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

Abstract

VÍTKOVÁ M., RAUCH P., FUKAL L. (2002): Optimisation of indirect competitive ELISAs of α-, β-, and κ-caseins for the recognition of thermal and proteolytic treatment of milk and milk products. Czech J. Food Sci., 20: 53-62.Polyclonal antibodies were raised against six immunogens (three native and three thermally treated casein fractions: α+β-casein, κ-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 α-, β-, and κ-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 κ-casein) but also its increase (1.5 times at α-casein).

Electrochemical immunosensor for the determination of β-casein

“…The concentration of β-casein used for the immobilization, the dilutions of the primary and secondary antibodies and the incubation parameters were optimized by ELISA based on a protocol developed by Vitkova et al [15]. The optimized values found for ELISA were tested on the biosensor and the electrochemical signal was optimized again adjusting the dilution of the primary and secondary antibodies.…”

Electrochemical Determination of β-Lactoglobulin Employing a Polystyrene Bead-Modified Carbon Nanotube Ink

“…The concentration of β-lactoglobulin used for the immobilization, the dilutions of the primary and secondary antibodies, the used buffers, the incubation time, and detection parameters were optimized by ELISA based on a protocol developed by Vitkova et al [13]. The optimized values found for ELISA were tested on the biosensor, and the dilutions of the primary and secondary antibodies were adjusted again to obtain the optimal electrochemical signal.…”