ELISA) plate for 2 h at 30 1C. 2.5. Preparation of colloidal gold probe About 1 ml of the same anti-E. coli O157:H7 mAb at concentrations of 25, 50, 100, 200, and 300 μg/ml were added into 10 ml of pH-adjusted CG solution and was agitated for 30 min. Afterward, 2 ml of 1% (w/v) BSA solution was added and was agitated for 15 min. The mixture was centrifuged at 9000g for 30 min. After centrifugation, the gold pellets were dissolved in 50 mM of Tris/ HCl buffer. The CG-labeled mAb compound was dried on an ELISA plate for 2 h at 30 1C.
2.8. Sensitivity comparison between FM-ICTS and CG-ICTS The E. coli O157:H7 culture was diluted from 2.0 102 to 1.6 106 CFU/ml by sterile PBS. About 100 μl aliquot of sample was pipetted into the ELISA well, which was added with FMlabeled mAb compound and CG-labeled mAb compound. The test was allowed 10 min for the antibody–antigen complex to form. Thereafter, the samples were transmitted into sample pads of FMICTS and CG-ICTS. After 10 min, the FM-ICTS was detected by the FM-ICTS reader, and the CG-ICTS was detected by the SkanFlexi BioAssay Reader. The properties of FM-ICTS were compared with that of CG-ICTS. All experiments were performed in triplicate. 2.9. Stability comparison between FM-ICTS and CG-ICTS The FM-ICTS and CG-ICTS were subjected to accelerated stability study. They were kept at 37 1C and taken out at different time intervals and tested using E. coli O157:H7 (ATCC 43888, approx. 105 CFU/ml). 2.10. Specificity comparison between FM-ICTS and CG-ICTS Five E. coli O157:H7 strains and 23 non-E. coli O157:H7 strains were tested by the FM-ICTS and CG-ICTS for evaluating the specificity of two methods. 2.11. Detection of food sample between FM-ICTS and CG-ICTS Ultra high temperature sterilized milk samples were purchased from a local supermarket in Nanchang. Each milk sample (25 g) transferred to a stomacher bag was inoculated with 0.1 ml of E. coli O157:H7 innocula (106, 105, 104, and 103 CFU per ml of milk). The samples were tested by the FM-ICTS and CG-ICTS, respectively.
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3. Results and discussion
Table 3 Signal intensity of T line of FM-ICTS and CG-ICTS.
3.1. Influence of different concentrations of mAb on the coupling rate with FMs The calibration curve was set up by the known concentration of mAb and its absorbance value (y¼ 0.790Ln(x)þ 4.022, R² ¼0.997). The concentration of residual mAb in supernatant corresponding to the absorbance value can be read from the calibration curve. The coupling rate of mAb with FMS can be identified according to the experimental data (Table 1). The results show that the coupling rate of mAb with FM decreased from 98.7% to 83.2% as mAb increased. The number of mAb conjugating on the FM surface also increased. 3.2. Influence of different concentrations of mAb on the coupling rate with CG The calibration curve was set up by the known concentration of mAb and its absorbance value (y¼0.664Ln(x) þ3.111, R² ¼0.993). The coupling rate of mAb with CG was irregular and generally low as mAb increased. However, the number of mAb conjugating on the CG surface increased (Table 2). 3.3. Comparison between FM-ICTS and CG-ICTS The sandwich immunoassay test was used. If E. coli O157:H7 is present in a sample, then the strain will be captured by the mAb – conjugated CG or the mAb – conjugated FMs. As the sample moves along the strip, some of the E. coli O157:H7-mAb-CG complexes or E. coli O157:H7-mAb-FMs complexes are captured by the goat antiE. coli O157:H7 polyclonal antibody coated on the test line. If the concentration of E. coli O157:H7 in the sample exceeds the cutoff level, a red or fluorescent T line will form. The T line will be detected by the reader, indicating a positive result. Some of the conjugated particles will continue to flow toward the control line and form a red-colored or fluorescent band regardless of the presence or absence of E. coli O157:H7. The two model assays were optimized with respect to analytical sensitivity with both types of reporter particles. The FM-ICTS had optimum sensitivity when the mAb content was 50 μg (Table S1). Increasing the mAb did not improve the ICTS sensitivity. Therefore,
Table 1 Effect of different concentrations of mAb on the coupling rate with mAb-FMs. Added mAb (μg)
