Immunogold for combined detection of multiple virus antigens, preparation method of immunogold, gold label pad, test strip and kit

Abstract

The invention relates to the field of rapid diagnosis, and particularly discloses immunogold for combined detection of multiple virus antigens, a preparation method of the immunogold, a gold-labeled pad, a test strip and a kit. Wherein the immunogold comprises the following raw materials: colloidal gold, a mycoplasma pneumoniae monoclonal antibody 1, an adenovirus monoclonal antibody 1, a syncytial virus monoclonal antibody 1 and sodium lauryl polyoxyethylene ether sulfate, the colloidal gold is prepared by a seed growth method, and the seed growth method takes hexadecyl trimethyl ammonium bromide and polyethylene glycol 20000 as stabilizers. The mass ratio of the hexadecyl trimethyl ammonium bromide to the polyethylene glycol 20000 is 1: (0.8-1.2). After the prepared immunogold is applied to the kit, respiratory syncytial virus, adenovirus and mycoplasma pneumoniae antigens can be detected at the same time, the kit has the advantages of being convenient to use, easy to operate and convenient to popularize, a detection sample is an oropharynx swab, non-invasive sampling is achieved, and cross infection caused by using modes such as blood sampling is avoided.

Description

Technical Field

[0001] The present application relates to the field of rapid diagnosis technology, and more specifically, to an immunogold for combined detection of multiple virus antigens and a preparation method thereof, as well as a gold label pad, a test strip and a test kit. Background Art

[0002] Respiratory syncytial virus (RSV) belongs to the genus Pneumovirus of the family Paramyxoviridae and has only one serotype. It mainly causes lower respiratory tract infections such as bronchiolitis and pneumonia in infants under 6 months old, and upper respiratory tract infections such as rhinitis and colds in older children and adults.

[0003] Adenovirus respiratory tract infection, pathogenic adenovirus types 3, 4, and 7 are closely related to pneumonia. Clinical manifestations of acute respiratory tract infection: fever, pharyngitis, cough without sputum, malaise, chills, myalgia and headache. Upper respiratory tract infection: acute fever, pharyngitis, tonsillitis, rhinitis, fever, cough, pharyngeal wall exudate, etc. Lower respiratory tract infection: bronchitis, pneumonia. The onset is slow, first manifested as fever, cough, etc., high fever (39-41°C) lasts for 8-14 days, dyspnea occurs 3-4 days after onset, and pulmonary signs appear later. Symptoms such as mental depression and drowsiness. Extrapulmonary symptoms such as kidney disease and hepatomegaly can be seen. It is the primary factor causing hospitalization of infants and young children due to viral respiratory tract infection, which seriously endangers children's health, especially for premature infants, infants and young children with congenital heart disease or primary immunodeficiency.

[0004] Mycoplasma pneumonia (M.Pneumonia) is the pathogen of human mycoplasma pneumonia. The pathological changes of mycoplasma pneumonia are mainly interstitial pneumonia, sometimes complicated by bronchopneumonia, which is called primary atypical pneumonia. It is mainly transmitted through droplets, with an incubation period of 2 to 3 weeks, and the highest incidence rate is in adolescents. The clinical symptoms are mild or even asymptomatic. If there are any, they are only general respiratory symptoms such as headache, sore throat, fever, cough, etc., but there are also individual deaths reported. It can occur all year round, but mostly in autumn and winter.

[0005] At present, the detection methods for respiratory syncytial virus, adenovirus, and mycoplasma pneumoniae used in clinical tests mainly include PCR method and colloidal gold method. Among them, the colloidal gold method has two indicators: antigen and antibody. Only fluorescent PCR methodology and colloidal gold antibody detection method can detect these three indicators at the same time, and there is no colloidal gold antigen detection method. Among them, the sample processing of the fluorescent PCR method is cumbersome and time-consuming, and it must be assisted by related instruments and equipment, and the operator must undergo professional training to be competent, which limits the use of ordinary users; the sample types of the colloidal gold antibody detection method are whole blood, serum, and plasma, and the window period is longer than that of the antigen detection method. It cannot be detected immediately after infection. In addition, blood testing is a traumatic test, and blood transmission is one of the three major transmission routes of infectious diseases such as AIDS and syphilis. The shortcomings of the blood testing method are: (1) the risk of medical staff being stabbed and infected during blood drawing; (2) the risk of infection caused by the waste and disposal of various related medical equipment after blood drawing; (3) patients cannot draw blood for testing by themselves to ensure personal privacy.

[0006] Therefore, the detection kits for simultaneous diagnosis of respiratory syncytial virus, adenovirus, and Mycoplasma pneumoniae produced in the prior art cannot meet the needs of rapid differential diagnosis. Summary of the invention

[0007] The present application provides immunogold for combined detection of multiple virus antigens and a preparation method thereof, as well as a gold label pad, a test strip and a test kit. After the immunogold prepared in the present application is used in the test kit, respiratory syncytial virus, adenovirus, and Mycoplasma pneumoniae antigens can be detected simultaneously. It has the advantages of being easy to use, simple to operate, and easy to promote. The test sample is an oropharyngeal swab, which is non-invasive and avoids cross infection caused by blood collection and other usage methods.

