A method for enhancing the detection sensitivity of colloidal gold immunochromatography and its application

Abstract

The present invention discloses a method and application for enhancing the detection sensitivity of colloidal gold immunochromatography. In the colloidal gold immunochromatography detection, the present invention uses a combination of colloidal gold of multiple different particle sizes, specifically using small-particle colloidal gold and large-particle colloidal gold to bind to antibodies respectively, and then mixing them for use, which can enhance the detection sensitivity of the colloidal gold immunochromatography. The optimal mixing ratio of colloidal gold of different particle sizes can significantly improve the sensitivity of the detection, avoid problems such as dragging, false positives, and steric hindrance; at the same time, concentrating colloidal gold of different particle sizes and then mixing them for use can further enhance the detection sensitivity of the immunochromatography, so that the colloidal gold can be more stably bound to the antibody and detected. The method provided by the present invention effectively improves the detection limit of the colloidal gold test strip while maintaining the original detection specificity, and the method is simple and easy to implement, low cost, and suitable for mass production.

Description

Technical Field

[0001] The present invention belongs to the technical field of colloidal gold detection, and more specifically, relates to a method and application for enhancing the detection sensitivity of colloidal gold immunochromatography. Background Art

[0002] Colloidal gold immunochromatography is an in vitro diagnostic technique that uses colloidal gold as a colorimetric medium, labels antibodies onto the colloidal gold, and exploits the specific binding of antigens and antibodies in immunology to detect antigens during chromatography. Currently, while colloidal gold immunochromatography is not as sensitive as other methods such as immunofluorescence and fluorescent PCR, it has applications in many fields, including medicine, food, and agriculture, due to its rapidity, simplicity, high specificity, stability, lack of complex instrumentation, and intuitive results.

[0003] The sensitivity of colloidal gold immunochromatography is limited, and many methods for improving its sensitivity are relatively cumbersome, hindering cost reduction and mass production. Prior art methods, such as using sensitizing reagents or optimizing detection system parameters, as well as optimizing antibody concentration and reaction rate, have been proposed to increase sensitivity. However, these methods suffer from poor stability, low labeling efficiency, susceptibility to false negative results, and susceptibility to background effects. In colloidal gold immunoassay applications, the concentration, particle size, and shape of the colloidal gold used can affect the sensitivity, specificity, and stability of the assay.

[0004] In existing technologies, colloidal gold particles with uniform particle size and shape, along with the optimal concentration, are used to improve detection sensitivity and stability. Existing research also shows that the sensitivity of colloidal gold test strips increases with particle size. However, blindly using large-particle colloidal gold often leads to carryover and false positives during testing, and can also cause steric hindrance, making it difficult for colloidal gold to bind to specific proteins. Furthermore, research shows that when the particle size of colloidal gold particles exceeds 40nm, the colloidal state of colloidal gold becomes unstable (Reference: Methods for Improving the Sensitivity of Colloidal Gold Immunochromatographic Assays, Zhao Xiaoming, 2019).

[0005] It is well known to those skilled in the art that colloidal gold particle size is when 10~50nm, itself has very good performance, its sensitivity is also better, is without the need to adjust and improve, but owing to adopt the colloidal gold solution molar extinction coefficient of 10~50nm particle size lower, color intensity is insufficient, so can cause final detection inaccurate.And the colloidal gold particle size commonly used in this area is 60nm, is the large particle size colloidal gold that can be used alone, unless applied to special detection preparation (need to prepare the colloidal gold of specific particle size), generally can not use the colloidal gold that exceeds 60nm particle size, because adopting the colloidal gold of larger particle size can cause serious dragging, long fading time, also can cause the problems such as false positive, its sensitivity also can not be enhanced along with the increase of particle size.In order to be able to better improve the sensitivity and stability of colloidal gold immunoassay, under the premise of maintaining original detection specificity, effectively improve the detection limit of colloidal gold detection test strip, need to develop and research out more simpler and more convenient, low-cost, high stability, high sensitivity raising colloidal gold detection more sensitive method, have great significance for the application of immunoassay and the development of detection product thereof. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the above-mentioned problems and provide a method and application for enhancing the detection sensitivity of colloidal gold immunochromatography. While maintaining the original detection specificity, the detection limit of the colloidal gold test strip is effectively improved, the cost is low, and it is easy to produce and apply.

[0007] The purpose of the present invention is to provide a method for enhancing the detection sensitivity of colloidal gold immunochromatography.

