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Measurement method

A measurement method and material technology, applied in the field of new measurement, can solve the problems of measurement error, hemolysis interference problem and so on.

Inactive Publication Date: 2002-09-25
SYSMEX CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in the measurement of clinical tests using enzyme reactions, the problem of measurement errors due to components in erythrocytes contained in the sample, that is, hemolytic interference, has not been sufficiently resolved.

Method used

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Embodiment 1

[0019] After collecting blood with a blood collection tube, the blood was centrifuged at 3000 rpm to obtain human red blood cells. The red blood cells were frozen at -20°C and then thawed at room temperature. As a result, blood cells are ruptured to obtain hemolyzed components. Dilute it with normal saline to obtain 500 mg / dL hemoglobin. Prepare the reagents shown below.

[0020] Dissolve 2.0mM NADP and 1.0mM 6PG into 0.1M triethanolamine buffer (pH7.5) to make reagent A; dissolve 2.0mM NAD and 1.0mM 6PG into 0.1M triethanolamine buffer (pH7.5) , dubbed reagent B.

[0021] After heating reagent A and reagent B at 37° C. for 5 minutes, 15 μL of the hemolyzed sample was added, and the absorbance was measured at a wavelength of 340 to 750 nm every 1 minute. When this hemolyzed solution was measured, as shown in FIG. 1 , no increase in absorbance was observed when NAD was applied. On the other hand, as shown in Fig. 2, when NADP is used as a coenzyme to react, the absorbance ...

Embodiment 2

[0023] Equipped with the reagents shown below for the determination of glucose (GLU).

[0024] (Reagent 1)

[0025] Tris 100mM

[0026] HK 3.5U / mL

[0027] G6PDH 5.0U / mL

[0028] β-NAD 3mM

[0029] Magnesium acetate 10mM

[0030] PH6.0

[0031] (Reagent 2)

[0032] Tris 200mM

[0033] ATP 6mM

[0034] PH9.0

[0035] In addition, the above-mentioned reagents were prepared in which β-NADP was substituted for β-NAD in the reagent composition. (How to operate)

[0036] 210 μL of reagent 1 and 70 μL of reagent 2 were added to 5 μL of the sample. Under the condition of dominant wavelength of 340 nm, the endpoint detection was performed with a Hitachi 7170 automatic analyzer, and the glucose concentration was calculated using the pre-made standard curve.

[0037] Prepare hemolyz...

Embodiment 3

[0039] Equipped with the reagents shown below for the determination of neutral fat (TG).

[0040] (1) Composition of Reagent 1

[0041] N-[Dihydroxyethyl]glycine 50mM

[0042] (Bicine)

[0043] Potassium chloride 100mM

[0044] Magnesium chloride 10mM

[0045] Non-ionic detergent A-10R 0.5%

[0046] (Nonion A-10R)

[0047] Triton X-100 0.2%

[0048] Sodium Azide 0.1%

[0049] PEP 4.0mM

[0050] ATP 3.0mM

[0051] G6PDH 4.5U / mL

[0052] PK 3.0U / mL

[0053] GK 3.0U / mL

[0054] PH8.5

[0055] (2) Composition of Reagent 2

[0056] MES 50mM

[0057] Oxalic acid 100mM

[0058] Glucose 80mM

[0059] Triton X-100 0.25%

[0060] β-NAD 7.0mM

[0061] Sodium Azide 0.1%

[0062] ADP-HK 10.0U / mL

[0063] Lipase 1500U / mL

[0064] pH6.5

[0065] In ad...

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Abstract

The present invention provides a method for measuring a substance to be measured in a biological sample through a reaction system capable of generating 6-phosphogluconic acid, which can obtain correct measured values. The present inventors focus on the final product in the reaction system for measuring glucose with coenzymes is 6-phosphogluconate, and 6PGDH (6-phosphogluconate dehydrogenase) in blood cells reacts with NADP as a coenzyme, and the results are given. The measured value brings positive errors, etc.; when the analyte in the biological sample is determined by the reaction system that can generate 6-phosphogluconate, the above-mentioned problem is solved by placing 6-phosphogluconate dehydrogenase outside the reaction system. topic.

Description

technical field [0001] The invention relates to a measuring method, which avoids measuring errors caused by hemolytic components contained in the sample when measuring the substance to be measured in a biological sample by using an enzyme reaction measuring reaction system. Background technique [0002] In the field of clinical testing, the change in absorbance is often used as an indicator of the change in coenzyme (NAD, NADP, NADH, NADPH, Thio-NAD Thio-NADP, Thio-NADH, Thio-NADPH) to quantify the target component. Serum is generally used as a test sample, but sometimes during the blood collection process to obtain serum, physical shocks or human errors may cause blood cells to rupture, resulting in hemolysis. In addition, when blood is collected from a patient suffering from a hemolytic disease, hemolysis due to the disease is sometimes seen. Furthermore, in order to shorten the detection time, there are recent attempts to perform measurement using whole blood as a detect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/54G01N33/66
CPCC12Q1/54G01N33/66
Inventor 岸浩司山下和昭
Owner SYSMEX CORP