JP2007232384A - Testing apparatus for chromatography, and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a testing apparatus for chromatography, which is equipped with a reference part for determining whether a sample is properly developed, even when the sample including avidin and/or biotin is used. <P>SOLUTION: The testing apparatus for chromatography for detecting a component to be detected in a biosample is equipped with a label holding member for holding a labeling material bondable to the component to be detected and a reference labeling material, and a chromatograph medium having a determination part for immobilizing a fixing material bondable to the component to be detected and a reference part for immobilizing a material bondable to the reference labeling material. In the testing apparatus for chromatography, the reference labeling material includes either one of a protein to which hapten having substantially no reactivity to a component in the biosample including the component to be detected, and an anti-hapten antibody bondable to hapten, and the other is immobilized on the reference part. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a chromatographic test device and a method for producing the same.

An example of a test device that detects a component to be detected using a substance that specifically binds to the component to be detected in a sample is a chromatographic test device. When a sample suspected of having a component to be detected is added to a part of this test device (sample addition part), the sample moves inside the sample addition part by capillary action and retains a labeling substance that can bind to the component to be detected. Reach the labeling substance holding part. When the component to be detected is present, the component to be detected binds to the labeling substance to form a complex. Further, as the sample is developed by capillary action, the detected component-labeled substance complex moves through the chromatographic medium and reaches the determination region where the fixing substance capable of binding to the detected component is immobilized. This complex binds to the immobilizing substance. As a result, a sandwich-like complex of the labeling substance, the component to be detected and the immobilizing substance is formed in the determination region.
When the labeling substance includes colored carrier particles (colored latex particles, colloidal metal particles, etc.), the determination area is colored when the labeling substance is captured in the determination area. By observing the color development in this determination region, the component to be detected in the sample can be detected.

  When detecting a component to be detected in a sample using chromatography, a control region is usually provided in the chromatographic medium in order to confirm whether or not the sample has been properly developed. By developing a control substance (labeled control substance) labeled with colored carrier particles together with the sample, the substance that can bind to the control substance immobilized on the control region and the labeled control substance are bound together, and the labeled control substance becomes It will be captured in the control area. Since the labeled control substance is colored, the control area develops color when captured by the control area. By observing the color development in the control region, it can be confirmed whether or not the sample has been properly developed.

  An example of a test device using chromatography is an apparatus described in Patent Document 1. Patent Document 1 describes that a combination of a hapten and a ligand specific to the hapten (for example, a combination of avidin and biotin) is used as an internal control. By comparing the color development in the internal control area of this apparatus with the color development in the determination area, the component to be detected in the sample can be quantified. Specifically, avidin is applied to a part of the nitrocellulose membrane of this apparatus to provide a control site, and the labeled particles are sensitized with biotin.

  As a control for confirming whether or not the sample has been properly developed, biotin and avidin can be used as in the internal control described in Patent Document 1. However, if a sample containing biotin, such as a sample prepared using human or avian stool, is used, the biotin in the sample binds to avidin immobilized on the control part. Can no longer bind to avidin. In addition, when a sample containing avidin such as a sample containing a component of a chicken egg that has been subjected to virus culture is used, the avidin in the sample binds to labeled biotin, and the labeled biotin cannot bind to avidin in the control part. Disappear. Therefore, in these cases, since the control part does not develop color or hardly develops color, it may not be possible to determine whether or not the sample has been properly developed.

Patent Document 1 describes that 2,4-dinitrophenol (hereinafter referred to as DNP) is adsorbed on particles, and an anti-DNP antibody can be immobilized on a chromatographic medium to provide an internal control. ing. However, since the binding force between the carrier particles and the chromatographic medium and the above-described hapten is weak, it is difficult to bind the hapten to these substances, and even if they are combined, they are easily separated. If the hapten separates from these substances during sample development, it may not be possible to confirm whether the sample has been properly developed.
JP 2001-33454 A

  In view of the above-mentioned present situation, the present invention is provided with a control part that can determine whether or not the sample has been properly developed even if a sample containing avidin and / or biotin is used, and a labeling substance or control in which the hapten is difficult to separate. An object of the present invention is to provide a thin-layer chromatography test device having a part.

Therefore, the present invention is a chromatographic test tool for detecting a component to be detected in a biological sample,
A label holding member for holding a labeling substance capable of binding to the detected component and a control labeling substance;
A chromatographic medium having a determination unit immobilizing a fixing substance capable of binding to the component to be detected and a control part immobilizing a substance capable of binding to the control labeling substance,
The control labeling substance includes any one of a protein to which a hapten having substantially no reactivity with a component in the biological sample containing the detected component is bound and an anti-hapten antibody capable of binding to the hapten. The remaining one is a chromatographic test device fixed to the control part.

