Development and evaluation of enzyme-linked immunosorbent assay for detection of molds in dairy products
Abstract
A double-sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting molds (Mucor circinelloides, Geotrichum candidum, Aspergillus versicolor, Penicillium chrysogenum and Cladosporium herbarum) in cheeses and yogurt. The detection limits for M. circinelloides, A. versicolor, P. chrysogenum, and C. herbarum were 1 $\mu$g dried mold/ml, and for G. candidum 1 ng dried mold/ml. Cross-reaction and competitive inhibition tests were done using forty-four different strains of molds and yeasts. The ELISAs for Mucor, Cladosporium, and Geotrichum were specific for the same genus; but Aspergillus and Penicillium showed cross-reactions between their antibodies and antigens. Citrate buffer gave the best recovery of molds in cheeses and yogurt. Although casein in cheeses and yogurt decreased ELISA responses for detecting molds, positive relationships between ELISA readings and mold concentrations were observed. When mold was inoculated into cheeses and yogurt and incubated at 5$\sp\circ$C, it could be detected within 4 to 7 days depending on the mold and its generation time. Antigens from the cultural filtrates, mycelia, and spores of molds were partially purified and characterized. The molecular weights for these antigens, as determined by size exclusion chromatography, ranged from 4.5 to 6.7 x 10$\sp5$ daltons. These antigens contained 13.3 to 82.6% neutral sugars, 8.3 to 50% protein, and some of them contained phosphate from 0.28 to 2%. D-Glucose, D-mannose and D-galactose were the main sugars for the antigens from Geotrichum, Aspergillus, Penicillium,, and Cladosporium. Mucor antigens contained these sugars plus L-fucose. The percentage of sugars differed among the mycelial, extracellular, and conidial antigens. Various enzymes were used to evaluate the immunodominant groups in these antigens. Protein was part of the active site. D-Mannose was the immunologically active sugar for Mucor antigens; however, D-glucosyl residues containing $\alpha$-1-4 and $\beta$-1-4 linkages to the D-mannosyl residues were also important. D-Galactosyl residues with $\beta$ linkages were immunodominant for Geotrichum, Aspergillus, Penicillium, and Cladosporium antigens. However, Geotrichum, and Cladosporium antigens differed from each other and from Aspergillus and Penicillium antigens. D-Galactose was also the immunologically active sugar in the conidial antigen from Penicillium species; however, it differed from the mycelial antigens for Penicillium species. The competitive inhibition tests using different D-glucose, D-mannose, and D-galactose derivatives further confirmed the results from the enzyme assays.
Degree
Ph.D.
Advisors
Cousin, Purdue University.
Subject Area
Food science|Agricultural chemicals
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