CHROMATOGRAPHIC STRIP COMPRISING MULTIPLE TEST LINES, DIAGNOSTIC KIT COMPRISING SAME, AND QUALITATIVE, SEMI-QUANTITATIVE OR QUANTITATIVE ANALYSIS METHOD COMPRISING MULTIPLE COMPETITIVE REACTION MEASUREMENT STEPS

Abstract

The present invention relates to: a chromatographic strip comprising a plurality of test lines for a competitive assay; a diagnostic kit comprising the chromatographic strip; and a method for qualitatively or quantitatively analyzing a target material within a specimen by using the same. By using the chromatographic strip of the present invention, qualitative analysis and highly reliable quantitative analysis can be rapidly and conveniently carried out with the naked eyes or a sensor even for a target material, which has one ligand binding position or is small. Furthermore, the present invention has an effect of improving the concentration measurement range of an analyte by the mutual supplementation of test lines and control lines.

Claims

1. A chromatographic strip comprising a conjugate pad and a detection pad, wherein: the conjugate pad contains a first ligand which has the ability to react with a target material and a labeling material which has the ability to generate a detectable signal and bind to the first ligand, wherein the first ligand and the labeling material are joined together as a first conjugate before or during sample migration, and the first conjugate has the ability to migrate to the detection pad during the sample migration; and the detection pad has two or more test lines which are separated from each other and, by containing an immobilized material the same as or similar to the target material, have the ability to react with a bare conjugate which is not bound to the target material present in the sample.

2. The chromatographic strip of claim 1, wherein: the conjugate pad contains a predetermined amount of the first ligand, a part of which reacts with the target material in the sample and forms a complex in which the target material is bound to the first conjugate including the first ligand and the labeling material; and as the concentration of the target material in the sample increases, the number of the formed complexes increases, whereas the number of the bare conjugates decreases.

3. The chromatographic strip of claim 2, wherein: the complex including the first ligand, the labeling material, and the target material is not captured by the test line; the bare conjugate, which does not bind to the target material present in the sample and includes the first ligand and the labeling material, is captured by the test line and produces a signal by reacting with a material, a predetermined amount of which has been immobilized in the test line and which is the same as or similar to the target material; and the chromatographic strip allows the determination of the presence and/or quantity of the target material in the sample based on the measurement of a signal generated by the labeling material in the test line.

4. The chromatographic strip of claim 3, wherein in all of the test lines immobilized to the detection pad, the intensity of a signal decreases as the concentration of the target material in the sample increases.

5. The chromatographic strip of claim 4, wherein in the test line, the intensity of a signal, which is maintained constant to a point where the concentration of the target material in the sample is M, is gradually decreased at higher concentrations until it finally converges to zero, wherein the M is the maximum concentration of the target material in a sample which results in the saturation of the test line with the bare conjugate, wherein the saturation of the test line refers to a case in which all of the predetermined amount of the material which has been immobilized in the test line and is the same as or similar to the target material has reacted with the bare conjugate.

6. The chromatographic strip of claim 5, wherein, since the number of the test lines used has been increased to N (N≥2) so that a plurality of the test lines are used, the intensity of a signal, which is maintained constant to a point where the concentration of the target material is Mn, is gradually decreased at higher concentrations until it finally converges to zero.

7. The chromatographic strip of claim 1, wherein: the conjugate pad contains a second ligand which does not react with the target material and a labeling material which has the ability to generate a detectable signal and bind to the second ligand, wherein the second ligand and the labeling material are joined together as a second conjugate before or during sample migration, and the second conjugate has the ability to migrate to the detection pad during the sample migration; and the detection pad further has a control line configured to allow the confirmation of sample migration, wherein the control line contains an immobilized material which does not react with the target material or with the first conjugate and has the ability to react with the second conjugate, wherein the second conjugate does not bind to the target material.

8. The chromatographic strip of claim 1, which includes: a sample pad into which a sample to be analyzed for the presence or absence of the target material is introduced; the conjugate pad, one end of which is connected to the sample pad; the detection pad, one end of which is connected to the other end of the conjugate pad; an absorbance pad, one end of which is connected to the other end of the detection pad and which provides a driving force for transporting the sample from the sample pad; and a solid-type support provided at the bottom portion of the chromatographic strip.

9. The chromatographic strip of claim 8, wherein the support is formed of a material selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), glass, and other polymeric materials.

10. The chromatographic strip of claim 1, wherein the ligand is a protein, an antigen, an antibody, DNA, RNA, PNA, or an aptamer.

11. The chromatographic strip of claim 1, wherein the labeling material is colloidal gold, latex particles, colored fine polystyrene particles, an enzyme, a fluorescent dye, a conductive polymer, a luminescent material, or magnetic particles.

12. The chromatographic strip of claim 1, wherein the target material has only one available binding site for the ligand.

13. The chromatographic strip of claim 1, wherein a dynamic range for target material concentration of a sample is from 1 ng/ml to 1 mg/ml.

14. A diagnostic kit comprising the chromatographic strip of claim 1 and an additional case in which the chromatographic strip is fixed, the diagnostic kit including: a lower portion which includes a guide and a strip supporting portion; and an upper portion which includes a sample inlet and, at the position corresponding to the test lines and the control line, a result display window.

15. The diagnostic kit of claim 14, which is provided with an accompanying signal data reference including a color reference table for the test lines and the control line, wherein the color reference table has been obtained for samples of various known target material concentrations.

