Development and Evaluation of an Enzyme-Linked Immunosorbent Assay Using a Nonpoisonous Extraction System for the Determination of Crustacean Protein in Processed Foods

Crustacean proteins are food allergens that cause severe allergic reactions in patients with food allergies; therefore, the identification of crustaceans such as shrimp, crab, and lobster as ingredients in processed food products is mandatory in Japan. We previously developed and validated an ELISA method coupled with an extraction process using the surfactant sodium dodecyl sulfate and the reductant 2-mercaptoethanol (2-ME) to quantify crustacean protein. However, 2-ME was designated as poisonous in Japan in 2008. Therefore, in this study, we developed and evaluated an ELISA method for detecting and quantifying crustacean protein that uses sodium sulfite (Na2SO3) in place of 2-ME for extraction. The proposed ELISA method showed high sensitivity, with an LOQ of 0.66 mu g protein/g food sample. Furthermore, the proposed method showed high specificity for the Decapoda order within the subphylum Crustacea, with recoveries ranging from 83.8 to 100.8% for model processed foods, as well as high reproducibility (infra- and interassay CVs of <= 8.2%) and high correlation with our previously validated ELISA method for processed foods (correlation coefficient of 0.996). The proposed ELISA method does not require the use of poisonous reagents, provides acceptable accuracy, and is useful for the routine monitoring of food products.