KR20190011643A - Diagnosis kit capable of identifying concentration of analyte in biological sample - Google Patents

Abstract

The present invention provides a diagnostic kit capable of analyzing a concentration of an analyte contained in a biological sample and an analyzing method using the same. The diagnostic kit comprises: a plurality of strips; a labeled auxiliary specific binding member coupled to each of the strips at different concentrations; and a reaction unit which is disposed on each of the strips and to which a fixing member, to which the auxiliary specific binding member is specifically coupled, is coupled.

Description

[0001] DIAGNOSIS KIT CAPABLE OF IDENTIFYING CONCENTRATION OF ANALYTITE IN BIOLOGICAL SAMPLE [0002]

The present invention relates to a bio-diagnostic kit, and more particularly, to a bio-diagnostic kit capable of analyzing the concentration of an analyte contained in a biological sample that accurately and quickly verifies not only the presence of an analyte present in a sample of blood, Kit.

Vitamin D is one of the fat-soluble vitamins that plays an important role in the metabolism of calcium and phosphorus, and is essential for the growth of organisms, especially calcium metabolism associated with bone development. Most of these vitamins are produced by sunlight and vitamin D is not consumed as food.

We need enough vitamin D in our bodies to keep our bones strong. Vitamin D is synthesized in the skin tissue in the sun when it gets sunlight. If the amount synthesized is less than the amount needed for metabolism, it should be ingested from food.

Children need a relatively large amount of vitamin D because their bones develop rapidly. In general, vitamin D deficiency criteria are sufficient for vitamin D blood levels above 30 ng / mL, and below 20 ng / mL for vitamin D deficiency. Vitamin D deficiency can lead to osteoporosis and can increase muscle weakness and risk of falls. Additional research is needed to determine the association of the immune system with some cancers. Severe vitamin D deficiency can result in rickets in children and osteomalacia in adults. However, excessive amounts of vitamin D accumulate in the body, so overdose of vitamin D on a continuous basis may cause toxic reactions. Thus, it is often important to determine the concentration of a large number of substances (hereinafter referred to as analytes), including vitamin D, in the blood.

Various diagnostic kits have been developed to identify existing analytes in the blood. These diagnostic kits include strips (or strips) made of the same material as nitrocellulose as shown in FIG. 1, and when a sample is loaded on one end of the strip, a reaction occurring by a predetermined binding reaction It is used to diagnose the infection or not by observing the result (for example, color development).

However, all of these conventional detection kits only confirm whether the analyte in the sample, such as blood, has exceeded the detection limit. Even if the concentration of the analyte is relatively high or low, the change amount of the phenotype according to the concentration marker is the same It is impossible to accurately and quickly confirm the difference in the concentration of the analyte in the sample in which the actual concentration is different.

Therefore, when the analyte is a specific factor in the blood that can confirm the presence of the disease, even if the concentration is very high in the blood, it may be a dangerous situation. However, when using the existing diagnostic kit, There is a limit to how much the progress of the disease can not be confirmed.

SUMMARY OF THE INVENTION Accordingly, the present invention has been made in order to solve the above-mentioned problems, and it is an object of the present invention to provide a biosensor capable of analyzing the concentration of an analyte to accurately and rapidly confirm not only the presence of an analyte present in a sample of blood, Lt; / RTI > kit.

The technical problem of the present invention as described above is achieved by the following means.

(1) a plurality of strips;

A labeled auxiliary specific binding member coupled to each of the strips at a different concentration; And

A reaction unit which is located on each of the strips and to which a fixing member to which the auxiliary specific binding member is specifically bound is coupled;

Wherein the analyte is capable of analyzing the concentration of the analyte contained in the biological sample.

(2) In the above (1)

Wherein the label is a member selected from the group consisting of a catalyst, an enzyme, an enzyme substrate, a fluorescent compound, a chemiluminescent compound, a radioactive element, a metal sol, a nonmetallic sol, a diol, a particulate latex, and a color indicator.

(3) In the above (1)

Wherein the analyte is heptene, an antibody, and combinations thereof.

(4) In the above (1)

Wherein the auxiliary specific binding member is one selected from the group consisting of antibody, protein G, protein A, and protein G / A.

(5) In the above (1)

And a color correction marker is provided on the cover.

(6) preparing a plurality of labeled coupled auxiliary specific binding members with different concentrations;

Loading a test sample on the prepared plurality of strips;

Binding an analyte contained in the test sample to the labeled auxiliary specific binding member and moving to a reaction part on the strip;

The labeled specific auxiliary binding member to which the analyte is bound is specifically bound to the immobilization member bound to the reaction unit; And

Analyzing the result of the reaction in the reaction part according to a mark having a different concentration in each of the strips, and calculating the concentration of the analyte;

And analyzing the concentration of the analyte contained in the biological sample using the diagnostic kit.

