- Email: [email protected]
Rapid detection of fumonisin B1 using a colloidal gold immunoassay strip test in corn samples
Toxicon 108 (2015) 210e215
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Rapid detection of fumonisin B1 using a colloidal gold immunoassay strip test in corn samples Sumei Ling a, Rongzhi Wang a, Xiaosong Gu a, Can Wen a, Lingling Chen a, Zhibin Chen a, Qing-Ai Chen b, Shiwei Xiao a, Yanling Yang a, Zhenhong Zhuang a, *, Shihua Wang a, * a Key Laboratory of Pathogenic Fungi and Mycotxins of Fujian Province, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China b College of Food Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 July 2015 Received in revised form 16 October 2015 Accepted 20 October 2015 Available online 23 October 2015
Fumonisin B1 (FB1) is the most common and highest toxic of fumonisins species, exists frequently in corn and corn-based foods, leading to several animal and human diseases. Furthermore, FB1 was reported that it was associated with the human esophageal cancer. In view of the harmful of FB1, it is urgent to develop a feasible and accuracy method for rapid detection of FB1. In this study, a competitive immunoassay for FB1 detection was developed based on colloidal gold-antibody conjugate. The FB1-keyhole limpet hemoeyanin (FB1-KLH) conjugate was embedded in the test line, and goat anti-mouse IgG antibody embedded in the control line. The color density of the test line correlated with the concentration of FB1 in the range from 2.5 to 10 ng/mL, and the visual limit detection of test for FB1 was 2.5 ng/mL. The results indicated that the test strip is specific for FB1, and no cross-reactivity to other toxins. The quantitative detection for FB1 was simple, only needing one step without complicated assay performance and expensive equipment, and the total time of visual evaluation was less than 5 min. Hence, the developed colloidal gold-antibody assay can be used as a feasible method for FB1 rapid and quantitative detection in corn samples. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Fumonisin B1 Hybridoma Colloidal gold immunoassay Quantitative detection
1. Introduction Fumonisins, exists frequently in corn and corn-based foods, are produced primarily by Fusarium moniliforme (Pagliuca et al., 2005; Mexiasalazar et al., 2008). FB1 was first discovered in 1988, and it is the most common and highest toxic of fumonisins species, often leading to several animal and human diseases, such as encephalomalacia in horses, pulmonary edema in pigs, and hepatic and renal toxicity in several species (Tardieu et al., 2009). Furthermore, FB1 was reported that it was associated with the human esophageal cancer. Due to its strong toxicity, several measures of regulation and prevention have been made by the Food and Drug Administration (FDA) and Commission Regulation of European Community (EC). For example, FDA has determined the limit of FB1 in different foods, as 2000e4000 mg/kg total fumonisins (FB1 þ FB2 þ FB3) for human
* Corresponding authors. E-mail addresses: [email protected] (Z. Zhuang), [email protected] (S. Wang). http://dx.doi.org/10.1016/j.toxicon.2015.10.014 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
foods, and 5000e10,0000 mg/kg for animal feeds (Wang et al., 2011), and the European Union maximum guidance level for fumonisins is 20 mg/kg for feeds (Antonissen et al., 2015), and 2e4 mg/kg for foods (Kadir and Tothill, 2010). In naturally contaminated samples, approximately 70% of the total detected fumonisins was FB1, especially in corn samples (Samapundo et al., 2006). Therefore, it is very important to develop effective methods to detect and quantify the contamination of FB1 in samples. Recently, several assays have been used for detection FB1 in different samples, such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography (LC) assays and so on. The detection limit of Immunoaffinity Extraction combined with Highperformance Liquid Chromatography (HPLC) with fluorescence for FB1 was 0.013 mg FB1/g in liver, kidney and muscle tissue. The minimum detection level of LC-MS method for FB1 in bovine milk was 0.1 mg/kg (Tardieu et al., 2008; Gazzotti et al., 2009). These methods are sensitive and reliable to determine the FB1 in samples, but these methods need expensive equipment, trained operators, and complicated sample preparation, making them
S. Ling et al. / Toxicon 108 (2015) 210e215
unsuitable for on-site inspection. Up to now, enzyme linked immunosorbent assay (ELISA) with high sensitivity and specificity based on monoclonal and polyclonal antibodies (mAb and pAb) have been widely used to detect FB1 in foods. The Limit detection of enhanced chemiluminescent (ECL)-ELISA method for quantification of FB1 was 0.09 mg/L (Quan et al., 2006). However, ELISA also has some drawback, including time-consuming, tedious washing and incubation steps. Therefore, the detection methods described above did not meet on-site testing. From 1980, colloidal gold immunoassay has been developed to detect the toxins in different samples in one step without other treatment, using a cellulose membrane as the carrier. To solve the problem of rapid assay for FB1 efficiently, a sensitive, specific and simple immunoassay for FB1 detection in corn samples was urgently needed. Our group had successfully obtained one hybrid cell lines 4G5 excreting monoclonal antibody specific against FB1, and then developed Ic-ELISA method for detection (Ling et al., 2014). In this study, colloidal gold immunoassay was established based on this McAb. The purified anti-FB1 McAb was conjugated to the prepared colloidal gold particles, and the formed colloidal gold-labeled IgG antibody was used as the tracer to detect FB1 on a test strip. The strip test can be used for rapid detection of FB1 in corn samples with high specificity and sensitivity, and the process of detection was simple, without any equipment and complicated handling procedures. Hence, the strip test has good application prospect for mycotoxin detection in samples. 2. Materials and methods 2.1. Reagents and buffers Nitrocellulose membranes were obtained from Whatman company (Middlesex, UK). Fumonisin B1 (the purity 98% by HPLC), goat anti-mouse IgG, bovine serum albumin (BSA), Chloroauric acid (HAuCl4), and trisodium citrate were purchased from Shanghai Chemical Reagents (Shanghai, China). All other chemicals were analytical grade and purchased from Beijing Chemical Reagent Co. (Beijing, China). 2.2. Preparation of anti-FB1 IgG antibody Hybridoma cells named 4G5 that secreted anti-FB1 IgG antibody were previously prepared in our laboratory (Ling et al., 2014). After injection of 4G5 hybridoma cells into mice for 2e3 weeks, the produced ascites was collected every other day. The specific IgG antibody was precipitated by ammonium sulfate, then, further purified by affinity chromatography with an immobilized protein G column. The titer of anti-FB1 McAb was determined by ELISA, and the absorbance was measured at 450 nm by Micro-plate reader. The concentration of purified IgG antibody was determined by the instruction of Enhanced BCA Protein Assay Kit. 2.3. Preparation of colloidal gold Colloidal gold particles were produced as described with minor modification (Chen et al., 2012; Biagini et al., 2006). Briefly, 100 mL of 0.01% chloroauric acid solution (HAuCl4) in distilled water was heated to the boiling point, and then 2.0 mL of 1% trisodium citrate solution was quickly added into this solution. After boiling for 5 min, the color of solution was changed from yellow to wine-red, then, allowed to cool the solution gradually to room temperature. Finally, the purified anti-FB1 IgG antibody was added into the solution for conjugation at 4 C for overnight. The conjugates were identified by a transmission electron microscope (TEM), and the absorbance peak of the conjugates was characterized by UVevisible
