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Abstract A fluorescence immunochromatographic strip was developed in this study for natamycin detection in food. The results showed that the best amount of labeled antibody was 10 μg, for every 50 μl of fluorescent microspheres with a 2.5% (w/v) concentration. This labeled antibody was diluted for 10 times, and the diluted solution was dispensed into conjugate pad at the amount of 3 μl/cm. The concentrations of natamycin labeled BSA for test line and goat antimouse IgG for control line were 2.0 and 1 mg/ml, respectively, which performed best. With the best conditions, the limit of detection was 1 ng/ml, the linearity ranged from 2 to 100 ng/ml, the recovery was about 80% to 120%, and the CV was below 23%.
Key words Food safety detection; Natamycin; Fluorescence immunochromatographic assay
Received: May 16, 2019Accepted: August 27, 2019
Supported by Ningbo EntryExit Inspection and Quarantine Bureau (Ningbo Customs) Science and Technology Project (YK072017).
Wei ZHENG (1981-), female, P. R. China, senior agronomist, devoted to research about food microbiology and plant quarantine.
*Corresponding author. Email: lixinyue1992@126.com.
In the processing of food, people usually add preservatives to the food to prolong the preservation time of food and prevent food from being contaminated by fungi. Natamycin, also known as natamycin or pimamycin, is a foodgrade preservative that is odorless, tasteless, and has the advantages of low dose and high safety. Although natamycin has been approved by the state for use in food production, as an antibiotic, the amount of natamycin added is also strictly controlled and longterm tracked［1-3］.
Fluorescence immunochromatographic assay uses fluorescent microspheres and fluorescent molecules instead of colloidal gold, and uses instruments to read fluorescent signals instead of naked eyes to observe color depth. It solves the limitations of colloidal gold immunochromatographic signal and dependence on individual judgment, and is widely used in the field of medical diagnosis［4-9］. After years of development, quantitative instruments for fluorescence immunochromatographic assay are becoming more and more miniaturized, and have the quality getting more and more reliable and gradually reducing cost. The application range of fluorescence immunochromatographic assay becomes wider and wider［10］. Due to its advantages of smallsized instruments, fast speed measurement and good quantitative effect, it is suitable for project inspection of various sample sizes as well as grassroots and onsite inspection. Materials and Methods
ATBSA Preparation
Adipic acid (15 mg) was dissolved in 5 ml of PBS 7.4+EDC hydrochloride (40 mg)+NHSsulfo (100 mg), followed by reaction at room temperature for 30 min. BSA (15 mg) was then added, followed by reaction at room temperature for 120 min. NAT (3 mg) was finally added. The reaction system was diluted to 3 ml with 0.02 M PB (7.4), and reacted for 2 h at room temperature. Dialysis was performed twice with 0.02 M PB (7.4). After recovery, the coupling efficiency was detected by ELISA.
Fluorescent microspherelabeled monoclonal antibody against NAT
At first, 50 μl of fluorescent microspheres (200 nm, 1%) was added, diluted with 450 μl of pure water, and washed once by centrifugation. NHSsuffo and EDC were freshly prepared with purified water to 10 mg/ml, and allowed to reaction for 30 min. Centrifugation was performed, obtaining a precipitate, which was finally redissolved to 500 μl. Next, 5/10/20 μg of NAT antibody was added to the microspheres, and the mixture was oscillated at room temperature for 2 h. Next, 50 μl of 10% BSA was added to the reaction system, followed by reaction for 2 h or overnight. The microspheres were centrifuged at 4 ℃ and resuspended and ultrasonically dispersed with pure water, which was repeated for 1 time. Finally, the washed microspheres were preserved with a preserving fluid to 200 μl (solid content: 0.25%).
Membrane dispensing
After membrane dispensing, the membrane was dried in an oven at 37 ℃ for 2 h.
Gold spraying
After gold spraying, the product was dried in an oven at 37 ℃ for 2 h.
Strip assembly and identification
The coated NC film, the goldlabeled antibody conjugate pad, the sample pad, the absorbent paper and the PVC bottom plate were assembled as required, and the prepared chromatographic card and the desiccant were placed in a foil pouch and packaged.
