HU207537B - Diagnostical composition for determining summa cholesterol in the serum - Google Patents

Abstract

The present invention relates to a diagnostic composition for the determination of total serum cholesterol content, comprising cholesterol esterase, cholesterol oxidase, peroxidase, and 0.5-1.5 mmol / l of 4-amino-antipyrine per meter of solution. chloride or hydrate, 5-15 mM sodium cholate, 0.25-0.35% w / v, enzyme-inducing emulsifier, 0.01-0.1% w / v, enzyme-friendly bactericide Contains 2-18 mmol of phenol compound and contains Trist.HCl buffer to adjust the pH to 6.7 to 8.7. The diagnostic composition of the present invention is a kit of lyophilized powder mixture and a solution, wherein the lyophilized powder mixture contains 0.040.1 IU / ml cholesterol esterase, 0.07-0.15 IU / ml cholesterol oxidase, 2.5- Containing 3.5 IU per ml of peroxidase and the amount of 4-amino-antipyrine given above, with 0.001 to 0.1% w / v of bovine serum albumin, and the solution contains the additional components listed above, and as a phenol compound m. containing at least two mixtures of cresol or 2,5-dimethylphenol or 3,4-dichlorophenol, phenol, m-cresol and 2,5-dimethylphenol. EN 207 537 Description range: 8 pages (including 3 sheets)

Description

The present invention relates to a diagnostic composition for the determination of total serum cholesterol.

It is known that abnormally elevated serum cholesterol is a significant risk factor for the development of coronary heart disease and myocardial infarction. In order to effectively prevent heart disease, diagnostic reagents and methods are needed to determine total serum cholesterol levels simply, quickly and accurately.

The most advanced analytical methods for determining cholesterol levels are enzymatic methods. The essence of enzymatic processes is to release cholesterol from cholesterol bound in the form of an ester by the action of a cholesterol esterase enzyme, then oxidize the cholesterol using the enzyme cholesterol oxidase and measure the amount of hydrogen peroxide formed during the oxidation. Such procedures are described, inter alia, in U.S. Patent Nos. 169,171 and 174541. Hungarian patent specification.

There are several analytical methods available to determine the amount of hydrogen peroxide. The most advantageous of these are photometric methods because they are easy to automate and allow for large number of sample analyzes.

Hydrogen peroxide in the presence of the enzyme peroxidase is known to form a colored product with 4-amino antipyrin and phenol, the amount of which can be measured photometrically [Ann. Clin. Biochem. 6, 24 (1969)].

Based on this knowledge, a number of diagnostic agents have been developed and marketed to determine total serum cholesterol. A common feature of known diagnostic formulations is that they contain cholesterol esterase, cholesterol oxidase, peroxidase, magnesium chloride and sodium cholate as the enzyme activator, an enzyme-inactive emulsifier, an enzyme-inactive bactericide, and a coloring agent7. in aqueous buffer solution. Known diagnostic formulations are marketed in the form of a dry powder mixture of the components, and the powder mixture is dissolved in the prescribed amount of water or aqueous buffer prior to use.

The known diagnostic compositions differ primarily in the amount and quantitative ratios of enzymes contained therein and in the quality of the coloring agents. Typical amounts of cholesterol esterase are 0.25-0.40 IU / ml standard solution, and typical amounts of cholesterol oxidase are 0.25-1.00 IU / ml standard solution. whereas a typical amount of peroxidase is 0.21.0 IU / ml standard solution (such as those marketed by Boehringer, Chemie Linz, Baker, and Heintel); the formulations thus contain a very large amount (sometimes more than ten times) of the two hard-to-access, costly and sensitive enzymes (cholesterol esterase and cholesterol oxidase), while the amount of peroxidase is in the order of the other enzymes. This is different from the Human preparation, which contains 0.15 IU / ml cholesterol esterase, 0.10 IU / ml cholesterol oxidase and 5.00 IU / ml peroxidase, relative to the standard solution, thus the excess of the costly and sensitive enzyme component the peroxidase content is significantly lower but extremely high.

