title: Validation of an extraction method for Cry1Ab protein from soil.
abstract: Corn expressing insecticidal proteins derived from Bacillus thuringiensis (Bt corn) has increased in usage in the United States from 8% of total corn acreage in 1996 to 67% in 2012. Because of this increase, it is important to be able to monitor the fate and transport of the insecticidal Bt proteins to evaluate environmental exposure and effects. Accurate and validated methods are needed to quantify these proteins in environmental matrices. A method to extract Bt Cry1Ab proteins from 3 soil types using a 10× phosphate-buffered saline with Tween buffer and a commercially available enzyme-linked immunosorbent assay (ELISA) was validated through a series of 6 tests. The validation process for Cry1Ab extractions in soil has not yet been reported in the scientific literature. The extraction buffer and each soil matrix were tested and validated for the ELISA. Extraction efficiencies were 41%, 74%, and 89% for the 3 soil types and were significantly correlated with the organic matter content of the soil. Despite low recoveries, consistent results with low coefficients of variation allowed for accurate measurements. Through validating this method with 3 different soils, a sensitive, specific, precise, and accurate quantification of Bt Cry1Ab was developed. The validation process can be expanded and implemented in other environmental matrices, adding consistency to data across a wide range of samples.
authors.affiliation: Center for Fisheries, Aquaculture, and Aquatic Sciences and Department of Zoology, Southern Illinois University, Carbondale, Illinois, USA.
authors.forename: Sara A
authors.initials: SA
authors.lastname: Mueting
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Strain
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Lydy
mesh_terms: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D004731:Endotoxins / Q000737:chemistry / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D019015:Geologic Sediments / Q000737:chemistry; D006460:Hemolysin Proteins / Q000737:chemistry / Q000302:isolation & purification*; D007087:Illinois; D010710:Phosphates / Q000737:chemistry; D011136:Polysorbates / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D045483:Rivers; D012965:Sodium Chloride / Q000737:chemistry; D012987:Soil / Q000737:chemistry; D012989:Soil Pollutants / Q000737:chemistry / Q000302:isolation & purification*; D013501:Surface-Active Agents / Q000737:chemistry; D014197:Trees; D003313:Zea mays
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D010710:Phosphates; D011136:Polysorbates; D017680:Potassium Compounds; D012987:Soil; D012989:Soil Pollutants; D013501:Surface-Active Agents; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D012965:Sodium Chloride; C013216:potassium phosphate; C018279:sodium phosphate
keywords: Bacillus thuringiensis; Cry1Ab; Enzyme-linked immunosorbent assay; Method validation; Transgenic research