  According to the present invention, it is possible to correctly perform the reaction of the control part even when using a sample, for example, a human stool sample, which has been difficult to perform the reaction of the control part correctly in the conventional chromatographic test device. Further, according to the present invention, there is provided a test device provided with a labeling substance or a control part in which a hapten is difficult to separate. Therefore, in the chromatographic test device of the present invention, the detection result can be made more reliable.

Examples of biological samples used in the chromatographic test device of the present invention include samples obtained from animals such as mammals and birds and samples obtained from plants. The biological sample is more preferably a sample obtained from an animal, particularly a sample obtained from a mammal including a human and a sample obtained from a bird. Samples obtained from mammals including humans include blood, serum, feces, urine, saliva, nasal aspirate, swabs and the like. Samples obtained from birds include blood, serum, eggs, stool, total excretory cavity wipes, and the like.
The “biological sample” in this specification includes not only a sample collected from a living body but also a sample prepared by subjecting this sample to pretreatment. In addition, “pretreatment” means that a sample collected from a living body is processed into a state suitable for detection. The pretreatment includes, for example, adding an appropriate pretreatment reagent to a sample collected from a living body and filtering it with a filter to obtain a filtrate. Moreover, when the component to be detected in a sample collected from a living body is a very small amount, the component to be detected can be amplified before detection as a pretreatment.

  The reagent for the pretreatment is not particularly limited, and purified water, physiological saline, phosphate buffer, tris (hydroxymethyl) aminomethane (Tris) buffer, N- (2-hydroxyethyl) piperazine-N ′ -(2-ethanesulfonic acid) (HEPES) solution, piperazine-N, N'-bis (2-ethanesulfonic acid) (PIBES) solution, 3- (cyanohexylamino) -1-propanesulfonic acid (CAPS) solution , 3- (morpholino) propanesulfonic acid (MOPS) solution, amino acid solution and the like.

The component to be detected that can be detected by the chromatographic test device of the present invention is not particularly limited as long as it can obtain an antibody against the component to be detected. Cells such as bacteria, protists and fungi, viruses, proteins And polysaccharides. For example, influenza virus, parainfluenza virus, RS virus, rotavirus, calcivirus, coronavirus, adenovirus, enterovirus, herpes virus, human immunodeficiency virus, hepatitis virus, pathogenic virus for severe acute respiratory syndrome (coronavirus), etc. Viruses such as Mycoplasma pneumoniae, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus spyogenes, malaria parasite, pathogens of various diseases such as digestive system diseases, central nervous system diseases, hemorrhagic fever, metabolites of these A tumor marker such as carcinoembryonic antigen or Shifura; a hormone or the like. One or more of these detected components may be contained in the biological sample.
In particular, the chromatographic test device of the present invention can be suitably used for detecting one or more viruses selected from influenza virus, coronavirus, rotavirus and adenovirus.

  In the chromatographic test device of the present invention, the control labeling substance is a protein bound to a hapten that has substantially no reactivity with a component in a biological sample (hereinafter referred to as “hapten-binding protein”) and One of the anti-hapten antibodies capable of binding to the hapten (hereinafter referred to as “anti-hapten antibody”) is included, and the other one is immobilized on the control part.

  The control labeling substance preferably contains one of the hapten-binding protein and the anti-hapten antibody and a carrier. The carrier is preferably in the form of particles, for example, agar, agarose, crosslinked agarose, crosslinked alginic acid, crosslinked guar gum, cellulose esters such as nitrocellulose and carboxyl cellulose, gelatin, crosslinked gelatin, latex, rubber, polyethylene, polypropylene, polystyrene, Natural or synthetic resins such as styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, and the like Particles made of inorganic materials such as derivatives, glass (e.g. activated glass), silica gel, kaolin, talc, silica-alumina, alumina, barium sulfate, etc. It can be exemplified labeled to obtain particles, magnetic particles, red blood cells, a metal colloid such as gold. Of these, latex particles and gold colloids having dye molecules, fluorescent molecules or magnetic particles are preferred.

  The method for labeling the hapten-binding protein or the anti-hapten antibody with the above-mentioned carrier is not particularly limited as long as it is a method that is usually used in the art. For example, physical methods such as ionic interaction, hydrophobic interaction, and covalent bond are used. This can be done by adsorption or chemical bonding using a suitable cross-linking agent.

  When latex is used as a carrier, the method for binding latex to a hapten-binding protein or anti-hapten antibody is not particularly limited. For example, a functional group for binding to a protein can be introduced on the latex surface using a crosslinking agent, and a hapten-binding protein or an anti-hapten antibody can be bound to this functional group. As a crosslinking agent, the below-mentioned crosslinking agent can be used.

  The hapten is not substantially reactive with components in the biological sample. Examples of components in the biological sample include amino acids, proteins, nucleic acids, hormones and the like in addition to avidin and biotin. Examples of haptens that are not substantially reactive with components present in the living body include 2,4-dinitrophenol (DNP) and digoxin. Since the control part or the labeling substance of the chromatographic medium includes such a hapten, it can be confirmed whether or not the sample has been properly developed regardless of the type of sample. Thereby, it is not necessary to redesign the control part of the test device for each animal species from which the sample is derived, and the production of the control part can be made common, so that the manufacturing cost can be reduced.

  The protein to which the hapten binds is not particularly limited as long as the protein has a functional group for binding to the hapten. Examples of the functional group for binding to the hapten include a sulfhydryl group, an amino group, and a carboxyl group. Therefore, the type of the protein is not particularly limited. As the above protein, albumin (for example, bovine serum albumin (BSA), egg albumin (OVA), etc.), casein, fibrinogen and the like are preferably used.

The above hapten and protein are preferably bound using a crosslinking agent. A crosslinking agent will not be specifically limited if it is a crosslinking agent normally used for the coupling | bonding of a hapten and protein. Specifically, a hapten and a protein can be bound by introducing a functional group into the hapten using a crosslinking agent and reacting the functional group with a functional group of the protein. Another functional group can also be introduced into the functional group of the protein using a crosslinking agent.
More specifically, a sulfhydryl group introduced into a terminal amino group of a protein with a crosslinking agent is reacted with a functional group highly reactive with the sulfhydryl group introduced into the hapten with a crosslinking agent, such as a maleimide group. Thus, the hapten and the protein can be bound, but the present invention is not limited to this form.

  Although it does not limit as a compound which can be used as said crosslinking agent, For example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC), N-hydroxysuccinimide (NHS), 2-iminothiolane, N, N ' -O-phenylene dimaleimide, N-succinimidyl S-acetylthioacetate (SATA), N-succinimidyl S-acetylthiopropionate (SATP), N-succinimidyl 3- (2-pyridyldithio) propionate, N-succinimidyl 4 -(N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl 6-maleimidohexanoate, N-succinimidyl 4-iodoacetylaminobenzoate, N-succinimidyl 3- (p-hydroxyphenyl) propiate Examples thereof include 1-onate, N-succinimidyl m-maleimidobenzoate, N-succinimidyl 4-maleimidobutyrate, N-succinimidyl (p-maleimidophenyl) acetate, N-succinimidyl 4- (p-maleimidophenyl) butyrate, etc. A seed may be used and two or more kinds may be used.

The anti-hapten antibody capable of binding to the hapten used in the chromatographic test device of the present invention can be obtained in accordance with ordinary techniques for antibody production. Specifically, the hapten is bound to an appropriate carrier, and the resulting conjugate is used to immunize animals such as horses, cows, sheep, rabbits, goats, rats, mice, etc., and serum is collected from the animals to obtain anti-hapten antibodies. The method of purifying can be used.
The anti-hapten antibody may be a polyclonal antibody or a monoclonal antibody, and these may be used as a mixture. Antibody fragments and derivatives thereof can also be used. As used herein, “antibody” includes antibody fragments and derivatives thereof. Antibody fragments and derivatives thereof include Fab, Fab ′, F (ab) ₂ and sFv fragments (Blazar et al., 1997, J. Immunol., 159: 5821-5833 and Bird et al., 1988, Science). , 242: 423-426). The subclass of the antibody is not limited to IgG, and may be IgM.

  In the chromatographic test device of the present invention, either the hapten-binding protein or the anti-hapten antibody is held on the label holding member, but the control labeling substance containing the anti-hapten antibody is held on the label holding member. preferable. When the control labeling substance containing the anti-hapten antibody is held on the label holding member, the hapten-binding protein is immobilized on the control part. Although it is difficult to bind only the hapten to the chromatographic medium, in the present invention, since the protein is bound to the hapten, physical action between the protein and the chromatographic medium (for example, ionic interaction, hydrophobic interaction, etc.) ) Makes it possible to easily fix the hapten-binding protein to the control part of the chromatographic medium.