[0008] Another object of the present invention is to provide a highly sensitive colloidal gold immunochromatographic detection method.

[0009] Another object of the present invention is to provide a colloidal gold-antibody reconstituted product combination that can improve the detection sensitivity of colloidal gold immunochromatography.

[0010] Another object of the present invention is to provide a highly sensitive colloidal gold immunoassay reagent strip or kit.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention provides a method for enhancing the detection sensitivity of colloidal gold immunochromatography. During the colloidal gold immunochromatography detection, a combination of colloidal gold particles with multiple different particle sizes is used. Specifically, a combination of colloidal gold particles with two particle sizes is used. Small-particle colloidal gold and large-particle colloidal gold particles are respectively combined with antibodies and then mixed for use. The particle size of the small-particle colloidal gold particles is 50-60 nm, and the particle size of the large-particle colloidal gold particles is 70-150 nm.

[0013] The present invention breaks through the existing stereotype. After a lot of research and exploration, it is found that the sensitivity of immunochromatographic detection can be improved by mixing colloidal gold of different particle sizes. When colloidal gold of large particle size and colloidal gold of small particle size are mixed in a certain proportion, there will be no problems of dragging, false positives, steric hindrance, and poor specific binding ability. At the same time, the colloidal gold of different particle sizes is concentrated and then mixed, which can further improve the sensitivity of immunochromatographic detection. The method provided by the present invention can significantly enhance the sensitivity of colloidal gold detection, effectively improve the detection limit of colloidal gold detection test strips, and the method is simple and easy to use. No additional substances need to be added to improve the sensitivity. It also avoids the problems of dragging, false positives, and steric hindrance in the detection of colloidal gold of large particle size, so that colloidal gold can be more stably combined with antibodies and detected, with low cost and suitable for mass production.

[0014] Preferably, the small-particle colloidal gold and large-particle colloidal gold used are a combination of 60nm and 90nm colloidal gold.

[0015] Furthermore, the volume ratio of the small-particle colloidal gold to the large-particle colloidal gold is 6-12:1, the concentration of the small-particle colloidal gold is 0.5-3 g / L, and the concentration of the large-particle colloidal gold is 0.5-2 g / L.

[0016] Furthermore, the volume ratio of the small-particle colloidal gold to the large-particle colloidal gold is 8:1.

[0017] The present invention provides a specific method for enhancing the detection sensitivity of colloidal gold immunochromatography:

[0018] S1. Colloidal gold of different particle sizes was taken, labeled with antibodies, and centrifuged to prepare antibody-labeled colloidal gold precipitates;

[0019] S2. The antibody-labeled colloidal gold precipitate obtained in step S1 above was redissolved using a reconstitution solution to obtain colloidal gold-antibody reconstituted products of different particle sizes;

[0020] S3. The colloidal gold-antibody reconstituted products of different particle sizes obtained in the above step S2 are mixed and used.

[0021] As a preferred embodiment, the present invention provides the following specific steps (taking 1 mL of colloidal gold as an example):

[0022] (1) Place 1 mL of colloidal gold in an EP tube, add 0.2 mol / L potassium carbonate solution, adjust the pH to 8.5-9.5, rotate to mix, and react at room temperature for 10 min;

[0023] (2) Add 10 μg of antibody to the solution in step (1), rotate to mix, and react at room temperature for 15 to 30 minutes;

[0024] (3) Add 50 μL of blocking solution to the solution in step (2), rotate and mix, and react at room temperature for 15 to 30 minutes. After the reaction is completed, centrifuge at low temperature and remove the supernatant to obtain the antibody-labeled colloidal gold precipitate;

[0025] (4) The above precipitate is redissolved to 10-100 μL using a redissolving solution (adjusting the concentration of colloidal gold). This redissolved product is the concentrated colloidal gold.

[0026] (5) Prepare colloidal gold of various particle sizes according to steps (1) to (4);

[0027] (6) Recombining large-particle colloidal gold-antibody resolubilization products and small-particle colloidal gold-antibody resolubilization products in a certain proportion can increase the detection sensitivity in colloidal gold immunochromatography.

[0028] The present invention provides a high-sensitivity colloidal gold immunochromatographic detection method, which is combined with the above method to perform colloidal gold immunochromatographic detection.

[0029] The present invention provides a colloidal gold-antibody resolubilized product combination capable of improving the detection sensitivity of colloidal gold immunochromatography, which contains colloidal gold-antibody resolubilized products of different particle sizes obtained by the above method.

