KR101157042B1 - Improved Lateral Flow Immunoassay And Device Therefor - Google Patents

Abstract

In the immunoassay method of immobilizing a capture molecule such as an antigen or an antibody on a solid phase, laterally moving a solution on the solid phase, and inducing an antigen-antibody reaction, the reaction of the capture molecule immobilized on a solid phase and an analyte in a sample By preferentially inducing and reacting the analyte bound to the solid capture molecule with the label conjugate later, thereby significantly increasing the sensitivity of the indirect immunoassay, which laterally shifts the solution and induces an antigen-antibody response. The present invention provides a method capable of improving the hook effect of an analyte and a novel immunochromatographic device capable of effectively performing such a method.

Description

Improved Lateral Shift Immunoassay and Device for It {Improved Lateral Flow Immunoassay And Device Therefor}

1 is an exploded schematic diagram of a conventional immunochromatography device consisting of an immunochromatography strip and a housing;

2 is an exploded schematic diagram of an immunochromatographic device according to one embodiment of the present invention;

3 is an exploded schematic diagram of an immunochromatographic device according to another embodiment of the present invention;

4 is an exploded schematic diagram of an immunochromatographic device according to another embodiment of the present invention;

5 is a result of testing the hepatitis C virus antibody positive sample that is not detected by conventional immunochromatography in Example 2;

FIG. 6 shows the results of testing for hepatitis C virus antibody positive 50 specimens in an experiment using the device of FIG. 2 (Example 3); FIG.

FIG. 7 shows the results of testing for hepatitis C virus antibody negative 50 specimens in an experiment using the device of FIG. 2 (Example 4). FIG.

The present invention relates to an improved lateral shift immunoassay and an immunochromatography device for this purpose, and more particularly, to immobilize capture molecules such as antigens or antibodies on a solid state and to laterally move the solution in such a solid state. In an immunoassay that induces an antibody response, the reaction of the capture molecules immobilized on the solid phase and the analyte in the sample is preferentially induced, and then the capture of the solid phase is carried out by removing the analyte that does not bind the solid phase in an appropriate manner. By later inducing a reaction between the analyte bound to the molecule and the label conjugate, it significantly improves the sensitivity of the indirect immunoassay, which laterally shifts the solution and induces an antigen-antibody response, and hooks with high concentrations of the analyte. How to improve the hook effect, and how to do it effectively Station relates to a chromatography device.

Immunoassay refers to an analytical method that detects an antigen or an antibody or measures its concentration. The immunoassay includes homogeneous immunoassays and solids in which a reaction proceeds only in a solution phase. Phase) Heterogeneous Immunoassay requires separation of analyte or label.

The most commonly used method is Enzyme-Linked Immunosorbent, which uses a 96 well plate made of polystyrene in solid form and an enzyme label as a conjugate. Assay) method. In the ELISA method, in addition to the 96-well plate, the solid form may include plastic tubes, plates, microparticles, beads, nitrocellulose membranes, and glass slides. In addition to the above-described enzymes, fluorescent substances, luminescent substances, gold colloids, silver colloids, or latexes can be used as the label.

On the other hand, a diagnostic method that can immediately check the patient's condition at the medical site without a separate device or equipment is called a point-of-care test (Rapid test) or rapid test (Rapid test). The immunological rapid test mainly uses a membrane such as nitrocellulose in solid form, uses a gold colloid as a label, and can be quickly tested at a relatively low cost.

In general, a rapid test using a nitrocellulose membrane is divided into a flow through method and an immunochromatography in which a lateral flow is carried out using a porous nitrocellulose membrane without passing through a solution. Can be.

The flow through method uses a porous membrane such as nitrocellulose, and is a method of dropping the solution by placing a filter paper, usually cellulose, under the porous membrane. The pass test consists of several steps, including washing, which makes the process complex and the rapid test in which the antigen / antibody reaction must occur in a very short time. It has the disadvantage of being worse than the chromatographic method. However, since the antigen-antibody reaction sequence is the same as that of the ELISA, that is, the capture molecules in the solid state and the analyte react first, and then the reactions between the label conjugates and the like have the following advantages.

