Establishing of a Method Combined Immunomagnetic Separation with Colloidal Gold Lateral Flow Assay and Its Application in Rapid Detection of Escherichia coli O157:H7 CUI Xi1, XIONG Qi-Rong1,2, XIONG Yong-Hua1, SHAN Shan1, LAI Wei-Hua1,* 1 State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China 2 Institute of Food Safety, Chinese Academy of Inspection and Quarantine, Beijing 100123, China
Abstract: In this study, immunomagnetic beads were prepared by coupling anti- Escherichia. coli O157:H7 monoclonal antibodies with magnetic beads. A colloidal gold test strip was composited with anti- E. coli O157:H7 monoclonal antibody marked by colloidal gold as the detection antibody, anti-E. coli O157:H7 polyclonal antibody as the test line, and donkey anti-mouse IgG secondary antibody as the control line. Immunomagnetic separation was coupled with colloidal gold lateral flow assay to detect E. coli O157:H7 rapidly. Results showed that 1 mL 7.6 × 103 colony-forming unit (CFU) mL�1 E. coli O157:H7 exhibited a positive outcome when colloidal gold lateral flow assay was performed after immunomagnetic separation. A food sample inoculated with 10 CFU/g E. coli O157:H7 after 6 h of enrichment could be detected within 72 min of immunomagnetic separation and colloidal gold lateral flow assay. The method proposed in this study could be used to on-site detect harmful substances in food significantly and rapidly. Key Words: Immunomagnetic separation; Colloidal gold; Immunochromatography; E. coli O157:H7
1 Introduction
Immunomagnetic beads are superparamagnetic particles which surface are chemically modified and coupled firmly with antibodies, and these beads can capture specific antigens. When immunomagnetic beads are added into a sample with corresponding antigens, the beads will capture the antigens and forms antigen-immunomagnetic bead complex. The antigen-immunomagnetic bead complex separates at proper magnetic field condition, and the antigen concentration enriched. For example, Sung et al[1] combined separation with the rapid colorimetric method to detect Staphylococcus aureus in milk. The sensitivity of this method is 1.5 × 105 colony- forming unit (CFU) mL�1. And Ken et al[2] combined separation with real-time polymerase chain reaction (PCR) to detect and isolate low levels of Escherichia coli O157:H7 in cilantro. The concentration of Escherichia coli O157:H7 can
be detected after enrichment for 5 h. Also Marcelo[3] combined separation with the use of a highly specific fiber optic immunosensor to detect low levels of Listeria monocytogenes and L. ivanovii. The sensitivity of this method is 3 × 102 CFU mL�1.
Colloidal gold immunochromatography, a simple and fast method, was applied in numerous fields after the pioneering work of Leuvering[4] in 1981. Later, Le[5] used a novel, colloidal gold-based lateral flow immunoassay to detect cyromazine and melamine in the foods of animal origin rapidly and simultaneously. The sensitivities of cyromazine and melamine were 0.22 and 0.26 ng mL�1, respectively. Jiheaet al[6] measured aflatoxin B1 via the lateral flow assay method. The substance could be detected in 10 min, and the sensitivity of the method was 10 ng mL�1.
E. coli O157:H7 is a primary serotype of enterohemorrhagic E. coli. People become ill with minimum 10 CFU of E. coli
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O157:H7[7]. Li et al[8] developed a magnetic nanoparticle microarray and combined it with PCR to detect E. coli O157:H7 and other three foodborne pathogens simultaneously. The sensitivity of this method was 316 CFU mL�1. Li et al[9] developed a method based on chemiluminescence and magnetic nanoparticles to detect and isolate E. coli O157:H7. This method has a sensitivity of 103 CFU mL�1 and 3 h detection time. Both techniques need skilled workers and high-quality equipments.
In the present study, immunomagnetic separation was combined with lateral flow assay to isolate and detect E. coli O157:H7. The immunomagnetic beads captured target bacteria from a sample, and the bacteria were added to the test strip after enriching. The influences of the background and other bacteria could be eliminated, and the sample was enriched and concentrated to an even smaller volume. Both specificity and sensitivity were improved significantly. Figure 1 shows the schematic diagram for the proposed method.
