KR100506165B1 - Non-continuous immunoassay device and immunoassay method using the same - Google Patents

Abstract

Discs for use in immunoassays such as immunochromatography are separated into two or more portions, and a non-continuous immunoassay apparatus for controlling the speed at which a mobile phase such as a liquid sample moves through the separated pads is disclosed. The immunoassay device may include a first pad including a sample pad for receiving a liquid sample; A second pad spaced apart from the first pad by a predetermined distance and the liquid sample is transferred; An upper case covering upper portions of the first and second pads; A lower case covering lower portions of the first and second pads; And a connection part formed on the upper case and / or the lower case and positioned between the first pad and the second pad to form a passage through which the liquid sample moves. When the liquid sample is injected into the sample pad through the sample inlet formed in the upper case of the immunoassay device, the liquid sample is transferred to the second pad including the porous membrane pad through the capillary passage formed by the connection part. The transported liquid sample reacts with the binder fixed to the porous membrane pad to generate a signal that can be detected using the naked eye or a sensor, and can be confirmed through the result confirmation window formed on the upper case.

Description

Non-continuous immunoassay device and immunoassay method using the same

The present invention relates to a discontinuous immunoassay device, and more particularly, pads used for immunoassay such as immunochromatography are separated into two or more portions, and a mobile phase such as a liquid sample through the separated pads. The present invention relates to a discontinuous immunoassay device capable of controlling the moving speed and an immunoassay method using the same.

Immunochromatography, also known as rapid test, is a method that can qualitatively and quantitatively test trace analytes using antigen-antibody reactions. It is used in various fields such as farming, animal husbandry, food, military and environment. For such immunochromatographic analysis, an assay device in the form of an assay strip or a device in which the assay strip is mounted inside a plastic case is generally used. 1 is a cross-sectional view of an assay strip used for conventional immunochromatographic analysis, as shown in FIG. 1, a conventional assay strip 10 using a sample pad 12, a naked eye or a sensor containing a liquid sample. Conjugate pad (14) containing a conjugate conjugated to a ligand such as an antigen, an antibody, or the like to generate a signal that can be detected, an analyte in a sample and / or a binder that specifically binds to the conjugate ( 16a, an antibody or antigen), and a porous membrane pad 16 fixed thereto, and a moisture absorbent pad 18 finally receiving a liquid sample. It is arranged continuously at the top. When the analysis strip 10 is mounted inside the plastic case and used, a sample inlet for dropping the sample on the upper portion of the case and a result confirmation window for checking the test result are formed.

In such an immunochromatographic assay using the assay strip 10, when a liquid sample is dropped onto the sample pad 12, the liquid sample moves through the conjugate pad 14 and the porous membrane pad 16 by capillary action. Finally, it is accommodated in the moisture absorption pad 18. At this time, the conjugate contained in the conjugate pad 14 also moves along with the liquid sample, and if analyte is present in the sample, the binder 16a fixed to the porous membrane pad 16 via the analyte. (Usually referred to as a "sandwich" reaction) or competitively with the binder 16a (commonly referred to as a "competition" reaction), to the presence of an analyte in the sample. Can be detected using. However, in such a conventional assay strip 10, in order to control the speed at which the mobile phase, such as a liquid sample, moves, it is necessary to adjust the pore size of the porous membrane pad 16. flow rate) and antigen-antibody reaction rate cannot be effectively controlled. That is, the reaction between the liquid sample and the measurement dilution diluting the sample, the reaction between the sample and the conjugate, and the antigen-antibody reaction between the binder 16a and the sample and / or the conjugate fixed to the porous membrane pad 16 are performed for a sufficient time. There is a disadvantage in that the sensitivity and specificity of the immunochromatographic assay are lowered. In addition, in the case of the assay strip 10 shown in FIG. 1, even if some of the pads fail during the manufacturing process, not only the entire assay strip 10 should be discarded, but the shape of the immunoassay device is in the form of a rod. There are limited disadvantages.

Accordingly, it is an object of the present invention to provide an immunoassay device having excellent sensitivity and specificity of immunochromatographic analysis.

Another object of the present invention is to provide an immunoassay device capable of controlling the speed at which a mobile phase such as a liquid sample moves through pads used for immunoassay.

Still another object of the present invention is to provide an immunoassay device capable of arbitrarily adjusting the antigen-antibody reaction time according to the type of antigen-antibody reaction.

Another object of the present invention is not only a simple manufacturing process of the pad used in the immunoassay, but also in the event of a failure of some pads, non-continuous immunoassay device that does not need to replace the entire pad and using the same It provides an immunoassay method.

Still another object of the present invention is to provide a discontinuous immunoassay device and an immunoassay method using the same, which can easily modify the structure of the immunoassay device, in order to facilitate sample insertion or reading.

