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.

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We have previously reported that CA19-9 values obtained with the enzyme immunoassay (EIA) method were higher than those obtained with radioimmunoassay (RIA) for patients with liver disorders. In this report the following findings are presented:
1) When screening of sera from 48 patients with liver disorders was performed using our criteria, 19 specimens were found to have more than 2-fold higher CA19-9 values as measured by EIA than as measured by RIA.
2) These 19 specimens with large discrepancies in CA19-9 values each had slightly elevated levels, in the range 100-500U/ml, of CA19-9 on measurement by EIA. They had been obtained from patients with chronic hepatitis or cirrhosis of the liver.
3) The molecular weight of CA19-9 was determined by high-performance liquid chromatography (HPLC). While the molecular weight of CA19-9 in two patients with carcinoma, which was used as a control, was approximately 1000kD, most of the sera with discrepancies in CA19-9 values noted above had CA19-9 of two molecular weights, one high, at 1300-1400kD, and one low, at 400-800kD.
4) When account is taken of the unsatisfactory degree of resolution in the high molecular weight region on HPLC, the above findings suggest that the low molecular weight components of CA19-9 react in EIA measurements, yielding false-positive results.

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A novel blocked type substrate for use in a human α-amylase assay, 2-chloro-4-nitrophenyl 65-azido-65-deoxy-β-maltopentaoside (6-N3G5CNP), was synthesized by the chemical modification of 2-chloro-4-nitrophenyl-β-maltopentaoside (G5CNP) at the 6 position of the non-reducing-end with an azido group. This new substrate possesses the following properties:
1. Due to the modification of its 6-position OH group with the azido group, there was no hydrolysis of 6-N3G5CNP by the coupled enzyme glucoamylase.
2. Compared with G5CNP, 6-N3G5CNP has higher affinities to human pancreatic α-amylase (HPA) and human salivary α-amylase (HSA), with smaller Km values of 0.15mmol/l and 0.21mmol/l, respectively. Due to the greater specificity of its hydrolytic position, more than 96% of the product of hydrolysis of 6-N3G5CNP was G2CNP, compared with 81% when G5CNP was used as a substrate.
3. The reactivities to HPA and HSA were 97 and 99%, compared with 100% in each case for the non-blocked substrate G5CNP the reactivities of the two substrates were thus considered to be the same.
When used in an automatic analyzer, the α-amylase assay with 6-N3G5CNP yielded good results, confirming its useability. 6-N3G5CNP has all the properties required for use in the α-amylase assay. It will, therefore, be quite useful for the measurement of α-amylase activity in routine laboratories.

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We describe a method using high-performance liquid chromatography (HPLC) for the simultaneous determination of uroporphyrin (UP)-I and -III in urine and whole blood. HPLC was performed on a reversed-phase (Zorbax-ODS) column with fluorometric detection (Ex 394nm/Em 624nm) and with a column switching system. Mobile phase (1mol/l ammonium acetate buffer, pH 5.2: acetonitrile, 865: 135, v/v) was isocratically eluted at a flow rate of 0.8 ml/min. The within-assay reproducibilities (CV) for urine and whole blood were 1.6-2.0% (n=21) and 5.8-5.9% (n=6), respectively.
Between-day reproducibilities were 4.9-5.7% (n=7) for urine and 9.4-9.9% (n=6) for whole blood. The calibration curves for UP-I and -III were linear over the range of 2-500ng/ml. The rates of recovery of UP-I and -III in urine and whole blood were 100-112% (1-160ng/ml) and 91-94% (2-40ng/ml), respectively. The lower limit of detection of UP-I or -III in urine or whole blood was estimated to be 2ng/ml.
In conclusion, the present method is simple and quick. Moreover, precision, accuracy and sensitivity are allowable for the routine clinical examination.

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We evaluated a method of assay for human α-amylase (Amy) which used? I-nitrophenyl β-Dgalactosyl-β-maltooligoside (Gal-GnPNP, n=4-7), in which D-galactose was β-(1-4)-linked (Gal-GnPNP-I, n=4-7) or β-(1-6)-linked (Gal-GnPNP-II, n=4-7) at the nonreducing end as a substrate.
Gal-GnPNP-I (n=4-7) proved superior to Gal-GnPNP-II (n=4-7) under the conditions of Km and relative activity. Of the Gal-GnPNP-I (n=4-7), Gal-G5PNP-I had the best ratio of activity between pancreatic amylase (p-Amy) and salivary amylase (s-Amy), and underwent catalyzed hydrolysis at two sites by each of the human Amy isozymes, 48.8, 51.2% of p-Amy and 49.3, 50.7% of s-Amy.
Our evaluation of the proposed method showed that it could obtain good results with a high degree of reproducibility; its CV was 0.31-0.71% (n=10), with non-interference except for hemolysis (3%). The coefficent of correlation between the proposed method and the previous method was high for both serum and urine (serum:β=0.999, y=0.68x+1.6, n=50; urine:β=0.999, y=0.70x+0.2, n=50).
These findings suggest that Gal-G5PNP-I is suitable for application in the human α-amylase assay used in the clinical chemistry laboratory.

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We devised a method featuring column-switching high-performance liquid chromatography for the determination of nicotinic acid (NiA) in human blood. Nicotinamide in blood was converted into NiA by heating with 10mol/l sodium hydroxide at 100°C for 50min. The reaction mixture was adjusted to pH 2.3 with hydrochloric acid, and applied to a Baker bond spe octadecyl column. NiA on the column was eluted with 60% methanol, and measured by HPLC. The calibration curve for NiA was linear over the range 10 to 1015 ng/injection. The lower limit of detection of NiA in blood was 0.5μg/ml. NiA (extract) was stable at room temperature in room light for at least 25 hr. The rate of recovery of NiA from blood was 89% in the range 1.01 to 10.08μg/ml blood. The reproducibilities (CV) for within-and between-day assays were 4.4% (n=10) and 6.7 % (n=5), respectively.

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Serum lipoprotein (a)(Lp (a)) concentration was measured by latex immunoassay (LIA) with Hitachi 7150 auto analyzer.
The range of assay was 10-1000mg/l and the lower limit was 6.4mg/l.
Within-run and day-to-day coefficients of variation were 1.64-3.14% at 74.0-456.1mg/l and 2.68-5.75% at 75.2-463.6mg/l, respectively.
There was no-interference from the high concentrations of hemoglobin, bilirubin and plasminogen. The normal range for serum Lp (a) established measuring sera from 187 healthy subjects including 94 women and 93 men, was 126.9±113.0mg/l.

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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.

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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.

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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.

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