To our knowledge, this combined method for detecting E. coli O157:H7 has not been previously reported. The method is simple, rapid, economical, and meaningful for screening and controlling E. coli O157:H7 primary level.
Materials: PM3-020 magnetic beads (180 nm) was purchased from Shanghai Allrun Nano Science & Technology
Fig.1 Schematic diagram of immunomagnetic separation combined with colloidal gold lateral flow assay method
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Co., Ltd. (China); Dynabeads E. coli O157:H7 immunomagnetic beads from Invitrogen Corporation (USA); anti-E. coli O157:H7 monoclonal antibody prepared by our laboratory; colloidal gold test strip for E. coli O157:H7 prepared by our laboratory; bovine serum albumin (BSA) from AMRESCO Corporation (USA); 4-morpholineethane- sulfonic acid (MES) from Beijing Suolaibao Technology Co., Ltd. (China); EDC-HCl, NHSS from Aladdin Reagent Database Inc. (China); and sorbitol MacConkey agar base and E. coli O157:H7 selective medium from Beijing Land Bridge Technology Co., Ltd. (China).
Instruments and equipment: Magnetic separation rack was purchased from Zodolabs Biotech Co., Ltd. (China); biohazard safety equipment from Suzhou Antai Airtech Co., Ltd. (China); and constant temperature incubator and constant temperature oscillation incubator from Shanghai Zhicheng Analytical Instrument Manufacturing Corporation (China), Rotation DH-II Ningbo Scientz Corporation (China).
2.2 Preparation and optimization of immunomagnetic beads
2.2.1 Activation and coupling of immunomagnetic beads
PM3-020 magnetic beads (5 mg) were washed four times
with MES buffer solution (0.01 M, pH 6.0). The supernatant was removed after magnetic separation for the first time.
EDC and NHSS solution (10 mg mL�1) was prepared by dissolving respect solute in MES buffer solution, and 0.5 mL of both solution were added to the sediments. The solution was then activated in a rotator at room temperature for 1 h (15 rpm). The supernatant was removed after magnetic separation for the second time. The sediments were resuspended with 1 mL MES. Activated magnetic beads (200 μL) were added in a centrifuge tube rapidly. The supernatant was removed again after magnetic separation for the third time.
Antibody (1 mL) was diluted by borate-saline buffer (0.01 M, pH 8.5). The solution was then reacted in a rotator at room temperature for 4 h (15 rpm). The supernatant was removed again after magnetic separation for the fourth time. BSA (1%, 1 mL) borate-saline buffer was added. The solution was then reacted in a rotator at room temperature for 2 h (15 rpm). The supernatant was removed again after magnetic separation for the fifth time. The sediments were washed three times with borate-saline buffer-Tween 20 (0.1%). The immunomagnetic beads were resuspended with 0.5 mL borate-saline buffer-Tween 20 (with 0.5% BSA). The immunomagnetic beads were preserved at 4 °C until use.
2.2.2 Determination of quantity of immunomagnetic beads required to capture 1 mL of 105 CFU mL�1 E. coli O157:H7 culture
1 mL of 105 CFU/mL E. coli O157:H7 culture was added
into each of the seven centrifuge tubes (2 mL). Then Immunomagnetic beads (0.005, 0.01, 0.02, 0.05, 0.1 and 0.2 mg) were added into centrifuge tubes 1 to 6, respectively. The ratio of the monoclonal antibody to the magnetic beads was 50 �g mg�1. Because tube 7 did not contain any immunomagnetic beads and served as the blank control. The solution was then placed in a rotator at room temperature for 45 min (10 rpm). The supernatant was removed after magnetic separation for 3 min. The sediments were washed twice with 1 mL borate-saline buffer-Tween 20 (0.02%). The supernatant was removed again after magnetic separation. The beads were resuspended in borate-saline buffer. The supernatant, scrubbing solution, resuspension solution, and blank control were diluted to the appropriate concentration. Approximately 100 �L of each solution mentioned above was dispersed to the E. coli O157:H7 selective culture medium. Three parallel experiments were performed for each concentration. The plates were cultivated at 37 °C for 12�16 h. CFUs ranging from 20 to 200 were selected for effective counting. The calculation of the capture rate is described in the following equation.