In order to achieve the above object, the present invention includes a first pad including a sample pad for receiving a liquid sample; A second pad spaced apart from the first pad by a predetermined distance and the liquid sample is transferred; An upper case covering upper portions of the first and second pads; A lower case covering lower portions of the first and second pads; And at least one of the upper case and the lower case, and positioned between the first pad and the second pad and including a connection part forming a passage through which the liquid sample moves. The present invention also provides a strip-shaped plastic support; Two or more immunoassay pads each spaced a predetermined distance apart from each other and positioned above the plastic support; An upper case covering an upper portion of the at least two immunoassay pads; A lower case covering a lower portion of the plastic support; And at least one of the upper case and the lower case, positioned between each pad spaced apart from the predetermined distance, and providing an immunoassay device including a connection part that forms a passage through which the liquid sample moves. In addition, the present invention comprises the steps of injecting a liquid sample to the sample pad; Moving the liquid sample through a capillary passage formed between the upper case and the lower case by a protrusion formed on at least one of the upper case and the lower case covering the sample pad; And it provides an immunoassay method comprising the step of detecting the liquid sample passed through the capillary passage in the antigen-antibody reaction.

Hereinafter, with reference to the accompanying drawings will be described in more detail the present invention.

2A and 2B are exploded perspective and side cross-sectional views of a discontinuous immunoassay device according to a first embodiment of the present invention, respectively. As shown in FIGS. 2A and 2B, the immunoassay device according to the first embodiment of the present invention includes a first pad 32 and a second pad 34 that are spatially isolated by a predetermined distance. The upper and lower portions of the first and second pads 32 and 34 are covered by the upper case 52 and the lower case 72, respectively. The first and second pads 32 and 34 may be fixed to the plurality of pad fixing guides 74 formed on the lower case 72, or fixedly coupled to the grooves formed on the upper or lower cases 52 and 72. In addition, the lower case 72 may be formed with a plate-shaped pad support portion 76 for supporting the first and second pads 32 and 34 as a cushioning member. As shown in FIG. 2B, the upper case 52 is provided with a connecting portion 60 fitted into the space 30 between the first and second pads 32 and 34. The connection part 60 may be a protrusion part spaced apart from the lower case 72 or the pad support part 76 formed as needed by a predetermined distance, and the liquid sample introduced into the first pad 32 may be connected to the connection part 60. Along the space between the lower case 72 or the pad support portion 76, the cap is moved by the capillary phenomenon and transferred to the second pad 34. Therefore, the connecting portion 60 forms a capillary passage through which the liquid sample moves in the space 30 between the first and second pads 32 and 34, and the connecting portion 60 is the upper case 52. It may have the form of a liquid impermeable protrusion formed in the), and may be formed in the lower case 72, or formed in both the upper case 52 and the lower case 72 as necessary.

In the immunoassay device according to the first embodiment of the present invention, the first and second pads 32 and 34 are (i) a liquid sample and / or a measurement dilution expected to contain an analyte. Acceptable sample pads 12, (ii) Labels that generate signals that can be detected using the naked eye or sensors (e.g., gold particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers, magnetic particles, etc.) ) Conjugate pads containing conjugates conjugated to ligands such as antigens and antibodies that react with the analyte, (iii) when whole blood is used as a specimen, to promote antigen-antibody reactions or to suppress non-specific reactions. A detection part containing a substance to be used, an auxiliary pad used as necessary, (iv) an analyte in a sample and / or a binding agent (antigen or antibody) that specifically binds to the conjugate (testing) An immunoassay pad such as a porous membrane pad 16 including at least one line), and a moisture absorption pad 18 for finally receiving a liquid sample. Here, the first pad 32 includes a sample pad for receiving a liquid sample, and the second pad 34 includes one or more pads to which the liquid sample is transferred. For example, as shown in FIG. 2A, the first pad 32 is a sample pad 12, and the second pad 34 is a porous membrane pad 16 and a moisture absorbing pad 18 connected to each other. ) May be included. In addition, the first pad 32 may be two or more pads overlapped or separated from each other, for example, (i) a sample pad 12 and a conjugate pad, (ii) a sample pad 12 for whole blood, various auxiliary pads, and a conjugate. The pad, and the second pad 34 may include the remaining pads for immunoassay. The auxiliary pad contains a substance which promotes an antigen-antibody reaction or suppresses a non-specific reaction. For example, when the adjuvant pad is mixed with a whole blood sample, the auxiliary pad contains dried reagents that lyse red blood cells. , Hemolyzed erythrocytes serve to prevent migration to the porous membrane pad 16. Such an auxiliary pad is preferably formed between the sample pad 12 and the conjugate pad, such that the liquid sample from which red blood cells are filtered by the sample pad 12 and the substance contained in the auxiliary pad are mixed. The porous membrane pad 16 may be made of nitrocellulose, glass fiber, polyethersulfone (PES), cellulose, nylon, and the like, and is preferably made of nitrocellulose having pores having a size of 5 to 15 μm. The immunoassay pads constituting the first and second pads 32 and 34 may be formed on two plastic supports 20a and 20b, respectively. And the second pads 32 and 34 are easy to manufacture and mount. The plastic supports 20a and 20b may be made of a polypropylene film, a polyester film, a polycarbonate film, an acrylic film, or the like, and preferably made of a polypropylene film.

