Recommended method of the Japanese Association of Medical Technologists for the determination of protein in urine by HPLC.
A recommended method for the determination of total urinary proteins as a designated comparative method (DCM) is described. The method is based on gel permeation high performance liquid chromatography (HPLC). A total of 20 microl of urine diluted 5-fold with saline solution were applied to a column, eluted with 0.13 mol/l phosphate buffer (pH 7.0) at a flow rate of 1.0 ml/min at 35 degrees C, and the absorbance is monitored at 220 nm. The fraction eluted in the void volume was quantified for urinary protein, using bovine serum albumin (NIST SRM 927) as the standard. The linear range of the assay was 10--5000 mg/l, and the precision was 1.5% in two samples with concentrations of 1037 and 4338, respectively. The analytical recovery was 96--104%. Low molecular weight substances such as glucose, ascorbic acid and bilirubin did not interfere the assay. The assay was satisfactorily applied to a variety of proteins regardless of their nature or molecular weights (12.4 kDa) including beta2-microglobulin and Bence Jones Proteins. Surveys in five clinical laboratories, employing different types of HPLC apparatus with the same column type, showed satisfactory measurement precision (CV 2.1%) for three urine samples with different protein concentrations.
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Japanese Association of Medical Technologists, Ichigaya Hoso Building, 4-1-5 Chiyoda-ku, Kudan-kita, Tokyo 102-0073, Japan.
D016477:Artifacts; D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D009101:Multiple Myeloma / Q000652:urine; D011507:Proteinuria / Q000652:urine*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity