Method for enhancing colloidal gold color development and kit thereof

Abstract

The invention provides a diluent for enhancing colloidal gold color development, a kit containing the diluent and a method for enhancing colloidal gold color development. Wherein the diluent comprises an enhancing reagent and an antigen stabilizing reagent, and the enhancing reagent is selected from one or more than two of 4-dimethylaminoantipyrine, p-acetamidophenol, gentamicin sulfate and 2-chloroacetamide. According to the method, by optimizing the diluent formula and increasing the copper ion deposition process, colloidal gold enhanced color development is achieved, and the sensitivity of the colloidal gold detection card is improved. Meanwhile, the method provided by the invention is relatively simple in use process, only one step of operation is added, the increased reaction time is also relatively short (1-5 minutes), and the operation complexity is not remarkably increased. Reagents used in the method are common chemicals, and special compounds do not need to be synthesized by themselves, so that the detection cost is not obviously increased.

Description

Technical Field

[0001] This application belongs to the field of detection technology. Specifically, it relates to a method for enhancing colloidal gold color development and its kit. Background Art

[0002] Lateral flow immunoassay (LFIA) is a common technology for rapid detection of analytes such as antigens, antibodies, or small molecules based on an immune platform. A typical application of this technology is the test strip, whose structure is mainly divided into four parts: a sample pad, a conjugate pad, a membrane, and an absorbent paper. Among them, the conjugate pad is fixed with an antigen or antibody labeled with a tracer, and the membrane is coated with an antigen or antibody for capture. During detection, the test sample is dropped on the sample pad, and the analyte in the sample will chromatograph along the direction of the membrane through capillary action, first bind to the antigen or antibody fixed on the conjugate pad, and then be captured by the antigen or antibody coated on the membrane, thereby forming a detectable signal at a fixed position on the membrane. Then, the corresponding signal is interpreted by the naked eye or an instrument, and the content of the analyte in the sample can be judged.

[0003] Common lateral flow immunoassays are mainly divided into three types according to different tracers: colloidal gold immunoassay chromatography, latex immunoassay chromatography, and fluorescence immunoassay chromatography, with colloidal gold immunoassay chromatography being the earliest developed. Colloidal gold immunoassay chromatography generally uses gold particles with a particle size in the range of 1 - 100 nm as tracers, labels antigens or antibodies through electrostatic adsorption, and the signal is a colored band, and the result is interpreted by visual inspection or by detecting the gray value with an instrument; latex immunoassay chromatography generally uses latex microspheres with a particle size in the range of 100 - 500 nm as tracers, labels antigens or antibodies through covalent bonding, and the signal is a colored band, and the result is also interpreted by visual inspection or by detecting the gray value with an instrument; fluorescence immunoassay chromatography generally uses fluorescence microspheres with a particle size in the range of 100 - 500 nm as tracers, labels antigens or antibodies through covalent bonding, and the signal is a fluorescent band with fixed excitation and emission wavelengths, and the result is interpreted by detecting the fluorescence intensity with an instrument.

[0004] Compared with the other two immunoassay chromatography methods, the colloidal gold method has poor sensitivity; at the same time, the colloidal gold particles label antibodies by electrostatic adsorption, and the stability of the binding between gold particles and antibodies is easily affected by factors such as solution pH and ionic strength, resulting in poor detection sensitivity. Summary of the Invention

[0005] Aiming at the problems existing in the prior art, this application provides a method for colloidal gold color development and its related kit.

[0006] Specifically, this application relates to the following aspects:

[0007] 1. A diluent for enhancing colloidal gold color development, comprising an enhancing reagent and an antigen stabilizing reagent, wherein the enhancing reagent is selected from one or more of 4-dimethylaminoantipyrine, p-acetaminophenol, gentamicin sulfate, and 2-chloroacetamide.

[0008] 2. The diluent according to item 1, wherein the content of the enhancing reagent in the diluent is 0.035 wt% - 6.2 wt%.

[0009] 3. The diluent according to item 1 or 2, wherein the antigen stabilizing reagent comprises one or more of a protective protein, a buffer system, a surfactant, a salt, and a preservative.