[0030] The present invention provides the use of a colloidal gold-antibody reconstituted product combination in preparing a high-sensitivity colloidal gold immunoassay reagent strip or kit.

[0031] The present invention also provides a high-sensitivity colloidal gold immunoassay reagent strip or kit containing a colloidal gold-antibody reconstituted product combination.

[0032] The present invention has the following beneficial effects:

[0033] The present invention discloses a method for enhancing the detection sensitivity of colloidal gold immunochromatography. The present invention changes the existing mindset that there is only one type of colloidal gold particle size. Mixing colloidal gold particles of multiple different particle sizes can significantly improve the detection sensitivity. Mixing large-particle colloidal gold particles and small-particle colloidal gold particles in a certain ratio can not only improve the detection sensitivity, but also avoid problems such as dragging, fading, false positives, and poor specific binding ability. At the same time, concentrating colloidal gold particles of different particle sizes and then mixing them can further enhance the detection sensitivity of the immunochromatography method. The present invention uses a mixture of large-particle colloidal gold particles and small-particle colloidal gold particles in the optimal ratio, which can effectively avoid steric hindrance, reduce the gaps between large particles of colloidal gold particles, and enable colloidal gold particles to bind to antibodies more stably, which is more conducive to the binding of colloidal gold particles to specific proteins and the detection of low-concentration samples.

[0034] The method provided by the present invention effectively improves the detection limit of the colloidal gold test strip while maintaining the original detection specificity, enhances the detection sensitivity of the colloidal gold immunochromatography, does not produce dragging, fading, or false positive results, and does not significantly increase production costs. The method is simple and easy to implement and is suitable for mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The sensitivity test results of Example 1 and Comparative Example 1 are shown;

[0036] Figure 2 The sensitivity test results of Example 2 and Comparative Example 1 are shown;

[0037] Figure 3 The sensitivity test results of Example 3 and Comparative Example 1 are shown;

[0038] Figure 4 The sensitivity test results of Examples 4 to 6 and Comparative Example 1 are shown;

[0039] Figure 5 The sensitivity test results of Examples 7, 8, 2, 9 and Comparative Example 1 are shown;

[0040] Figure 6 The sensitivity test results of Comparative Example 2 and Comparative Example 1 are shown in FIG.

[0041] Figure 7 The sensitivity test results of Examples 1, 2, 4 and Comparative Example 1 are shown;

[0042] Note: Figures 1 to 9 are the corresponding test results of the colloidal gold immunochromatographic test strips prepared in Examples 1 to 9, CK is comparative example 1, and CK2 is comparative example 2. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0044] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0045] Example 1

[0046] (1) Place 1 mL of 60 nm colloidal gold in a 1.5 mL centrifuge tube, add 0.2 mol / L K2CO3 to adjust the pH to 9.0, and rotate and mix at room temperature for 10 min.

[0047] (2) Add 10 μg of monoclonal antibody SC2N-118 and incubate with rotation at room temperature for 15 min;

[0048] (3) Add 50 μL of 1% casein solution and rotate at room temperature for 15 min to block excess binding sites;

[0049] (4) Centrifuge at 6000 g for 15 min at 4°C and discard the supernatant. Add 50 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 60 nm colloidal gold-antibody reconstitution solution (concentration of 1 g / L) and store at 4°C for later use.

[0050] (5) Treat 90 nm colloidal gold according to steps (1) to (3) above, then centrifuge at 4°C, 4,000 g for 15 min and discard the supernatant. Add 25 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 90 nm colloidal gold-antibody reconstitution solution (concentration of 2 g / L), which is stored at 4°C until use.

[0051] (6) The two prepared colloidal gold-antibody complex solutions were mixed in a ratio of 8:1 (60 nm:90 nm) and evenly sprayed onto an 8 mm wide glass cellulose membrane. The membrane was dried at 37°C overnight to prepare a conjugate pad.

[0052] Example 2

[0053] (1) Place 1 mL of 60 nm colloidal gold in a 1.5 mL centrifuge tube, add 0.2 mol / L K2CO3 to adjust the pH to 9.0, and rotate and mix at room temperature for 10 min.

[0054] (2) Add 10 μg of monoclonal antibody SC2N-118 and incubate with rotation at room temperature for 15 min;

[0055] (3) Add 50 μL of 1% casein solution and rotate at room temperature for 15 min to block excess binding sites;

[0056] (4) Centrifuge at 6000 g for 15 min at 4°C and discard the supernatant. Add 100 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 60 nm colloidal gold-antibody reconstitution solution (concentration of 0.5 g / L) and store at 4°C for later use.

