Method for reducing HOOK effect in colloidal gold immunochromatography detection and application

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a method for reducing a HOOK effect in colloidal gold immunochromatography detection and application. The upstream of the T line is provided with the B line, the B line is coated with the first antibody which is not marked, the combination pad is coated with the first antibody which is marked by colloidal gold, and the first antibody which is marked by colloidal gold and the first antibody which is not marked can be specifically combined with an analyte antigen in a sample to be detected; in addition, the T line is coated with a second antibody, the second antibody and a first antibody marked by colloidal gold, and the unmarked first antibody is a pair of antibodies capable of performing specific immunoreaction with different epitopes of the same antigen. According to the invention, due to the arrangement of the B line, the competitive strength of a large number of free analyte antigens and gold-labeled antibody-analyte antigen compounds on the binding site of the T line is favorably reduced, so that the HOOK effect is reduced, and the detection accuracy is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical detection, and specifically relates to a method for reducing the HOOK effect in colloidal gold immunochromatographic detection and its application. Background Art

[0002] Colloidal gold immunochromatographic test kits are an important means for screening various diseases. However, when measuring samples containing a relatively high concentration of analyte, only a weak T-line signal or even no T-line signal is observed. This phenomenon of weakening or disappearance of the T-line signal at high concentrations of the test sample is called the "hook effect", that is, the HOOK effect.

[0003] The HOOK effect is a common phenomenon in various immunochromatographic detections. The existence of this effect weakens or disappears the signal of the test line T-line, resulting in a low test result value or even a false negative situation, thus leading to misdiagnosis by doctors and causing serious consequences.

[0004] At present, in order to reduce the adverse effects brought by the HOOK effect during immunochromatographic detection, domestic and foreign researchers have made a lot of efforts.

[0005] For example, in Patent CN110596380A, a detection H-line coated with the target to be detected is set between the detection T-line and the quality control C-line of the immunochromatographic test card. According to the signal situation of the H-line during detection, it is judged whether the concentration of the detected substance is too high, and then the sample is diluted according to the actual situation to avoid misdiagnosis caused by the HOOK effect. However, this technology can only identify whether the HOOK effect occurs and cannot weaken or eliminate the HOOK effect; Patent CN108204959A is based on the washing-free and reaction homogeneity of the photochemiluminescence platform (luminescent oxygen channel). By reading the signal value multiple times during the reaction process and observing the change of the signal, the true concentration of the sample is judged to exclude the influence of the HOOK effect. In addition, in Patent CN107389955A, the HOOK effect is identified through the built-in standard curve of the detection device, thereby expanding the detection range.

[0006] Although the technical solutions provided by the above patents can all achieve the identification of the HOOK effect, they still cannot directly detect samples with relatively high concentrations and need to dilute the samples before detection.

[0007] Of course, at the current stage, there are also studies on reducing the impact of the HOOK effect by improving the structure of the test kit. For example, in patent CN100476437A, a chromatography region with multiple microporous particles is provided on the detection device. By means of the microporous particles, the principle that larger-sized analyte / probe complexes reach the detection region before the uncomplexed analyte is utilized to inhibit the uncomplexed analyte from competing with the complex for the binding sites in the detection region, thereby avoiding the occurrence of the HOOK effect. However, this technique may have the problem that "for different analytes, the size of the particle micropores needs to be precisely controlled, which undoubtedly increases the difficulty of the production process of the test kit, and due to the limited filtration capacity of the chromatography region, high-concentration analytes will still exhibit the HOOK effect" during detection.

[0008] Therefore, for colloidal gold immunochromatographic test kits, it is of great significance to provide a low-cost and easy-to-operate method for reducing the HOOK effect in colloidal gold immunochromatographic detection. Summary of the Invention

[0009] To solve the above technical problems, the present invention provides a method and application for reducing the HOOK effect in colloidal gold immunochromatographic detection.

[0010] In the first aspect of the present invention, a method for reducing the HOOK effect in colloidal gold immunochromatographic detection is provided. Specifically, a colloidal gold-labeled first antibody is loaded on the conjugate pad of the colloidal gold immunochromatographic test strip. A B line is set "upstream" of the test line T line, and the B line is loaded with a first antibody without any label. Both the colloidal gold-labeled first antibody and the unlabeled first antibody can specifically bind to the analyte antigen in the sample to be detected to form a first antibody-analyte antigen complex. A second antibody is coated on the test line T line. The second antibody and the first antibody are a pair of antibodies that can specifically immunoreact with different epitopes of the same antigen. Here, the first antibody includes the colloidal gold-labeled first antibody and the unlabeled first antibody. Therefore, the second antibody can specifically bind to the colloidal gold-labeled first antibody-analyte antigen complex migrating "upstream" in front of the test line T line to form a complex system.

[0011] In the present invention, to more clearly describe the positional relationship of the test strip, the direction of chromatographic migration of the sample to be detected is defined as from "upstream" to "downstream" herein for better description and understanding, that is, the sample to be detected undergoes chromatographic migration from "upstream" to the test line T line and finally chromatographic migration to the quality control line C line set "downstream" of the test line T line.

[0012] In the above method provided by the present invention, preferably, the number of B lines is set to 1 to 3, and the width of the B line is 1 to 1.5 times the width of the test line T line.

[0013] More preferably, the B line is set to 1.

[0014] Preferably, the concentration of the unlabeled first antibody loaded on the B line is 1.0 - 2.2 mg / mL.

[0015] The second aspect of the present invention is to provide the application of the above method for reducing the HOOK effect in colloidal gold immunochromatographic detection in a fecal occult blood colloidal gold immunochromatographic detection kit.

