KR20020032752A - A method for detecting several target materials simultaneously by the multiple immune reaction and a kit therefor - Google Patents

Abstract

PURPOSE: A method for detection target materials and kit for detection is provided to obtain a large volume of information during a short analysis time, while allowing a wide variety of types of target materials to be simultaneously and easily analyzed. CONSTITUTION: A method comprises a first step of injecting a sample for analysis, into a capillary passage(31) where first antigens of two or more types with respect to the target materials of two or mote types are fixed; a second step of performing a first antigen-antibody reaction by shifting the sample to a reaction space where antigens of two or more types are fixed; a third step of removing non-reactive sample solution from the capillary passage, and washing the capillary passage; a fourth step of performing a secondary antigen-antibody reaction by injecting a second antibody to be coupled to the target material, into the capillary passage and shifting the second antibody to the reaction space; a fifth step removing non-reactive second antibody from the capillary passage, and washing the capillary passage; a sixth step of performing color developing precipitation reaction with the color developing enzyme attached to the capillary passage; and a seventh step of checking the existence of the target material in the sample by measuring the degree of color developing precipitation reaction.

Description

A method for detecting several target materials simultaneously by the multiple immune reaction and a kit therefor}

The present invention relates to a method for simultaneously detecting several kinds of target substances through multiple immune responses and a kit for detection therefor.

When a specific substance is to be analyzed in a sample such as blood collected from a test subject for the presence or absence of pathogenic microorganisms in the body, infectious diseases, and immunity, current microbial plate analysis methods are performed in a microwell plate. Analytical and immunochromatographic techniques, which are performed by the EIA technique, are utilized. The features and disadvantages of these methods are as follows.

EIA technique of the above method is to fix the antigen (or antibody) for the target material on the inner surface of each well of the micro-well plate, inject the sample to the immune response, and then the enzyme-attached secondary antibody After the reaction, and then adding a coloring reagent to the color reaction by the enzyme is carried out to determine the presence of the target material in the sample by measuring the degree of color development using analytical equipment and the like.

In addition, immunochromatography immobilizes an antigen (or antibody) to a target substance on a membrane, and injects a sample having a primary antigen-antibody reaction with a gold powder to which an antigen (or antibody) is attached. As the solution component moves the surface of the membrane by chromatographic effect, the target material in the sample undergoes a secondary antigen-antibody reaction with an immobilized antigen (or antibody) on the membrane, forming the band represented by the gold powder. By checking the degree, it is a method of analyzing the target substance in a sample.

The conventional methods described above require a skilled technician, analytical equipment, and the like, take a long time to obtain a result, and can not simultaneously test several proteins in one experiment. In order to analyze several kinds of target substances, the same process has to be performed several times separately for each of them, which is problematic in terms of inconvenience, time and cost.

Accordingly, an object of the present invention is to provide a method for easily detecting a plurality of target substances in a short time using a capillary passage in a single step, and a simple detection in which ordinary people can easily perform sample analysis by the method of the present invention. To provide a kit.

1 is an exploded perspective view showing the configuration of a detection kit according to the present invention.

Figure 2a is a longitudinal sectional view showing a state in which a sample is injected into the sample injection means constituting the detection kit according to the present invention.

Figure 2b is a longitudinal sectional view showing a state driven by the first drive means after the sample is injected into the sample injection means constituting the detection kit according to the present invention.

Figure 3a to Figure 3d is a longitudinal cross-sectional view showing the operating state of the second antibody storage means and the color precipitation reagent storage means constituting the detection kit according to the present invention.

Figure 4a to 4b is a cross-sectional view showing the operating state of the second antibody storage means and the color precipitation reagent storage means constituting the detection kit according to the present invention.

Figure 5 is a perspective view showing the appearance of a drive device for driving a detection kit according to the present invention.

Figure 6 is a longitudinal cross-sectional view of the coupling kit in a state where the detection kit according to the present invention is installed in the main body constituting the drive device.

Fig. 7 is a block diagram showing the configuration of a drive device for driving a detection kit according to the present invention.