211
spectra at 400e660 nm. 2.4. Optimization of test strips One milliliter of colloidal gold solution was added separately to the series of 1.5 mL tubes. The pH of colloidal gold solution was adjusted with potassium carbonate. The same concentration of the anti-FB1 IgG antibody diluted with phosphate buffer was added into the solution. After incubating the mixture for 5 min, 0.02 mL of 10% NaCl was added into the tube, and the minimum pH that keep the red color in tube without change was considered as the optimal pH. Different volume of purified anti-FB1 IgG antibody solution (from 0 to 9.5 mL) was added separately to 1 mL of the colloidal gold solution. After incubating the mixture for 5 min, 0.02 mL of 10% NaCl was added to the solution, and the absorbance of solution was measured at 525 nm. The optimal concentration of purified antiFB1 IgG antibody was determined according to the standard curve drawn from the concentration and the absorbance. 2.5. Preparation of colloidal gold-antibody conjugates Colloidal gold solution was adjusted to optimal pH with potassium carbonate. Then, 1 mL of purified anti-FB1 IgG antibody was diluted with phosphate buffer to the optimal concentration, and then added into the 100 mL colloidal gold solution with gentle stirring for 60 min. After reaction, 1 mL of 1% (w/v) BSA solution was dropped into the mixture with gentle stirring for 30 min. The solution was incubated at 4 C for overnight, then, centrifuged at 2000 g for 30 min. The supernatant was centrifuged at 10,000 g for 60 min, and the precipitated colloidal gold-antibody conjugates was re-suspended with conjugate dilution buffer (0.01 M Tris, 5% BSA, 2% sucrose, 0.87% NaCl and 0.1 M sodium azide) and stored at 4 C for further use. 2.6. Preparation of colloidal gold immunoassay test strips Colloidal gold immunoassay test strip consisted of three pads (sample, conjugate, and absorbent pads) and one nitrocellulose (NC) membrane with test and control zones (Liu et al., 2014). The colloid gold strip test device was constructed as follows: As FB1 is a small molecular hapten, it cannot bind directly on the surface of nitrocellulose membrane. So the FB1-KLH conjugate and rabbit anti-mouse IgG antibody were embedded onto nitrocellulose (NC) membrane, respectively. The distance between the two lines was about 5 mm. The treated nitrocellulose membrane was dried at room temperature for 10 min. Subsequently, the absorption pad, nitrocellulose (NC) membrane, conjugation pad, and sample application pad were assembled into a laminated sheet. Finally, the sheet was cut into 5 mm wide strips for further use. If both the test and control lines turn red, the sample is recorded as negative. When the control line but not the test line was colored, it is considered as positive. If no visible red line is present in the control area, the test strip is considered invalid regardless of whether an red line appears in the test area or not. 2.7. Determination of cross-reactivity and sensitivity of the test strips To evaluate the cross-reactivity of the test strips, different toxins including Aflatoxin B1 (AFB1), HT-2 toxin (HT-2), Ochratoxin A (OTA), Ochratoxin B (OTB), T-2 toxin (T-2), Citreoviridin (CIT), were allowed to react with the colloidal goldeFB1 McAb conjugate which was pipetted into glass fiber paper (Wang et al., 2014a). The mixture then moves upward on the nitrocellulose membrane. After incubation at room temperature for 5 min, the detection results
212
S. Ling et al. / Toxicon 108 (2015) 210e215
could be observed by the naked eye. To further determine the sensitivity of the test strips, FB1-KLH conjugates prepared in our laboratory was sprayed to the test zone, and rabbit anti-mouse IgG antibody sprayed to the control zone. In the study, the series concentrations of FB1 toxin from 0 to 10 ng/mL were allowed to react with the colloidal gold-antibody (anti-FB1 IgG antibody) conjugates, and then FB1 test strips results could be observed by the naked eye. 2.8. Assay of FB1 in corn samples Corn related samples, including corn, corn steamed bread, corn kernels, and corn sheath, were purchased and collected from supermarket. After samples treated with methyl alcohol, the test strips were used to detect the FB1 in corn samples. FB1-KLH conjugates prepared in our laboratory was immobilized to the test zone and rabbit anti-mouse IgG antibody immobilized to the control zone on the nitrocellulose membrane, respectively. Subsequently, the extracted sample solution was migrated on the membrane. The strip test was allowed to develop red color for 5e10 min. If both the test and control lines turn red, the sample is recorded as negative. When the control line but not the test line was colored, it is considered as positive. 3. Results
immunoassay for detecting FB1 because of the easy preparation of the required size. 2 mL of 1% sodium citrate was added into 100 mL 0.01% chloroauric acid solutions. After boiling, the color of solution was changed to stable wine-red color (Fig. 2A). The result derived from TEM image showed that the average diameter of these particles was approximately 40 nm with well-dispersed colloidal gold particles (Fig. 2B), which provided a good basis for preparation of colloidal gold-antibody conjugates. Different volumes of 0.01 M potassium carbonate were added into colloidal gold solution to determine the optimal pH condition, and the same concentration of the anti-FB1 McAb was added into solution. After incubating the mixture for 5 min, 0.02 mL of 10% NaCl was added into the tubes. The minimum pH keep the red color in tube without change was regarded as the optimal pH, and then further confirmed by absorbance measurement at 525 nm (Fig. 2C). According to the curve, 450 mL of 0.01 M K2CO3 solution was considered as the best condition of solution addition, and the optimal pH is 8.5. The optimal amount of anti-FB1 McAb required to stabilize the colloidal gold was determined just like that of the optimal pH selection. According to the curve drawn from the concentration of anti-FB1 McAb and the absorbance, the optimal concentration of purified anti-FB1 McAb was determined to be 2.0 mL purified anti-FB1 McAb (the concentration of anti-FB1 McAb is 2.7 mg/mL), which was enough to stabilize the colloidal gold, and the color of solution is red (Fig. 2D).
3.1. Purification of anti-FB1 IgG antibody The anti-FB1 IgG antibody (Positive clone 4G5) was collected from ascites, and further purified by affinity chromatography with an immobilized protein G column. SDS-PAGE was preformed to analysis the purified anti-FB1 IgG antibody As shown in Fig. 1, the purified anti-FB1 IgG antibody had a band of heavy chain at 50 kDa and a band of light chain at 27 kDa (Fig. 1A), indicating that the antiFB1 IgG antibody was prepared and purified successfully. The indirect ELISA showed that the titer of purified anti-FB1 IgG antibody secreted by 4G5 hybridoma cells was above 2.56 104 (Fig. 1B). These results indicated that the anti-FB1 McAb had a high titer, and it could be used to further develop the colloidal gold immunoassay for detecting Fumonisin B1. 3.2. Characterization of gold nanoparticles and optimization of test strips Gold nanoparticles were used as a probe in colloidal gold
Fig. 2. Characterization of gold nanoparticles and optimization of test strips. (A) The solution of colloidal gold; (B) The TEM images of gold nanoparticle. (C) Curve drawn from the different concentration of potassium carbonate (K2CO3) and the absorbance; (D) Curve drawn from the different concentration of anti-FB1 McAb and the absorbance.
