JP2000310640A - Treatment of specimen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a specimen treatment method for preventing non-specific reaction of latex particles in an immunological analyzing method using latex particles to achieve the enhancement of measuring sensitivity, the reduction of an error and the enhancement of reliability. SOLUTION: A speciment treatment method in an immunological analyzing method using latex particles is characterized by that a carbohydrate is contained in a specimen in an amt. of 2-40 pts.wt. per 100 pts.wt. of the specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for treating a sample in an immunological analysis using latex particles, and in particular, to improve the measurement sensitivity by including a saccharide in the sample. The present invention relates to a sample processing method.

[0002]

2. Description of the Related Art Conventionally, there has been known an immunological analysis method for analyzing a specific antigen or antibody in a sample by utilizing a specific reaction by an antigen-antibody. In particular, the immunological assay method using a carrier such as latex particles can be used for any of a test for detecting an analyte by simple visual inspection and a quantitative analysis method using a measuring device. So it has become the mainstream of immunological analysis.

An immunoassay using a carrier such as latex particles as described above is a method using latex particles to which an antibody or an antigen to a substance to be analyzed is bound. Immunochromatography (US Pat. Nos. 4,168,146 and 409)
No. 4847, No. 42356601, No. 4361537
No.) are known.

However, since latex particles have a hydrophobic surface, they tend to cause non-specific reactions in the measurement system. As a result, in the immunoagglutination measurement method, there was a problem that latex particles caused nonspecific aggregation in a solution, resulting in a decrease in measurement sensitivity and a large error. In immunochromatography,
Latex particles generally have a larger particle size than other carriers such as metal colloids, and cause clogging in the porous membrane due to non-specific reactions, preventing the particles from flowing, resulting in reduced sensitivity and accurate There was a serious problem that the judgment could not be made. In particular, when serum or plasma was used as a specimen, these nonspecific reactions were large, greatly affecting the measurement results, and causing a large error.

[0005] In order to solve the above problems, attempts have been made to reduce the influence on the measurement by reducing the particle diameter, but at the same time, there has been a problem that the measurement sensitivity is reduced. Further, in the immunochromatography method, various studies such as increasing the pore size of the porous membrane have been performed,
When the pore size is increased, the developing speed of the specimen or the like is increased, and there is a fear that the detection sensitivity is reduced. In analyzing serum, plasma, etc., a method using a development-promoting solution, a diluting solution, or the like has been studied. However, there has been a problem in that the sample is diluted so that the measurement sensitivity is reduced.

[0006]

In view of the above, the present invention provides an immunological analysis method using latex particles.
It is an object of the present invention to provide a method for treating a sample for preventing non-specific reaction of latex particles, improving measurement sensitivity, reducing errors, and improving reliability.

[0007]

According to the first aspect of the present invention,
A method for treating a sample in an immunological analysis method using latex particles, wherein the sample contains saccharide, and the content of the saccharide is 2 to 40 parts by weight with respect to 100 parts by weight of the sample. A method for processing a specimen, characterized in that:

The invention according to claim 2 is the method for treating a specimen according to claim 1, wherein the immunological analysis is an immunoagglutination assay or an immunochromatography.

The invention according to claim 3 is that the saccharide is at least one selected from the group consisting of monosaccharides, disaccharides and oligosaccharides.
3. The method for treating a specimen according to claim 1, wherein the specimen is a seed.

[0010] The invention according to claim 4 is characterized in that the saccharide is at least one selected from the group consisting of glucose, fructose, mannose, sucrose, trehalose, maltose, lactose and cellobiose. 2 is a method for processing a sample according to 2.

The present invention relates to a method for treating a sample in an immunological analysis method using latex particles, wherein the sample contains a carbohydrate, and the content of the carbohydrate is 2 parts per 100 parts by weight of the sample. -40 weight parts. Hereinafter, the present invention will be described.

Examples of the immunological analysis include an immunoagglutination assay using an agglutination reaction in a solution, and an immunochromatography method using a development transfer by capillary action in a solid phase. However, the present invention is not particularly limited thereto as long as it is an immunological analysis method using latex particles.

