KR20190058357A - A wide range of in-vitro diagnostic kit capable of quantitating target substance in high concentration specimen - Google Patents

Abstract

The present invention relates to a wide range in-vitro diagnosis kit capable of quantifying a target substance in a high concentration sample. The present invention includes: a diagnosis strip including at least two test lines; and a diagnosis kit body accommodating the diagnosis strip in an internal space. The diagnosis strip includes: a support; a sample pad which is attached to the upper part of the support, and to which a sample including a target material is injected; a conjugation pad which is attached to the upper part of the support in a shape of being sequentially connected to the sample pad, and includes a first antibody having a target material adhered thereto by being specifically bound to the target material; and a detection film which is attached to the upper part of the support in a form of being sequentially connected to the conjugation pad, and includes a first detection line having a second antibody, that is specifically combined with the target material specifically combined with the first antibody, fixated thereto, and a second detection line having a third antibody, that is specifically combined with the target material specifically combined with the first antibody, fixated at a position separated from the first detection line.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a wide-range in vitro diagnostic kit capable of quantifying a target substance in a high-

The present invention relates to an extracorporeal diagnostic kit capable of quantifying a target substance in a high concentration sample and applicable to a wide range of diagnostic objects.

Recently, a variety of simple test reagents, diagnostic agents, and diagnostic devices for diagnosing the presence or absence of harmful substances such as pesticide residues in specific raw materials such as the presence or absence of a pathogen infection by a virus or bacteria, the presence of pregnancy, Is being developed.

In particular, in vitro diagnostic technology is a technique to rapidly diagnose diseases from outside the body using substances derived from human body such as blood, manure, body fluids and saliva, plays an important role in clinical decision making and becomes an essential and specialized element in patient treatment have.

An in vitro diagnostic device using the same is used as a medical device used for a test using a specimen collected from a human body, such as blood, urine, etc. for the purpose of diagnosing a disease, determining a prognosis, evaluating a health condition, Refers to a device containing a reagent, and diagnosis is made outside the human body, so that it is less burdensome than a medical device requiring insertion into the human body.

In vitro diagnostic kits using Lateral Flow Immunochromatographic Assay (LFIA) were designed to test immobilized target substances in various samples in a short time using an antigen-antibody reaction (immune reaction). One-step method to analyze qualitatively and quantitatively.

In recent years, in vitro diagnostic kits using a lateral immunochromatographic assay have been continuously developed and developed in a form specific to the particular type for rapid diagnosis of various diseases. Prior art documents related to the related arts include Korean Patent Laid- -2017-0127833 (hereinafter referred to as " Prior Art 1 ").

However, the in vitro diagnostic kit using the conventional side immunochromatographic assay including the conventional technique 1 focuses on improvement of characteristics such as the diagnostic speed and accuracy specific to a specific diagnosis object, and further, by using the in vitro diagnostic kit, the quantification level of the target substance Which had many problems.

Specifically, in quantifying a target substance in a specimen using an in vitro diagnostic kit, quantification can not be accurately performed when the specimen is prepared in a high concentration state, thereby reducing the reliability of the result.

In addition, in vitro diagnostic kits using a lateral immunochromatographic assay are undergoing various efforts to achieve functional enhancement or addition through various structural modifications. In this regard, in the related art, a plurality of test lines in an in- A prior art for the technology is disclosed in Korean Patent Laid-Open Publication No. 10-2017-0096619 entitled " Method and kit for simultaneously detecting three-dimensional protein nanoparticle probe-based multiple diseases " (hereinafter referred to as "prior art 2").

However, the in vitro diagnostic kit characterizing the conventional structural modification including the conventional art 2 merely functions as a means for simultaneously diagnosing a plurality of diseases. In the conventional technique 1, quantification of a target substance in a high concentration is performed There is a problem that the inhibition of the resultant reliability that occurs is not solved.

The object of the present invention is to solve the above problems, and it is an object of the present invention to provide an in vitro diagnostic kit in which the concentration of a sample in a wide range of concentrations ranging from a low concentration to a high concentration is applied to an in vitro diagnostic kit, And to provide a technique capable of accurately performing quantification.

It is a further object of the present invention to provide a method and apparatus for measuring the amount of a target substance in a sample using a diagnostic result, The present invention provides a technique capable of preparing a wide variety of diagnostic targets applicable to an in vitro diagnostic kit that can be performed.

In order to attain the above object, the present invention provides a broad-spectrum in vitro diagnostic kit capable of quantifying a target substance in a high-concentration sample, comprising: a diagnostic strip including at least two test lines; And a diagnostic kit body for receiving the diagnostic strip in an inner space, the diagnostic strip comprising: a support; A sample pad attached to an upper portion of the supporter and into which a sample containing a target material is injected; A conjugation pad attached to an upper portion of the supporter in the form of being sequentially connected to the sample pad, the first antibody being specifically bound to the target material and having a labeling substance bound thereto; And a first detection line attached to the upper portion of the support in the form of being sequentially connected to the conjugation pad, wherein the second antibody specifically bound to the target substance specifically bound to the first antibody is immobilized, And a detection membrane including a second detection line in which the third antibody specifically binding to the target substance specifically bound to the first antibody is immobilized at a position spaced apart from the first detection line.

Here, the detection membrane may further comprise a control line in which a fourth antibody to which the first antibody specifically binds is immobilized at a position spaced apart from the second detection line, and the detection membrane is provided on the conjugation pad side The first detection line, the second detection line, and the control line are sequentially formed as a reference.

