JP2009186359A - Specimen processing reagent composition for immunological measurement - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a specimen processing reagent composition for correctly performing immunological measurement by suppressing a nonspecific reaction without reducing the sensitivity. <P>SOLUTION: This specimen processing reagent composition for immunological measurement contains polyoxyethylene/polyoxypropylene block copolymer having an alkyl group at one terminal. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a specimen processing reagent composition for immunological measurement. More specifically, the present invention relates to a treatment reagent composition for treating an immunologically measured specimen such as an influenza virus specimen before measurement.

When using mucosal components such as nasal discharge, sputum, and pharyngeal wiping fluid such as influenza virus specimens as samples, even if no detected substance is present in the specimen, it is judged as if the detected substance is present May end up.
As a cause of such false positives, protein components other than the detected substance contained in mucous membrane components such as nasal discharge, sputum, throat swab, etc., antibodies labeled with colloidal gold, latex, enzymes, etc. Non-specific binding can be mentioned.
As a method of suppressing non-specific reactions that are not immune reactions that occur between contaminants such as protein components other than the substance to be detected contained in the specimen and labeled antibodies, non-ionic A sample pretreatment solution containing both a surfactant and an alkali metal ion of 0.3 M or more and a washing solution for immunoassay containing a water-soluble polyoxyalkylene alcohol and a higher alcohol nonionic surfactant are known. (See Patent Document 1 and Patent Document 2).
However, even if the sample pretreatment liquid is used, a nonspecific reaction may not be suppressed, it may be difficult to respond depending on the concentration of mucosal components, or a large amount of pretreatment liquid may be used for one measurement. I sometimes needed it. In addition, as will be described later, there are similar problems with substances to be detected other than influenza viruses.
JP 2005-24323 A Japanese Patent Laid-Open No. 9-80048

  An object of the present invention is to provide a specimen treatment reagent composition for immunological measurement that can suppress non-specific reaction without lowering the measurement sensitivity in view of the above prior art.

  Another object of the present invention is to provide a specimen treatment for immunoassay containing certain nonionic surfactants and containing less than 0.3M alkali metal ions and exhibiting superior advantages as described above. It is to provide a reagent composition.

  Still other objects and advantages of the present invention will become apparent from the following description.

According to the present invention, the above objects and advantages of the present invention are:
It is achieved by an analyte treatment reagent composition for immunological measurement, which comprises a polyoxyethylene / polyoxypropylene block copolymer having an alkyl group at one end.

  According to the present invention, sensitivity is obtained by dispersing mucosal components such as nasal discharge, sputum and pharyngeal wiping liquid in a sample treatment liquid containing a polyoxyethylene / polyoxypropylene block copolymer having an alkyl group at one end. Thus, immunological measurement can be performed accurately by suppressing non-specific reactions without lowering.

  The sample processing reagent composition of the present invention contains a polyoxyethylene / polyoxypropylene block copolymer having an alkyl group at one end. The alkyl group at one end may be linear or branched, and is preferably an alkyl group having 1 to 18 carbon atoms, more preferably an alkyl group having 10 to 18 carbon atoms. In the polyoxyethylene / polyoxypropylene block copolymer having hydroxyl groups at both ends, denaturation of contaminants such as glycoproteins that cause non-specific reactions is inhibited, so that the solubility and dispersibility in the processing solution are reduced. Alternatively, non-specific reaction cannot be suppressed and accurate determination cannot be performed because the electrostatic or hydrophobic affinity of the labeled antibody or detection antibody cannot be controlled. In addition, in the case of an alkyl group at one end having more than 18 carbon atoms, formation of a complex due to the reaction between a detection target substance required for measurement and a labeled antibody is inhibited, and sufficient sensitivity cannot be obtained. Furthermore, the ratio of the oxyethylene repeating unit of the polyoxyethylene block and the oxypropylene repeating unit of the polyoxypropylene block is preferably 0.1 to 1.5, preferably 0.15 to 1.1 in terms of the molar ratio of the former to the latter. More preferred. The alkyl group at one end is preferably bonded to the polyoxyethylene block. If the molar ratio of the above repeating units is less than 0.1, denaturation of contaminants such as glycoproteins that cause non-specific reactions is inhibited, solubility or dispersibility in the processing solution is reduced, or labeled antibodies or Non-specific reaction cannot be suppressed and accurate determination cannot be made due to reasons such as electrostatic or hydrophobic binding affinity with the detection antibody being uncontrollable. On the other hand, if the ratio exceeds 1.5, formation of a complex due to the reaction between the target substance to be measured and the labeled antibody is inhibited, and sufficient sensitivity cannot be obtained.