First, in noncompetitive assays for specific antibodies, immunochromatography requires very high amounts of capture molecules or label conjugates, whereas immune transit methods respond in much smaller amounts. Not only is this possible, but it is also possible to use them efficiently. Second, in the case of using a high concentration of analyte, the immunochromatography method may cause the label conjugate to occupy all the binding sites of the analyte. In some cases, immune transit can be used to overcome this problem.

In this regard, immunochromatography can be said to be a test method that combines the specific immune response of antigen-antibody with the progress of chromatography. This method can be performed using specific binding of antigen-antibodies, transfer of reactants on the Lateral Flow Membrane, mobility of gold colloids to which antigens or antibodies are bound, and color development by aggregation. As a method, this method has the advantage that the test can be performed in only one or two procedures without a separate washing step. That is, the reaction and washing of the sample with the capture molecule or the label conjugate are performed by the chromatographic method itself rather than a separate process. Each antigen-antibody reaction can be regulated primarily by the rate of chromatography, and also by the amount of capture molecules such as antigens or antibodies immobilized on the label conjugate or nitrocellulose membrane.

Despite these advantages, immunochromatography is characterized by the fact that antigen-antibody reactions are performed differently from the sequence of ELISA, so that the analytes react first with the label conjugate rather than with the capture molecules in the solid phase. The analyte or label conjugate bound to the solid capture molecule by means of In heterogeneous immunoassays, when chromatography is used to separate analytes or label conjugates in solution using solid phases, it is difficult to wash or separate efficiently as in ELISA. Therefore, the non-competitive assays for Specific Antibodies (Noncompetitive Assays for Specific Antibodies) is not good sensitivity, there is a disadvantage that easily exposed to the hook effect (Hook Effect) by a high concentration of analyte. However, in spite of these drawbacks, immunochromatography, which is simpler to use and more sensitive than pass-through assay, is preferred as a field test.

Devices for performing such immunochromatography generally consist of a structure in which a chromatography strip is wrapped in a pair of housings. In the chromatography strip, a solid phase for moving a solution is mounted on a plate-like support, and a conjugate pad and a sample pad are sequentially stacked so as to contact one side of the solid phase. The absorbent pads are stacked on the opposite side of the solid so as to be in contact with each other, and the inspection line and the control line having the capture molecules attached to the middle portion of the solid are located. The upper housing includes a sample addition part punctured to expose the sample pad, and a confirmation window through which the test line and the control line are exposed, and the lower housing includes a fixing part for positioning the strip.

A representative example of an immunochromatographic device of such a configuration is disclosed in FIG. 1. Referring to FIG. 1, the chromatography strip 110 includes a sample pad 101, a conjugate pad 102, a nitrocellulose membrane 103 as a solid form, and an absorbent pad. 106) and an adhesive laminate card 107 as a support.

The sample pad 101 is a site to which a sample is added, and allows the sample to be chromatographed at a constant speed and distribution. The sample pad 101 is treated with a blocking protein, a solvent, a buffer component, a salt and a viscosity increasing substance. It may also play a role in controlling the rate of migration, controlling the solubility of the label conjugate, blocking nonspecific reactions, and the like.

The conjugate pad 102 contains a detection material such as a gold-labeled conjugate, and when the sample reaches the conjugate pad 102 after passing through the sample pad 101, in the case of a positive sample, the sample binds to the detection material to form nitrocellulose. Move to membrane 103.

The nitrocellulose membrane 103 is a porous membrane in which an antigen or an antibody is immobilized on an inspection line 104 and a control line 105 on one side of an upper surface thereof. When the label conjugate and the sample move along the nitrocellulose membrane in a laterally flow (Lateral Flow) manner to reach the test line 104, the label conjugate and the sample bind to the antigen or antibody on the test line 104 if positive, and negative. The inspection line 104 is too much as it is.

Absorption pad 106 absorbs the chromatographic material and allows the solution to continuously move.

Adhesive laminate card 107 serves as a support for the components.

