Development of Polyclonal Antibody‐Based Indirect Enzyme‐Linked Immunosorbent Assay for the Detection of Alicyclobacillus Strains in Apple Juice

Abstract: A sort of specific polyclonal anti‐ Alicyclobacillus antibody was generated by immunizing New Zealand white rabbits, and a sensitive indirect enzyme‐linked immunosorbent assay (ELISA) was developed for Alicyclobacillus detection in apple juice. A set of experimental parameters such as concentration of antigen, dilutions of the antibody and goat anti‐rabbit IgG‐horseradish peroxidase conjugate, selection of the blocking reagent, incubation time, and temperature was optimized. The cross‐reactivity of the antibody was evaluated by ELISA and the result was consistent with Western blot analysis. The detection limit of the ELISA was about 10 5 colony forming units (CFU)/mL in apple juice samples. Samples were detected by ELISA and conventional culture method, and the ELISA results gave a good agreement with the results obtained by plating on Alicyclobacillus acidoterrestris medium agar. ELISA takes a total detection time of 6 to 7 h, which is less than the time of conventional techniques requiring more than 24 to 48 h. These results indicated that the established ELISA was a potential useful analytical method for detection of Alicyclobacillus in apple juice.