  When the anti-hapten antibody is immobilized on the control part, it is preferable that the control labeling substance containing the hapten-binding protein and the carrier is held on the label holding member. Although it is difficult to bind only the hapten to the carrier, in the present invention, since the protein is bound to the hapten, as described above, the hapten-binding protein can be easily bound to the carrier. As described above, by using the hapten-binding protein, the hapten can be firmly bound to the carrier or the chromatographic medium, so that the development of the sample can be appropriately confirmed without separation of the hapten.

  When immobilizing the hapten-binding protein on the control part of the chromatographic medium, the immobilization method includes applying a hapten-binding protein solution in which the hapten-binding protein is dissolved in an appropriate solvent to the chromatographic medium and drying it appropriately. Can be mentioned. The concentration of the hapten-binding protein solution and the amount to be applied are not particularly limited, and can be appropriately changed according to the desired sensitivity of the chromatographic test device. The drying time and temperature are not particularly limited as long as they do not affect the hapten-binding protein, and are, for example, 30 to 55 ° C. for 20 minutes to 1 hour.

  The chromatographic test device of the present invention preferably includes a sample addition member. The sample addition member can be formed of various materials such as rayon, glass fiber, and cellulose fiber.

  The chromatographic test device of the present invention includes a label holding member that holds a labeling substance capable of binding to a component to be detected and a control labeling substance. The label holding member can be formed of various materials such as glass fiber, cellulose fiber, and plastic (for example, polyester, polypropylene, polyethylene, etc.) fiber.

  The labeling substance capable of binding to the component to be detected held on the label holding member is preferably one that specifically binds to the component to be detected, and more preferably one in which an antibody against the component to be detected is labeled with a carrier. preferable. The antibody against the component to be detected can be obtained by a method similar to the method for obtaining an anti-hapten antibody. Specifically, the component to be detected is bound to an appropriate carrier protein as necessary, and animals such as horses, cows, sheep, rabbits, goats, rats, and mice are immunized, and serum is collected from the animals to obtain antibodies. A purification method can be used. The antibody that binds to the component to be detected may be a polyclonal antibody, but is preferably a monoclonal antibody.

  As the above-mentioned carrier, the carrier exemplified for the above-mentioned control labeling substance can be suitably used. As a method for labeling the antibody against the component to be detected with a carrier, the same method as the method for labeling a hapten-binding protein or anti-hapten antibody with a carrier can be used.

  The labeling substance and the control labeling substance that can be bound to the component to be detected held on the label holding member may be labeled in different colors or in the same color.

  Examples of a method for holding a labeling substance capable of binding to the component to be detected or a control labeling substance on the label holding member include a method in which the label holding member is impregnated with a buffer solution containing these labeling substances and dried appropriately.

  The chromatographic test device of the present invention includes a chromatographic medium having a determination part in which an immobilizing substance capable of binding to a component to be detected is immobilized and a control part in which a substance capable of being bound to a reference labeling substance is immobilized. The chromatographic medium only needs to be formed of a material that can bind to a protein by a physical action such as electrostatic action or hydrophobic interaction and can develop a sample by capillary action. Examples of such materials include nitrocellulose, nylon (for example, modified nylon having an amino group which may have a carboxyl group or an alkyl group as a substituent), polyvinylidene difluoride (PVDF), cellulose acetate, and the like. Can be mentioned.

  In the chromatographic medium, a fixing substance that can bind to the component to be detected is fixed to the determination unit. The immobilizing substance recognizes the detected component at a site different from the site where the labeling substance that can bind to the detected component recognizes the detected component. As the immobilization substance, it is preferable to use an antibody that can specifically bind to the component to be detected. Specifically, the substance to be detected is different from the site to which the labeling substance that can bind to the component to be detected binds. Antibodies that bind can be used.

  The chromatographic medium may include only one determination unit or two or more according to the type of component to be detected. When two or more are provided, a fixing substance that can bind to different types of components to be detected is immobilized in each determination unit.

As a method for immobilizing the immobilizing substance capable of binding to the component to be detected in the determination unit, the same method as the method for immobilizing the control labeling substance on the control unit can be used.
The determination part and the contrast part of the chromatographic medium can be formed in a line shape, a circular shape, a square shape, or the like.

  In the chromatographic test device of the present invention, the sample addition member and the label holding member are preferably in contact with each other, and either or both of the sample addition member and the label holding member are preferably in contact with the chromatographic medium. Specifically, (a) the sample addition member and the label holding member are in contact with each other, and the label holding member and the chromatographic medium are in contact with each other; (b) the sample addition member and the label holding member are in contact with each other; A form in which the sample addition member and the chromatographic medium are in contact; and (c) the sample addition member and the label holding member are in contact, the sample addition member and the chromatographic medium are in contact, and the label holding member and the chromatograph. And a form in contact with the medium. An example of each of the forms (a) to (c) is shown in FIG.

