Purpose: The objectives of this study are to confirm a thermal-stable soluble protein (TSSP) in peanut, and to develop and characterize monoclonal antibodies (MAbs) specific to peanut using TSSP as an immunogen.
Methods: TSSP in peanut was extracted by non-heating and heating treatment and confirmed by SDS-PAGE. TSSP was used as an immunogen, and the mice showing high titer were used for cell fusion and cloning. The characterization of MAbs produced from hybridoma cells obtained were performed by indirect ELISA and Western blot.
Results: SDS-PAGE protein pattern with raw peanut extracted by non-heating treatment showed major protein bands at 100 kDa, 65 kDa and 10 to 40 kDa, whereas the roasted, boiled peanuts and peanut butter extracts obtained by heating treatments showed major bands at 65 kDa and 10 to 40 kDa. Six mice were immunized with roasted or boiled peanut extracts obtained by heating treatment, and four MAbs (RP 5F9-23, RP 6G4-30, RP 4C12-10 and BP 3A1-12) were developed. The RP 5F9-23, RP 6G4-30 and BP 3A1-12 MAbs were confirmed to be a specific MAb to peanut without cross-reaction to other nuts and food allergen, whereas the RP 4C12-10 MAb was reacted soybean in the indirect ELISA and Western blot analyses. The ELISA based on the MAbs assay can sensitively detect 0.005 % peanut protein.
Significance: These results support that the MAbs developed can be sufficiently used as a bio-receptor in the development of immunoassay for the simple and rapid detection of peanut in food products.