16. The diagnostic kit of claim 15, which allows a qualitative analysis or a semi-quantitative analysis to be implemented through the visual inspection of the test lines and the control line for the presence/absence and intensity of a signal generated by a labeling material.

17. The diagnostic kit of claim 15, which allows a quantitative analysis to be implemented through the determination of the presence/absence and intensity of a signal generated by a labeling material in the test lines and the control line using a medical device including a reader.

18. An analysis method of qualitatively or quantitatively determining a target material in a sample using the chromatographic strip of claim 1, the method comprising: a first step of introducing the sample into the conjugate pad or a pad preceding the conjugate pad and allowing the sample to migrate; a second step of determining the presence/absence and intensity of a signal generated by a labeling material in the test lines and the control line; and a third step of determining the amount of the target material by comparing the intensity of signals detected from the test lines and the control line with respect to a signal data reference, wherein the signal data reference has been obtained by carrying out the first step and the second step with respect to samples of various known target material concentrations.

19. The analysis method of claim 18, wherein in the second step, the determination of the presence/absence and intensity of a signal generated by a labeling material in the test lines and the control line is carried out using a densitometer.

Description

DESCRIPTION OF DRAWINGS

[0072] FIG. 1 is a cross-sectional overview of a conventional assay strip used for chromatographic analysis.

[0073] FIG. 2 is a cross-sectional overview of an assay strip used for the chromatographic analysis of the present invention.

[0074] FIG. 3 is a schematic diagram of a chromatographic strip according to an overview of the present invention.

[0075] FIG. 4 is an overview illustrating how the multiple test lines and the control line of a chromatographic strip of the present invention appear before and after the introduction of a sample.

[0076] FIG. 5 is a schematic diagram illustrating a method using a dip strip according to one exemplary embodiment of the present invention.

[0077] FIG. 6 is an image comparing the results of analyzing samples of various analyte concentrations according to one exemplary embodiment of the present invention. Here, rabbit IgG was used as the analyte.

[0078] FIG. 7 is a quantitative graph derived by multiple test lines according to an overview of the present invention.

[0079] FIG. 8 is a quantitative graph based on the results of an experiment of the present invention, in which the signal intensity of each one of multiple test lines is plotted as a function of the analyte concentration and the dynamic range is indicated.

BEST MODE

[0080] Hereinafter, the present invention will be described in more detail with reference to exemplary embodiments. These embodiments are provided to explain the present invention more specifically, and the scope of the invention is not limited to the embodiments. The embodiments utilized a dip-strip method, which is one of the various chromatographic analysis methods.

EXAMPLE 1

Preparation of Chromatographic Dip Strip

[0081] A. Preparation of Detection Pad Having Test Lines

[0082] Three test lines were formed on a nitrocellulose membrane. The nitrocellulose membrane was laminated onto a polymeric card using a laminator. Subsequently, test lines 1, 2, and 3 spaced apart from one another at regular intervals were formed by applying, as the analyte or an analyte analog, rabbit IgG (Arista Biologicals Inc., USA) to corresponding positions using an automatic dispenser, and dried at a temperature of 25° C. to 30° C. for two days (i.e., 48 hours).

[0083] B. Preparation of Absorbance Pad

[0084] The absorbance pad, the moisture of which had been completely removed in a dryer, was used as it was without any further treatment, and was cut to an appropriate size prior to its use.

[0085] C. Fabrication of Chromatographic Strip

[0086] The detection pad and the absorbance pad prepared through each of the above-described processes were assembled according to the configuration illustrated in FIG. 5.

[0087] That is, the detection pad was attached to an adhesive applied to a polymeric support, and the absorbance pad was attached such that one end of the detection pad was overlapped with one end of the absorbance pad. About 2±1.0 mm of the resultant was cut out using a cutter, and thereby a strip as shown in FIG. 3 was obtained.

[0088] In FIG. 5, each reference numeral denotes the following:

[0089] 1: Absorbance pad; 18±4 mm×4±2 mm

[0090] 2: Nitrocellulose detection pad; 25±5 mm×4±2 mm

[0091] 3: Test lines 1, 2, and 3 containing immobilized rabbit IgG

[0092] 4: Polymeric support

EXAMPLE 2

Analysis Using Chromatographic Dip Strip

[0093] One hundred microliters of a sample solution containing rabbit IgG as the analyte and PBS was dispensed into each well of a 96-well plate. The sample solutions were prepared so as to contain the rabbit IgG at concentrations of 0, 5, 10, 50, 100, 500, 1000, and 5000 μg/ml. Subsequently, a first predetermined amount of a solution containing a conjugate consisting of a labeling material (fluorescent material) and a first ligand (anti-rabbit IgG) was added to the sample solutions.

[0094] Then, the chromatographic dip strips fabricated in Example 1 were dipped into the sample solutions contained in the 96-well plate to allow sample migration for 10 minutes.

[0095] FIG. 6 is an image showing the result of migration in chromatographic strips at various rabbit IgG concentrations.

[0096] It was confirmed that the signal intensity of the test lines 1, 2, and 3 varies with the analyte concentration and that a different quantitative curve for the analyte concentration can be obtained for each test line.

[0097] In addition, it was confirmed that the chromatographic strip exhibited a wide dynamic range for the analyte concentration, from 5 μg/ml or less to 5,000 μg/ml or more.