  By contacting each member as described above, the sample added to the sample addition member comes into contact with the labeling substance capable of binding to the component to be detected and the labeling substance for control, and can be developed into a chromatographic medium. . In addition, it is possible to promote the development of the sample on the sample addition member, the label holding member, and the chromatographic medium. Between the sample addition member and the label holding member, between the label holding member and the chromatographic medium, or between the sample addition member and the chromatographic medium, there may be a member that can further promote the development of the sample.

  The method for bringing the sample addition member, the label holding member, and the chromatographic medium into contact with each other as described above is not particularly limited. For example, by placing these members in contact with each other as appropriate on an adhesive substrate. Can be connected. As the base material, various materials such as plastic, paper, and glass can be used.

The chromatographic test device of the present invention can include an absorbing member in addition to the chromatographic medium, the sample addition member, and the label holding member. The absorbent member can be formed of various materials such as cellulose and glass fiber. By having the absorbing member, the developing speed of the sample is increased.
The above-mentioned chromatographic medium, sample addition member, label holding member and optionally absorbing member can be of various structures that can develop the sample by capillary action, in addition to the nonwoven fabric and porous body. .

The chromatographic test device of the present invention is:
A step of immobilizing a hapten-bound protein that does not react with a component in a sample to the chromatographic medium, and a control labeling substance containing an anti-hapten antibody capable of binding to the hapten is attached to the label holding member. It can be manufactured by a method including a holding step.
The order in which these steps are performed is not particularly limited.

  Preferably, the manufacturing method further includes a step of connecting the label holding member to the sample addition member and a step of connecting a chromatographic medium to either or both of the sample addition member and the label holding member. The order in which these steps are performed is not particularly limited. These steps are preferably performed after the above two steps.

  The present invention also uses as a control reaction the reaction between a protein bound to a hapten that is not substantially reactive to the components in the biological sample and an anti-hapten antibody that can specifically bind to the hapten. It is also a method for detecting by chromatography a component to be detected in a biological sample, including use.

The present invention will be described in detail according to the following examples, but the present invention is not limited to the forms of these examples.
The buffer solutions used in the following production examples and comparative production examples are as follows.
Washing buffer: 10 mM phosphate buffered saline, pH 6.0
Binding buffer: 10 mM phosphate buffered saline, pH 7.0
Blocking buffer: 10 mM BSA-containing 10 mM phosphate buffered saline, pH 7.0
Storage buffer: 7% BSA, 10% sucrose, 10 mM phosphate buffered saline, pH 8.0

Production Example 1: Production of chromatographic test device using reaction of anti-DNP antibody-DNP (A) Preparation of anti-DNP antibody-sensitized latex (1) Red latex particles (particle size 0.19 μm, JSR G0103R, polystyrene) 1.9 mL of a washing buffer solution was added to 0.1 mL of a 10% dispersion of (latex particles), and the mixture was stirred and mixed.
(2) Using an ultrasonic crusher (Ultrasonic homogenizer US-600T, Nippon Seiki Seisakusho) while cooling the resulting dispersion in ice water, irradiation for 30 seconds under the conditions of V-LEVEL: 200 μA, tip: φ7 mm And 30 cycles of 30 second intervals.
(3) 1-Ethyl 3- (3-dimethylaminopropylcarbodiimide) (EDAC) and N-hydroxysuccinimide (NHS) were dissolved in a washing buffer so as to be 0.05M and 0.027M, respectively.
(4) 0.1 mL of the obtained EDAC / NHS solution was quickly added to the dispersion treated in (2) and gently stirred at room temperature for 15 minutes.
(5) The latex particles were washed by the following method.
Washing method: 30,000 g for 10 minutes in a high-speed centrifuge to precipitate latex particles. The supernatant is removed, 2 mL of binding buffer is added, and latex particles are redispersed using an ultrasonic crusher under the same conditions as in (2) above.
(6) The above step (5) was performed again.
(7) To the latex particle dispersion, 100 μL of an anti-DNP antibody solution (mouse IgG monoclonal antibody, antibody concentration 5 mg / mL) was added with gentle stirring, followed by stirring at 25 ° C. for 2 hours.
(8) The above step (5) was performed.
(9) 2 mL of blocking buffer was added with gentle stirring and incubated at 37 ° C. for 60 minutes.
(10) Centrifugation at 30,000 g for 10 minutes in a high-speed centrifuge to precipitate latex particles, remove the supernatant, add 2 mL of storage buffer, and under the same conditions as in (2) above, The latex particles were redispersed using an ultrasonic crusher.
(11) The solution was adjusted with a storage buffer so that the latex concentration was 0.2%, and stored at 2-8 ° C. until use.