[0016] Furthermore, the specific application is that a colloidal gold-labeled first antibody is loaded on the conjugate pad of the fecal occult blood colloidal gold immunochromatographic test strip, and an unlabeled first antibody is introduced "upstream" of the test line T line. Both the colloidal gold-labeled first antibody and the unlabeled first antibody can specifically bind to the analyte antigen in the sample to be detected to form a first antibody-analyte antigen complex; a second antibody is coated on the test line T line. The second antibody and the first antibody are a pair of antibodies that can specifically immunoreact with different epitopes of the same antigen. Here, the first antibody includes the colloidal gold-labeled first antibody and the unlabeled first antibody. Therefore, the second antibody can specifically bind to the colloidal gold-labeled first antibody-analyte antigen complex coming from "upstream" of the test line T line to form a complex system. Among them, when the analyte in the sample to be detected is hemoglobin (Hb), the first antibody is hemoglobin antibody 1 and the second antibody is hemoglobin antibody 2; when the analyte in the sample to be detected is transferrin (TF), the first antibody is transferrin antibody 1 and the second antibody is transferrin antibody 2.

[0017] The method of the present invention is applicable to detecting the hemoglobin content in fecal samples in the range of 50 ug / mL - 2000 ug / mL and transferrin in the range of 20 ug / mL - 400 ug / mL.

[0018] In addition, the third aspect of the present invention is to provide a fecal occult blood colloidal gold immunochromatographic kit, which is specifically prepared by the method described above.

[0019] Furthermore, the fecal occult blood colloidal gold immunochromatographic kit includes a PVC bottom plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and a blotting paper. The conjugate pad is loaded with a colloidal gold-labeled first antibody. The nitrocellulose membrane is provided with a test line (T line) and a quality control line (C line), and a B line is arranged "upstream" of the test line (T line); the B line is loaded with an unlabeled first antibody, and the test line (T line) is coated with a second antibody. The second antibody and the first antibody are a pair of antibodies that can specifically immunoreact with different epitopes of the same antigen, and there is no or only a weak steric hindrance when forming a double-antibody sandwich complex. The quality control line is coated with a goat anti-mouse IgG antibody. Among them, the first antibody can specifically bind to the analyte antigen in the sample to be detected, and the second antibody can specifically bind to the colloidal gold-labeled first antibody-analyte antigen complex that comes before chromatography "upstream" of the test line (T line), forming a complex system.

[0020] When the analyte in the sample to be detected is hemoglobin, the first antibody is hemoglobin antibody 1, and the second antibody is hemoglobin antibody 2; when the analyte in the sample to be detected is transferrin, the first antibody is transferrin antibody 1, and the second antibody is transferrin antibody 2.

[0021] The beneficial effects of the present invention are as follows: By arranging one or more B lines "upstream" of the test line (T line) of the conventional colloidal gold immunochromatographic test strip card, and loading the first antibody that can specifically bind to the analyte antigen in the sample to be detected on the B line, to a certain extent, the competition intensity of a large amount of free analyte antigen and the colloidal gold-labeled first antibody-analyte antigen complex for the binding site of the T line is reduced, thereby reducing the occurrence of the HOOK effect. The experimental results of the present invention show that the method of the present invention is applied to the fecal occult blood colloidal gold immunochromatographic detection process, significantly improving the detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic structural diagram of the reagent card provided in Embodiment 1 of the present invention; Among them, 1 - sample pad, 2 - conjugate pad, 3 - B line, 4 - test line (T line), 5 - quality control line (C line), 6 - blotting paper, 7 - nitrocellulose membrane, 8 - PVC bottom plate; Figure 2 It is a mechanism diagram of reducing the HOOK effect of colloidal gold immunochromatographic detection provided by the present invention; Figure 3 It is a chromaticity diagram of the colorimetric card provided in Embodiment 1 of the present invention; Figure 4 It is a detection signal result diagram of Hb and TF of the first group of samples in Embodiment 1 of the present invention; Figure 5It is the Hb and TF detection signal result diagram of the second group of samples in Example 1 of the present invention; Figure 6 It is the Hb and TF detection signal result diagram of the sixth group of samples in Example 1 of the present invention. Detailed implementation manners

[0023] In order to enable those skilled in the art to better understand the present invention, the present invention will be further elaborated below in combination with specific implementation manners.

[0024] Example 1 The schematic structural diagram of the reagent card for reducing the HOOK effect in colloidal gold immunochromatographic detection provided by the present invention is as shown in the appendix Figure 1 shown, wherein, 1 - sample pad, 2 - conjugate pad, 3 - B line, 4 - test line T line, 5 - control line C line, 6 - absorbent paper, 7 - nitrocellulose membrane, 8 - PVC bottom plate.

[0025] The structure of this kit is a conventional structure, only a B line is provided "upstream" of the test line T line.

[0026] When using the above-mentioned kit to detect the sample to be tested, its mechanism of action is as shown in the appendix Figure 2 shown.

[0027] Appendix Figure 2 In (a) of the appendix, it is the mechanism of action of setting the B line when the hemoglobin content is low, in (b) it is the mechanism of generating the HOOK effect when the B line is not set when the hemoglobin content is high, and in (c) it is the principle of the kit provided by the present invention to reduce the HOOK effect.

[0028] Here, only the detection of hemoglobin in fecal samples is taken as an example to further elaborate the present invention in detail, but the present invention is not limited thereto, nor can the scope of the present invention be limited thereby. Materials, reagents, etc. used in the examples can be obtained from commercial channels without special instructions.

[0029] When the kit of the present invention is used to detect fecal occult blood, when the fecal sample to be detected contains hemoglobin, as the test solution migrates, the hemoglobin therein first binds to the gold-labeled antibody on the conjugate pad, that is, the first antibody labeled with colloidal gold, to form a colloidal gold-labeled first antibody-hemoglobin complex. (1) When the hemoglobin content is small or appropriate, no HOOK effect will occur, and the unlabeled first antibody loaded on the B line will not affect the test result. This is because when the hemoglobin content is small or appropriate, after the formation of the colloidal gold-labeled first antibody-hemoglobin complex, there is no remaining hemoglobin, and since the first antibody binding site on the hemoglobin antigen in the complex has been occupied by the colloidal gold-labeled first antibody, the unlabeled hemoglobin antibody 1 loaded on the B line set "upstream" of the test line T line will no longer intercept the colloidal gold-labeled first antibody-hemoglobin complex. The colloidal gold-labeled first antibody-hemoglobin complex can then smoothly chromatograph to the test line T line and bind to hemoglobin antibody 2 to form a colloidal gold-labeled first antibody-hemoglobin-antibody 2 complex, thus forming a red band at the position of the test line T line, as shown in Figure 2 Figure (a) in the appendix. In addition, the remaining colloidal gold-labeled first antibody or colloidal gold-labeled first antibody-hemoglobin complex continues to chromatograph to the position of the control line C line "downstream" of the test line and binds to the goat anti-mouse IgG antibody coated thereon to form a red band.

