The Validation of the Wheat Gluten ELISA Kit.

Background: It is important to analyze the presence of wheat/gluten in food to avoid wheat allergy or celiac disease. Objective: The Wheat/Gluten ELISA kit was developed to measure total wheat protein or gluten content in wheat, barley, and rye cereals as raw materials, and processed foods. Validation as to whether this kit is suitable for quantifying total wheat protein/gluten was carried out. Methods: The Wheat/Gluten ELISA kit was designed as a sandwich ELISA based on antigliadin polyclonal antibody. Selectivity, interference study, matrix study including incurred food, robustness, stability, and lot-to-lot consistency studies were conducted for the Wheat/Gluten ELISA kit. Incurred matrix studies were also conducted in an independent laboratory. Results: The analysis of 38 different substances revealed no cross-reactivity above the LOQ except for oats. Recoveries of the spiked samples were mostly in the range of 75-140%, including an independent laboratory result. The LOD of the ELISA was found to be 0.02-0.16 mg/kg. Robustness testing proved that extraction time and incubation time of first reaction and enzyme reaction had no significant influence on quantified value. The stability at 2-8°C was found to exceed 12 months. Good lot-to-lot consistency was observed. Conclusions: The Wheat/Gluten ELISA kit showed good analytical performance in the quantitative analysis of total wheat protein/gluten in the identified food products using the AOAC Performance Tested Method(s)SM program. Highlights: The Wheat/Gluten ELISA kit was validated and showed good analytical performance in the quantitative analysis of total wheat protein/gluten in food.

Interlaboratory evaluation of two enzyme-linked immunosorbent assay kits for the detection of egg, milk, wheat, buckwheat, and peanut in foods.

The labeling of 5 major allergenic ingredients (egg, milk, wheat, buckwheat, and peanut) is mandatory in Japan, and 2 series of enzyme-linked immunosorbent assay (ELISA) kits have been established as official screening methods. However, these official methods have not provided the necessary sensitivity, due in part to poor extraction efficiency. To address this need, 2 novel ELISA kits have been developed: the FASTKIT ELISA Ver. II Series and the FASPEK Allergenic Substances Detection Kit. The new kit systems use an improved extraction buffer that can extract insoluble proteins produced by processing and feature new antibodies that bind to the denatured proteins extracted with the new extraction buffer. The analytical performances of the 2 new ELISA kit series were evaluated in an interlaboratory study. Ten laboratories participated in the study and determined the major allergenic ingredients contained in 5 types of model processed food. The 2 ELISAs displayed fairly good reproducibility and sufficient recovery.

Novel ELISA for the detection of raw and processed egg using extraction buffer containing a surfactant and a reducing agent.

Enzyme-linked immunosorbent assay (ELISA) has been considered extremely useful for the detection of markers of allergenic substances in food, because it is simple, offers a suitable sensitivity, and is useful in providing quantitative results. Allergenic protein present in processed food can be denatured or altered, hindering therefore their possibility to be extracted and detected. This paper reports the development of an ELISA method that can be used for the determination of allergenic proteins in buffer solutions containing SDS, a surfactant, and 2-mercaptoethanol, a reducing agent. Measurement by ELISA in solutions containing 1% SDS and 7% 2-mercaptoethanol has been made possible by using an antibody prepared through immunization with an antigen denatured with SDS and 2-mercaptoethanol. This ELISA technique can be used to measure proteins in food that have been denatured by various manufacturing processes. An example is egg white albumin, which is susceptible to heat denaturation and has been difficult to recover from food in the past. Its recovery was improved 10- to 100-fold by the new ELISA method as compared with previous methods. This means that allergenic substances in food can now be detected quantitatively. This method can be very useful in allergy prevention and control strategies.