An indirect ELISA system for the detection of heat-stable Pseudomonas endopeptidases (AprX) in milk
نویسندگان
چکیده
Heat-stable endopeptidases in raw milk, especially the alkaline metallopeptidase AprX secreted by Pseudomonas spp., are a well-known challenge for dairy industry. They can withstand UHT treatment and may cause quality defects over shelf life of milk products. Therefore, we established an indirect ELISA detection milk. We developed 2-step sample contaminated with to avoid interference proteins system. First, casein micelles were destabilized detraction Ca2+ using trisodium citrate; then, was concentrated 10-fold hydrophobic interaction chromatography. The recovery spiked samples after 43 ± 0.1%. Specific antibodies purified from lactis produced establish ELISA. Western blot experiments showed that binding affinity these depended on sequence homology P. several other spp. ELISA, which completed 6 7 h, had limit 21.0 ng mL?1 quantification 25.7 mL?1. Milk or endogenous peptidases not detected antibodies. high precision, CV between 0.2 0.8% measured same day (intraday) 5.6 6.8% 5 separate days (interday). different activity levels [7.5–150 nkat Na-caseinate/o-phthalaldehyde (OPA) mL?1] evaluated 92.3 1.6 105 4.7%. lowest quantifiable 500 pkat Na-caseinate/OPA proof concept detect heat-stable established.
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ژورنال
عنوان ژورنال: Journal of Dairy Science
سال: 2021
ISSN: ['0022-0302', '1525-3198', '1529-9066']
DOI: https://doi.org/10.3168/jds.2020-19790