[0010] 4. The diluent according to item 1 or 2, wherein the protective protein is bovine serum albumin or casein, the buffer system is selected from one or more of Tris-HCl buffer system, MES buffer system, phosphate buffer system, boric acid buffer system, and acetate buffer system, the surfactant is selected from one or more of lauryl alcohol ether, Triton X-100, Tween-20, and sodium dodecyl sulfate, the salt is selected from one or more of sodium chloride, potassium chloride, and sodium deoxycholate, and the preservative is Proclin300.

[0011] 5. The diluent according to item 4, wherein the antigen stabilizing reagent comprises tris(hydroxymethyl)aminomethane, sodium chloride, bovine serum albumin, lauryl alcohol ether, Triton X-100, sodium deoxycholate, and Proclin300.

[0012] 6. A kit for enhancing colloidal gold color development, comprising the diluent according to any one of items 1-5, and a copper ion solution.

[0013] 7. The kit according to item 6, wherein the copper ion solution comprises copper sulfate and PEG, wherein PEG is PEG10000 - PEG50000.

[0014] 8. The kit according to item 6 or 7, wherein the content of divalent copper ions in the copper ion solution is 6.25 mM - 625 mM, and the content of PEG is 1 g / L - 100 g / L.

[0015] 9. The kit according to any one of items 6-8, wherein the kit further comprises a colloidal gold test strip.

[0016] 10. A method for enhancing colloidal gold color development, the method comprising reacting an enhancing reagent with divalent copper ions, wherein the enhancing reagent is selected from one or more of 4-dimethylaminoantipyrine, p-acetaminophenol, gentamicin sulfate, and 2-chloroacetamide.

[0017] 11. The method according to item 10, wherein the method comprises:

[0018] Diluting the sample to be tested with a diluent containing the enhancement reagent,

[0019] Dropping the diluted sample to be tested onto a colloidal gold test strip,

[0020] Contacting the test strip with a copper ion solution containing divalent copper ions to reduce the divalent copper ions to copper.

[0021] 12. The method according to item 11, wherein the diluent is the diluent described in any one of items 1-5.

[0022] 13. The method according to item 11, wherein the copper ion solution comprises copper sulfate and PEG, and PEG is PEG10000 - PEG50000.

[0023] 14. The method according to item 13, wherein the content of divalent copper ions in the copper ion solution is 6.25 mM - 625 mM, and the content of PEG is 1 g / L - 100 g / L.

[0024] 15. Use of an enhancement reagent in enhancing the color development of colloidal gold, wherein the enhancement reagent is selected from one or more of 4-dimethylaminoantipyrine, p-acetaminophenol, gentamicin sulfate, and 2-chloroacetamide.

[0025] 16. The use according to item 15, wherein the enhancement reagent is used to enhance the reduction of metal ions on colloidal gold. Preferably, the metal ion is divalent copper ion.

[0026] The present application provides a method for enhancing the color development of colloidal gold and its kit. The method of the present application realizes the enhanced color development of colloidal gold and improves the sensitivity of the colloidal gold test card by optimizing the diluent formula and adding a copper ion deposition process. At the same time, the operation process of the method of the present application is relatively simple, only adding one step of operation, and the increased reaction time is also short (1-5 minutes), which will not significantly increase the operation complexity. The reagents used in the method of the present application are all common chemicals and do not require self-synthesis of special compounds, so the detection cost will not be significantly increased. Detailed implementation mode

[0027] The following further illustrates the present application with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not used to limit the present application.

[0028] Unless otherwise defined, the technical and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art. Although methods and materials similar or equivalent to those described herein can be used in experiments or practical applications, the materials and methods are described hereinafter. In case of conflict, the present specification, including its definitions, shall prevail. In addition, the materials, methods, and examples are for illustrative purposes only and are not restrictive. The present application will be further described below in conjunction with specific embodiments, but it is not intended to limit the scope of the present application.

[0029] In view of the problem of poor sensitivity in colloidal gold detection in the prior art, the present application provides a diluent for enhancing colloidal gold color development, a kit for enhancing colloidal gold color development, and a method for enhancing colloidal gold color development.

