Optimisation of indirect competitive ELISAs of a-, b-, and k-caseins for the recognition of thermal and proteolytic treatment of milk and milk products

Polyclonal antibodies were raised against six immunogens (three native and three thermally treated casein fractions: a+b-casein, k-casein and whole casein). Using these antibodies the procedures of an indirect competitive enzyme immunoassay (ELISA) were constructed, optimised and characterised for determination of each immunogen. It was found that ELISA of caseins is very specific without any interferences of whey proteins and with proportionally low cross-interactions between caseins. Detection limits for a-, b-, and k-caseins and whole casein were 110, 49, 58 and 505 ng/ml, respectively. The coefficient of variation was lower than 12% in intra-assay and lower than 9% in inter-assay. The developed ELISA format was used to study changes in casein immunoreactivity during heat-treatment and proteolytic hydrolysis. Heating below 100°C did not change the milk immunoreactivity but heating above 100°C caused its significant changes. Depending on type of proteolytic treatment (with enzyme preparation Pancreatin or with microbial cultures of Lactobacillus helveticus and Lactococcus) a decrease and an increase in casein immunoreactivity were observed. While Pancreatin reduced the casein immunoreactivity substantially (5-1000 times in dependence on the casein type), the more gentle proteolysis by bacteria caused not only its reduction (even 100 times at k-casein) but also its increase (1.5 times at a-casein).