3 is an enlarged front view (A), bottom view (B) and side view (C) of the connecting portion 60 formed in the upper case 52 and / or lower case 72, as shown in FIG. The connecting portion 60 may have a shape of a protrusion 64 formed with shoulders 62a, 62b and shoulders in the direction of the first and second pads 32 and 34, where the shoulders 62a and 62b are formed. ) Serves to press and fix the ends of the first and second pads 32 and 34, respectively, and the protrusion 64 forms a capillary passage between the first and second pads 32 and 34. Do it. Accordingly, the protrusion 64 preferably has a shape that fits into the space between the first and second pads 32 and 34. Preferably, the shoulders 62a and 62b are provided with a plurality of fine protrusions 66 to facilitate fixing of the first and second pads 32 and 34, and the protrusions 64 may include protrusions. It is preferable that a plurality of fine protrusions 68 are formed to maintain a uniform distance between the 64 and the pad support 76 to form a uniform capillary passage. When the minute protrusions 68 are formed on the protrusions 64 as described above, even when the minute protrusions 68 contact the pad supporters 76, capillary passages may be formed around the minute protrusions 68. As shown in FIG. 3, the fine protrusions 66 and 68 may have a straight protrusion shape formed in the moving direction of the liquid sample, and may have various shapes such as hemispherical protrusions and hexahedral protrusions. The distance between the connecting portion 60, specifically, the protrusion 64 and the pad support 76 or the lower case 72 may be adjusted by changing the height of the protrusion 64, the fine protrusions 68, and the like. In accordance with this, the moving speed of the mobile phase such as a liquid sample passing through the connecting portion 60 can be adjusted, and thus the antigen-antibody reaction rate of the immunoassay can be controlled. In the present invention, the distance between the first and second pads 32 and 34 separated from each other, and the height of the capillary passage formed between the upper and lower cases 52 and 72, are used for the antigen-antibody reaction, the sample used. The distance between the first and second pads 32 and 34 may be 0.5 to 5 mm, preferably 1 to 3 mm, more preferably about 2 mm, depending on the type of the capillary passage. The height is 0.005 to 1 mm, preferably 0.01 to 0.5 mm, more preferably 0.01 to 0.2 mm. If the distance and height are too large or too small, there is a fear that the movement of the liquid sample may not be performed smoothly.

The connecting portion 60 may be treated with a hydrophobic or hydrophilic material to control the antigen-antibody reaction rate. This is because the aqueous solution does not easily pass through the hydrophobic region, and when a specific component in the aqueous solution binds to the hydrophobic region, and the hydrophobic region is modified into a hydrophilic region, the aqueous solution is facilitated to pass. Therefore, when the connection part 60 is treated hydrophobicly or hydrophilically, the first pad 32 reacts by controlling the time that a mobile phase such as a liquid sample moves from the first pad 32 to the second pad 34. It can serve as a reaction chamber that holds the reaction mixture for a certain time. As a method of treating the connecting portion 60 to be hydrophobic or hydrophilic, the connecting portion 60 may be treated (for example, coated) with hydrophobic or hydrophilic latex particles or a compound, or a hydrophobic or hydrophilic group may be connected using a plasma. ) May be used. In this case, a polymer such as polystyrene, polypropylene, polyethylene or polyester may be used as the hydrophobic latex particles, and a polymer having a hydrophilic group such as a carboxyl group, an amine group or a hydroxyl group may be used as the hydrophilic latex particle. have. As the hydrophobic compound, long fatty acid compounds such as palmitic acid, stearic acid, oleic acid, and the like can be used, and as the hydrophilic compound, a surfactant, glycerol, and polyvinyl alcohol can be used. Hydrophilic polymers, such as these, can be used. As a method of attaching the hydrophilic group to the connecting portion 60 using plasma, plasma graft polymerization of hydrophilic monomers such as acrylic, methacryl, unsaturated amide, diene, and triene monomers is performed on the surface of the connecting portion 60. Alternatively, oxygen or nitrogen may be treated with hydrogen plasma on the surface of the connecting portion 60 to introduce a carboxyl group or an amine group, which is a hydrophilic functional group, on the surface of the connecting portion 60. In the method of attaching the hydrophobic group to the connecting portion 60 using plasma, hexafluoropropene (CF ₂ = CF-CF ₃ ) is plasma polymerized on the surface of the connecting portion 60 to connect the thin film having the CF bond to the connecting portion. (60) The method of forming in the surface can be used.

