# Effect of sonication and protease inhibitors on Elisa quantification of selected proteins in bovine udder tissue homogenates

## TL;DR

This study shows that sample preparation methods like sonication and protease inhibitors significantly affect protein measurements in bovine udder tissue using ELISA.

## Contribution

The study demonstrates that combining mechanical homogenization with sonication and protease inhibitors reduces protein yields in ELISA.

## Key findings

- Protease inhibitors and sonication reduced protein concentrations, with the lowest values seen when combined with mechanical homogenization.
- Alkaline phosphatase was more sensitive to processing than alpha-casein.
- Mechanical homogenization alone yielded the highest protein concentrations.

## Abstract

The protocols for preparing ELISA samples vary in terms of the use of mechanical homogenization (MH) alone or the additional use of protease inhibitors (PI) and/or sonication (S). It is unclear whether the type of protocol can affect the final protein yield; thus, we evaluated how different preparation methods and their combination affect the alpha-casein (CSN1), lactoferrin (LTF), and alkaline phosphatase (ALP) concentrations in udder parenchyma using a sandwich ELISA, and tested whether the udder health state (coagulase-positive staphylococcal ​​infection) affects the protocol efficacy. Samples from 22 cows (11 healthy, 11 infected) were processed using the four protocols. PI and S reduced protein concentrations, with the combination of MH + PI+S giving the lowest ​​values for all three proteins. Unlike CSN1, ALP was the most sensitive to processing. CSN1 levels were higher in infected samples, while lactoferrin and ALP were not influenced by infection. All preparation methods affected the protein levels, irrespective of health state. The highest protein concentrations were obtained using MH alone. Our findings highlight the need for careful optimization and standardization of sample preparation to ensure reliable and reproducible ELISA results.

The online version contains supplementary material available at 10.1038/s41598-026-38653-x.