Development of an immunoradiometric assay for quantitative determination of CrylA(b) protein in transgenic sugarcane plants.
An immunoradiometric assay (IRMA) system was performed to quantify the recombinant CrylA(b) protein produced by transgenic sugarcane lines. The method allowed detection of 0.1-1 ng CrylA(b) per 25 micrograms of soluble protein in leaf extracts from plants transformed with an expression vector containing a truncated version of the CrylA(b) gene from Bacillus thuringiensis. The technique was based upon the use of radioiodinated immunopurified antibodies specific to natural CrylA proteins in a one-step sandwich procedure by direct simultaneous incubation of the leaf extracts with the detecting antibody solution. This IRMA system provides a simple routine method to quantify the CrylA proteins in transgenic plants with different expression levels. We suggest that the methodology presented herein may become an efficient tool to quantify heterologous or native plant proteins, present at low levels in tissue extracts.
Vázquez
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Plant Division, Centre for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba. griva@serverdos.cigb.edu.cu
Prieto
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De la Riva
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Selman-Housein
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D001413:Bacillus thuringiensis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D001427:Bacterial Toxins; D004731:Endotoxins / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D006460:Hemolysin Proteins; D015592:Immunoradiometric Assay / Q000379:methods*; D010936:Plant Extracts / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D030821:Plants, Genetically Modified / Q000737:chemistry* / Q000276:immunology*; D006109:Poaceae / Q000235:genetics* / Q000276:immunology
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D010936:Plant Extracts; C030956:insecticidal crystal protein, Bacillus Thuringiensis