The Use of Chicken Igy in a Double Antibody Sandwich Elisa for the Quantification of Melittin in Bee Venom and Bee Venom Melittin Content in Cosmetics

Abstract

Abstract Two enzyme-linked immunosorbent assay (ELISA) - based detection systems: indirect competitive ELISA and biotinylated double antibody sandwich ELISA (DAS-ELISA) were developed to determine the melittin concentration in honeybee (Apis mellifera) venom and the melittin concentration in cosmetics which contain bee venom. The indirect competitive ELISA employed chicken anti-melittin IgY. The biotinylated DAS-ELISA employed anti-melittin monoclonal antibody (MAb) and biotinylated anti-melittin IgY. To produce anti-melittin IgY; Sigma melittin was emulsified with Freund‘s incomplete adjuvant and immunised to Leghorn laying chickens intramuscularly at four different sites (50 μg/mL, 0.25 mL per site) of the breast muscles. After 5 to 8 weeks of the immunisation, anti-melittin IgY was extracted and analysed by ELISA. The anti-melittin IgY antibody produced was highly specific to melittin and did not cross-react with other bee venom proteins, as examined by ELISA and a western-blot assay. Indirect competitive ELISA demonstrated a higher range of melittin detection (2.5 to 80 μg/mL). Double antibody sandwich ELISA using MAb as the capture antibody and biotinylated polyclonal IgY as the detection antibody, provided a lower range of detection (2.5 - 40 ng/mL), which has a 1000 times higher sensitivity than that of indirect competitive ELISA. Therefore, indirect competitive ELISA is a useful tool to measure the concentration of melittin in bee venom as a raw material. Biotinylated DAS-ELISA, on the other hand, is more suitable for nanoscale quantification of melittin in commercial products.