JP7082544B2 - Method for eliminating bubbles generated in the reagent for measuring complement value and the reagent for measuring complement value - Google Patents

Description

The present invention relates to a reagent for measuring complement value and a method for eliminating bubbles generated in the reagent for measuring complement value.

Complement-binding antibodies against pathogens such as viruses and bacteria increase or decrease in complement titer when infected with the pathogen. The measurement of complement titer is important as an index of diagnosis, judgment of therapeutic effect and immune ability in various diseases.

In the conventional complement valence measurement method, sensitized erythrocyte erythrocytes in which an anti-sheep erythrocyte antibody is bound to sheep erythrocytes and a solution containing complement gradually diluted to the optimum multiple (serum, etc.) are mixed and mixed at 37 ° C. 6 The Mayer method is generally used to calculate the hemolytic rate of erythrocytes from a graph and obtain the complement value by measuring the hemoglobin in the erythrocytes that leaks into the solution as a result of the rupture of the erythrocytes due to the action of complement after reacting for a minute by the absorbance method. It was a target. In recent years, instead of measuring hemoglobin, an automatic method of measuring the hemolysis rate of red blood cells by optically measuring the turbidity of residual red blood cells and measuring the complement value with an automatic chemical analyzer has become common. (Patent Document 1). In the automatic method, a reagent that has been stirred in advance is set in an automatic chemical analyzer for measurement, but if the time from setting the reagent to measurement is long, the red blood cells in the reagent gradually settle. As a result, when the automatic analyzer collected the reagent as it was, the predetermined amount of red blood cells required for the measurement could not be collected and the measurement could not be performed accurately. Therefore, in order to perform accurate measurement, it is necessary to stir the reagent immediately before the measurement or when there is an interval between measurements, which makes the measurement work complicated.

When the reagent is agitated for each measurement, bubbles are generated in the reagent. As a result, bubbles are generated on the liquid surface when the reagent is collected, so that the automatic chemical analyzer cannot correctly detect the liquid level of the reagent, and the predetermined amount of reagent required for measurement may not be accurately collected. In addition, there is a problem that the detection sensitivity and reproducibility are deteriorated due to the residual foam in the analysis cell.

Japanese Unexamined Patent Publication No. 2004-301612

An object of the present invention is to provide a reagent for measuring complement value, which can quickly eliminate bubbles generated when the reagent is stirred.

As a result of diligent research, the inventors of the present invention have found that by adding polyalkylene glycol to a conventional reagent for measuring complement value, the reagent is stirred and even if foam is generated, it is quickly eliminated. Was completed.

That is, the present invention provides the following.
(1) A reagent for measuring complement value containing sensitized erythrocytes or sensitized liposomes suspended in a buffer solution, which comprises polyalkylene glycol (excluding polyethylene glycol) . ..
(2) The reagent according to (1), wherein the polyalkylene glycol is polyoxypropylene glycol.
(3) The reagent according to (1) or (2), wherein the concentration of the polyalkylene glycol is 0.0005% by weight or more based on the total amount of the reagent (excluding sensitized erythrocytes or sensitized liposomes).
(4) A method for eliminating foam generated in a complement titer measuring reagent containing sensitized erythrocytes or sensitized liposomes suspended in a buffer solution, which comprises adding polyalkylene glycol as a defoaming agent to the buffer solution.
(5) The method according to (4), wherein the polyalkylene glycol is polyoxypropylene glycol.
(6) The method according to (4) or (5), wherein the concentration of the polyalkylene glycol is 0.0005% by weight or more based on the total amount of the reagent (excluding sensitized erythrocytes or sensitized liposomes).
(7) A diluted solution for measuring complement value, which contains polyalkylene glycol (excluding polyethylene glycol ) in a buffer solution.
(8) The sample diluent according to (7), wherein the polyalkylene glycol is polyoxypropylene glycol.
(9) The sample diluent according to (7) or (8), wherein the concentration of the polyalkylene glycol is 0.0005% by weight or more based on the total amount of the sample diluent.