(B) Preparation of DNP-bound BSA as hapten-binding protein Preparation of DNP-maleimide (1) N-DNP-L-lysine (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter referred to as “DNP-lysine”) 16 mg, 50% dimethylformamide 0.45 mL was added and stirred to dissolve DNP-lysine.
(2) 0.45 mL of 50% dimethylformamide was added to 15 mg of N- (6-maleimidocaproyloxy) succinimide (EMCS, manufactured by Doujin Chemical Co., Ltd.) and stirred to dissolve the EMCS.
(3) 0.23 mL of EMCS solution was added to the above DNP-lysine solution and stirred.
(4) 1.6 mL of 0.1 M sodium phosphate buffer was added and stirred.
(5) Reaction was performed at 30 ° C. for 1 hour to obtain a DNP-maleimide solution.

Preparation of DNP-bound BSA (1) Bovine albumin (protease-free, manufactured by Intergen) 0.052 g was dissolved in 2.6 mL of 0.1 M phosphate buffer (pH 7.0) to obtain an albumin solution.
(2) 16 mg of N-succinimidyl-S-acetylthioacetate (SATA, manufactured by Pierce) was dissolved in 2 mL of 50% dimethylformamide to obtain a SATA solution.
(3) 0.1 mL of the SATA solution was added to the total amount of the albumin solution, stirred, and reacted at 30 ° C. for 30 minutes.
(4) 0.26 mL of 1 M Tris (pH 7.0), 0.13 mL of 0.1 M EDTA (pH 7.0) and 0.31 mL of 1 M hydroxylamine (pH 7.0) were added to the obtained reaction solution in this order. The reaction was allowed to stand for 10-20 minutes at ambient temperature (20-30 ° C.).
(5) 1.6 mL of the above DNP-maleimide solution was added and stirred, and allowed to react at 30 ° C. for 30 minutes.
(6) The obtained solution was applied to a PD-10 column (Amersham) to remove DNP that did not bind to albumin.
(7) The recovered DNP-bound BSA was adjusted to a concentration of 0.5 mg / mL with 10 mM phosphate buffered saline (pH 7.0).

(C) Preparation of anti-adenovirus antibody-sensitized latex (1) 0.1 mL of a 10% dispersion of blue latex particles (particle size 0.3 μm, manufactured by JSR, polystyrene latex particles) was added to a binding buffer solution of 1.9 mL. The mixture was stirred and mixed.
(2) Using an ultrasonic crusher (Ultrasonic homogenizer US-600T, Nippon Seiki Seisakusho) while cooling the resulting dispersion in ice water, irradiation for 30 seconds under the conditions of V-LEVEL: 200 μA, tip: φ7 mm And 30 cycles of 30 second intervals.
(3) Glutaraldehyde was added so that it might become 0.1%, and it was made to react at 25 degreeC for 1 hour.
(4) The latex particles were washed by the following method.
Washing method: 30,000 g for 10 minutes in a high-speed centrifuge to precipitate latex particles. The supernatant is removed, a binding buffer is added, and latex particles are redispersed using an ultrasonic crusher under the same conditions as in (2) above.
(5) To this latex dispersion, an anti-adenovirus antibody (mouse IgG monoclonal antibody) was added at 100 μg / mL and reacted at 25 ° C. for 2 hours.
(6) The above step (4) was performed again.
(7) The obtained dispersion is centrifuged, the supernatant is removed, PBS containing 3% taurine (pH 8.0) is added to the remaining sediment, and a blocking buffer is added with gentle stirring. Incubate overnight at 37 ° C.
(8) Centrifugation at 30,000 g for 10 minutes in a high-speed centrifuge to precipitate latex particles, remove the supernatant, add 2 mL of storage buffer, and under the same conditions as in (2) above, The latex particles were redispersed using an ultrasonic crusher.
(9) The latex was adjusted with a storage buffer so that the latex concentration was 0.2%, and stored at 2-8 ° C. until use.

