Enzyme-Linked Immunosorbent Assay for Detection of Molds in Cheese and Yogurt

Abstract

An enzyme-linked immunosorbent assay was developed for the detection of molds in dairy products. New Zealand White female rabbits were immunized with .45 mg of partially purified extracellular antigen from freeze-dried culture filtrates of Aspergillus versicolor, Cladosporium herbarum, Geotrichum candidum, Mucor circinelloides, and Penicillium chrysogenum. Blood was drawn at various intervals, and antibodies were separated and purified. Antibody-peroxidase conjugates were prepared with the following ratios being the optimum ones: A. versicolor 1020; C. herbarum 5: 10; G. candidum 1:10; M. circinelloides 5:5; and P. chrysogenum 10:10. The assays were sensitive within a range of 1 ng to 1 μg/ml, depending on the mold used. Inhibition tests were done for each mold with concentrations of 0 to 5000 μg/ml of antigen. The enzyme-linked immunosorbent assay tests for Cladosporium, Geotrichum, and Mucor were only inhibited by antigens from other species of the same genus; whereas there was crossreaction between antibodies and antigens of species of Penicillium and of Aspergillus. Citrate buffer was best for extracting the mold from cheese and yogurt. The extract was adjusted to pH 7.2 and ELISA was performed. Results showed that these molds can be detected in Cheddar and cottage cheeses and yogurt within 2 d, which is before mold growth is visible in these products.