JPH0580056A - Fractioning liquid and analysis method for analysis of high specific gravity lipoprotein cholesterol - Google Patents

Abstract

PURPOSE:To achieve higher accuracy of a quantitative analysis by a method wherein an analysis element is used having a reagent layer and a development layer on one side of a support to determine a calibration curve employing as calibration liquid containing a top serum obtained by fractioning serum based on human serum and a surfactant and protein or the like is assayed using the calibration curve. CONSTITUTION:An analysis element which has a reagent layer on one side of a light transmitting and non-water permeable support and a porous development layer thereon is used to perform a quantitative analysis on protein in a body liquor employing a fractioning liquid containing a top serum obtained by fractioning serum based on human serum and a surfactant as control liquid for accuracy management. The serum is refined from a defatted serum based on serum derived from a human being. The fractioning liquid is obtained by processing the serum by a sedimentation method using a fractioning agent composed of polyanions and bivalent cations.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a calibration curve for quantitative analysis of high-density lipoprotein cholesterol (hereinafter referred to as HDL cholesterol) in a biological fluid such as blood, especially when quantitative analysis is carried out by a dry method multi-layer analytical element. The present invention relates to a fractionation liquid that can be used as a calibration liquid for preparation and a control liquid for quality control.

[0002]

BACKGROUND OF THE INVENTION Since quantitative analysis of high-density lipoprotein cholesterol (hereinafter referred to as HDLC) in a biological fluid such as blood is useful for diagnosis of coronary heart disease, ischemic heart disease, cerebral infarction, etc., Along with the measurement of total cholesterol, it has been particularly emphasized in recent years. The measurement is based on HDL rather than lipoprotein.
It is composed of two steps, an operation of fractionating C and an operation of measuring cholesterol in HDL. The method of fractionation includes ultracentrifugation method, gel filtration method, polyanion-divalent metal precipitation method, electrophoresis method, etc. Is used. Currently HDLC
The precipitation method is often used as the measurement method for the method because it is easy to operate and does not require any special equipment. An enzyme method is widely used for measuring cholesterol.

By the way, the automation of the analyzer has been advanced, and the HDLC measurement has been incorporated into the automatic analyzer, and the cholesterol concentration of the fractionated supernatant serum is usually measured. A reagent for measuring cholesterol or the like is used for the measurement, and a “cholesterol standard solution” or the like is used for calibrating the reagent used. This standard solution is prepared by dissolving standard cholesterol in alcohol such as ethanol. Further, as the control solution for quality control, the above standard solution or a commercially available control serum fractionated is used.

In contrast to the above-mentioned wet method, an analytical element by a dry method has been developed and put into practical use in recent years. This analytical element is a multi-layer analytical material consisting of at least one reagent layer on one side of a light-permeable, water-impermeable support and a porous development layer on the uppermost layer. Quantitative analysis of specific components in body fluids is possible. Currently H
An analytical element for DLC has also been put into practical use, and it is possible to quantitatively analyze the cholesterol concentration of a fractionated specimen.

By the way, when the calibration is performed in the quantitative analysis, the same standard solution as in the wet method cannot be used for the calibration of the analytical element. The reason for this is that the calibration curve used in the analysis element was created based on a human serum sample (supernatant sample fractionated using a fractionating agent). In other words, the calibration curve used to convert the obtained optical density to the substance concentration was created based on a human serum sample (supernatant sample fractionated using a fractionating agent). In the case of a liquid sample having physical properties different from serum, for example, the above-mentioned cholesterol standard solution, the color reaction is different because the solvent is alcohol.

Therefore, there is a drawback that the calibration curve prepared using this standard solution as a calibration solution does not match the true calibration curve for human serum samples. Therefore, when pooled serum prepared by pooling a large number of sera is used, the above-mentioned drawbacks are solved, but the composition is not constant depending on the lot, and especially the long-term storage stability is unstable. There is. Further, when a commercially available control serum is used instead of the pooled serum, the HDLC concentration is generally low, so that it is difficult to obtain a sample in the concentration range necessary for calibration.