In addition, the second antibody immobilized on the first detection line and the third antibody immobilized on the second detection line may be provided with the same antibody.

The second antibody immobilized on the first detection line and the third antibody immobilized on the second detection line may be provided with different antibodies and specifically bind to the first antibody of the second antibody Specific binding site for the target substance may differ from the specific binding site for the target substance specifically bound to the first antibody of the third antibody.

Here, the specific binding affinity of the target antibody to the target substance specifically bound to the first antibody of the second antibody is determined by the binding affinity of the target substance specifically bound to the first antibody of the third antibody Specific binding affinity for the < / RTI >

In addition, the saturation of the specific binding between the second antibody immobilized on the first detection line and the target substance specifically bound to the first antibody is detected by the third antibody immobilized on the second detection line, 1 < / RTI > antibody specifically bound to the target substance.

The diagnostic strip may further include an absorption pad attached to an upper portion of the support in a form of being sequentially connected to the detection membrane, and the remaining analyte absorbed by the detection membrane is absorbed.

The present invention has the following effects.

First, even if two or more detection lines that specifically bind to the same target substance in the sample are provided on the diagnostic strip in the extensive in vitro diagnostic kit, the quantification of the target substance in the sample is performed with high reliability even if the in vitro diagnosis is performed using the high concentration sample .

Secondly, two or more detection lines specifically binding to the same target substance in a sample can be provided on a diagnostic strip in a broad-spectrum in vitro diagnostic kit, so that the concentration range of a sample that can be accurately quantified can be widened widely from a low concentration to a high concentration.

In addition, a wide range of in vitro diagnostic kits can be used to diagnose inflammation based on C-reactive protein (CRP), canine C-reactive protein (C-CRP) Feline serum amyloid A) -based diagnosis of cat inflammation and the like.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a cross-sectional perspective view showing the structure of a broad-spectrum in vitro diagnostic kit capable of quantifying a target substance in a high-concentration sample of the present invention. FIG.
FIG. 2 is a reference diagram for explaining an antibody binding structure of a conventional in vitro diagnostic kit and a diagnostic progression type according to injection of a specimen.
FIG. 3 is a graph showing the intensity of a labeling substance according to the concentration of a target substance in a sample when performing a pregnancy diagnosis using a conventional in vitro diagnostic kit. FIG.
FIG. 4 is a graph showing a result of a standard calibration curve using a result graph of FIG. 3 when conducting a pregnancy diagnosis using a conventional IVC kit.
FIG. 5 is a reference diagram for explaining an antibody binding structure of a wide-range in vitro diagnostic kit capable of quantifying a target substance in a high-concentration sample of the present invention and a diagnostic progression type upon injection of a sample.
FIG. 6 is a graph showing the intensity of a labeled substance according to the concentration of a target substance in a sample during a pregnancy diagnosis using a broad-spectrum in vitro diagnostic kit capable of quantifying the target substance in the high-concentration sample of the present invention.
FIG. 7 is a graph showing a standard calibration curve derived from the result graph of FIG. 6 when performing a pregnancy diagnosis using a broad-spectrum in vitro diagnostic kit capable of quantifying a target substance in a high-concentration sample of the present invention.
FIG. 8 is a graph showing the relationship between the concentration of a labeling substance and the concentration of a labeling substance in a sample according to the concentration of a target substance in a sample, when performing a CRP (C-reactive protein) -based inflammation diagnosis using a wide range of in vitro diagnostic kits capable of quantifying a target substance in a high- Fig.
FIG. 9 is a graph showing the results of a standard calibration curve derived from the result graph of FIG. 8 when performing a CRP (C-reactive protein) -based inflammation diagnosis using a broad in vitro diagnostic kit capable of quantifying a target substance in a high- Graph.
FIG. 10 is a graph showing the expression of a labeling substance according to the concentration of a target substance in a sample when performing a diagnosis of canine C-reactive protein (c-CRP) -based inflammation using a broad in vitro diagnostic kit capable of quantifying the target substance in a high- The graph shows the intensity.
FIG. 11 is a graph showing the results of a calibration curve (calibration) using the result graph of FIG. 10 when performing diagnosis of canine C-reactive protein (c-CRP) based on a wide- curve.
FIG. 12 is a graph showing the expression of a labeling substance according to the concentration of a target substance in a sample when performing a diagnosis of cat inflammation based on F-SAA (Feline serum amyloid A) using a broad in vitro diagnostic kit capable of quantifying the target substance in a high concentration sample of the present invention (Intensity).
FIG. 13 is a graph showing the results of the F-SAA (Feline serum amyloid A) -based cat inflammation diagnosis using a broad-spectrum in vitro diagnostic kit capable of quantifying the target substance in the high-concentration sample of the present invention, ).

The preferred embodiments of the present invention will be described in more detail with reference to the accompanying drawings, in which the technical parts already known will be omitted or compressed for the sake of brevity.

1. Description of the extensive in vitro diagnostic kit

1 and 5, the inventive broad-spectrum in vitro diagnostic kit 100 includes a diagnostic kit body 110; And a diagnostic strip (120).

The diagnostic kit body 110 is a housing configuration for accommodating the diagnostic strip 120 in a separate receiving space provided therein as shown in FIG.

