IMMUNOCHROMATOGRAPHY STRIP FOR PREGNANCY DIAGNOSIS WITH MULTIPLE TEST LINES, AND PREGNANCY DIAGNOSIS KIT COMPRISING SAME

Abstract

The present invention relates to a chromatography strip for diagnosing pregnancy having multiple test lines, a pregnancy diagnosis kit having the chromatography strip, and a method for diagnosing pregnancy using the same.

The present invention overcomes the problem of false negatives due to hCG variants such that pregnancy can be determined more accurately and conveniently by the naked eye. Furthermore, there is an effect of improving the concentration measurement range of hCG or a variant thereof by using a chromatography strip for diagnosing pregnancy which is constituted by multiple test lines and control lines.

Claims

1. A chromatography strip for diagnosing pregnancy, comprising: a conjugate pad; and a detection pad, wherein the conjugate pad comprises a first anti-human chorionic gonadotropin (hCG) monoclonal antibody and a signal detection labeling material, and wherein the detection pad is provided with test line 1 and test line 2 that are isolated, the test line 1 comprises a second anti-hCG monoclonal antibody, and the test line 2 comprises human chorionic gonadotropin (hCG).

2. The chromatography strip of claim 1, wherein the first anti-hCG monoclonal antibody recognizes a β-core fragment site of human chorionic gonadotropin (hCG), and wherein the second anti-hCG monoclonal antibody recognizes a different site of hCG which is not the β-core fragment site of hCG.

3. The chromatography strip of claim 1, wherein the first anti-hCG monoclonal antibody is linked to the signal detection labeling material before or during the development of a sample to form a first conjugate, wherein a complex in which hCG or a variant thereof is bound to the first conjugate is formed when hCG or a variant thereof is present in the sample, and wherein as the concentration of hCG or a variant thereof increases in the sample, the number of the formed complexes increases, and conversely, the number of bare conjugates which are not bound to hCG in the sample decreases.

4. The chromatography strip of claim 3, wherein a complex in which normal hCG is bound to the first conjugate is captured on test line 1 to indicate a signal, and a complex in which a variant is bound to the first conjugate is not captured on test line 1, and wherein a bare conjugate which is not bound to hCG or a variant thereof in the sample reacts with hCG immobilized at a certain amount on test line 2, thereby being captured on test line 2 to indicate a signal.

5. The chromatography strip of claim 3, wherein the variant comprises an hCG β core fragment (hCGβcf).

6. The chromatography strip of claim 1, wherein the signal detection labeling material is colloidal gold, a latex particle, a colored polystyrene microparticle, an enzyme, a fluorescent dye, a conductive polymer, a luminescent material, or a magnetic particle.

7. The chromatography strip of claim 1, wherein the detection pad further comprises a control line for confirming the development of a sample.

8. A method for diagnosing pregnancy using the chromatography strip for diagnosing pregnancy according to claim 1, comprising: injecting a sample into the conjugate pad or a pad positioned before to develop; and confirming the presence or absence of a signal of the signal detection labeling material from test line 1, test line 2, and a control line.

9. The method of claim 8, wherein the control line and test line 2 appear in the case of non-pregnancy, and in the case of pregnancy, test line 1, test line 2, and the control line all appear at a low concentration of hCG, and test line 1 and the control line appear, or only the control line appears at a high concentration of hCG.

10. A pregnancy diagnosis kit, in which the chromatography strip for diagnosing pregnancy according to claim 1 is additionally fixed in a case, wherein a guide and a strip support are provided in a lower case, and a sample input port is provided in an upper case; and a result confirmation window is provided at a position corresponding to the test line or the control line.

Description

DESCRIPTION OF DRAWINGS

[0039] FIG. 1 is a schematic diagram showing a cross-section of an immunochromatography strip used in a conventional pregnancy diagnostic kit.

[0040] FIG. 2 is a schematic diagram showing an immunochromatography strip for diagnosing pregnancy with multiple test lines used in the pregnancy diagnostic kit of the present invention.