Known diagnostic compositions include 4-amino antipyrine and phenol, o-dianizidine.HCl and phenol or o-dianizidine.HCl and methanol as coloring agents.

Known diagnostic compositions have several disadvantages. A common disadvantage is that the absorbance of the color produced during the reaction remains unchanged for a short time (30-60 minutes according to our own measurements). This low color stability is particularly detrimental to large-scale serial analysis, where it may take several hours between sample preparation and actual photometric measurement, which can lead to false results and thus incorrect diagnosis. A further disadvantage is that the formulations have an inadequate storage stability and in some cases the measurement sensitivity is not satisfactory.

The reason for the inadequate storage stability of the formulations is that the enzyme components in the formulations, in particular cholesterol esterase and cholesterol oxidase, are continuously deactivated during storage. This explains why, in the vast majority of known diagnostic formulations, cholesterol esterase and cholesterol oxidase are used in excess of what is needed. However, this high excess of enzymes adversely affects the time course of the first two enzymatic reactions (ester cleavage by cholesterol esterase and subsequent oxidation of cholesterol), especially since serum always contains free cholesterol in addition to the ester bound cholesterol, which is oxidized immediately. so that the two reactions occur partly in parallel and partly in succession. Immediate and quantitative conversion of hydrogen peroxide requires a large excess of peroxidase, and the excess peroxidase is not disturbing, but the vast majority of known diagnostic formulations do not contain peroxidase in excess of other enzymes. According to our studies, the insufficient color stability of the known formulations in diagnostic formulations using high cholesterol esterase and excess cholesterol oxidase is primarily due to the uneven duration of enzymatic reactions. Diagnostic formulations using low cholesterol esterase and excess cholesterol oxidase result in uneven deactivation of enzymes (resulting in an uneven slowing of the two enzymatic reactions) leading to inadequate color stability.

Our experiments have shown that when cholesterol esterase, cholesterol oxidase and peroxidase are co-lyophilized in the presence of bovine serum albumin as one of the coloring components and the lyophilizate is used in this form until further use, Separated from its components - stored, the enzymes stabilize, ie retain their original activity for a long time. That's our realization

EN 207 537 Β also allows the use of small amounts of cholesterol esterase and cholesterol oxidase enzymes, and eliminates factors that lead to deterioration in color stability.

Attempts have also been made to improve the sensitivity of the photometric measurement. The phenolic component of the 4-amino-antipyrin-phenol color forming reagent pair was replaced with other derivatives. No. 4226713. U.S. Pat. However, the description does not include any valid comparative figures for the phenolic derivatives; in the specific examples, only phenol is mentioned as the chromophore agent in addition to 4-amino-anti-pyrin.

In our experiments we found that m-cresol or 2,5-dimethylphenol or 3,4-dichlorophenol, phenol, m-cresol and 2,5-dimethylphenol instead of phenol instead of phenol as the second coloring agent in addition to 4-amino-antipyrin If a mixture of at least two of 20 is used, the sensitivity of the measurement is increased by leaps and bounds.

The present invention therefore relates to a diagnostic composition for the determination of total serum cholesterol, which comprises cholesterol esterase, cholesterol oxidase, peroxidase and 0.51,5 mmol / 14-amino-antipyrin, 20-60 mmol / l magnesium chloride or hydrate, 5-15 mmol / l sodium cholate, 0.25-0.35% w / v, enzyme inert emulsifier, 0.01-0.1% w / v, enzymes containing inactivated bactericidal material, 2-18 mM phenol compound and Tris.HCl buffer to adjust pH to 6.7-8.7. The diagnostic composition of the present invention is a kit consisting of a lyophilized powder mixture and 35 one solution, wherein the lyophilized powder mixture is 1.04-0.1 IU / ml cholesterol esterase, 0.07-0.15 IU / ml cholesterol oxidase, , 5-3.5 IU / ml peroxidase and the above-stated amount of 4-amino-antipyrin, 0.001-0.1% w / v bovin serum albumin, relative to standard solution 40, and the solution contains the other components listed above and, as the phenolic compound, m-cresol or 2,5-dimethylphenol, or a mixture of at least two of 3,4-dichlorophenol, phenol, m-cresol and 2,5-dimethylphenol.