A specimen injection port 111H for providing an open space through which a specimen in the sample pad 121 of the diagnostic strip 120 is injected is formed on one side of the upper portion of the diagnostic kit body 110. [

A detection line observation port 112H for exposing the detection result through the first detection line 125 and the second detection line 126 in the detection membrane 124 to the outside is provided on one side of the upper portion of the diagnostic kit body 110, A control line observation port 113H for exposing the control comparison result through the control line 127 in the detection film 124 to the outside can be formed.

The diagnostic strip 120 is configured to directly perform diagnosis using a lateral flow immunochromatographic assay (LFIA) of the specimen which is accommodated and injected in the diagnostic kit body 110. The diagnostic strip 120 is used to quantify a target substance in a high- Unlike the prior art, includes at least two detection lines (Test Line).

First, the diagnostic strip 120 includes a support 121, a sample pad 122, a conjugation pad 123, a detection membrane 124 and an absorbent pad 128, and a lateral flow assay (LFA) ) Diagnostic strips.

1 and 5, the support 121 includes a sample pad 122, a conjugation pad 123, a detection membrane 124, and an absorption pad 128 which are sequentially attached Lt; / RTI >

Specifically, on the support 121, the conjugation pad 123, the detection membrane 124, and the absorption pad 128 are arranged in order starting from the sample pad 122 as shown in Figs. 1 and 5 And the sample pad 122 and the conjugation pad 123 in close proximity to each other in order, the conjugation pad 123 and the detection membrane 124, the detection membrane 124 and the absorption pad 128 are tangentially connected to each other, The sample injected into the pad 122 is provided with a path through which the capillary phenomenon can finally reach the absorption pad 128 via the conjugation pad 123 and the detection membrane 124.

First, the sample pad 122 is attached to the upper part of the support 121 as shown in FIG. 5 (a), and a sample (serum, plasma, urine, swear, other bio fluid, etc.) And is located at the tip of the path along the capillary phenomenon of the specimen including the target substance (TB).

Next, the conjugation pad 123 is attached in such a manner that it is sequentially connected to the upper part of the support 121 and next to the sample pad 122 as shown in FIG. 5 (a), and the first antibody A1, And a labeling substance (AB) to which the labeling substance (L) is bonded.

Here, the labeling substance (L) conjugated to the first antibody (A1) can induce the fluorescence expression of the intensity of specific light intensity according to the degree of binding saturation. Thus, qualitative labeling of the diagnostic result and quantitative labeling of the diagnostic level are possible - Gold-nano particles. Fluorescent molecules such as fluorescence beads, poly-L-lysine-fluorescein isothiocyanate (FITC), or chromogenic enzymes such as HRP (horseradish peroxidase), and the like.

In addition, in one embodiment, the labeling substance (L) to be bound to the first antibody (A1) may be provided by the same substance as FSD 647 NHS ester (BioActs).

The target substance TB in the sample injected into the sample pad 122 is transferred to the conjugation pad 123 and the first antibody AB conjugated with the labeling substance L inside the conjugation pad 123 And an antigen-antibody reaction to bind specifically to each other.

In this way, the target substance (TB) is labeled in the conjugation pad (123), and the first antibody (AB) to which the labeling substance (L) is conjugated is bound.

5 (a), the detecting film 124 is attached to the upper side of the supporting body 121 in such a manner as to be sequentially connected after the conjugation pad 123, and the first detecting line 125, 2 detection line 126 and a control line 127. In this embodiment,

Here, the detection film 124 is a porous film that can be formed of a polymer material such as NC (nitrocellulose), PVDF (polyvinylidene fluoride), charge modified Nylon or PES (Polyethersulfone) The binding form of the antibody can be applied correspondingly to electrostatic or hydrophobic binding.

5 (b), some of the first antibodies AB to which the labeling substance L in the conjugation pad 123 is conjugated are labeled with the target substance TB 124 and the first antibody AB conjugated with the remaining labeling substance L moves to the detection membrane 124 as it is without binding the target substance TB.

 Thus, the target substance TB moved into the detection membrane 124 is labeled as shown in Fig. 5 (b), and the first antibody AB conjugated with the labeling substance L is detected by the detection membrane 124, The first detection line 125 or the second detection line 126 to which the second antibody or the third antibody is applied.

The target substance TB moved into the detection membrane 124 is not labeled as shown in FIG. 5 (b), and the first antibody AB conjugated only to the label substance L is detected by the detection membrane 124, Through the first detection line 125 and the second detection line 126 applied in the control line 127, to react with the fourth antibody.

In this case, the first detection line 125 may be configured such that the target-derived target substance (TB) specifically bound to the first antibody (AB) to which the labeling substance (L) is conjugated causes an antigen-antibody reaction to specifically bind The second antibody A2 is fixedly installed.

In addition, the second detection line 126 may be configured such that the target-derived target substance (TB) specifically bound to the first antibody (AB) to which the labeling substance (L) is conjugated causes an antigen-antibody reaction to specifically bind And is provided at a position spaced apart from the first detection line 125 in the detection membrane 124. The third antibody A '

In addition, the control line 127 is configured such that the original antibody (AB) in the original state in which the specimen-derived target substance (TB) is not bound and the labeling substance (L) is conjugated causes an antigen- And a fourth antibody (A3) capable of binding to the second detection line (126) is fixed and provided at a position apart from the second detection line (126) in the detection film (124).