<Description of the symbols for the main parts of the drawings>

30: body

31: Moses passage

31a: entrance

31b: exit section

60: sample injection means

50: second antibody storage means

70: coloring precipitation reagent storage means

The method of the present invention for achieving the above object comprises the following steps:

(1) injecting a sample to be analyzed into a capillary passage in which two or more first antigens (or antibodies) for two or more target substances are fixed at regular intervals on an inner surface thereof,

(2) transferring the injected sample to two or more first antigens (or antibodies) to a fixed reaction space to achieve a primary antigen-antibody reaction,

(3) removing the unreacted sample solution from the capillary passage, followed by washing with a washing solution,

(4) A second antibody capable of specifically binding to the target substance, to which the chromophore is attached, is injected into the capillary passage and moved to the reaction space to perform a primary antigen-antibody conjugate and a secondary antigen-antibody reaction. Steps to achieve,

(5) removing the unbound second antibody from the capillary passage, followed by washing with a washing solution,

(6) injecting and transferring a color precipitation reagent into the reaction space of the capillary passage to achieve a color precipitation reaction with a color enzyme attached to the capillary passage;

(7) confirming the presence of the target substance in the sample by measuring the degree of the color precipitation reaction resulting from the reaction of step (6).

The method of the present invention is a method of simultaneously detecting two or more different target substances through multiple antigen-antibody immune responses, wherein the first antigen (or antibody) and the second antibody used are determined according to the target substance to be analyzed. .

If the biological material to be analyzed in the blood sample is different kinds of antibodies, various antigens that can specifically bind to the antibody are attached to different positions on the inner surface of the capillary passage, and after the reaction with the sample, the capillary passage In order to identify an antibody that has a binding reaction with an antigen attached thereto, a chromophoric enzyme is attached to an antibody capable of specific immune binding reaction to a human antibody as a second antibody.

In addition, when the substance to be analyzed in the blood sample is different kinds of antigens, as the first antibody, various antibodies capable of specifically binding to the substances to be analyzed are attached to the inner surface of the capillary passage at each position. By reacting with the sample to make the binding between the antigen in the sample and the antibody attached to the capillary passage, and as a second antibody to confirm the binding reaction is used by attaching a chromophoretic enzyme to other types of antibodies to the antigen.

In addition, the detection kit of the present invention for performing the above method is formed through the capillary passage in the longitudinal direction therein, at least two first antigens (or two or more first antigens for two or more target substances on the rear inner surface of the capillary passage) Antibodies) bodies of which are fixed at regular intervals; A sample injection means provided in the body corresponding to the inlet side of the capillary passage so that a sample can be injected into the capillary passage; A second antibody storage means provided in the body corresponding to the inlet side of the capillary passage to store a second antibody for injecting a second antibody against the target substance with a color-encoding enzyme into the capillary passage; and; A color precipitation reagent storage means provided in the body corresponding to the inlet side of the capillary passage to store a color precipitation precipitate reagent for injecting a color precipitation reagent that reacts with the color enzyme to cause color precipitation into the capillary passage Characterized in that consisting of.

Hereinafter, with reference to the accompanying drawings, preferred embodiments of the kit according to the present invention will be described in detail as follows.

1 is an exploded perspective view showing the configuration of a detection kit according to the present invention, Figure 2a is a longitudinal sectional view showing a state in which a sample is injected into the sample injection means constituting the detection kit according to the present invention, Figure 2b Figure 3a to 3d is a longitudinal sectional view showing a state driven by the first drive means after the sample is injected into the sample injection means constituting the detection kit according to the present invention, Figure 3a to 3d 4 is a longitudinal cross-sectional view showing an operating state of the second antibody storage means and the color precipitation reagent storage means, and FIGS. 4A to 4B are operating states of the second antibody storage means and the color precipitation reagent storage means constituting the detection kit according to the present invention. 5 is a perspective view showing an appearance of a driving device for driving a detection kit according to the present invention, and FIG. 6 is a driving kit for a detection kit according to the present invention. Fig. 7 is a block diagram showing the configuration of a drive device for driving a detection kit according to the present invention in a state where it is installed in the main body constituting the tooth.

As shown, the detection kit according to the present invention comprises a body 30, a sample injection means 60, a second antibody storage means 50, and a color precipitation reagent storage means 70.

First, the body 30 is made of a transparent material, the capillary passage 31 is penetrated in the longitudinal direction therein, at least two kinds of agents for two or more target substances on the rear inner surface of the capillary passage 31. One antigen (or antibody) is composed of fixed intervals.

More preferably, the body 30 is composed of a rectangular upper plate 20 and lower plate 25 having the same size.

A capillary passage 21 having a semi-circular cross section in the longitudinal direction is formed at the center of the upper surface of the upper plate 20 from one end to the other end, and a capillary passage 26 having a semi-circular cross section in the longitudinal direction at the center of the lower surface of the lower plate 25. Is formed, and at the rear end side of the capillary passages 21 and 26 of the upper and lower plates 20 and 25, two or more first antigens (or antibodies) to two or more target substances (1) (2) (3) are fixed at regular intervals.