Fig. 1. SDS-PAGE and titer analysis of purified anti-FB1 IgG antibody secreted by 4G5 hybridoma cells. (A) SDS-PAGE analysis of purified anti-FB1 McAb. Lane M: standard protein markers; Lane 1: the purified McAb of 4G5; Lane 2: Ascites fluid. (B) The titers of ascites and purified anti-FB1 McAb were determined by indirect ELISA.
S. Ling et al. / Toxicon 108 (2015) 210e215
213
Table 1 Results of visual FB1 detection by the test strip in spiked samples. Samples
Different concentration of FB1 (ng/mL)
Visual results (n ¼ 3)
Corn
0 1.25 2.5 5.0 10.0 0 1.25 2.5 5.0 10.0
þ, þ, þ þ, þ, þ ±, ±, ± , , , , þ, þ, þ þ, þ, þ ±, ±, ± , , , ,
Corn kernels
Note: þ means negative result; ± means positive result (the detection limit of colloidal gold strip test for FB1); means positive result. Fig. 3. Colloidal gold immunoassay strip for detecting fumonisin B1. Details were described in text. (A) Positive control: Only the control line appears red color; (B) Negative control: the visual color appear both on the test line and control line; (C) and (D) invalid: no visible red line is present in the control test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3. Preparation of colloidal gold immunoassay test strips Anti-FB1 IgG antibody was applied to develop an effective colloidal gold immunoassay test strip, in which the FB1-KLH conjugates were embedded in the test line to compete with FB1 in the sample solution for limited colloidal gold-antibody conjugate. The description of the colloidal gold strip is shown in Fig. 3. If the concentration of FB1 in sample is sufficient, the gold-labeled anti-FB1 IgG antibody will bind to the FB1 from sample, further preventing any binging to the FB1-KLH conjugates on the test line. Therefore, the resulted test strip showed no red color on the test line, the sample is regarded as the FB1 positive (A). In contrast, if there is no FB1 in sample, the colloidal gold-antibody conjugate was trapped by FB1KLH embedded in the test line, resulted in developing of a visible red color in the test line. Therefore, the sample is defined as the FB1 negative (B). Control test embedded with goat anti-mouse IgG was applied to verify whether the assay has been performed properly, and this control zone should always show a red color under an accurate operation regardless of the presence or the absence of FB1 in the detection samples. If no visible red line is present in the control test, the test strip is considered invalid regardless of whether red line appears in the test area or not (C) and (D).
3.4. Characterization of the test strips Cross-reactivity and sensitivity are very important characters for colloidal gold strips. The cross-reactivity of the test strip was
analyzed as described in the parts of materials and methods. Different toxins including Aflatoxin B1 (AFB1), HT-2 toxin (HT-2), Ochratoxin A (OTA), Ochratoxin B (OTB), T-2 toxin (T-2), Citreoviridin (CIT) were tested. As shown in Fig. 4A, only one red band appeared clearly on the control line if sample containing FB1 toxin. However, the colloidal gold strip test showed red color in control and test line if samples containing other toxins. This result showed that no cross-reactivity was observed, indicating that the colloidal gold strip test had specific binding ability to FB1. To determine the sensitivity of the test strips, various concentrations of FB1 standard solution (2.5e10 ng/mL) were subjected to the test strips respectively. The result showed that the detection limit of the test strip for FB1 was 2.5 ng/mL (Fig. 4B). The whole assay can be completed in less 5 min.
3.5. Detection of spiked and real samples Corn samples were spiked with different concentration of FB1 ranged 0e10 ng/mL, and the colloidal gold test strips were applied to detect the FB1 contamination in corn samples. The extracted sample was migrated onto the membrane. The results in Table 1 showed that the detection limit of colloidal gold strip test for FB1 is 2.5 ng/mL, and the color density of the test lines was significantly reduced when the concentration of FB1 in spiked samples exceeds 2.5 ng/mL. Finally, the colloid gold strip test was used to detect the FB1 contamination in real samples. Four samples purchased from supermarket (corn, corn steamed bread, corn kernels, and corn sheath) were detected by colloidal gold strip after treatment with acetic acid. The result showed that the color density of the test lines just the same as the control line in all samples (Table 2), indicating that there were no detected FB1 (>2.5 ng/mL TTX) in these samples.
Fig. 4. Cross-reactivity and sensitivity of the test strips. (A) The cross-reactivity analysis of the test strips with others toxins, such as Aflatoxin B1 (AFB1), HT-2 toxin (HT-2), Ochratoxin A (OTA), Ochratoxin B (OTB), T-2 toxin (T-2), Citreoviridin (CIT). (B) The sensitivity of the test strips. A series of dilutions (0e10 ng/mL) of standard FB1 were dissolved in PBS. A higher concentration containing more than 2.5 ng/mL of FB1 was found to cause a disappearance of red band on the test line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
214
S. Ling et al. / Toxicon 108 (2015) 210e215
Table 2 Detection result of real samples by colloidal gold immunoassay. Samples
Corn
Corn steamed bread
Corn kernels
Corn sheath
Visual results (n ¼ 3) Content of FB1 (mg/kg)
þ, þ, þa ndb
þ, þ, þ nd
þ, þ, þ nd
þ, þ, þ nd
a b
Note: þ, þ, þ means the color density of the test line is the same as that of the control line, meaning negative result. nd, not detected.
4. Discussion
Ethical statement
Fumonisin B1 (FB1), one of the most common mycotoxins associated with corn and corn-based foods, is mainly produced by Fusarium genus. It is well known that FB1 is toxic and harmful to human and animals, leading to the occurrence of several toxic diseases in animals, especially esophageal cancer in humans. The International Agency for Research on Cancer (IARC) have evaluated the potential of FB1 for carcinogens, and the FB1 was classified as “probable human carcinogen” (class 2B) (Stockmann-Juvala et al., 2008). Therefore, it was necessary to develop a rapid and simple detection method with high specificity and sensitivity to detect the FB1 contamination in real samples. Recently, colloidal gold-based test strip is available for on-site and one-step testing (Chen et al., 2012), which has become a good selected choice to detect FB1 quickly in corn and corn-based foods. A hybridoma cell line named 4G5 generated mAb specifically against FB1 was obtained in our laboratory (Ling et al., 2014). Thus, anti-FB1 McAb with high titer (above 2.56 104) was purified from ascites for further experiment. In immunoassay test device, preparation of high-quality colloid gold solution is a key step to ensure the sensitivity of the test strip, because colloidal gold particles was used as an indicator in test device (Lin et al., 2011; Matsui et al., 2010). Appropriate colloidal gold particles were successfully prepared in the study. The wine-red solution color could be seen by naked eyes and those particles were approximately 40 nm dispersed uniformly under TEM image, which provided a good basis for preparation of colloidal gold-antibody conjugate. The optimal pH and concentrations of anti-FB1 McAb are great important for best strip test. Our study showed that 2.0 mL of purified anti-FB1 McAb (2.7 mg/mL) that added into solution (the optimal pH, 8.5) was enough to stabilize the colloidal gold, and the color of solution is red. The cross-reactivity and sensitivity of test strip was also dependent on the antigen used in immunoassay test device. Fumonisin B1 is a hapten with low small molecular weight (721). To embed the FB1 onto the surface of nitrocellulose membrane, KLH protein was used as a carrier to form the FB1-KLH conjugates. Then, the conjugated FB1-KLH complete antigen can be embedded in the test line, and the goat anti-mouse IgG antibody was embedded in the control line. Our experiment showed that the test strip for onsite FB1 determination was established with a detection limit of 2.5 ng/mL FB1 and no cross-reactivity to other toxins. Compared to instrumental methods such as HPLC and LC-MS, colloidal gold immunoassay strip test could be accomplished within 5 min in only one-step without any equipment, trained operators, and complicated sample preparation. The test strip developed in the study was very simple and rapid for FB1 detection in food samples (Engler et al., 2002; Wang et al., 2014b), indicating a good on-site method for mycotoxins detection.