As a result, the first detection line 125, the second detection line 126, and the control line 127 in the detection film 124 start the side of the conjugation pad 123 as shown in Figs. 1 and 5 The first detection line 125, the second detection line 126, and the control line 127 are sequentially formed in this order.

The first antibody AB binding the target substance TB among the first antibodies AB to which the labeling substance L moving from the conjugation pad 123 side into the detection membrane 124 is conjugated, The first detection line 125 is subjected to a preferential binding reaction with respect to the line 126 and accordingly the saturation of the antigen-antibody binding in the detection line proceeds first in the first detection line 125.

The second antibody A2 fixed to the first detection line 125 and the third antibody A'2 fixed to the second detection line 126 are immobilized on the target substance TB May be prepared with the same antibody or provided with different antibodies on the assumption that they are the same.

Therefore, when the second antibody A2 fixed to the first detection line 125 and the third antibody A'2 fixed to the second detection line 126 are provided with different antibodies, the second antibody A2, The specific binding site for the target substance (TB) specifically bound to the first antibody (A1) of the third antibody (A'2) is the target substance (TB) specifically bound to the first antibody ). ≪ / RTI >

In other words, when the second antibody (A2) immobilized on the first detection line (125) and the third antibody (A'2) immobilized on the second detection line (126) are provided with different antibodies, the target substance ), But the binding sites are different.

Furthermore, the specific binding affinity for the target substance (TB) specifically bound to the first antibody (A1) of the second antibody (A2) is higher than that of the first antibody Is higher than the specific binding affinity for the target substance (TB) specifically bound to the antibody (A1).

That is, the first detection line 125 in the detection film 124 is located at the tip of the second detection line 126 relative to the movement path from the sample pad 122 of the sample to the absorption pad 128, (A1) of the second antibody (A2) and the third antibody (A'2) and a characteristic of preferentially binding reaction with the target substance (TB) specifically bound to the first antibody Based on the characteristic of the specific binding affinity difference to the bound target substance (TB), specificity between the second antibody (A2) immobilized on the first detection line (125) and the target substance (TB) Saturation of the red binding occurs earlier than the saturation of the specific binding between the third antibody (A'2) immobilized on the second detection line 126 and the target substance (TB).

Finally, the absorbent pad 128 is attached to the top of the support 121 in a sequential manner following the detection membrane 124, as shown in FIG. 5 (a), and the detection membrane 124 is removed, The first antibody (AB) to which the labeling substance (L) is conjugated is absorbed.

In addition, the absorbent pad 128 serves to provide power for various moving materials due to the capillary phenomenon including the specimen.

 The broad-spectrum in vitro diagnostic kit 100 of the present invention having such a constitutional feature can quantitate the target substance (TB) in the sample with high reliability even if the in vitro diagnosis is performed using the high-concentration sample, The concentration range of the specimen can be widened widely from low concentration to high concentration.

2. Explanation of examples and quantitative analysis of extensive in vitro diagnostic kits

The extensive in vitro diagnostic kit 100 according to the present invention can be applied to various diagnostic fields so as to provide the above-described constitutional features and effects. I want to explain.

(1) Conventional in vitro diagnostic kit and quantitative analysis type

First, in the case of the conventional in vitro diagnostic kit 10, the diagnostic strip 12 is provided with a sample pad 12b, a conjugation pad 12c, a detection membrane 12b, The second detection line 12d, the first detection line 12e, the control line 12f, and the absorption pad 12g were connected to each other and connected.

When the specimen is injected into the sample pad 12b of the diagnostic strip 12 in the conventional in vitro diagnostic kit 10 as shown in Fig. 2 (a), the target substance TB in the specimen is transferred to the conjugation pad 12c are bound to each other through an antigen-antibody reaction with the first antibody (AB) to which the labeling substance (L) of the first antibody (12c) is conjugated.

Here, when the concentration of the injected sample is at a low level, the target substance TB bound to the first antibody AB conjugated with the labeling substance L as shown in FIG. 2 (b) The second antibody A2 fixed to the first detection line 12e in the detection membrane 12d causes a color reaction for a positive label and the fourth antibody 12a in the detection membrane 12d reacts with the second antibody A2 fixed to the first detection line 12e in the detection membrane 12d, The antibody (A3) is bound to the first antibody (AB) conjugated with the labeling substance (L) to cause color development for labeling of confirmation.

However, when a sample of high concentration is injected into the sample pad 12b of the diagnostic strip 12 in the conventional in vitro diagnostic kit 10, an excessive level of the target substance TB as shown in FIG. 2 (c) Inhibits the binding reaction between the target substance TB bound to the first antibody AB conjugated with the substance L and the second antibody A2 fixed to the first detection line 12e in the detection membrane 12d And a hook effect (prozone effect) in which the target substance (TB) is present but the color is not developed for the positive label is generated.

Therefore, the person conducting the diagnosis can not confirm the accurate diagnosis result when a high concentration sample is injected into the sample pad 12b of the diagnostic strip 12 in the conventional in vitro diagnostic kit 10, (TB) can not be quantified.

