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

Published online by Cambridge University Press:  23 April 2013

Akinori Kume*
Food Science Research Labs, R&D Div. Meiji Co., Ltd, Japan
Akina Sasayama
Food Science Research Labs, R&D Div. Meiji Co., Ltd, Japan
Tetsuo Kaneko
Food Science Research Labs, R&D Div. Meiji Co., Ltd, Japan
Junichi Kurisaki
The Department of Food and Nutrition, Jumonji University, Japan
Munehiro Oda
Graduate School of Bioresource Sciences, Nihon University, Fujisawa, Japan
*For correspondence; e-mail:


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.

Research Article
Copyright © Proprietors of Journal of Dairy Research 2013 

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