All animal studies were carried out according to institutional guidelines in Fujian Agriculture and Forestry University (China). All authors declare no conflict of interest statement.
Conflict of interest statement All authors declare that they have no conflict of interest statement.
Acknowledgment This work was supported by the Program from Key Scientific and Technology Project of Fujian Province of China (No. 2014YZ0001-1, 2013Y0004, 2014Y4001), and The Program for New Century Excellent Talents in Fujian Province University (JA13087), and Agricultural Five-new Engineering Projects of Fujian Development and Reform Commission (exploitation on rapid-detection kits for significant mycotoxins). References Antonissen, G., Croubels, S., Pasmans, F., Ducatelle, R., Eeckhaut, V., Devreese, M., Verlinden, M., Haesebrouck, F., Eeckhout, M., Saeger, S.D., Antlinger, B., Novak, B., Martel, A., Immerseel, F.V., 2015. Fumonisins affect the intestinal microbial homeostasis in broiler chickens, predisposing to necrotic enteritis. Vet. Res. 46 (1), 98. Biagini, R.E., Sammons, D.L., Smith, J.P., MacKenzie, B.A., Striley, C.A.F., Snawder, J.E., Robertson, S.A., Quinn, C.P., 2006. Rapid, sensitive, and specific lateral-flow immunochromatographic device to measure anti-anthrax protective antigen immunoglobulin G in serum and whole blood. Clin. Vaccine Immunol. 13 (5), 541e546. Chen, K., Zhao, K., Song, D., He, W.Q., Gao, W., Zhao, C.B., Wang, C.L., Gao, F., 2012. Development and evaluation of an immunochromatographic strip for rapid detection of porcine hemagglutinating encephalomyelitis virus. J. Virol. 9, 172. Engler, K.H., Efstratiou, A., Norn, D., Kozlov, R.S., Selga, I., Glushkevich, T.G., Tam, M., Melnikov, V.G., Mazurova, I.K., Kim, V.E., Tseneva, G.Y., Titov, L.P., George, R.C., 2002. Immunochromatographic strip test for rapid detection of diphtheria toxin: description and multicenter evaluation in areas of low and high prevalence of diphtheria. J. Clin. Microbiol. 40, 80e83. Gazzotti, T., Lugoboni, B., Zironi, E., Barbarossa, A., Serraino, A., Pagliuca, G., 2009. Determination of fumonisin B1 in bovine milk by LCeMS/MS. Food Control 20, 1171e1174. Kadir, M.K.A., Tothill, I.E., 2010. Development of an electrochemical immunosensor for fumonisins detection in foods. Toxins Basel 2 (4), 382e398. Lin, T., Shao, J.J., Du, J.Z., Cong, G.Z., Gao, S.D., Chang, H.Y., 2011. Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease. J. Virol. 8, 418. Ling, S.M., Pang, J., Yu, J.J., Wang, R.Z., Liu, L.C., Ma, Y.L., Zhang, Y.M., Jin, N., Wang, S.H., 2014. Preparation and identification of monoclonal antibody against fumonisin B1 and development of detection by Ic-ELISA. Toxicon 80, 64e72. Liu, L.Q., Luo, L.J., Suryoprabowo, S., Peng, J., Kuang, H., Xu, C.L., 2014. Development of an immunochromatographic strip test for rapid detection of ciprofloxacin in milk samples. Sensors 14 (9), 16785e16798. Matsui, H., Hanaki, H., Inoue, M., Akama, H., Nakae, T., Sunakawa, K., Omura, S., 2010. Development of an immunochromatographic strip for simple detection of penicillin-binding protein 2'. Clin. Vaccine Immunol. 18, 248e253. Mexiasalazar, A., Hernandezlopez, J., Burgoshernandez, A., Cortezrocha, M., Castrolongoria, R., Ezquerrabrauer, J., 2008. Role of fumonisin B1 on the immune system, histopathology, and muscle proteins of white shrimp (Litopenaeus vannamei). Food Chem. 110, 471e479. Pagliuca, G., Zironi, E., Ceccolini, A., Materaa, R., Serrazanetti, G.P., Pivac, A., 2005. Simple method for the simultaneous isolation and determination of fumonisin B1 and its metabolite aminopentol-1 in swine liver by liquid chromatographyefluorescence detection. J. Chromatogr. B 819, 97e103. Quan, Y., Zhang, Y., Wang, S., Lee, N., Kennedyc, I.R., 2006. A rapid and sensitive chemiluminescence enzyme-linked immunosorbent assay for the determination of fumonisin B1 in food samples. Anal. Chim. Acta 580, 1e8. Samapundo, S., De, M.B., De, M.N., Debevere, J., Devlieghere, F., 2006. Influence of experimental parameters on the fluorescence response and recovery of the high-performance liquid chromatography analysis of fumonisin B1.
S. Ling et al. / Toxicon 108 (2015) 210e215 J. Chromatogr. A 1109, 312e316. Stockmann-Juvala, H., Alenius, H., Savolainen, K., 2008. Effects of fumonisin B1 on the expression of cytokines and chemokines in human dendritic cells. Food Chem. Toxicol. 46, 1444e1451. Tardieu, D., Auby, A., Bluteau, C., Bailly, J.D., Guerre, P., 2008. Determination of fumonisin B1 in animal tissues with immunoaffinity purification. J. Chromatogr. B 870, 140e144. Tardieu, D., Bailly, J.D., Benlashehr, I., Auby, A., Jouglar, J.Y., Guerre, P., 2009. Tissue persistence of fumonisin B1 in ducks and after exposure to a diet containing the maximum European tolerance for fumonisins in avian feeds. Chemico Biol.
215
Interact. 182, 239e244. Wang, Y., He, C.H., Zheng, H., Zhang, H.B., 2011. Characterization and comparison of fumonisin B1-protein conjugates by six methods. Int. J. Mol. Sci. 13, 84e96. Wang, Z.Z., Zhi, D.J., Zhao, Y., Zhang, H.L., Wang, X., Ru, Y., Li, H.Y., 2014a. Lateral flow test strip based on colloidal selenium immunoassay for rapid detection of melamine in milk, milk powder, and animal feed. Int. J. Nanomed. 9, 1699e1707. Wang, Y., Wang, L.F., Zhang, J.W., Wang, G.X., Chen, W.B., Chen, L., Zhang, X.L., 2014b. Preparation of colloidal gold immunochromatographic strip for detection of Paragonimiasis skrjabini. Plos One 9, e92034.