Referring again to FIG. 2A, a sample inlet 55 is formed at a position corresponding to the sample pad 12 in the upper case 52, and a result is provided at a position corresponding to the detector of the porous membrane pad 16. A confirmation window 56 is formed, and a separate sample diluent input unit 54 is formed at a lower end of the sample pad 12, or a ventilation port 58 is formed at a position corresponding to the moisture absorption pad 18. There may be. In this way, if the sample diluent injecting section 54 is formed separately, it is preferable to reduce the test deviation and improve the reaction specificity and sensitivity. Ventilation port 58 formed on the upper side of the moisture absorbing pad 18 facilitates the ventilation of the moisture absorbing pad 18 and prevents the liquid sample and the conjugate from flowing back toward the result confirmation window 56, thereby providing a background clearance. clearance, which facilitates reading of test results. Such a ventilation port 58 may be formed in the side direction of the moisture absorption pad 18, and may be formed in both the upper surface and the side direction. In the upper or lower cases 52 and 72, a plurality of guides, protrusions, etc. may be formed to properly arrange and fix or compress various pads for immunoassay, and the upper or lower cases 52 and 72 may be formed. ) May be combined by conventional means such as coupling grooves, coupling projections, or in some cases may be integrally formed.

Referring to the operation of the immunoassay device according to the first embodiment of the present invention, when the liquid sample is injected into the sample pad 12 through the sample inlet 55 of the upper case 52, the liquid sample is the upper case 52 Is connected to the second pad 34 composed of the porous membrane pad 16 and the moisture absorption pad 18 through the capillary passage formed between the upper and lower cases 52 and 72 by the connecting portion 60 formed in the do. In this case, since the size of the capillary passage is controlled in advance according to an antigen-antibody reaction performed by an analyte, a conjugate, a binder of the porous membrane pad 16, and the like, the antigen-antibody reaction in the first pad 32 is performed. After this is sufficiently performed, the next zone is moved to the second pad 34, where the antigen-antibody reaction is performed. Thus, the linker 60 serves as a reaction barrier or antigen-antibody reaction rate control unit that induces a flow delay in the mobile phase. The liquid sample and / or the conjugate transferred to the porous membrane pad 16 reacts with the binder fixed to the porous membrane pad 16 to generate a signal that can be detected by the naked eye or using a sensor.

4 is a side cross-sectional view of a non-continuous immunoassay device according to a second embodiment of the present invention, wherein the immunoassay device according to the present embodiment has a first distance with a predetermined distance on two plastic supports 20a and 20b. To third pads 32, 34, 36 are sequentially formed, and the space 30a between the first and second pads 32, 34 and the space between the second and third pads 34, 36. Except that the first connection portion 60a and the second connection portion 60b are formed at 30b, the configuration is substantially the same as in the first embodiment. In the present embodiment, the first pad 32 is the sample pad 12, the second pad 34 is the conjugate pad 14 or the auxiliary pad, and the third pad 36 is the porous membrane pad 16 on which the detector is formed. ) And a moisture absorbing pad 18, wherein the first and second pads 32 and 34 are formed on the first plastic support 20a, and the third pad 36 is the second plastic support 20b. It can be formed on top. In the same manner, three pads, for example, a sample pad 12, an auxiliary pad, and a conjugate pad 14, are formed at a predetermined distance on the first plastic support 20a, and the second plastic support ( 20b) After forming the porous membrane pad 16 on the top, it is possible to connect them using a total of three connections. As such, when two or more connecting portions 60a and 60b are used, the movement speed of the mobile phase passing through each pad can be individually controlled, thereby not only improving the sensitivity of the immunoassay, but also the plurality of pads. Even if any of the defects occur, there is an advantage that the replacement of the defective pads is easy. When the conjugate pad 14 is used, when the immunoassay method is a sandwich assay, when a liquid sample is dropped into the sample pad 12, the sample moves to the conjugate pad 14 by capillary action. The analyte in the sample and the ligand site of the conjugate form an immunocomplex by an immunological response. The immunocomplex moves to the porous membrane pad 16 along the flow of the sample and is captured by an immunologically specific binding reaction at the detection part (inspection line), which is a site where the binder is immobilized. In the case of a competitive or inhibition assay, the porous membrane pad 16 has a detection portion formed of a binder that reacts with the ligand site of the conjugate, and the binding agent is the same as the analyte. It may be an analog of this. In the immunoassay, since the intensity of the signal generated by the label of the conjugate is proportional to the amount of analyte, the presence of the analyte can be detected qualitatively such as "positive" or "negative", and further, the intensity of the signal. The amount of analyte can be measured quantitatively by comparing with a standard colorimetric table.

