Preparation and application of colloidal gold test paper

Abstract

The application belongs to the technical field of immune colloidal gold, and particularly relates to preparation and application of colloidal gold test paper. The application provides a colloidal gold test paper, which is prepared by adding a specially treated reaction pad and a filter pad, so that the prepared colloidal gold test paper has high specificity and high sensitivity. In the colloidal gold test paper, the filter pad is added with an anti-heterophilic antibody to eliminate interference of the heterophilic antibody and improve the specificity of detection, and the reaction pad is specially treated, so that the hydrophilic performance and liquid wetting time of the reaction pad are controlled. Therefore, the sensitivity is improved by controlling the sample reaction time and flow speed physical parameters in the lateral flow. The preparation method is simple, the prepared colloidal gold test paper has high specificity, high sensitivity and good usability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of immune colloidal gold, and particularly relates to preparation and application of colloidal gold test paper. BACKGROUND

[0002] The immune colloidal gold technology has the characteristics of simplicity, rapidness and no need of auxiliary equipment. The technology is currently developing rapidly and has a wide application in the field of biological detection. The classic immune colloidal gold technology includes a sample pad, a conjugate pad, a coated membrane, a water absorption pad and a PVC bottom plate. The coated membrane is coated with proteins on a nitrocellulose membrane in advance, the colloidal gold marker in the sample and the conjugate pad is reacted first, and then completes the reaction with the coated proteins on the nitrocellulose membrane in the process of lateral flow, and whether the substance to be detected exists in the sample is determined according to the coated line.

[0003] The immune colloidal gold technology has the disadvantages of insufficient sensitivity and interference of heterophilic antibodies. The sensitivity is related to various factors, including the flow speed of the sample, the reaction time, the marker, the background and the new biological amplification system. For some projects with high sensitivity requirements, such as infectious projects, the low sensitivity often causes missed detection and brings confusion in application. In the detection, especially in the detection by the double antibody sandwich method, the existence of heterophilic antibodies often causes false positives. The common method for eliminating heterophilic antibodies is to add anti-heterophilic antibodies in the sample pad, and the sample pad filters the initial sample preliminarily, only filters the obvious flocculent or particulate substances, and the reaction with the heterophilic antibodies is not sufficient, so the interference caused by the heterophilic antibodies cannot be completely eliminated.

[0004] In addition, researchers try various methods to improve the sensitivity of the immune colloidal gold. The patent with the application number 200880132189.2 "Method for signal amplification in immunochromatographic tests and immunochromatographic kit using the method" uses different markers to realize cascade reaction of two kinds of conjugates to achieve signal amplification and improve the detection sensitivity. The patent with the application number 201210507386.6 "Method for improving the detection sensitivity of colloidal gold immunochromatographic test strip" drops transparent reagent on the test paper after reaction to reduce the background of the test strip and improve the sensitivity. The patent with the application number 201710046695.0 "High-sensitivity colloidal gold test paper and preparation method and application thereof" adds avidin in the sample pad and biotin-labeled antibody in the conjugate pad, and uses the biotin and avidin system to improve the sensitivity. The above patents use marker cascade amplification, reduce the background signal, and use the biotin and avidin amplification system to improve the sensitivity, and do not involve optimization of the physical parameters such as the reaction time and flow speed of the sample in lateral flow. In addition, the existing immunochromatographic test strip also has some disadvantages. SUMMARY

[0005] Therefore, the present application aims to provide a preparation and application of colloidal gold test paper.

[0006] The present application provides a colloidal gold test paper, which comprises a nitrocellulose membrane, a sample pad, a filter pad, a binding pad, a reaction pad and a water absorption pad arranged in sequence.

[0007] The preparation raw material of the filter pad comprises a filter pad matrix and a filter pad treatment solution; the filter pad matrix is selected from glass fiber or polyester fiber; and the filter pad treatment solution comprises water and Tris, casein, bovine serum albumin and anti-heterophilic antibody.

