A simple competitive enzyme-linked immunosorbent assay for the specific detection of the multiphosphorylated 1–25 β-casein fragment

Abstract

A specific and simple competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine bovine β-casein phosphopeptides (β-CPP) in casein phosphopeptides (CPP) or CPP complexes such as casein phosphopeptide amorphous calcium phosphate complexes added into dairy products. The method combines sample pretreatment designed for CPP enrichment and anti-β-CPP(f(1-25)) monoclonal antibody 1A5 (mAb 1A5). The mAb 1A5 bound specifically to the tryptic phosphopeptides from β-casein but not from αs1- or αs2-casein. Reactivity was also influenced by the extent of the phosphorylated form of serine residues. Based on the sequence-specific recognition and contribution of phosphorylated serine residues, the epitope of mAb 1A5 was found to reside within the cluster motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu and the surrounding residues in β-CPP. The competitive ELISA developed here can be used as an alternative to specialised and expensive techniques such as mass spectrometry. In particular, it is suitable for the measurement of CPP or CPP complexes in dairy products, which contain closely related endogenous molecular species.

Two phase feature-ranking for new soil dataset for Coxiella burnetii persistence and classification using machine learning models

“…Detecting C. burnetii in the soil can help prevent Q-fever disease outbreaks. The approaches generally used for recognizing C. burnetii are ELISA 18 , PCR 19 , and mass spectroscopy (MS) 20 . Although these tests are expensive, we can reduce their operational cost by assessing only those specimens that are more likely to be positive for C. burnetii.…”