5A and 5B are side cross-sectional views of a discontinuous immunoassay device according to a third embodiment of the present invention, wherein the immunoassay device according to the present embodiment includes first and second portions on one strip-shaped plastic support 20. Pads 32 and 34 are formed to be spaced apart from each other by a predetermined distance, and a buffer groove for uniformly deploying or moving the liquid sample under the space between the first and second pads 32 and 34 as needed. Except that 78) is further formed, it has substantially the same configuration as the first embodiment. The buffer groove 78 is formed to have a size larger than the distance between the first and second pads 32 and 34, so that the ends of the first and second pads 32 and 34 are the shoulders of the connection portion 60. When pressed by 62a and 62b, the ends of the first and second pads 32 and 34 move toward the buffer groove 78 so that the liquid sample moves uniformly. In the immunoassay device shown in FIG. 5A, the sample pad 12 may be used as the first pad 32, and the porous membrane pad 16 and the moisture absorption pad 18 may be used as the second pad 34. . In addition, as illustrated in FIG. 5B, a plurality of pads may be formed on one strip-shaped plastic support 20, and the plurality of pads are spaced at a predetermined distance from at least two parts. In the immunoassay device shown in FIG. 5B, the first to fourth pads 32, 33, 34, and 36 are formed to be separated from each other on one strip-shaped plastic support 20, and these three connecting portions 60a and 60b are formed. , 60c). In the example shown in FIG. 5, the first to fourth pads 32, 33, 34, and 36 each overlap the specimen pad 12, the auxiliary pad 13, the conjugate pad 14, and the moisture absorption pad 18. Porous membrane pad 16. Here, the use of the auxiliary pad 13 is optional as described above.

6A and 6B are exploded perspective and side cross-sectional views of a discontinuous immunoassay device according to a fourth embodiment of the present invention, respectively. 6A and 6B, the immunoassay device according to the fourth embodiment of the present invention includes first, second and third pads 32, 34, and 36 spaced apart from each other by a predetermined distance. The first and second pads 32 and 34 are formed on the first plastic support 20a, and the third pad 36 is formed on the second plastic support 20b. Since the first and second pads 32 and 34 and the third pad 36 are formed completely independently, similarly to the first embodiment of the present invention, the upper case 52 and the lower case 72 may be formed. It can be modified as needed. 6A and 6B, a first pad 32 serving as a sample pad 12 into which a liquid sample is introduced, and a second pad 34 serving as a conjugate pad 14 or an auxiliary pad are provided. The upper and lower cases 52 and 72 which cover are configured to be parallel to the ground, and the upper and lower cases 52 and 72 which cover the third pad 36 serving as the porous membrane pad 16 in which the detector is formed. If the part is inclined at a predetermined angle with respect to the first pad 32, for example, 30 to 50 degrees with respect to the ground, the inspector can easily read the detection result through the result confirmation window 56. In the present embodiment, the first connection part 60a connecting the first pad 32 and the second pad 34 has the same structure as described in the first embodiment of the present invention. 7 is an enlarged front view (A) and a bottom view (B) of a second connection portion 60c for connecting the third pad 36 and the first and second pads 32 and 34 inclined at a predetermined angle. And as a side view (C), as shown in Figure 7, the second connecting portion (60c) is formed in the first inclined protrusion (64a) and the upper case 52 formed in the lower case 72, the second It consists of the 2nd inclined protrusion 64b which has the inclined surface corresponding to the inclined surface of the 1 inclined protrusion 64a. Here, the shoulder portion 62a may be further formed on the side surface of the second inclined protrusion 64b to press and fix the end of the second pad 34. The inclined surfaces of the first inclined protrusion 64a and the second inclined protrusion 64b are spaced apart by a predetermined distance from each other so as to form a capillary passage therebetween. As in the first embodiment according to the present invention, the first and second inclined protrusions 64a and 64b are preferably located exactly in the space between the second pad 34 and the third pad 36. In order to facilitate the fixing of the second pad 34, the shoulder part 62a has a linear fine protrusion 66 formed thereon, and inclined surfaces of the first and second inclined protrusions 64a and 64b. It is preferable that the linear fine protrusions 68 are formed in the same to form a uniform capillary passage. In addition, two or more pads may be formed on the first plastic support 20a, and as illustrated in FIG. 8, three pads 32, 33, and 34 are disposed on the first plastic support 20a. It is formed separately, and the spaces 30a and 30b therebetween may be connected by two connection parts 60a and 60b. In the example shown in FIG. 8, the three pads 32, 33, 34 function as the sample pad 12, the auxiliary pad 13, and the conjugate pad 14, respectively.

