The goal of this proposed research is to provide a convenient and reliable analytical tool for the enforcement of the new Food Allergen Labeling and Consumer Protection Act in order to protect fish-allergic consumers from ingestion of undeclared fish allergen. The research will develop two types of monoclonal antibody (Mab)-based immunoassays for the detection of a) finfish ingredient and b) the already identified major fish allergen (parvalbumin) present in fresh and heat-processed food. This will be accomplished by, firstly, identifying a suitable biomarker protein present in all finfish as the antigen (analyte) and characterize the parvalbumin, followed by developing Mabs against these two thermal-stable fish marker proteins, and lastly, developing rapid immunoassays for sensitive detection of these fish marker proteins in raw and processed foods.<P>
Specific Objectives: <OL> <LI> To identify and characterize heat-stable marker common proteins or peptides in all food fish species <LI> To characterize and purify the major fish allergen, parvalbumin <LI> To develop Mabs against a) the selected fish marker protein (FMP) identified from Specific Objective 1, as well as b) fish parvalbumin. <LI>To develop Mab-based immunoassays for the detection of a) fish ingredients and b) fish parvalbumin, in both fresh and processed foods with desired specificity, sensitivity and robustness. from Specific Objective 1, as well as b) fish parvalbumin.</OL>
Expected Outcomes: Upon the completion of this project, firstly, new fish TSMPs will have been explored. Information obtained from this study on TSMPs in fish tissue will add to our understanding of the biological, biochemical, and physiological implications of these proteins. Secondly, panels of Mabs against the marker proteins for the detection of either fish tissue or the fish allergen will be produced. Once developed, it will also be possible to employ these Mabs, either individually or collectively, in a wide range of immunochemical systems such as lateral flow strip tests, 96-well microplate assays, magnetic beads, immunosensors, and microarray chips for biological and physiological studies of the target fish proteins or allergens, and for a wide range of other detection purposes. While characterizing each Mab, the experimental conditions for each assay will be optimized, thus facilitating subsequent immunoassay development. Lastly, sandwich ELISA incorporating two selected Mabs will be developed. Based on our earlier experience with the development of Mab-based ELISAs, we expect the detection limit of the analysis to achieve at a level of 0.1% (w/w) of the target material, regardless of the degree of heat treatment. This research is both demanding and labor-intensive.
NON-TECHNICAL SUMMARY: This proposal addresses the program (71.1) priority #2 and fits the program's long term goals to develop new technologies to produce better foods and promote health. Fish allergy is an important health issue in the U.S. and around the world. Undeclared fish as contamination or intentional adulteration into food products are a major risk for fish allergic consumers. There is a need for improved control methods that will prevent the introduction of undeclared fish allergens into the food supply. The Food Allergen Labeling and Consumer Protection Act of 2004, effective January 1, 2006, requires labeling of major eight allergenic foods or food types that cause most allergic reactions. Fish is one of the "Big 8" allergen food. Currently no methods are available for the detection of the presence of fish protein or any fish allergen in processed food, this project will develop fish-specific immunoassays to meet the urgent need for food allergen labeling law enforcement. The specific objectives and approaches of the proposal are to 1) identify a heat-stable marker protein (TSMP) in all finfish as the antigen; 2) characterize and purify the already identified major fish allergen, parvalbumin; 3) develop finfish-specific monoclonal antibodies (Mabs) against the identified TSMP and paralbumin; and 3) develop and validate the two types of finfish-specific immunoassays using the Mabs developed. Based on the heat-stable analytes used, this assay will enable the detection of fish allergen or contaminants in either raw or processed food materials. This research will provide a convenient tool to protect fish-allergic consumers from ingestion of undeclared fish allergen.
<P>APPROACH: 1). Identify and characterize a heat-stable common marker protein: To examine the thermal-stable proteins (TSPs), we will first heat each fish sample flesh with or without pressure for 30 minutes. Soluble proteins will be extracted from tissues from all fish species collected. The protein banding profiles of the samples will be evaluated by SDS-PAGE and isoelectric focusing (IEF). A fish marker protein (FMP) will be selected based on its molecular size (>10 kDa to ensure the antigenicity) and the presence and even distribution in all fish species. This selected FMP will be further characterized by 2-D gel electrophoresis, eluted from the gel, dialyzed, and then stored at -20C for future use as the immunogen for Mab development.<P>
2) Characterize and purify the major fish allergen, parvalbumin: We will isolate both and paralbumin isoforms from yellowfin tuna and salmon and characterize them by comparing their quantity, molecular weight, PI., and isoforms with paralbumin purified from other selected fish species by SDS-PAGE, IEF and 2-D electrophoresis. A previously developed anti-parvalbumin Mab 3E1 will be tested against each of these purified isoforms using indirect and competitive ELISAs and Western blot. In addition, the salmon paralbumin will be used as the immunogen for new anti-parvalbumin Mab development. Only the positive hybridoma clones that secrete Mabs reacted with all or most fish species will be selected. Information about the presence of parvalbumin in various fish will be revealed. <P>
3). Develop Mabs against a) the selected FMP as well as against b) the purified parvalbumin: The selected FMP and the purified tuna parvalbumin contain both á and â isoforms, will be used as immunogens for the development of Mabs. Selection of hybridomas will be achieved by cloning and further screening cloned hybridomas with the same antigen from various fish and other animal species as well as protein additives commonly used in foods using indirect ELISA. Only those clones that secrete Mabs without cross-reactivity will be stabilized for further testing. The antigenic proteins will be confirmed using Wester blot. Mabs showing the desired specificity (fish tissue-specific or paralbumin-specific) will be selected to develop an assay<P>
4) Develop Mab-based immunoassays for the detection of a) fish ingredients and b) fish parvalbumin: The optimal dilution of each Mab will be determined by two-dimensional titrations using indirect ELISA. Other conditions that need to be optimized include pH for coating, blocking materials, sample extraction and dilution, and incubation time. We will select a pair of suitable Mabs in order to develop a sandwich ELISA for each target antigen by conjugating one Mab to biotin as the detecting Mab and coating the other on the microtiter plates as the capturing Mab. Laboratory formulated samples spiked with known amounts of fish tissue in pure meat samples will be tested. The effects of factors such as fat content, matrix effect, heat treatment conditions, and storage duration of both the laboratory formulated and commercial food samples will be examined to assess the performance of the final assay.