[0057] (5) Treat 90 nm colloidal gold according to steps (1) to (3) above, then centrifuge at 4°C, 4,000 g for 15 min and discard the supernatant. Add 100 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 90 nm colloidal gold-antibody reconstitution solution (concentration 0.5 g / L), which is stored at 4°C until use.

[0058] (6) The prepared colloidal gold-antibody complex solution was mixed in a ratio of 8:1 (60 nm:90 nm) and evenly sprayed on an 8 mm wide glass cellulose membrane, and dried at 37°C overnight to prepare a conjugate pad.

[0059] Example 3

[0060] (1) Place 1 mL of 60 nm colloidal gold in a 1.5 mL centrifuge tube, add 0.2 mol / L K2CO3 to adjust the pH to 9.0, and rotate and mix at room temperature for 10 min.

[0061] (2) Add 10 μg of monoclonal antibody SC2N-118 and incubate with rotation at room temperature for 15 min;

[0062] (3) Add 50 μL of 1% casein solution and rotate at room temperature for 15 min to block excess binding sites;

[0063] (4) Centrifuge at 6000 g for 15 min at 4°C and discard the supernatant. Add 100 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 60 nm colloidal gold-antibody reconstitution solution (concentration of 0.5 g / L) and store at 4°C for later use.

[0064] (5) Treat 150 nm colloidal gold according to steps (1) to (3) above, then centrifuge at 3,000 g for 15 min at 4°C and discard the supernatant. Add 100 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 150 nm colloidal gold-antibody reconstitution solution (concentration of 0.5 g / L), which is stored at 4°C until use.

[0065] (6) The prepared colloidal gold-antibody complex solution was mixed in a ratio of 8:1 (60 nm:150 nm) and evenly sprayed on an 8 mm wide glass cellulose membrane, and dried at 37°C overnight to prepare a conjugate pad.

[0066] Example 4

[0067] (1) Take 1 mL of 60 nm colloidal gold in a 1.5 mL centrifuge tube, add 0.2 mol / L K2CO3 to adjust the pH to 9.0, and rotate and mix at room temperature for 10 min;

[0068] (2) Add 10 μg of monoclonal antibody SC2N-118 and incubate with rotation at room temperature for 15 min;

[0069] (3) Add 50 μL of 1% casein solution and rotate at room temperature for 15 min to block excess binding sites;

[0070] (4) Centrifuge at 6000 g for 15 min at 4°C and discard the supernatant. Add 50 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 60 nm colloidal gold-antibody reconstitution solution (concentration of 1 g / L) and store at 4°C for later use.

[0071] (5) The prepared colloidal gold-antibody complex solution was evenly sprayed on an 8 mm wide glass cellulose membrane and dried at 37°C overnight to prepare a conjugate pad.

[0072] Example 5

[0073] (1) Take 1 mL of 60 nm colloidal gold in a 1.5 mL centrifuge tube, add 0.2 mol / L K2CO3 to adjust the pH to 9.0, and rotate and mix at room temperature for 10 min;

[0074] (2) Add 10 μg of monoclonal antibody SC2N-118 and incubate with rotation at room temperature for 15 min;

[0075] (3) Add 50 μL of 1% casein solution and rotate at room temperature for 15 min to block excess binding sites;

[0076] (4) Centrifuge at 6000 g for 15 min at 4°C and discard the supernatant. Add 25 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 60 nm colloidal gold-antibody reconstitution solution (concentration of 2 g / L), which is stored at 4°C for later use.

[0077] (5) The prepared colloidal gold-antibody complex solution was evenly sprayed on an 8 mm wide glass cellulose membrane and dried at 37°C overnight to prepare a conjugate pad.

[0078] Example 6

[0079] (1) Take 1 mL of 60 nm colloidal gold in a 1.5 mL centrifuge tube, add 0.2 mol / L K2CO3 to adjust the pH to 9.0, and rotate and mix at room temperature for 10 min;

[0080] (2) Add 10 μg of monoclonal antibody SC2N-118 and incubate with rotation at room temperature for 15 min;

[0081] (3) Add 50 μL of 1% casein solution and rotate at room temperature for 15 min to block excess binding sites of the colloid;

[0082] (4) Centrifuge at 6,000 g for 15 min at 4°C and discard the supernatant. Add 16.7 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 60 nm colloidal gold-antibody reconstitution solution (concentration of 3 g / L). Store at 4°C until use.