(D) Preparation of chromatographic medium (1) Anti-adenovirus antibody as a fixing substance capable of binding to the component to be detected (mouse IgG monoclonal antibody, adenovirus at a site different from the above-mentioned anti-adenovirus antibody labeled with latex) Was adjusted with 10 mM phosphate buffered saline (pH 7.2) to 1 mg / mL.
(2) 0.5 mg / mL DNP obtained in (B) above using XYZ3000 (manufactured by Biodot) on a nitrocellulose membrane (Millipore, high flow membrane HF135, width 1 cm × length 30 cm) The bound BSA solution and the anti-adenovirus antibody solution of (1) were each applied in a line and dried at 50 ° C. for 2 hours using a dry heat dryer.
(3) The obtained membrane was immersed in 1% bovine albumin-10 mM phosphate buffer (pH 7.0) for 1 minute.
(4) The membrane was immersed in 0.01% SDS-5% sucrose-10 mM phosphate buffer (pH 7.0) for 5 minutes. This process was performed three more times with the buffer exchanged.
(5) The membrane was dried at 40 ° C. for 2 hours using a dry heat dryer.

(E) Production of label holding member (1) Anti-adenovirus antibody-sensitized latex produced in (C) above and anti-DNP antibody-sensitized latex produced in (A) above were mixed 40: 1 (latex). In a weight ratio).
(2) After impregnating this liquid into a glass fiber filter paper (20 cm × 1 cm, manufactured by Whatman), it was dried under reduced pressure at 30 ° C. for 2 hours.
(F) Manufacture of chromatographic test device Chromatographic medium manufactured in (D) above, label holding member manufactured in (E), sample addition member (manufactured by Whatman, glass fiber filter paper GF-AVA) and absorption A member (manufactured by Whatman, glass fiber filter paper WF1.5) was attached to the mount in this order from the side closer to the mount and cut into strips of 6 cm length and 6 mm width. This was stored in a plastic case.

Comparative Production Example 1: Production of test device using streptavidin-biotin reaction (A) Preparation of streptavidin-sensitized latex In Production Example 1 (A), a streptavidin solution (2 mg / mL) was used instead of the anti-DNP antibody solution. A streptavidin-sensitized latex was prepared in the same manner as in (A) of Production Example 1 except that (1) was used.

(B) Preparation of biotin-conjugated BSA Preparation of biotin-maleimide (1) Biotin (manufactured by Sigma) 15 mg was added with 0.4 mL of 0.1 M phosphate buffer.
(2) To 10 mg of N- (6-maleimidocaproyloxy) succinimide (EMCS, manufactured by Doujin Chemical Co., Ltd.), 0.32 mL of 50% dimethylformamide was added and stirred to dissolve EMCS.
(3) 0.2 mL of EMCS solution was added to the total amount of the above biotin solution and stirred.
(4) 1.4 mL of 0.1 M sodium phosphate buffer was added and stirred.
(5) The mixture was reacted at 30 ° C. for 1 hour to obtain a biotin-maleimide solution.

Preparation of biotin-conjugated BSA (1) Bovine albumin (protease-free, manufactured by Intergen) 0.027 g was dissolved in 2.6 mL of 0.1 M phosphate buffer (pH 7.0) to obtain an albumin solution.
(2) 10 mg of N-succinimidyl-S-acetylthioacetate (SATA, manufactured by Pierce) was dissolved in 0.42 mL of 50% dimethylformamide to obtain a SATA solution.
(3) 0.08 mL of the SATA solution was added to the total amount of the albumin solution, stirred, and reacted at 30 ° C. for 30 minutes.
(4) 0.26 mL of 1 M Tris (pH 7.0), 0.13 mL of 0.1 M EDTA (pH 7.0) and 0.31 mL of 1 M hydroxylamine (pH 7.0) were added to the obtained reaction solution in this order. The reaction was allowed to stand for 10-20 minutes at ambient temperature (20-30 ° C.).
(5) 1.6 mL of the above biotin-maleimide solution was added and stirred, and reacted at 30 ° C. for 30 to 40 minutes.
(6) The resulting solution was applied to a PD-10 column (Amersham) to remove biotin that did not bind to albumin.
(7) The recovered biotin-bound BSA was adjusted to a concentration of 1.0 mg / mL with 10 mM phosphate buffered saline (pH 7.0).

(C) Preparation of anti-adenovirus antibody-sensitized latex In the same manner as in Production Example 1 (C), anti-adenovirus antibody-sensitized latex was prepared.

(D) Preparation of chromatographic medium In (D) of Production Example 1, (D) of Production Example 1 was repeated using a biotin-conjugated BSA solution instead of the DNP-conjugated BSA solution, and an anti-adenovirus antibody and biotin-conjugated BSA were A chromatographic medium in which was immobilized.

(E) Production of Label Holding Member In (E) of Production Example 1, (E) of Production Example 1 is repeated using streptavidin-sensitized latex instead of anti-DNP antibody-sensitized latex, and anti-adenovirus antibody sensitization A holding member holding the prepared latex and the streptavidin-sensitized latex was obtained.