The strip 100 may be used by putting the strip itself into a test tube or the like, but may also be assembled into a housing. The upper housing 110 includes a sample adding unit 111, a result confirming window 112 to read a result, and a pin 114 structure to be coupled to the lower housing. Various materials such as polystyrene, polyethylene, polyvinyl chloride, and the like may be used in the housing. When using a plastic of transparent material, the result confirmation window 112 does not need to be punched as shown in the drawing.

The lower housing 120 is composed of a fixing part 121 for fixing the strip 100, a pinhole 122 that can be combined with the upper housing 110, and the like.

In a non-competitive assay for detecting specific antibodies among immunoassay methods using immunochromatography, a very large amount of labeled antibody conjugate is required because the labeled antibody conjugate and the sample react first, but are not easy to satisfy in reality. For example, if a substance such as Protein A is conjugated to a label such as a gold colloid, and if the label conjugate does not bind all of the IgG in the sample, a specific IgG that does not bind to the label conjugate may And all kinds of IgG molecules can bind to the control line. This competitively inhibits the binding of the labeled conjugate-IgG complex to the control line or the control line. Normally, the concentration of IgG in blood is known to be 10 mg / ml or more. For more effective immunochromatography, very large amounts of label conjugates or capture molecules must be used. However, the protein binding capacity of the nitrocellulose membrane is limited, and in the case of the label conjugate, the amount thereof is limited when immobilized on a pad or a solid, and there is a possibility that problems such as inducing a non-specific reaction due to excessive use may occur. Cannot use the required amount of label conjugate or capture molecule. Therefore, the sensitivity is not good compared to other test methods. In immunoassays such as ELISA, this problem does not occur because the antibody and the antigen immobilized on the solid phase are reacted first and the unreacted antibody is removed by washing. However, the conventional method of immunochromatography first reacts a sample with a label conjugate, so that a large number of IgG positive molecules which are not bound in this reaction bind to the antigen of the test line and competitively prevent the binding of the label conjugate. . In the case of the control line, IgG molecules not bound to the label conjugate bind and interfere with the binding of the label conjugate.

Therefore, indirect immunoassay for the detection of specific antibodies may be said to be more suitable for immune transfection than immunochromatography. In addition, in the immunoassay method of inducing antigen-antibody reactions using capillary phenomena of microplastic structures, such as the method mentioned in USP 6767510 of Biosite, the reaction between the fluorescent label conjugate and the analyte is induced first, Indirect immunoassay is bound to decrease the sensitivity.

On the other hand, in the immunoassay, when a high concentration of analyte is present in a sample, when the sample and the label conjugate react with the solid antigen or antibody at the same time, the hook effect that the analyte binds to all positions where the label conjugate can bind ( Hook Effect) occurs. In order to avoid this, in immunoassays such as ELISA, analytes and solid antigens or antibodies are first reacted, and excess analytes are removed by washing, and then a label conjugate is reacted. However, there is no method to effectively overcome the high-dose hook effect in conventional immunochromatography using a membrane or immunoassay using a capillary phenomenon of a microplastic structure disclosed in USP 6,767,510 of Biosite. . Therefore, there is a high need for an immunoassay that solves the conventional problem of the non-competitive assay for detecting specific antibodies and does not cause a high-dose hook effect due to a high concentration of analyte.

The present invention aims to solve these problems of the prior art and technical problems that have been requested from the past.

The inventors of the present invention require a very large amount of label conjugates as described above, in which a sample is first reacted with a label conjugate in a method of inducing an immune response by laterally moving a solution using conventional chromatography or capillary phenomenon. If there is a high concentration of analyte in the sample, there is a problem that a hook effect is likely to occur because the analyte is bound to all positions where the label conjugate can bind, so that the label conjugate cannot bind to the solid. It was confirmed.

Therefore, the inventors of the present invention, after in-depth research and various experiments to develop an improvement method, induce an immune response that first reacts analyte in a sample with a solid capture molecule and then reacts a label conjugate. It has been found that the indirect immunoassay for antibody detection is effective and prevents the hook effect by high concentration analytes. The present invention has been completed based on this finding.

