# Comparing reverse phase high-performance liquid chromatography and enzyme-linked immunosorbent assay techniques for the quantification of κ-casein B in bovine milk

## TL;DR

This study compares two methods for measuring κ-casein B in milk and finds they agree well, except at very high or low levels.

## Contribution

The study demonstrates that ELISA can be a cost-effective alternative to RP-HPLC for routine κ-casein B analysis in bovine milk.

## Key findings

- RP-HPLC and ELISA showed strong correlation for κ-casein B measurements.
- ELISA has limitations in quantifying very high and low κ-casein B concentrations.
- ELISA is suitable for routine screening and large-scale studies due to its speed and lower cost.

## Abstract

Summary: This study evaluated the agreement between the reference chromatographic method, reverse phase high performance liquid chromatography (RP-HPLC), and a rapid enzyme-linked immunosorbent assay (ELISA) technique for the quantification of κ-casein B (κ-CN B) in individual bovine milk samples. Results showed substantial agreement between the 2 methods, except for some discrepancies mainly due to the limitations of ELISA in quantifying very high and low concentrations of κ-CN B. Overall, ELISA can serve as a technique for routine screening or large-scale studies, thus enabling faster processing at lower costs compared with more complex analytical techniques.

Summary: This study evaluated the agreement between the reference chromatographic method, reverse phase high performance liquid chromatography (RP-HPLC), and a rapid enzyme-linked immunosorbent assay (ELISA) technique for the quantification of κ-casein B (κ-CN B) in individual bovine milk samples. Results showed substantial agreement between the 2 methods, except for some discrepancies mainly due to the limitations of ELISA in quantifying very high and low concentrations of κ-CN B. Overall, ELISA can serve as a technique for routine screening or large-scale studies, thus enabling faster processing at lower costs compared with more complex analytical techniques.

•Chromatographic and immunoenzymatic measures for κ-CN B were strongly correlated.•The immunoenzymatic technique showed limits at high and low κ-CN B concentrations.•Improving the sensitivity of the immunoenzymatic technique would result in greater accuracy.

Chromatographic and immunoenzymatic measures for κ-CN B were strongly correlated.

The immunoenzymatic technique showed limits at high and low κ-CN B concentrations.

Improving the sensitivity of the immunoenzymatic technique would result in greater accuracy.

Among κ-CN variants in milk, κ-CN B is the most reactive to chymosin activity, enhancing the quality, profitability, and sustainability of the final cheese product. The aim of this study was to assess the agreement between reverse phase HPLC (RP-HPLC) reference method and a rapid ELISA technique for the quantification of κ-CN B in individual bovine milk samples. Chromatographic and immunoenzymatic analyses were performed on individual milk samples from 933 Brown Swiss cows, with κ-CN B expressed as (1) milligrams per milliliter of milk, (2) percentage of κ-CN B over total milk protein content, and (3) grams of κ-CN B yielded on a milking event. The agreement between κ-CN B phenotypes measured through RP-HPLC and ELISA was evaluated through r and z-scores. Results suggested a general agreement between the 2 techniques, with r ranging from 0.88 for κ-CN B expressed in milligrams per milliliter and as a percentage to 0.90 for κ-CN B expressed in grams. This is further supported by relatively low z-scores (<0.5), which suggested the absence of significant differences between the values obtained from RP-HPLC and ELISA. Observed discrepancies were likely because ELISA does not provide quantitative results for concentrations of κ-CN B >10 mg/mL, and to the limited sensitivity of the ELISA at low concentrations of κ-CN B. Overall, findings of the present study demonstrated a strong agreement between the 2 techniques.