JP2023110104A - Biological sample pretreatment method, filtration member for pretreatment, and immunological analysis method - Google Patents

Abstract

To enable quantitative measurement or detection of the presence of a detection target substance using immunoassay while ensuring reproducibility and suppressing occurrence of non-specific reactions, and preventing clogging of a filter by solid components.SOLUTION: The above challenge is cleared by providing a method of pretreating a biological sample to be subjected to immunoassay for detecting a detection target substance, the method comprising filtering the biological sample using a filtration member having at least two filters containing glass fiber with a retention particle diameter of 0.3-1.1 μm, where each of the at least two filters contains an organic binder.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a method for pretreatment of a biological sample to be subjected to immunoassay for detecting a substance to be detected, and a filter member for pretreatment. The invention also relates to an immunological analysis method.

Immunoassays are methods that utilize antigen-antibody reactions to assay the amount or presence of analytes in biological samples such as serum and urine. Among immunoassays, rapid assays such as dipstick assays and lateral flow assays have been used in numerous diagnostic and laboratory methods for many years. In particular, as one method for detecting a substance to be detected in a sample by antigen-antibody reaction, a measurement method using an immunochromatographic test strip is widely used.

When immunochromatography is used to measure the amount of a substance to be detected or to detect its presence, the biological sample used for immunochromatography may contain solid components. In this case, there is a risk that the biological sample, and thus the substance to be detected contained therein, will spread unevenly on the immunochromatographic test strip. As a result, reproducibility of measurement or detection is reduced. In addition, substances in solid components contained in biological samples may cause non-specific reactions and erroneous determinations. To prevent this from happening, it is preferable to pre-filter the biological sample. However, if the pore size of the filter is made too small to sufficiently remove the solid components of the sample, there is a problem that the filter will be clogged with the solid components derived from the biological sample, making it impossible to secure the amount of sample required for testing. rice field.

Patent Document 1 discloses a simple membrane assay method for an analyte, which is characterized in that the analyte sample is filtered using a filtration filter and then dropped onto a membrane to detect or quantify the presence of the analyte in the analyte sample. disclose. Patent Literature 2 discloses a sample pretreatment method in which a sample obtained by pretreating a collected sample is filtered using a filtration member in which at least two types of membranes having different pore sizes are layered. However, even if these techniques are used, the reproducibility of the measurement or detection of the substance to be detected cannot be guaranteed, resulting in non-specific reactions and/or clogging of the filter with solid components derived from biological samples. there were.

Japanese Patent Application Laid-Open No. 2016-153811 Japanese Patent Application Laid-Open No. 2005-024325

An object of the present invention is to suppress the occurrence of non-specific reactions by ensuring reproducibility when measuring the amount of a substance to be detected or detecting its presence using immunoassay, and to suppress the occurrence of non-specific reactions by solid components. To prevent clogging and to achieve both.

The present inventors performed pretreatment of a biological sample using a filter member having the following configuration to suppress the occurrence of non-specific reaction in immunoassay for detecting a substance to be detected, In addition, the inventors have found that the solid component can prevent clogging of the filter, and have completed the present invention.
Specifically, the present invention is as follows.
<1> A method for pretreating a biological sample to be subjected to immunoassay for detecting a substance to be detected,
filtering the biological sample with a filtering member comprising at least two filters comprising glass fibers having a retained particle size of 0.3 μm to 1.1 μm;
and wherein the at least two filters both contain an organic binder.
<2> The pretreatment method according to <1>, wherein the organic binder is an acrylic resin binder.
<3> The pretreatment method according to <1> or <2>, wherein the amount of the biological sample after pretreatment is 50% by mass or more of the amount of the biological sample before pretreatment.
<4> The pretreatment method according to any one of <1> to <3>, wherein the biological sample is a respiratory secretion.
<5> The pretreatment method according to any one of <1> to <4>, wherein each of the at least two filters has a thickness of 0.1 mm to 0.7 mm.
<6> The at least two filters are composed of a first filter close to the opening side of the filtering member and a second filter close to the tip side of the filtering member,
The pretreatment method according to any one of <1> to <5>, wherein the thickness of the first filter is smaller than the thickness of the second filter.
<7> The pretreatment method according to any one of <1> to <6>, wherein the immunoassay is immunochromatography.
<8> Immunological analysis method for detecting a substance to be detected, including the following steps (1) to (3), by the pretreatment method according to any one of <1> to <7>, (2) contacting the sample supply portion of the test strip with the pretreated biological sample obtained in step (1); and (3) detecting a signal derived from the label.
<9> A filter member for pretreatment of a biological sample to be subjected to immunoassay for detecting a substance to be detected,
The filtering member for pretreatment, comprising at least two filters containing glass fibers having a retained particle size of 0.3 μm to 1.1 μm, both of the at least two filters containing an organic binder.
<10> The filter member for pretreatment according to <9>, wherein the organic binder is an acrylic resin binder.
<11> The pretreatment filtration member according to <9> or <10>, wherein the biological sample is respiratory secretions.
<12> The filter member for pretreatment according to any one of <9> to <11>, wherein each of the at least two filters has a thickness of 0.1 mm to 0.7 mm.
<13> The at least two filters are composed of a first filter close to the opening side of the pretreatment filter member and a second filter close to the tip side of the pretreatment filter member,
The pretreatment filter member according to any one of <9> to <12>, wherein the thickness of the first filter is smaller than the thickness of the second filter.
<14> The pretreatment filter member according to any one of <9> to <13>, wherein the immunoassay is immunochromatography.