At this time, the type of the first antigen (or antibody) to be fixed to the inner surface of the capillary passage is determined according to the type of the target material to be analyzed, which is illustrated as three types in FIG.

When the upper and lower plates 20 and 25 having the structure as described above are bonded to each other and bonded to each other, the capillary passages 21 and 26 have a body 30 in which one capillary passage 31 having a circular cross section is formed. do.

A tapered surface is formed on the inner side of both ends of the capillary passage 31.

On the other hand, the sample injection means 60 is provided in the interior of the body 30 corresponding to the inlet portion (31a) side of the capillary passage 31 so that the sample can be injected into the capillary passage 31 Do it.

Detailed configuration thereof will be described.

A sample accommodating chamber (81) communicating at right angles with the capillary passage (31), a piston (82) slidably installed in the sample accommodating chamber (81) for advancing the sample into the capillary passage (31), The lower end is fixed to the piston 82 and the upper end protrudes to the upper surface of the body 30 and the agent for advancing the piston 82 by injecting air into the sample receiving chamber 81 from the outside. It consists of an air supply path 84 and the opening-and-closing board 85 which is provided in the upper end of the reverse lever 83, and opens and closes the sample accommodating chamber 81. As shown in FIG.

Referring to the accompanying drawings, the operation of the sample injection means 60 configured as described above is as follows.

First, the operator pushes the opening and closing plate 85 in the direction of the arrow as shown in FIG. 2 to open the sample receiving chamber 81 and then injects the sample into the sample receiving chamber 81 using a separate syringe or the like. .

Thereafter, as shown in FIG. 2B, when air is injected from the outside through the first air supply path 84, the piston 82 is advanced by the pressure of the air, and the sample injected into the sample receiving chamber 81 is capillary. It moves into the passage 31.

On the other hand, the second antibody storage means 50 is provided in the interior of the body 30 corresponding to the inlet portion (31a) side of the capillary passage 31 for the target material attached to the color development enzyme 2 to store the second antibody to inject the antibody into the capillary passage 31, the chromogenic precipitation reagent storage means 70 is the body corresponding to the inlet (31a) side of the capillary passage 31 ( It is provided to the inside of 30) to be able to store the coloration precipitation reagent for injecting the coloration precipitation reagent that reacts with the coloration enzyme to cause color precipitation into the capillary passage (31).

The capillary passage 31 is formed at a position adjacent to the inlet portion 31a side of the capillary passage 31 at a predetermined depth having a rectangular shape, and the middle of the long side of the slide chamber 59. And first and second connection paths 54 and 54a connecting the slide chamber 59 and the first connection path 54 and the second connection path 54a in line with each other. 2, a slide member 51 having an antibody storage hole 52 and a chromogenic precipitation reagent storage hole 53, which is slidably installed in the slide chamber 59, and the second antibody storage hole 52 comprises: In order to push the slide member 51 so that the color settling reagent storage hole 53 is in line with the connecting passage 54 and the second connecting passage 54a is formed to supply air from the outside 2 The air supply path 55 and the slide chamber 59 is moved so that the second antibody storage hole 52 is the first A third air supply passage 56 formed to supply air from the outside to allow the second antibody in the second antibody storage hole 52 to move into the capillary passage 31 in a state in which the condensation 54 is aligned; The slide chamber 51 is moved so that the color settling reagent storage hole 53 is in line with the second connection path 54a so that the color settling reagent moves from the outside to the capillary passage 31. A fourth air supply path 57 formed therein and an agent configured to supply air from the outside to push the slide member 51 to return to its original position after the second antibody and the color development precipitation reagent move into the capillary passage 31; It consists of five air supply paths (58).

Here, the fifth air supply path 58 may be selectively formed, and may not necessarily be provided in the embodiment of the present invention.

Referring to the accompanying drawings the operation of the second antibody storage means 50 and the color precipitation reagent storage means 70 configured as described above are as follows.

First, as shown in FIGS. 3A and 4A, when air is supplied from the outside through the second air supply path 55, the second antibody storage hole 52 and the color precipitation reagent storage hole of the slide member 51 ( As shown in FIGS. 3B and 4B in the closed state 53, the second antibody storage hole 52 and the color precipitation precipitation reagent storage hole 53 are connected to the first connection path as the slide member 51 is moved. 54) and the second connection passage 54a, respectively, to be in a state in which the second antibody and the color development precipitation reagent in the capillary passage 31 can be injected.