[0008] The preparation raw material of the reaction pad comprises a reaction pad matrix and a reaction pad treatment solution; the reaction pad matrix comprises polyester glass fiber; and the reaction pad treatment solution comprises water and a hydrophobic surfactant, a hydrophilic surfactant, tris-hydroxymethyl aminomethane, an amino acid, casein, gelatin and bovine serum albumin.

[0009] In the present application, the nitrocellulose membrane is coated with a quality control antibody and a target protein antibody. Further, the quality control antibody comprises an IgG antibody, and the IgG antibody comprises goat anti-mouse IgG, goat anti-rabbit IgG, goat anti-human IgG, goat anti-chicken IgG, rabbit anti-human IgG, anti-goat IgG, anti-dog IgG, anti-mouse IgG, anti-chicken IgG, anti-cow IgG, anti-pig IgG; in the embodiment of the present application, the quality control antibody uses goat anti-mouse IgG antibody, and the concentration is 1.5 mg / ml.

[0010] In the present application, the target protein antibody is selected according to the detection target, and needs to be adaptively changed according to the target when the detection target changes. The detection target includes but is not limited to virus, bacteria, fungus and animal; the virus includes but is not limited to herpes virus, hepatitis B virus, influenza virus, measles virus, SARS virus, poliomyelitis virus, dengue fever virus, cytomegalovirus and new coronavirus; the target protein antibody of the new coronavirus includes new coronavirus N protein antibody and new coronavirus S protein antibody; the present application uses new coronavirus N protein mouse IgG antibody as the target protein antibody for detection of new coronavirus, and the concentration is 1.0 μl / cm.

[0011] In the present application, the material of the sample pad is selected from polyester glass fiber, glass fiber and non-woven fabric. The present application uses polyester glass fiber as the material of the sample pad, which is more conducive to uniform chromatography of the sample at a certain speed and concentration and then flows to the nitrocellulose membrane.

[0012] In the present application, the material of the binding pad is selected from the group consisting of glass fiber, non-woven fabric, dust-free paper, reactive film polyester film or polymer composite. The present application uses glass fiber as the material of the binding pad, which, together with other necessary technical features of the colloidal gold test paper according to the present application, is used for the preparation of colloidal gold test paper with high specificity and sensitivity.

[0013] In the present application, the filter pad treatment solution comprises water and (w / w): 6.0% Tris, 0.2% casein, 0.1% bovine serum albumin and 0.005% anti-heterophilic antibody, pH 8.3.

[0014] The reaction pad treatment solution comprises water and (w / w): 0.01-0.5% hydrophobic surfactant, 0.01-0.5% hydrophilic surfactant, 6.0% tris, 2% amino acid, 0.5% casein, 0.2% gelatin and 0.1% bovine serum albumin, pH 8.3.

[0015] Further, the anti-heterophilic antibody is preferably used with a coated labeled homologous antibody. Experimental results show that the use of anti-heterophilic antibody with a coated labeled homologous antibody can eliminate or reduce non-specific reactions. The anti-heterophilic antibody is selected from anti-murine antibody, anti-rabbit antibody, anti-ovine antibody or anti-chicken antibody. Further, the present application preferably uses anti-murine antibody, which is used with murine IgG antibody. When the system IgG antibody changes, the corresponding anti-heterophilic antibody needs to be replaced.

[0016] In the present application, the surfactant comprises hydrophilic surfactant and hydrophobic surfactant. In the present application, HBL represents the hydrophilic-lipophilic balance value of the hydrophilic group and the lipophilic group. The larger the HBL value of the surfactant, the stronger the hydrophilicity of the molecule.

