Establishment of Dipstick Development Technology for Detection of Cry1Ac in Transgenic Plants

Establishment of Dipstick Development Technology for Detection of Cry1Ac in Transgenic Plants ( Vol-2,Issue-6,November - December 2017 )

Author: Muhammad Irfan, Muhammad Asif, Aftab Bashir, Kauser Abdullah Malik

ijeab doi crossref DOI: 10.22161/ijeab/2.6.3

Keyword: Nitrocellulose membrane, Cry1Ac, Dipstick, Nano-colloidal gold particles.

Abstract: The insecticidal Bt Cry1Ac protein is, currently used for transgene expression in numerous crops or deliberating resistance against lepidopteron pests. Since the introduction of Bt cotton in Pakistan. It has been demonstrated that the technology has achieved the goal of providing an effective tool for lepidopteron control. In this study, single step, sensitive and specific dipstick strip test for the revealing of recombinant Cry1Ac protein in the transgenic plants was established. Anti-Bt-Cry1Ac antibodies and goat anti-rabbit IgG antibodies were used in test and control lines, respectively. The distance betweenthese lines were optimized as 0.5 cm. Polyclonal rabbit anti Bt-Cry1Ac antibody conjugated to nanocolloidal gold (20 nm of OD 15 and 40 nm; OD 1 in separate experiments) at pH 9.2 was used to serve as a probe for detecting Cry1Ac protein in transgenic Bt cotton samples. Both conjugate solutions were coated on separate polyester conjugate pads (0.7 cm × 0.5 cm). The total size of strip was 7.5 cm × 0.5 cm. For 20 nm gold conjugated strip, purple color test line and for 40 nm gold red color test line indicated the binding of gold labeled antibodies to antigen. The assay was corroborated with transgenic cotton samples with protein extraction buffer 1X PBS of pH 7. This on-site test offers fast screening for any genetically modified crop devouring Cry1Ac transgenic protein.