[0030] (2) When the amount of hemoglobin in the sample is relatively large, as the sample gradually migrates from "upstream" to "downstream" on the test strip, the antigen of hemoglobin in the sample binds to the gold-labeled antibody on the conjugate pad to form a colloidal gold-labeled first antibody-hemoglobin complex. At this time, there is still a large amount of free hemoglobin remaining. If there is no B line set "upstream" of the test line T line and coated with unlabeled hemoglobin antibody 1, then the free hemoglobin will reach the test line T line and bind to hemoglobin antibody 2, thereby weakening or even preventing the binding of hemoglobin antibody 2 on the test line T line to the colloidal gold-labeled first antibody-hemoglobin complex, resulting in the HOOK effect, that is, as shown in Figure 2 Figure (b) in the appendix; however, by introducing a B line and coating hemoglobin antibody 1 before the test line, the presence of this hemoglobin antibody 1 will intercept the free hemoglobin to a certain extent, thereby reducing or eliminating the HOOK effect, that is, as shown in Figure 2 Figure (c) in the appendix.

[0031] In this embodiment, a fecal occult blood and transferrin combined detection kit is taken as an example for illustration. The concentration of the first antibody sprayed on the B line is 2.2 mg / mL, and the B line is set 6 mm "upstream" of the test line T line. Other preparation processes are exactly the same as those of the existing kits and will not be elaborated. After preparation, the kit is tested.

[0032] In this embodiment, the hemoglobin and transferrin standards used are analytical pure drugs purchased from SIGMA Corporation.

[0033] The method for preparing the samples is as follows: A series of artificial samples with gradient concentrations are prepared by mixing the hemoglobin and transferrin standards with the sample diluent of the test kit. Among them, the highest gradient concentration of hemoglobin is 5000 ug / mL, and the low concentration is close to the lowest detection limit; the highest gradient concentration of transferrin is 800 ug / mL, and the low concentration is close to the lowest detection limit.

[0034] Detection method: All diluted gradient artificial samples are detected using the test kit. Each sample is detected 3 times. Record the concentration of the detected artificial sample and the colorimetry card chromaticity corresponding to the signal intensity of the detection line T. Analyze the relationship between the concentration of the detection target and the signal intensity to determine the hook effect of the test kit. The test results are shown in Tables 1-2 respectively.

[0035] Table 1 List of experimental results of the correspondence between the color of the colorimetry card and the hemoglobin concentration Sample Number Sample Concentration T-line Signal Ratio Chromaticity <![CDATA[S 1 > 5000 ug / mL 3C8 <![CDATA[S 2 > 4500 ug / mL 3C7 <![CDATA[S 3 > 4400 ug / mL 1C7, 2C6 <![CDATA[S 4 > 4350 ug / mL 3C6 <![CDATA[S 5 > 4300 ug / mL 3C6 <![CDATA[S 6 > 4250 ug / mL 3C6 <![CDATA[S 7 > 4200 ug / mL 1C5, 2C6 <![CDATA[S 8 > 4150 ug / mL 3C5 <![CDATA[S 9 > 4100 ug / mL 3C5 <![CDATA[S 10 > 4050 ug / mL 3C5 <![CDATA[S 11 > 4000 ug / mL 3C5 <![CDATA[S 12 > 3950 ug / mL 3C5 <![CDATA[S 13 > 3900 ug / mL 3C5 <![CDATA[S 14 > 3800 ug / mL 3C5 <![CDATA[S 15 > 3700 ug / mL 3C5 <![CDATA[S 16 > 3600 ug / mL 3C5 <![CDATA[S 17 > 3400 ug / mL 3C6 <![CDATA[S 18 > 3200 ug / mL 3C6 <![CDATA[S 19 > 3000 ug / mL 3C5 <![CDATA[S 20 > 2800 ug / mL 3C5 <![CDATA[S 21 > 2600 ug / mL 3C5 <![CDATA[S 22 > 2400 ug / mL 3C4 <![CDATA[S 23 > 2200 ug / mL 3C3 <![CDATA[S 24 > 2000 ug / mL 3C1 <![CDATA[S 25 > 1800 ug / mL 3C1 <![CDATA[S 26 > 1600 ug / mL 3C1 <![CDATA[S 27 > 1400 ug / mL 3C1 <![CDATA[S 28 > 1200 ug / mL 3C1 <![CDATA[S 29 > 1000 ug / mL 3C1 <![CDATA[S 30 > 800 ug / mL 3C1 <![CDATA[S 31 > 600 ug / mL 3C1 <![CDATA[S 32 > 400 ug / mL 3C1 <![CDATA[S 33 > 200 ug / mL 3C1 <![CDATA[S 34 > 100 ug / mL 3C1 <![CDATA[S 35 > 50 ug / mL 3C1 <![CDATA[S 36 > 10 ug / mL 3C2 <![CDATA[S 37 > 5 ug / mL 3C2 <![CDATA[S 38 > 2 ug / mL 3C3 <![CDATA[S 39 > 1 ug / mL 3C3 <![CDATA[S 40 > 0.5 ug / mL 3C4 <![CDATA[S 41 > 0.2 ug / mL 3C5 <![CDATA[S 42 > 0.1 ug / mL 3C8 <![CDATA[S 43 > 0.05 ug / mL 3B Table 2 List of experimental results of the correspondence between the color of the colorimetry card and the transferrin concentration Sample Number Sample Concentration T-line Signal Ratio Chromaticity <![CDATA[T 1 > 800 ug / mL 3C8 <![CDATA[T 2 > 650 ug / mL 3C8 <![CDATA[T 3 > 600 ug / mL 2C6, 1C7 <![CDATA[T 4 > 550 ug / mL 3C5 <![CDATA[T 5 > 500 ug / mL 3C3 <![CDATA[T 6 > 450 ug / mL 3C2 <![CDATA[T 7 > 400 ug / mL 3C1 <![CDATA[T 8 > 350 ug / mL 3C1 <![CDATA[T 9 > 300 ug / mL 3C1 <![CDATA[T 10 > 200 ug / mL 3C1 <![CDATA[T 11 > 100 ug / mL 3C1 <![CDATA[T 12 > 50 ug / mL 3C1 <![CDATA[T 13 > 40 ug / mL 3C1 <![CDATA[T 14 > 30 ug / mL 3C1 <![CDATA[T 15 > 20 ug / mL 3C1 <![CDATA[T 16 > 10 ug / mL 3C2 <![CDATA[T 17 > 8 ug / mL 3C2 <![CDATA[T 18 > 6 ug / mL 3C3 <![CDATA[T 19 > 4 ug / mL 3C3 <![CDATA[T 20 > 2 ug / mL 3C3 <![CDATA[T 21 > 1 ug / mL 2C3, 1C4 <![CDATA[T 22 > 0.5 ug / mL 3C4 <![CDATA[T 23 > 0.3 ug / mL 3C4 <![CDATA[T 24 > 0.1 ug / mL 3C5 <![CDATA[T 25 > 0.08 ug / mL 3C5 <![CDATA[T 26 > 0.06 ug / mL 3C6 <![CDATA[T 27 > 0.04 ug / mL 2C8, 1B <![CDATA[T 28 > 0.02 ug / mL 3B The detection results in Table 1 show that when the test kit is used to detect artificial samples with gradient concentrations from low to high, for hemoglobin detection, in the concentration range of 0.1 ug / mL - 5000 ug / mL, the T-line signal changes in a stepped parabola from C8 to C1 and then to C8. When the concentration of the hemoglobin detection target is between 50 ug / mL - 2000 ug / mL, the detection signal is the strongest. When it exceeds 2000 ug / mL, the hook effect starts to appear, and the detection signal decreases as the concentration further increases. However, when the concentration is 5000 ug / mL, it can still be detected as positive.