[0030] In a first aspect, the present application provides a diluent for enhancing colloidal gold color development, comprising an enhancing reagent and an antigen stabilizing reagent, wherein the enhancing reagent is selected from one or more of 4-dimethylaminoantipyrine, paracetamol, gentamicin sulfate, and 2-chloroacetamide.

[0031] In a specific embodiment, the enhancing reagent is 4-dimethylaminoantipyrine.

[0032] In a specific embodiment, the enhancing reagent is paracetamol.

[0033] In a specific embodiment, the enhancing reagent is gentamicin sulfate.

[0034] In a specific embodiment, the enhancing reagent is 2-chloroacetamide.

[0035] In a specific embodiment, the enhancing reagent is 4-dimethylaminoantipyrine and paracetamol, and the components can exist in any mass ratio.

[0036] In a specific embodiment, the enhancing reagent is 4-dimethylaminoantipyrine and gentamicin sulfate, and the components can exist in any mass ratio.

[0037] In a specific embodiment, the enhancing reagent is 4-dimethylaminoantipyrine and 2-chloroacetamide, and the components can exist in any mass ratio.

[0038] In a specific embodiment, the enhancing reagent is paracetamol and gentamicin sulfate, and the components can exist in any mass ratio.

[0039] In a specific embodiment, the enhancing reagent is paracetamol and 2-chloroacetamide, and the components can exist in any mass ratio.

[0040] In a specific embodiment, the enhancing reagent is gentamicin sulfate and 2-chloroacetamide, and the components can be present in any mass ratio.

[0041] In a specific embodiment, the enhancing reagent is 4-dimethylaminoantipyrine, paracetamol, and gentamicin sulfate, and the components can be present in any mass ratio.

[0042] In a specific embodiment, the enhancing reagent is 4-dimethylaminoantipyrine, paracetamol, and 2-chloroacetamide, and the components can be present in any mass ratio.

[0043] In a specific embodiment, the enhancing reagent is 4-dimethylaminoantipyrine, gentamicin sulfate, and 2-chloroacetamide, and the components can be present in any mass ratio.

[0044] In a specific embodiment, the enhancing reagent is paracetamol, gentamicin sulfate, and 2-chloroacetamide, and the components can be present in any mass ratio.

[0045] In a specific embodiment, the enhancing reagent is 4-dimethylaminoantipyrine, paracetamol, gentamicin sulfate, and 2-chloroacetamide, and the components can be present in any mass ratio.

[0046] In a specific embodiment, the content of the enhancing reagent in the diluent is 0.035 wt% - 6.2 wt%, for example, it can be 0.035 wt%, 0.05 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.1 wt%, 5.2 wt%, 5.3 wt%, 5.4 wt%, 5.5 wt%, 5.6 wt%, 5.7 wt%, 5.8 wt%, 5.9 wt%, 6 wt%, 6.1 wt%, 6.2 wt%, and any range between these values.

[0047] Wherein, the content of the enhancing reagent in the diluent refers to the total content of all enhancing reagents in the diluent. For example, when the diluent contains one enhancing reagent, the content of the enhancing reagent in the diluent refers to the content of one enhancing reagent. When the diluent contains two enhancing reagents, the content of the enhancing reagent in the diluent refers to the total content of these two enhancing reagents.

[0048] In a specific embodiment, the diluent includes 4-dimethylaminoantipyrine, paracetamol, gentamicin sulfate, 2-chloroacetamide. Among them, in the diluent, the content of 4-dimethylaminoantipyrine is 0.01 wt% - 2 wt%, the content of paracetamol is 0.01 wt% - 2 wt%, the content of gentamicin sulfate is 0.005 wt% - 0.2 wt%, and the content of 2-chloroacetamide is 0.01 wt% - 2 wt%.

[0049] Among them, the antigen stabilizing reagent can be a component in a commonly used antigen diluent for colloidal gold detection known in the art. For example, it can include one or more of a protective protein, a buffer system, a surfactant, a salt, and a preservative. The content of the antigen stabilizing reagent in the diluent can also be a commonly known content in the art.