As experimentally shown by the following Examples and Comparative Examples, when the reagent for measuring complement value of the present invention is used, bubbles generated when the reagent is stirred can be quickly eliminated. Therefore, when the reagent of the present invention is used, it is possible to avoid the problem that the automatic chemical analyzer cannot correctly detect the liquid level of the reagent due to the generation of foam on the liquid surface and cannot accurately collect the predetermined amount of the reagent required for measurement. In addition, since foam does not remain in the analysis cell, it is possible to prevent deterioration of detection sensitivity and reproducibility due to foam, which is advantageous for automatic measurement of complement value.

The calibration curve obtained in Example 5 below is shown. The calibration curve obtained in Example 6 below is shown. The calibration curve obtained in Example 7 below is shown. The calibration curve obtained in Example 8 below is shown. The calibration curve obtained in Example 9 below is shown. The calibration curve obtained in Example 10 below is shown.

As described above, the reagent for measuring complement value of the present invention contains polyalkylene glycol in the reagent so that even if the reagent is stirred immediately before the measurement and foam is generated, it can be quickly eliminated. It is characterized by that. As the polyalkylene glycol, polyoxypropylene glycol is preferable. Since polyoxypropylene glycol itself is well known and commercially available, a commercially available product can be preferably used. For example, it is commercially available from Sanyo Chemical Industries under the trade name of "Kararin", and among them, "Kararin 102" (trade name) can be mentioned as a preferable example.

The concentration of the polyalkylene glycol to be blended is preferably 0.0005% by weight or more based on the total amount of the reagent containing no sensitized erythrocytes or sensitized liposomes in the buffer solution, considering the effect of quickly eliminating foam. If more remarkable defoaming effect is desired, 0.0025% by weight or more is preferable. On the other hand, considering the influence on the performance of the reagent, it is preferably less than 0.01% by weight, more preferably 0.0095% by weight or less, still more preferably 0.009% by weight or less. Any combination of the lower limit value and the upper limit value of these preferable ranges may be used.

Except for containing the above-mentioned polyalkylene glycol, the composition of the reagent and the measuring method using the same may be the same as those conventionally used. That is, after the sample containing complement is diluted with a diluted solution, a sample diluted with a reagent in which sensitized erythrocytes are suspended in a buffer solution is added and reacted. Since the number of erythrocytes depending on the complement value in the sample is hemolyzed, the absorbance of the supernatant is measured after centrifugation, or the hemolyzed erythrocytes lose their turbidity in proportion to the complement value in the sample. By measuring the turbidity at a wavelength of 600 to 700 nm, the complement value in the sample can be measured. In automatic chemical analyzers, the latter method of measuring turbidity is widely adopted. The sensitized erythrocyte suspension buffer is not particularly limited, but a phosphate buffer solution containing albumin and glucose is used, and this buffer solution can also be preferably used in the present invention. In addition, sensitized liposomes may be used instead of sensitized erythrocytes. Methods using sensitized liposomes are also well known in the art.

When the complement value in the sample is measured using the above-mentioned complement value measuring reagent of the present invention, the reagent is vigorously stirred to generate bubbles in order to uniformly mix the red blood cells in the reagent (erythrocyte suspension). Even so, it will be erased promptly. By blending the above polyalkylene glycol, bubbles generated in the reagent are eliminated. Accordingly, the present invention also arises in complement titer measurement reagents comprising sensitized erythrocytes or sensitized liposomes suspended in a buffer, comprising adding polyalkylene glycol as an antifoaming agent to the buffer. It also provides a method of eliminating foam.

Further, as specifically shown in the following examples, when a buffer solution containing polyalkylene glycol is used as a diluent, the method of the present invention can be easily carried out. Therefore, the present invention has a polyalkylene glycol in the buffer solution. Also provided is a diluted solution for measuring complement titer, which comprises.

Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to the following examples. In addition, "%" means all by weight%.

