JPS58141797A - Reagent for automatic analysis - Google Patents

Abstract

PURPOSE:To prevent the abnormal positive error in the determination of cholesterol, triglyceride and uric acid by an autoanalyzer, by adding a proper amount of a chelating agent to the reagent for colorimetry. CONSTITUTION:In the determination of total cholesterol, free cholesterol, triglyceride and uric acid by an autoanalyzer, a proper ampount, e.g. >=10<-4>M concentration, of a chelating agent such as ethylenediamine-tetraacetic acid disodium salt, glycol ether diamine tetraacetic acid, etc. is added to a conventional colorimetric reagent which induces the generation of hydrogen peroxide from the above compounds, conjugates the produced hydrogen peroxide with other redox enzyme and oxidizes the chromogen to effect color development.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to automated analytical measurement reagents, and in particular to reagents related to the measurement of total and free cholesterol, triglycerides, and uric acid.

For example, to explain cholesterol as an example, the components of conventional reagents used for cholesterol measurement are:
Cholesterol esters Hydrol 7 (1 of total cholesterol. Cholesterol A oxidase, bar oxidase (oxidoreductase) and 3-methyl-N-ethyl-N
-(β-hydromucdiethyl-aniline, 4-aminoantipyrine (chromogen).

When applying these conventional reagents to an automatic analyzer and examining their long-term reproducibility, most of the measured values were ±2 m
It shows good reproducibility in the g/di range. However, 1
20 mg/d 1 at a rate of about 1 in 00 tests
In some cases, higher measured values may not be obtained. Furthermore, when the above-mentioned reagents are applied to a small automatic analyzer that dispenses different reagents one after another with -1 dispensers,
It has been found that when total cholesterol or free cholesterol is measured immediately after protein is measured using a biuret reagent containing copper ions, a significant positive difference occurs. However, the cause is unknown and rare.

A similar phenomenon was found for triglycerides and uric acid.

An object of the present invention is to provide a reagent that can prevent the above-mentioned errors in measuring total cholesterol, free cholesterol, triglycerides, and acids using an automatic analyzer.

To achieve this goal, we investigated the causes of errors in the measurements of the above compounds, and found that most of them were:
It turned out that this was due to the promotion of the oxidative coloring reaction of the chromogen by metal ions. Therefore, the present invention is based on the discovery that chelating agents do not affect the reaction.

The present invention will be described in detail below using Examples.

To investigate the cause, each component of the above-mentioned biuret reagent was added to the total cholesterol reagent, and the influence * on the measured value was examined. As a result, it was found that the addition of copper sulfate increased the measured value of total cholesterol by about 10. 1
A zero error is significant. It has also been found that 4 positive differences occur due to ferric ions in addition to copper ions.

The coloring reaction of cholesterol is shown by the following formula. Hydrogen peroxide produced by the reaction is conjugated with peroxidase, an oxidoreductase enzyme, to densify the chromogen, and the resulting color is compared.

Deca fatty acid oxytase HwOt + diethylaniline + 4-aminoantipyrine → blue-violet dye In general, methods using peroxidase are susceptible to the negative effects of reducing substances, as well as the positive effects of oxidizing substances. it is conceivable that. Considering the reaction in the above equation, the chromogen is produced by the copper ions contained in the biuret reagent and the ferric ions derived from the stainless steel alloy of automatic analyzers such as serum pipetting systems and reagent dispensing systems. It is presumed that the rate of color development increases because the oxidation of

In view of the above, in the present invention, a chelating agent is added to the reagent in order to mask heavy metal ions. An example is shown in Clothing 1.

Shishi 1 Candy Cholesterol Reagent Composition The conventional reagent composition has the concentration of EDTA/2Na in Table 1 as 0
It is expressed by the composition as follows.

In order to compare the reagent of the present invention with a conventional reagent, simultaneous determination of total protein and total cholesterol was performed using both reagents using the above-mentioned compact automatic analyzer. Table 2 shows the results.
Show your friends. With the reagent of the present invention, no increase in total cholesterol measurements is observed immediately after measuring total protein. In contrast, with conventional reagents, an increase of about 1096 is observed.

Table 2 Comparison results using a small automatic analyzer Next, EDTA and 2Nat were added to the total cholesterol test strip using a chelating agent. The effect on total cholesterol measurements was examined. EDTA, 2Na Reika (reagent of the present invention),
Correlation experiments were performed on a four-apparatus using no additives (conventional reagents). The results are shown in FIG. No adverse T#I#1 was observed due to the addition of a chelating agent.

Additionally, the amounts of EDTA and 2Na added were investigated. As a result, the effect of the present invention was recognized at s degree of 10 −4 M or higher.

Furthermore, in addition to ED'l''A and 2Na, GEDTA (glycol ether cyamine tetraacetic acid), DPTA-OH (
A similar effect was also observed with chelating agents such as cyaminopropanol tetraacetate.

Free cholesterol, tricrycerite and acid can also be reduced by adding a chelating agent to the
It had the same effect as the total cholesterol mentioned above.

As described above, by mixing the appropriate chelating agent in the reagent, the purpose of preventing the normal negative difference in total cholesterol, free cholesterol, triglyceride, and acid measurements by automatic analyzers cannot be completely achieved. Ta.

Moreover, since the chelating agent added is extremely thin, the cost remains the same as before.

The present invention is particularly effective in the above-mentioned small-sized automatic analyzer.

[Brief explanation of the drawing]

FIG. 1 is a correlation diagram of total cholesterol measurements when using a reagent with a conventional composition and a reagent of the present invention.

Claims (1)

[Claims] 1. In connection with the measurement of total cholesterol, free cholesterol, triglyceride, and uric acid using an automatic analyzer, the production of hydrogen peroxide is induced from the above compound, and the produced hydrogen peroxide is conjugated with other redox enzymes to produce color. A reagent for automatic analysis and measurement whose percentage sign is that an appropriate amount of a chelating agent is mixed with a reagent that uses a colorimetric method to oxidize the original substance to develop color.