JPS6047552B2 - C-reactive protein measurement reagent - Google Patents

Description

[Detailed description of the invention] The present invention relates to C-reactive protein (hereinafter referred to as CRP).

) Regarding measurement reagents. Conventionally, CRP refers to a protein that has an affinity for a polysaccharide called the C fraction of pneumococci, and is known to have a weak affinity with various other polysaccharides such as dextran sulfate, heparin, etc., or with the same acid. .

CRP utilizing this affinity with CRP(7)C polysaccharide
Although a measurement method has been proposed, it is not used today because the reagents used in this method are unstable and the measurement results are inconsistent. In recent years, research has progressed on the affinity between CRP and C-polysaccharide, and it is also known that phosphorylcholine residues have an important significance in the affinity with CRP, and that calcium ions are essential for this reaction. It became like that. Since CRP has strong species-specific antigenicity,
Conventionally, immunochemical measurement methods using anti-CRP antibodies have been put into practical use. However, the most common method of this method, which uses capillary precipitation reaction, is complicated, requires a long time to judge the results, and requires a long time to produce a specific anti-CRP antibody. There was a problem. The present invention aims to improve the above-mentioned difficulties, overcomes the manufacturing difficulties by using lecithin, which is relatively easily obtained, as a raw material, and has extremely simple measurement techniques, and can be determined in a short time of less than m minutes. The present invention provides reagents for slide agglutination reactions that can be used for slide agglutination reactions.

The reagent for measuring CRP of the present invention is based on the report that choline phosphoratiides having this phosphorylcholine residue, such as lecithin and sphingomyelin, also show a strong affinity for CRP in the presence of calcium ions. , was developed. The reagent for measuring CRP of the present invention includes a first solution in which lecithin, cholesterol, and a fat-soluble dye are uniformly suspended in a buffer containing 5 to 40% choline chloride together with a surfactant; It is characterized in that it consists of a second physiological saline sluice containing 2M calcium and magnesium ions. Also,
0.2 to the first solution and physiological saline containing 0.02 to 0.2 rV1 of calcium and magnesium ions.
It is characterized by consisting of a second liquid to which ~1.0% of sodium polyanethole sulfonate is added.

That is, the present invention stabilizes the reagent by adding choline chloride to a suspension of lecithin and cholesterol, and adding calcium and magnesium ions, which are thought to be factors that destabilize lecithin, to a separate solution. It has become.

Normally, the reaction between a suspension of lecithin or cholesterol and CRP appears in the form of aggregation of the particles, and is generally observed with a microscope or by optical observation such as turbidimetry. If this reagent is used, cholesterol is stained with a fat-soluble dye, which creates a state similar to an agglutination reaction on the slide, making it easy to observe with the naked eye.

Please note that the above procedure may cause the measured values to vary and may not necessarily match the measured values for immunochemical assays.
As a result of research into the cause, the present inventor discovered that it lies in complement activity in serum. For this purpose, either inactivate the test serum by heating it at 56°C for 3 minutes, or add 0.2 to 2.0% sodium polyanethole sulfonate to the second reagent solution to increase its complement activity. By pressing
CRP measurements according to the present invention now match those of immunochemical assays. The preparation of the CRP measurement reagent of the present invention is, for example, as follows.

Preparation of O first solution Cholesterol 0.8-1.2%, Lecithin 0.2-0
．． 6%, Span 60 (Product name (Zorbitan Monostearate) 0.05%, Stan Black 0.1-0.2
Gradually mix 1 volume of alcoholic solution containing 1% to 9 volumes of veronal buffer (pH 8.6) containing 5-40% choline chloride to homogeneously suspend.

During storage, add preservatives such as sodium azide as appropriate. Preparation of second solution Dissolve 0.02-0.2M of calcium chloride and magnesium chloride in physiological saline or further dissolve 0.2-1.0M of calcium chloride and magnesium chloride.
% sodium polyanethole sulfonate.