According to the pretreatment method or the pretreatment filter member of the present invention, it is possible to suppress the occurrence of non-specific reactions and prevent filter clogging due to solid components in immunoassay for detecting a substance to be detected. can.

1 is a schematic diagram showing the appearance of one embodiment of a filtration member used in the pretreatment method of the present invention. FIG. 1 is a schematic diagram showing a cross section (dotted line in FIG. 1) of one embodiment of a filtration member used in the pretreatment method of the present invention. FIG. 10 is a graph showing the results (total amount of dripping liquid) of a sample filtration test using filter members 1 to 5. FIG. 10 is a graph showing the results (droplet weight average value) of a sample filtration test using filter members 1 to 5. FIG. FIG. 10 is a graph showing the results of sample filtration tests using filter members 1 to 5 (variation in weight between droplets); FIG. 1 is a schematic diagram showing one embodiment of a test strip that can be used after performing the pretreatment method of the present invention; FIG.

(substance to be detected)
Substances to be detected in the present invention include physiologically active substances such as viruses and proteins. Examples of substances to be detected in the present invention are as follows.
myocardial markers such as CK-MB, H-FABP, troponin, myoglobin; coagulation such as fibrin degradation products (eg D-dimer), soluble fibrin, TAT (thrombin-antithrombin complex), PIC (plasmin-plasmin inhibitor complex) Fibrinolytic markers; circulation-related markers such as oxidized LDL and BNP (brain natriuretic peptide); metabolism-related markers such as adiponectin; CEA (carcinoembryonic antigen), AFP (α-fetoprotein), CA19-9, CA125, PSA Tumor markers such as (prostate specific antigen); inflammation-related markers such as CRP (C-reactive protein), IgA, IgG, and IgM; influenza, HIV (human immunodeficiency virus), HBV (hepatitis B virus), HCV (C hepatitis virus), toxoplasma, chlamydia, syphilis, staphylococcus aureus, and E. coli.

A respiratory infection virus is preferable as a substance to be detected in the present invention. As used herein, a respiratory infection virus is a virus that causes respiratory diseases. The respiratory infection virus in the present invention is not particularly limited as long as it is a virus that can be detected using an antigen-antibody reaction, and coronaviruses such as coronavirus 229E, coronavirus OC43, and coronavirus NL63; Influenza viruses such as influenza virus type B, influenza virus type B, influenza virus type C, parainfluenza virus 1, parainfluenza virus 2, and parainfluenza virus 3; respiratory syncytial virus such as respiratory syncytial virus type A and respiratory syncytial virus type B; adenovirus rhinovirus; metapneumovirus; cytomegalovirus; and bocavirus. Among them, respiratory syncytial viruses such as RS virus type A and RS virus type B, adenoviruses, and/or influenza viruses such as influenza type A virus and influenza type B virus are preferably used as substances to be detected.
As used herein, the term "respiratory system" is a general term for organs related to respiration, and includes organs from the nasal vestibule to the alveoli (lungs) via the nasal cavity, pharynx, larynx, trachea, bronchi, and bronchioles. say.

(biological sample)
Examples of biological samples that may contain a substance to be detected, which are used in the present invention, include substances mainly derived from living organisms (living organisms) such as body fluids, extracts obtained by extracting substances to be detected from them, and the like. be done. Specific examples of biological (organism)-derived substances include blood, urine, feces, and respiratory secretions. When the substance to be detected is a respiratory infection virus, the biological sample includes respiratory secretions, specifically, nasal discharge, sputum, saliva, etc. derived from the nostrils, nasal cavities, pharynx, nasopharynx, etc. is preferred, more preferably nasal mucus or throat swab. The biological (organism)-derived substance or its extract may be used as a sample as it is, or may be diluted with a sample diluent as appropriate and used as a sample. Also, the sample may be prepared by performing other pretreatment such as mixing of additives. In addition, in this specification, a biological sample may be simply referred to as a "sample".