In the immunoassay according to the present invention for achieving the above object, in the immunoassay that immobilizes a capture molecule such as an antigen or an antibody on a solid phase and induces an antigen-antibody reaction while laterally moving a solution containing a sample on the solid phase, Preferentially induce a reaction between the capture molecule immobilized on the solid phase and the analyte in the sample, and later induce a reaction between the analyte (capture molecule-analyte complex) and the label conjugate bound to the solid capture molecule It is.

The term "immunoassay" is an indirect immunoassay that induces an antigen-antibody response while moving a solution laterally, and detects specific antibodies such as IgA, IgG, IgM, IgD, etc. using a secondary antibody or an antibody-specific protein. Non-competitive methods of analysis.

In the immunoassay, chromatography, capillary phenomenon, electrophoresis, pump, centrifugation, magnetic field, or the like may be used as a method of inducing the movement of a solution including a sample.

A feature of the present invention is that, as described above, the immunoassay consists of first inducing the reaction of the capture molecule and the analyte and then inducing the reaction of the capture molecule-analyte complex and the label conjugate. Due to this structural feature, the indirect immunoassay, which moves the solution laterally and induces an antigen-antibody response, greatly enhances the sensitivity and improves the hook effect by the high concentration of the analyte.

The solid phase is a mediator capable of immobilizing capture molecules such as antigens or antibodies that bind to the analyte in the sample and laterally shifting the solution. For example, nitrocellulose, polyvinylidene fluoride (PVDF), Absorbent materials such as nylon, porous plastics, plate-shaped members having microstructures, and the like may be used.

As a label in the label conjugate, a metal label such as gold or silver, a color developing enzyme such as peroxidase, a fluorescent substance, a luminescent material, a chromosome (Dye), or the like can be used.

The manner of inducing the reaction of the capture molecule-analyte complex and the label conjugate after the binding reaction between the analyte and the capture molecule can be achieved by various configurations. For example, a sample containing (or likely to contain) an analyte may be contacted directly or indirectly with a capture molecule to induce a reaction, and over time to the capture molecule-analyte conjugate obtained through this reaction. The method may induce a reaction between the capture molecule-analyte conjugate and the label conjugate by contacting the label conjugate through lateral movement.

This continuous reaction over time can also be implemented through conventional immunochromatographic devices as disclosed in FIG. 1.

For example, the continuous reaction as described above may be possible by adding a buffer to the sample adding unit 111 after adding the sample to the result confirmation window 112. Specifically, when the analyte in the sample added first reacts with the capture molecule, and then the buffer is added, the label conjugate of the conjugate pad 102 moves with the buffer and pushes the sample toward the absorbent pad 106, thus, the label. The conjugate does not react with the sample but with the capture molecule-analyte complex bound to solid 103.

While the above method using the conventional immunochromatography device as shown in FIG. 1 is naturally included in the scope of the present invention, when the above device is used, the movement of the solution in the solid state when the insoluble material is included in the sample components is prevented. It has a disadvantage that can be disturbed.

Accordingly, the present invention provides a variety of immunochromatographic devices capable of more efficiently performing the above-described continuous reaction.

The first preferred example of such an immunochromatographic device consists of a structure wrapped around a chromatography strip in a pair of housings,

In the chromatography strip, a solid phase for moving a solution is mounted on a plate-like support, and a conjugate pad and a buffer pad are sequentially stacked so as to contact one side of the solid, and absorbed on the opposite side of the solid. The pads are stacked so as to be in contact with each other, and a test line and a control line to which capture molecules are attached are located at the intermediate portion of the solid, and a sample pad is stacked on the solid between the absorbent pad and the control line;

The upper housing includes a buffer addition portion drilled to expose the buffer pad, a sample addition portion drilled to expose the specimen pad, and a confirmation window to which the inspection line and the control line are exposed;

The lower housing includes a fixing portion for positioning the strip.

In one preferred embodiment, an interference member for inhibiting the movement of the solution for a certain time may be further included between the absorbent pad and the solid state.

