As recommended by IFCC, the biuret method is generally used to determine the total protein asa reference method. In order to achieve accurate measurement of the total protein by biuret method, a detailed description of establishment, validation and uncertainty evaluation of this method is proposed in the metrological view in this paper. Then the method was used to determine the total protein in RELA serum samples and the results of 2015-RELA sample A and B were 73.9g·L−1 and 58.9g·L−1, with the RSD of 1.18% and 1.07%, respectively. The uncertainty was evaluated in detail fully following the Guide to the Expression of Uncertainty in Measurement, and the expanded uncertainty of RELA sample A and B were 2.0 and 1.2g·L−1, respectively. The results have good agreement with those from other research institutions. The proposed procedure gives a good example to realize an accurate reference method with strict metrological control, which can benefit not only total protein measurement but also other biological measurement by reference methods.
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