(F) Manufacture of test device In (F) of Production Example 1, using the chromatographic medium produced in (D) of this Comparative Production Example and the label holding member produced in (E), (F) of Production Example 1 ) Was repeated to produce a test device.

Example 1 and Comparative Example 1
A supernatant obtained by suspending 100 mg of human stool in 10 mL of phosphate buffer and centrifuging at 6000 rpm was used as a biological sample. Five specimens collected from five humans were used. In addition, it has been confirmed that these biological samples are free of adenovirus by a virus separation test.
Five drops of a biological sample were dropped on the test devices obtained in Production Example 1 and Comparative Production Example 1 using a dropper, and the coloring of the judgment part and the control part was visually observed. The results are shown in Table 1.

In Table 1, “+” indicates that the coloration can be confirmed in a line shape, and “−” indicates that the color cannot be confirmed.
From Table 1, it can be seen that in the test device of Comparative Production Example 1, no color development in the control part was confirmed with any specimen. This is considered to be because biotin contained in the biological sample (stool) was bound to the streptavidin-sensitized latex and the streptavidin-sensitized latex could not be bound to biotin in the control part. On the other hand, in the test device of Production Example 1, color development of the control part was confirmed regardless of which specimen was used. This is because both the substance immobilized on the control part and the control labeling substance were substances that were not substantially reactive with the components in the biological sample, so that the control labeling substance was properly captured by the control part. It shows that the development of the sample was confirmed.

Production Example 2: Production of chromatographic test device using reaction of anti-DNP antibody-DNP In production example 1 (C), an anti-influenza antibody (mouse IgG monoclonal antibody) was used instead of the anti-adenovirus antibody. In D), an anti-influenza antibody (mouse IgG monoclonal antibody, which recognizes influenza at a site different from the anti-influenza antibody used in (C)) was used to repeat Production Example 1 to produce a chromatographic test device.

Example 2
A total excretory cavity wipe of chicken was used as a biological sample. In these samples, 5 samples were confirmed to be influenza positive (sample numbers 1 to 5) and 5 samples were confirmed to be influenza virus negative (sample numbers 6 to 10) by a virus separation test.
Five drops of a biological sample were dropped onto the test device obtained in Production Example 2 using a dropper, and the coloring of the judgment part and the control part was visually observed. The results are shown in Table 2.

In Table 2, “+” indicates that the coloration can be confirmed in a line shape, and “−” indicates that the color cannot be confirmed.
From Table 2, it can be seen that coloring of the control part was confirmed using any of the influenza virus positive samples (sample numbers 1 to 5) and the influenza virus negative samples (sample numbers 6 to 10). This indicates that the sample was properly developed regardless of which sample was used. In addition, when the influenza virus positive sample was used, the coloration of the judgment part was confirmed, and when the influenza virus negative sample was used, the coloration of the judgment part was not confirmed. It can be seen that it was detected.

The example of the form of the test device for chromatography of this invention is shown.

Explanation of symbols

1 Sample addition member 2 Label holding member 3 Chromatographic medium 4 Absorbing member

Claims (6)

A chromatographic test device for detecting a component to be detected in a biological sample,
A label holding member for holding a labeling substance capable of binding to the detected component and a control labeling substance;
A chromatographic medium having a determination unit immobilizing a fixing substance capable of binding to the component to be detected and a control part immobilizing a substance capable of binding to the control labeling substance,
The control labeling substance includes any one of a protein to which a hapten having substantially no reactivity with a component in the biological sample containing the detected component is bound and an anti-hapten antibody capable of binding to the hapten. A chromatographic test device, the other of which is fixed to the control part.   The chromatographic test device according to claim 1, wherein the protein to which the hapten is bound is immobilized on the control part, and the control labeling substance contains the anti-hapten antibody.   The chromatographic test device according to claim 1 or 2, wherein the hapten is dinitrophenol.   The chromatographic test device according to any one of claims 1 to 3, wherein the protein is albumin.   The chromatographic test device according to any one of claims 1 to 4, wherein the component to be detected is at least one selected from the group consisting of influenza virus, coronavirus, rotavirus and adenovirus. A method for producing a chromatographic test device comprising a label holding member and a chromatographic medium,
A step of immobilizing a hapten-bound protein that does not react with a component in a sample to the chromatographic medium, and a control labeling substance containing an anti-hapten antibody capable of binding to the hapten is attached to the label holding member. A method for producing a chromatographic test device comprising a step of holding.

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