9 is a partial cutaway plan view of a non-continuous immunoassay device according to a fifth embodiment of the present invention. In the immunoassay device according to the present embodiment, the first and second pads 32 and 34 have a predetermined distance from each other. It is formed separately, the first and second pads (32, 34) at a predetermined angle around the space 30, that is, the connection portion 60 between the first and second pads (32, 34), Figure 9 Has the same configuration as the first embodiment of the present invention except that they are formed spaced apart from each other at right angles. Therefore, a capillary passage is formed between the connecting portion 60 formed in the upper case 52 and the lower case 72 in the space 30 between the first and second pads 32 and 34, and the capillary tube A mobile phase, such as a liquid sample, is transferred from the first pad 32 to the second pad 34 along the passage. As shown in FIG. 9, in the immunoassay device according to the present invention, the respective pads 32 and 34 are formed independently of each other, and the respective pads 32 and 34 can be connected by using a linker. There is an advantage in that the shape of the analysis device can be variously modified.

10 is an exploded perspective view of a discontinuous immunoassay device according to a sixth embodiment of the present invention. In the immunoassay device according to the present embodiment, instead of forming a sample inlet in the upper case 52, the sample pad 12 is used. Except for mounting a wick 38 (wick) that penetrates the upper and / or lower cases 52 and 72 in the direction of the first pad 32 serving as a role of connecting the first pad 32 to the outside. Has the same configuration as the first embodiment of the present invention. In such an immunoassay device, when the wick 38 is immersed in a liquid sample, the liquid sample flows into the first pad 32 along the wick 38, and the first pad 32 and the second pad. It is introduced into the second pad 34 through a linker (not shown) formed between the 34, to perform an immunoassay by the antigen-antibody reaction. The immunoassay device shown in FIG. 10 may use a large amount of liquid samples, and does not require a separate device such as a dropper dropping them into the sample inlet.

The immunoassay device according to the present invention uses body fluids such as plasma, serum, urine, runny nose, tears, and saliva as specimens to confirm the presence of pregnancy, ovulation, influenza virus, rota virus, avian influenza virus, chlamydia, and whole blood. Use as a sample to confirm the presence of malaria, AIDS, hepatitis C, hepatitis B, syphilis, gastric ulcer bacteria, cancer markers (AFP, PSA, CEA), tuberculosis, SARS, dengue, leprosy, opium, morphine, methamphetamine Whether or not drugs such as hiropan, amphetamines, cocaine, and cannabis, cholera, staphylococcal enterotoxin B (SEB), botulinum, lysine, anthrax, confirmation of biochemical terrorism caused by detection of brussela, bexonia, salmonella, yato, etc. It can be used for various rapid diagnostics.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited by the following Examples.

Experimental Example 1 Influenza Virus Test Using Immunoassay System

(A) Preparation of Antibody Immobilized Nitrocellulose Pads

Dilution of monoclonal antibodies against nucleocapsid antigens of influenza virus type A and type B with phosphate buffer, followed by test line 1 and test line on a nitrocellulose pad (25 mm wide, pore size: 10-12 μm) using an injector Each was sprayed in 2 positions. In addition, anti-mouse immunoglobulin antibodies obtained by immunizing mouse immunoglobulins with goats were diluted with phosphate buffer, sprayed to the control position of the nitrocellulose pad, and dried and immobilized in a 37 ° C thermostat. The remaining space of the nitrocellulose pad except the fixed part of the antibody was sprayed with a phosphate buffer containing 0.05% by weight of bovine serum albumin, 4% by weight of sucrose and 0.0625% by weight of an ionic surfactant, and then sealed in a 30 ° C thermostat. Dried for 120 minutes. The nitrocellulose pads were attached to a polypropylene plate backing plate coated with an adhesive, and a hygroscopic pad (absorbent pad, manufactured by Millipore, USA) was attached to the upper portion of the nitrocellulose pad by 1 mm.

(B) Sample pad and antibody-gold conjugate pad attachment strip preparation

Minimize the amount of gold colloid stabilization by adding monoclonal antibodies against influenza virus type A and type B nucleocapsid antigens to each 1 ml of colloidal gold solution, and then adding 150 mM aqueous sodium chloride solution equal to 1/10 of the volume of gold colloid solution. Were obtained, mixed and conjugated in the ratio determined here, and then blocked with 1% by weight bovine serum albumin. The conjugate solution was centrifuged four times at 800 rpm in a centrifuge to remove the supernatant and resuspended with 1% by weight bovine serum albumin phosphate buffer to an absorbance of 10. The prepared gold colloidal antibody conjugate solution was diluted with absorbance 2 using distilled water containing 0.5 wt% sucrose, and then dispensed into a glass fiber pad at a ratio of 10 μl / 25 mm ² . The pad containing the gold colloidal antibody conjugate was rapidly frozen with liquid nitrogen, dried in a lyophilizer for 20 hours, and the dried pad was cut into a size of 0.7 × 30 mm. The obtained antibody-gold conjugate pad and sample pad were separated at 1 mm intervals and attached to a polypropylene plate support coated with an adhesive.