Thereafter, when air is supplied through the third air supply passage 56, the second antibody in the second antibody storage hole 52 moves into the capillary passage 31 as shown in FIG. 3C, and the fourth air When air is supplied through the supply passage 57, the colored precipitated reagent in the colored precipitated reagent storage hole 53 is moved into the capillary passage 31 as shown in FIG. 3D.

Subsequently, when air is supplied through the fifth air supply path 58, the slide member 51 moves to the initial position, and eventually the second antibody storage hole 52 and the color precipitation reagent storage hole 53 are closed. Will be.

The description so far has described the configuration of the detection kit, and hereinafter, the description will be given of the driving device for driving the kit.

The driving device is a main body 90 having a body accommodating portion 90a on the upper and lower sides so that the body 30 can be accommodated, and a sample injection means 60 for driving the sample to move into the capillary passage 31. A first driving means 95, a second driving means 96 for driving the second antibody storage means 50 to move the second antibody into the capillary passage 31, and the body 30 Washing liquid supply means 98 for supplying the washing liquid to the inlet portion 31a of the capillary passage 31, and for driving the coloring precipitation reagent storage means 70 to move the color precipitation reagent into the capillary passage 31. The third driving means 97, the washing liquid and the unreacted solution injected into the capillary passage 31 are discharged to the outlet portion 31b of the capillary passage 31, and provided on the inner wall of the capillary passage 31. Air to transfer the sample, the second antibody and the color precipitation reagent to two or more regions of the first antibody (or antigen) Control to control the operation of the air supply means 99 for supplying, the first, second, third drive means (95, 96, 97) and the cleaning liquid supply means (98) and the air supply means (99) And a means 94.

In carrying out the method of the present invention using the detection kit of the present invention configured as described above, specific actions will be described as follows.

First, the user purchases a detection kit from a purchase place, and then stores the body 30 in the body accommodating portion 90a of the main body 90 as shown in FIG. 5.

When the body 30 is completely received in the body accommodating part 90a as described above, as shown in FIG. 6, the inlet part 31a of the capillary passage 31 and the first, second, third, and third parts thereof. 4, the supply pipe 91 for supplying air to the tapered ends of the fifth air supply passage (84, 55, 56, 57, 58) is automatically coupled.

Thereafter, the operator pulls the opening and closing plate 85 to secure a space in the sample accommodating chamber 81 so that the sample can be injected from the outside as shown in FIG. 2A.

Thereafter, the worker injects a sample such as blood into the sample receiving chamber 81 using an injection device such as a syringe.

Subsequently, as shown in FIG. 7, when an operator applies power to the power supply unit 93, the first driving unit 95 is operated by the control unit 94, and by the first driving unit 95. The piston 82 in the sample receiving chamber 81 is advanced.

When the piston 82 is advanced as shown in FIG. 2B as described above, the sample moves from the sample receiving chamber 81 to the capillary passage 31.

Then, the control means 94 is controlled to operate the air supply means 99, the air is injected from the air supply means 99 to the inlet portion 31a of the capillary passage 31 by this control signal The sample inside the capillary passage 31 by the injected air is a reaction in which two or more first antigens (or antibodies) are fixed at each position along the capillary passage 31 to two or more target substances. It moves to space.

If a target substance is present in the sample, the target substance will specifically attach to the inner surface of the capillary passage by primary antigen-antibody reaction with the first antigen (or antibody).

Thereafter, the control means 94 controls the air supply means 99 to operate after a reaction time of about 2 minutes has elapsed, and the inlet of the capillary passage 31 from the air supply means is controlled by this control signal. Air is injected into 31a and the unreacted sample is discharged to the outlet portion 31b of the capillary passage 31.

Thereafter, the control means 94 is controlled to operate the washing liquid supply means 98, by the control signal a predetermined amount of the washing liquid from the washing liquid supply means 98 at a predetermined pressure inlet of the capillary passage 31 When injected into the portion 31a, blood components and the like remaining in the capillary passage 31 are completely washed and removed. PBS-T (10 mM phosphate buffer pH7.4, 150 mM NaCl, 0.05% Tween20) is used as the washing solution.

Here, the control means 94 performs a process of injecting the washing solution about three times to remove the unreacted blood components and the like as described above.

