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

作者：Kume Akinori^*; Sasayama Akina; Kaneko Tetsuo; Kurisaki Junichi; Oda Munehiro

来源：Journal of Dairy Research, 2013, 80(3): 326-333.

DOI：10.1017/S0022029913000162

• Casein phosphopeptide
• amorphous calcium phosphate
• monoclonal antibody
• competitive enzyme-linked immunosorbent assay

A specific and simple competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine bovine beta-casein phosphopeptides (beta-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-beta-CPP(f(1-25)) monoclonal antibody 1A5 (mAb 1A5). The mAb 1A5 bound specifically to the tryptic phosphopeptides from beta-casein but not from alpha(s1)- or alpha(s2)-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 beta-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.

• 出版日期2013-8