CE (%) = (C1 – C2 –C3) × 100/C1 (1) where, CE is capture efficiency; C1 is total amount of colonies in the blank; C2 is total amount of colonies in supernatant; C3 is total amount of colonies in scrubbing solution. 2.2.3 Capture effect in food sample
Ground beef or raw milk (25 g or mL) was weighed and
added into a sterile triangular flask. E. coli O157:H7 was added at a concentration of 102 CFU g�1 (CFU mL�1). 225 mL of modified E. coli (mEC) broth was then added. The solution was cultivated in a table concentrator (160 rpm) at 37 °C for 8 h. 1 mL of the supernatant was added into a centrifuge tube. Immunomagnetic beads prepared in our laboratory (0.05 mg) or commercial Dynabeads E. coli O157:H7 immunomagnetic beads (0.05 mg) were added into the tubes. The tubes were reactived in a rotator at room temperature for 30 min and magnetically separated for 3 min. The supernatant, scrubbing solution, resuspension solution, and blank control were diluted to the appropriate concentration. Approximately 100 �L of each solution was dispersed to the E. coli O157:H7 selective culture medium. Three parallel experiments were performed for each concentration. The plates were cultivated at 37 °C for 12�16 h. CFUs ranging from 20 to 200 were selected for effective counting. The capture rate was calculated.
2.3 Preparation and evaluation of collidal gold test strip 2.3.1 Preparation of collidal gold test strip
The collidal gold test strip was prepared using the method
established in the previous study[10]. The colloidal gold test
CUI Xi et al. / Chinese Journal of Analytical Chemistry, 2013, 41(12): 1812–1816
strip was composited with anti-E. coli O157:H7 monoclonal antibody marked by colloidal gold as the detection antibody, anti-E. coli O157:H7 polyclonal antibody as the test line, and donkey anti-mouse IgG secondary antibody as the control line. Test line become red when E. coli O157:H7 in sample exceeded the threshold. Control line present red when the test strip is effective. 2.3.2 Specificity of collidal gold test strip
Five E. coli O157:H7 strains and 36 non-E. coli O157:H7
strains were used in the specificity test. Approximately 100 �L 108 CFU mL�1 culture was added to the collidal gold test strip. The results were obtained after 10 min.
2.4 Establishment of combined immunomagnetic separation with lateral flow aassay mmethod
2.4.1 Detection of E. coli O157:H7 in pure culture
E. coli O157:H7 strain was cultivated overnight and diluted to 103, 104, 105, 106 and 107 CFU mL�1, respectively. Approximately 1 mL of each culture was added into two groups of 2 mL centrifuge tubes. The immunomagnetic beads prepared in our laboratory were added into the tubes in Group 1, whereas no bead was added to the tubes in Group 2 which served as blank control. The tubes were incubated in a rotator at room temperature for 30 min. After the immunomagnetic separation, the beads were resuspended by borate-saline buffer. The tubes were placed in water bath at 85 °C for 15 min. The supernatant was removed for the next step after magnetic separation. 100 �L of the supernatant and the broth in Group 2 were added into the collidal gold test strip. The results were obtained after 15 min.
2.4.2 Detection of E. coli O157:H7 in inoculated food sample
E. coli O157:H7 was added to 25 g ground beef (10 CFU g�1). The inoculated ground beef was added to 225 mL mEC. The mixed liquid was incubated at 37 °C. After 4, 6, 8 and 10 h, 1 mL liquid was removed and added into two centrifuge tubes (2 mL). Group 1 was managed by immunomagnetic separation, whereas Group 2 was not. The supernatant in Group 1 (100 �L) and the culture in Group 2 (100 �L) were added to the collidal gold test strip. The results were obtained after 15 min.