In order to confirm such a problem in the case of pregnancy diagnosis, for example, an anti-HCG monoclonal antibody (Medix, Anti mouse hCG antibody 5006) is attached to the first detection line 12e in the detection membrane 12d, (Arista Biologicals product) was fixed with the fourth antibody (A3) to the control line (12f) in the detection membrane (12d), and the sample was immobilized to the concentration of the target substance (TB) in the sample The concentration of each labeling substance was measured by using a spectrophotometer at a wavelength of 280 nm, and the concentration of each labeling substance was measured using a spectrophotometer after dividing into 0 mIU / mL, 100 mIU / mL (low concentration), 5000 mIU / mL and 10000 mIU / The absorbance of each line was measured and the results as shown in Fig. 3 were obtained (when the OD280 = 1.35, the antibody concentration was 1 mg / ml)

Here, the X-axis in the result graph of FIG. 3 is a value obtained by setting the range of the diagnostic kit reading interval to the number of steps, and the Y-axis represents the intensity of the fluorescent substance (L) emitting fluorescence.

As shown in FIG. 3, in the case of the conventional in vitro diagnostic kit 10, as the concentration of the sample increases from 0 mIU / mL to 5000 mIU / mL, the level of the expression level of the labeling substance (L) , 10000 mIU / mL (high concentration), the expression level of the labeling substance (L) is not increased but rather lowered.

As shown in FIG. 3, in the case of the conventional in vitro diagnostic kit 10, if the concentration of the specimen is set at a high concentration, the accuracy of the diagnosis result is inhibited due to the occurrence of the hook effect (prozone effect) And further, the quantification of the target substance (TB) in the sample using the diagnosis result can not be performed.

Specifically, if a calibration curve is derived using the resultant graph showing the intensity of the labeling substance (L) according to the concentration of the sample shown in FIG. 3, the problem is more clearly shown in FIG. It can be judged.

Here, the X-axis in the result graph of FIG. 4 represents the level of expression of the labeling substance (L) in the first detection line 12e in the detection membrane 12d and the labeling substance (L) in the control line 12f in the detection membrane 12d L) expression level, and the Y axis represents the concentration level (unit: mIU / mL) of the target substance (TB) in the sample.

As shown in FIG. 4, in the case of the conventional in vitro diagnostic kit 10, when the concentration of the specimen is provided at a high concentration, the linearity of the standard quantitative curve is not shown and the proportional relationship is not established.

Therefore, in the case of the conventional in vitro diagnostic kit 10, there is a problem that quantification of the target substance (TB) when the concentration of the sample is provided at a high concentration is difficult or inaccurate.

(2) First embodiment (diagnosis of pregnancy) and quantitative analysis form of extensive in vitro diagnostic kit

The inventive extensive in vitro diagnostic kit 100 according to the present invention, in which the above-described conventional in vitro diagnostic kit 10 can provide a quantifiable effect based on a high concentration sample that can not be solved, is typically applicable to the diagnosis of pregnancy.

Specifically, in the case of the extensive in vitro diagnostic kit 100 according to the first embodiment (pregnancy diagnosis) of the present invention, the first detection line 125 of the detection membrane 124 in the diagnostic strip 120 is provided with an anti-HCG monoclonal antibody (Medix, Anti mouse hCG antibody 5006) is fixed to the second antibody (A2) at a concentration level of 2.0 mg / ml, and the second detection line 126 of the detection membrane 124 in the diagnostic strip 120 The HCG monoclonal antibody (Medix, anti mouse hCG antibody 5008) was immobilized with the third antibody (A'2) at a concentration level of 1.0 mg / ml and the control line 127 of the detection membrane 124 in the diagnostic strip 120 ), Chicken IgY antibody (product of Arista Biologicals) is fixed with 4th antibody (A3) at a concentration level of 1.0 mg / ml.

Next, the specimen was incubated with 0 mIU / mL, 100 mIU / mL (low concentration), 5000 mIU / mL, 10000 mIU / mL depending on the concentration of human chorionic gonadotrophin (hCG) (high concentration). Then, each specimen is injected into the sample pad 122 in the diagnostic strip 120 of the broad-spectrum ex vivo diagnostic kit 100 according to the first embodiment of the present invention (diagnosis of pregnancy) Diagnosis was performed.

Thereafter, the detection line 124 of the detection strip 124 in the diagnostic strip 120 of the broad-spectrum in vitro diagnostic kit 100 according to the first embodiment (pregnancy diagnosis) of the present invention, The intensity of the labeling substance (L) by the second detection line (125) and the second detection line (126) was measured using a spectrophotometer at a wavelength of 280 nm to obtain the results as shown in FIG. 6 (OD280 = 1.35, the antibody concentration was 1 mg / ml)

The X axis in the result graph of FIG. 6 is a value obtained by setting the reading range of the diagnostic kit within the range of the number of steps, and the Y axis represents the intensity of the fluorescent substance (L) The first detection line 125 is located at a position (far from the origin on the X axis), and the second detection line 126 is located at a position where the number of steps is relatively small (near the origin on the X axis)

As shown in FIG. 6, in the case of the extensive in vivo diagnostic kit 100 according to the first embodiment (diagnosis of pregnancy) of the present invention, as the concentration of the sample increases from 0 mIU / mL to 5000 mIU / mL, The level of expression of the labeling substance L in the line 125 gradually increases and reaches 10000 mIU / mL (high concentration), the level of the expression level of the labeling substance L in the first detection line 125 becomes stagnant And the expression level of the labeling substance (L) in the second detection line (126) is higher than that of the second detection line (126), and the level of expression of the labeling substance (L) .

