title: Laboratory and field validation of a Cry1Ab protein quantitation method for water.
abstract: The widespread planting of crops expressing insecticidal proteins derived from the soil bacterium Bacillus thuringiensis (Bt) has given rise to concerns regarding potential exposure to non-target species. These proteins are released from the plant throughout the growing season into soil and surface runoff and may enter adjacent waterways as runoff, erosion, aerial deposition of particulates, or plant debris. It is crucial to be able to accurately quantify Bt protein concentrations in the environment to aid in risk analyses and decision making. Enzyme-linked immunosorbent assay (ELISA) is commonly used for quantitation of Bt proteins in the environment; however, there are no published methods detailing and validating the extraction and quantitation of Bt proteins in water. The objective of the current study was to optimize the extraction of a Bt protein, Cry1Ab, from three water matrices and validate the ELISA method for specificity, precision, accuracy, stability, and sensitivity. Recovery of the Cry1Ab protein was matrix-dependent and ranged from 40 to 88% in the validated matrices, with an overall method detection limit of 2.1 ng/L. Precision among two plates and within a single plate was confirmed with a coefficient of variation less than 20%. The ELISA method was verified in field and laboratory samples, demonstrating the utility of the validated method. The implementation of a validated extraction and quantitation protocol adds consistency and reliability to field-collected data regarding transgenic products.
authors: Center for Fisheries, Aquaculture, and Aquatic Sciences and Department of Zoology, Southern Illinois University Carbondale, Carbondale, IL 62901, USA. Electronic address: katie.strain@siu.edu.
authors: Katherine E
authors: KE
authors: Strain
authors: Center for Fisheries, Aquaculture, and Aquatic Sciences and Department of Zoology, Southern Illinois University Carbondale, Carbondale, IL 62901, USA. Electronic address: saraannwhiting@gmail.com.
authors: Sara A
authors: SA
authors: Whiting
authors: Center for Fisheries, Aquaculture, and Aquatic Sciences and Department of Zoology, Southern Illinois University Carbondale, Carbondale, IL 62901, USA. Electronic address: mlydy@siu.edu.
authors: Michael J
authors: MJ
authors: Lydy
mesh_terms: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D018556:Crops, Agricultural / Q000235:genetics / Q000378:metabolism*; D004731:Endotoxins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D004784:Environmental Monitoring / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D060587:Groundwater / Q000032:analysis / Q000737:chemistry; D006460:Hemolysin Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D030821:Plants, Genetically Modified; D055550:Protein Stability; D015203:Reproducibility of Results; D045483:Rivers / Q000737:chemistry; D012987:Soil / Q000737:chemistry; D013696:Temperature; D014873:Water Pollutants / Q000032:analysis; D062066:Water Resources / Q000032:analysis*; D014881:Water Supply / Q000032:analysis
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D012987:Soil; D014873:Water Pollutants; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Bacillus thuringiensis; Cry1Ab; ELISA; Method validation; Water