The polyoxyethylene / polyoxypropylene block copolymer having an alkyl group at one end is preferably contained in the specimen treatment reagent composition at 0.05 to 2% by weight, more preferably 0.3 to 1.5% by weight. Can be made.
The sample processing reagent composition of the present invention desirably contains substantially no nonionic surfactant other than the polyoxyethylene / polyoxypropylene block copolymer having an alkyl group at one end.

The sample processing reagent composition of the present invention can contain components that can be commonly used by those skilled in the art, in addition to the polyoxyethylene / polyoxypropylene block copolymer having an alkyl group at one end.
For example, it can contain bovine serum albumin at a concentration of 1% by weight or more, components constituting a buffer solution that can be maintained at 5 to 9 at an optimum pH, and other suitable organic acids. The sample may be processed according to a normal method, for example, by adding 0.1 to 0.2 mL of the sample to 0.5 to 1.0 mL of the sample processing reagent composition, and well shaken and mixed. be able to. Similarly, a cotton swab from which a sample such as nasal discharge is collected is immersed in the sample processing reagent composition, and the sample processing reagent composition and the sample are mixed well.

Samples to be processed in the present invention include, for example, a solution in which a pharyngeal or nasal wiping solution is suspended in the sample processing reagent composition of the present invention, nasal aspirate, fecal suspension, plasma, serum, urine, saliva, tears Biological samples such as sac secretions, corneal scrapings, vaginal contents or uterine contents, prostate fluid, semen, amniotic fluid, cerebrospinal fluid, pus, organ extracts, various tissue extracts and the like.
Examples of the substance to be detected from the specimen include virus antigens such as influenza virus antigen, adenovirus antigen, RS virus antigen, HA antigen, HBc antigen, HCV antigen, HIV antigen, EBV antigen, and NLV antigen, Chlamydia tracho Matis antigen, Streptococcus antigen, Bordetella pertussis antigen, Helicobacter pylori antigen, Leptospira antigen, Treponema paridum antigen, Toxoplasma gondii antigen, Borrelia antigen, Bacillus anthracis antigen, MRSA antigen and other tumor antigens, PSA, CEA, AFP and other tumors Examples include, but are not limited to, markers, anti-HIV antibodies, anti-HBV antibodies, anti-HCV antibodies, anti-mite allergen antibodies, immunoglobulins such as anti-cedar pollen allergen antibodies, and the like. These specimens can be collected by a method known per se.

The sample obtained by processing with the sample processing reagent composition according to the present invention can be examined for identification and quantification of viruses and the like by antigen-antibody reaction using conventional immunochromatography.
Although the technique of immunochromatography is known, its principle is schematically shown in FIG.
An antigen or antibody (labeled antigen, labeled antibody) labeled with an enzyme, metal colloid particles, latex particles, or the like is held at the labeled substance holding site (2) in FIG. 1, and the antigen or antibody is immobilized at the detection site (4). .
The treated specimen is dropped as a sample on the sample addition site (1) in FIG. 1, and the sample is developed in the direction of the absorption site (5) through the chromatography medium (3). When the target substance to be detected is mixed in the sample, the target substance reacts with the labeled antibody or the labeled antigen, and these complexes are captured at the detection site (4), and a colored band appears. . The amount of the substance to be detected contained in the sample can be roughly grasped by the color tone of the band appearing in (4).
The antigen or antibody used for the labeling substance holding site (2) and the antigen or antibody used for the detection site (4) may be those that bind different sites of the substance to be detected.