6) of the detection membrane 124 in the diagnostic strip 120 of the broad-spectrum ex vivo diagnostic kit 100 according to the first embodiment (pregnancy diagnosis) of the present invention by the concentration of the sample shown in Fig. 6 The calibration curve was derived using the resultant graph showing the intensity of the labeling substance (L) by the second detection line (125) and the second detection line (126). The result of the standard calibration curve is shown in FIG. It can be judged more clearly.

Here, the X-axis in the result graph of FIG. 7 represents the sum of the level of expression of the summed label substance (L) in the first detection line 125 and the second detection line 126 in the detection membrane 124, And the Y axis represents the concentration level (unit: m IU / mL) of hCG as the target substance (TB) in the sample.

As shown in FIG. 7, in the case of the broad-spectrum in vitro diagnostic kit 100 according to the first embodiment (pregnancy diagnosis) of the present invention, the linearity of the standard quantitative curve is shown and the proportional relationship is established, Accurate quantification is possible.

(3) Second embodiment of extensive in vitro diagnostic kit (human inflammation diagnosis) and quantitative analysis form

The extensive in vitro diagnostic kit 100 according to the present invention, in which the above-described conventional in vitro diagnostic kit 10 can provide a quantifiable effect based on a high concentration sample that can not be solved, is typically applicable to diagnosis of human inflammation.

Specifically, in the case of the extensive in vitro diagnostic kit 100 according to the second embodiment of the present invention (diagnosis of inflammation), the first detection line 125 of the detection membrane 124 in the diagnostic strip 120 is provided with an anti- mouse CRP antibody 13300 antibody was immobilized with the second antibody (A2) at a concentration level of 1.5 mg / ml and the second detection line 126 of the detection membrane 124 in the diagnostic strip 120 was immobilized with an anti mouse CRP antibody 13300 antibody was fixed with a third antibody (A'2) at a concentration level of 1.5 mg / ml and a control line 127 of the detection membrane 124 in the diagnostic strip 120 was coated with a Chicken IgY antibody (Arista Biologicals Product) is fixed with the fourth antibody (A3) at a concentration level of 1.0 mg / ml.

Next, the sample was incubated with 0 mIU / mL, 10 mIU / mL (low concentration), 100 mIU / mL, and 300 mIU / mL depending on the concentration of C-reactive protein (CRP) (High concentration). Then, each sample is injected into the sample pad 122 in the diagnostic strip 120 of the broad-spectrum in vitro diagnostic kit 100 according to the second embodiment of the present invention (diagnosis of inflammation) Respectively.

The detection line 124 in the diagnostic strip 120 of the broad-spectrum in vitro diagnostic kit 100 according to the second embodiment of the present invention (inflammation diagnosis) The intensities of the labeling substances (L) according to the second detection line (125) and the second detection line (126) were measured using a spectrophotometer at 280 nm wavelength to obtain the results as shown in FIG. 8 (OD280 = 1.35, the antibody concentration was 1 mg / ml)

The X axis in the result graph of FIG. 8 is a value obtained by setting the range of the diagnostic kit reading interval to the number of steps, and the Y axis represents the intensity of the fluorescent substance (L) The first detection line 125 is located at a position (far from the origin on the X axis), and the second detection line 126 is located at a position where the number of steps is relatively small (near the origin on the X axis)

As shown in FIG. 8, in the case of the extensive in vitro diagnostic kit 100 according to the second embodiment of the present invention (diagnosis of inflammation), as the concentration of the sample increases from 0 mIU / mL to 100 mIU / mL, When the level of expression of the labeling substance L in the line 125 is gradually increased to reach 300 mIU / mL (high concentration), the expression level of the labeling substance L in the first detection line 125 is also stagnant And the expression level of the labeling substance (L) in the second detection line (126) is higher than that of the second detection line (126), and the level of expression of the labeling substance (L) .

8) of the detection membrane 124 in the diagnostic strip 120 of the broad-spectrum ex vivo diagnostic kit 100 according to the second embodiment of the present invention (inflammation diagnosis) according to the concentration of the sample shown in Fig. 8 The calibration curve was derived using the resultant graph showing the intensity of the labeling substance (L) by the second detection line (125) and the second detection line (126). The result of the standard calibration curve is shown in FIG. It can be judged more clearly.

The X-axis in the result graph of FIG. 9 indicates the degree of expression of the summed levels of the labeled substance (L) in the first detection line 125 and the second detection line 126 in the detection membrane 124, And the Y axis represents the concentration level (unit: m IU / mL) of the target substance (TB) of CRP in the control line (127).

As shown in FIG. 9, in the case of the extensive in vitro diagnostic kit 100 according to the second embodiment of the present invention (diagnosis of inflammation), the linearity of the standard quantitative curve is shown and the proportional relationship is established, Accurate quantification is possible.

(4) Third embodiment of extensive in vivo diagnostic kit (diagnosis of dog inflammation) and quantitative analysis form

The broad-spectrum in vitro diagnostic kit 100 of the present invention, in which the above-described conventional in vitro diagnostic kit 10 can provide a quantifiable effect based on a high concentration sample that can not be solved, is typically applicable to diagnosis of dog inflammation.