[0013] The reagent for measuring the immunoagglutination reaction used in the above-described immunoagglutination reaction method or the immunochromatography apparatus used in the above-mentioned immunochromatography method comprises:
There is no particular limitation, and known ones can be used.

The latex particles are not particularly restricted but include, for example, homopolymers and copolymers of vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylates and methacrylates; styrene-butadiene copolymers. Fine particles such as a polymer and a butadiene-based copolymer such as a methyl methacrylate-butadiene copolymer are exemplified, and a styrene-based one is particularly preferable.

The particle size of the latex particles is not particularly limited, but is preferably 50 to 5000 nm.

The specimen is not particularly limited as long as it is a biological sample such as whole blood, serum, or plasma, but is more preferably serum or plasma.

In the present invention, the sample contains a saccharide. In addition, the content is 100 parts by weight of the sample,
2 to 40 parts by weight. As a result, non-specific reactions of latex particles are prevented, improving measurement sensitivity, reducing errors,
It is possible to improve the reliability. In addition, the more preferable content of the saccharide in the sample is 4 to 20 parts by weight based on 100 parts by weight of the sample.

The above-mentioned saccharide may be contained in the sample before the immunological analysis is performed, and the method for containing the saccharide is not particularly limited. Here, before the immunological analysis is performed, in the above-mentioned immunoagglutination measurement method, means before or at the time when the immunoagglutination measurement reagent is added to the sample, and the immunochromatography method is used. Refers to the time before or at the time of addition of the sample to the immunochromatography apparatus.

Therefore, in the method for treating a sample of the present invention, the above-mentioned saccharide is contained in the sample directly by dissolving a predetermined amount of saccharide in a liquid such as a buffer solution or water. Examples of the method include a method of adding a saccharide, and a method of adding a predetermined amount of a saccharide to a container or the like in which a specimen is stored in advance.

The saccharide may be a substance generally classified as a saccharide, such as a polyhydric alcohol, a monosaccharide, a disaccharide, and an oligosaccharide. In particular, monosaccharides, disaccharides and oligosaccharides are used. Is preferred. In addition, polyhydric alcohol, monosaccharide,
The disaccharides / oligosaccharides also include these modifications.

Examples of the polyhydric alcohol include sorbitol and mannitol.

Examples of the above monosaccharide include glucose, fructose, mannose, xylose, erythritol and the like.

The disaccharides include, for example, sucrose, trehalose, maltose, lactose, cellobiose and the like.

Examples of the above oligosaccharides include fructooligosaccharides, galactooligosaccharides, polydextrose and the like.

Among the above saccharides, it is particularly preferable to use at least one selected from glucose, fructose, mannose, monosaccharides, sucrose, trehalose, maltose, lactose and cellobiose, which are monosaccharides.

In the method for treating a sample according to the present invention, a blocking agent, a buffer, a blood coagulation promoter, a blood anticoagulant, a surfactant, and / or a carbohydrate may be contained in the sample before, after, or at the same time as containing the carbohydrate in the sample. Etc. may be added.

[0027]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. (A) Sample treatment method in immunoagglutination assay [Example 1] 1) Preparation of immunoagglutination assay reagent Latex particle dispersion (latex N-400 10%)
(W / v), particle diameter 400 nm, manufactured by Sekisui Chemical Co., Ltd.)
100 mM phosphate buffer (pH 7.5) 9 in 1.0 ml
ml, and centrifuged at 15000 rpm for 20 minutes. 0.5 mg / ml of HBs was added to the obtained sediment.
1 ml of a 10 mM Tris buffer (pH 7.4) containing the antigen was added, mixed well, and stirred at room temperature for 1 hour.
15,000 rpm to remove unreacted HBs antigen
The precipitate was suspended in 2.5 ml of 100 mM phosphate buffer (pH 7.5) and centrifuged again. Furthermore, a 100 mM phosphate buffer (pH: 1%) containing bovine serum albumin at a concentration of 1% (w / v) was used.
7.5) Suspend this sediment in 2.5 ml and add 1 ml at room temperature.
After stirring for an hour, a blocking treatment was performed. Then, 15
After centrifugation at 000 rpm for 20 minutes, 1% (w
/ V) concentration of bovine serum albumin and 0.01% (w /
v) The precipitate was suspended in 2 ml of a 100 mM phosphate buffer (pH 7.5) containing sodium azide at a concentration, and refrigerated.