[0017] In the present application, the HLB value of the hydrophilic surfactant is >24, which comprises pure lauryl sodium sulfate, N-hexadecyl-N-ethyl morpholinyl ethyl sulfate cation, ethylenediamine-oxalate block copolymer (Brij 35), (polyoxyethylene-polyoxypropylene) block copolymer (Pluronic F68), preferably Pluronic F68, which is used at a concentration of 0.01-0.5%. 1307) and (polyoxyethylene-polyoxypropylene) block copolymer (Pluronic F68), preferably Pluronic F68, which is used at a concentration of 0.01-0.5%. RF68), preferably RF68, which is used at a concentration of 0.01-0.5%.

[0018] In the present application, the HLB value of the hydrophobic surfactant is <5, which comprises glyceryl monostearate, hydroxylated lanolin, propylene glycol fatty acid ester, sorbitan monooleate, diethylene glycol fatty acid ester, octyl phenol ethoxylate (Triton X-100), sorbitan monostearate (Span 60), preferably Span 60, which is used at a concentration of 0.01-0.5%. CA210) and sorbitan monostearate (Span 60), preferably Span 60, which is used at a concentration of 0.01-0.5%. 60), preferably 60, using a concentration of 0.01-0.5%.

[0019] In the present application, the amino acid includes glycine and charged polar amino acid; the polar amino acid includes aspartic acid, glutamic acid, cysteine, arginine, lysine, histidine. The present application preferably glycine. Experiments show that the addition of amino acid increases the sensitivity of the reaction.

[0020] In the present application, when the filter pad treatment liquid is (w / w): 6.0% Tris, 0.2% casein, 0.1% bovine serum albumin and 0.005% anti-mouse antibody, pH 8.3; the reaction pad treatment liquid is (w / w): 0.01% (polyoxyethylene-polyoxypropylene) block copolymer, 0.5% sorbitan monostearate, 6.0% tris-hydroxymethyl aminomethane, 2% glycine, 0.5% casein, 0.2% gelatin and 0.1% bovine serum albumin, pH 8.3; the performance of the prepared test strip is more excellent.

[0021] In the present application, it is found in experiments that the improvement of the performance of the colloidal gold test strip is closely related to the material selection and reagent treatment steps of each component of the test strip, and various factors support each other and interact, ultimately making the prepared colloidal gold test strip specific and high in sensitivity.

[0022] The present application provides a preparation method of colloidal gold test strip, and the steps include:

[0023] Step 1, coupling colloidal gold with marker N protein antibody to obtain colloidal gold-marker antibody conjugate;

[0024] Step 2, spraying the colloidal gold-marker antibody conjugate on the binding pad;

[0025] Step 3, soaking the filter pad in the filter pad treatment liquid and drying;

[0026] Step 4, soaking the reaction pad in the reaction pad treatment liquid and drying;

[0027] Step 5, laying the nitrocellulose membrane on the pvc bottom plate and drying, and then laying the sample pad, filter pad, binding pad, reaction pad and water absorption pad on it in sequence to prepare the colloidal gold test strip.

[0028] In the preparation method of the present application, the average diameter of colloidal gold in step 1 is 20-60 nm; the preparation method of colloidal gold is: adding 0.01% (mass unit) of trisodium citrate solution into the boiling tetrachloroauric acid solution.

[0029] In step 1 of the present application, the colloidal gold needs to be adjusted to pH 8.0 using 0.2M K2CO3 before adding the label N protein antibody, and the concentration of the label N protein antibody is 10 μg / mL. After the colloidal gold reacts with the label N protein antibody for 30 min, the colloidal gold-label antibody conjugate is obtained by blocking with BSA at a concentration of 0.01 g / mL for 30 min.

[0030] In the preparation method of the present application, the obtained colloidal gold-label antibody conjugate is stored in 0.05M PB containing 5mg / ml BSA and 1mg / ml arginine at pH 7.4.

[0031] In step 2 of the present application, the colloidal gold-label antibody conjugate is sprayed on the binding pad at a concentration of 5.0 μl / cm.

[0032] In the present application, the drying refers to drying at room temperature or 37℃, and the suitable temperature is conducive to the maintenance of the activity of the material.