One preferred example of such a chromatography device is disclosed in FIG. 2.

Referring to FIG. 2, the sample adding portion 213 and the buffer adding portion 211 are separated from each other in the upper housing 210, and the sample pad 208 attached to the strip 200 is provided with a control line ( Located between 205 and absorbent pad 206, the strip 200 and the upper and lower housings 210 and 220 can be assembled to complete the device.

Referring to the method of performing a continuous reaction according to the present invention using the device of Figure 2 as follows. When a sample is added to the sample adding portion 213 of the device, the sample reacts first with the control line 205 and the inspection line 204 of the solid 203 where the capture molecules are fixed before the sample reacts with the conjugate pad 202. The binding reaction between the analyte and the capture molecule in the sample occurs first. Subsequently, when the buffer is added to the buffer addition unit 201, the buffer dissolves the labeled conjugate dried on the conjugate pad 202 through the buffer pad 201 and moves to the solid 203, and the buffer and the labeled conjugate are high. After moving to the shape 203 for some time, the sample meets the sample and moves the sample toward the absorbent pad 206. At this time, the label conjugate that is initially released is in contact with the sample to some extent, but continues to push the sample toward the absorbent pad 206 while moving laterally, so that after some time, the label conjugate does not react with the sample and becomes solid (203). Reacts with the capture molecule-analyte complex bound to). That is, the released label conjugate is complexed in response to the test line 204 and the control line 205 to which the capture molecule and the analyte are bound, and a red or purple band is formed when a detection material such as gold is used.

The sample pad 208 may be bonded to the solid form 203 using various adhesive materials or adhesive tapes. In addition, the amount of the sample to be added should be used in consideration of the size of the sample pad 208, etc. For example, the size of the solid 203 is 4 mm x 25 mm and the sample pad 208 is 4 In the case of mm x 5 to 10 mm, the amount of sample is preferably about 10 to 50 μl. If the amount of the sample exceeds 50 μl, the sample moves along the solid 203 toward the conjugate pad 202 before the buffer is added. If the amount is less than 10 μl, the sample does not move to the test line 204. You may not. In addition, in order to make the transfer effective by using the buffer, it is preferable that the amount of the buffer is larger than the amount of the sample.

In some cases, the absorbent pad 206 and the solid state 203 may be used to prevent the sample from moving toward the absorbent pad 206 and to move the solution toward the inspection line 204 and the control line 205 of the solid state 203. The flow of the sample may be directed toward the solid state 203 via the interference member therebetween. Preferred examples of such an interference member may be a water-soluble film made of polyvinyl alcohol or the like, a hydrophobic film or glass fiber. In addition, a small amount of buffer may be added to the sample pad 208 to facilitate movement of the sample.

A second preferred example of an immunochromatographic device according to the invention consists of a structure in which a chromatography strip is wrapped in a pair of housings,

In the chromatography strip, a solid phase for moving a solution is mounted on a plate-like support, and a conjugate pad and a buffer pad are sequentially stacked so as to contact one side of the solid, and absorbed on the opposite side of the solid. The pads are stacked so as to be in contact with each other, and a test line and a control line to which capture molecules are attached are located at a solid intermediate portion, and a sample pad is stacked on a solid between the conjugate pad and the test line;

The upper housing includes a buffer addition portion drilled to expose the buffer pad, a sample addition portion drilled to expose the specimen pad, and a confirmation window to which the inspection line and the control line are exposed;

The lower housing includes a fixing portion for positioning the strip.

In one preferred embodiment, an interference member for inhibiting the movement of the solution for a certain time may be further included between the conjugate pad and the solid state.

One preferred example of such chromatography device is disclosed in FIG. 3.

Referring to FIG. 3, compared to the device of FIG. 2, a sample pad 308 attached to the strip 300 is positioned between the conjugate pad 302 and the inspection line 304, and the conjugate pad 302 and the solid shape. There is a difference in that the interference member 309 is interposed between 303 so that the sample and the buffer proceed in one direction.