Further, these sera need to be fractionated using a fractionating agent, which makes the calibration operation complicated, and is susceptible to the fractionation operation error caused by performing fractionation every time, and the calibration is stable. It lacks sex. Further, when pooling fractions of these sera collectively, there is a drawback that the long-term storage stability is unstable. Further, when the above-mentioned standard solution or a commercially available controlled serum is used as a control solution used for daily quality control, there is a similar problem and there is a drawback that it cannot be controlled accurately.

[0008]

DISCLOSURE OF THE INVENTION It is an object of the present invention to quantitatively analyze HDLC in a biological fluid, particularly HD in a biological fluid using a multilayer analytical element.
It is intended to improve the accuracy of LC quantitative analysis. That is, in the quantitative analysis of HDLC using a multi-layer analytical element, when a calibration curve is created using a calibration solution, the calibration curve obtained by the calibration is the same response as when a human serum is used as a sample, and The aim is to provide a fractionated solution that matches the standard curve of. Another object is to provide a calibration solution and a control solution that can be used for a long period of time.

The above-mentioned object of the present invention is a fractionation liquid used for quantifying high-density lipoprotein cholesterol in a biological fluid, which is obtained by fractionating human serum-based serum and supernatant serum and surface activity. And a high-density lipoprotein cholesterol-analyzing fraction. Further, it is an analytical method for quantifying high density lipoprotein cholesterol concentration in biological fluid, comprising at least one reagent layer on one side of a light-permeable and water-impermeable support and a porous development layer above it. Using an analytical element, a calibration curve is obtained by using a fractionated solution containing supernatant serum obtained by fractionating human serum-based serum and a surfactant as a calibration solution, and using the calibration curve, a biological fluid It is achieved by a method for analyzing high-density lipoprotein cholesterol, which comprises quantifying high-density lipoprotein cholesterol in the medium.

Further, there is provided an analytical method for quantifying high density lipoprotein cholesterol concentration in a biological fluid, which comprises at least one reagent layer on one side of a light-permeable and water-impermeable support and a porous layer above the reagent layer. Using an analytical element having a layer, a fractionated liquid containing supernatant serum obtained by fractionating human serum-based serum and a surfactant was used as a control liquid for quality control, and was It is achieved by a method for analyzing high-density lipoprotein cholesterol, which comprises performing a quantitative analysis of high-density lipoprotein cholesterol.

In the above invention, the serum is preferably serum prepared from delipidated serum and lipid serum based on human-derived serum, and the fraction is polyanion and divalent cation. It is preferably carried out by a precipitation method using a fractionating agent composed of Hereinafter, the present invention will be described in more detail.

The human serum-based delipidated serum used in the present invention is obtained by delipidating an appropriate serum. Regarding lipid sera, a high concentration of lipid sera can be obtained by measuring blood cholesterol and triglyceride. .. It is also possible to select from commercially available raw materials for control serum. As these raw materials, for example, those having the following compositions and concentrations are used. An example of the control serum is shown below, but the control serum used in the present invention is not limited to these.

[Example of Controlled Serum Used] 1) Degreased Serum ・ Origin: Human Serum ・ Cholesterol: 10 mg / dl ・ Triglyceride: 15 mg / dl ・ Protein concentration: 7.5 g / dl ・ pH: 7.2 2) Lipid Serum-Origin: Human serum-Cholesterol: 1020 mg / dl-Triglyceride: 569 mg / dl-Protein concentration: 1.7 g / dl-pH: 7.1 Heparin, dextran sulfate, phosphotungstic acid as polyanions constituting the fractionating agent Examples of divalent cations include Mn ²⁺ , Mg ²⁺ and Ca ²⁺ ,
A combination of these methods can be used. For example, phosphotungstic acid-Mg ²⁺ , dextran sulfate-
Fractionating agents such as Mg ²⁺ , heparin-Mn ²⁺ , heparin-Ca ²⁺ can be used. A preferred fractionating agent is phosphotungstic acid-Mg ²⁺ .

Then, after the serum having a desired concentration is prepared using the above-mentioned serum, the serum is fractionated with a fractionating agent composed of a polyanion and a divalent cation. The supernatant is collected and the surfactant as described below is contained therein to obtain the fractionated solution of the present invention. The fractionation method used is a general method, and serum and the fractionating agent are mixed 1:
After mixing in 1 and sufficiently reacting, the precipitate and the supernatant are separated by centrifugation.