[0033] The present application provides the use of the colloidal gold test paper prepared by the preparation method in the preparation of a virus detection reagent or kit.

[0034] Further, the present application also provides a virus detection method, which comprises using the colloidal gold test paper of the present application. The detection mode can be visual inspection or machine reading, and the present application does not limit the same.

[0035] The present application provides a colloidal gold test paper, which is prepared by adding a specially treated reaction pad and a filter pad, so that the prepared colloidal gold test paper has high specificity and high sensitivity. In the colloidal gold test paper of the present application, the filter pad is added with an anti-heterophilic antibody to eliminate the interference of the heterophilic antibody and improve the specificity of the detection, and the reaction pad is specially treated to control the hydrophilic performance and liquid wetting time of the reaction pad. Thus, the physical parameters of the sample reaction time and flow rate in the lateral flow are controlled to improve the sensitivity. The preparation method is simple, and the prepared colloidal gold test paper has high specificity, high sensitivity and good usability. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The colloidal gold test paper is shown in the schematic diagram, wherein 1 is a sample pad, 2 is a filter pad, 3 is a binding pad, 4 is a reaction pad, 5 is a nitrocellulose membrane, and 6 is a water absorption pad. DETAILED DESCRIPTION

[0037] The present application provides the preparation and application of colloidal gold test paper, the person skilled in the art can learn from the content of this paper, and the process parameters are improved appropriately. It is particularly pointed out that all similar substitutions and changes are obvious to the person skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application of this paper without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0038] In the present application, each technical feature supports each other in function, interacts with each other, and affects the technical effect of the present application as a whole, and is protected as a whole.

[0039] The test materials used in the present application are all ordinary market products and can be purchased in the market.

[0040] The present application will be further described below in combination with examples:

[0041] Example 1 Preparation of colloidal gold test paper

[0042] (1) Preparation of colloidal gold: According to the method of Frens (Frens, G., Preparation of gold dispersions varying particle size: controlled nucleation for the regulation of the particle size in monodisperse gold suspensions in Nature: Physical Science 241 (1973), 20-22), colloidal gold with an average particle diameter of about 40 nm was prepared by adding 0.01% (mass unit) of trisodium citrate solution to the boiling tetrachloroauric acid solution.

[0043] (2) Preparation of colloidal gold-labeled antibody complex

[0044] Take 10ml colloidal gold, adjust the required pH value to 8.0 using 0.2M K2CO3, and add 100μg of label N protein antibody, react for 30min, then add 0.1g BSA for blocking for 30min, centrifuge the reaction and discard the supernatant, to obtain colloidal gold-labeled antibody conjugate. Store in 2ml 0.05M PB pH7.4 (containing 5mg / ml BSA and 1mg / ml arginine). Use at 4 degrees.

[0045] (3) Preparation of binding pad

[0046] Colloidal gold-labeled antibody conjugate was sprayed on glass fiber pad 30 cm x 0.5 cm at 5.0 μl / cm and dried at 37°C for standby.

[0047] Sheep anti-mouse IgG antibody and mouse IgG antibody against new crown N protein were coated on nitrocellulose membrane at 1.5 mg / ml and 1.0 μl / cm, and the nitrocellulose membrane was dried at 37°C on a pvc bottom plate for standby. On the dried nitrocellulose membrane with a pvc bottom plate, the sample pad, the binding pad coated with the labeled antibody, and the 30 cm long and 2 cm wide water absorption pad were pasted in turn, and the components were tightly connected to each other to make an immunochromatography test paper large plate. The plastic plate was cut into 4 mm wide immunochromatography test strips by a strip cutting machine, and was placed in a detection card. The sample to be tested was added to one end of the sample pad, and the result was observed after 15 minutes. The test line and the control line were recorded as positive if color lines appeared, and the test line was recorded as negative if no color lines appeared.