(7) In the above (6)

Wherein the label is one selected from the group consisting of a catalyst, an enzyme, an enzyme substrate, a fluorescent compound, a chemiluminescent compound, a radioactive element, a metal sol, a nonmetallic sol, a diol, a particulate latex, and a color indicator.

(8) In the above (6)

Wherein the analyte is heptene, an antibody, and combinations thereof.

(9) In the above (6)

Wherein the auxiliary specific binding member is one selected from the group consisting of antibody, protein G, protein A, and protein G / A.

(10) In the above (6)

Wherein a color correction marker is provided on one side of the cover to eliminate the influence on illumination using the color correction marker.

As described above, according to the present invention, it is possible to provide a diagnostic kit that accurately and quickly confirms not only the presence of an analyte present in a sample such as blood, but also the concentration of the analyte.

1 is a structural diagram of a diagnostic kit according to the prior art.
2 is a structural view of a diagnostic kit according to the present invention.

According to an aspect of the present invention, there is provided a diagnostic kit including: a plurality of strips; A labeled auxiliary specific binding member coupled to each of the strips at a different concentration; And a reaction unit which is disposed on the strip and to which a fixing member to which the auxiliary specific binding member is specifically coupled is coupled.

The method of analyzing the concentration of an analyte using the diagnostic kit according to the present invention may further comprise the steps of preparing a plurality of strips, each of which is bound to a labeled auxiliary specific binding member at different concentrations; Loading a test sample on the prepared plurality of strips; Binding an analyte contained in the test sample to the labeled auxiliary specific binding member and moving to a reaction part on the strip; The labeled specific auxiliary binding member to which the analyte is bound is specifically bound to the immobilization member bound to the reaction unit; And calculating the concentration of the analyte by analyzing the result of the reaction occurring in the reaction part according to a mark having a different concentration in each of the strips.

In the above, the test sample is preferably a body fluid to be secreted out of the body, and includes blood, urine, tears, saliva, milk and the like.

Also, the analyte included in the sample to be analyzed includes any substance having a specific binding site that can be naturally formed or artificially imparted, for example, an antigen presenting substance, heptene, an antibody , And combinations thereof. In the present invention, the analyte is preferably vitamin D or an allergy symptom-related antibody.

Fig. 2 shows the structure of a diagnostic kit exemplified as a preferred embodiment used in the present invention.

The diagnostic kit includes a plurality of strips (1) and a housing. The housing is required for sample port 2, development reagent inlet 3 (when spotting a sample to a filter pad (or die pad: a pad containing a labeled detection reagent)), a test result And a main body 7 provided with a strip fixing part 6 for fixing the strips 1 in place.

The cover 5 and the main body 7 are connected to each other via the coupling member 8 and are required to fix the strip 1 and prevent contact or contamination with the reaction part, Nonreactive material that does not react excessively is preferable. An example of such a non-reactive material is plastic.

It is preferable that a color correction marker (for example, a gray card) 15 is provided in a predetermined shape on one side of the cover 5 so that the color is not affected by the illumination. For example, when the gray card is mounted in the shape of a quadrangle at a predetermined size in the vicinity of the display portion, the position of the gray card is discriminated through software operation at the time of photographing by using a camera means such as a cellular phone The markers for position identification are displayed on at least three corners of the gray card), and if white balancing is performed by using the discriminated gray card color information programmatically, it is possible to precisely determine the chromaticity by eliminating the influence of the color of the illumination.

2, when the test sample is spotted on the filter pad (also serving as the die pad) 11 through the sample input port 2, a separate developing reagent input port 3 is required, The developing unit 3 is preferably formed in a cup shape so as to be able to accommodate a developing reagent in a predetermined amount and the lower end is brought into close contact with the storage pad 10 so that the developing reagent does not flow out to other places in the apparatus.

Unlike the configuration shown in FIG. 2, when the sample inlet is positioned directly above the storage pad constituting the strip and the sample is dropped directly onto the storage pad (in this case, the storage pad has a function of the filter pad Or a separate filter pad must be provided after the storage pad. In this case, a separate developing reagent inlet is not necessary), and the sample inlet preferably has a curved portion in a cup shape to receive a predetermined amount of sample And the lower end is in close contact with the storage pad to prevent the sample from flowing out to other places in the apparatus.

Further, unlike the case shown in FIG. 2, it is needless to say that the sample and the developing reagent input port can be made into one. That is, even if several concentrations are measured at a time, only one sample and development reagent input port may be present. This allows the sample and development reagent to chemically and physically flow uniformly in each line, It is because the concentration can be distinguished by the difference of coloring degree by adding a reagent (for example, conjugated gold, etc.).