(filter)
The filtering member used in the pretreatment method of the present invention includes at least two filters containing glass fibers having a retained particle size of 0.3 μm to 1.1 μm. The retained particle size is preferably 0.4 μm to 1.0 μm, more preferably 0.45 μm to 0.9 μm, still more preferably 0.5 μm to 0.8 μm, still more preferably 0.5 μm to 0.7 μm, still more preferably is between 0.55 μm and 0.65 μm. In this specification, when "0.3 μm to 1.1 μm" is described, the values at both ends thereof are included within the range. In this case, the range includes 0.3 μm and 1.1 μm. If the retained particle size is smaller than 0.3 μm, the filter may be clogged, and if the retained particle size is larger than 1.1 μm, the solid content cannot be completely removed, which may cause non-specific reactions. be.
The retained particle size in the present invention is a particle size that can retain 90% or more of the seven kinds of powder dispersion water specified in JIS Z 8901;

In this specification, the filter close to the opening side of the filtering member (the side to which the untreated liquid is introduced) is called the first filter, and the filter close to the tip side of the filtering member (the side from which the filtered liquid is obtained) is called the second filter. called. In the present invention, the retained particle sizes of the first filter and the second filter may differ between the first filter and the second filter, but are preferably substantially the same. For example, the retained particle size of the first filter is preferably 0.5 to 1.5 times the retained particle size of the second filter, preferably 0.8 to 1.2 times, 0 More preferably, it is between 0.9 and 1.1 times.

(filter thickness)
In the present invention, the thickness of the first filter and the second filter can be adjusted as long as the effect of the present invention can be obtained, but both are preferably 0.1 mm to 0.7 mm, and 0.15 mm to 0.60 mm is more preferred.
In the present invention, the thickness of the second filter with respect to the first filter (second filter/first filter) can be adjusted as long as the effects of the present invention can be obtained. is preferably smaller than the thickness of the second filter. Specifically, the thickness of the second filter with respect to the first filter is preferably 1.1 to 4.0, more preferably 1.5 to 3.5, and 2.0 to 3.5. 0 is more preferred.
As specific thicknesses, the thicknesses of the first filter and the second filter are 0.1 mm to 0.5 mm and 0.3 mm to 1.1 mm, 0.1 mm to 0.4 mm and 0.3 mm to 0.3 mm, respectively. .9 mm, 0.1 mm to 0.3 mm and 0.3 mm to 0.7 mm, 0.15 mm to 0.25 mm and 0.4 mm to 0.6 mm, or 0.15 mm to 0.25 mm and 0.45 mm to 0.45 mm. It can be 55 mm.

In the present invention, each of the first filter and the second filter is made of glass fiber. Other materials (for example, nitrocellulose, paper, nitrocellulose, silica fiber, cellulose ester, polyethersulfone, polysulfone, tetrafluoroethylene resin, vinylidene fluoride resin, polycarbonate, polypropylene, etc., as long as the effects of the present invention can be obtained) , polyamide, etc.) may also be used. Usage ratio of glass fiber to other materials (mass of glass fiber used/mass of glass fiber used + mass of other fiber used) is 50% or more, 60% or more, 70% or more, 80% or more, 90% % or higher, 95% or higher, 99% or higher, or 100%.

In the present invention, a commercially available filter can be used. For example, GS25, DP-70 or GC-90 manufactured by ADVANTEC can be used, GS25 can be used as the first filter and DP-70 can be used as the second filter. The surface of each filter may be provided with protrusions or recesses. The surface of each filter may be surface-treated.

(organic binder)
The organic binder used in the present invention refers to an organic compound that has the effect of binding substances such as additives to carriers such as fibers. Organic binders used in the present invention include melamine compounds, glyoxal compounds, urethane compounds, blocked isocyanate compounds, silicone compounds, acrylic resins, polyester resins, and the like. The organic binder is preferably an acrylic resin. The organic binders may be used singly or in combination of two or more. If the filter does not contain an organic binder, the pressure during filtration deforms the pores, increasing the possibility of clogging.

Examples of acrylic resins include polyethyl methacrylate, polybutyl methacrylate, butyl methacrylate/methyl methacrylate copolymer, butyl methacrylate/ethyl methacrylate copolymer, 2-ethylhexyl methacrylate/ethyl methacrylate copolymer, and lauryl methacrylate/ethyl methacrylate copolymer. (Meth)acrylate polymers or copolymers, such as polymers. These acrylic resins may be used alone or in combination of two or more.