[0048] Example 2 Colloidal gold test paper preparation condition 2

[0049] (1) Preparation of colloidal gold: According to the method of Frens (Frens, G., Preparation of gold dispersions varying particle size: controlled nucleation for the regulation of the particle size in monodisperse gold suspensions in Nature: Physical Science 241 (1973), 20-22), colloidal gold with an average particle diameter of about 40 nm was prepared by adding 0.01% (mass unit) of trisodium citrate solution to a boiling tetrachloroauric acid solution.

[0050] (2) Preparation of colloidal gold-labeled antibody complex

[0051] Take 10 ml of colloidal gold, adjust the required pH value to 8.0 using 0.2M K2CO3, and add 100 μg of labeled N protein antibody, react for 30 min, then add 0.1 g of BSA for blocking for 30 min, centrifuge the reaction mixture and discard the supernatant to obtain colloidal gold-labeled antibody conjugate. Store in 2 ml of 0.05M PB pH 7.4 (containing 5 mg / ml BSA and 1 mg / ml arginine). Store at 4°C for standby.

[0052] (3) Preparation of binding pad

[0053] Colloidal gold-labeled antibody conjugate was sprayed on glass fiber pad 30 cm x 0.5 cm at 5.0 μl / cm and dried at 37°C for standby.

[0054] (4) Preparation of filter pad treatment solution (w / w):

[0055]

[0056] Adjust pH to 8.3 ± 0.1 with 1M hydrochloric acid

[0057] (5) Preparation of filter pad: Glass fiber pad with a length of 30 cm x width of 0.5 cm was soaked in 3 ml of filter pad treatment solution, and after the polyester fiberglass was completely wet, it was taken out, dried at 37°C, and then stored at room temperature after sealing.

[0058] (6) Preparation of reaction pad treatment solution (w / w):

[0059]

[0060] Adjust pH to 8.3 ± 0.1 with 1M hydrochloric acid

[0061] (7) Preparation of reaction pad: Polyester fiberglass pad with a thickness of 2 mm, length of 30 cm x width of 1 cm was soaked in 5 ml of reaction pad treatment solution, and after the polyester fiberglass was completely wet, it was taken out, dried at 37°C, and then stored at room temperature after sealing.

[0062] Sheep anti-mouse IgG antibody and mouse IgG antibody against new crown N protein were coated on a nitrocellulose membrane at a concentration of 1.5 mg / ml and 1.0 μl / cm, and the nitrocellulose membrane was dried on a pvc bottom plate at 37°C for standby. On the dried nitrocellulose membrane, the sample pad, filter pad containing anti-mouse antibody, binding pad coated with labeled antibody, and treated reaction pad were pasted in turn, and a 30 cm long and 2 cm wide water absorption pad was pasted at the end, and the components were tightly connected to each other to form an immunochromatography test paper plate. The plastic plate was cut into 4 mm wide immunochromatography test strips vertically by a cutting machine, and was placed in a detection card. The sample was added to the sample pad, and the result was observed after 15 minutes. If the detection line and the quality control line both appeared color lines, it was recorded as positive, and if the detection line did not appear color lines, it was recorded as negative.

[0063] Example 3: Preparation of colloidal gold test paper under condition 3

[0064] (1) Preparation of colloidal gold: According to the method of Frens (Frens, G., Preparation of gold dispersions varying particle size: controlled nucleation for the regulation of the particle size in monodisperse gold suspensions in Nature: Physical Science 241 (1973), 20-22), colloidal gold with an average particle diameter of about 40 nm was prepared by adding 0.01% (mass unit) of trisodium citrate solution to a boiling solution of tetrachloroauric acid.

[0065] (2) Preparation of colloidal gold-labeled antibody complex

[0066] Take 10 ml of colloidal gold, adjust the required pH value to 8.0 using 0.2M K2CO3, and add 100 μg of label N protein antibody, react for 30 min, then add 0.1 g of BSA for blocking for 30 min, centrifuge the reaction and discard the supernatant to obtain colloidal gold-labeled antibody complex. Store in 2 ml of 0.05M PB pH 7.4 (containing 5 mg / ml BSA and 1 mg / ml arginine). Use at 4 degrees.