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Rapid detection of fumonisin B1 using a colloidal gold immunoassay strip test in corn samples Sumei Ling a, Rongzhi Wang a, Xiaosong Gu a, Can Wen a, Lingling Chen a, Zhibin Chen a, Qing-Ai Chen b, Shiwei Xiao a, Yanling Yang a, Zhenhong Zhuang a, *, Shihua Wang a, * a Key Laboratory of Pathogenic Fungi and Mycotxins of Fujian Province, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China b College of Food Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 July 2015 Received in revised form 16 October 2015 Accepted 20 October 2015 Available online 23 October 2015
Fumonisin B1 (FB1) is the most common and highest toxic of fumonisins species, exists frequently in corn and corn-based foods, leading to several animal and human diseases. Furthermore, FB1 was reported that it was associated with the human esophageal cancer. In view of the harmful of FB1, it is urgent to develop a feasible and accuracy method for rapid detection of FB1. In this study, a competitive immunoassay for FB1 detection was developed based on colloidal gold-antibody conjugate. The FB1-keyhole limpet hemoeyanin (FB1-KLH) conjugate was embedded in the test line, and goat anti-mouse IgG antibody embedded in the control line. The color density of the test line correlated with the concentration of FB1 in the range from 2.5 to 10 ng/mL, and the visual limit detection of test for FB1 was 2.5 ng/mL. The results indicated that the test strip is specific for FB1, and no cross-reactivity to other toxins. The quantitative detection for FB1 was simple, only needing one step without complicated assay performance and expensive equipment, and the total time of visual evaluation was less than 5 min. Hence, the developed colloidal gold-antibody assay can be used as a feasible method for FB1 rapid and quantitative detection in corn samples. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Fumonisin B1 Hybridoma Colloidal gold immunoassay Quantitative detection
1. Introduction Fumonisins, exists frequently in corn and corn-based foods, are produced primarily by Fusarium moniliforme (Pagliuca et al., 2005; Mexiasalazar et al., 2008). FB1 was first discovered in 1988, and it is the most common and highest toxic of fumonisins species, often leading to several animal and human diseases, such as encephalomalacia in horses, pulmonary edema in pigs, and hepatic and renal toxicity in several species (Tardieu et al., 2009). Furthermore, FB1 was reported that it was associated with the human esophageal cancer. Due to its strong toxicity, several measures of regulation and prevention have been made by the Food and Drug Administration (FDA) and Commission Regulation of European Community (EC). For example, FDA has determined the limit of FB1 in different foods, as 2000e4000 mg/kg total fumonisins (FB1 þ FB2 þ FB3) for human
* Corresponding authors. E-mail addresses: [email protected] (Z. Zhuang), [email protected] (S. Wang). http://dx.doi.org/10.1016/j.toxicon.2015.10.014 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
foods, and 5000e10,0000 mg/kg for animal feeds (Wang et al., 2011), and the European Union maximum guidance level for fumonisins is 20 mg/kg for feeds (Antonissen et al., 2015), and 2e4 mg/kg for foods (Kadir and Tothill, 2010). In naturally contaminated samples, approximately 70% of the total detected fumonisins was FB1, especially in corn samples (Samapundo et al., 2006). Therefore, it is very important to develop effective methods to detect and quantify the contamination of FB1 in samples. Recently, several assays have been used for detection FB1 in different samples, such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography (LC) assays and so on. The detection limit of Immunoaffinity Extraction combined with Highperformance Liquid Chromatography (HPLC) with fluorescence for FB1 was 0.013 mg FB1/g in liver, kidney and muscle tissue. The minimum detection level of LC-MS method for FB1 in bovine milk was 0.1 mg/kg (Tardieu et al., 2008; Gazzotti et al., 2009). These methods are sensitive and reliable to determine the FB1 in samples, but these methods need expensive equipment, trained operators, and complicated sample preparation, making them
S. Ling et al. / Toxicon 108 (2015) 210e215
unsuitable for on-site inspection. Up to now, enzyme linked immunosorbent assay (ELISA) with high sensitivity and specificity based on monoclonal and polyclonal antibodies (mAb and pAb) have been widely used to detect FB1 in foods. The Limit detection of enhanced chemiluminescent (ECL)-ELISA method for quantification of FB1 was 0.09 mg/L (Quan et al., 2006). However, ELISA also has some drawback, including time-consuming, tedious washing and incubation steps. Therefore, the detection methods described above did not meet on-site testing. From 1980, colloidal gold immunoassay has been developed to detect the toxins in different samples in one step without other treatment, using a cellulose membrane as the carrier. To solve the problem of rapid assay for FB1 efficiently, a sensitive, specific and simple immunoassay for FB1 detection in corn samples was urgently needed. Our group had successfully obtained one hybrid cell lines 4G5 excreting monoclonal antibody specific against FB1, and then developed Ic-ELISA method for detection (Ling et al., 2014). In this study, colloidal gold immunoassay was established based on this McAb. The purified anti-FB1 McAb was conjugated to the prepared colloidal gold particles, and the formed colloidal gold-labeled IgG antibody was used as the tracer to detect FB1 on a test strip. The strip test can be used for rapid detection of FB1 in corn samples with high specificity and sensitivity, and the process of detection was simple, without any equipment and complicated handling procedures. Hence, the strip test has good application prospect for mycotoxin detection in samples. 2. Materials and methods 2.1. Reagents and buffers Nitrocellulose membranes were obtained from Whatman company (Middlesex, UK). Fumonisin B1 (the purity 98% by HPLC), goat anti-mouse IgG, bovine serum albumin (BSA), Chloroauric acid (HAuCl4), and trisodium citrate were purchased from Shanghai Chemical Reagents (Shanghai, China). All other chemicals were analytical grade and purchased from Beijing Chemical Reagent Co. (Beijing, China). 2.2. Preparation of anti-FB1 IgG antibody Hybridoma cells named 4G5 that secreted anti-FB1 IgG antibody were previously prepared in our laboratory (Ling et al., 2014). After injection of 4G5 hybridoma cells into mice for 2e3 weeks, the produced ascites was collected every other day. The specific IgG antibody was precipitated by ammonium sulfate, then, further purified by affinity chromatography with an immobilized protein G column. The titer of anti-FB1 McAb was determined by ELISA, and the absorbance was measured at 450 nm by Micro-plate reader. The concentration of purified IgG antibody was determined by the instruction of Enhanced BCA Protein Assay Kit. 2.3. Preparation of colloidal gold Colloidal gold particles were produced as described with minor modification (Chen et al., 2012; Biagini et al., 2006). Briefly, 100 mL of 0.01% chloroauric acid solution (HAuCl4) in distilled water was heated to the boiling point, and then 2.0 mL of 1% trisodium citrate solution was quickly added into this solution. After boiling for 5 min, the color of solution was changed from yellow to wine-red, then, allowed to cool the solution gradually to room temperature. Finally, the purified anti-FB1 IgG antibody was added into the solution for conjugation at 4 C for overnight. The conjugates were identified by a transmission electron microscope (TEM), and the absorbance peak of the conjugates was characterized by UVevisible