[0036] In addition, as shown in Table 2, when the transferrin concentration changes in the gradient range of 0.04 ug / mL - 800 ug / mL, the T-line signal changes in a stepped parabola from C8 to C1 and then to C8. When the concentration of the detection target is between 20 ug / mL - 400 ug / mL, the detection signal is the strongest. When it exceeds 400 ug / mL, the hook effect starts to appear, and the detection signal decreases as the concentration increases. However, when the concentration is 800 ug / mL, it can still be detected as positive.

[0037] It can be seen that after setting the B-line "upstream" of the T-line, the hook effect of the test kit does not appear until a relatively high concentration, and when the hemoglobin concentration is 5000 ug / mL and the transferrin concentration is 800 ug / mL, it can still be detected as positive.

[0038] Furthermore, Table 3 shows the sample group numbers, sample numbers, sample concentrations, and signal results of some parallel tests in this embodiment, attached Figure 3 is the chromaticity diagram of the colorimetric card provided by the present invention; attached Figures 4 - 6 shows the Hb and TF detection T-line signal chromaticity conditions of the samples in groups 1-2 and group 6 in Table 3.

[0039] Table 3 Sample Concentration Corresponding to Detected T-Line Signal Group Number Sample Number Hb Sample Concentration (ug / mL) T-line Signal Ratio Chromaticity Sample Number TF Sample Concentration (ug / mL) T-line Signal Ratio Chromaticity 1 <![CDATA[H 1 H 30 H 59 > 2500 9C5 <![CDATA[T 1 T 13 T 25 > 500 9C3 2 <![CDATA[H 2 H 31 H 60 > 2400 9C4 <![CDATA[T 2 T 14 T 26 > 400 9C1 3 <![CDATA[H 3 H 32 H 61 > 2300 9C4 <![CDATA[T 3 T 15 T 27 > 300 9C1 4 <![CDATA[H 4 H 33 H 62 > 2200 9C3 <![CDATA[T 4 T 16 T 28 > 200 9C1 5 <![CDATA[H 5 H 34 H 63 > 2100 9C2 <![CDATA[T 5 T 17 T 29 > 100 9C1 6 <![CDATA[H 6 H 35 H 64 > 2000 9C1 <![CDATA[T 6 T 18 T 30 > 50 9C1 7 <![CDATA[H 7 H 36 H 65 > 1900 9C1 <![CDATA[T 7 T 19 T 31 > 20 9C1 8 <![CDATA[H 8 H 37 H 66 > 1800 9C1 <![CDATA[T 8 T 20 T 32 > 10 9C2 9 <![CDATA[H 9 H 38 H 67 > 1700 9C1 <![CDATA[T 9 T 21 T 33 > 1 9C3 10 <![CDATA[H 10 H 39 H 68 > 1600 9C1 <![CDATA[T 10 T 22 T 34 > 0.5 9C4 11 <![CDATA[H 11 H 40 H 69 > 1500 9C1 <![CDATA[T 11 T 23 T 35 > 0.2 9C4 12 <![CDATA[H 12 H 41 H 70 > 1400 9C1 <![CDATA[T 12 T 24 T 36 > 0.1 9C5 13 <![CDATA[H 13 H 42 H 71 > 1300 9C1 14 <![CDATA[H 14 H 43 H 72 > 1200 9C1 15 <![CDATA[H 15 H 44 H 73 > 1100 9C1 16 <![CDATA[H 16 H 45 H 74 > 1000 9C1 17 <![CDATA[H 17 H 46 H 75 > 900 9C1 18 <![CDATA[H 18 H 47 H 76 > 800 9C1 19 <![CDATA[H 19 H 48 H 77 > 700 9C1 20 <![CDATA[H 20 H 49 H 78 > 600 9C1 21 <![CDATA[H 21 H 50 H 79 > 500 9C1 22 <![CDATA[H 22 H 51 H 80 > 200 9C1 23 <![CDATA[H 23 H 52 H 81 > 50 9C1 24 <![CDATA[H 24 H 53 H 82 > 20 9C2 25 <![CDATA[H 25 H 54 H 83 > 10 9C2 26 <![CDATA[H 26 H 55 H 84 > 5 ug / mL 9C2 27 <![CDATA[H 27 H 56 H 85 > 1 ug / mL 9C3 28 <![CDATA[H 28 H 57 H 86 > 0.5 ug / mL 9C4 29 <![CDATA[H 29 H 58 H 87 > 0.2 ug / mL 9C5 The results of Table 3 and attached Figures 4 - 6 It can be seen that the Hb sample concentration shows a HOOK effect only when it is greater than 2 mg / mL, while the TF sample concentration shows a HOOK effect only when it is greater than 400 μg / mL.