[0041] FIG. 3 is a mimetic diagram of an immunochromatography strip for diagnosing pregnancy according to an exemplary embodiment of the present invention.

[0042] FIG. 4 is a result of analyzing hCG standard solutions at various concentrations using the pregnancy diagnostic kit of the present invention.

[0043] FIG. 5 is a result of analyzing sample solutions in which hCG and hCGβcf are present at various concentrations using the pregnancy diagnostic kit of the present invention.

MODES OF THE INVENTION

[0044] Hereinafter, the present invention will be described in more detail through exemplary embodiments. These exemplary embodiments are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these exemplary embodiments.

Example 1

[0045] Manufacture of Immunochromatography Strips

[0046] 1-1. Manufacture of Detection Pad Formed with Test Line. Heterogeneous Test Line, and Control Line

[0047] Three analysis lines were dispensed on a nitrocellulose membrane. The nitrocellulose film was laminated on a plastic card using a laminator. Afterwards, as a second anti-human chorionic gonadotropin antibody of test line 1, a monoclonal anti-human chorionic gonadotropin antibody (monoclonal anti-hCG 5014, Medix, Finland) was used, and human chorionic gonadotropin (hCG) was used for test line 2, and an anti-mouse immunoglobulin antibody from goat (anti-mouse antibody from goat, Arista, USA) was used as a ligand for the control line. Each of these was dispensed using an automatic dispenser and then dried at 25° C. to 30° C. for 2 days (48 hours).

[0048] 1-2. Manufacture of Conjugate Pad

[0049] A conjugate pad was pre-treated by soaking a pad sufficiently with Tris buffer (10 mM, pH 8.5) containing 0.5% polyvinyl alcohol (PVA) and thoroughly drying in a dryer.

[0050] A first conjugate solution, in which colloidal gold particles with a diameter of about 40 nm were combined with a complex clone anti-human chorionic gonadotropin antibody (monoclonal anti-hCG 5006, Medix, Finland) as a first anti-chorionic gonadotropin antibody, was prepared, and a second conjugate solution, in which colloidal gold particles with a diameter of about 40 nm were combined with mouse immunoglobulin (mouse IgG, Arista. USA), was prepared. The first conjugate solution and the second conjugate solution were dispensed on a pre-treated conjugate pad, and after drying completely, it was prepared by cutting it to an appropriate size.

[0051] 1-3. Manufacture of Sample Pad

[0052] A sample pad was sufficiently soaked in a 0.08 M borate buffer solution containing 1% Triton X-100, 0.5% NaN.sub.3, and 0.1% BSA, and after drying completely in a dryer, it was prepared by cutting it into an appropriate size.

[0053] 1-4. Manufacture of Absorbance Pad

[0054] The absorbance pad was used as it was without any treatment in a state where moisture was completely dried in a dryer, and it was prepared by cutting it into an appropriate size.

[0055] 1-5. Manufacture of Immunochromatography Strip

[0056] The detection pad, the conjugate pad, the sample pad, and the absorbance pad prepared through each of the above processes were assembled as shown in FIG. 4.

[0057] That is, the sample pad was attached so as to overlap with one end of the conjugate pad, one end of the detection pad was attached so as to overlap with the other end of the conjugate pad, and the other end of the detection pad and one end of the absorbance pad were attached to overlap each other. It was cut to about 4±2.0 mm using a cutter to prepare a strip as shown in FIG. 4.

[0058] In FIG. 4, the meanings of each reference numeral are as follows.

[0059] 1: Sample pad; 16±4×4±2 mm

[0060] 2: Conjugate pad of first anti-hCG antibody and gold particles;

[0061] 6±1.0×4±2 mm

[0062] 3: Nitrocellulose detection pad; 25±5×4 f 2 mm

[0063] 4: Plastic solid support

[0064] 5: Absorbance pad; 18 4×4 2 mm

[0065] 6: Test line 1 with immobilized second anti-hCG antibody

[0066] 7: Control line with immobilized anti-mouse immunoglobulin

[0067] 8: Test line 2 with immobilized hCG

[0068] 1-6. Assembly of Case

[0069] After inserting the manufactured immunochromatography strip for diagnosing pregnancy into the fixed position of a strip in a plastic lower case, the upper case with a sample input port and a result confirmation window was inserted, and then assembled by pressing with a press.