The preferred composition of the lyophilized powder mixture (with reference to the standard solution) is as follows:

4-aminoantipyrine 1.0 mM Cholesterol esterase 0.15 IU / ml cholesterol oxidase 0.09 IU / ml peroxidase 3.3 IU / ml bovin serum albumin 0.1% w / v The second component of the kit is enzymes

for example, Triton 55 X 100 as an emulsifier in vitro and sodium azide as an enzyme inert bactericide. The pH of the solution is particularly preferably from 7.2 to 7.3.

Particularly preferred phenolic compounds in the second component of the kit include the following:

4-7 mmol (preferably 6 mmol) m-cresol,

4-7 mmol (preferably 6 mmol) of 2,5-dimethylphenol, mmol of 3,4-dichlorophenol + 6 mmol of phenol, mmol of 3,4-dichlorophenol + 6 mmol of m-cresol, mmol of 3,4-dichloro -phenol + 6 mmol 2,5-dimethylphenol, mmol mmol 3,4-dichlorophenol + 6 mmol phenol + 6 mmol m-cresol, mmol mol-3,4-dichlorophenol + 6 mmol phenol + 6 mmol 2,5- dimethylphenol, mmol of phenol + 6 mmol of m-cresol.

The most important advantages of the kit according to the invention are:

- lyophilisate containing enzyme components can be stored for a long time without loss of activity,

- the presence of significantly less cholesterol esterase and cholesterol oxidase than is known,

- the measuring solution obtained by mixing the lyophilisate and the buffered solution is stable for at least 3 weeks when stored at a temperature between -2 ° C and -8 ° C,

- the color development during the reaction is at least 5 hours, which is several times the color stability of the known reagents,

- the sensitivity of the measurement is high.

In addition, the reagent kit of the present invention retains the favorable properties of known reagent compositions, i

- allow linear measurement over a wide range of concentrations,

- enables very accurate and well reproducible measurement.

The invention is illustrated in more detail by the following examples.

Example 1

A kit of reagents having the following composition was prepared (figures given are values for the standard solution):

Lyophilized powder mixture: 4-amino-antipyrine 1.0 mM Bovin serum albumin 0.1% w / v Cholesterol esterase 0.05 IU / ml Cholesterol oxidase 0.09 IU / ml peroxidase 3.3 IU / ml Buffered aqueous solution. Tris HCl 0.1 mol / L (pH 7.7) MgCl ₂ .6 H ₂ O 40 mmol / l Sodium cholate 10 mM Phenol 6 mM 3,4-dichlorophenol 4 mmol / l Triton X 100 0.3% w / v Sodium azide 0.1% w / v The standard composition for measurements is as follows solution was used: Cholesterol linoleum 5.17 mmol / l Triton X 100 1.0% w / v Sodium azide 0.001% w / v The lyophilized powder mixture and buffered aqueous solution by mixing to form the standard solution.

HU 207 537 Β

Cholesterol was determined as follows.

Venous blood was used for testing. Following blood collection, the blood sample was centrifuged at 4000 rpm for 20 minutes. Serum was used for the assays. The serum can be stored at 2-8 ° C for 3 days, -20 ° C for 6 months.

For each series of measurements, 3-3 control serum samples were used in the normal, pathological and lipemic ranges. RocheN, RocheP and RocheL control sera were used for the measurements.