[0040] Example 2 Different from Example 1, the concentration of the first antibody sprayed on line B is 1.5 mg / mL, and line B is set 5 mm "upstream" of the detection line T line.

[0041] The sample concentration gradient, preparation method, and detection method in this embodiment are exactly the same as those in Example 1.

[0042] The test results of this embodiment are shown in Tables 4-5 respectively.

[0043] Table 4 Experimental Result List of the Corresponding Relationship between Colorimetric Card Color and Hemoglobin Concentration Sample Number Sample Concentration T-line Signal Ratio Chromaticity <![CDATA[S 1 > 5000 ug / mL 3B <![CDATA[S 2 > 4500 ug / mL 3C8 <![CDATA[S 3 > 4400 ug / mL 1C6, 2C7 <![CDATA[S 4 > 4350 ug / mL 3C6 <![CDATA[S 5 > 4300 ug / mL 3C6 <![CDATA[S 6 > 4250 ug / mL 3C6 <![CDATA[S 7 > 4200 ug / mL 3C6 <![CDATA[S 8 > 4150 ug / mL 3C5 <![CDATA[S 9 > 4100 ug / mL 3C5 <![CDATA[S 10 > 4050 ug / mL 3C5 <![CDATA[S 11 > 4000 ug / mL 3C5 <![CDATA[S 12 > 3950 ug / mL 3C5 <![CDATA[S 13 > 3900 ug / mL 3C5 <![CDATA[S 14 > 3800 ug / mL 3C5 <![CDATA[S 15 > 3700 ug / mL 3C5 <![CDATA[S 16 > 3600 ug / mL 3C5 <![CDATA[S 17 > 3400 ug / mL 3C6 <![CDATA[S 18 > 3200 ug / mL 3C6 <![CDATA[S 19 > 3000 ug / mL 3C5 <![CDATA[S 20 > 2800 ug / mL 3C5 <![CDATA[S 21 > 2600 ug / mL 3C5 <![CDATA[S 22 > 2400 ug / mL 3C4 <![CDATA[S 23 > 2200 ug / mL 3C3 <![CDATA[S 24 > 2000 ug / mL 3C2 <![CDATA[S 25 > 1800 ug / mL 3C2 <![CDATA[S 26 > 1600 ug / mL 3C1 <![CDATA[S 27 > 1400 ug / mL 3C1 <![CDATA[S 28 > 1200 ug / mL 3C1 <![CDATA[S 29 > 1000 ug / mL 3C1 <![CDATA[S 30 > 800 ug / mL 3C1 <![CDATA[S 31 > 600 ug / mL 3C1 <![CDATA[S 32 > 400 ug / mL 3C1 <![CDATA[S 33 > 200 ug / mL 3C1 <![CDATA[S 34 > 100 ug / mL 3C1 <![CDATA[S 35 > 50 ug / mL 3C1 <![CDATA[S 36 > 10 ug / mL 3C2 <![CDATA[S 37 > 5 ug / mL 3C2 <![CDATA[S 38 > 2 ug / mL 3C3 <![CDATA[S 39 > 1 ug / mL 3C3 <![CDATA[S 40 > 0.5 ug / mL 3C4 <![CDATA[S 41 > 0.2 ug / mL 3C5 <![CDATA[S 42 > 0.1 ug / mL 3C8 <![CDATA[S 43 > 0.05 ug / mL 3B Table 5 Experimental Result List of the Corresponding Relationship between Colorimetric Card Color and Transferrin Concentration Sample number Sample concentration T-line signal ratio chromaticity <![CDATA[T 1 > 800 ug / mL 3B <![CDATA[T 2 > 650 ug / mL 3C8 <![CDATA[T 3 > 600 ug / mL 2C6, 1C7 <![CDATA[T 4 > 550 ug / mL 3C5 <![CDATA[T 5 > 500 ug / mL 3C3 <![CDATA[T 6 > 450 ug / mL 3C2 <![CDATA[T 7 > 400 ug / mL 3C2 <![CDATA[T 8 > 350 ug / mL 3C1 <![CDATA[T 9 > 300 ug / mL 3C1 <![CDATA[T 10 > 200 ug / mL 3C1 <![CDATA[T 11 > 100 ug / mL 3C1 <![CDATA[T 12 > 50 ug / mL 3C1 <![CDATA[T 13 > 40 ug / mL 3C1 <![CDATA[T 14 > 30 ug / mL 3C1 <![CDATA[T 15 > 20 ug / mL 3C1 <![CDATA[T 16 > 10 ug / mL 3C2 <![CDATA[T 17 > 8 ug / mL 3C2 <![CDATA[T 18 > 6 ug / mL 3C3 <![CDATA[T 19 > 4 ug / mL 3C3 <![CDATA[T 20 > 2 ug / mL 3C3 <![CDATA[T 21 > 1 ug / mL 3C4 <![CDATA[T 22 > 0.5 ug / mL 3C4 <![CDATA[T 23 > 0.3 ug / mL 3C4 <![CDATA[T 24 > 0.1 ug / mL 3C5 <![CDATA[T 25 > 0.08 ug / mL 3C5 <![CDATA[T 26 > 0.06 ug / mL 3C6 <![CDATA[T 27 > 0.04 ug / mL 1C8, 2C7 <![CDATA[T 28 > 0.02 ug / mL 3B From the detection results in Tables 4-5, for the test kit detection experiment on artificial samples with a gradient concentration from low to high, the hemoglobin detection results show that in the concentration range of 0.1 ug / mL - 5000 ug / mL, the T-line signal changes in a stepped parabola from C8 to C1 and then to C8 and B. When the concentration of the detected substance is between 50 ug / mL - 1600 ug / mL, the detection signal is the strongest. When it exceeds 1600 ug / mL, the HOOK effect starts to appear and the signal decreases as the concentration increases. When the concentration is higher than 4500 ug / mL, no positive result can be detected.