Specifically, in the case of the broad-spectrum ex vivo diagnostic kit 100 according to the third embodiment of the present invention (diagnosis of dog inflammation), the first detection line 125 of the detection membrane 124 in the diagnostic strip 120 is provided with anti- mouse mouse canine CRP antibody 1 antibody was immobilized with a second antibody (A2) at a concentration level of 1.0 mg / ml and the second detection line 126 of the detection membrane 124 in the diagnostic strip 120 was immobilized with anti mouse A canine CRP antibody antibody was immobilized with a third antibody (A'2) at a concentration level of 1.0 mg / ml and a control line 127 of the detection membrane 124 in the diagnostic strip 120 contained a Chicken IgY antibody (Arista Biologicals Product) is fixed with the fourth antibody (A3) at a concentration level of 1.0 mg / ml.

Next, the specimens were incubated with 0 mIU / mL, 10 mIU / mL (low concentration), 100 mIU / mL, and 10 μM, depending on the concentration of canine C-reactive protein (TBP) And the sample pad 122 in the diagnostic strip 120 of the wide-scope external diagnostic kit 100 according to the third embodiment of the present invention (diagnosis of dog inflammation) And the diagnosis was made.

The first detection line 124 of the detection membrane 124 in the diagnostic strip 120 of the wide-scope in vitro diagnostic kit 100 according to the third embodiment of the present invention (diagnosis of dog inflammation) The intensity of the labeling substance L by the second detection line 125 and the second detection line 126 was measured using a spectrophotometer at a wavelength of 280 nm to determine the absorbance of each line as shown in FIG. = 1.35, the antibody concentration was 1 mg / ml)

The X-axis in the result graph of FIG. 10 is a value obtained by setting the range of the diagnostic kit reading interval to the number of steps, and the Y-axis represents the intensity of the fluorescent substance (L) The first detection line 125 is located at a position (far from the origin on the X axis), and the second detection line 126 is located at a position where the number of steps is relatively small (near the origin on the X axis)

As shown in FIG. 10, in the case of the extensive in vitro diagnostic kit 100 according to the third embodiment of the present invention (diagnosis of dog inflammation), as the concentration of the sample increases from 0 mIU / mL to 100 mIU / mL, The level of expression of the labeling substance L in the detection line 125 gradually increases to a level of 300 mIU / mL (high concentration), and the level of expression of the labeling substance L in the first detection line 125 also reaches the peak level The level of expression of the labeling substance (L) in the second detection line (126) becomes higher than that of the labeling substance, and the expression level of the labeling substance (L) see.

10, the detection line 124 in the diagnostic strip 120 of the broad-spectrum ex vivo diagnostic kit 100 according to the third embodiment of the present invention (diagnosis of dog inflammation) according to the concentration of the sample shown in Fig. The calibration curve was derived using the resultant graph showing the intensities of the labeling substance L for each of the first detection line 125 and the second detection line 126. The results of the standard calibration curve are shown in FIG. It is possible to judge more clearly.

Here, the X-axis in the result graph of FIG. 11 indicates the degree of expression of the summed labeling substance (L) in the first detection line 125 and the second detection line 126 in the detection membrane 124, And the Y axis represents the concentration level (unit: mIU / mL) of the c-CRP as the target substance (TB) in the sample.

As shown in FIG. 11, in the case of the extensive in vitro diagnostic kit 100 according to the third embodiment of the present invention (diagnosis of dog inflammation), the linearity of the standard quantitative curve is shown and the proportional relationship is established, Accurate quantification is possible.

(5) Fourth Embodiment of Extensive In Vitro Diagnostic Kit (Diagnosis of Cat Inflammation) and Quantitative Analysis Form

The inventive extensive in vitro diagnostic kit 100 of the present invention, in which the above-described conventional in vitro diagnostic kit 10 can provide a high-concentration sample-based quantifiable effect, is typically applicable to diagnosis of inflammation in cats.

Specifically, in the case of the extensive in vitro diagnostic kit 100 according to the fourth embodiment of the present invention (diagnosis of cat inflammation), the first detection line 125 of the detection membrane 124 in the diagnostic strip 120 is provided with anti- mouse mouse feline SAA antibody 19 antibody was immobilized with a second antibody (A2) at a concentration level of 1.5 mg / ml and the second detection line 126 of the detection membrane 124 in the diagnostic strip 120 was immobilized with anti mouse feline SAA antibody 19 antibody was immobilized with a third antibody (A'2) at a concentration level of 1.0 mg / ml and a control line 127 of the detection membrane 124 in the diagnostic strip 120 contained a Chicken IgY antibody (Arista Biologicals product) is fixed with the fourth antibody (A3) at a concentration level of 1.5 mg / ml.

Then, the sample was incubated with 0 mIU / mL, 10 mIU / mL (low concentration), 100 mIU / mL and 200 mIU / mL (high concentration) depending on the concentration of feline serum amyloid A (feline SAA) Diagnosis was performed by injecting each sample into the sample pad 122 in the diagnostic strip 120 of the broad-spectrum in vitro diagnostic kit 100 according to the fourth embodiment of the present invention (diagnosis of cat inflammation) .