At the beginning of the measurement, 20-20 μΐ of serum was added to the test tubes and 2.0 to 2.0 ml of measuring solution was added to the serum samples. The contents of the test tubes were thoroughly mixed and incubated for 15 minutes at 37 ° C. Subsequently, the absorbance of the reaction mixture was measured on a spectrophotometer at a wavelength of 1 cm at a wavelength of 500 nm for 5 hours. 3-3 replicates of each test sample were prepared. One reagent blank and three standards were prepared for each series of measurements; 20 μ reag of distilled water for reagent blank and 20 μΐ of cholesterol linoleate solution as above for standard solution were added to the 2.0 ml standard solution.

The absorbance was measured for 5 hours to determine the color development of the reaction. The curve plotted at 5.17 mmol / l cholesterol linoleate at 25 ° C is shown in Figure 1 a and the curve plotted at 37 ° C with the same standard is plotted in Figure 1 b. The figure shows that the color intensity remains constant for 5 hours.

The calibration line plotted for measuring the absorbance of known cholesterol-containing samples is illustrated in Figure 2. Equation of the line: y = 0.068x + 0.001, where y is the absorbance (nm), x is the cholesterol concentration (mmol / l). For 360 samples, the regression coefficient was 0.9997. It can be seen from the figure that the absorbance varies linearly with concentration over the concentration range of 1.29 to 15.55 mmol / l. Adult human serum has a normal cholesterol level of 3.88-6.46 mmol / l; Thus, the reagent kit of the present invention can be used to measure 2.5 times the normal range along the linear region.

Cholesterol content of standard sera with known cholesterol content was measured to check the accuracy of measurement. The results are shown in Table 1.

/. spreadsheet

Name of the serum Cholesterol content in mmol / l scatter Relative standard deviation,% theoretical because Roche N 2.14 (2.00 to 2.28) 2.14 0,012 0.56 Richge P 2.43 (2.24-2.62) 2.36 0,015 0.64 Roche L 7.24 (6.91 to 7.60) 7.12 0,013 0:18

From the data in Table 1, it can be concluded that the reagent kit of the present invention allows a very accurate measurement.

According to our studies, the stability of the measuring solution is 28 weeks when stored at 28 ° C. The above measurements were repeated for 8 months using a lyophilized powder mixture stored at room temperature. There was no change in color stability or measurement accuracy.

For reproducibility testing, the cholesterol content of 15 different clinical serum samples was also tested in an independent reference laboratory using the reagent kit of Example 1. The results are shown in Table 2.

Table 2

Serial number of the sample Cholesterol content in mmol / l Deviation% reference measurement own measurement First 5.75 5.56 3.40 Second 4.38 4.30 1.91 Third 7.35 7.12 3.21 4th 9.52 9.32 2.20 5th 8.72 8.85 1.00 6th 7.12 7.47 1.05 7th 3.57 3.43 4.00 8th 7.04 7.10 0.85 9th 5.13 5.20 1.36 10th 4.57 4.60 0.66 11th 3.19 3.23 1.25 12th 2.07 2.12 2.42 13th 5.23 5.26 0.57 14th 6.47 6.57 0.77 15th 7.45 7.52 0.94

It can be seen that the difference between the measurements in the two different laboratories is within the 5% allowed, so that the reagent kit according to the invention allows a well reproducible measurement.

Example 2

Examination of measurement sensitivity

Reagent kits were used to test the sensitivity of the assay in which the composition of the lyophilized powder mixture was the same as that described in Example 1, but the quantity and quality of the phenolic compounds in the buffered solution were changed as follows:

Sample 1: 4 mmol of 3,4-dichlorophenol + 6 mmol of phenol (composition of Example 1)