[0050] Among them, the protective protein can be bovine serum albumin or casein.

[0051] The buffer system can be selected from one or more of Tris-HCl buffer system, MES buffer system, phosphate buffer system, boric acid buffer system, and acetate buffer system.

[0052] The surfactant can be selected from one or more of lauryl alcohol ether, Triton X-100, Tween-20, and sodium dodecyl sulfate.

[0053] The salt can be selected from one or more of sodium chloride, potassium chloride, and sodium deoxycholate.

[0054] The preservative is Proclin300.

[0055] In a specific embodiment, the antigen stabilizing reagent includes one or more of tris(hydroxymethyl)aminomethane, sodium chloride, bovine serum albumin, lauryl alcohol ether, Triton X-100, sodium deoxycholate, and Proclin300.

[0056] In a specific embodiment, the antigen stabilizing reagent includes Tris-HCl buffer system, sodium chloride, bovine serum albumin, lauryl alcohol ether, Triton X-100, sodium deoxycholate, and Proclin300.

[0057] In a specific embodiment, the diluent includes 4-dimethylaminoantipyrine, paracetamol, gentamicin sulfate, 2-chloroacetamide, Tris-HCl buffer system, sodium chloride, bovine serum albumin, lauryl alcohol ether, Triton X-100, sodium deoxycholate, and Proclin300. Among them, in the diluent, the content of 4-dimethylaminoantipyrine is 0.01 wt% - 2 wt%, the content of paracetamol is 0.01 wt% - 2 wt%, the content of gentamicin sulfate is 0.005 wt% - 0.2 wt%, and the content of 2-chloroacetamide is 0.01 wt% - 2 wt%.

[0058] In a second aspect, the present application provides a kit for enhancing colloidal gold color development, including a diluent and a copper ion solution. The diluent can be any of the above diluents for enhancing colloidal gold color development.

[0059] The copper ion solution is a solution containing divalent copper ions. In a specific embodiment, the copper ion solution includes copper sulfate and PEG, where PEG is PEG10000 - PEG50000, that is, any PEG with a molecular weight between 10000 and 50000.

[0060] In a specific embodiment, the copper ion solution comprises copper sulfate and PEG20000.

[0061] In a specific embodiment, the content of divalent copper ions in the copper ion solution is 6.25 mM to 625 mM, for example, it can be 6.25 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 625 mM, and any range between these values; the content of PEG is 1 g / L to 100 g / L, for example, it can be 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, 100 g / L, and any range between these values.

[0062] In a specific embodiment, the content of divalent copper ions in the copper ion solution is 6.25 mM to 50 mM, and the content of PEG is 1 g / L to 10 g / L.

[0063] In a specific embodiment, the content of divalent copper ions in the copper ion solution is 31.25 mM, and the content of PEG is 2 g / L.

[0064] Those skilled in the art can understand that in the kit, the diluent and the copper ion solution are stored in different containers and can be taken separately when needed.

[0065] Furthermore, the kit of the present application may further include a colloidal gold test strip.

[0066] According to actual needs, the kit of the present application may further include an instruction manual, an extraction tube, or a swab, etc.

[0067] In a third aspect, the present application provides a method for enhancing the color development of colloidal gold, the method comprising reacting an enhancing reagent with divalent copper ions, wherein the enhancing reagent is selected from one or more of 4-dimethylaminoantipyrine, p-acetaminophen, gentamicin sulfate, and 2-chloroacetamide.

[0068] In a specific embodiment, the method comprises: diluting the sample to be tested with a diluent containing the enhancing reagent; dropping the diluted sample to be tested onto a colloidal gold test strip; and contacting the test strip with a copper ion solution containing divalent copper ions to reduce the divalent copper ions to copper.

[0069] The diluent is any one of the diluents mentioned above, and the copper ion solution can be any one of the copper ion solutions mentioned above.