[0083] (5) The prepared colloidal gold-antibody complex solution was evenly sprayed on an 8 mm wide glass cellulose membrane and dried at 37°C overnight to prepare a conjugate pad.

[0084] Example 7

[0085] (1) Place 1 mL of 60 nm colloidal gold in a 1.5 mL centrifuge tube, add 0.2 mol / L K2CO3 to adjust the pH to 9.0, and rotate and mix at room temperature for 10 min.

[0086] (2) Add 10 μg of monoclonal antibody SC2N-118 and incubate with rotation at room temperature for 15 min;

[0087] (3) Add 50 μL of 1% casein solution and rotate at room temperature for 15 min to block excess binding sites;

[0088] (4) Centrifuge at 6000 g for 15 min at 4°C and discard the supernatant. Add 100 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 60 nm colloidal gold-antibody reconstitution solution (concentration of 0.5 g / L) and store at 4°C for later use.

[0089] (5) Treat 90 nm colloidal gold according to steps (1) to (3) above, then centrifuge at 4°C, 4000 g for 15 min, and discard the supernatant. Add 100 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 90 nm colloidal gold-antibody reconstitution solution (concentration of 0.5 g / L), which is stored at 4°C for later use.

[0090] (6) The prepared colloidal gold-antibody complex solution was mixed in a ratio of 6:1 (60 nm:90 nm) and evenly sprayed on an 8 mm wide glass cellulose membrane, and dried at 37°C overnight to prepare a conjugate pad.

[0091] Example 8

[0092] (1) Place 1 mL of 60 nm colloidal gold in a 1.5 mL centrifuge tube, add 0.2 mol / L K2CO3 to adjust the pH to 9.0, and rotate and mix at room temperature for 10 min.

[0093] (2) Add 10 μg of monoclonal antibody SC2N-118 and incubate with rotation at room temperature for 15 min;

[0094] (3) Add 50 μL of 1% casein solution and rotate at room temperature for 15 min to block excess binding sites;

[0095] (4) Centrifuge at 6000 g for 15 min at 4°C and discard the supernatant. Add 100 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 60 nm colloidal gold-antibody reconstitution solution (concentration of 0.5 g / L) and store at 4°C for later use.

[0096] (5) Treat 90 nm colloidal gold according to steps (1) to (3) above, then centrifuge at 4°C, 4000 g for 15 min, and discard the supernatant. Add 100 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 90 nm colloidal gold-antibody reconstitution solution (concentration of 0.5 g / L), which is stored at 4°C for later use.

[0097] (6) The prepared colloidal gold-antibody complex solution was mixed in a ratio of 10:1 (60 nm:90 nm) and evenly sprayed on an 8 mm wide glass cellulose membrane, and dried at 37°C overnight to prepare a conjugate pad.

[0098] Example 9

[0099] (1) Take 1 mL of 60 nm colloidal gold in a 1.5 mL centrifuge tube, add 0.2 mol / L K2CO3 to adjust the pH to 9.0, and rotate and mix at room temperature for 10 min;

[0100] (2) Add 10 μg of monoclonal antibody SC2N-118 and incubate with rotation at room temperature for 15 min;

[0101] (3) Add 50 μL of 1% casein solution and rotate at room temperature for 15 min to block excess binding sites;

[0102] (4) Centrifuge at 6000 g for 15 min at 4°C and discard the supernatant. Add 100 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 60 nm colloidal gold-antibody reconstitution solution (concentration of 0.5 g / L) and store at 4°C for later use.

[0103] (5) Treat 90 nm colloidal gold according to steps (1) to (3) above, then centrifuge at 4°C, 4000 g for 15 min, and discard the supernatant. Add 100 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 90 nm colloidal gold-antibody reconstitution solution (concentration of 0.5 g / L), which is stored at 4°C for later use.

[0104] (6) The prepared colloidal gold-antibody complex solution was mixed in a ratio of 12:1 (60 nm:90 nm) and evenly sprayed on an 8 mm wide glass cellulose membrane and dried at 37°C overnight to prepare a conjugate pad.

[0105] Comparative Example 1 Traditional Process

[0106] (1) Take 1 mL of 60 nm colloidal gold in a 1.5 mL centrifuge tube, add 0.2 mol / L K2CO3 to adjust the pH to 9.0, and rotate and mix at room temperature for 10 min;

[0107] (2) Add 10 μg of monoclonal antibody SC2N-118 and incubate with rotation at room temperature for 15 min;

[0108] (3) Add 50 μL of 1% casein solution and rotate at room temperature for 15 min to block excess binding sites of the colloid;

[0109] (4) Centrifuge at 6000 g for 15 min at 4°C and discard the supernatant. Add 100 μL of reconstitution solution and gently pipette to reconstitute the colloidal gold-antibody complex to obtain a 60 nm colloidal gold-antibody reconstitution solution (concentration of 0.5 g / L) and store at 4°C for later use.