Sample detection
Into 27 centrifuged tubes, 5.00 g of cake was accurately added. Then, 10, 50 and 200 μg of natamycin were added into 9 tubes, respectively. The samples were stood at room temperature for 2 h. Next, 30 ml of methanol was added into each centrifuge tube, followed by ultrasonic extraction for 30 min. After the addition of 10 ml of water, centrifugation was performed at 2 000 g for 5 min. Each supernatant was filtered with 0.45 and 0.22 μm filter sequentially. Next, 0.20 ml of each extract was taken and added with 9.8 ml of PB solution, and 50 μl of each obtained test solution was taken for the determination of concentration. The recovery was finally calculated. Results and Analysis
ELISA analysis results
The coupled NATBSA was able to bind to the antiNAT antibody, and it was confirmed that the NAT was successfully labeled. Meanwhile, after adding NAT, 1 ng/ml NAT showed significant inhibition on ELISA signal, indicating that the affinity of the antibody was high, which is conducive to the development of kits.
Strip optimization test
The conditions such as antibody concentration, amount of sprayed gold, and NATBSA concentration were optimized.
For the labeling of 10 μg of antibody, the C line and T line were both clear, and showed approximately the same brightness, so a ratio of 10 μg∶50 μl was chosen for labeling.
For the dilution gradients of 1∶10 and 1∶5, the resolution ratio was still good at the low concentration of 1 ng/ml. Taking cost into account, the 1∶10 dilution gradient was chosen.
As the final concentration increasing, the value of T/C was also too large, but as the concentration for the T line increased, the sensitivity decreased slightly. The stability at 1.2 mg/ml was better than that at 0.8 mg/ml, so the concentration of 1.2 mg/ml was chosen as the final concentration.
Sample testing
Construction of standard curve
With the relative fluorescence intensity value (T/C) plotted on the ordinate and the NAT standard concentration plotted on the abscissa, the standard working curve was drawn with OriginPro 8 using nonlinear fourparameter logistic fitting. The IC50 (halfinhibitory concentration) and LOD (limit of detection) were determined. The IC50 was the concentration of NAT at an inhibitory rate of 50%, and the LOD was the concentration of NAT at an inhibitory rate of 10%.
Recovery and variable coefficient
According to the national standard, into 5.0 g of blank cake, 10, 40 and 200 μg of NAT were added, respectively, followed by extraction, dilution and determination of concentration. The tests were done in nine replicates, and the results are shown in Table 1.
It can be seen from the results that the method had a high recovery (>95%) and a low CV (<15%), indicating high precision.
Conclusions and Discussion
Factors affecting the detection stability include ovendrying time, antibody stability, dryness of the conjugate pad, environmental humidity and assembly environment, which require further research.
In the field of food safety testing, there are often false negatives and false positives. The reasons for this may be: First, there are natural surfactants or added surfactants in food, which interfere with the detection process, causing false negatives or false positives. Second, there is similar molecular competition in food, causing false positives. There were no false negatives and false positives in this project. The main reason may be that the concentration of natamycin was higher, and the kit was more sensitive and can be diluted in a large proportion, reducing the interference by related interferents. References
［1］ AGO YR, WANG XP. Property of natanycin: A natural antiepiphyte biopreservative［J］. Farm Products Processing, 2009, (5): 7-13. (in Chinese)
［2］ SUN CX, ZHANG FJ, WANG YP. Research progress of application and detection method of natamycin［J］. China Food Additives, 2017, (3): 178-182. (in Chinese)
［3］ YUE HB, YUE XQ, LI J, et al. The properities and application of natamycin and its production and advanced research［J］. Food Science and Technology, 2007, (3): 162-166. (in Chinese)
［4］ LI YQ, YANG RF. Rapid test of immunocolloidal gold with membrane as solid phase carrier［J］. Progress in Microbiology and Immunology, 2003, 31(1): 74-78. (in Chinese)
［5］ WU G, JIANG ZM, HUO GC, et al. Application of immunochromatography in food determination［J］. Science and Technology of Food Industry, 2007, (12): 216-218. (in Chinese)
［6］ YAN H, SHEN HF. Application and prospect of colloidal gold immunochromatography ［J］. Progress in Microbiology and Immunology, 2005, 33(3): 86-89. (in Chinese)
［7］ ZHANG FX, WANG XL. Analysis of the affecting factors of immune colloidal gold technique［J］. Chinese Animal Husbandry and Veterinary Medicine, 2009, 36(5): 199-202. (in Chinese)
［8］ ZHANG ZJ, WANG FH. The Enzymelinked immunosorbent assay technology and its application in food safety determination［J］. Food Research and Development, 2011, 32(1): 157-160. (in Chinese)
［9］ KONG LW. Study on Immunoassay of chloramphenicol by lateral flow immunochromatography assay［D］. Zhoushan: Zhejiang Ocean University, 2012. (in Chinese)
［10］ CONNOLLY L, FODEY TL, CROOKS SRH, et al. The production and charaacterisation of dinitrocarbanilide antibodies raised using antigen mimics［J］. Journal of Immunological Methods, 2002, 264(12): 45-51. (in Chinese)
Key words Food safety detection; Natamycin; Fluorescence immunochromatographic assay
Received: May 16, 2019Accepted: August 27, 2019
Supported by Ningbo EntryExit Inspection and Quarantine Bureau (Ningbo Customs) Science and Technology Project (YK072017).