Surfactants added to the supernatant include ionic (anionic: sodium lauryl sulfate,
Lauryl sulfate triethanolamine, POE (2) sodium lauryl ether sulfate, POE (2) lauryl ether sulfate triethanolamine, etc., cationic: coconut alkyltrimethylammonium chloride, benzalkonium chloride, etc.) or nonionic I can, but
Nonionic surfactants are preferred. Specific examples of the nonionic surfactant preferable for the present invention are shown below. still,
It is not limited to these.

POE (10) octyl phenyl ether, POE (15) octyl phenyl ether, POE (30) octyl phenyl ether, POE (12) nonyl phenyl ether, POE (20) nonyl phenyl ether, POE (20) sorbitan mono Laurate-POE (20) sorbitan monooleate-POE (20) sorbitan tristearate-POE (4) trioleate-POE (30) stearate-POE (40) stearate-POE (100) stearate-PEG ( 400) Monostearate-PEG (400) monolaurate-PEG (1000) dilaurate-PEG (1540) distearate-Lauryl alcohol EO 6 mol condensate-Lauryl alcohol EO 10 mol condensate-La Uryl alcohol EO 30 mol condensate-Oleyl alcohol EO 20 mol condensate-Cetyl alcohol EO 20 mol condensate-Triethanolamine oleate (Note) POE: Polyethylene oxide PEG: Polyethylene glycol EO: Ethylene oxide Condensation of ethylene oxide unit The additive concentration of surfactant is 0.03 to the calibration solution.
It is preferably used within the range of 1.0% by weight.

The fractionation liquid of the present invention can be used in any of quantitative analysis methods such as a wet method and a dry method, but it is disclosed in JP-A-64-35369, JP-A-2-6751 and JP-A-2-6751. No. 2-55954, JP-A-2-2102
It is particularly useful for the multilayer analysis elements described in Japanese Patent Application Laid-Open No. 65, JP-A-2-222699, and the like.

[0018]

EXAMPLES The present invention will be described in more detail below with reference to examples. [Example 1] A dry multi-layer analytical element and a fractionation solution were prepared for the purpose of measuring the HDLC concentration in serum and plasma.

1) Preparation of HDLC Analytical Element An HDLC analytical element was produced by sequentially coating a reagent layer, an adhesive layer and a developing layer having the following composition on a transparent undercoated polyethylene terephthalate support having a film thickness of 180 μm and drying. .. [Reagent layer] Gelatin 12.10 (g / m ² ) 7-chloro-3- (2-hexyldecylsulfonylethyl) -6-methyl-pyrazolo [3,2-c] -s-triazole 1.76 (g / M ² ) dibutyl phthalate 0.95 (g / m ² ) peroxidase 12100 (U / m ² ) ascorbate oxidase 4500 (U / m ² ) N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid 3 .71 (g / m ² ) potassium hydroxide 0.81 (g / m ² ) sodium triisopropylnaphthalenesulfonate 0.52 (g / m ² ) 1,2-bis (vinylsulfonyl) ethane 0.05 (g / M ² ) Sodium azide 0.15 (g / m ² ) [Adhesive layer] N-vinylpyrrolidone-vinyl acetate copolymer (polymerization ratio 2: 8) 4.57 (g / m ² ). [Development layer] b Paper raw material fiber 99.20 (g / m ² ) styrene-glycidyl methacrylate copolymer (polymerization ratio 9: 1) 24.73 (g / m ² ) polyoxyethylene lauryl ether 11.41 ( g / m ² ) Hydrochloric acid-4-N, N-diethylamino-2 (2′-methanesulfonamidoethyl) aniline 0.17 (g / m ² ) 5,5-dimethyl-1,3-cyclohexanedione 1. 47 (g / m ² ) ascorbate oxidase 4500 (U / m ² ) cholesterol esterase 1620 (U / m ² ) cholesterol oxidase 1620 (U / m ² ) bovine serum albumin 2.85 (g / m ² ) Note; ascorbine Acid oxidase, cholesterol esterase, and cholesterol oxidase are dissolved in water together with bovine serum albumin and then frozen. A powdered product is used after drying.