Sample 2: 4 mmol 3,4-dichlorophenol + 2 mmol m-cresol

Sample 3: 4 mmol of 3-4-dichlorophenol + 3 mmol of m-cresol

Sample 4: 4 mmol 3,4-dichlorophenol + 4 mmol m-cresol

HU 207 537 Β

Sample 5: 4 mmol 3,4-dichlorophenol + 5 mmol m-cresol

Sample 6: 4 mmol of 3-4-dichlorophenol + 6 mmol of m-cresol

Sample 7: 4 mmol 3,4-dichlorophenol + 8 mmol m-cresol

Sample 8: 6 mM m-cresol

Sample 9: 6 mmol 2,5-dimethylphenol

Sample 10: 4 mmol phenol + 6 mmol m-cresol

Sample 11: 4 mmol 3,4-dichlorophenol + 6 mmol 2,5-dimethylphenol

Sample 12: 4 mmol 3,4-dichlorophenol + 6 mmol phenol + mmol m-cresol

Sample 13: 4 mmol 3,4-dichlorophenol + 6 mmol phenol + mmol 2,5-dimethylphenol Sample A: 6 mmol 2,4-dimethylphenol

Sample B: 6 mmol 3,5-dimethylphenol

Sample C: 4 mmol 3,4-dichlorophenol + 6 mmol 2,4-dimethylphenol

Sample D: 4 mmol 3,4-dichlorophenol + 6 mmol 3,5-dimethylphenol

1-13. sample is the composition of the invention, while sample A-D serves as a comparison to illustrate that the quality of the phenolic compound is critical to the measurement sensitivity.

Measurement sensitivity was characterized by the sensitivity factor F (ratio of cholesterol concentration to absorbance). The lower the F value, the more sensitive the measurement. The results are shown in Table 3.

Claims (2)

PATENT CLAIMS A diagnostic composition for the determination of total serum cholesterol, which comprises 0.5-1.5 mmol / L 4-aminoantipyrin, 20-60 mmol / L magnesium chloride or its hydrate, cholesterol esterase, cholesterol oxidase, peroxidase, 15 mM sodium cholate, 0.25-0.35% w / v, enzymatically inactive emulsifier, 0.01-0.1% w / v, enzymatically inoculated bactericide, 218 mmol / l phenol and Tris.HCl buffer for the adjustment of pH from 6.7 to 8.7, characterized in that the diagnostic composition is a kit consisting of a lyophilized powder mixture and a solution, wherein the lyophilized powder mixture is 0.04-0. , Containing 1 IU / ml cholesterol esterase, 0.07-0.15 IU / ml cholesterol oxidase, 2.5-3.5 IU / ml peroxidase and the amount of 4-amino-antipyrin indicated in the standard solution. containing 0.001 to 0.1% w / v bovin serum albumin, and the solution contains the other components listed above, and the phenol compound is m-cresol or 2,5-dimethylphenol or 3,4-dichlorophenol, phenol , a mixture of at least two of m-cresol and 2,5-dimethylphenol. (Priority: July 26, 1991) 2. Diagnostic composition for the determination of total serum cholesterol, containing 0.3% w / v Triton X 100 emulsifier, 1.0 mmol / L 4-amino antipyrine, 40 mmol / L in cholesterol esterase, cholesterol oxidase, peroxidase and assay solution. containing magnesium chloride hexahydrate, 10 mM sodium cholate, 0.01% w / v sodium azide, 10 mM phenol and Tris.HCl buffer to adjust pH to 6.9 to 8.2 characterized in that the diagnostic composition is a reagent kit consisting of a lyophilized powder mixture and a solution, wherein the lyophilized powder mixture is 0.05 IU / ml cholesterol esterase, 0.09 IU / ml cholesterol oxidase, 3.3 IU / ml ml of peroxidase and the amount of 4-amino-antipyrin indicated in the standard solution, together with 0.001% w / v bovine serum albumin, in the standard solution, and Contains the other components listed in Circular 5A and a 6: 4 molar mixture of phenol and 3,4-dichlorophenol as the phenolic compound. (Elsőbbsé-