JPH0334590B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a reagent for measuring plasma hemoglobin. Generally, when red blood cells are destroyed and hemolysis occurs, the supply of oxygen to various parts of the body decreases, resulting in anemia. Since hemoglobin is contained only in red blood cells, it serves as an indicator of hemolysis. Diseases that cause hemolytic anemia include hereditary spherocytosis, red blood cell glucose-6-phosphate dehydrogenase deficiency, and autoimmune hemolytic anemia, and it can also be caused by bacterial infection or chemicals such as lead and penzene. As a test for hemolysis, urinary hemoglobin is generally measured, but it is not detected in mild cases. In other words, 100 to 150 mg/dl of haptoglobin exists in plasma, and when red blood cells are destroyed and hemoglobin is eluted into plasma, it immediately combines to form a haptoglobin-hemoglobin conjugate, which is transferred to the renal glomerulus. urinary hemoglobin cannot be detected. However, there is a quantitative upper limit for haptoglobin, and urinary hemoglobin is generally detected when the plasma hemoglobin value reaches 135 mg/dl or higher. Therefore, in order to measure mild hemolysis, it is necessary to measure hemoglobin in plasma. The normal range of plasma hemoglobin in healthy people is 0 to 5 mg/dl, and since a measurement range of 10 mg/dl or more is clinically important, plasma hemoglobin concentration has been measured by the benzidine method for many years. However, since the carcinogenicity of benzidine began to be seen as a problem,
A method using tetramethylbenzidine (TMB) or dicarboxybenzidine (DCB) as a coloring agent instead of benzidine (hereinafter, the method using the former will be referred to as
The method using the TMB method and the latter method are called the DCB method. )
However, although the DCB method has good sensitivity,
The TMB method is generally used because the measurement range is narrow. However, with the TMB method, the values are low due to the influence of plasma, making it difficult to obtain linearity of the calibration curve. Another drawback is that the standard solution and sample must be treated using different methods and repeated operations over time. and,
Since TMB and DCB are derivatives of benzidine, they are suspected of being carcinogenic. Therefore, the present inventors investigated 2,2'-azinobis (3-ethylbenzothiazoline-6-
As a result of intensive research, we were able to develop a reagent for this assay that is more sensitive than the TMB method, is unaffected by plasma, and is easy to operate. The gist of the present invention is to provide a reagent for measuring hemoglobin in plasma for measuring the amount of hemoglobin by exhibiting an oxidized blue color of ABTS due to the peroxidase-like action of hemoglobin in the presence of hydrogen peroxide. That is, the oxidized form of ABTS is shown by the reaction formula below. Furthermore, the addition of disodium ethylenediaminetetraacetate (EDTA) helps stabilize the color development. In addition, since the pH of the reaction solution in the present invention is 2 to 3, plasma proteins may aggregate;
This effect can be removed by centrifuging at 2000 xg for 5 minutes or more. In the present invention, the absorbance of a plasma hemoglobin amount of 10 mg/dl is approximately 0.12, and the calibration curve is from 2 mg/dl to
It shows good linearity up to 100mg/dl and has high reproducibility. The reaction is completed within 90 minutes at room temperature. Next, side reactions gradually proceed, and the absorbance also gradually increases (ΔA=0.0005/min). But TMB
Compared to the method of temporarily increasing the absorbance of the reaction solution and then gradually decreasing it, the present invention is less prone to errors, and the error is only about 0.005 mg/dl if measured within 20 minutes after the reaction. The measurement reagent of the present invention is a 1.0-4.0mM ABTS acetic acid solution, a 0.5-3.0% hydrogen peroxide solution, and a 0.4-4.0mM acetic acid solution.
It consists of 0.05-0.2M phosphate buffer (PH6-8) containing 1.2mMEDTA. These reagents are prepared separately and mixed in sequence when quantifying hemoglobin in plasma. Next, the preparation method of each reagent will be shown. ABTS solution: ABTS50~205mg 80~100%
Dissolve in 100ml of acetic acid solution. Hydrogen peroxide solution: 30% hydrogen peroxide solution 0.5-3
Adjust the total volume to 30ml with purified water. Phosphate buffer containing EDTA: 0.4~
2 parts of 0.05~0.2M phosphate-potassium solution containing 1.2mM MEDTA, 0.05~0.2M containing EDTA at the same concentration
After mixing 1-3 parts of 0.2M disodium phosphate solution, adjust the pH to 6-8 with 1N sodium hydroxide solution.
Match. 2-3 parts of ABTS solution, 1 part of hydrogen peroxide solution,
Mix 5 to 15 parts of phosphate buffer containing EDTA thoroughly, add plasma, leave for 30 to 90 minutes at a temperature between room temperature and 50°C, and then centrifuge at 1200 to 2000 x g for 5 minutes or more. Measure the absorbance of the supernatant at a wavelength of 400-430 nm. The preparation method and measurement method of the measurement reagent of the present invention will be specifically illustrated in the following Examples. Example (1) Preparation of reagent ABTS solution: ABTS 150mg 90% acetic acid 100%
Dissolve in ml. Hydrogen peroxide solution: Mix 1 ml of 30% hydrogen peroxide with 29 ml of purified water. Phosphate buffer containing EDTA: Phosphate buffer containing EDTA: After mixing 128 ml of 0.1 M monopotassium phosphate solution containing 32 mg EDTA and 100 ml of 0.1 M disodium phosphate solution containing 25 mg EDTA, mix with 1 N sodium hydroxide solution. Adjust to PH7.6. Hemoglobin standard solution: 1 ml of red blood cells is 0.9
Wash twice with % sodium chloride solution. next
After hemolyzing the mixture by adding 10 ml of purified water and freezing it at -30°C, it is allowed to stand at room temperature to thaw. The solution was centrifuged at 4000 x g for 10 minutes, and the amount of hemoglobin in the supernatant was measured by the cyanmethemoglobin method.
Dilute to mg/ml and use as hemoglobin standard solution. (2) Determination of plasma hemoglobin: 2.5 ml of ABTS solution, 1 ml of hydrogen peroxide solution,
Put 8 ml of phosphate buffer containing EDTA into a test tube, mix well, add 5 μl of hemoglobin standard solution or plasma, and mix well. After standing at room temperature for 90 minutes, centrifuge at 1500 x g for 5 minutes and remove the supernatant.
Measure the absorbance at 410nm. Conversion from absorbance to hemoglobin amount is performed by reading from a calibration curve prepared using a hemoglobin standard solution. Simultaneous reproducibility and daily variation in experimental examples are shown in the table.

【table】

【table】 [Brief explanation of drawings]

The drawing is a graph showing a standard curve in an example of the present invention, in which the amount of standard hemoglobin at a known concentration is shown on the horizontal axis and the absorbance is shown on the vertical axis.

Claims (1)

[Claims] 1 1.0-4.0mM2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in acetic acid solution, 0.5-3.0% hydrogen peroxide solution, 0.4-
A reagent for measuring plasma hemoglobin consisting of 0.05-0.2M phosphate buffer (PH6-8) containing 1.2mM disodium ethylenediaminetetraacetate.