Stability Of Cystatin Research Articles

Determination of Cystatin C in human urine by isotope dilution tandem mass spectrometry

This work presents the development of a methodology for the accurate and precise quantification of the renal biomarker Cystatin C in human urine by Isotope Dilution Mass Spectrometry (IDMS). The procedure is based on the addition of a known quantity of the proteotypic peptide ALDFAVG*EYNK labelled with 13C2-glycine to the urine sample followed by protein hydrolysis using trypsin. Then, preconcentration and purification of the isotope diluted peptide was carried out by a selective monoclonal antibody bound to magnetic beads and final measurement was done after injection of the sample in a HPLC-MS/MS triple quadrupole instrument. The isotopic distribution of the isotope diluted proteotypic peptide was measured by low resolution selected reaction monitoring. Using this aquisition mode, the bandpass of the first quadrupole was widened (FWHM =13 u) so the whole isotopic clusters for both the natural abundance and the labelled peptides entered the collision cell. The proposed acquisition mode provided similar accuracy and precision than the regular SRM mode (FWHM =0.7 u) but a higher sensitivity was observed. The purification of the sample by antibody based enrichment of the target peptide was shown to remove interfering compounds more efficiently in comparison with a sample purification based on semipreparative liquid chromatography. Using 5 ng of the labelled peptide it was possible to quantify Cystatin C in human urine in patients with normal and impaired renal function. Recoveries from 100 to 104% were obtained in samples containing from 90 to 700 μg L−1 of Cystatin C with relative standard deviations from 0.5 to 6%. The stability of Cystatin C in urine samples was evaluated under different storage conditions showing that only when the urine samples were stored at room temperature during more than 10 days, a significant degradation of Cystatin C was observed.

Evaluation of the Dade Behring N Latex Cystatin C assay on the Dade Behring Nephelometer II System

The Dade Behring N Latex Cystatin C assay, a particle-enhanced nephelometric immunoassay for measuring serum cystatin C, was evaluated on the Dade Behring Nephelometer II. The assay time was 6 min and the throughput was 75 samples per hour. The sample volume was 40 microL and the measuring range was 0.25-7.90 mg/L. Imprecision studies revealed within-run CVs < 1.8% and between-run CVs < 1.8% in the concentration range 0.87-4.63 mg/L. Recovery was 92.4-101.3%. Linearity studies showed excellent correlation between the theoretical and obtained values. No interferences were detected from haemoglobin < 1.0 mmol/ L, bilirubin <512 micromol/L and Intralipid <20 g/L. Stability of cystatin C in serum was 7 days at temperatures from 20 degrees C to 20 degrees C and 6 months at -80 degrees C. Measurements of cystatin C in heparin-plasma and EDTA-plasma did not differ significantly from cystatin C measured in serum. Fifty patient samples run on the Dade Behring Nephelometer II (y) were compared to the Dako Cystatin C assay (x). The Passing-Bablok regression analysis revealed y = 1.105x - 0.340. In conclusion, the Dade Behring N Latex Cystatin C assay was precise and correlated with the Dako Cystatin C assay.