As the antibody, an antibody that specifically binds to the substance to be detected is used. Antibodies include humans, mice, rats, rabbits, goats, horses and the like as production animal species, each of which has a predetermined range of immunoglobulins. Any of IgG, IgM, IgA, IgE, and IgD may be used, and monoclonal antibodies, polyclonal antibodies, and fragments thereof (having antigen-binding ability; for example, H chain, L chain, Fab, F (ab ′) ₂ ) and the like. ).
Specifically, anti-PSA antibody, anti-AFP antibody, anti-CEA antibody, anti-adenovirus antibody, anti-influenza virus antibody, anti-HCV antibody, anti-IgG antibody, anti-human IgE antibody, etc., and fragments thereof (antigen-binding ability) Any of F (ab) ′ 2 or Fab ′) may be used. ).
As the antigen, an antigen that specifically binds to the substance to be detected is used. For example, HIV antigen, HBV antigen, HCV antigen, mite allergen, cedar pollen allergen and the like can be mentioned.

Furthermore, examples of insoluble carriers for labeling antigens and antibodies include insoluble metal colloid particles such as gold colloid particles, non-metal colloid particles such as selenium colloid particles, colored resin particles such as latex particles, dye colloid particles and colored liposomes. Particulate materials and the like are listed. Examples of enzymes that label antigens and antibodies include peroxidase, alkaline phosphatase, glucose oxidase and the like.
The membrane carrier for the chromatographic medium is not particularly limited as long as it can absorb and flow the sample specimen by capillary action. For example, nitrocellulose, cellulose acetate, nylon, polyethersulfone, polyvinyl alcohol, polyester, glass fiber, polyolefin, cellulose, or an artificial polymer composed of a mixed fiber thereof is selected.

  Hereinafter, the present invention will be described in more detail by way of examples. The present invention is not limited in any way by the examples.

Example 1
(1) Preparation of determination part on chromatography medium A sheet made of nitrocellulose (manufactured by Millipore, trade name: HF120, 300 mm × 25 mm) was used as a membrane. The mouse-derived anti-influenza A monoclonal antibody (first antibody) was diluted with a phosphate buffer solution (pH 7.4) containing 5% by weight of isopropyl alcohol to a concentration of 1.0 mg / mL. 150 μL of this solution was applied on the membrane with a width of 1 mm and dried at 50 ° C. for 30 minutes. After drying, blocking was performed by immersing in 100 mL of a phosphate buffer solution (pH 7.4) containing 0.5% by weight of casein (manufactured by Wako Pure Chemical Industries, Ltd.) at room temperature for 30 minutes. After blocking, it was washed with a phosphate buffer (pH 7.4) containing 0.05% by weight of Tween 20, and dried overnight at room temperature to prepare a chromatography medium.

(2) Preparation of labeling substance solution 0.5 mL of colloidal gold dispersion (manufactured by Tanaka Kikinzoku Kogyo Co., Ltd .: LC 40 nm) diluted with phosphate buffer (pH 7.4) to a concentration of 0.1 mg / mL 0.1 mL of the mouse-derived anti-influenza A monoclonal antibody (second antibody) was added and allowed to stand at room temperature for 10 minutes. Next, 0.1 mL of a phosphate buffer solution (pH 7.4) containing 10% by weight of bovine serum albumin (hereinafter BSA) was added, and the mixture was further allowed to stand at room temperature for 10 minutes. Then, after sufficiently stirring, centrifugation was performed at 8,000 × g for 15 minutes, and the supernatant was removed. Then, 0.1 mL of a phosphate buffer (pH 7.4) containing 1 wt% BSA was added. The labeling substance solution was prepared by the above procedure.

(3) Preparation of immunochromatographic test strip A 15 mm × 300 mm glass fiber pad (Millipore) made by adding 300 μL of 10 wt% trehalose aqueous solution and 1.8 mL of distilled water to 300 μL of the prepared labeling substance solution. After being added uniformly, it was dried with a vacuum dryer to produce a label holding member. Next, on the base material composed of a backing sheet, the above-prepared chromatography medium, the labeling substance holding member, the sample pad used for adding the sample (Millipore: 300 mm × 30 mm), the developed sample and the surplus label of the reaction An absorbent pad for absorbing the substance was bonded. And it cut | judged so that a width | variety might be set to 5 mm with the cutter, and it was set as the test piece for immunochromatography.