The content of the organic binder in the filter used in the present invention is not limited as long as the effect of the present invention can be obtained. parts, or 1 to 15 parts by weight.

(Filtration member)
The filtering member used in the present invention includes at least two filters containing glass fibers having a retained particle size of 0.3 μm to 1.1 μm. The filtering member supplies the biological sample solution from the open side, filters the biological sample solution through at least two filters inside, and discharges the filtered biological sample solution from the tip side. is a member of
FIG. 1 illustrates one embodiment of the filtering member used in the present invention. The shape of the filtering member is not particularly limited as long as the effects of the present invention can be obtained, but preferably has a cylindrical shape.
In the present invention, the filtering member can include a holding material for holding these filters on the opening side of the first filter and/or the tip side of the second filter. The holding material is not particularly limited as long as it can hold the filter and allow the biological sample to pass through, and any shape, thickness and material can be used. For the holding material, for example, one having a holding particle size of 50 μm to 300 μm and a thickness of 0.5 mm to 3 mm can be used.
The filtering member can include members other than the filter and the retainer, such as a cap. A nozzle or the like may be connected to the tip as needed.

The filtration member used in the present invention is any filter having a smaller or larger retained particle size than the first filter or the second filter, as long as the effect of the present invention can be obtained between the first filter and the second filter. Can contain filters. However, from the viewpoint of ensuring reproducibility and preventing clogging of the filter, it is preferable not to include such a filter. In particular, it is preferred not to include any filter between the first filter and the second filter that has a smaller retained particle size than the first or second filter.
The filtration member used in the present invention is a material different from the first filter or the second filter (for example, nitrocellulose, paper, nitrocellulose, silica fiber, cellulose ester) between the first filter and the second filter. , polyethersulfone, polysulfone, tetrafluoroethylene, vinylidene fluoride, polycarbonate, polypropylene, polyamide, etc.). However, from the viewpoint of ensuring reproducibility and preventing clogging of the filter, it is preferable not to include such a filter.
Similarly, the filtering member used in the present invention can also include a filter made of the same material as the first filter or the second filter, ie glass fiber, between the first filter and the second filter. , preferably not included.

The filtration member used in the present invention has a retention particle size larger than that of the first filter or the second filter, as long as the effect of the present invention is obtained on the opening side of the first filter and/or the tip side of the second filter. Any filter, small or large, can be included. However, from the viewpoint of ensuring reproducibility and preventing clogging of the filter, it is preferable not to include such a filter. In particular, it is preferred that the opening side of the first filter and/or the tip side of the second filter not include any filter having a smaller retained particle size than the first filter or the second filter.
Similarly, the filtering member used in the present invention has a material different from that of the first filter or the second filter (for example, nitrocellulose, paper, nitrocellulose, silica fibers, cellulose esters, polyethersulfone, polysulfone, ethylene tetrafluoride resin, vinylidene fluoride resin, polycarbonate, polypropylene, polyamide, etc.). However, from the viewpoint of ensuring reproducibility and preventing clogging of the filter, it is preferable not to include such a filter.
Similarly, the filter member used in the present invention is provided on the opening side of the first filter and/or the tip side of the second filter, and is made of the same material as the first filter or the second filter, that is, glass fiber. can also be included, but preferably not.

(Total amount of dripping liquid)
As used herein, the total amount of dripping liquid means the total amount of the filtered biological sample discharged from the tip side of the filtering member. In the pretreatment method of the present invention, by preventing clogging of the filter with solid components, it is possible to obtain the total amount of dripping liquid necessary for inspection. In the pretreatment method of the present invention, the total drop amount is 50% by mass or more, preferably 55% by mass or more, more preferably 60% by mass or more of the amount of the biological sample before pretreatment. Specific ranges for the total amount of dripping liquid are 50-90% by weight, 55-85% by weight, or 60-80% by weight of the amount of the biological sample before pretreatment. In addition, the pretreatment method of the present invention can reduce weight variations among droplets of the filtered biological sample discharged from the tip side of the filtering member. Reducing the weight variation between droplets allows a constant amount of sample to be introduced into the measurement system, ensuring reproducibility of the test.
If the total amount of dropped liquid drops significantly or if the variation between drop weights increases, there is a possibility that clogging has occurred.

As used herein, immunoassay utilizes antigen-antibody reactions to assay the amount or presence of a substance to be detected in biological samples such as respiratory secretions, serum and urine. means how to Immunoassays include electrochemiluminescence immunoassay (ECL method), enzyme immunoassay (ELISA method), latex agglutination immunoassay (LTIA method), chemiluminescence immunoassay, fluorescent antibody method), immunochromatography, etc. but not limited to these. The immunological analysis method of the present invention is preferably immunochromatography.