In the kit of the present invention, the display window 4 is for observing a change occurring in the reaction part 13 and the control part 14 on the transporting film 9 constituting the strip 1 from the outside, And is provided on the housing cover (5) so as to be positioned directly above the vertebrae and the vertebrae.

Hereinafter, the structure of the strip constituting the diagnostic apparatus of the present invention will be described with reference to FIG.

The strip 1 according to the present invention is basically composed of a transfer section 9, a storage pad 10, a filter pad (also serving as a die pad) 11, an absorption pad 12, 13, and a verification unit 14. A base member (not shown) for fixing the strip to the fixing portion 6 of the housing main body 7 is attached to the bottom surface of the strip 1.

The filter pad 11 is brought into contact with the lower end of the transfer membrane 9 for chromatography to form a connection path and the storage pad 10 is brought into contact with the lower end of the filter pad, . The absorbing pad 12 is attached to the upper end of the front opening. A reaction part 13 to which a fixing member for specifically binding with an analyte to be detected is fixed and a verification part 14 for determining whether the kit is normally operated are separately installed in a predetermined region of the strip.

The storage pad 10 is required to have sufficient pore and volume to accommodate the developing reagent (or sample solution). Materials which can be used as such storage pads are cellulose, polyester, polyurethane, glass fibers having a pore size of 0.45 to 60 mu m, and the like, preferably low molecular weight protein binding materials.

The filter pad 11 filters out unwanted components in the sample and can include a detection reagent (in this case, the filter pad also serves as a die pad). When the detection reagent is contained in the filter pad, there is an advantage that the step of preliminarily mixing the detection reagent into the sample for inspection can be reduced. Examples of the material that can be used as the filter pad 11 include polyurethane, polyacetate, cellulose, glass fiber, nylon having a pore size of 0.45 to 60 mu m, and the like.

The detection reagent is made of a predetermined label so that the antibody to be examined can be identified from the outside. Such labels include, for example, catalysts, enzymes (e.g., phosphatase, peroxidase, etc., more specific examples include basic phosphatase and amphoteric peroxidase used in combination with enzyme substrate), enzymatic substrate For example, nitro blue tetrazolium, 3,5 ', 5,5'-tetranitrobenzidine, 4-methoxy 1 naphthol, 4 chloro 1 naphthol, 5 bromo 4 chloro 3 indolyl phosphate, (Such as fluorescein, phycobiliprotein, rhodamine, and derivatives and analogs thereof), chemiluminescent compounds, fluorescent compounds, and the like, Metal sols, radioactive elements, metal sols including nano gold particles, Dye sol, Particulate latex, Color Indicator, Color matter contained in liposome, Carbon sol And non-metallic sols such as selenium.

The markers form an auxiliary specific binding member and a conjugate having a property of easily binding to an analyte to be examined. Examples of these auxiliary specific binding members include, but are not limited to, an antibody that specifically binds to the vitamin when the analyte is a vitamin corresponding to heptene (for example, only one kind exists for vitamin D) Protein G, protein A, protein G / A, and other antibodies known as substances that bind easily to an antibody when water is an antibody, and other antibodies known to bind well to IgG and the like.

In this case, the detection reagent does not necessarily have to be included in the filter pad 11. The detection reagent may be provided at any point between the reaction part 13 on the strip on which the detection result appears and the sample injection point. This detection reagent can be put in a dry state or in a lyophilized state on top of or inside the filter pad 11 or at any point on the transfer membrane 9. At this time, auxiliary agents such as buffer, detergent, anticoagulant, etc. may be added to increase the sensitivity and reactivity of the test as needed.

The strip 1 may further include a predetermined control reagent to determine whether the kit is normally operated. The control reagent may be provided at any point between the reaction part 13 on the filter pad 11 or the upstream membrane 9 and the sample injection point as in the detection reagent. The control reagent may be selected from a labeled protein, an antigen, an antibody, or the like that specifically binds to a fixative (such as a protein, an antigen, or an antibody) forming a control portion (or a control band) on the strip. Such fixatives and control reagents are well known to those of ordinary skill in the art. The label that can be included in the control reagent can be equally applied to the above detection reagent. Examples of the receptor include, but are not limited to, biotin, which binds easily to a solid body, for example, when the solid body is avidin.

A fixing member (or a capture reagent) is an unlabeled specific binding member that specifically binds to the analyte, a label, an auxiliary specific binding member, and the like and captures the analyte, and is directly or indirectly fixed do.

The preform 9 should have sufficient voids to be able to absorb a substantial portion of the sample that has passed through the filter pad 11. Examples of materials that can be used as the preliminary film include nylon, cellulose, polysulfone, polyvinylidene difluoride, cellulose acetate, polyurethane, glass fiber, nitrocellulose, and the like.