[0044] When the transferrin concentration varies within the gradient range of 0.04 ug / mL - 800 ug / mL, the T-line signal changes in a stepped parabola from C8 to C1, then to C8 and then to B. When the concentration of the analyte is between 20 ug / mL - 350 ug / mL, the detection signal is the strongest. When it exceeds 350 ug / mL, the HOOK effect starts to appear, and the detection signal decreases as the concentration increases. When the concentration exceeds 650 ug / mL, no positive result can be detected.

[0045] From the above results, it can be seen that after setting the B-line "upstream" of the T-line, the influence of the HOOK effect can be appropriately reduced, and it can still be detected as positive when the hemoglobin concentration is 4500 ug / mL, and it can still be detected as positive when the transferrin concentration is 650 ug / mL. As the concentration of the antibody coated on the B-line decreases, the ability to reduce the HOOK effect also weakens.

[0046] Example 3 Different from Example 1, the concentration of the first antibody sprayed on the B-line is 1.0 mg / mL, and the B-line is set at 5 mm "upstream" of the detection line T-line.

[0047] The sample concentration gradient, preparation method, and detection method in this example are exactly the same as those in Example 1.

[0048] The test results of this example are shown in Tables 6 - 7 respectively.

[0049] Table 6 List of experimental results of the correspondence between the color of the colorimetric card and the hemoglobin concentration Sample number Sample concentration T-line signal ratio chromaticity <![CDATA[S 1 > 5000 ug / mL 3B <![CDATA[S 2 > 4500 ug / mL 3B <![CDATA[S 3 > 4400 ug / mL 3B <![CDATA[S 4 > 4350 ug / mL 2C8 1B <![CDATA[S 5 > 4300 ug / mL 2C7 1C8 <![CDATA[S 6 > 4250 ug / mL 3C7 <![CDATA[S 7 > 4200 ug / mL 3C6 <![CDATA[S 8 > 4150 ug / mL 3C6 <![CDATA[S 9 > 4100 ug / mL 3C6 <![CDATA[S 10 > 4050 ug / mL 3C5 <![CDATA[S 11 > 4000 ug / mL 3C5 <![CDATA[S 12 > 3950 ug / mL 3C5 <![CDATA[S 13 > 3900 ug / mL 3C5 <![CDATA[S 14 > 3800 ug / mL 3C5 <![CDATA[S 15 > 3700 ug / mL 3C5 <![CDATA[S 16 > 3600 ug / mL 3C5 <![CDATA[S 17 > 3400 ug / mL 3C6 <![CDATA[S 18 > 3200 ug / mL 3C6 <![CDATA[S 19 > 3000 ug / mL 3C5 <![CDATA[S 20 > 2800 ug / mL 3C5 <![CDATA[S 21 > 2600 ug / mL 3C5 <![CDATA[S 22 > 2400 ug / mL 3C4 <![CDATA[S 23 > 2200 ug / mL 3C3 <![CDATA[S 24 > 2000 ug / mL 3C3 <![CDATA[S 25 > 1800 ug / mL 3C3 <![CDATA[S 26 > 1600 ug / mL 3C2 <![CDATA[S 27 > 1400 ug / mL 3C2 <![CDATA[S 28 > 1200 ug / mL 3C1 <![CDATA[S 29 > 1000 ug / mL 3C1 <![CDATA[S 30 > 800 ug / mL 3C1 <![CDATA[S 31 > 600 ug / mL 3C1 <![CDATA[S 32 > 400 ug / mL 3C1 <![CDATA[S 33 > 200 ug / mL 3C1 <![CDATA[S 34 > 100 ug / mL 3C1 <![CDATA[S 35 > 50 ug / mL 3C1 <![CDATA[S 36 > 10 ug / mL 3C2 <![CDATA[S 37 > 5 ug / mL 3C2 <![CDATA[S 38 > 2 ug / mL 3C3 <![CDATA[S 39 > 1 ug / mL 3C3 <![CDATA[S 40 > 0.5 ug / mL 3C4 <![CDATA[S 41 > 0.2 ug / mL 3C5 <![CDATA[S 42 > 0.1 ug / mL 3C8 <![CDATA[S 43 > 0.05 ug / mL 3B Table 7 List of experimental results of the correspondence between the color of the colorimetric card and the transferrin concentration Sample number Sample concentration T-line signal ratio chromaticity <![CDATA[T 1 > 800 ug / mL 3B <![CDATA[T 2 > 650 ug / mL 3B <![CDATA[T 3 > 600 ug / mL 3C8 <![CDATA[T 4 > 550 ug / mL 2C7 1C6 <![CDATA[T 5 > 500 ug / mL 3C5 <![CDATA[T 6 > 450 ug / mL 3C5 <![CDATA[T 7 > 400 ug / mL 3C3 <![CDATA[T 8 > 350 ug / mL 3C2 <![CDATA[T 9 > 300 ug / mL 3C1 <![CDATA[T 10 > 200 ug / mL 3C1 <![CDATA[T 11 > 100 ug / mL 3C1 <![CDATA[T 12 > 50 ug / mL 3C1 <![CDATA[T 13 > 40 ug / mL 3C1 <![CDATA[T 14 > 30 ug / mL 3C1 <![CDATA[T 15 > 20 ug / mL 3C1 <![CDATA[T 16 > 10 ug / mL 3C2 <![CDATA[T 17 > 8 ug / mL 3C2 <![CDATA[T 18 > 6 ug / mL 3C3 <![CDATA[T 19 > 4 ug / mL 3C3 <![CDATA[T 20 > 2 ug / mL 3C3 <![CDATA[T 21 > 1 ug / mL 3C4 <![CDATA[T 22 > 0.5 ug / mL 3C4 <![CDATA[T 23 > 0.3 ug / mL 3C4 <![CDATA[T 24 > 0.1 ug / mL 3C5 <![CDATA[T 25 > 0.08 ug / mL 3C5 <![CDATA[T 26 > 0.06 ug / mL 3C6 <![CDATA[T 27 > 0.04 ug / mL 1C8, 2C7 <![CDATA[T 28 > 0.02 ug / mL 3B The detection results in Tables 6 - 7 show that when testing the test kit with artificial samples of gradient concentrations from low to high, within the concentration range of 0.1 ug / mL - 5000 ug / mL of hemoglobin, the T-line signal changes in a stepped parabola from C8 to C1, then to C8 and then to B. When the concentration of the analyte is between 50 ug / mL - 1200 ug / mL, the detection signal is the strongest. When it exceeds 1200 ug / mL, the HOOK effect starts to appear, and the detection signal decreases as the sample concentration increases. When the concentration is higher than 4350 ug / mL, no positive result can be detected.