(Immunochromatography)
As used herein, the term "immunochromatography" means an immunoassay method that applies the property (capillary action) in which an analyte slowly flows while dissolving a reagent on a porous material such as a cellulose membrane. In immunochromatography, an antibody or antigen against an antigen or an antibody, which is a substance to be detected, is immobilized on an insoluble membrane carrier, which is a chromatographic medium, to prepare a detection part, which is a stationary phase. Then, using as a mobile phase a label sensitized by an antibody or antigen capable of binding to the substance to be detected, which is a conjugate (detection reagent), the substance to be detected and the conjugate, which is a mobile phase, react specifically. Let Furthermore, in the detection part, which is the stationary phase, the substance to be detected bound to the conjugate is caused to specifically react with the antibody or antigen immobilized on the detection part. Fluorescent substances, colloidal metal particles such as colloidal gold, or colored latex particles are usually used as labels. In the detection section, the presence or, depending on the case, the amount of the substance to be detected in the biological sample is detected from the signals derived from these labels.

(sample pad)
An immunochromatographic test strip that can be used after carrying out the pretreatment method of the present invention will be described. In immunochromatographic test strips, a pad-like porous material can be used as a sample pad. A part of the sample pad has a sample supply section. In addition to the sample pad, a conjugate pad that holds a conjugate can also be provided downstream of the sample supply section. There is also a porous material portion downstream of the sample supply.
The sample supply unit is a part that supplies a pretreated biological sample. This sample supply part is formed in a part of the porous material, and the sample supply part is upstream of the sample pad.
The total length of the sample pad, ie, the length from the upstream end to the downstream end of the sample pad, can be adjusted appropriately by those skilled in the art. Specifically, it can be adjusted to 5-40 mm, 10-30 mm, or 10-25 mm. When a conjugate pad is provided, the length from the upstream end of the sample pad to the downstream end of the conjugate pad may be adjusted to an appropriate length.

(Insoluble membrane carrier)
In the immunochromatographic test strip, the insoluble membrane carrier has at least one detection part for capturing and detecting the substance to be detected. It is preferable that the detection part is linear. Antibodies or antigens that immunologically react with the substance to be detected are immobilized on the detection portion. Antibodies or antigens that immunologically react with the substance to be detected can be immobilized on the insoluble membrane carrier by a conventionally known method.
Both the lateral flow method and the dipstick method can be used for immunochromatographic test strips.

As the membrane constituting the insoluble membrane carrier used in the immunochromatographic test strip, known membranes conventionally used as insoluble membrane carriers for test strips can be used. Examples thereof include membranes composed of fibers made of polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, and ceramics. Specific examples include glass fiber filter paper and nitrocellulose membranes commercially available from Sartorius, Merck, Toyo Roshi, Whatman, and the like. The average pore size or retained particle size of the insoluble membrane carrier is not limited to these, but can be 0.1 μm to 20 μm, more preferably 0.5 μm to 16 μm, and 0.7 μm to 10 μm. is more preferred. In terms of flow rate, for example, 55 to 300 seconds can be used, preferably 90 to 225 seconds, more preferably 90 to 180 seconds. The flow rate means the number of seconds required for water to develop at one end of the insoluble membrane support and for the development to progress by 4 cm.
The total length of the insoluble membrane carrier, that is, the length from the upstream end to the downstream end of the insoluble membrane carrier can be appropriately adjusted by those skilled in the art. Specifically, the length can be adjusted to 15-40 mm, 18-35 mm, or 20-30 mm.
The thickness of the insoluble membrane carrier can be adjusted appropriately by those skilled in the art. Specifically, the thickness can be adjusted to 120-400 μm, 180-320 μm, or 240-280 μm.

(Conjugate holding part)
As used herein, the term “conjugate holding portion” refers to a portion in which a detection reagent that specifically reacts with a substance to be detected is held dry. The conjugate holding part has a function of forming a complex between the substance to be detected in the sample and the detection reagent when the sample passes through the conjugate holding part. The conjugate holding section may be arranged downstream from the sample supply section, and may be arranged downstream from the sample supply section on the sample pad or upstream from the detection section on the insoluble membrane carrier. It is desirable to form the conjugate holding portion in a line-like shape perpendicular to the developing direction of the sample.

The conjugate holding portion may be fabricated as a conjugate pad independently of other members and placed in fluid connection with the sample pad and the insoluble membrane carrier upstream from the detection portion. Suitable materials for conjugate pads include, but are not limited to, paper, cellulose blends, nitrocellulose, polyesters, acrylonitrile copolymers, glass fibers or non-woven fibers such as rayon. Glass fiber pads are preferably used.