Examples 1 to 4, Comparative Example 1
Caralin 102 (trade name) was added to Veronal buffer (concentration: calcium 0.0022%, magnesium 0.011%) containing 0.1% gelatin, calcium ions and magnesium ions as a sample diluent, as shown in Table 1 below. Added to concentration. Next, each sample diluted solution was dispensed into a plastic bottle and then sealed. Each bottle was shaken violently by hand to generate bubbles, and then the time until the bubbles disappeared was followed up.

×：消泡性なし、○：消泡性良好、◎：消泡性極めて良好

×: No defoaming property, ○: Good defoaming property, ◎: Very good defoaming property

The defoaming effect was obtained when the concentration of Caralin 102 (trade name) was 0.0005% or more. In particular, when the concentration of Caralin 102 (trade name) was 0.0025% or more, the effect was that the time until defoaming was short.

Examples 5 to 10, Comparative Example 2
Sensitive erythrocytes (concentration about 2%) in which an antibody was bound to sheep erythrocytes were suspended in a phosphate buffer solution containing albumin (concentration 0.5%) and glucose (concentration 1%). After stirring this sensitized sheep erythrocyte suspension, it was set in an automatic chemical analyzer (Hitachi 7180 type), and the standard complement HC "Seiken", which is the standard solution of Auto CH50-L, was used to measure 1.5 μl of the sample. 195 μl of the sample diluent of Example 1 was added to dilute the sample, 23 μl of the above erythrocyte suspension was further added, and the mixture was reacted at 37 ° C. for 6 minutes, and then the turbidity at a wavelength of 660 nm was measured and a calibration line was drawn.

When the concentration of Caraline 102 (trade name) in the sample diluent was 0% (Comparative Example 2), the foam did not disappear, so the automatic chemical analyzer could not correctly detect the liquid level of the reagent, resulting in an error and measurement impossible. Met. On the other hand, the caraline 102 (trade name) concentration was 0.0005% (Example 5), 0.0025% (Example 6), 0.009% (Example 7), 0.0095% (Example 8). In the case of 0.01% (Example 9) or 0.015% (Example 10), the foam disappeared and measurement by an automatic chemical analyzer was possible. The concentration of cararin 102 (trade name) was 0.095%, which slightly affected the reactivity, and 0.01%, which affected the measured value. It is considered that this is because when the caralin 102 (trade name) is contained in a high concentration, it has a function of inhibiting the complement hemolysis reaction, and since the caralin 102 (trade name) itself is a cloudy liquid, the concentration is high. It is probable that the addition caused turbidity in the diluted solution, which affected the turbidity measurement.

Claims (9)

A reagent for measuring complement value, which comprises polyalkylene glycol (excluding polyethylene glycol) in a reagent for measuring complement value containing sensitized erythrocytes or sensitized liposomes suspended in a buffer solution. The reagent according to claim 1, wherein the polyalkylene glycol is polyoxypropylene glycol. The reagent according to claim 1 or 2, wherein the concentration of the polyalkylene glycol is 0.0005% by weight or more based on the total amount of the reagent (excluding sensitized erythrocytes or sensitized liposomes). A method for eliminating foam generated in a complement titer measuring reagent containing sensitized erythrocytes or sensitized liposomes suspended in a buffer solution, which comprises adding polyalkylene glycol as a defoaming agent to the buffer solution. The method according to claim 4, wherein the polyalkylene glycol is polyoxypropylene glycol. The method according to claim 4 or 5, wherein the concentration of the polyalkylene glycol is 0.0005% by weight or more based on the total amount of the reagent (excluding sensitized erythrocytes or sensitized liposomes). A sample diluent for measuring complement titer, which contains polyalkylene glycol (excluding polyethylene glycol) in the buffer solution. The sample diluent according to claim 7, wherein the polyalkylene glycol is polyoxypropylene glycol. The sample diluent according to claim 7 or 8, wherein the concentration of the polyalkylene glycol is 0.0005% by weight or more based on the total amount of the sample diluent.

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