211
spectra at 400e660 nm. 2.4. Optimization of test strips One milliliter of colloidal gold solution was added separately to the series of 1.5 mL tubes. The pH of colloidal gold solution was adjusted with potassium carbonate. The same concentration of the anti-FB1 IgG antibody diluted with phosphate buffer was added into the solution. After incubating the mixture for 5 min, 0.02 mL of 10% NaCl was added into the tube, and the minimum pH that keep the red color in tube without change was considered as the optimal pH. Different volume of purified anti-FB1 IgG antibody solution (from 0 to 9.5 mL) was added separately to 1 mL of the colloidal gold solution. After incubating the mixture for 5 min, 0.02 mL of 10% NaCl was added to the solution, and the absorbance of solution was measured at 525 nm. The optimal concentration of purified antiFB1 IgG antibody was determined according to the standard curve drawn from the concentration and the absorbance. 2.5. Preparation of colloidal gold-antibody conjugates Colloidal gold solution was adjusted to optimal pH with potassium carbonate. Then, 1 mL of purified anti-FB1 IgG antibody was diluted with phosphate buffer to the optimal concentration, and then added into the 100 mL colloidal gold solution with gentle stirring for 60 min. After reaction, 1 mL of 1% (w/v) BSA solution was dropped into the mixture with gentle stirring for 30 min. The solution was incubated at 4 C for overnight, then, centrifuged at 2000 g for 30 min. The supernatant was centrifuged at 10,000 g for 60 min, and the precipitated colloidal gold-antibody conjugates was re-suspended with conjugate dilution buffer (0.01 M Tris, 5% BSA, 2% sucrose, 0.87% NaCl and 0.1 M sodium azide) and stored at 4 C for further use. 2.6. Preparation of colloidal gold immunoassay test strips Colloidal gold immunoassay test strip consisted of three pads (sample, conjugate, and absorbent pads) and one nitrocellulose (NC) membrane with test and control zones (Liu et al., 2014). The colloid gold strip test device was constructed as follows: As FB1 is a small molecular hapten, it cannot bind directly on the surface of nitrocellulose membrane. So the FB1-KLH conjugate and rabbit anti-mouse IgG antibody were embedded onto nitrocellulose (NC) membrane, respectively. The distance between the two lines was about 5 mm. The treated nitrocellulose membrane was dried at room temperature for 10 min. Subsequently, the absorption pad, nitrocellulose (NC) membrane, conjugation pad, and sample application pad were assembled into a laminated sheet. Finally, the sheet was cut into 5 mm wide strips for further use. If both the test and control lines turn red, the sample is recorded as negative. When the control line but not the test line was colored, it is considered as positive. If no visible red line is present in the control area, the test strip is considered invalid regardless of whether an red line appears in the test area or not. 2.7. Determination of cross-reactivity and sensitivity of the test strips To evaluate the cross-reactivity of the test strips, different toxins including Aflatoxin B1 (AFB1), HT-2 toxin (HT-2), Ochratoxin A (OTA), Ochratoxin B (OTB), T-2 toxin (T-2), Citreoviridin (CIT), were allowed to react with the colloidal goldeFB1 McAb conjugate which was pipetted into glass fiber paper (Wang et al., 2014a). The mixture then moves upward on the nitrocellulose membrane. After incubation at room temperature for 5 min, the detection results
212
S. Ling et al. / Toxicon 108 (2015) 210e215
could be observed by the naked eye. To further determine the sensitivity of the test strips, FB1-KLH conjugates prepared in our laboratory was sprayed to the test zone, and rabbit anti-mouse IgG antibody sprayed to the control zone. In the study, the series concentrations of FB1 toxin from 0 to 10 ng/mL were allowed to react with the colloidal gold-antibody (anti-FB1 IgG antibody) conjugates, and then FB1 test strips results could be observed by the naked eye. 2.8. Assay of FB1 in corn samples Corn related samples, including corn, corn steamed bread, corn kernels, and corn sheath, were purchased and collected from supermarket. After samples treated with methyl alcohol, the test strips were used to detect the FB1 in corn samples. FB1-KLH conjugates prepared in our laboratory was immobilized to the test zone and rabbit anti-mouse IgG antibody immobilized to the control zone on the nitrocellulose membrane, respectively. Subsequently, the extracted sample solution was migrated on the membrane. The strip test was allowed to develop red color for 5e10 min. If both the test and control lines turn red, the sample is recorded as negative. When the control line but not the test line was colored, it is considered as positive. 3. Results
immunoassay for detecting FB1 because of the easy preparation of the required size. 2 mL of 1% sodium citrate was added into 100 mL 0.01% chloroauric acid solutions. After boiling, the color of solution was changed to stable wine-red color (Fig. 2A). The result derived from TEM image showed that the average diameter of these particles was approximately 40 nm with well-dispersed colloidal gold particles (Fig. 2B), which provided a good basis for preparation of colloidal gold-antibody conjugates. Different volumes of 0.01 M potassium carbonate were added into colloidal gold solution to determine the optimal pH condition, and the same concentration of the anti-FB1 McAb was added into solution. After incubating the mixture for 5 min, 0.02 mL of 10% NaCl was added into the tubes. The minimum pH keep the red color in tube without change was regarded as the optimal pH, and then further confirmed by absorbance measurement at 525 nm (Fig. 2C). According to the curve, 450 mL of 0.01 M K2CO3 solution was considered as the best condition of solution addition, and the optimal pH is 8.5. The optimal amount of anti-FB1 McAb required to stabilize the colloidal gold was determined just like that of the optimal pH selection. According to the curve drawn from the concentration of anti-FB1 McAb and the absorbance, the optimal concentration of purified anti-FB1 McAb was determined to be 2.0 mL purified anti-FB1 McAb (the concentration of anti-FB1 McAb is 2.7 mg/mL), which was enough to stabilize the colloidal gold, and the color of solution is red (Fig. 2D).
3.1. Purification of anti-FB1 IgG antibody The anti-FB1 IgG antibody (Positive clone 4G5) was collected from ascites, and further purified by affinity chromatography with an immobilized protein G column. SDS-PAGE was preformed to analysis the purified anti-FB1 IgG antibody As shown in Fig. 1, the purified anti-FB1 IgG antibody had a band of heavy chain at 50 kDa and a band of light chain at 27 kDa (Fig. 1A), indicating that the antiFB1 IgG antibody was prepared and purified successfully. The indirect ELISA showed that the titer of purified anti-FB1 IgG antibody secreted by 4G5 hybridoma cells was above 2.56 104 (Fig. 1B). These results indicated that the anti-FB1 McAb had a high titer, and it could be used to further develop the colloidal gold immunoassay for detecting Fumonisin B1. 3.2. Characterization of gold nanoparticles and optimization of test strips Gold nanoparticles were used as a probe in colloidal gold
Fig. 2. Characterization of gold nanoparticles and optimization of test strips. (A) The solution of colloidal gold; (B) The TEM images of gold nanoparticle. (C) Curve drawn from the different concentration of potassium carbonate (K2CO3) and the absorbance; (D) Curve drawn from the different concentration of anti-FB1 McAb and the absorbance.