[0050] In the gradient range of transferrin concentration from 0.04 ug / mL to 800 ug / mL, the T-line signal changes in a stepped parabolic shape from C8 to C1, then to C8, and then to B. When the concentration of the analyte is between 20 ug / mL and 300 ug / mL, the detection signal is the strongest. When it exceeds 300 ug / mL, the HOOK effect starts to appear, and the signal decreases as the concentration increases. When the concentration exceeds 600 ug / mL, no positive result can be detected.

[0051] It can be seen that after setting the B-line "upstream" of the T-line, the kit can appropriately reduce the influence of the HOOK effect, and it can still be detected as positive when the hemoglobin concentration is 4350 ug / mL and the transferrin concentration is 600 ug / mL. As the concentration of the antibody coated on the B-line further decreases, the ability to reduce the HOOK effect is further weakened, but it still has a certain effect.

[0052] Comparative Example 1 Taking the fecal occult blood and transferrin combined detection kit as an example, different from Example 1, in this comparative example, the B-line is not set "upstream" of the T-line, and the preparation process is the same as that of the original kit. After preparation, the kit is tested. The sample preparation and detection methods in this comparative example are the same as those in Example 1, and the detection results are shown in Tables 8 - 9.

[0053] Table 8 List of experimental results of the correspondence between the color of the colorimetric card and the hemoglobin concentration Sample Number Sample Concentration T-line Signal Ratio Chromaticity <![CDATA[S 1 > 5000 ug / mL 3B <![CDATA[S 2 > 4500 ug / mL 3B <![CDATA[S 3 > 4400 ug / mL 3B <![CDATA[S 4 > 4350 ug / mL 3B <![CDATA[S 5 > 4300 ug / mL 3B <![CDATA[S 6 > 4250 ug / mL 3B <![CDATA[S 7 > 4200 ug / mL 3B <![CDATA[S 8 > 4150 ug / mL 3B <![CDATA[S 9 > 4100 ug / mL 3B <![CDATA[S 10 > 4050 ug / mL 3B <![CDATA[S 11 > 4000 ug / mL 3C8 <![CDATA[S 12 > 3950 ug / mL 1C7 2C8 <![CDATA[S 13 > 3900 ug / mL 1C7 2C6 <![CDATA[S 14 > 3800 ug / mL 3C5 <![CDATA[S 15 > 3700 ug / mL 3C5 <![CDATA[S 16 > 3600 ug / mL 3C5 <![CDATA[S 17 > 3400 ug / mL 3C6 <![CDATA[S 18 > 3200 ug / mL 3C6 <![CDATA[S 19 > 3000 ug / mL 3C5 <![CDATA[S 20 > 2800 ug / mL 3C5 <![CDATA[S 21 > 2600 ug / mL 3C5 <![CDATA[S 22 > 2400 ug / mL 3C4 <![CDATA[S 23 > 2200 ug / mL 3C4 <![CDATA[S 24 > 2000 ug / mL 3C3 <![CDATA[S 25 > 1800 ug / mL 3C3 <![CDATA[S 26 > 1600 ug / mL 3C3 <![CDATA[S 27 > 1400 ug / mL 3C2 <![CDATA[S 28 > 1200 ug / mL 3C2 <![CDATA[S 29 > 1000 ug / mL 3C2 <![CDATA[S 30 > 800 ug / mL 3C1 <![CDATA[S 31 > 600 ug / mL 3C1 <![CDATA[S 32 > 400 ug / mL 3C1 <![CDATA[S 33 > 200 ug / mL 3C1 <![CDATA[S 34 > 100 ug / mL 3C1 <![CDATA[S 35 > 50 ug / mL 3C1 <![CDATA[S 36 > 10 ug / mL 3C2 <![CDATA[S 37 > 5 ug / mL 3C2 <![CDATA[S 38 > 2 ug / mL 3C3 <![CDATA[S 39 > 1 ug / mL 3C3 <![CDATA[S 40 > 0.5 ug / mL 3C4 <![CDATA[S 41 > 0.2 ug / mL 3C5 <![CDATA[S 42 > 0.1 ug / mL 3C8 <![CDATA[S 43 > 0.05 ug / mL 3B Table 9 List of experimental results of the correspondence between the color of the colorimetric card and the transferrin concentration Sample Number Sample Concentration T-line Signal Ratio Chromaticity <![CDATA[T 1 > 800 ug / mL 3B <![CDATA[T 2 > 650 ug / mL 3B <![CDATA[T 3 > 600 ug / mL 3B <![CDATA[T 4 > 550 ug / mL 3B <![CDATA[T 5 > 500 ug / mL 3B <![CDATA[T 6 > 450 ug / mL 3C8 <![CDATA[T 7 > 400 ug / mL 2C7 1C8 <![CDATA[T 8 > 350 ug / mL 3C7 <![CDATA[T 9 > 300 ug / mL 3C5 <![CDATA[T 10 > 200 ug / mL 3C4 <![CDATA[T 11 > 100 ug / mL 3C3 <![CDATA[T 12 > 50 ug / mL 3C3 <![CDATA[T 13 > 40 ug / mL 3C1 <![CDATA[T 14 > 30 ug / mL 3C1 <![CDATA[T 15 > 20 ug / mL 3C1 <![CDATA[T 16 > 10 ug / mL 3C2 <![CDATA[T 17 > 8 ug / mL 3C2 <![CDATA[T 18 > 6 ug / mL 3C3 <![CDATA[T 19 > 4 ug / mL 3C3 <![CDATA[T 20 > 2 ug / mL 3C3 <![CDATA[T 21 > 1 ug / mL 3C4 <![CDATA[T 22 > 0.5 ug / mL 3C4 <![CDATA[T 23 > 0.3 ug / mL 3C4 <![CDATA[T 24 > 0.1 ug / mL 3C5 <![CDATA[T 25 > 0.08 ug / mL 3C5 <![CDATA[T 26 > 0.06 ug / mL 3C6 <![CDATA[T 27 > 0.04 ug / mL 1C8, 2C7 <![CDATA[T 28 > 0.02 ug / mL 3B The detection results in Tables 8 - 9 show that when testing the kit with artificial samples of gradient concentrations from low to high, in the concentration range of hemoglobin from 0.1 ug / mL to 5000 ug / mL, the T-line signal changes in a stepped parabolic shape from C8 to C1, then to C8, and then to B. When the concentration of the analyte is between 20 ug / mL and 800 ug / mL, the detection signal is the strongest. When it exceeds 800 ug / mL, the HOOK effect starts to appear, and the signal decreases as the concentration increases. When the concentration is higher than 4000 ug / mL, no positive result can be detected.

