Quantification of pepsin in rennet using a monoclonal antibody-based inhibition ELISA

- A high specific monoclonal antibody (mAb) against pepsin was produced.
- The mAb recognized pepsin in rennets from calf, kid and lamb sources.
- An inhibition enzyme-linked immnunosorbent assay (ELISA) for pepsin in rennet was developed.
- ELISA was validated by comparing analytical results with the standard chromatographic method.
- ELISA showed a good agreement with the chromatographic method with a fixed bias.

Pepsin and chymosin are milk-clotting enzymes found in rennets. They differ in their pH and temperature sensitivities and their milk-clotting activity (MCA)/proteolytic activity ratio which impact cheese technology. Therefore, characterization of rennet should not be limited to its total MCA, but also its enzymatic composition. Monoclonal antibodies against pepsin, obtained from mice immunized with purified pepsin, were characterized. A specific inhibition enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of pepsin in rennets. The limit of quantification was 143 ng pepsin/mL. The precision within runs ranged from 7.0 to 9.4%, for rennets with low and high pepsin concentrations, respectively. The precision among runs also ranged from 8.8 to 11.4%. Satisfying recovery, from 84.7 to 100.3%, was found with spiked samples. The applicability of the developed inhibition ELISA for pepsin quantification was assessed by analyzing commercial rennets, and compared to the standard chromatographic method 110A of International Dairy Federation. The relation and agreement between methods were evaluated using Deming regression analysis and Bland-Altman plot. A good agreement was found with a fixed bias. By means of bias subtraction, inhibition ELISA could be a reliable alternative for rapid and sensitive determination of pepsin in rennet.