Fig. 1. SDS-PAGE and titer analysis of purified anti-FB1 IgG antibody secreted by 4G5 hybridoma cells. (A) SDS-PAGE analysis of purified anti-FB1 McAb. Lane M: standard protein markers; Lane 1: the purified McAb of 4G5; Lane 2: Ascites fluid. (B) The titers of ascites and purified anti-FB1 McAb were determined by indirect ELISA.
S. Ling et al. / Toxicon 108 (2015) 210e215
213
Table 1 Results of visual FB1 detection by the test strip in spiked samples. Samples
Different concentration of FB1 (ng/mL)
Visual results (n ¼ 3)
Corn
0 1.25 2.5 5.0 10.0 0 1.25 2.5 5.0 10.0
þ, þ, þ þ, þ, þ ±, ±, ± , , , , þ, þ, þ þ, þ, þ ±, ±, ± , , , ,
Corn kernels
Note: þ means negative result; ± means positive result (the detection limit of colloidal gold strip test for FB1); means positive result. Fig. 3. Colloidal gold immunoassay strip for detecting fumonisin B1. Details were described in text. (A) Positive control: Only the control line appears red color; (B) Negative control: the visual color appear both on the test line and control line; (C) and (D) invalid: no visible red line is present in the control test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3. Preparation of colloidal gold immunoassay test strips Anti-FB1 IgG antibody was applied to develop an effective colloidal gold immunoassay test strip, in which the FB1-KLH conjugates were embedded in the test line to compete with FB1 in the sample solution for limited colloidal gold-antibody conjugate. The description of the colloidal gold strip is shown in Fig. 3. If the concentration of FB1 in sample is sufficient, the gold-labeled anti-FB1 IgG antibody will bind to the FB1 from sample, further preventing any binging to the FB1-KLH conjugates on the test line. Therefore, the resulted test strip showed no red color on the test line, the sample is regarded as the FB1 positive (A). In contrast, if there is no FB1 in sample, the colloidal gold-antibody conjugate was trapped by FB1KLH embedded in the test line, resulted in developing of a visible red color in the test line. Therefore, the sample is defined as the FB1 negative (B). Control test embedded with goat anti-mouse IgG was applied to verify whether the assay has been performed properly, and this control zone should always show a red color under an accurate operation regardless of the presence or the absence of FB1 in the detection samples. If no visible red line is present in the control test, the test strip is considered invalid regardless of whether red line appears in the test area or not (C) and (D).
3.4. Characterization of the test strips Cross-reactivity and sensitivity are very important characters for colloidal gold strips. The cross-reactivity of the test strip was
analyzed as described in the parts of materials and methods. Different toxins including Aflatoxin B1 (AFB1), HT-2 toxin (HT-2), Ochratoxin A (OTA), Ochratoxin B (OTB), T-2 toxin (T-2), Citreoviridin (CIT) were tested. As shown in Fig. 4A, only one red band appeared clearly on the control line if sample containing FB1 toxin. However, the colloidal gold strip test showed red color in control and test line if samples containing other toxins. This result showed that no cross-reactivity was observed, indicating that the colloidal gold strip test had specific binding ability to FB1. To determine the sensitivity of the test strips, various concentrations of FB1 standard solution (2.5e10 ng/mL) were subjected to the test strips respectively. The result showed that the detection limit of the test strip for FB1 was 2.5 ng/mL (Fig. 4B). The whole assay can be completed in less 5 min.
3.5. Detection of spiked and real samples Corn samples were spiked with different concentration of FB1 ranged 0e10 ng/mL, and the colloidal gold test strips were applied to detect the FB1 contamination in corn samples. The extracted sample was migrated onto the membrane. The results in Table 1 showed that the detection limit of colloidal gold strip test for FB1 is 2.5 ng/mL, and the color density of the test lines was significantly reduced when the concentration of FB1 in spiked samples exceeds 2.5 ng/mL. Finally, the colloid gold strip test was used to detect the FB1 contamination in real samples. Four samples purchased from supermarket (corn, corn steamed bread, corn kernels, and corn sheath) were detected by colloidal gold strip after treatment with acetic acid. The result showed that the color density of the test lines just the same as the control line in all samples (Table 2), indicating that there were no detected FB1 (>2.5 ng/mL TTX) in these samples.
Fig. 4. Cross-reactivity and sensitivity of the test strips. (A) The cross-reactivity analysis of the test strips with others toxins, such as Aflatoxin B1 (AFB1), HT-2 toxin (HT-2), Ochratoxin A (OTA), Ochratoxin B (OTB), T-2 toxin (T-2), Citreoviridin (CIT). (B) The sensitivity of the test strips. A series of dilutions (0e10 ng/mL) of standard FB1 were dissolved in PBS. A higher concentration containing more than 2.5 ng/mL of FB1 was found to cause a disappearance of red band on the test line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
214
S. Ling et al. / Toxicon 108 (2015) 210e215
Table 2 Detection result of real samples by colloidal gold immunoassay. Samples
Corn
Corn steamed bread
Corn kernels
Corn sheath
Visual results (n ¼ 3) Content of FB1 (mg/kg)
þ, þ, þa ndb
þ, þ, þ nd
þ, þ, þ nd
þ, þ, þ nd
a b
Note: þ, þ, þ means the color density of the test line is the same as that of the control line, meaning negative result. nd, not detected.
4. Discussion
Ethical statement
Fumonisin B1 (FB1), one of the most common mycotoxins associated with corn and corn-based foods, is mainly produced by Fusarium genus. It is well known that FB1 is toxic and harmful to human and animals, leading to the occurrence of several toxic diseases in animals, especially esophageal cancer in humans. The International Agency for Research on Cancer (IARC) have evaluated the potential of FB1 for carcinogens, and the FB1 was classified as “probable human carcinogen” (class 2B) (Stockmann-Juvala et al., 2008). Therefore, it was necessary to develop a rapid and simple detection method with high specificity and sensitivity to detect the FB1 contamination in real samples. Recently, colloidal gold-based test strip is available for on-site and one-step testing (Chen et al., 2012), which has become a good selected choice to detect FB1 quickly in corn and corn-based foods. A hybridoma cell line named 4G5 generated mAb specifically against FB1 was obtained in our laboratory (Ling et al., 2014). Thus, anti-FB1 McAb with high titer (above 2.56 104) was purified from ascites for further experiment. In immunoassay test device, preparation of high-quality colloid gold solution is a key step to ensure the sensitivity of the test strip, because colloidal gold particles was used as an indicator in test device (Lin et al., 2011; Matsui et al., 2010). Appropriate colloidal gold particles were successfully prepared in the study. The wine-red solution color could be seen by naked eyes and those particles were approximately 40 nm dispersed uniformly under TEM image, which provided a good basis for preparation of colloidal gold-antibody conjugate. The optimal pH and concentrations of anti-FB1 McAb are great important for best strip test. Our study showed that 2.0 mL of purified anti-FB1 McAb (2.7 mg/mL) that added into solution (the optimal pH, 8.5) was enough to stabilize the colloidal gold, and the color of solution is red. The cross-reactivity and sensitivity of test strip was also dependent on the antigen used in immunoassay test device. Fumonisin B1 is a hapten with low small molecular weight (721). To embed the FB1 onto the surface of nitrocellulose membrane, KLH protein was used as a carrier to form the FB1-KLH conjugates. Then, the conjugated FB1-KLH complete antigen can be embedded in the test line, and the goat anti-mouse IgG antibody was embedded in the control line. Our experiment showed that the test strip for onsite FB1 determination was established with a detection limit of 2.5 ng/mL FB1 and no cross-reactivity to other toxins. Compared to instrumental methods such as HPLC and LC-MS, colloidal gold immunoassay strip test could be accomplished within 5 min in only one-step without any equipment, trained operators, and complicated sample preparation. The test strip developed in the study was very simple and rapid for FB1 detection in food samples (Engler et al., 2002; Wang et al., 2014b), indicating a good on-site method for mycotoxins detection.