Next, the procedure for CRP measurement using the reagent of the present invention is, for example, as follows.

Procedure for measuring 0CRP Take 0.05ml of the serum or body fluid to be tested on a slide plate, add 0.05ml of the second liquid to it, mix it, then add one drop of the first liquid and let it react for 8 minutes to check for agglutination. Determine.

The CRP measuring reagent of the present invention will be specifically explained below with reference to Examples.

Example 1 Cholesterol 0.9%, lecithin 0.3%, Span 6
0 (trade name) 0.5% and Stan black 0.1% were gradually mixed into 9 volumes of veronal buffer (pH 8.6) containing 10% choline chloride. The first solution was combined with a second solution of physiological saline containing 0.02 M calcium chloride and magnesium chloride, and CRP was measured for 15 serum samples heated at 56° C. for 30 minutes according to the CRP measurement procedure described above.

The state of aggregation was measured according to the following criteria. C2ll standard test...Large aggregates are observed+...
・Soil where medium-sized aggregates are observed - Many small aggregates are observed + Soil where numerous extremely small aggregates are observed: Agglomerates are unclear Table 1 shows the results in which no agglomerates were observed.

As shown in Table 1, when inactivated test serum was measured using the reagent of the present invention, it was found that the results were in good agreement with the precipitation method, which is a conventional immunoassay method.

Example 2 Cholesterol 0.9%, Lecithin 0.45%, Span 80 (trade name) 0.5%, Stan Black 0.15%
A first solution prepared by gradually mixing 1 volume of an alcoholic solution containing 10% choline chloride with 9 volumes of veronal buffer (pH 8.6) containing 10% choline chloride, and 0.03M calcium chloride,
A second solution containing 0.02 M magnesium chloride and 0.2% sodium polyanethole sulfonate was combined, and CRP was measured for fresh serum 1C@ and 5 cases of cerebrospinal fluid according to the procedure for CRP measurement described above.

The judgment criteria are the same as in Example 1. The results are shown in Table 2. As shown in Table 2, even in specimens with strong complement activity (fresh serum, cerebrospinal fluid, etc.), the reagent of the present invention containing sodium polyanethole sulfonate is effective for CRP. It was found that the measured values were in good agreement with the conventional sedimentation method even if the inactivation operation was omitted.

Example 3 Cholesterol 1.2%, lecithin 0.6%,
Span 60 (product name) 0.5%, Oil Red 0.5
The first solution was prepared by gradually mixing 1 volume of an alcoholic solution containing 15% choline chloride with 9 volumes of veronal buffer (pH 8.6) containing 15% choline chloride, 0.05r1-4 calcium chloride, 0. A second solution containing 0.2 M magnesium chloride and 0.3% sodium polyanethole sulfonate was combined, and CRP was quantitatively measured for five types of serum according to the CRP measurement procedure described above.

The test serum was diluted with physiological saline according to the dilution ratio shown in Table 3 below. The results are shown in Table 3.

As shown in Table 3, it was found that the final dilution concentration for agglutination using the reagent of the present invention was in good agreement with the final dilution concentration for sedimentation using the capillary sedimentation method.

As described above, the reagent for CRP measurement of the present invention stabilizes the reagent as a two-part reagent, reduces the variation in measured values, and can match well with the measured values by immunochemical measurement.

Claims (1)

[Claims] 1. A first solution in which lecithin, cholesterol and a fat-soluble dye are uniformly suspended together with a surfactant in a buffer containing 5 to 40% choline chloride; A reagent for measuring C-reactive protein, comprising a second solution of physiological saline containing calcium and magnesium ions. 2. A first solution containing lecithin, cholesterol and a fat-soluble dye, together with a surfactant, uniformly suspended in a buffer containing 5-40% choline chloride, and 0.02-0.20M calcium and magnesium ions. C, characterized in that it consists of a second liquid in which 0.2 to 1.0% sodium polyanethole sulfonate is added to physiological saline.
Reagent for measuring reactive proteins.