[0054] In the gradient range of transferrin concentration from 0.04 ug / mL to 800 ug / mL, the T-line signal changes in a stepped parabolic shape from C8 to C1, then to C8, and then to B. When the concentration of the analyte is between 20 ug / mL and 40 ug / mL, the detection signal is the strongest. When it exceeds 40 ug / mL, the HOOK effect has already started to appear, and the signal decreases as the concentration increases. When the concentration exceeds 450 ug / mL, no positive result can be detected.

[0055] Obviously, the kit without improvement in Comparative Example 1 is severely affected by the HOOK effect compared with the improved kits in each Example. When the hemoglobin concentration is higher than 4000 ug / mL, a positive result cannot be detected. When the transferrin concentration is higher than 450 ug / mL, a positive result cannot be detected. The upper limit concentration of the detection is relatively low, and the detection accuracy of the kit is poor.

Claims

1. A method for reducing the HOOK effect in colloidal gold immunochromatography detection, characterized in that: A colloidal gold-labeled first antibody is loaded on the binding pad of the colloidal gold immunochromatographic test paper card, and a B line is set "upstream" of the detection line T line, and an unlabeled first antibody is loaded on the B line. The colloidal gold-labeled first antibody and the unlabeled first antibody can both specifically bind to the analyte antigen in the sample to be detected to form a first antibody-analyte antigen complex; The detection line T is coated with a second antibody, the second antibody and the first antibody are a pair of antibodies that can react specifically with different epitopes of the same antigen, and the first antibody includes a colloidal gold-labeled first antibody and an unlabeled first antibody.

2. A method for reducing the HOOK effect in colloidal gold immunochromatography detection as claimed in claim 1, characterized in that: The concentration of the unlabeled first antibody loaded on the B line is 1.0-2.2 mg / mL.

3. A method for reducing the HOOK effect in colloidal gold immunochromatography detection as claimed in claim 1, characterized in that: The B lines are set to 1 to 3, and the width of the B lines is 1 to 1.5 times the width of the detection line T line.

4. Use of the method according to claim 1 in a fecal occult blood colloidal gold immunochromatography detection kit, characterized in that: When the analyte in the sample to be detected is hemoglobin, the first antibody is hemoglobin antibody 1 and the second antibody is hemoglobin antibody 2; when the analyte in the sample to be detected is transferrin, the first antibody is transferrin antibody 1 and the second antibody is transferrin antibody 2; the first antibody includes a colloidal gold-labeled first antibody and an unlabeled first antibody.

5. A fecal occult blood colloidal gold immunochromatography kit, characterized in that: The method as claimed in claim 1 is used for preparation.

6. The fecal occult blood colloidal gold immunochromatography kit according to claim 5, characterized in that: The kit comprises a PVC base plate, a sample pad, a conjugation pad, a nitrocellulose membrane, and absorbent paper; the conjugation pad is loaded with a first antibody labeled with colloidal gold; a detection line T line and a quality control line C line are arranged on the nitrocellulose membrane; a B line is arranged "upstream" of the detection line T line; the B line is loaded with an unlabeled first antibody; the detection line T line is coated with a second antibody; and the quality control line is coated with a goat anti-mouse IgG antibody, wherein the second antibody and the first antibody are a pair of antibodies that can undergo specific immune reactions with different epitopes of the same antigen; When the analyte in the sample to be detected is hemoglobin, the first antibody is hemoglobin antibody 1 and the second antibody is hemoglobin antibody 2; when the analyte in the sample to be detected is transferrin, the first antibody is transferrin antibody 1 and the second antibody is transferrin antibody 2; the first antibody includes a colloidal gold-labeled first antibody and an unlabeled first antibody.

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