Then, the detection line 124 of the detection strip 124 in the diagnostic strip 120 of the broad-spectrum in vitro diagnostic kit 100 according to the fourth embodiment of the present invention (cat inflammation diagnosis) The intensity of the labeling substance L by the second detection line 125 and the second detection line 126 was measured using a spectrophotometer at a wavelength of 280 nm to determine the absorbance of each line as shown in FIG. = 1.35, the antibody concentration was 1 mg / ml)

The X-axis in the result graph of FIG. 12 is a value obtained by setting the range of the diagnostic kit reading interval to the number of steps, and the Y-axis represents the intensity of the fluorescent substance (L) The first detection line 125 is located at a position (far from the origin on the X axis), and the second detection line 126 is located at a position where the number of steps is relatively small (near the origin on the X axis)

As shown in FIG. 12, in the case of the extensive in vivo diagnostic kit 100 according to the fourth embodiment of the present invention (diagnosis of cat inflammation), as the concentration of the sample increases from 0 mIU / mL to 100 mIU / mL, The level of expression of the labeling substance L in the detection line 125 gradually increases and reaches 200 mIU / mL (high concentration), the level of the expression of the labeling substance L in the first detection line 125 also reaches the peak level The level of expression of the labeling substance (L) in the second detection line (126) becomes higher than that of the labeling substance, and the expression level of the labeling substance (L) see.

12, the detection line 124 in the diagnostic strip 120 of the broad-spectrum in vitro diagnostic kit 100 according to the fourth embodiment of the present invention (diagnosis of cat inflammation) according to the concentration of the sample shown in FIG. The calibration curve is derived from the resultant graph showing the intensity of the labeling substance L by the second detection line 125 and the second detection line 126. The results of the standard calibration curve are shown in FIG. It is possible to judge more clearly.

Here, the X-axis in the result graph of FIG. 13 indicates the degree of expression of the summed labeling substance (L) in the first detection line 125 and the second detection line 126 in the detection membrane 124, And the Y axis represents the concentration level (unit: m IU / mL) of the feline SAA as the target substance (TB) in the sample.

As shown in FIG. 13, in the case of the extensive in vitro diagnostic kit 100 according to the fourth embodiment of the present invention (diagnosis of cat inflammation), the linearity of the standard quantitative curve is shown and the proportional relation is established, Accurate quantification is possible.

The embodiments disclosed in the present invention are not intended to limit the scope of the present invention but to limit the scope of the technical idea of the present invention. The scope of protection is to be construed in accordance with the following claims, and all technical ideas within the scope of equivalents thereof should be construed as being included in the scope of the present invention.

100: Extensive in vitro diagnostic kit
110: Diagnostic kit body
111H: specimen injection port 112H: detection line observation port
113H: Control line observation port
120: diagnostic strip
121: support 122: sample pad
123: Conjugation pad 124: Detection membrane
125: first detection line 126: second detection line
127: contrast line 128: absorption pad
TB: target substance in a sample
AB: First antibody conjugated with a labeling substance
L: Label substance
A1: 1st antibody
A2: Second antibody
A'2: Third antibody
A3: fourth antibody

Claims (7)

A diagnostic strip comprising at least two detection lines (Test Line); And
And a diagnostic kit body for accommodating the diagnostic strip in an inner space,
The diagnostic strip,
A support;
A sample pad attached to an upper portion of the supporter and into which a sample containing a target material is injected;
A conjugation pad attached to an upper portion of the supporter in the form of being sequentially connected to the sample pad, the first antibody being specifically bound to the target material and having a labeling substance bound thereto; And
A first detection line attached to an upper portion of the support in the form of being sequentially connected to the conjugation pad, the first detection line to which the second antibody specifically binding to the target substance specifically bound to the first antibody is immobilized, And a second detection line immobilized at a position spaced apart from the first detection line, wherein the third antibody to which the target substance specifically bound to the first antibody specifically binds is specifically bound
Extensive in vitro diagnostic kit capable of quantifying target substances in high concentration samples.
The method according to claim 1,
Wherein the detection membrane further comprises a control line in which a fourth antibody to which the first antibody specifically binds is immobilized at a position spaced apart from the second detection line,
Wherein the detection film is formed such that the first detection line, the second detection line, and the control line are sequentially formed with reference to the conjugation pad side
Extensive in vitro diagnostic kit capable of quantifying target substances in high concentration samples.
3. The method of claim 2,
Wherein the second antibody immobilized on the first detection line and the third antibody immobilized on the second detection line are provided with the same antibody
Extensive in vitro diagnostic kit capable of quantifying target substances in high concentration samples.
3. The method of claim 2,
Wherein the second antibody immobilized on the first detection line and the third antibody immobilized on the second detection line are provided as different antibodies and are immobilized on the target substance specifically bound to the first antibody of the second antibody Wherein the specific binding site is different from the specific binding site for the target substance specifically bound to the first antibody of the third antibody
Extensive in vitro diagnostic kit capable of quantifying target substances in high concentration samples.
5. The method of claim 4,
The specific binding affinity for the target substance specifically bound to the first antibody of the second antibody is determined by the binding affinity of the target antibody specifically bound to the first antibody of the third antibody Specific binding affinity < RTI ID = 0.0 >
Extensive in vitro diagnostic kit capable of quantifying target substances in high concentration samples.
5. The method according to any one of claims 3 to 4,
Wherein saturation of a specific binding between the second antibody immobilized on the first detection line and the target substance specifically bound to the first antibody is fixed to the second detection line and the first antibody Of the target substance, and the saturation of the specific binding between the target substance specifically bound to the target substance
Extensive in vitro diagnostic kit capable of quantifying target substances in high concentration samples.
The method according to claim 1,
The diagnostic strip,
And an absorption pad attached to the upper portion of the support in a form that is sequentially connected to the detection membrane and absorbing the remaining specimen through the detection membrane.
Extensive in vitro diagnostic kit capable of quantifying target substances in high concentration samples.

Images