Wei ZHENG (1981-), female, P. R. China, senior agronomist, devoted to research about food microbiology and plant quarantine.
*Corresponding author. Email: lixinyue1992@126.com.
In the processing of food, people usually add preservatives to the food to prolong the preservation time of food and prevent food from being contaminated by fungi. Natamycin, also known as natamycin or pimamycin, is a foodgrade preservative that is odorless, tasteless, and has the advantages of low dose and high safety. Although natamycin has been approved by the state for use in food production, as an antibiotic, the amount of natamycin added is also strictly controlled and longterm tracked［1-3］.
Fluorescence immunochromatographic assay uses fluorescent microspheres and fluorescent molecules instead of colloidal gold, and uses instruments to read fluorescent signals instead of naked eyes to observe color depth. It solves the limitations of colloidal gold immunochromatographic signal and dependence on individual judgment, and is widely used in the field of medical diagnosis［4-9］. After years of development, quantitative instruments for fluorescence immunochromatographic assay are becoming more and more miniaturized, and have the quality getting more and more reliable and gradually reducing cost. The application range of fluorescence immunochromatographic assay becomes wider and wider［10］. Due to its advantages of smallsized instruments, fast speed measurement and good quantitative effect, it is suitable for project inspection of various sample sizes as well as grassroots and onsite inspection. Materials and Methods
ATBSA Preparation
Adipic acid (15 mg) was dissolved in 5 ml of PBS 7.4+EDC hydrochloride (40 mg)+NHSsulfo (100 mg), followed by reaction at room temperature for 30 min. BSA (15 mg) was then added, followed by reaction at room temperature for 120 min. NAT (3 mg) was finally added. The reaction system was diluted to 3 ml with 0.02 M PB (7.4), and reacted for 2 h at room temperature. Dialysis was performed twice with 0.02 M PB (7.4). After recovery, the coupling efficiency was detected by ELISA.
Fluorescent microspherelabeled monoclonal antibody against NAT
At first, 50 μl of fluorescent microspheres (200 nm, 1%) was added, diluted with 450 μl of pure water, and washed once by centrifugation. NHSsuffo and EDC were freshly prepared with purified water to 10 mg/ml, and allowed to reaction for 30 min. Centrifugation was performed, obtaining a precipitate, which was finally redissolved to 500 μl. Next, 5/10/20 μg of NAT antibody was added to the microspheres, and the mixture was oscillated at room temperature for 2 h. Next, 50 μl of 10% BSA was added to the reaction system, followed by reaction for 2 h or overnight. The microspheres were centrifuged at 4 ℃ and resuspended and ultrasonically dispersed with pure water, which was repeated for 1 time. Finally, the washed microspheres were preserved with a preserving fluid to 200 μl (solid content: 0.25%).
Membrane dispensing
After membrane dispensing, the membrane was dried in an oven at 37 ℃ for 2 h.
Gold spraying
After gold spraying, the product was dried in an oven at 37 ℃ for 2 h.
Strip assembly and identification
The coated NC film, the goldlabeled antibody conjugate pad, the sample pad, the absorbent paper and the PVC bottom plate were assembled as required, and the prepared chromatographic card and the desiccant were placed in a foil pouch and packaged.
Sample detection
Into 27 centrifuged tubes, 5.00 g of cake was accurately added. Then, 10, 50 and 200 μg of natamycin were added into 9 tubes, respectively. The samples were stood at room temperature for 2 h. Next, 30 ml of methanol was added into each centrifuge tube, followed by ultrasonic extraction for 30 min. After the addition of 10 ml of water, centrifugation was performed at 2 000 g for 5 min. Each supernatant was filtered with 0.45 and 0.22 μm filter sequentially. Next, 0.20 ml of each extract was taken and added with 9.8 ml of PB solution, and 50 μl of each obtained test solution was taken for the determination of concentration. The recovery was finally calculated. Results and Analysis
ELISA analysis results
The coupled NATBSA was able to bind to the antiNAT antibody, and it was confirmed that the NAT was successfully labeled. Meanwhile, after adding NAT, 1 ng/ml NAT showed significant inhibition on ELISA signal, indicating that the affinity of the antibody was high, which is conducive to the development of kits.
