Determination of Cry9C Protein in Corn-Based Foods by Enzyme-Linked Immunosorbent Assay: Interlaboratory Study

Abstract

The performance of a commercially available enzyme-linked immunosorbent assay kit (Enviro-Logix) was assessed for the determination of Cry9C protein, which is produced by the genetically modified corn StarLink, in 8 types of corn-based foods (starch, refined oil, soft tortillas, tortilla chips, corn flakes, corn puffs, corn muffins, and corn bread) in an interlaboratory study involving 7 laboratories in the United States. The assay kit is a double antibody sandwich and is based on the specific interaction between antibody and antigen. The Cry9C protein analyte is sandwiched between 2 antibodies, one to capture the analyte and the other is conjugated to the enzyme, horseradish peroxidase. The enzyme uses tetramethylbenzidine/peroxide for color development. A strong acid stopping reagent is then used to change the color from blue to a stable yellow. The intensity of the color is proportional to the concentration of the Cry9C protein. In this study blind duplicates of control samples (blank material prepared from non- StarLink corn), spiked samples (blank material with the addition of Cry9C protein), and samples containing incurred analyte (products prepared with StarLink corn) were analyzed. Cry9C protein from 2 different sources was used to spike the food products. Cry9C protein produced and purified from a bacterial host was used to prepare spiked test samples at 2.72 and 6.8 ng/g. Cry9C protein from StarLink corn flour was used to prepare spiked samples at 1.97 ng/g. Average recoveries for samples spiked with corn flour Cry9C protein at 1.97 ng/g ranged from 73 to 122%, within-laboratory relative standard deviations (RSDr) ranged from 6 to 22%, and between-laboratories relative standard deviations (RSDR) ranged from 16 to 56%. Average recoveries for samples spiked with bacterial Cry9C protein at 2.72 and 6.8 ng/g ranged from 27 to 96% and from 32 to 113%, respectively; RSDr values ranged from 10 to 35% and from 7 to 38%, respectively; and the RSDR ranged from 28 to 84% and 15 to 75%, respectively. The incurred test samples were found to contain Cry9C protein at levels ranging from 0.8 to 3187 ng/g depending on the product, RSDr values ranged from 5 to 16% and RSDR values ranged from 11 to 71%. Results of the statistical analysis indicate that this method is applicable to the determination of Cry9C protein in the 8 types of collaboratively studied corn-based products containing Cry9C protein (from StarLink) at levels of > or =2 ng/g.