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A highly sensitive method for quantifying gallocatechin gallate and its epimer using a catechin-specific peptide

Miyamoto, Takahisa, Mekada, Youko, Kurahachi, Masahiro, Umeno, Mai, Nakayama, Motokazu, Shigemune, Naofumi, Tsugukuni, Takashi, Tokuda, Hajime, Tachibana, Hirofumi, Honjoh, Ken-ichi
Food control 2013 v.29 no.1 pp. 162-166
absorbance, bovine serum albumin, catechin, epigallocatechin, equipment, laminin, pH, peroxidase, receptors, temperature, washing
We have developed a method for quantifying gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) using a catechin-binding peptide (part of the 67-kDa laminin receptor). Using micro titer plates, we investigated various conditions, including the quantifiable range of EGCg concentrations, the optimal concentration of the catechin-binding peptide, and the optimal reaction conditions. In this microplate assay, after each well was coated with bovine serum albumin, sample containing GCg and EGCg was added at pH 8.0, and allowed to stand at 37 °C for 2 h. After washing, biotinylated-peptide solution was added at 1 μg mL−1 and allowed to react for 1 h at 37 °C. Each well was added with streptavidin–horseradish peroxidase conjugate, followed by chromogenic reaction for 25 min at room temperature. After the reaction, absorbance was measured at 405 nm. Our method is capable of quantifying EGCg in the range of approximately 0.1–2.0 mg L−1 with a high degree of sensitivity and a high correlation (R2 = 0.98) between EGCg concentration and absorbance. The method was specific to GCg and EGCg and seems capable of estimating GCg and EGCg contents in the presence of other catechin compounds. The method is simple and highly sensitive for quantitative GCg and EGCg measurement that requires no special equipment or operation and can measure multiple samples simultaneously.