A fluorescent enzyme immunoassay (EIA) for measurement of cholecystokinin (CCK) in human plasma was developed. An alkaline phosphatase (Alp)-labeled CCK-33 conjugate was used as a tracer in the EIA. The bound and free fractions were separated with the use of a second antibody (goat anti-rabbit IgG antibody) coated to beads. The Alp-labeled CCK-33 conjugate bound to beads was measured flurophotometrically using 4-methylumbelliferyl phosphate as a substrate. The range of measurement and the fifty percent inhibitory concentration (IC50) for CCK-8 were 0.4 to 100fmol/assay and 5.3fmol/assay, respectively. The sensitivity of this assay was 5 times higher than that using Alp-labeled CCK-8 and radioimmunoassay (RIA) using 125I-CCK-39 as a tracer. The measurement of CCK in plasma samples required pretreatment by immuno-affinity columm extraction of CCK in order to achieve separation from interfering substances. The rate of recovery of CCK-8 using the immuno-affinity column was 97.4±14.8% (n=36). The coefficient of correlation between values of CCK plasma concentration obtained by the fluorescent EIA and RIA was 0.940 (n=71). The plasma CCK concentration of normal subjects and patients with various diseases could accurately by measured with the EIA we have proposed.
The ion selective electrode method (ISE) for concentration measurement of the sodium, potassium and chloride has been widely used in field method. However, the measured values from antomated analysis with the ISE could not be corrected accurately. The accuracy transferring in the accuracy-based measurement system could be transfered by definitive methods, ion-exchange gravimetry for sodium, isotope dilution mass spectrometry for potassium and chloride, to the primary serum reference material, and reference method, secondary serum, reference material and ISE for field method. The certified serum reference material (CRM) conforms to the golden standard of undiluted and diluted potentiometry, and it can be solved the problems which results in differences in the measured values between device or facilities. Furthermore, it can be maintained the measured values accuralely by which manufacturers and users can standardize their instruments to give results in concentration which are verifiable to the CRM. This document offers a protocol the theory of setting the CRM, the data correction, the accuracy control algorithm and the allowable limit.
The ion selective electrode method (ISE) for concentration measurement of the ionized calcium has an effect on the measured sample's matrix being large in the same manner as sodium and potassium measurement. Accordingly, measured values by the ISE have differences due to the measurement system. The certified serum reference material (CRM), as the certified matrix reference material, was established for the correction of results of ISE to utilize the ISE in a better way. The certified value of the ionized calcium is measured by using the Covington-Umemoto cell as the reference standard cell system. This CRM can be solved the problems which results in differences in the measured values between device or facilities. This document offers a protocol the theory of setting the CRM, the data correction, sampling, transport, storage and clinical application.
Recommended method for determination of uric acid in serum was established by HPLC. For deproteinization of 0.2 ml of the test serum, various deproteinizing agents were tested. Among them, 0.3 mol/1 percholric acid was used in this recommended method. After the deproteinization, 0.3 ml of the supernatant was mixed with 0.3 ml of 0.2mol/1 Na2HPO4 solution, and 100μl of the mixture was applied to the ODS column of HPLC. The HPLC determination was completed in 10 min.