Thereafter, the control means 94 controls the second driving means 96 to operate, and the second driving means 96 is driven by the control signal, whereby the second antibody storage means 50 The driven second antibody is attached to the reaction space (1) (2) (3) of the capillary passage (31).

The second antibody attached to the chromophore transferred to the reaction space is bound by the secondary antigen-antibody reaction with the primary antigen-antibody conjugate already attached to the inner surface of the capillary passage.

At this time, the color developing enzyme to be used is an enzyme that reacts with the color settling reagent to be injected to cause color precipitation. It is preferable to use alkaline phosphatase.

Thereafter, the control unit 94 controls the washing liquid supplying means 98 to operate after approximately 2 minutes has elapsed, and a predetermined amount of the washing liquid is supplied from the washing liquid supplying means 98 by this control signal. When injected into the inlet portion 31a of the capillary passage 31 by pressure, the unbound secondary antibody remaining in the capillary passage 31 is removed.

Here, the control means 94 performs a process of injecting the washing solution about three times to completely wash and remove the unbound secondary antibody as described above.

Thereafter, the control means 94 controls the third driving means 97 to operate, and the third driving means 97 is driven by this control signal, and thus the third driving means 97 is driven. As a result, the color precipitated reagent solution moves to the reaction spaces 1, 2, and 3 of the capillary passage 31.

At this time, the chromogenic precipitation reagent is a substrate solution for the chromogenic enzyme, which is transferred to the reaction space to react with the chromogenic enzyme to cause chromogenic precipitation. In the case of using alkaline phosphatase as the color development enzyme, it is preferable to use NBT or BCIP as the coloration precipitation reagent.

Thereafter, the control unit 94 generates a color for each antigen (or antibody) attached to the capillary passage 31 by operating the reading means 92 such as a digital camera after a reaction time of about 2 minutes has elapsed. By analyzing the degree of precipitation reaction, it is determined whether the target substance is present in the sample.

The kit used in the present invention is made of a transparent, durable material such as glass or plastic, and the inside of the BSA (bovine serum albumin) or other hydrophilic substance after immobilization of antigen or antibody to prevent binding of non-specific proteins. It is preferable to coat with.

A technique for attaching an antigen or an antibody to the inner surface of the capillary passage is a surface of plastic or the like using a conventionally known protein attachment technique, for example, EDC [1-Ethyl-3- (3-dimethylaminopropyl) -carbodiimide]. A technique of activating a carboxyl group, followed by binding reaction with an amino group of a protein to attach it (see Bioorg Khim. Ivanovskaia MG, Naryshkin NA, Shabarova ZA. 21 (6): 454-60. Russian (1995, 6)] or streptavidin-biotin technology (Methods in Enzymology, Green. N. M., Vol. XVIII, p148 (1970).

In the above described the configuration and operation of the detection kit according to the present invention, hereinafter will be described for the embodiment for simultaneously detecting a variety of target substances according to an embodiment of the present invention.

Example 1 Blood HCV, HBV and HIV Antibody Identification Test Using the Method and Kit of the Present Invention

According to the method of the present invention, the antigenic proteins of HCV, HBV and HIV are immobilized on the inner surface of the capillary passage, and a blood sample is injected thereto and reacted at room temperature for about 2 minutes to be present in the antigen and blood immobilized on the surface of the capillary passage. After allowing the antibody to be analyzed to undergo the first antigen-antibody binding reaction, unbound blood samples were discharged to the outside using air pressure. Then, to remove the blood components remaining in the capillary passage was washed three times (PBS-T: 10mM phosphate buffer pH7.4, 150mM NaCl, 0.05% Tween20) three times. Alkaline phosphatase is attached to the secondary antibody specific to human antibodies as a color developing enzyme and sent to the reaction space to achieve a secondary antigen-antibody reaction for 2 minutes at room temperature, and then unbound secondary antibody using air pressure and washing solution. Was removed. NBT was injected into the reaction part with the coloration precipitation reagent, and the coloration precipitation reaction by the enzyme was carried out for 2 minutes, and the color level of each color in the capillary path was measured with a digital camera (ccd camera) to give a specific immune response to each antigen. Bound antibodies were analyzed.

As a result, it was confirmed that antibodies to HCV and HBV existed in the test sample, and therefore, it was determined that the test subjects were infected with two types of hepatitis C virus.

