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.

Immunomagnetic Separation Combined with Polymerase Chain Reaction for the Detection of Alicyclobacillus acidoterrestris in Apple Juice

“…Although the recovery proportion of IMS was actually lost with the concentration of bacteria more than 10 4 CFU/mL, the total numbers of isolated bacteria cells still increased and could meet the requirement of detection. Furthermore, high numbers of A. acidoterrestris would never be encountered in commercialized apple juice, and when the concentration was above 10 5 CFU/mL, the juice samples can be detected directly by PCR, ELISA or other methods [4,9,19]. …”

“…IMPs were prepared by immobilizing the specific polyclonal anti- Alicyclobacillus IgG antibody onto the surface of the amino-silane modified magnetic nanoparticles (SMNPs) as recommended in our previous study [4,16]. The obtained IMPs were resuspended in 0.01 mol/L phosphate buffer sodium (PBS, pH 7.4) plus 5% bovine serum albumin (BSA) and 0.05 % Tween-20 and stored at 4 °C for use.…”

“…As it can survive in highly acidic environments of pH 2.5-6.0 at temperatures of 25-60 °C, and its spore can germinate and proliferate in acidic fruit juices or beverages, A. acidoterrestris is extremely difficult to be inactivated completely by conventional pasteurization [4,5]. A. acidoterrestris -related spoilage is characterized by the formation of medicinal or antiseptic off-odors.…”

“…To expeditiously detect this bacterium in food samples, a number of rapid methods have been developed, including polymerase chain reaction (PCR), Enzyme-linked immunosorbent assay (ELISA), Fourier transform infrared (FT-IR), electronic nose and 16S rDNA gene sequence analysis [4,9–11]. However, the complex background interference of the food components and the non-target flora has a negative impact on the detection of target bacteria [7,12].…”

Wash-free colorimetric homogeneous immunoassay for Zygosaccharomyces rouxii

“…The antigens were stored at À20 C until use. 18 Preparation of immunogold colorimetric probe Antibody production. Immunization was performed according to reported methods.…”