Referring to the method of performing a continuous reaction according to the present invention using the device of Figure 3 as follows. When the sample is added to the sample pad 308 through the sample adding unit 313, the sample moves through the test line 304 and the control line 305 toward the absorbent pad 306. At this time, the interference member 309 is interposed between the conjugate pad 302 and the solid shape 303 to prevent the sample from reacting with the label conjugate. Preferred examples of such an interference member include a water-soluble film. That is, since it takes time for the sample to melt the water-soluble film, the sample does not directly contact the label conjugate within a predetermined time, and most of the sample moves along the solid form 303. When the buffer is added to the buffer addition unit 311 at a certain time interval after the sample is added, the buffer is absorbed through the buffer pad 301 to release the label conjugate of the conjugate pad 302, and then the water-soluble film 309. Melt is moved to the solid state 303, and some remaining sample is moved while pushing toward the absorbent pad 306. Thus, after some time, the label conjugate does not react with the sample but with the capture molecule-analyte complex bound to the solid state 303.

In addition to the water-soluble film, the interference member 309 may be a film having a hydrophobic property such as polyvinylidene fluoride (PVDF), glass fiber, or the like. Because of the hydrophobic nature of these components, the sample does not come into contact with the label conjugate, but if a component such as a solvent is used in the buffer, the solution may migrate through the membrane or glass fiber. Therefore, the size of the hole, lattice, etc. of the interference material must be enough to allow the protein to pass through.

In some cases, a buffer may be added to the sample addition unit 308 for the flow of the sample or the washing of the sample components bound to the solid phase 303.

A third preferred example of an immunochromatographic device according to the invention consists in a structure in which a chromatography strip is wrapped in a pair of housings,

In the chromatography strip, a solid state for moving a solution is mounted on a plate-like support, an absorbent pad is laminated on one side of the solid state, and a capture molecule is attached to an intermediate portion of the solid state. A line and a control line are positioned, and a sample pad is laminated on a solid on the opposite side to the absorbent pad about the inspection line;

The upper housing includes a buffer addition portion perforated to expose the buffer pad, a sample addition portion perforated to expose the sample pad, and a confirmation window to which the test line and the control line are exposed, and the buffer addition portion is formed from the main body of the housing. Are separated;

The lower housing includes a fixing portion for positioning the strip, wherein a portion of the strip in which the conjugate pad and the buffer pad are sequentially stacked is hingedly coupled to the lower housing on the opposite side of the strip and opposite the high It is supported by the temporary support in the state spaced apart from the shape side.

One preferred example of such chromatography device is disclosed in FIG. 4.

Referring to FIG. 4, the conjugate pad 402 and the buffer pad 401 are connected to the lower housing 420 in a state of being fixed on the hinge support 409. The opposite side of the hinge support 409 is supported by a Y-shaped fixing pin 423 as a temporary support. The fixing pin 423 is made of, for example, soft plastic that can be easily broken by physical force. The strip 400 is configured such that the sample pad 408 is positioned below the test line 404, and the conjugate pad 402 is not directly connected, and thus the buffer addition portion in the upper housing 410 is disposed. 411 is separated from the sample adding portion 414.

After the housings 410 and 420 and the strip 400 are assembled, the sample is added to the sample adding portion 414, and after some time of movement, a buffer is added to the buffer adding portion 411, and then the opposite side of the hinge is pressed. As the fixing pin 423 breaks, the conjugate pad 402 comes into contact with the solid phase 403 and the buffer and the label conjugate move toward the solid phase 403. This method can expect the same effect as using the interference member in the device of FIG.

In the above, the structure of the preferred device capable of performing the method of the present invention has been described by way of example, but those skilled in the art can conceive a variety of devices based on the above information All of these should be construed as being included in the scope of the present invention.