(C) Preparation of immunoassay device

After mounting the strips prepared in the steps A and B at 2 mm intervals in the lower case, respectively, connecting the antibody-gold conjugate pad and the sample pad and between the antibody-gold conjugate pad and the nitrocellulose pad 2 An immunoassay device according to a fourth embodiment of the present invention (see FIG. 6A) was prepared by covering an upper case in which four connection parts were formed. One influenza virus type A and B positive and negative specimens were tested using the completed immunoassay device, and it was confirmed that the analysis was performed with good sensitivity.

Experimental Example 2 Syphilis Bacteria Test Using Immunoassay System

Instead of using monoclonal antibodies against the nucleocapsid antigen of influenza virus, nitrocellulose pads and antigen-gold conjugate pads were prepared in the same manner as in Experimental Example 1 except that the recombinant syphilis antigen was used. The nitrocellulose pad was attached to an adhesive-coated polypropylene plate support, and a hygroscopic pad (Millipore, USA) was attached to the upper part of the nitrocellulose pad so as to overlap 1 mm, and the whole blood sample pad, auxiliary pad, and antigen- The gold conjugate pads were attached to a separate polypropylene plate support with an adhesive spacing of 1 mm each. After mounting the prepared two strips in the lower case at 2mm intervals, and covering the upper case formed with three connection parts connecting the whole blood sample pad, auxiliary pad, antigen-gold conjugate pad and nitrocellulose pad, the present invention An immunoassay device was prepared in accordance with Example 4 (see FIG. 8). Each of the positive and negative syphilis samples were tested using the completed immunoassay device, and it was confirmed that the analysis was performed with good sensitivity.

Experimental Example 3 HGC Test Using Immunoassay Device

Instead of using monoclonal antibodies against the nucleocapsid antigen of influenza virus, nitrocellulose pads and antibody-gold conjugate pads were prepared in the same manner as in Experiment 1 except that monoclonal antibodies against alpha HCG antigens were used. The sample pad, the antibody-gold conjugate pad, and the nitrocellulose pad were attached to the polypropylene plate support coated with an adhesive at intervals of 1 mm, respectively, and a hygroscopic pad (manufactured by Millipore, USA) was placed on top of the nitrocellulose pad. 1 mm overlapped. After mounting the prepared strip on the lower case, and covers the upper case formed with two connecting portions connecting the sample pad, the antibody-gold conjugate pad and the nitrocellulose pad, the immunoassay device according to the third embodiment of the present invention Prepared. As a result of testing each of the HGC positive and negative specimens using the completed immunoassay device, it was confirmed that the analysis is performed with good sensitivity.

Experimental Example 4 HIV Virus Test Using Immunoassay Device

Experimental Example 1 except that the monoclonal antibody against the nucleocapsid antigen of influenza virus was diluted with phosphate buffer, instead of diluting HIV virus type 1 and type 2 antigen antigens gp4l and gp36 with carbonate buffer. Nitrocellulose pads and antigen-gold conjugate pads were prepared in the same manner as The whole blood sample pad, the auxiliary pad, the antigen-gold conjugate pad, and the nitrocellulose pad are attached to one polypropylene plate membrane coated with an adhesive at intervals of 1 mm each, and are absorbed on top of the nitrocellulose pad. Pads (Millipore, USA) were attached in 1 mm overlap. After the prepared strips were mounted on the lower case at 2 mm intervals, the upper case was covered with an upper case in which three connection parts connecting the whole blood sample pad, the auxiliary pad, the antigen-gold conjugate pad, and the nitrocellulose pad were formed. An immunoassay device was prepared according to Example 3 (see FIG. 5B). One positive and negative sample of HIV virus type 1 and type 2 was tested using the completed immunoassay device, and it was confirmed that the analysis was performed with good sensitivity.

Experimental Example 5 HGC test using an immunoassay device

An immunoassay device was manufactured in the same manner as in Experimental Example 3, except that a wick connected to the outside was mounted on one end of the sample pad to prepare an immunoassay device according to the sixth embodiment of the present invention (see FIG. 10). It was. Liquid samples were injected into the immunoassay device through the wick, and each of the HGC positive and negative samples was tested, and it was confirmed that the analysis was performed with good sensitivity.