[0067] (3) Preparation of binding pad

[0068] Spray colloidal gold-labeled antibody complex at 5.0 μl / cm on glass fiber pad 30 cm x 0.5 cm, dry at 37 degrees for standby.

[0069] (4) Preparation of filter pad treatment solution (w / w):

[0070]

[0071] Adjust the pH to 8.3 ± 0.1 with 1M hydrochloric acid

[0072] (5) Preparation of filter pad: Glass fiber pad with a length of 30 cm x width of 0.5 cm is soaked in 3 ml of filter pad treatment solution, and after the polyester fiber is completely wet, it is taken out, dried at 37°C, and then stored at room temperature after sealing.

[0073] (6) Preparation of reaction pad treatment solution (w / w):

[0074]

[0075] Adjust the pH to 8.3 ± 0.1 with 1M hydrochloric acid

[0076] (7) Preparation of reaction pad: polyester fiberglass pad with thickness of 2 mm, length of 30 cm and width of 1 cm was soaked in 5 ml of reaction pad treatment solution. After the polyester fiberglass was completely wet, it was taken out and dried at 37°C, and then stored at room temperature after sealing.

[0077] Sheep anti-mouse IgG antibody and mouse IgG antibody against new crown N protein were coated on a nitrocellulose membrane at an amount of 1.5 mg / ml and 1.0 μl / cm, respectively, and the nitrocellulose membrane was dried on a pvc bottom plate at 37°C for standby. On the dried nitrocellulose membrane, a sample pad, a filter pad containing an anti-mouse antibody, a binding pad coated with a labeled antibody, and a treated reaction pad were sequentially pasted, and a water absorption pad with a length of 30 cm and a width of 2 cm was pasted at the end, and each component was tightly connected to form an immunochromatography test paper plate. The plastic plate was cut into 4 mm wide immunochromatography test strips by a strip cutting machine, and was placed in a detection card. The sample to be tested was added to the sample pad, and the result was observed after 15 minutes. The test line and the control line were recorded as positive if color lines appeared, and the test line was recorded as negative if no color lines appeared.

[0078] Example 4 Preparation conditions of colloidal gold test paper 4

[0079] (1) Preparation of colloidal gold: According to the Frens method (Frens, G., Preparation of gold dispersions varying particle size: controlled nucleation for the regulation of the particle size in monodisperse gold suspensions in Nature: Physical Science 241 (1973), 20-22), colloidal gold with an average particle diameter of about 40 nm was prepared by adding 0.01% (mass unit) of trisodium citrate solution to a boiling tetrachloroauric acid solution.

[0080] (2) Preparation of colloidal gold-labeled antibody complex

[0081] Take 10 ml of colloidal gold, adjust the required pH value to 8.0 using 0.2M K2CO3, and add 100 μg of labeled N protein antibody, react for 30 min, then add 0.1 g of BSA for blocking for 30 min. Centrifuge the reaction mixture and discard the supernatant to obtain a colloidal gold-labeled antibody conjugate. Store in 2 ml of 0.05M PB pH 7.4 (containing 5 mg / ml BSA and 1 mg / ml arginine). Store at 4°C.

[0082] (3) Preparation of binding pad

[0083] Colloidal gold-labeled antibody conjugate was sprayed on a glass fiber pad 30 cm x 0.5 cm at 5.0 μl / cm and dried at 37°C for standby.

[0084] (4) Preparation of filter pad treatment solution (w / w):

[0085]

[0086] Adjust pH to 8.3 ± 0.1 with 1M hydrochloric acid

[0087] (5) Preparation of filter pad: A glass fiber pad with a length of 30 cm x width of 0.5 cm was soaked in 3 ml of filter pad treatment solution, and after the polyester fiber was completely wet, it was taken out, dried at 37°C, and then stored at room temperature after sealing for standby.