All animal studies were carried out according to institutional guidelines in Fujian Agriculture and Forestry University (China). All authors declare no conflict of interest statement.
Conflict of interest statement All authors declare that they have no conflict of interest statement.
Acknowledgment This work was supported by the Program from Key Scientific and Technology Project of Fujian Province of China (No. 2014YZ0001-1, 2013Y0004, 2014Y4001), and The Program for New Century Excellent Talents in Fujian Province University (JA13087), and Agricultural Five-new Engineering Projects of Fujian Development and Reform Commission (exploitation on rapid-detection kits for significant mycotoxins). References Antonissen, G., Croubels, S., Pasmans, F., Ducatelle, R., Eeckhaut, V., Devreese, M., Verlinden, M., Haesebrouck, F., Eeckhout, M., Saeger, S.D., Antlinger, B., Novak, B., Martel, A., Immerseel, F.V., 2015. Fumonisins affect the intestinal microbial homeostasis in broiler chickens, predisposing to necrotic enteritis. Vet. Res. 46 (1), 98. Biagini, R.E., Sammons, D.L., Smith, J.P., MacKenzie, B.A., Striley, C.A.F., Snawder, J.E., Robertson, S.A., Quinn, C.P., 2006. Rapid, sensitive, and specific lateral-flow immunochromatographic device to measure anti-anthrax protective antigen immunoglobulin G in serum and whole blood. Clin. Vaccine Immunol. 13 (5), 541e546. Chen, K., Zhao, K., Song, D., He, W.Q., Gao, W., Zhao, C.B., Wang, C.L., Gao, F., 2012. Development and evaluation of an immunochromatographic strip for rapid detection of porcine hemagglutinating encephalomyelitis virus. J. Virol. 9, 172. Engler, K.H., Efstratiou, A., Norn, D., Kozlov, R.S., Selga, I., Glushkevich, T.G., Tam, M., Melnikov, V.G., Mazurova, I.K., Kim, V.E., Tseneva, G.Y., Titov, L.P., George, R.C., 2002. Immunochromatographic strip test for rapid detection of diphtheria toxin: description and multicenter evaluation in areas of low and high prevalence of diphtheria. J. Clin. Microbiol. 40, 80e83. Gazzotti, T., Lugoboni, B., Zironi, E., Barbarossa, A., Serraino, A., Pagliuca, G., 2009. Determination of fumonisin B1 in bovine milk by LCeMS/MS. Food Control 20, 1171e1174. Kadir, M.K.A., Tothill, I.E., 2010. Development of an electrochemical immunosensor for fumonisins detection in foods. Toxins Basel 2 (4), 382e398. Lin, T., Shao, J.J., Du, J.Z., Cong, G.Z., Gao, S.D., Chang, H.Y., 2011. Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease. J. Virol. 8, 418. Ling, S.M., Pang, J., Yu, J.J., Wang, R.Z., Liu, L.C., Ma, Y.L., Zhang, Y.M., Jin, N., Wang, S.H., 2014. Preparation and identification of monoclonal antibody against fumonisin B1 and development of detection by Ic-ELISA. Toxicon 80, 64e72. Liu, L.Q., Luo, L.J., Suryoprabowo, S., Peng, J., Kuang, H., Xu, C.L., 2014. Development of an immunochromatographic strip test for rapid detection of ciprofloxacin in milk samples. Sensors 14 (9), 16785e16798. Matsui, H., Hanaki, H., Inoue, M., Akama, H., Nakae, T., Sunakawa, K., Omura, S., 2010. Development of an immunochromatographic strip for simple detection of penicillin-binding protein 2'. Clin. Vaccine Immunol. 18, 248e253. Mexiasalazar, A., Hernandezlopez, J., Burgoshernandez, A., Cortezrocha, M., Castrolongoria, R., Ezquerrabrauer, J., 2008. Role of fumonisin B1 on the immune system, histopathology, and muscle proteins of white shrimp (Litopenaeus vannamei). Food Chem. 110, 471e479. Pagliuca, G., Zironi, E., Ceccolini, A., Materaa, R., Serrazanetti, G.P., Pivac, A., 2005. Simple method for the simultaneous isolation and determination of fumonisin B1 and its metabolite aminopentol-1 in swine liver by liquid chromatographyefluorescence detection. J. Chromatogr. B 819, 97e103. Quan, Y., Zhang, Y., Wang, S., Lee, N., Kennedyc, I.R., 2006. A rapid and sensitive chemiluminescence enzyme-linked immunosorbent assay for the determination of fumonisin B1 in food samples. Anal. Chim. Acta 580, 1e8. Samapundo, S., De, M.B., De, M.N., Debevere, J., Devlieghere, F., 2006. Influence of experimental parameters on the fluorescence response and recovery of the high-performance liquid chromatography analysis of fumonisin B1.
S. Ling et al. / Toxicon 108 (2015) 210e215 J. Chromatogr. A 1109, 312e316. Stockmann-Juvala, H., Alenius, H., Savolainen, K., 2008. Effects of fumonisin B1 on the expression of cytokines and chemokines in human dendritic cells. Food Chem. Toxicol. 46, 1444e1451. Tardieu, D., Auby, A., Bluteau, C., Bailly, J.D., Guerre, P., 2008. Determination of fumonisin B1 in animal tissues with immunoaffinity purification. J. Chromatogr. B 870, 140e144. Tardieu, D., Bailly, J.D., Benlashehr, I., Auby, A., Jouglar, J.Y., Guerre, P., 2009. Tissue persistence of fumonisin B1 in ducks and after exposure to a diet containing the maximum European tolerance for fumonisins in avian feeds. Chemico Biol.
215
Interact. 182, 239e244. Wang, Y., He, C.H., Zheng, H., Zhang, H.B., 2011. Characterization and comparison of fumonisin B1-protein conjugates by six methods. Int. J. Mol. Sci. 13, 84e96. Wang, Z.Z., Zhi, D.J., Zhao, Y., Zhang, H.L., Wang, X., Ru, Y., Li, H.Y., 2014a. Lateral flow test strip based on colloidal selenium immunoassay for rapid detection of melamine in milk, milk powder, and animal feed. Int. J. Nanomed. 9, 1699e1707. Wang, Y., Wang, L.F., Zhang, J.W., Wang, G.X., Chen, W.B., Chen, L., Zhang, X.L., 2014b. Preparation of colloidal gold immunochromatographic strip for detection of Paragonimiasis skrjabini. Plos One 9, e92034.