Examples of developing reagents that may be used when developing reagents are phosphate buffers, saline, Tris-HCl, water, and the like. The developing reagent is required when the sample inlet 2 is located directly above the filter pad 11 and it is desired to spot treat the sample. Therefore, it is not particularly required when the sample input port is located on the storage pad and a certain amount of sample is to be dropped into the storage pad.

The complex labeled by the detection reagent combines with the fixing member located in the reaction part 13 on the transport membrane 9 to cause an apparent color change in appearance.

The immobilizing member associated with the control reagent forms a reference site at a different location from the reaction site. Since various reagents and fixing members applied to other diagnostic kits currently available on the market can be used, detailed description thereof will be omitted.

In the present invention, the contents of the labeled auxiliary specific binding member and the fixing member, which are respectively coupled to the plurality of strips, are configured differently. For example, when two identical strips (1, 1 ') are arranged in parallel as shown in FIG. 2, the content of the auxiliary specific binding member and the fixing member labeled on one strip (1) ), The content of the auxiliary specific binding member and the fixing member is set to 2 (unit is omitted) in the other strip 1 '.

In this manner, when the auxiliary specific binding member and the fixing member labeled with different concentrations (to contents) are formed on a plurality of strips, not only the presence but also the concentration of the analyte contained in the sample can be confirmed.

Assuming, for example, that the two strips all have the same red band (where the chromaticity value is defined as A), the concentration of the analyte contained in the sample can be viewed as 1, If the concentration of the analyte contained is 2, one of the two strips will have a chromaticity value of A, and the other will have a chromaticity value of 2A, so the two are distinguished.

As described above, according to the configuration of the present invention, not only the presence or absence of the analyte can be confirmed, but also the concentration of the analyte in the sample can be confirmed, so that when the analyte is related to the diagnosis of the disease, Prescription becomes possible.

In the embodiment of the present invention, two strips are arranged in parallel. However, it is also possible to apply three or more strips with different concentrations of the labeled auxiliary specific binding member and the fixing member.

Hereinafter, the content of the present invention will be described with reference to specific examples for specifying the content of vitamin D in a sample.

In a preferred embodiment of the present invention, the case where A: B = 1: 5 is used to set the detection concentration for each line using two lines (A line and B line) for measuring vitamin D in the sample.

In this case, the measurement result is as shown in Table 1 below, and the judgment result for each case may be deficient, deficient, normal, or excessive [1: red, 0: colorless]

Line A Line B Measurement concentration result 0 0 Deficiency: less than 1 One 0 Tribe: 1 to less than 5 One One Normal to over: 5 or more

If both line A and line B are colored in the above table, the chromaticity value is measured to measure the exact concentration of vitamin D. In this case, the exact concentration of vitamin D can be calculated depending on the value of the color of the line B.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. It can be understood that

Claims (10)

A plurality of strips;
A labeled auxiliary specific binding member coupled to each of the strips at a different concentration; And
A reaction unit which is located on each of the strips and to which a fixing member to which the auxiliary specific binding member is specifically bound is coupled;
Wherein the analyte is capable of analyzing the concentration of the analyte contained in the biological sample. The method according to claim 1,
Wherein the label is a member selected from the group consisting of a catalyst, an enzyme, an enzyme substrate, a fluorescent compound, a chemiluminescent compound, a radioactive element, a metal sol, a nonmetallic sol, a diol, a particulate latex, and a color indicator. The method according to claim 1,
Wherein the analyte is heptene, an antibody, and combinations thereof. The method according to claim 1,
Wherein the auxiliary specific binding member is one selected from the group consisting of antibody, protein G, protein A, and protein G / A. The method according to claim 1,
And a color correction marker is provided on one side of the cover. Preparing a plurality of strips to which labeled secondary specific binding members are bound at different concentrations;
Loading a test sample on the prepared plurality of strips;
Binding an analyte contained in the test sample to the labeled auxiliary specific binding member and moving to a reaction part on the strip;
The labeled specific auxiliary binding member to which the analyte is bound is specifically bound to the immobilization member bound to the reaction unit; And
Analyzing the result of the reaction in the reaction part according to a mark having a different concentration in each of the strips, and calculating the concentration of the analyte;
And analyzing the concentration of the analyte contained in the biological sample using the diagnostic kit. The method according to claim 6,
Wherein the label is one selected from the group consisting of a catalyst, an enzyme, an enzyme substrate, a fluorescent compound, a chemiluminescent compound, a radioactive element, a metal sol, a nonmetallic sol, a diol, a particulate latex, and a color indicator. The method according to claim 6,
Wherein the analyte is heptene, an antibody, and combinations thereof. The method according to claim 6,
Wherein the auxiliary specific binding member is one selected from the group consisting of antibody, protein G, protein A, and protein G / A. The method according to claim 6,
Wherein a color correction marker is provided on one side of the cover to eliminate the influence on illumination using the color correction marker.

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