[0088] (6) Preparation of reaction pad treatment solution (w / w):

[0089]

[0090] Adjust pH to 8.3 ± 0.1 with 1M hydrochloric acid

[0091] (7) Preparation of reaction pad: A polyester fiber pad with a thickness of 2 mm, a length of 30 cm x width of 1 cm was soaked in 5 ml of reaction pad treatment solution, and after the polyester fiber was completely wet, it was taken out, dried at 37°C, and then stored at room temperature after sealing for standby.

[0092] Sheep anti-mouse IgG antibody and mouse IgG antibody against new crown N protein were coated on a nitrocellulose membrane at 1.5 mg / ml and 1.0 μl / cm, and the nitrocellulose membrane was dried at 37°C on a pvc bottom plate for standby. On the dried nitrocellulose membrane, the sample pad, the conjugate pad coated with the labeled antibody, and the treated reaction pad were pasted in turn, and finally a water absorption pad with a length of 30 cm and a width of 2 cm was pasted, and the components were tightly connected to each other to form an immunochromatography test paper large plate. The plastic plate was cut into 4 mm wide immunochromatography test strips vertically by a cutting machine, and then placed in a detection card. The sample to be tested was added to one end of the sample pad, and the result was observed after 15 minutes. If both the detection line and the quality control line appeared color lines, it was recorded as positive, and if the detection line did not appear color lines, it was recorded as negative.

[0093] Example 5 Colloidal gold test paper preparation condition 5

[0094] (1) Preparation of colloidal gold: According to the method of Frens (Frens, G., Preparation of gold dispersions varying particle size: controlled nucleation for the regulation of the particle size in monodisperse gold suspensions in Nature: Physical Science 241 (1973), 20-22), colloidal gold with an average particle diameter of about 40 nm was prepared by adding 0.01% (mass unit) of trisodium citrate solution to a boiling tetrachloroauric acid solution.

[0095] (2) Preparation of colloidal gold-labeled antibody complex

[0096] Take 10 ml of colloidal gold, adjust the required pH value to 8.0 using 0.2M K2CO3, and add 100 μg of label N protein antibody, react for 30 min, then add 0.1 g of BSA for blocking for 30 min, centrifuge the reaction to discard the supernatant, and obtain the colloidal gold-labeled antibody complex. Store in 2 ml of 0.05M PB pH 7.4 (containing 5 mg / ml BSA and 1 mg / ml arginine). Use at 4 degrees.

[0097] (3) Preparation of binding pad

[0098] Spray colloidal gold-labeled antibody complex at 5.0 μl / cm on glass fiber pad 30 cm x 0.5 cm, dry at 37 degrees for standby.

[0099] (4) Preparation of reaction pad treatment solution (w / w):

[0100]

[0101] Adjust the pH to 8.3 ± 0.1 with 1M hydrochloric acid

[0102] (5) Preparation of reaction pad: Soak the polyester fiber pad with a thickness of 2 mm; length 30 cm x width 1 cm in 5 ml of reaction pad treatment solution, take it out after the polyester fiber is completely wet, dry at 37°C, and store at room temperature after sealing for standby.

[0103] Sheep anti-mouse IgG antibody, new crown N protein mouse IgG antibody is coated on a nitrocellulose membrane at an amount of 1.5 mg / ml, 1.0 μl / cm, and the nitrocellulose membrane is dried on a pvc bottom plate at 37 degrees for standby. On the dried nitrocellulose membrane, a sample pad, a binding pad coated with a labeled antibody, a treated reaction pad are pasted in turn, and finally a 30 cm long and 2 cm wide water absorption pad is pasted, and the components are tightly connected between each other to form an immunochromatography test paper plate, and the pasted plastic plate is cut into 4 mm wide immunochromatography test strips by a strip cutting machine, and placed in a detection card. Add the sample to be tested at one end of the sample pad, and observe the results after 15 minutes. If both the detection line and the quality control line appear color lines, it is recorded as positive, and if the detection line does not appear color lines, it is recorded as negative.