[0070] In the method of the present application, in the conventional colloidal gold immunochromatography process, for positive samples, colloidal gold will be fixed on the test line (T line) and the quality control line (C line); for negative samples, colloidal gold will only be fixed on the quality control line (C line). Then the chromatographic strip is immersed in a diluent containing an enhancement reagent, and the copper ions can be reduced to copper in the presence of a specific reagent, and the colloidal gold fixed on the T line and the C line is used as a crystal nucleus to precipitate, showing a brown-red color, so that the T line and C line fixed with colloidal gold are enhanced in color, while the T line without colloidal gold is not enhanced in color.

[0071] The method of the present application optimizes the diluent formula and increases the copper ion deposition process to achieve colloidal gold enhanced color development and improve the sensitivity of the colloidal gold detection card. At the same time, the method of the present application is relatively simple to use, only one step is added, and the added reaction time is also short (1-5 minutes), which will not significantly increase the complexity of the operation. The reagents used in the method of the present application are all common chemicals, and there is no need to synthesize special compounds by themselves, so the detection cost will not be significantly increased.

[0072] In a fourth aspect, the present application provides the use of an enhancing reagent in enhancing the color development of colloidal gold, wherein the enhancing reagent is selected from one or more of 4-dimethylaminoantipyrine, acetaminophenol, gentamicin sulfate, and 2-chloroacetamide.

[0073] The enhancing agent is used to enhance the reduction of metal ions on colloidal gold, and any metal ions can be reduced on colloidal gold and enhance the color development of colloidal gold.

[0074] In a specific embodiment, the metal ion is a divalent copper ion.

[0075] Example

[0076] Example 1

[0077] The diluent was prepared according to the formula in Table 1, with the remainder being water, and the total amount of the diluent was 1000 mL.

[0078] Table 1

[0079] Component Dosage Tris(hydroxymethyl)aminomethane 6.06g Concentrated hydrochloric acid 0.5mL 4-Dimethylaminoantipyrine 0.1g 4-Acetamidophenol 0.1g Sodium chloride 20g Bovine serum albumin 20g 2-Chloroacetamide 0.1g Lauryl alcohol ether 0.5g Triton X-100 3ml Sodium deoxycholate 1g Proclin300 0.3ml Gentamicin sulfate 10mL

[0080] Prepare a copper ion solution, which includes CuSO 4 and PEG20000, with the balance being water, where the content of divalent copper ions is 31.25 mM and the content of PEG is 2 g / L.

[0081] Add the single components in the dilution solution formula in Table 1 above, except for Tris-HCl buffer, bovine serum albumin, sodium chloride, surfactant, and preservative, to the Tris-HCl buffer respectively, and perform a color development enhancement operation on the colloidal gold test strip after chromatography using the copper ion solution.

[0082] Specifically: Take a parainfluenza virus type 3 (PIV-3) test strip, first add 100 μl of positive control solution, and perform normal chromatography for 10 minutes. Use GD-010 to detect the gray value of the T line, then soak it in the above-prepared copper ion solution containing copper ions and PEG20000 for 1 minute, and then soak it in the above Tris-HCl buffer containing different single components for 1.5 minutes respectively. Use GD-010 to detect the gray value of the T line again, and use the blank Tris-HCl buffer as a control. For the single components showing an enhancement effect in the preliminary test, repeat the test for verification.

[0083] The test gray value results are shown in Table 2.

[0084] Table 2

[0085]

[0086]

[0087] As can be seen from the results, compared with the Tris-HCl buffer control, the components with obvious color development enhancement effects are 4-dimethylaminoantipyrine, p-acetaminophen, gentamicin sulfate, and 2-chloroacetamide. The above several substances can all reduce the Cu 2+ in copper sulfate to achieve enhanced color development of colloidal gold.

[0088] Example 2

[0089] The dilution solution used in this example is the dilution solution prepared according to the formula in Table 1 in Example 1.

[0090] (1) Experiment 1

[0091] Assemble the copper ion solution with the dilution solution, test card, extraction tube, instruction manual, swab, tube holder, and biosafety bag to obtain a PIV-3 test kit (colloidal gold method).

[0092] Prepare PIV-3 natural antigen solutions with different concentrations using the diluent in Example 1, with dilution ratios of 1:100, 1:500, 1:1000, and a blank control (without antigen), as samples for enhanced color development.

