Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco.
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.
Geada
Déborah
D
Tobacco Research Institute, Tumbadero Road 8.5 km, San Antonio de los Baños, Havana, La Havana, Cuba. dgeadalop@yahoo.com.mx
Valdés
Rodolfo
R
Escobar
Arturo
A
Ares
Dulce M
DM
Torres
Edel
E
Blanco
Reinaldo
R
Ferro
Williams
W
Dorta
Dayamí
D
González
Marcos
M
Alemán
María R
MR
Padilla
Sigifredo
S
Gómez
Leonardo
L
Del Castillo
Norma
N
Mendoza
Otto
O
Urquiza
Dioslaida
D
Soria
Yordanka
Y
Brito
José
J
Leyva
Alberto
A
Borroto
Carlos
C
Gavilondo
Jorge V
JV
D000348:Aflatoxins / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000706:statistics & numerical data; D004340:Drug Contamination; D004797:Enzyme-Linked Immunosorbent Assay; D006510:Hepatitis B Antibodies / Q000235:genetics / Q000302:isolation & purification*; D006514:Hepatitis B Surface Antigens / Q000276:immunology; D006801:Humans; D051379:Mice; D046549:Plantibodies / Q000235:genetics / Q000302:isolation & purification*; D030821:Plants, Genetically Modified; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis*; D014026:Nicotiana / Q000235:genetics / Q000276:immunology
D000348:Aflatoxins; D000911:Antibodies, Monoclonal; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D046549:Plantibodies; D012273:Ribulose-Bisphosphate Carboxylase