Yu-Rong Zhou1,2,†, Chao-Nan He1,†, Geng Ni1, Xiaodong Zheng1, Wen-Wen Zhou1,*
1College of Biosystems Engineering and Food Science, Fuli Institute of Food Science, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang University, Hangzhou 310058, Zhejiang, China.
2School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu, China.
Crystal proteins (Cry) from Bacillus thuringiensis (Bt) are well-known pesticidal proteins in genetically modified (GM) crops and often used as the target proteins in detection of GM food. Instead of the classic mouse hybridoma, new rabbit monoclonal antibodies (McAb) were first produced in detection of GM food. Rabbit McAb and polyclonal antibodies (PcAb) were prepared using Bt-Cry1Ac protein as the immune antigen (Ag). Sandwich ELISA with either PcAb as the coating antibody and peroxidase horseradish (HRP) labeled McAb as the detecting antibody (PcAb-Ag-McAb), or McAb as the coating antibody and HRP labeled PcAb as the detecting antibody (McAb-Ag-PcAb), was developed for detecting Bt-Cry1Ac protein. The analytical sensitivity of McAb-Ag-PcAb was higher with the linear detection range of 0.97-62.50 ng mL-1 and the limit of detection (LOD) of 0.24 ng mL-1. There was no cross-reaction with Cry1Ab and Cry1c proteins and the mean recovery was 103.35% in corn leaves. The LOD and the linear detection range of the proposed ELISA are more satisfactory than other ELISA systems using an anti-Cry1Ac PcAb or two Cry1Ac-specific McAb for detection of the target protein and the research provides an effective instrument for detecting Cry1Ac protein in GM crops and their derivatives.
Cry1Ac; Sandwich ELISA; Rabbit monoclonal antibody; Genetically modified food; Detection
Yu-Rong Zhou, Chao-Nan He, Geng Ni, Xiaodong Zheng, Wen-Wen Zhou. A novel sandwich ELISA system using rabbit monoclonal and polyclonal antibodies for rapid detection of Bt-Cry1Ac protein. International Journal of New Developments in Engineering and Society (2019) Vol.3, Issue 3: 25-39. https://doi.org/10.25236/IJNDES.19303.
Allen, R. C., Rogelj, S., Cordova, S. E., & Kieft, T. L. (2006). An immuno-PCR method for detecting Bacillus thuringiensis Cry1Ac toxin. Journal of Immunological Methods 308:109-115.
Asensio, L., Gonzalez, I., Garcia, T., & Martin, R. (2008). Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA). Food Control 19:1-8.
Berdal, K. G., & Holst, J. A. (2001). Roundup Ready soybean event-specific real-time quantitative PCR assay and estimation of the practical detection and quantification limits in GMO analyses. European Food Research and Technology 213: 432-438.
Chaouachi, M., Chupeau, G., Berard, A., Mckhann, H., Romaniuk, M., Giancola, S., Laval, V., Bertheau, Y., & Brunel, D. (2008). A high-throughput multiplex method adapted for GMO detection. Journal of Agricultural and Food Chemistry 56:11596-11606.
Dong, W., Yang, L., Shen, K., Kim, B., Kleter, G. A., Marvin, H. J. P., Guo, R., Liang, W., & Zhang, D. B. (2008). GMDD: a database of GMO detection methods. BMC Bioinformatics 9:260.
Feng, L., Wang, X., & Jin, H. (2011). Rabbit monoclonal antibody: potential application in cancer therapy. American Journal of Translational Research 3:269-274.
Gruber, H., Paul, V., Meyer, H. D., & Muller, M. (2008). Validation of an enzyme immunoassay for monitoring Cry1Ab toxin in soils planted with Bt-maize (MON810) in a long-term field trial on four South German sites. Journal für Verbraucherschutz und Lebensmittelsicherheit, 3: 22-25.
Guertler, P., Paul, V., Albrecht, C., & Meyer, H. H. D. (2009). Sensitive and highly specific quantitative real-time PCR and ELISA for recording a potential transfer of novel DNA and Cry1Ab protein from feed into bovine milk. Analytical and Bioanalytical Chemistry 393:1629-1638.
Harry, A. K., Esther, J. K., & Karl, H. E. (2003). Exploitation of molecular profiling techniques for GM food safety assessment. Current Opinion in Biotechnology 14:238-243.
Hori, H., Takahashi, Y., Takahashi, M., & Wada, Y. (2000). Detection of the Bacillus thuringiensis serovar japonenis strain Buibui protoxin with enzyme-linked immunosorbent assay and its application to detection of the protoxin in soil. Applied Entomology and Zoology 35:401-441.