2) Treatment of Sample 1 μl of 100 mM phosphate buffer (pH 7.5) containing 2 μg / μl of sucrose was added to 50 μl of HBs antibody negative serum. (3.9 parts by weight of sucrose per 100 parts by weight of serum. However, the specific gravity of serum was calculated as 1.028)

3) Immunological analysis HBs antigen-sensitized latex particles at a concentration of 0.5% (w / v), bovine serum albumin at a concentration of 1% (w / v) and 0.0
10 containing 1% (w / v) concentration of sodium azide
50 μl of 0 mM phosphate buffer (pH 7.5) and a sample to which sucrose was added in the above 2) were mixed, and the mixture was gently stirred for 3 minutes with an agglutination reaction measurement plate (manufactured by Sekisui Chemical Co., Ltd.). Observed by visual inspection, 0 that does not aggregate
A sample having a remarkable agglomeration was rated on a scale of 6 as 5. The average value of the three judges was taken as the cohesive strength, and the results are shown in Table 1.

Example 2 Example 2 was repeated except that the sucrose content was changed to 39 parts by weight per 100 parts by weight of serum. Table 1 shows the results
It was shown to.

[Comparative Example 1] The procedure of Example 1 was repeated, except that the sucrose content was changed to 1.3 parts by weight per 100 parts by weight of serum. The results are shown in Table 1.

[Comparative Example 2] The procedure of Example 1 was repeated, except that the sucrose content was changed to 50 parts by weight per 100 parts by weight of serum. Table 1 shows the results
It was shown to.

(B) Method of treating specimen in immunochromatography [Example 3] 1) Preparation of immunochromatography apparatus Hi-Flow ^™ nitrocellulose membrane (SRH)
F: Millipore Co.) was cut into a width of 5 mm and a length of 50 mm.
1 μl of 10 mM Tris-HCl buffer (pH 7.4) containing the HBs antigen at a concentration was applied in a circular shape and dried at room temperature for 2 hours. Thereafter, the plate was immersed in 100 mM phosphoric acid (pH 7.5) containing bovine serum albumin (manufactured by DIFCO) at a concentration of 1% (w / v) for 1 hour to perform a blocking treatment. (V) Washed with 100 mM phosphate buffer (pH 7.5) containing sodium laurylbenzenesulfonate at a concentration and dried at room temperature.

Blue colored latex particle dispersion (color latex C-300B, 10% (W / V), particle size 30)
0 nm, manufactured by Sekisui Chemical Co., Ltd.) and 9 ml of 100 mM phosphate buffer (pH 7.5) were added to 1.0 ml, and 15,000 r
Centrifugation was performed at pm for 20 minutes. In the obtained sediment,
1 ml of 10 mM Tris-HCl buffer (pH 7.4) containing 0.5 mg / ml HBs antigen was added, mixed well, and stirred at room temperature for 1 hour. To remove unreacted HBs antigen, centrifuge at 15000 rpm for 20 minutes, and wash the sediment with 100 mM phosphate buffer (pH 7.5).
The suspension was suspended in 2.5 ml and centrifuged again. 1%
100m containing (w / v) concentration of bovine serum albumin
The precipitate was suspended in 2.5 ml of M phosphate buffer (pH 7.5) and stirred at room temperature for 1 hour to perform a blocking treatment. Thereafter, centrifugation was performed at 15000 rpm for 20 minutes, and 100 mM containing 1% (w / v) of bovine serum albumin and 0.01% (w / v) of sodium azide.
The precipitate was suspended in 2 ml of a phosphate buffer (pH 7.5) and stored in a refrigerator.