As described above, the immunoassay device according to the present invention can adjust the moving speed of the mobile phase such as a liquid sample through the pads used in the immunoassay, and according to the type of the antigen-antibody reaction, the antigen-antibody reaction time Can be arbitrarily controlled, and has excellent advantages in sensitivity, detection limit and specificity of immunochromatographic analysis. In addition, the immunoassay device according to the present invention is not only a simple manufacturing process of the pad used in the immunoassay, but even if some of the pad failure occurs, there is no need to replace the entire pad, the structure of the immunoassay device It can be easily modified.

1 is a cross-sectional view of an assay strip used in a conventional immunoassay device.

2A and 2B are exploded perspective and side cross-sectional views of a discontinuous immunoassay device according to a first embodiment of the present invention, respectively;

Figure 3 is an enlarged front view (A), bottom view (B) and side view (C) of the connecting portion used in the discontinuous immunoassay device according to the first embodiment of the present invention.

Figure 4 is a side cross-sectional view of a discontinuous immunoassay device according to a second embodiment of the present invention.

Figure 5 is a side cross-sectional view of a discontinuous immunoassay device according to a third embodiment of the present invention.

6A and 6B are exploded perspective and side cross-sectional views of a discontinuous immunoassay device according to a fourth embodiment of the present invention, respectively;

Figure 7 is an enlarged front view (A), bottom view (B) and side view (C) of the connecting portion used in the discontinuous immunoassay device according to a fourth embodiment of the present invention.

8 is a modification of the non-continuous immunoassay device according to a fourth embodiment of the present invention.

Figure 9 is a partial cutaway plan view of a discontinuous immunoassay device according to a fifth embodiment of the present invention.

10 is an exploded perspective view of a discontinuous immunoassay device according to a sixth embodiment of the present invention.

Claims (17)

A first pad including a sample pad receiving a liquid sample; A second pad spaced apart from the first pad by a predetermined distance and the liquid sample is transferred; An upper case covering upper portions of the first and second pads; A lower case covering lower portions of the first and second pads; And Is formed in the upper case, the immunoassay device comprising a connection portion which is located between the first pad and the second pad, to form a passage for the liquid sample to move. The immunoassay device of claim 1, wherein the connection part is a liquid impermeable protrusion formed on the upper case, and the protrusion forms a capillary passage through which a liquid sample moves between the lower case and the upper case. The immunoassay device according to claim 1, wherein the lower case is provided with a plate-shaped pad support for supporting the first and second pads. The immunoassay device according to claim 1, wherein the connection portion is a protrusion formed with a shoulder portion for pressing and fixing the ends of the first and second pads. The immunoassay device of claim 2, wherein a plurality of circular microprojections are formed on the protrusion. The immunoassay device of claim 1, wherein the linking portion is treated with a hydrophobic or hydrophilic material. The immunoassay device of claim 6, wherein the treating of the connecting portion with the hydrophobic or hydrophilic material comprises coating the connecting portion with hydrophobic or hydrophilic latex particles or compounds, or binding a hydrophobic or hydrophilic group to the connecting portion using a plasma. . The immunoassay device of claim 1, wherein the first pad is a sample pad, and the second pad includes a porous membrane pad and a moisture absorption pad, which overlap each other. The immunoassay device of claim 1 wherein the first and second pads are each formed on a separate plastic support. The porous membrane pad of claim 1, wherein the first pad includes a specimen pad and a conjugate pad formed on the first plastic support at a predetermined distance, and the second pad is formed on the second plastic support. And immunoassay device comprising a hygroscopic pad. The immunoassay device of claim 1, wherein the upper and lower case portions covering the first pad are inclined at a predetermined angle with respect to the upper and lower case portions covering the first pad. The immunoassay device of claim 1, wherein the first and second pads are spaced at a predetermined angle about the connection portion. Strip-shaped plastic supports; Two or more immunoassay pads positioned on the plastic support, spaced a predetermined distance apart from each other; An upper case covering an upper portion of the at least two immunoassay pads; A lower case covering a lower portion of the plastic support; And Is formed in the upper case, the immunoassay device comprising a connection portion which is located between each pad spaced apart from the predetermined distance, to form a passage for the liquid sample to move. The immunoassay device according to claim 13, wherein the two or more immunoassay pads comprise a porous membrane pad having a sample pad, a conjugate pad, and a detector. Injecting a liquid sample into the sample pad; Moving the liquid sample through a capillary passage formed between the upper case and the lower case by a protrusion formed in an upper case covering the sample pad; And An immunoassay method comprising the step of detecting the liquid sample passed through the capillary passage by the antigen-antibody reaction. The immunoassay device according to claim 1 or 13, wherein the lower case further includes a connecting portion positioned between the pads spaced apart from the predetermined distance and forming a passage through which the liquid sample moves. The immunoassay method according to claim 15, wherein the lower case further includes a protrusion forming a capillary passage between the upper case and the lower case.