(absorbent pad)
As used herein, an absorbent pad is a liquid-absorbent portion that controls sample development by absorbing the sample as it migrates and passes through a porous membrane. The absorbent pad may be provided on the most downstream side of the immunochromatographic test strip. As the absorbent pad, for example, filter paper can be used, but it is not limited to this. The total length of the absorbent pad, that is, the length from the upstream end of the absorbent pad to the downstream end of the absorbent pad, can be adjusted to an appropriate length by those skilled in the art. , 5-100 mm, 20-80 mm, or 20-60 mm.

(test strip)
Immunochromatographic test strips are prepared by fixing each member on a solid phase support such as a plastic adhesive sheet. The solid support is composed of a material that does not impede capillary flow of the sample and conjugate. The adhesive for fixing each member to the plastic pressure-sensitive adhesive sheet may be any substance that does not hinder the capillary flow of the sample and conjugate due to its components. It is also possible to laminate a polyester film or the like on the test strip for the purpose of increasing the mechanical strength of the insoluble membrane carrier and preventing evaporation (drying) of water during the assay. The test strip is used by storing and mounting it in an appropriate container (housing) considering the size of the test strip, the method and position of adding the sample, the position of forming the detection part of the insoluble membrane carrier, the method of detecting the signal, etc. The state of being stored and mounted in this way is called a "device".
In addition, the immunochromatographic test strip that can be used in the present invention includes a conjugate-holding portion and a detection portion, and may further include other reagents and configurations depending on measurement conditions and samples. Other reagents include, for example, blocking agents to prevent non-specific reactions, and other configurations include, for example, additional pads for removing components unnecessary for measurement in the sample. For example, an additional pad can be placed between the sample pad and the insoluble membrane to allow the substance to be detected or a complex containing the substance to be detected to smoothly spread through the insoluble membrane carrier.
FIG. 6 shows one embodiment of a test strip that can be used in the present invention.

(others)
An immunochromatographic test strip that can be used in the present invention can be prepared by a method known to those skilled in the art.

(Immunological analysis method by immunochromatography)
After performing the pretreatment method of the present invention, an immunological analysis method for a substance to be detected using the immunochromatographic test strip as described above can be performed. Specifically, the following procedures (A) and (D) can be performed.
(A) Bringing the biological sample into contact with the sample supply (D) Measuring the signal from the conjugate.
Also, the following steps (B) and (C) can be included between (A) and (D).
(B) A substance to be detected in the biological sample is contacted with the conjugate to form a first complex.
(C) The first complex and an antibody that immunologically reacts with the substance to be detected are brought into contact to form a second complex.

The pretreatment filter member of the present invention can also be manufactured and sold as an immunochromatography kit together with an immunochromatography test strip. The kit also contains reagents necessary for detection (e.g., detection reagents containing conjugates), sample diluents, test tubes, swabs for obtaining biological samples, instructions for use, and a housing for storing test strips. and so on.

As used herein, "detection" includes not only qualitative detection but also quantitative detection for a substance to be detected that can be quantified.
As used herein, the term "non-specific reaction" refers to the fact that a signal derived from the conjugate is detected and determined to be positive even though the substance to be detected does not exist or is substantially absent in the sample. (so-called false positive), and the fact that the signal derived from the conjugate is not detected and judged to be negative despite the presence of a considerable amount of the substance to be detected in the sample (so-called false negative). In the case of quantitative detection, it includes that the measured value fluctuates to a low value (false low) or to a high value (false high).

EXAMPLES Next, the present invention will be specifically described with reference to examples, but these are not intended to limit the scope of the present invention. In addition, % means % by mass unless otherwise specified.

[Preparation example of filter for sample filtration]
Each material was inserted into a plastic nozzle in the order of filter membrane holding material, filter 1, filter 2, and filter membrane holding material from the nozzle opening side toward the tip side to obtain a filter member for sample pretreatment. Table 1 shows the retained particle size, thickness, and material of each filter. Table 2 shows the configuration of each filter member.

[Test strip preparation example] Preparation of test strip for immunochromatography 1) Preparation of colloidal gold-labeled RS virus monoclonal antibody (conjugate) 1 PBS containing 100 μg/mL anti-RS virus monoclonal antibody to 20 mL of OD/mL colloidal gold solution was added and stirred for 10 minutes at room temperature. Subsequently, 2 mL of 10% bovine serum albumin (BSA) aqueous solution was added to the colloidal gold solution and stirred for 5 minutes. The obtained solution was centrifuged at 10,000 rpm for 45 minutes at 10° C. and the supernatant was removed. The residue was suspended in Conjugate Dilution Buffer (manufactured by Scripps) to obtain a conjugate.