0.1% (w / v) of blue latex particles sensitized with HBs antigen and 1% (w / v) of bovine serum albumin
/ V), a 100 mM phosphate buffer (pH 7.5) containing 0.01% (w / v) of sodium azide was prepared,
20 μl of this solution was impregnated in a glass fiber filter (GFF: manufactured by Millipore) having a width of 5 mm and a length of 15 mm, and dried at room temperature.

A nitrocellulose membrane sensitized with the HBs antigen was adhered to an adhesive substrate having a length of 5 mm and a length of 60 mm. The immunochromatography apparatus was prepared by mounting so as to overlap each other and pressure bonding with a tape.

2) Treatment of Sample 2 μl of 100 mM phosphate buffer (pH 7.5) containing 2 μg / μl sucrose was added to 100 μl of HBs antibody negative serum. (3.9 parts by weight of sucrose per 100 parts by weight of serum, but calculated with a specific gravity of serum of 1.028)

3) Immunological analysis The sucrose-added sample was added to the glass fiber filter of the immunochromatography apparatus, and it was determined whether a blue spot was detected. In addition, the clogged and unmoved blue particles were visually observed on the membrane at the portion overlapping with the glass fiber filter, and those that were not clogged were evaluated as 0, and those that were significantly clogged were evaluated as a 6-point scale. The clogging strength was calculated. The clogging strength was calculated by an average value of three evaluators, and the results are shown in Table 2.

Example 4 The procedure of Example 3 was repeated except that the content of sucrose was changed to 19.5 parts by weight per 100 parts by weight of serum. The results are shown in Table 2.

Example 5 The same procedure as in Example 3 was carried out except that the content of sucrose was changed to 39.0 parts by weight per 100 parts by weight of serum. The results are shown in Table 2.

Example 6 The procedure of Example 3 was repeated except that trehalose was used in place of sucrose and the content of trehalose was changed to 19.5 parts by weight per 100 parts by weight of serum. . The results are shown in Table 2.

Example 7 The procedure of Example 3 was repeated except that trehalose was used in place of sucrose and the content of trehalose was 39.0 parts by weight per 100 parts by weight of serum. . The results are shown in Table 2.

Comparative Example 3 The procedure of Example 3 was repeated, except that the sucrose content was changed to 1.3 parts by weight per 100 parts by weight of serum. The results are shown in Table 2.

Comparative Example 4 The procedure of Example 3 was repeated, except that the content of sucrose was changed to 50 parts by weight per 100 parts by weight of serum. The results are shown in Table 2.

Comparative Example 5 The procedure of Example 3 was repeated except that trehalose was used in place of sucrose and the trehalose content was changed to 1.3 parts by weight per 100 parts by weight of serum. . The results are shown in Table 2.

[Comparative Example 6] In the above 2), trehalose was used instead of sucrose, and the content of trehalose was changed to 50 parts by weight per 100 parts by weight of serum.
Performed in the same manner as in Example 3. The results are shown in Table 2.

[0047]

[Table 1]

[0048]

[Table 2]

[0049]

According to the method for treating a sample of the present invention, a non-specific reaction of latex particles can be prevented in an immunological analysis method using latex particles, and as a result, measurement sensitivity can be improved. Thus, it is possible to reduce errors and improve the reliability of analysis results.

Claims (4)

[Claims] 1. A method for treating a sample in an immunological analysis method using latex particles, wherein the sample contains a saccharide, and the content of the saccharide is 2 to 100 parts by weight of the sample.
A method for treating a specimen, comprising 40 parts by weight. 2. The method for treating a specimen according to claim 1, wherein the immunological analysis is an immunoagglutination assay or an immunochromatography. 3. The method according to claim 1, wherein the saccharide is at least one selected from the group consisting of monosaccharides, disaccharides and oligosaccharides. 4. The method according to claim 1, wherein the carbohydrate is at least one selected from the group consisting of glucose, fructose, mannose, sucrose, trehalose, maltose, lactose and cellobiose. Method.