Strip optimization test
The conditions such as antibody concentration, amount of sprayed gold, and NATBSA concentration were optimized.
For the labeling of 10 μg of antibody, the C line and T line were both clear, and showed approximately the same brightness, so a ratio of 10 μg∶50 μl was chosen for labeling.
For the dilution gradients of 1∶10 and 1∶5, the resolution ratio was still good at the low concentration of 1 ng/ml. Taking cost into account, the 1∶10 dilution gradient was chosen.
As the final concentration increasing, the value of T/C was also too large, but as the concentration for the T line increased, the sensitivity decreased slightly. The stability at 1.2 mg/ml was better than that at 0.8 mg/ml, so the concentration of 1.2 mg/ml was chosen as the final concentration.
Sample testing
Construction of standard curve
With the relative fluorescence intensity value (T/C) plotted on the ordinate and the NAT standard concentration plotted on the abscissa, the standard working curve was drawn with OriginPro 8 using nonlinear fourparameter logistic fitting. The IC50 (halfinhibitory concentration) and LOD (limit of detection) were determined. The IC50 was the concentration of NAT at an inhibitory rate of 50%, and the LOD was the concentration of NAT at an inhibitory rate of 10%.
Recovery and variable coefficient
According to the national standard, into 5.0 g of blank cake, 10, 40 and 200 μg of NAT were added, respectively, followed by extraction, dilution and determination of concentration. The tests were done in nine replicates, and the results are shown in Table 1.
It can be seen from the results that the method had a high recovery (>95%) and a low CV (<15%), indicating high precision.
Conclusions and Discussion
Factors affecting the detection stability include ovendrying time, antibody stability, dryness of the conjugate pad, environmental humidity and assembly environment, which require further research.
In the field of food safety testing, there are often false negatives and false positives. The reasons for this may be: First, there are natural surfactants or added surfactants in food, which interfere with the detection process, causing false negatives or false positives. Second, there is similar molecular competition in food, causing false positives. There were no false negatives and false positives in this project. The main reason may be that the concentration of natamycin was higher, and the kit was more sensitive and can be diluted in a large proportion, reducing the interference by related interferents. References
［1］ AGO YR, WANG XP. Property of natanycin: A natural antiepiphyte biopreservative［J］. Farm Products Processing, 2009, (5): 7-13. (in Chinese)
［2］ SUN CX, ZHANG FJ, WANG YP. Research progress of application and detection method of natamycin［J］. China Food Additives, 2017, (3): 178-182. (in Chinese)
［3］ YUE HB, YUE XQ, LI J, et al. The properities and application of natamycin and its production and advanced research［J］. Food Science and Technology, 2007, (3): 162-166. (in Chinese)
［4］ LI YQ, YANG RF. Rapid test of immunocolloidal gold with membrane as solid phase carrier［J］. Progress in Microbiology and Immunology, 2003, 31(1): 74-78. (in Chinese)
［5］ WU G, JIANG ZM, HUO GC, et al. Application of immunochromatography in food determination［J］. Science and Technology of Food Industry, 2007, (12): 216-218. (in Chinese)
［6］ YAN H, SHEN HF. Application and prospect of colloidal gold immunochromatography ［J］. Progress in Microbiology and Immunology, 2005, 33(3): 86-89. (in Chinese)
［7］ ZHANG FX, WANG XL. Analysis of the affecting factors of immune colloidal gold technique［J］. Chinese Animal Husbandry and Veterinary Medicine, 2009, 36(5): 199-202. (in Chinese)
［8］ ZHANG ZJ, WANG FH. The Enzymelinked immunosorbent assay technology and its application in food safety determination［J］. Food Research and Development, 2011, 32(1): 157-160. (in Chinese)
［9］ KONG LW. Study on Immunoassay of chloramphenicol by lateral flow immunochromatography assay［D］. Zhoushan: Zhejiang Ocean University, 2012. (in Chinese)
［10］ CONNOLLY L, FODEY TL, CROOKS SRH, et al. The production and charaacterisation of dinitrocarbanilide antibodies raised using antigen mimics［J］. Journal of Immunological Methods, 2002, 264(12): 45-51. (in Chinese)