2) Preparation of Fraction Solution Based on the following materials and preparation method, a fraction solution having an arbitrary concentration was prepared. [Materials] Serum A: PENTEX HUMAN SERUM DELIPIDIZED (* 2) Serum B: PENTEX HUMAN CHOLESTEROL CONCENTRATE (* 3) Fractionation agent: SERA-PAK (* 4) (* 2) Miles degreased serum (* 3) Miles Lipid (cholesterol-concentrated) serum (Note 4) Miles Italia HDLC fractionating agent (phosphotungstic acid, magnesium chloride) [Preparation] 1. To determine the concentration of serum A and B, fractionation is performed using a fractionating agent. Equivalent amounts of each serum and fractionating agent (for example, 2
00 μl) mix and centrifuge at a minimum of 1500 g for 15 minutes. After centrifugation, collect the supernatant and use HDL with Hitachi 7050
The C was measured. As a result, serum A: 3.5 mg / d
1, serum B: 217 mg / dl.

2. The mixing ratio at any concentration was calculated using the following formula. Ratio (%) of serum B having an arbitrary concentration = (C−D) / (E−
D) C: arbitrary concentration (mg / dl) D: concentration of serum A (mg / dl) E: concentration of serum B (mg / dl) 10 mg / dl, 25 mg / dl as arbitrary concentration
45 mg / dl, 65 mg / dl, 90 mg / dl 5
The levels are mixed in the proportions shown in Table 1 below, fractionated using the above fractionating agent, and the supernatant is collected. A surfactant (POE (10) octylphenyl ether: trade name Triton X-100) was added to this to a concentration of 0.1% by weight to prepare a fractionated solution.

Table 1 Level Target Concentration Serum A Serum B 1 10 mg / dl 0.970 0.030 2 25 mg / dl 0.899 0.101 3 45 mg / dl 0.806 0.194 4 65 mg / dl 0.698 0 .302 5 90 mg / dl 0.571 0.429 3. For comparison, 5 levels having the same concentration as above were prepared by using a cholesterol standard product and preparing a cholesterol standard solution using ethanol as a solvent.

The fractionated solution of the present invention, 20 samples of human serum and the cholesterol standard solution of the comparative example were dropped on each of the two HDLC analysis elements, and the reflection density was measured using Konica Dry Lab 80M (manufactured by Konica Corporation). (Dr) was measured.
A calibration curve obtained by plotting the average Dr of two sheets and the data of the control method (Hitachi 7050) is shown in FIG.

As is clear from FIG. 1, the calibration curve obtained by using the fractionated solution of the present invention as the calibration solution is in good agreement with human serum as compared with the calibration curve using the cholesterol standard solution for comparison. , The same response as when using human serum as a sample,
It can be seen that the calibration curve obtained by calibration agrees with the true calibration curve for human serum samples. [Example 2] In order to investigate the preservability of the fractionated solution of the present invention, the fractionated solution of the present invention (containing 0.03% of Triton X-100) and the fractionated solution containing no surfactant (Comparative Example) Was prepared and stored under frozen conditions (-10 ° C or lower), and immediately after 1 month and 2
Table 2 shows the results measured with the above-mentioned HDLC analysis element and Konica Dry Lab 80M after 4 months and 6 months.
Shown in. Table 3 shows the results of measuring the absorbance at 600 nm using a Hitachi spectrophotometer (U-2000) in order to observe the state of the fractionated liquid.

Table 2 (Measurement by Konica Dry Lab 80M, unit mg / dl) Concentration Immediately 1 month later 2 months 4 months 6 months 25 mg / dl The present invention 25.3 24.8 25.1 24.8 24 .7 Comparative Example 25.1 23.9 22.4 21.2 20.4 90 mg / dl Invention 89.8 90.1 90.7 89.2 89.5 Comparative Example 90.2 86.3 81.1 74.4 72.7 Table 3 (Measurement by U-2000, absorbance) Concentration Immediately 1 month later 2 months 4 months 6 months 25 mg / dl Present invention 0.039 0.039 0.040 0.042 0.045 Comparative example 0.041 0.063 0.078 0.089 0.104 90 mg / dl Present Invention 0.036 0.037 0.040 0.044 0.049 Comparative Example 0.039 0.077 0.101 0.142 0.199 As shown in Table 2, the fraction-containing solution containing no surfactant (Comparative Example) shows a change in the measured value even during frozen storage. Further, as shown in Table 3, precipitates were observed over time in the fractionation liquid containing no surfactant (Comparative Example). On the other hand, the measured value of the fractionated liquid of the present invention containing a surfactant was stable, and precipitation (white turbidity) was not observed.

