Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco |
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Authors: | D borah Geada, Rodolfo Vald s, Arturo Escobar, Dulce M. Ares, Edel Torres, Reinaldo Blanco, Williams Ferro, Dayamí Dorta, Marcos Gonz lez, Marí a R. Alem n, Sigifredo Padilla, Leonardo G mez, Norma del Castillo, Otto Mendoza, Dioslaida Urquiza, Yordanka Soria, Jos Brito, Alberto Leyva, Carlos Borroto,Jorge V. Gavilondo |
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Affiliation: | 2. Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba;4. Center for Genetic Engineering and Biotechnology, Circunv. Norte, P.O. Box 60200, Sancti Spíritus, Cuba |
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Abstract: | Antibodies have been one of the proteins widely expressed in tobacco plants for pharmaceutical purposes, which demand contaminant free preparations. Rubisco constitutes 40-60% of tobacco leaf soluble proteins; therefore it is the major potential protein contaminant of plantibodies, while mycotoxins are toxic compounds that could be introduced during the biomass production and post-harvest stages with important consequences to human health. The objective of this paper was to investigate whether Rubisco and mycotoxins are present in Plantibody HB-01 preparations used in the immunopurification of the hepatitis B surface antigen. Rubisco was purified from Nicotiana tabacum yielding 154 microg of protein per gram of leaves and purity over 95%. Among mouse monoclonal antibodies generated against this enzyme, the CBSS.Rub-2 was selected for its immunodetection. It recognizes a conserved sequential epitope of Rubisco large subunit with an affinity constant of 0.13 x 10(8)M(-1). Rubisco quantification limit was 1 microg spreading to the measurement of this contaminant less than 4% of plantibodies samples. Additionally, according to a Reverse Phase-HPLC used to measure the level of adventitiously introduced contaminants, it can be concluded that aflatoxins B1, B2, G1 and G2 were undetected in the purified Plantibody HB-01 samples. |
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Keywords: | Aflatoxins Monoclonal antibody Nicotiana tabacum Plantibody Rubisco |
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