Example 2 Confirmation of the Presence of Pathogenic Microorganisms Using the Method and Kit of the Present Invention

In accordance with the method of the present invention, antibodies specific for Mycobacterium tuberculosis (M.TB), Chlamydia, Malaria, and Streptococcus were immobilized by position at predetermined positions in the prepared capillary passages. The blood sample of the patient was injected into the capillary passage and the immune reaction was performed at room temperature for 2 minutes so that the antigen (pathogenic microorganism) present in the sample and the antibody inside the capillary passage could be combined by the immune response. Unbound samples were removed by air pressure and washed three times with a wash solution (PBS-T: 10 mM phosphate buffer pH7.4, 150 mM NaCl, 0.05% Tween20). An alkaline phosphatase as a color developing enzyme was attached to the secondary antibody specific for each microorganism and injected into the capillary passage to form a secondary immune response with the primary antigen-antibody conjugate bound to the capillary passage. NBT was injected into the coloration precipitation reagent to achieve colorimetric precipitation reaction by the enzyme for 2 minutes, and microorganisms bound to specific antibodies for each antibody were analyzed by measuring the degree of coloration by position in the capillary passage with a digital camera.

As a result, it was confirmed that Mycobacterium tuberculosis was present in the test sample, and therefore the subject was determined to be a tuberculosis patient.

Although the preferred embodiments of the present invention have been illustrated and described above, the present invention is not limited to the above-described embodiments, and the present invention is not limited to the above-described embodiments without departing from the spirit of the present invention as claimed in the claims. Various modifications can be made by those skilled in the art, and such modifications are within the scope of the appended claims.

As can be seen from the above, when the specific material in the sample is analyzed using the method and kit of the present invention, the operation method is simple and the time required for obtaining the result is short, so that non-experts can use the simple operation equipment. A lot of information can be obtained easily during a short analysis time, and compared to the conventional method that each of them was carried out through separate experimental procedures for analyzing different types of target substances, only one injection of several kinds of target substances was performed at the same time. It is easy to know the analysis result, which is advantageous in terms of time and cost.

Claims (5)

(1) injecting a sample to be analyzed into a capillary passage in which two or more first antigens (or antibodies) for two or more target substances are fixed at regular intervals on an inner surface thereof, (2) transferring the injected sample to two or more first antigens (or antibodies) to a fixed reaction space to achieve a primary antigen-antibody reaction, (3) removing the unreacted sample solution from the capillary passage, followed by washing with a washing solution, (4) A second antibody capable of specifically binding to the target substance, to which the chromophore is attached, is injected into the capillary passage and moved to the reaction space to perform a primary antigen-antibody conjugate and a secondary antigen-antibody reaction. Steps to achieve, (5) removing the unbound second antibody from the capillary passage, followed by washing with a washing solution, (6) injecting and transferring a color precipitation reagent into the reaction space of the capillary passage to achieve a color precipitation reaction with a color enzyme attached to the capillary passage; (7) detecting the presence of the target material in the sample by measuring the degree of color precipitation reaction resulting from the reaction of step (6), the method for detecting several types of target material in the sample. The method according to claim 1, wherein when the target substance is two or more kinds of human antibodies, an antibody capable of specifically binding to a human antibody having a coloring enzyme attached as a second antibody is used. The method according to claim 1, wherein when the target material is two or more pathogenic microorganisms, a mixture of antibodies capable of specifically binding to these microorganisms, to which a colorizing enzyme is attached, is used as the second antibody. The method according to any one of claims 1 to 3, wherein the chromogenic enzyme is alkaline phosphatase and the chromogenic precipitation reagent is NBT or BCIP. A capillary passage 31 is formed in the longitudinal direction therein, and at least two first antigens or first antibodies against two or more target substances are fixed on the rear inner surface of the capillary passage 31 at regular intervals. A body 30 of material; A sample injection means (60) provided inside the body (30) corresponding to the inlet portion (31a) side of the capillary passage (31) to allow a sample to be injected into the capillary passage (31); It is provided inside the body 30 corresponding to the inlet portion 31a side of the capillary passage 31 to inject the second antibody against the target substance to which the chromophore is attached into the capillary passage 31. A second antibody storage means (50) for storing a second antibody; It is provided in the body 30 corresponding to the inlet portion (31a) side of the capillary passage (31) is a coloration precipitation reagent for the enzyme that reacts with the chromogenic enzyme to cause color precipitation into the capillary passage (31) A detection kit for simultaneously detecting a plurality of target substances through multiple immune reactions, characterized in that it comprises a chromogenic precipitation reagent storage means 70 for storing the chromogenic precipitation reagent for injection.