Huang, B., Yin, Y., Lu, L., Ding, H., Wang, L., Yu, T., Zhu, J., Zheng, X., & Zhang, Y. (2010). Preparation of high-affinity rabbit monoclonal antibodies for ciprofloxacin and development of an indirect competitive ELISA for residues in milk. Journal of Zhejiang University Science B 11: 812-818.
Liang, J., Wu, Y., Liu, C., Cao, Y., Liu, J., & Lin, Y. (2017). Preparation of high stable core/shell magnetic nanoparticles and application in Bacillus thuringiensis Cry1Ac proteins detection. Sensor and Actuators B:Chemical 241:758-764.
Liu, N., Han, Z., Lu, L., Wang, L., Ni, G., Zhao, Z., Wu, A., & Zheng, X. (2013). Development of a new rabbit monoclonal antibody and its based competitive indirect enzyme-linked immunosorbent assay for rapid detection of sulfonamides. Journal of the Science of Food and Agriculture 93:667-673.
Margarit, E., Reggiardo, M. I., Vallejos, R. H., & Permingeat, H. R. (2006). Detection of BT transgenic maize in foodstuffs. Food Research International 39:250-255.
Markoulatos, P., Siafakas, N., Papathoma, A., Nerantzis, E., Betzios, B., Dourtoglou, V., & Moncany, M. (2004). Qualitative and quantitative detection of protein and genetic traits in genetically modified food. Food Research International 20:275-296.
Nelson, P. N., Reynolds, G. M., Waldron, E. E., Ward, E., Giannopoulos, K., & Murray, P. G. (2000). Monoclonal antibodies. Journal of Clinical Pathology-Molecular Pathology 53:111-117.
Paul, V., Steinke, K., & Meyer, H. H. D. (2008). Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810). Analytica Chimica Acta 607:106-113.
Querci, M., Bulcke, M. V., Zel, J., Eede, V. G., & Broll, H. (2010). New approaches in GMO detection. Analytical and Bioanalytical Chemistry 396:1991-2002.
Shan, G., Embrey, S. K., & Schafer, B. W. (2007). A highly specific enzyme-linked immunosorbent assay for the detection of Cry1Ac insecticidal crystal protein in transgenic WideStrike cotton. Journal of Agricultural and Food Chemistry 55:5974-5979.
Shelton, A. M., Zhao, J. Z., & Roush, R. T. (2002). Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Annual Review of Entomology 47:845-881.
Sheng, W., Yang, L., Wang, J., Zhang, Y., & Wang, S. (2013). Development of an enzyme-linked immunosorbent assay for the detection of gentamycin residues in animal-derived foods. LWT-Food Science and Technology 50:204-209.
Szekacs, A., Lauber, E., Takacs, E., & Darvas, B. (2010). Detection of Cry1Ab toxin in the leaves of MON 810 transgenic maize. Analytical and Bioanalytical Chemistry 396:2203-2211.
Takahashi, Y., Hori, H., Furuno, H., Kawano, T., Takahashi, M., & Wada, Y. (1998). Enzyme-linked immunosorbent assays for rapid and quantitative detection of insecticidal crystal proteins of Bt pesticides. Journal of Pesticide Science 23:386-391.
Tapp, G., & Stotzky, G. (1995). Dot blot enzyme-linked immunosorbent assay for monitoring the fate of insecticidal toxins from Bacillus thuringiensis in soil. Applied and Environmental Microbiology 61:602-609.
Walschus, U., Witt, S., & Wittmann, C. (2002). Development of monoclonal antibodies against Cry1Ab protein from Bacillus thuringiensis and their application in an ELISA for detection of transgenic Bt-Maize. Food and Agricultural Immunology 14:231-240.
Wang, S., Guo, A. Y., Zheng, W. J., Zhang, Y., Qiao, H., & Kennedy, I. R. (2007). Development of ELISA for the determination of transgenic Bt-cotton using antibodies against Cry1Ac Protein from Bacillus thuringiensis HD-73. Engineering in Life Sciences 7:149-154.
Wang, Y. H., Li, G. C., & Zhou, X. F. (2005). Antibody theory and technology. Science Press Inc., Beijing, China, 90-137.
Zhong, J., Hu, X., Zhang, X., Liu, Y., Xu, C., Zhang, C., Lin, M., & Liu, X. (2018). Broad specificity immunoassay for detection of Bacillus thuringiensis Cry toxins through engineering of a single chain variable fragment with mustagenesis and screening. International Journal of Biological Macromolecules 107:920-928.
Zhu, X., Chen, L., Shen, P., Jia, J., Zhang, D. B., & Yang, L. (2011). High sensitive detection of Cry1Ab protein using a quantum dot-based fluorescence-linked immunosorbent assay. Journal of Agricultural and Food Chemistry 59:2184-2189.