2) Preparation of gold colloid particle-labeled adenovirus monoclonal antibody (conjugate) 1 1 mL of PBS containing 100 μg/mL anti-adenovirus monoclonal antibody solution was added to 20 mL of OD/mL colloidal gold solution, and the mixture was stirred at room temperature for 10 minutes. . Subsequently, 2 mL of 10% bovine serum albumin (BSA) aqueous solution was added to the colloidal gold solution and stirred for 5 minutes. The obtained solution was centrifuged at 10,000 rpm for 45 minutes at 10° C. and the supernatant was removed. The sediment was suspended in Conjugate Dilution Buffer (manufactured by Scripps) to obtain a conjugate.

3) Preparation of sample pad The conjugate prepared in 1) and 2) above was mixed with a 1.33% casein, 4% sucrose solution (pH 7.5) to prepare a conjugate solution. A glass fiber pad was coated with the conjugate liquid in a line having a width of 4 mm, dried by heating at 70° C. for 30 minutes, and a sample pad containing the conjugate part was obtained.

4) Preparation of insoluble membrane carrier (antibody-immobilized membrane) to which anti-RS virus and anti-adenovirus monoclonal antibodies are immobilized 1.0 mg/mL anti-RS virus monoclonal antibody, 10 mmol/L phosphoric acid containing 2.5% sucrose A buffer solution (pH 7.2) and a 10 mmol/L phosphate buffer solution (pH 7.2) containing 1.0 mg/mL anti-adenovirus monoclonal antibody and 2.5% sucrose were prepared as test line coating solutions. . A 10 mmol/L phosphate buffer (pH 7.2) containing 1.0 mg/mL anti-mouse IgG monoclonal antibody and 2.5% sucrose was prepared and used as a control line coating solution. Using a dispenser for immunochromatography "XYZ3050" (manufactured by BIO DOT), the test line coating solution and the control line coating solution were each applied onto the nitrocellulose membrane at 1.0 µL/cm. It was dried by heating at 70° C. for 45 minutes to obtain an antibody-immobilized membrane. At this time, antibodies were placed in the order of anti-RS virus monoclonal antibody (c1), anti-adenovirus monoclonal antibody (c2), and control antibody (c3) from the sample pad side of the test strip. The nitrocellulose membrane used has a thickness of 240-280 μm and a flow rate of 90-180 s/40 mm.

5) Preparation of immunochromatographic test strip The sample pad obtained in 3), the antibody-immobilized membrane obtained in 4), and the absorbent pad were attached to a plastic adhesive sheet to prepare an immunochromatographic test strip. At this time, a part of the sample pad overlaps with the antibody-immobilized membrane at one end of the antibody-immobilized membrane, and a part of the absorption pad at the other end of the antibody-immobilized membrane overlaps with the antibody-immobilized membrane. bottom.

Example 1 Sample Filtration Test Using Filtration Members 1 to 5 A sample filtration test was performed using each of the filtration members 1 to 5 produced in the sample filtration filter production examples described above.

1. Test method (1) sample
A sample was prepared by suspending nasal mucus of a healthy subject free of respiratory syncytial virus and adenovirus in sample diluent.

(2) Procedure Each filter member was attached to a test tube into which 500 μL of a sample was dispensed. One drop of the sample was dropped by squeezing the test tube with the tip side of the filtering member facing downward, and the weight of the drop was measured. This operation was repeated until no more droplets could be dropped.

(3) Results Table 3 shows the operability felt by each operator when pushing the test tube.
In Condition 1, none of the operators felt the hardness, and the operability was evaluated as good. Similar results were obtained under condition 5 as well. In condition 2, all operators felt a little harder than in condition 1, but it was judged to be practical. In each of conditions 3 and 4, all or most of the operators evaluated that it was hard, resulting in poor operability.

FIG. 3 shows the total drip amount when each filter member is used. FIG. 4 shows the droplet weight average value when each filter member is used. FIG. 5 shows variation in the amount of droplets (variation in weight between droplets when the same sample is continuously dropped) when each filter member is used. In normal lateral flow measurement, it is desirable that 100 to 150 μL of sample can be dropped with 3 to 5 drops. In addition, it is preferable to secure a sample amount that can be measured twice or more in preparation for re-measurement and measurement of multiple items.
Under any conditions, it was possible to drop a sample that exceeded the sample amount in normal measurement. However, in conditions 3 and 4, the total amount of dripping liquid was small in some samples. Furthermore, under conditions 3 and 4, there was a difference in the average weight of droplets between samples, and the variation in the amount of droplets dropped was extremely large, making it difficult to drop a constant amount of sample. Also, under conditions 3 and 4, there were some samples that could not drop 3 drops, which is the drop amount in normal measurement. Filter clogging appeared to occur during the filtration of samples 2 and 3 in condition 3 and during the filtration of samples 1 and 2 in condition 4. Under Condition 5, the droplet weight average value was constant among the samples, but a large variation was observed in the droplet amount. Under conditions 1 and 2, the average value of droplet weight between samples was constant, the variation coefficient of droplet weight was about 10%, and the dispersion of the droplet amount was small.