[0093] Prepare PIV-3 natural antigen solutions with different concentrations using Tris-HCl buffer, with dilution ratios of 1:100, 1:500, 1:1000, and a blank control (without antigen), as non-enhanced control samples. The non-enhanced control refers to only performing conventional chromatography without being immersed in the copper ion solution and the enhanced color development solution.

[0094] The specific experimental steps are as follows:

[0095] 1. Take 100 μl of each of the enhanced color development samples with different concentrations and add them to the sample addition port of the test card. After chromatography for 15 minutes, use GD-010 to detect the gray value of the T line (i.e., group 1), and measure each concentration 2 times;

[0096] 2. Take 1 chromatographed test card for each concentration of the enhanced color development sample, immerse it in the copper ion solution containing copper ions and PEG20000 prepared above for 1 minute, then immerse it in the full-component diluent listed in Table 2 for 5 minutes respectively, and use GD-010 to detect the gray value of the T line again (i.e., group 2).

[0097] (2) Experiment 2

[0098] 1. Take 100 μl of each of the non-enhanced control samples with different concentrations and add them to the sample addition port of the test card. After chromatography for 15 minutes, use GD-010 to detect the gray value of the T line (i.e., group 1), and measure each concentration 2 times;

[0099] 2. Take 1 chromatographed test card for each concentration of the non-enhanced control sample, immerse it in the copper ion solution containing copper ions and PEG20000 prepared above for 1 minute, then immerse it in the full-component diluent listed in Table 2 for 1 minute respectively, and use GD-010 to detect the gray value of the T line again (i.e., group 2).

[0100] 3. Do a reduction control with sodium ascorbate (VcNa). Take 100 μl of each of the non-enhanced control samples with different concentrations and add them to the sample addition port of the test card. After chromatography for 15 minutes. Take the chromatographed test card, immerse it in the copper ion solution containing copper ions and PEG20000 prepared above for 5 minutes, and then wash it with purified water. Then immerse it in 1 mM VcNa solution for 2 minutes respectively, and then use GD-010 to detect the gray value of the T line (i.e., group 3)

[0101] The gray value detection results of Experiment 1 and Experiment 2 are shown in Table 3.

[0102] Table 3

[0103]

[0104] The process and required time of the method of this application (i.e., the method in (2) of Experiment 2) were compared with the process and required time of the method in the literature, and the results are shown in Table 4.

[0105] Table 4

[0106]

[0107]

[0108] As can be seen from Table 4:

[0109] Compared with the literature (Yaofeng Zhou, et al. Biosensors and Bioelectronics 171(2021)112753), this application has obvious improvements both in terms of the usage process and the detection time, being shortened from 4 steps and 22 minutes in the original literature to 2 steps and 16 minutes.

[0110] When comparing the color development intensity of colloidal gold, the diluent of this application can achieve enhanced color development without the need to additionally add a reducing agent; if the reducing agent VcNa used in the literature is introduced into the solution of this application, it will have the opposite effect of reducing color development.

[0111] Example 3

[0112] In this example, the steps of Experiment 1 in Example 2 were adopted to perform the enhanced color development operation. The difference between this example and Example 2 lies in the use of different diluents.

[0113] The difference between the diluent used in Group 1 and the diluent used in Example 2 is that it does not include 4-dimethylaminoantipyrine, p-acetaminophen, gentamicin sulfate, and 2-chloroacetamide.

[0114] The difference between the diluent used in Group 2 and the diluent used in Example 2 is that it does not include gentamicin sulfate and 2-chloroacetamide, and the content of 4-dimethylaminoantipyrine is 0.03 wt%, and the content of p-acetaminophen is 1.0 wt%.

[0115] The difference between the diluent used in Group 3 and the diluent used in Example 2 is that it does not include 4-dimethylaminoantipyrine and p-acetaminophen, and the content of gentamicin sulfate is 1.0 wt%, and the content of 2-chloroacetamide is 0.03 wt%.

[0116] The diluent used in Group 4 is different from the diluent used in Example 2 in that it does not include 4-dimethylaminoantipyrine and gentamicin sulfate, and the content of p-acetaminophen is 0.03 wt%, and the content of 2-chloroacetamide is 1.0 wt%.