[1] Brada, D., & Roth, J. (1984). “Golden blot”—Detection of polyclonal and monoclonal antibodies bound to antigens on nitrocellulose by protein A-gold complexes. Analytical biochemistry, 142(1), 79-83.
[2] Campbell, R. L., Wagner, D. B., & O'connell, J. P. (1987). Solid phase assay with visual readout: Google Patents.
[3] Chandler, J., Gurmin, T., & Robinson, N. (2000). The place of gold in rapid tests. IVD technology, 6(2), 37-49.
[4] Fren, G. (1973). Preparation of gold dispersions of varying particle size: Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature Physics, 241, 20-22.
[5] Frens, G. (1973). Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature, 241(105), 20-22.
[6] Grothaus, G. D., Bandla, M., Currier, T., Giroux, R., Jenkins, G. R., Lipp, M., . . . Pantella, V. (2006). Immunoassay as an analytical tool in agricultural biotechnology. Journal of AOAC international, 89(4), 913-928.
[7] Gupta, A. K., & Chandak, V. (2005). Agricultural biotechnology in India: ethics, business and politics. International Journal of Biotechnology, 7(1-3), 212-227.
[8] Hansen Jesse, L. C., & Obrycki, J. J. (2000). Field deposition of Bt transgenic corn pollen: lethal effects on the monarch butterfly. Oecologia, 125(2), 241-248.
[9] Henderson, K., & Stewart, J. (2002). Factors influencing the measurement of oestrone sulphate by dipstick particle capture immunoassay. Journal of immunological methods, 270(1), 77-84.
[10] Herring, R. J. (2008). Opposition to transgenic technologies: ideology, interests and collective action frames. Nature Reviews Genetics, 9(6).
[11] Herring, R. J. (2008). Whose numbers count? Probing discrepant evidence on transgenic cotton in the Warangal district of India. International Journal of Multiple Research Approaches, 2(2), 145-159.
[12] Holst-Jensen, A. (2008). GMO testing—trade, labeling or safety first? Nature biotechnology, 26(8), 858-859.
[13] James, C. (2010). A global overview of biotech (GM) crops: adoption, impact and future prospects. GM crops, 1(1), 8-12.
[14] Jones, K. D. (1999). Troubleshooting protein binding in nitrocellulose membranes, Part 1: Principles. IVD Technol, 5(2), 32-41.
[15] Katrukha, A. G., Bereznikova, A. V., Esakova, T. V., Pettersson, K., Lövgren, T., Severina, M. E., . . . Gusev, N. B. (1997). Troponin I is released in bloodstream of patients with acute myocardial infarction not in free form but as complex. Clinical chemistry, 43(8), 1379-1385.
[16] Kumar, K. S., & Kang, S. H. (2007). Ultra‐fast simultaneous analysis of genetically modified organisms in maize by microchip electrophoresis with LIF detector. Electrophoresis, 28(22), 4247-4254.
[17] Leimanis, S., Hernandez, M., Fernandez, S., Boyer, F., Burns, M., Bruderer, S., . . . Philipp, P. (2006). A microarray-based detection system for genetically modified (GM) food ingredients. Plant Molecular Biology, 61(1), 123-139.
[18] Li, Y., Hou, L., Ye, J., Liu, X., Dan, H., Jin, M., Cao, S. (2010). Development of a convenient immunochromatographic strip for the diagnosis of infection with Japanese encephalitis virus in swine. Journal of virological methods, 168(1), 51-56.
[19] Lin, T., Shao, J.-j., Du, J.-z., Cong, G.-z., Gao, S.-d., & Chang, H. (2011). Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease. Virology journal, 8(1), 418.
[20] May, K., Prior, M. E., & Richards, I. (1997). Capillary immunoassay and device therefor comprising mobilizable particulate labelled reagents: Google Patents.
[21] McCabe, A. F., Eliasson, C., Prasath, R. A., Hernandez-Santana, A., Stevenson, L., Apple, I., . . . Corish, P. (2006). SERRS labelled beads for multiplex detection. Faraday discussions, 132, 303-308.
[22] McCabe, A. F., Eliasson, C., Prasath, R. A., Hernandez-Santana, A., Stevenson, L., Apple, I., . . . Corish, P. (2006). SERRS labelled beads for multiplex detection. Faraday Discuss., 132(0), 303-308.
[23] Ponti, J. S. (2009). Material platform for the assembly of lateral flow immunoassay test strips Lateral Flow Immunoassay (pp. 1-7): Springer.
[24] Samra, Z. Q., Aslam, M. S., Shaukat, H., Dar, N., & Athar, M. A. (2007). Development of diagnostic dip strip immunoassay using antibodies of PreS~ 2 region of hepatitis B surface antigen. PAKISTAN JOURNAL OF ZOOLOGY, 39(3), 185.
[25] Singer, J. M., & Plotz, C. M. (1956). The latex fixation test:: I. Application to the serologic diagnosis of rheumatoid arthritis. The American journal of medicine, 21(6), 888-892.
[26] Takahashi, A., Uchiyama, S., Kato, Y., Yuhi, T., Ushijima, H., Takezaki, M., . . . Miyahara, T. (2009). Immunochromatographic assay using gold nanoparticles for measuring salivary secretory IgA in dogs as a stress marker. Science and technology of advanced materials, 10(3), 034604.
[27] Thobhani, S., Attree, S., Boyd, R., Kumarswami, N., Noble, J., Szymanski, M., & Porter, R. A. (2010). Bioconjugation and characterisation of gold colloid-labelled proteins. Journal of Immunological Methods, 356(1-2), 60-69.
[28] Vermij, P. (2006). Liberty Link rice raises specter of tightened regulations: Nature Publishing Group.
[29] Xu, Y., Huang, Z.-B., He, Q.-H., Deng, S.-Z., Li, L.-S., & Li, Y.-P. (2010). Development of an immunochromatographic strip test for the rapid detection of deoxynivalenol in wheat and maize. Food chemistry, 119(2), 834-839.
[30] Yu, C. Y., Ang, G. Y., Chua, A. L., Tan, E. H., Lee, S. Y., Falero-Diaz, G., . . . Acosta, A. (2011). Dry-reagent gold nanoparticle-based lateral flow biosensor for the simultaneous detection of Vibrio cholerae serogroups O1 and O139. Journal of microbiological methods, 86(3), 277-282.

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