Further, it can be seen that HDLC can be accurately quantitatively analyzed when the fractionated liquid of the present invention is used as a control liquid for daily quality control. [Example 3] In the same manner as in Example 2, polyoxyethylene sorbitan monolaurate (trade name: Tween 20) was used as a surfactant to prepare a fractionated solution of the present invention. The content concentration of Tween 20 was 1.
It is 0% by weight.

As a comparative example, a calibration solution containing no surfactant was prepared and stored under frozen conditions (-10 ° C. or below), and immediately after 1 month, 2 months, 4 months, 6 months, the above-mentioned HDL.
Table 4 shows the results of measurement using the C analysis element and Konica Dry Lab 80M. Table 5 shows the results of measuring the absorbance at 600 nm using a Hitachi spectrophotometer (U-2000) in order to observe the state of the fractionated liquid.

Table 4 (Measurement by Konica Dry Lab 80M, unit mg / dl) Concentration Immediately 1 month 2 months 4 months 6 months 25 mg / dl Invention 24.7 24.4 24.8 24.2 23.9 Comparative Example 25.1 23.7 22.4 21.2 20.4 90 mg / dl Invention 90.4 89.6 90.1 91.6 89.6 Comparative Example 90.2 86.3 81.1 74.4 72 .7 Table 5 (Measurement by U-2000, absorbance) Concentration Immediately 1 month 2 months 4 months 6 months 25 mg / dl Invention 0.038 0.038 0.040 0.041 0.044 Comparative Example 0.041 0.063 0.078 0.089 0.104 90 mg / dl Invention 0.037 0.038 0.041 0.044 0.048 Comparative Example 0.039 0.077 0.101 0.142 0.199 As shown in Tables 4 and 5, the fractionated liquid of the present invention containing Tween 20 had stable measured values and precipitation (white turbidity).
It turns out that it is not admitted.

Further, it can be seen that HDLC can be accurately quantitatively analyzed when the fractionated liquid of the present invention is used as a control liquid for daily quality control.

[Brief description of drawings]

FIG. 1 is a graph showing a calibration curve.

Claims (5)

[Claims] 1. A fractionation liquid used for quantifying high-density lipoprotein cholesterol in a biological fluid, comprising a supernatant serum obtained by fractionating human serum-based serum and a surfactant. A fractionated liquid for high-density lipoprotein cholesterol analysis, which comprises: 2. The fractionated liquid for high-density lipoprotein cholesterol analysis according to claim 1, wherein the serum is a serum prepared from defatted serum and lipid serum based on human serum. 3. The fraction for high-density lipoprotein cholesterol analysis according to claim 1, wherein the fractionation was performed by a precipitation method using a fractionating agent composed of a polyanion and a divalent cation. liquid. 4. An analytical method for quantifying high specific gravity lipoprotein cholesterol concentration in a biological fluid, comprising at least one reagent layer on one side of a light-permeable and water-impermeable support and a porous layer above the reagent layer. Using an analytical element having a layer, a calibration curve is obtained using a fractionated solution containing a supernatant serum obtained by fractionating human serum-based serum and a surfactant as a calibration solution, and the calibration curve is used. A method for analyzing high-density lipoprotein cholesterol, which comprises quantifying high-density lipoprotein cholesterol in a biological fluid. 5. An analytical method for quantifying high specific gravity lipoprotein cholesterol concentration in a biological fluid, comprising at least one reagent layer on one side of a light-permeable and water-impermeable support and a porous development above the reagent layer. Using an analytical element having a layer, a fractionated liquid containing supernatant serum obtained by fractionating human serum-based serum and a surfactant was used as a control liquid for quality control, and was A method for analyzing high-density lipoprotein cholesterol, which comprises quantitatively analyzing high-density lipoprotein cholesterol.