[0104] The results are as follows:

[0105]

[0106]

[0107] The sensitivity of the colloidal gold test paper prepared in Examples 1-5 was detected under different concentrations of detection targets (new crown N protein or N protein) and different interference backgrounds. Different concentrations of N protein were added to the sample diluent to prepare detection targets with different concentrations; different concentrations of HAMA (HAMA is human anti-mouse antibody) were added to the sample diluent to prepare interference samples with different HAMA concentrations; the sample diluent was: 0.02M Tris-HCl pH 7.4, 0.2% Triton X-100 (w / w), 0.9% NaCl (w / w).

[0108] Result analysis: by comparing with the control scheme, comparing schemes 3 and 5, the specific improvement of the filter pad is obvious; comparing schemes 3 and 4, the addition of specific amino acids increases the sensitivity of the reaction; comparing the effects of different hydrophilic and hydrophobic treatments of schemes 2 and 3 on the detection results; comparing schemes 1 and 3, the conventional chromatography technology scheme is compared with the technical scheme of the present application, and the sensitivity of the detection is improved by about 20 times by adding the treated filter pad and reaction pad.

[0109] The above is only a preferred embodiment of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A colloidal gold test paper, comprising a nitrocellulose membrane, a sample pad, a filter pad, a binding pad, a reaction pad and a water absorption pad arranged in sequence. The filter pad is prepared from a filter pad substrate and a filter pad treatment solution: the filter pad substrate is selected from glass fiber or polyester fiber; the filter pad treatment solution comprises water and (w / w): 6.0% Tris, 0.2% casein, 0.1% bovine serum albumin and 0.005% anti-mouse antibody, pH 8.3; The reaction pad is prepared from a reaction pad substrate and a reaction pad treatment solution: the reaction pad substrate comprises polyester fiberglass; the reaction pad treatment solution comprises water and (w / w): 0.5% sorbitan monostearate, 0.01% polyoxyethylene-polyoxypropylene block copolymer, 6.0% tris-hydroxymethyl aminomethane, 2% glycine, 0.5% casein, 0.2% gelatin and 0.1% bovine serum albumin, pH 8.

3.

2. The colloidal gold test paper according to claim 1, characterized in that, The nitrocellulose membrane is coated with a quality control antibody and a target protein antibody.

3. The colloidal gold test paper according to claim 2, characterized in that, The quality control antibody is a goat anti-mouse IgG antibody; the target protein antibody is a new crown N protein IgG antibody.

4. The colloidal gold test paper of claim 1, wherein The sample pad is made of polyester fiberglass; The binding pad is made of glass fiber.

5. A method for preparing the colloidal gold test paper of any one of claims 1-4, comprising the following steps: Step 1, coupling colloidal gold with marker N protein antibody to obtain colloidal gold-marker antibody conjugate; Step 2, spraying the colloidal gold-marker antibody conjugate on the binding pad; Step 3, soaking the filter pad in the filter pad treatment solution and drying; Step 4, soaking the reaction pad in the reaction pad treatment solution and drying; Step 5, placing the nitrocellulose membrane on a pvc base plate and drying, then placing the sample pad, filter pad, binding pad, reaction pad and water absorption pad on it in sequence to obtain the colloidal gold test paper.

6. The preparation method of claim 5, wherein The average diameter of the colloidal gold is 20-60 nm; The concentration of the marker N protein antibody is 10 μg / mL; The spraying concentration of the colloidal gold-marker antibody conjugate on the binding pad is 5.0 μl / cm.

7. Use of the colloidal gold test paper of any one of claims 1-4 and the colloidal gold test paper prepared by the preparation method of claim 5 or 6 in the preparation of a viral detection reagent or kit.

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