[0110] (5) The prepared colloidal gold-antibody was re-dissolved and evenly sprayed on an 8 mm wide glass cellulose membrane and dried at 37°C overnight to prepare a conjugate pad.

[0111] Comparative Example 2

[0112] The preparation method and steps are the same as those in Comparative Example 1, with the only difference being that the colloidal gold particles used have a diameter of 90 nm (concentration of 0.5 g / L).

[0113] Application Example 1

[0114] 1. Assembly and detection method of colloidal gold immunochromatographic test strips

[0115] (1) Coating of nitrocellulose membrane: 1.5 mg / mL SC2N-113 monoclonal antibody and 1.0 mg / mL goat anti-rabbit IgG antibody were sprayed onto nitrocellulose membrane into uniform lines to form the test line and quality control line, respectively. The membrane was dried at 50°C overnight to obtain antibody-coated nitrocellulose membrane.

[0116] (2) Assembly of colloidal gold immunochromatographic test strips: First, affix the coated nitrocellulose membrane to the middle of the PVC base plate, with the quality control line above the test line. Paste absorbent paper on the top of the nitrocellulose membrane, and first paste the binding pad and the nitrocellulose membrane on the bottom, overlapping each other before pasting the sample pad. The parts on the PVC base plate overlap each other by 1-2 mm, and finally cut into 3 mm wide test strips. Use the binding pads prepared in Examples 1-9 and Comparative Examples 1-2 above to assemble colloidal gold immunochromatographic test strips for subsequent testing.

[0117] (3) Detection method: Take the recombinant SARS-CoV-2 NP protein (from the Brazilian strain, Nanjing Baikang Biotechnology Co., Ltd.) and dilute it using the two-fold dilution method. Add 50 μL of the diluted antigen to the sample pad of the test paper and let it stand for 15 to 30 minutes. After the test results appear, compare the detection line intensity of the different colloidal gold immunochromatographic test strips obtained above.

[0118] 2. Sensitivity determination of colloidal gold immunochromatographic test strips

[0119] The conjugate pads prepared in Examples 1 to 9 and Comparative Documents 1 to 2 were assembled into colloidal gold immunochromatographic test strips, and the sensitivity of the antigens at different dilution ratios (1:8000, 1:16000, 1:32000, and 1:64000) was determined. Example 1 is a group of colloidal gold particles concentrated and then mixed, Examples 2 to 3 are a group of colloidal gold particles mixed, Examples 4 to 6 are groups of colloidal gold particles concentrated, and Examples 7 to 9 are groups of colloidal gold particles mixed at different ratios. The specific detection method is the same as that described in 1 and (3) above.

[0120] The results of the colloidal gold immunochromatographic test strips using the conventional process of Example 1 and Comparative Example 1 are as follows: Figure 1 The results are shown in Table 1, which show that Example 1 has a more significant detection line than Comparative Example 1, and the detection sensitivity is doubled. This shows that mixing 60nm colloidal gold concentrated twice (1g / L) after antibody labeling with 90nm colloidal gold concentrated four times (2g / L) after antibody labeling can improve detection sensitivity and readability, and the detection results will not produce problems such as dragging, fading, and false positives.

[0121] Table 1 Sensitivity test of Example 1 and Comparative Example 1

[0122] Antigen concentration Negative 1:8000 1:16000 1:32000 1:64000 Example 1 - +++ ++ + - / + Comparative Example 1 - ++ + - / + -

[0123] “-” indicates negative; “- / +” indicates that the T line is sometimes present and sometimes absent; “+” indicates a clear weak positive; “++” indicates positive; and “+++” indicates a strong positive.

[0124] The results of the colloidal gold immunochromatographic test strips using the conventional process of Example 2 and Comparative Example 1 are as follows: Figure 2 The results are shown in Table 2, which show that Example 2 has a more obvious detection result than Comparative Example 1 at an antigen dilution factor of 1:32000, and its detection results do not cause problems such as dragging, fading, and false positives. This shows that mixing labeled 90nm colloidal gold and labeled 60nm colloidal gold can improve detection sensitivity.