(4) Preparation of specimen treatment reagent Polyoxyethylene / polyoxypropylene-alkyl ether so as to be 0.3% by weight in 10 mL of Tris-HCl buffer solution (pH 8.0) containing 1% by weight of BSA and 150 mM NaCl. (Nippon Yushi Co., Ltd .: trade name Nonion (registered trademark) MN-811, carbon number of alkyl group = 14, molar ratio of oxypropylene / molar ratio of oxyethylene = 1.1) A reagent for treating a specimen such as a throat swab was used.

(5) Measurement Using the prepared immunochromatographic test piece, the presence or absence of influenza A virus in the sample was measured by the following method.
That is, one tube of the suction trap was inserted into the suction pump and the other tube was inserted to the back of the nasal cavity, and the suction pump was set to a negative pressure to collect nasal discharge. The collected nasal discharge was diluted 100 times with the above-described sample treatment reagent, and this was used as a negative sample sample. Moreover, what added the influenza A virus inactivated so that protein concentration might be 2 ng / mL, 5 ng / mL, and 10 ng / mL to the negative sample sample was made into the positive sample sample. 150 μL of both the negative specimen sample and the positive specimen sample was placed on the sample pad of the immunochromatographic test piece and developed, and visually judged after 15 minutes. The test line that can confirm the red line is “+”, the red line can be confirmed, but the light color is “±”, and the red line that cannot be confirmed is “−”. Table 1 shows the results.

Comparative Example 1
Other than using nonionic MN-811 polyoxyethylene / polyoxypropylene block copolymer surfactant (trade name Pluronic L-44, manufactured by Asahi Denka Kogyo Co., Ltd.) having hydroxyl groups at both ends instead of nonionic MN-811 Were measured in the same manner as in Example 1. The results are shown in Table 1.

Example 2
Nonion TA-411 (manufactured by Nippon Oil & Fats Co., Ltd .: mixture of alkyl group carbon number = 11-18, molar ratio of oxypropylene / molar ratio of oxyethylene = 0.15) was used instead of nonionic MN-811. The measurement was performed in the same manner as in Example 1 except that. The results are shown in Table 1.

Example 3
Except for using Adekatol LB-53B (Asahi Denka Kogyo Co., Ltd .: carbon number of alkyl group = 12, molar ratio of oxypropylene / molar ratio of oxyethylene = 0.3) instead of Nonion MN-811 The measurement was performed in the same manner as in Example 1. The results are shown in Table 1.

Example 4
Measurement was carried out in the same manner as in Example 1 except that the concentration of Nonion MN-811 was 1.5% by weight. The results are shown in Table 1.

Schematic of the test piece for immunochromatography.

Explanation of symbols

1 Sample addition site 2 Labeled substance holding site 3 Chromatographic medium 4 Detection site 5 Absorption site

Claims (7)

  A specimen treatment reagent composition for immunological measurement, comprising a polyoxyethylene / polyoxypropylene block copolymer having an alkyl group at one end.   A specimen treatment reagent composition for immunological measurement, comprising a polyoxyethylene / polyoxypropylene block copolymer having an alkyl group having 1 to 18 carbon atoms at one end.   A polyoxyethylene / polyoxypropylene block having an alkyl group at one end and a molar ratio of the oxypropylene repeating unit of polyoxypropylene to the oxyethylene repeating unit of polyoxyethylene in the range of 0.1 to 1.5 A specimen processing reagent composition for immunological measurement, comprising a copolymer.   A polyoxygen having an alkyl group having 1 to 18 carbon atoms at one end and a molar ratio of the oxypropylene repeating unit of polyoxypropylene to the oxyethylene repeating unit of polyoxyethylene in the range of 0.1 to 1.5, A specimen treatment reagent composition for immunological measurement, comprising an oxyethylene / polyoxypropylene block copolymer.   The specimen treatment reagent composition according to any one of claims 1 to 4, wherein the concentration of the polyoxyethylene / polyoxypropylene block copolymer having an alkyl group at one end is 0.05 to 2% by weight.   The specimen treatment reagent composition according to any one of claims 1 to 4, comprising bovine serum albumin at a concentration of 1% by weight or more.   The specimen treatment reagent composition according to any one of claims 1 to 4, wherein the specimen for immunological measurement is nasal discharge, sputum or a wiping fluid.

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