Determination of κ-casein B in bovine milk through HPLC and ELISA techniques

Among milk casein fractions, κ-casein (κ-CN) plays a central role in cheese making process since it is the target compound of rennet. The κ-CN exists in different genetic variants, with κ-CN B being the most reactive toward coagulant activity, thus leading to greater cheese making efficiency and improved cheese quality. Therefore, there is great interest in the application of analytical techniques which are able to quantify κ-CN B. High pressure liquid chromatography (HPLC) is the reference analytical method for both qualitative and quantitative determination of κ-CN variants, being characterized by high sensitivity and accuracy but also by high labor demand and analytical costs. On the other hand, enzyme-linked immunosorbent assay (ELISA) is an alternative technique for fast and cheap quantification of κ-CN B. On this background, the aim of this study was to gauge the agreement between HPLC and ELISA techniques for the quantification of κ-CN B in bovine milk. Individual milk samples (n = 975) of Italian Brown Swiss cows were collected during evening milking in 50 herds located in the north-central area of Italy. Samples were analyzed through reverse phase HPLC to identify and quantify κ-CN B. The same samples were analyzed through ELISA commercial kit for the quantification of κ-CN B. The agreement between the two meth-ods was estimated through Pearson’s correlation coefficients and z-score (z). According to the latter indicator, results from 2 experimental conditions may be considered equal when |z| ≤ 2, similar when 2 < |z| ≤ 3, and different when |z| > 3. Chromatographic analyses highlighted that 68% and 4% of the cows were homozygous for the allele of κ-CN B (genotype BB) and κ-CN A (genotype AA), respectively. The remaining 28% of the cows were heterozygous (genotype AB). Pearson’s correlation coefficients (z-scores) calculated for concentrations of κ-CN B measured through HPLC and ELISA techniques were 0.88 (0.29) when considering BB and AB genotypes together, 0.60 (0.59) when considering only BB genotypes, and 0.40 (4.11) when considering only AB genotypes. Results indicate that ELI-SA is consistent with HPLC technique when κ-CN B is represented in relatively great concen-trations (i.e. when including BB genotypes). Poor agreement was observed for milk samples with low concentrations of κ-CN B (i.e. AB genotypes).