[0125] Table 2 Sensitivity test of Example 2 and Comparative Example 1

[0126] Antigen concentration Negative 1:8000 1:16000 1:32000 1:64000 Example 2 - ++ + + - Comparative Example 1 - ++ + - / + -

[0127] “-” indicates negative; “- / +” indicates that the T line is sometimes present and sometimes absent; “+” indicates a clear weak positive; “++” indicates positive; and “+++” indicates a strong positive.

[0128] The results of the colloidal gold immunochromatographic test strips using the conventional process of Example 3 and Comparative Example 1 are as follows: Figure 3The results are shown in Table 3, which shows that the detection results of Example 3 and Comparative Example 1 are basically the same at different antigen concentrations, indicating that mixing 150nm colloidal gold with 60nm colloidal gold cannot effectively improve sensitivity.

[0129] Table 3 Sensitivity test of Example 3 and Comparative Example 1

[0130] Antigen concentration Negative 1:8000 1:16000 1:32000 1:64000 Example 3 - ++ + - / + - Comparative Example 1 - ++ + - / + -

[0131] “-” indicates negative; “- / +” indicates that the T line is sometimes present and sometimes absent; “+” indicates a clear weak positive; “++” indicates positive; and “+++” indicates a strong positive.

[0132] The results of the colloidal gold immunochromatographic test strips of Examples 4 to 6, which are different colloidal gold concentration groups, and the conventional colloidal gold immunochromatographic test strips of Comparative Example 1 are shown in FIG. Figure 4 The results are shown in Table 4, which shows that the detection sensitivity of Examples 4, 5, and 6 is higher than that of Comparative Example 1. By adjusting the concentration of the colloidal gold resolubilizer and concentrating it, the detection sensitivity can be improved, among which Examples 4 and 5 have the best effects. This shows that when the labeled 60nm colloidal gold is concentrated to improve the detection sensitivity, the best sensitization effect can be achieved by concentrating it at least twice (1g / L).

[0133] Table 4 Sensitivity test of Examples 4 to 6 and Comparative Example 1

[0134]

[0135]

[0136] “-” indicates negative; “- / +” indicates that the T line is sometimes present and sometimes absent; “+” indicates a clear weak positive; “++” indicates positive; and “+++” indicates a strong positive.

[0137] The mixing ratio of the colloidal gold used in Example 2 is different from that in Examples 7 to 9. The test results are shown in Table 1. Figure 5 As shown in Table 5, the statistical results show that Example 2 can obtain a definite positive test result under an antigen dilution factor of 1:32000, while the test result after mixing colloidal gold with other different particle sizes is poorer than that of Example 2. The mixing of colloidal gold with large particle size and colloidal gold with small particle size using the best ratio can well avoid steric hindrance, so that the small particle size colloidal gold can be evenly dispersed in the middle of the large particle size colloidal gold, reduce the gaps generated between the large particle size colloidal gold, and make the colloidal gold more stably bound to the antibody, which is more conducive to the binding of colloidal gold to specific protein and the detection of low concentration samples. The best sensitization effect can be achieved by mixing 60nm labeled colloidal gold and 90nm labeled colloidal gold in an 8:1 ratio.

[0138] Table 5 Sensitivity test of Examples 2, 7, 8, 9 and Comparative Example 1

[0139] Antigen concentration Negative 1:8000 1:32000 Example 2 - +++ + Example 7 - ++ - / + Example 8 - +++ - / + Example 9 - +++ - / + Comparative Example 1 - ++ - / +

[0140] “-” indicates negative; “- / +” indicates that the T line is sometimes present and sometimes absent; “+” indicates a clear weak positive; “++” indicates positive; and “+++” indicates a strong positive.

[0141] Comparative Example 2 uses a larger colloidal gold particle size of 90nm to mark the test strips, and the comparative test results of Comparative Example 1 using a colloidal gold particle size of 60nm using the traditional process are as follows: Figure 6 As shown in Table 6, the statistical results show that the detection effect of Comparative Example 2 is worse than that of Comparative Example 1, and no definite positive detection result can be obtained at an antigen dilution multiple of 1:8000, indicating that the 90nm colloidal gold is not suitable for use alone in colloidal gold immunoassay; similarly, the detection sensitivity of colloidal gold with a larger particle size such as 120nm and 150nm is worse than that of the 90nm colloidal gold, and is not suitable for use alone in colloidal gold immunoassay.