Experimental Example 1

[0070] Evaluation of Chromatography Strip for Diagnosing Pregnancy Using hCG Standard Solutions

[0071] Human hCG standard materials were spiked in non-pregnant urine to prepare standard samples of chorionic gonadotropin and subjected to quantitative and qualitative tests. The prepared standard sample solutions were dispensed by 100 μL into the sample input port of the case and then developed for 5 minutes. The sample solutions were prepared to have normal human chorionic gonadotropin concentrations at 0, 12.5, 25, 200, 5,000, 50,000, 500,000, 1,000,000, and 2,000,000 mIU/mL, respectively.

[0072] FIG. 4 is a diagram showing the development result of the immunochromatography strip for diagnosing pregnancy according to the concentration of normal human chorionic gonadotropin (hCG). Furthermore, the intensity of signals appearing on the control line, test line 1, and test line 2 was confirmed by the naked eye, the intensity of the control line was set to ++, and the generation intensity of generation after the reaction was shown in [Table 1] below.

TABLE-US-00001 TABLE 1 Sample number 1 7 3 4 5 6 7 8 9 Normal hCG 0 12.5 25 200 5,000 50,000 500,000 1,000,000 2,000,000 concentration (mIU/mL) Test line 1 − +/− + ++ +++ +++ ++ + +/− intensity Test line 2 +++ +++ +++ +++ + +/− − − − intensity Control line ++ ++ ++ ++ ++ ++ ++ ++ ++ intensity

[0073] As shown in [Table 1] and [FIG. 4], when only normal hCG was present, a positive response was observed clearly at 25 mIU/mL or more, and at 200 mIU/mL, it showed a reaction of high intensity of ++.

Experimental Example 2

[0074] Evaluation of chromatography strip for diagnosing pregnancy using sample solutions with hCG and hCGcf

[0075] Next, in order to confirm the accuracy of the analysis method of the present invention when pregnancy was diagnosed with urine including a β-core fragment, sample solutions were prepared that were spiked with a β-core fragment and a normal hCG standard material in non-pregnant urine, and these were developed for 5 minutes after dispensing by 100 μL. The sample solutions were prepared as shown in [Table 2] below.

[0076] FIG. 5 is a diagram showing the comparison of analysis results depending on the concentrations in samples in which both normal hCG and hCGcf were present. Furthermore, the intensity of signals appearing on the control line, test line 1, and test line 2 was confirmed by the naked eye, the intensity of the control line was set to ++, and the generation intensity of the test line after the reaction was shown in [Table 2] below.

TABLE-US-00002 TABLE 2 Sample 1 2 3 4 5 6 Normal hCG 0 200 0 0 200 200 concentration (mIU/mL) β-core 0 0 30 250 30 125 fragment (pmol/mL) Test line 1 − ++ − − +/− − intensity Test line 2 +++ +++ +/− − +/− − intensity Control line ++ ++ ++ ++ ++ ++ intensity

[0077] As shown in [Table 2] and [FIG. 5], in sample 2 in which only hCG was present, it showed a positive response clearly on test line 1 as in Experimental Example 1, but in samples 5 and 6 in which hCGcf was present, even when hCG was at 200 mIU/mL, almost no color was developed on test line 1. From this, it can be confirmed that in the conventional pregnancy diagnosis kit in which only one test line is present, when hCGcf is present, there may be a possibility of false negatives. Since the pregnancy diagnostic kit of the present invention is additionally provided with test lines, pregnancy may be diagnosed by referring to the result of test line 2, when hCGcf is present. In addition, when not only test line 1 shows color development, but also when test line 2 is not visible or is shown at an intensity of the control line or below, it is determined as positive, and thus, it is possible to diagnose pregnancy more accurately.