Hereinafter, the content of the present invention will be described with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 Preparation of Immunochromatography Strips for Hepatitis C Virus Antibody Testing

Nitrocellulose membrane (SRHF Nitrocellulose Membrane from Millipore) was prepared and inspected for contaminants and cut to a size of 25 mm x 20 cm. The solution dispenser (Biodot 3000) was prepared in the absence of foreign substances by washing well with purified water before antigen / antibody dispensing. After placing the nitrocellulose membrane in the dispenser, the test line was dispensed with a mixture of the recombinant hepatitis C virus antigen (Core antigen, NS3 antigen, NS4 antigen, NS5 antigen, 1.0 mg / ml) at 1.0 μl / cm. In the control line, goat anti human immunoglobulin (1.0 mg / ml) was dispensed at 1.0 μl / cm. The nitrocellulose membrane onto which the antigen was adsorbed was then dried to dry the dispensed solution and attached to the laminate card after drying.

The gold colloidal label conjugate was used as a protein A-gold label conjugate and prepared by referring to Bioconjugate Techniques (author: Greg T. Hermanson), pp. 593-602, published by Academic Press. The protein A-gold label conjugate was adsorbed onto glass fibers of Millipore, and the solution was dried and cut into 5 mm lengths.

Absorbent pads were cut to 20 mm length using Millipore's cellulose pads. The sample pad was cut to a length of 5 mm using a cellulose pad manufactured by Millipore. In addition, in the case of using a buffer pad, Millipore's cellulose pad was cut to a size of 1.6 mm and used.

Each element was attached to a laminate card and cut to a size of 4 mm with a cutter to complete the immunochromatography strip, as in FIGS. 1, 2 and 3. Each strip was assembled to a white or transparent housing and used for inspection.

Example 2: Hepatitis C Virus Antibody Test

Immunochromatography strip for hepatitis C virus antibody test was prepared as in Example 1, bound to the housing, and diluted hepatitis C virus antibody positive sample with serum of normal person, 20 μl was added to the sample addition buffer, and then the buffer solution (phosphate 100 μl of buffer, 1.0% bovine serum albumin) was added and developed. The sample used was a positive sample that was negative by the conventional immunochromatography under the same conditions as in Example 1, and did not react in the control line.

In the immunochromatography device having the structure of FIG. 1, the sample was added to the 111 (FIG. 1) site, and the sample was added to the 211 site (FIG. 2) with the immunochromatography device of the FIG. In the case, no reaction occurred in the test line or the control line (501, 509 in Fig. 5). However, in the immunochromatography device of FIG. 2, when the sample was added to the 213 site and the buffer was added to the 211 site, a strong positive reaction occurred, and the sample was 1/10 to 1/640 (Fig. 502 to 508 of 5) was positively recognized up to 1/320 dilution ratio when diluted in 1/2 successively (507 of FIG. 5). Therefore, in the case of indirect immunoassay in which the specific antibody is tested in the immunoassay in which the solution is moved in a solid state and tested, the analyte reacts first with a capture molecule immobilized on the solid state to increase the concentration of the antigen or antibody or the label conjugate. It was confirmed that the sensitivity can be greatly improved without increasing.

Example 3: Tests for Hepatitis C Virus Antibody Negative and Positive Specimens

Tests were performed on hepatitis C virus antibody negative and positive samples to see if the method according to the invention could actually be used for clinical testing. Among the strip devices prepared in Example 1, a hepatitis C virus antibody test device having the structure of FIG. 2 was used. The housing used a transparent polystyrene material and the inspection method used the same method as Example 2. As a result of the test for the positive 50 specimens and the negative 50 specimens, all of the positive 50 specimens were positive and all the negative 50 specimens were determined as negative, and these results are shown in FIGS. 6 and 7.

As shown in these figures, desired results were obtained, respectively.

As described above, according to the present invention, by first inducing the reaction of the capture molecule and the analyte and later inducing the reaction of the capture molecule-analyte complex and the label conjugate, the solution is moved laterally and the antigen-antibody reaction is induced. Indirect immunoassay can greatly improve sensitivity and improve hook effect by high concentrations of analytes.

Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims (14)

In an immunoassay that induces an antigen-antibody reaction by immobilizing a capture molecule such as an antigen or an antibody on a solid phase and laterally moving a solution containing a sample on the solid phase, a reaction between the capture molecule immobilized on a solid phase and the analyte in the sample Immuno-assay comprising preferentially inducing and later inducing a reaction of an analyte (capture molecule-analyte complex) bound to a solid capture molecule with a label conjugate. The immunoassay according to claim 1, wherein the immunoassay is a non-competitive assay for detecting a specific antibody using a secondary antibody or an antibody-specific protein. 2. The immunoassay according to claim 1, wherein chromatography, capillary action, electrophoresis, pump, centrifugation or magnetic field are used in a manner to induce the movement of the solution. 4. The immunoassay according to claim 3, wherein the method uses chromatography. The immunoassay according to claim 1, wherein the solid state is an absorptive material, a porous plastic, or a plate member in which a microstructure is designed. 6. The immunoassay method according to claim 5, wherein the solid phase is made of nitrocellulose, polyvinylidene fluoride (PVDF) or nylon. The immunoassay according to claim 1, wherein the label in the label conjugate is a metal label, a chromogenic enzyme, a fluorescent substance, a luminescent substance, or a chromosome (Dye). An immunochromatographic device for performing an immunoassay according to claim 1, comprising a structure in which a chromatography strip is wrapped in a pair of housings composed of an upper housing and a lower housing, In the chromatography strip, a solid phase for moving a solution is mounted on a plate-like support, and a conjugate pad and a buffer pad are sequentially stacked so as to contact one side of the solid, and absorbed on the opposite side of the solid. The pads are stacked so as to be in contact with each other, and a test line and a control line to which capture molecules are attached are located at the intermediate portion of the solid, and a sample pad is stacked on the solid between the absorbent pad and the control line; The upper housing includes a buffer addition portion drilled to expose the buffer pad, a sample addition portion drilled to expose the specimen pad, and a confirmation window to which the inspection line and the control line are exposed; And the lower housing includes a fixture for positioning the strip. The device of claim 8, further comprising an interference member between the absorbent pad and the solid state to suppress movement of the solution for a predetermined time. 10. A device according to claim 9, wherein the interference member is made of a water-soluble film, a hydrophobic film or glass fiber. An immunochromatographic device for performing an immunoassay according to claim 1, comprising a structure in which a chromatography strip is wrapped in a pair of housings composed of an upper housing and a lower housing, In the chromatography strip, a solid phase for moving a solution is mounted on a plate-like support, and a conjugate pad and a buffer pad are sequentially stacked so as to contact one side of the solid, and absorbed on the opposite side of the solid. The pads are stacked so as to be in contact with each other, and a test line and a control line to which capture molecules are attached are located at a solid intermediate portion, and a sample pad is stacked on a solid between the conjugate pad and the test line; The upper housing includes a buffer addition portion drilled to expose the buffer pad, a sample addition portion drilled to expose the specimen pad, and a confirmation window to which the inspection line and the control line are exposed; And the lower housing includes a fixture for positioning the strip. 12. The device of claim 11, further comprising an interference member between the assembly pad and the solid state to suppress movement of the solution for a predetermined time. 13. The device of claim 12, wherein the interference member is made of a water-soluble film, a hydrophobic film or glass fiber. An immunochromatographic device for performing an immunoassay according to claim 1, comprising a structure in which a chromatography strip is wrapped in a pair of housings composed of an upper housing and a lower housing, In the chromatography strip, a solid state for moving a solution is mounted on a plate-like support, an absorbent pad is laminated on one side of the solid state, and a capture molecule is attached to an intermediate portion of the solid state. A line and a control line are positioned, and a sample pad is laminated on a solid on the opposite side to the absorbent pad about the inspection line; The upper housing includes a buffer addition portion perforated to expose the buffer pad, a sample addition portion perforated to expose the sample pad, and a confirmation window to which the test line and the control line are exposed, and the buffer addition portion is formed from the main body of the housing. Are separated; The lower housing includes a fixing portion for positioning the strip, wherein a portion of the strip in which the conjugate pad and the buffer pad are sequentially stacked is hingedly coupled to the lower housing on the opposite side of the strip and opposite the high An immunochromatography device supported by a temporary support while being spaced apart on the shape side.

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