[Example 2] Respiratory syncytial virus and adenovirus detection test Using the immunochromatographic test strip prepared in the above test strip preparation example, the sample solution obtained using each filter member of conditions 1, 2, and 5 A detection test for respiratory syncytial virus and adenovirus was performed.

1. Test method (1) sample
Samples were prepared by suspending nasal mucus of healthy subjects free from respiratory syncytial virus and adenovirus in sample diluent.

(2) Procedure Each filter member of conditions 1, 2, and 5 was attached to a test tube into which 500 μL of the sample was dispensed, and the sample was filtered. 120 μL of the filtered sample was dropped onto the test strip, and after 10 minutes, it was visually determined whether the test line on the antibody-immobilized membrane developed color.

(3) Results Table 4 shows the test results of respiratory syncytial virus. Table 5 shows the adenovirus test results. All conditions 1, 2 and 5 were negative for respiratory syncytial virus. Regarding adenovirus, under condition 5, sample 3 showed non-specific reaction. Under conditions 1 and 2, no non-specific reaction was observed. Therefore, by using a filter member having the configuration of condition 1 or 2, in immunochromatographic analysis for detecting a substance to be detected, the occurrence of non-specific reactions is suppressed and clogging of the filter by solid components is prevented. thought it was possible.

According to the present invention, when measuring the amount of a substance to be detected or detecting its presence using immunochromatography, it is possible to suppress the occurrence of non-specific reactions by ensuring reproducibility and to filter the filter with solid components. clogging prevention can both be achieved.

a backing sheet b antibody-immobilized membrane c1 anti-RS virus monoclonal antibody (test line 1)
c2 anti-adenovirus monoclonal antibody (test line 2)
c3 control antibody (control line)
d conjugate holder e sample pad f absorbent pad

Claims (14)

A method for pretreating a biological sample for immunoassay to detect a substance to be detected, comprising:
filtering the biological sample with a filtering member comprising at least two filters comprising glass fibers having a retained particle size of 0.3 μm to 1.1 μm;
and wherein the at least two filters both contain an organic binder. 2. The pretreatment method according to claim 1, wherein the organic binder is an acrylic resin binder. 3. The pretreatment method according to claim 1 or 2, wherein the amount of the biological sample after pretreatment is 50% by mass or more of the amount of the biological sample before pretreatment. The pretreatment method according to any one of claims 1 to 3, wherein the biological sample is respiratory secretions. The pretreatment method according to any one of claims 1 to 4, wherein each of the at least two filters has a thickness of 0.1 mm to 0.7 mm. The at least two filters are composed of a first filter close to the opening side of the filtering member and a second filter close to the tip side of the filtering member,
The pretreatment method according to any one of claims 1 to 5, wherein the thickness of the first filter is smaller than the thickness of the second filter. The pretreatment method according to any one of claims 1 to 6, wherein the immunoassay is immunochromatography. (1) an immunological analysis method for detecting a substance to be detected, comprising the following steps (1) to (3); (2) contacting the sample supply portion of the test strip with the pretreated biological sample obtained in step (1) to detect a substance to be detected; forming a complex with the conjugate; and (3) detecting the signal from the label. A filter member for pretreatment of a biological sample to be subjected to immunoassay for detecting a substance to be detected,
The filtering member for pretreatment, comprising at least two filters containing glass fibers having a retained particle size of 0.3 μm to 1.1 μm, both of the at least two filters containing an organic binder. 10. The pretreatment filter member according to claim 9, wherein the organic binder is an acrylic resin binder. The pretreatment filtering member according to claim 9 or 10, wherein the biological sample is respiratory secretions. The pretreatment filtration member according to any one of claims 9 to 11, wherein each of the at least two filters has a thickness of 0.1 mm to 0.7 mm. The at least two filters are composed of a first filter close to the opening side of the pretreatment filter member and a second filter close to the tip side of the pretreatment filter member,
The pretreatment filtration member according to any one of claims 9 to 12, wherein the thickness of the first filter is smaller than the thickness of the second filter. The pretreatment filtering member according to any one of claims 9 to 13, wherein the immunoassay is immunochromatography.

Images