[0117] The diluent used in Group 5 is different from the diluent used in Example 2 in that it does not include gentamicin sulfate, and the content of 4-dimethylaminoantipyrine is 0.03 wt%, the content of p-acetaminophen is 0.5 wt%, and the content of 2-chloroacetamide is 0.5 wt%.

[0118] The gray values of the T lines of the test cards under different conditions are shown in Table 5. (Blank conditions in Table 5)

[0119] The test cards under different conditions

[0120] Table 5

[0121]

[0122] It can be seen from the results that the diluent used in this application can achieve enhanced color development without the need to additionally add a reducing agent; single-component, different component combinations, and all components have color development effects.

[0123] Enhanced color development of the FIT-HP kit (colloidal gold method) in Example 4

[0124] Prepare the FIT-HP kit in the same method as in Example 3

[0125] Use the prepared FIT-HP kit for the qualitative detection of FIT-HP.

[0126] Specifically, after collecting feces according to the instructions of the manual and extracting with the diluent, add 3 - 4 drops of the extract to the sample pad of the test card, and measure 2 test cards for each sample. 10 minutes after adding the sample, take 1 test card from each concentration and soak it in the copper ion solution for 1 minute, and then insert the test cards for enhanced color development and control into the immunochromatographic analyzer (GD-010) to detect the gray value of the T line. The gray values of the T lines obtained from different samples are shown in Table 6.

[0127] Table 6

[0128]

[0129] It can be seen from the results that after enhanced color development, the gray values of the T lines of all weakly positive samples are significantly enhanced, and on average, the color development gray values of weakly positive samples can be enhanced by more than 2 times, while the gray values of the T lines of negative samples are still below the cut-off value. Therefore, the method described in this application will not bring non-specific signals. At the same time, the diluent provided in this application has a color development effect on test strips of different types, indicating its universality.

Claims

1. A diluent for enhancing colloidal gold color development, comprising an enhancing reagent and an antigen stabilizing reagent, wherein the enhancing reagent is selected from one or more of 4-dimethylaminoantipyrine, p-acetaminophen, gentamicin sulfate, and 2-chloroacetamide.

2. The diluent according to claim 1, wherein the content of the enhancing reagent in the diluent is 0.035 wt% - 6.2 wt%.

3. The diluent according to claim 1 or 2, wherein the antigen stabilizing reagent comprises one or more of a protective protein, a buffer system, a surfactant, a salt, and a preservative.

4. The diluent according to claim 1 or 2, wherein the protective protein is bovine serum albumin or casein, the buffer system is selected from one or more of Tris-HCl buffer system, MES buffer system, phosphate buffer system, boric acid buffer system, and acetate buffer system, the surfactant is selected from one or more of lauryl alcohol ether, Triton X-100, Tween-20, and sodium dodecyl sulfate, the salt is selected from one or more of sodium chloride, potassium chloride, and sodium deoxycholate, and the preservative is Proclin300.

5. The diluent according to claim 4, wherein the antigen stabilizing reagent comprises Tris-HCl buffer system, sodium chloride, bovine serum albumin, lauryl alcohol ether, Triton X-100, sodium deoxycholate, and Proclin300.

6. A kit for enhancing colloidal gold color development, comprising the diluent according to any one of claims 1-5, and a copper ion solution.

7. The kit according to claim 6, wherein the copper ion solution comprises copper sulfate and PEG, wherein PEG is PEG10000 - PEG50000.

8. The kit according to claim 6 or 7, wherein the content of divalent copper ions in the copper ion solution is 6.25 mM - 625 mM, and the content of PEG is 1 g / L - 100 g / L.

9. The kit according to any one of claims 6-8, wherein the kit further comprises a colloidal gold test strip.

10. A method for enhancing colloidal gold color development, the method comprising reacting an enhancing reagent with divalent copper ions, wherein the enhancing reagent is selected from one or more of 4-dimethylaminoantipyrine, p-acetaminophen, gentamicin sulfate, and 2-chloroacetamide.

Images