[0142] Table 6 Sensitivity test of Comparative Example 2 and Comparative Example 1

[0143] Antigen concentration Negative 1:8000 1:16000 1:32000 1:64000 Comparative Example 2 - - / + - - - Comparative Example 1 - ++ + - / + -

[0144] “-” indicates negative; “- / +” indicates that the T line is sometimes present and sometimes absent; “+” indicates a clear weak positive; “++” indicates positive; and “+++” indicates a strong positive.

[0145] The results of the colloidal gold immunochromatographic test strips using the conventional process of Examples 1, 2, 4 and Comparative Example 1 are shown in FIG. Figure 7 The results are shown in Table 7, which shows that the detection sensitivity of Examples 1, 2, and 4 is higher than that of Comparative Example 1, where the sensitivity is from high to low as Example 1, Example 4, and Example 2. This shows that appropriate concentration before mixing particles of different sizes can further improve the detection sensitivity.

[0146] Table 7 Sensitivity test of Examples 4 to 6 and Comparative Example 1

[0147] Antigen concentration Negative 1:8000 1:16000 1:32000 1:64000 Example 2 - ++ + - / + - / + Example 4 - +++ ++ + - / + Example 1 - +++ ++ + + Comparative Example 1 - + - / + - / + -

[0148] “-” indicates negative; “- / +” indicates that the T line is sometimes present and sometimes absent; “+” indicates a clear weak positive; “++” indicates a positive result; due to different experimental batches, there are slight errors in each batch, which are within the acceptable range for technical personnel in this field.

[0149] The above results show that the present invention can significantly improve the sensitivity of immunochromatography detection by mixing colloidal gold of multiple different particle sizes; the sensitivity of immunochromatography detection can be further improved by concentrating colloidal gold of different particle sizes and then mixing them. The use of colloidal gold of large particle size and colloidal gold of small particle size mixed with the optimal ratio can well avoid steric hindrance, reduce the gaps generated between large particles of colloidal gold, and enable colloidal gold to bind to antibodies more stably, which is more conducive to the binding of colloidal gold and specific proteins and the detection of low-concentration samples. The present invention provides a method for mixing colloidal gold of multiple different particle sizes to increase detection sensitivity, which can effectively improve sensitivity. The method is simple and easy to implement. After the large particle size colloidal gold and the small particle size colloidal gold are reasonably mixed and used in a certain ratio, there will be no problems of dragging, fading, false positives and poor specific binding ability. No additional reagents need to be added to improve sensitivity. The cost is low and it is suitable for mass production.

[0150] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for enhancing the detection sensitivity of colloidal gold immunochromatography, characterized in that: In colloidal gold immunochromatography testing, a combination of two colloidal gold particles of different particle sizes is used; specifically, small-particle colloidal gold and large-particle colloidal gold are respectively combined with antibodies and then concentrated more than twice, and then mixed for use; the particle size of the small-particle colloidal gold is 50-60 nm, and the particle size of the large-particle colloidal gold is 70-90 nm; the volume ratio of the small-particle colloidal gold to the large-particle colloidal gold is 8-12:1; the concentration of the small-particle colloidal gold is 1-3 g / L, and the concentration of the large-particle colloidal gold is 2 g / L.

2. The method according to claim 1, characterized in that The small-particle colloidal gold is 60nm colloidal gold, and the large-particle colloidal gold is 90nm colloidal gold.

3. The method according to claim 1 or 2, characterized in that The specific method is as follows: S1. Prepare antibody-labeled colloidal gold precipitates by labeling small colloidal gold particles (50-60 nm) and large colloidal gold particles (70-90 nm). S2. The antibody-labeled colloidal gold precipitate obtained in step S1 was redissolved using a reconstitution solution to obtain colloidal gold - antibody reconstituted products of different particle sizes; S3. The colloidal gold-antibody reconstituted products of different particle sizes obtained in step S2 are mixed and used.

4. A highly sensitive colloidal gold immunochromatographic detection method, characterized in that: Combine the method according to any one of claims 1 to 3 to perform colloidal gold immunochromatographic detection.

5. A colloidal gold-antibody reconstituted product combination capable of improving the detection sensitivity of colloidal gold immunochromatography, characterized in that: Contains colloidal gold-antibody reconstituted products of different particle sizes obtained by the method of claim 3.

6. Use of the colloidal gold-antibody reconstituted product combination according to claim 5 in the preparation of a highly sensitive colloidal gold immunoassay reagent strip or kit.

7. A highly sensitive colloidal gold immunoassay reagent strip or kit, characterized in that: Contains the colloidal gold-antibody reconstituted product combination according to claim 5.

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