3 Results and discussion 3.1 Determination of quantity of immunomagnetic
beads required to capture 1 mL of 105 CFU mL�1 E. coli O157:H7 culture
When the amount of immunomagnetic beads exceeded 0.05 mg, the capture rate of 1 mL 105 CFU mL�1 E. coli O157:H7 exceeded 99%, whenas the immunomagnetic separation was effective (Fig.2).
3.2 Capture effect in the food sample The capture rates of immunomagnetic beads prepared in our
laboratory were 94.4% and 99.8% in ground beef and raw milk, respectively. The capture rates of Dynabeads E. coli O157:H7 immunomagnetic beads were 72.8% and 95.8% in ground beef and raw milk, respectively. These results indicated that the beads prepared in our laboratory were better than commercial beads.
3.3 Specificity of colloidal gold test strip
The specificity of colloidal gold test strip was estimated. As
shown in Fig.3, Nos. 1–5 are E. coli O157:H7 strains that were preserved. No. 6 to 41 are non-E. coli O157:H7 strains. No. 42 is phosphate bufferd saline (PBS). The results showed that the specificity of the collidal gold test strip was suitable. The five strains of E. coli O157:H7 were detected successfully, and no cross reaction with other strains or PBS were observed.
3.4 Combined immunomagnetic separation and lateral
flow assay method
3.4.1 Detection of E. coli O157:H7 in pure culture 100 �L of the bacteria solution without immunomagnetic
separation was added to the colloidal gold test strip. The results were achieved after 15 min. The concentration of 7.6 × 105 and 7.6 × 106 CFU mL�1 presented strong positive results. The concentration of 7.6 × 104 CFU mL�1 presented a weak positive result. As shown in Fig.4A, the detection limit of the colloidal gold test strip was 7.6 × 104 CFU mL�1. After immunomagnetic separation, the concentration of 7.6 × 104 CFU mL�1 presented a positive result. The concentration of
Fig.2 Capture efficiency with different doses of immunomagnetic beads
CUI Xi et al. / Chinese Journal of Analytical Chemistry, 2013, 41(12): 1812–1816
Fig.3 Specificity of the colloidal gold test strip Fig.4 Comparison of detection effect in pure culture of two methods (A) Pure culture detected directly by colloidal gold test strip; (B) Pure culture detected by the colloidal gold test strip after immunomagnetic separation. The concentrations of culture Nos. 1 to 5 were 0, 7.6 × 103, 7.6 × 104, 7.6 × 105, and 7.6 × 106 CFU mL�1, respectively 7.6 × 103 CFU mL�1 presented a weak positive result. These results showed that a 7.6 × 103 CFU mL�1 bacteria solution could be detected by the colloidal gold test strip after immunomagnetic separation (Fig.4B). The sensitivity of the combined method is 10 times higher than that of the colloidal gold test strip.
3.4.2 Detection of E. coli O157:H7 in inoculated food sample
Further experiments were conducted to compare with or
without immunomagnetic seperation (IMS) before detection by gold colloidal lateral flow assay. The results are listed in Table 1. The bacteria solution cultivated in mEC at 37 °C for 6 h without IMS presented a negative result. On the contrary, the solution presented a positive result after immunomagnetic separation. When the food matrix with E. coli O157:H7 broth was added to the colloidal gold test strip, the food sample moved slowly because food particles and fats influence the
Table 1 Comparison of test effects of two methods for ground beef Culture time Detection result of the gold colloidal lateral flow assay
Without IMS before detection With IMS before detection
4 6 + 8 + ++
10 ++ ++ Note: “–” stands for negative, “+” stands for positive, and “++” stands for strong positive.
effect of the colloidal gold test strip. This phenomenon did not occur after immunomagnetic separation. Immunomagnetic separation eliminates undesirable influences and improves detection sensitivity.
In this study, we established a method by combining immunomagnetic separation with colloidal gold lateral flow assay to detect E. coli O157:H7 rapidly. Using this method, a food sample inoculated with 10 CFU g�1 E. coli O157:H7 after 6 h of enrichment could be detected within 72 min. The cost of the immunomagnetic separation and the test strip is as low as 20 RMB, also these methods can be performed without professional skill. Overall, the method proposed in this study could be used to on-site detect harmful substances in food significantly.
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