Enzyme-linked immunosorbent assay for quantitation of neomycin phosphotransferase II in genetically modified cotton tissue extracts

• Published in: Journal of agricultural and food chemistry Vol. 40; no. 8; pp. 1453 - 1458
• Main Authors: Rogan, Glen J, Ream, Joel E, Berberich, Sharon A, Fuchs, Roy L
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.08.1992
• Subjects: Agronomy. Soil science and plant productions; ANALISIS CUANTITATIVO; ANALYSE QUANTITATIVE; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biotechnology; ELISA; Enzymes and enzyme inhibitors; EXTRAIT TISSULAIRE; Fundamental and applied biological sciences. Psychology; Genetic engineering applications; Genetics and breeding of economic plants; Gossypium; GOSSYPIUM HIRSUTUM; Invertebrates; MUESTRA DE TEJIDO; NEOMICINA; NEOMYCINE; Plant breeding: fundamental aspects and methodology; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; TEST ELISA; Transferases; Malvaceae; Seeds; Gossypium; Bacillaceae; Fiber crop; Immunological method; Assay; Tissue extract; Bacillus thuringiensis var. kurstaki; Bacillales; Dicotyledones; Angiospermae; Bacteria; Transgenic plant; Quantitative analysis; Method study; Plant tissue; Biochemical analysis; Enzyme; Transferase; Plant leaf; Analysis method; Plant protein; Spermatophyta; ELISA assay
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf00020a033

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed and validated to accurately quantitate neomycin phosphotransferase II (NPTII) levels in genetically modified cotton seed and leaf tissue. This assay provides a safer and more efficient alternative than the existing methods to measure NPTII levels in cotton tissues from genetically modified plants that contain NPTII as a selectable marker. Parallelism studies using purified bacterially-expressed NPTII and plant-expressed NPTII in the ELISA established that these proteins are immunologically and conformationally equivalent. Data on sensitivity, accuracy, and precision substantiated the utility of this ELISA for analysis of NPTII in genetically modified plant tissue. NPTII was extracted from cotton tissue using an aqueous buffer providing maximum recovery of NPTII and minimizing tissue interference. Expression levels of NPTII measured in three different genetically modified cotton lines ranged from 0.040 to 0.044% and from 0.009 to 0.019% of extractable protein for leaf and seed extracts, respectively