Coupling rate (%)
Labeled mAb (μg)
25 50 100 200 300
98.7 96.2 89.5 86.7 83.2
24.7 48.1 89.5 173.5 249.5
Table 2 Effect of different concentrations of mAb on the coupling rate with mAb-CG. Added mAb (μg)
Coupling rate (%)
Labeled mAb (μg)
25 50 100 200 300
72.1 52.2 57.7 62.9 58.3
18.0 26.1 57.7 125.8 175.0
Concentration Signal intensity of FM-ICTS in (CFU/ml) T line
2
2.0 10 4.0 102 8.0 102 1.6 103 3.1 103 6.3 103 1.25 104 2.5 104 5.0 104 1.0 105 2.0 105 4.0 105 8.0 105 1.6 106
Signal intensity of CG-ICTS in T line
Mean
SD
CV
Mean
SD
CV
No No No 63 145 196 335 693 822 1301 1586 1665 1703 2012
No No No 3 8 9 14 44 34 63 78 75 75 79
No No No 4.8% 5.5% 4.6% 4.2% 6.3% 4.1% 4.8% 4.9% 4.5% 4.4% 3.9%
No No No No No No 126 298 412 503 614 669 713 797
No No No No No No 21 45 59 70 75 77 80 82
No No No No No No 16.7% 15.1% 14.3% 13.9% 12.2% 11.5% 11.2% 10.2%
excess mAb bonds on the surface of FM could not improve the sensitivity of the ICTS. With the FM-ICTS reader detection, the test line value increased with the increase in the concentration of E. coli O157: H7. The CV for each concentration was less than 7%. The mean and standard deviation of the blank sample was zero. Therefore, calculating the limit of detection (LOD) of the FM-ICTS according to mean plus threefold standard deviation of the blank sample is unsuitable (Zhu et al., 2011). The FM-ICTS sensitivity of the E. coli O157:H7 assay was 1.6 103 CFU/ml, and the CV was 4.8% (Table 3). The CG-ICTS had optimum sensitivity when the mAb content was 200 μg (Table S2). With the SkanFlexi BioAssay Reader detection, the test line value increased with the increase in the concentration of E. coli O157:H7. The CG-ICTS sensitivity of the E. coli O157:H7 assay was 1.25 104 CFU/ml, and the CV was 16.7%. It was concluded that the FM-ICTS and CG-ICTS can be stored at room temperature for 12 months according to accelerated stability study (Table S3). Five E. coli O157:H7 strains and 23 non-E. coli O157:H7 strains were tested. The result showed that the FM-ICTS and CG-ICTS are specific to the E. coli O157:H7 strains (Table S4). Milk sample inoculated with E. coli O157:H7 were tested by the FM-ICTS and CG-ICTS. The FM-ICTS sensitivity was 104 CFU/ml while the CG-ICTS sensitivity was 105 CFU/ml. When E. coli O157:H7 was used as model analyte, the results indicate that the fluorescent test strips were about eight times more sensitive than the gold-based test strips are. Furthermore, the mAb was used on every FM-ICTS and CG-ICTS with values of 0.1 and 0.4 μg, respectively.
4. Conclusions The development of labels or markers for lateral flow immunoassay has gone hand in hand with the advances in detection methodology and instrumentation. Sensitive assays by using fluorescent labels have appeared in recent years (Raphael and Harley, 2009). In the study, FM-ICTS and CG-ICTS were developed for the detection of E. coli O157:H7. The two test strips were compared. The fluorescent microsphere immunochromatographic lateral flow assay has advantages over CG immunochromatographic lateral flow assay in terms of coupling rate, sensitivity, CV, and antibody needed. Other important comparison between FM-ICTS and CG-ICTS including the steric incumbrance and total detection time should be studied in the future.
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Acknowledgements We are grateful to the Research Program of State Key Laboratory of Food Science and Technology, Nanchang University (Project No. SKLF-ZZB-201307), the National Natural Science Foundation of China (No. 30960301), Jiangxi province main science and technology leader project (20113BCB22007), The Jiangxi education bureau technology put into use project (KJLD13009), and the Nanchang Technological Program (2012-CYH-DW-SP-001) for financial support. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.11.002. References Chalinan, P., Parin, C., Siwaporn, L., Paisarn, S., 2013. Biosens. Bioelectron. 42, 229–235. Chandler, J., Gurmin, T., Robinson, N, 2000. IVD Technol. 〈http://www.devicelink. com/ivdt/archive/00/03/004.html〉. Chen, X.L., Xu, H.Y., Lai, W.H., Chen, Y., Yang, X.H., Xiong, Y.H., 2012. Food Addit. Contam. 29, 383–391. Chou, S.F., 2013. Analyst 138, 2620–2623.
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