[0008] In the first aspect, the present application provides an immunogold for combined detection of multiple virus antigens, using the following technical solution:

[0009] An immunogold for combined detection of multiple virus antigens, wherein the raw materials of the immunogold include colloidal gold, 1 monoclonal antibody to Mycoplasma pneumoniae, 1 monoclonal antibody to adenovirus, 1 monoclonal antibody to syncytial virus, and sodium lauryl alcohol polyoxyethylene ether sulfate;

[0010] The colloidal gold simultaneously labels Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 to form an antibody conjugate, and the ratio of the mass amount (g) of the sodium lauryl polyoxyethylene ether sulfate to the volume amount (ml) of the colloidal gold is (0.01-0.05): 1;

[0011] The colloidal gold is prepared by a seed growth method, and the seed growth method uses hexadecyltrimethylammonium bromide and polyethylene glycol 20000 as stabilizers, and the mass ratio of the hexadecyltrimethylammonium bromide to the polyethylene glycol 20000 is 1:(0.8-1.2).

[0012] Furthermore, the preparation method of the colloidal gold comprises the following steps:

[0013] S1: Add ultrapure water to the chloroauric acid solution, heat and stir under reflux until boiling, and add sodium citrate solution; the mass ratio of chloroauric acid to sodium citrate in the mixed solution is 1:(1.6-2), and the concentration of chloroauric acid is 0.19-0.2g / L; after the solution turns into wine red, continue heating and reflux for 10-15min, then stop heating, and wait for the solution to cool naturally to room temperature to obtain a small particle gold seed solution;

[0014] S2: Add ultrapure water to the chloroauric acid solution, at which time the chloroauric acid concentration in the mixed solution is 0.09-0.1 g / L, and then add the gold seed solution, the ratio of the mass amount of chloroauric acid (g) to the volume amount of gold seed solution (L) in S2 is 1:(0.1-0.2); add a stabilizer, heat to 70-90°C, add a reducing agent to react, and naturally cool to room temperature to obtain colloidal gold.

[0015] Furthermore, in the stabilizer, the mass ratio of hexadecyltrimethylammonium bromide to chloroauric acid in S2 is (80-120):1.

[0016] Furthermore, in step S2, a certain amount of reducing agent is added every 9-11 minutes for 54-66 minutes.

[0017] Further, the reducing agent is a mixture of hydroxylamine hydrochloride and sodium citrate or a mixture of p-phenylenediamine phenol and sodium citrate. Further, the reducing agent is a solution containing 40mM hydroxylamine hydrochloride and 10g / L sodium citrate or a solution containing 0.3M p-phenylenediamine phenol and 10g / L sodium citrate; the ratio of the mass amount (g) of chloroauric acid in S2 to the volume amount (L) of the reducing agent each time is 1: (0.015-0.02).

[0018] In a second aspect, the present application provides a method for preparing immunogold for combined detection of multiple viral antigens, using the following technical solution:

[0019] A method for preparing immunogold for combined detection of multiple virus antigens comprises the following steps: taking a certain amount of colloidal gold in a beaker, adding sodium lauryl polyoxyethylene ether sulfate, wherein the ratio of the mass amount (g) of sodium lauryl polyoxyethylene ether sulfate to the volume amount (ml) of colloidal gold is (0.01-0.05):1; adjusting the pH value to 7.2-7.8, adding 1 monoclonal antibody of Mycoplasma pneumoniae, 1 monoclonal antibody of adenovirus, and 1 monoclonal antibody of syncytial virus to the colloidal gold, wherein the ratio of the mass amount (ug) of each antibody to the volume amount (ml) of colloidal gold is (50-75):(4-6), and reacting at room temperature with stirring for 8-15 minutes;

[0020] After centrifugation, the supernatant is discarded, and blocking solution is added for blocking, the volume ratio of colloidal gold to blocking solution is (10-20):1; after centrifugation, the supernatant is discarded, and preservation solution is added for re-dissolution to obtain colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 conjugate, namely immunogold.

[0021] In a third aspect, the present application provides a gold label pad, which adopts the following technical solution:

[0022] A gold-labeled pad is prepared by diluting an antibody conjugate and then soaking a blank pad, and then drying to obtain the gold-labeled pad.

[0023] Furthermore, the antibody conjugate was diluted 20 times and then soaked in a blank gasket.

[0024] In a fourth aspect, the present application provides a test strip for combined detection of multiple virus antigens, which adopts the following technical solution:

[0025] A test strip for combined detection of multiple virus antigens comprises a base plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorbent pad, wherein the sample pad, the gold label pad, the nitrocellulose membrane and the water absorbent pad are overlapped and pasted on the base plate in sequence according to the chromatography direction, the nitrocellulose membrane is coated with a quality control line and a detection line, the quality control line is coated with sheep anti-mouse IgG, the detection lines comprise detection line T1, detection line T2 and detection line T3, the detection line T1 is coated with a mycoplasma pneumoniae monoclonal antibody 2, the detection line T2 is coated with an adenovirus monoclonal antibody 2, and the detection line T3 is coated with a syncytial virus monoclonal antibody 2.

[0026] In a fifth aspect, the present application provides a kit for combined detection of multiple viral antigens, which adopts the following technical solution:

[0027] A kit for combined detection of multiple virus antigens, comprising a test strip and a lysis solution.

[0028] Furthermore, the lysate includes a blocking agent, and the blocking agent is dithiothreitol or TCEP hydrochloride. Still further, in the lysate, the concentration of the blocking agent is 0.1-0.5 g / L.

[0029] In summary, this application has the following beneficial effects:

[0030] 1. This application uses the antibody sandwich principle to qualitatively detect respiratory syncytial virus, adenovirus, and mycoplasma pneumonia antigens in oral pharyngeal swab samples. When respiratory syncytial virus, adenovirus, and mycoplasma pneumonia antigens are present in the sample during detection, the antigen in the sample reacts with the colloidal gold-labeled mycoplasma pneumonia monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 on the conjugate pad to form a labeled antigen-antibody complex, which is chromatographed upward by capillary action and captured by the corresponding detection line coated on the nitrocellulose membrane, and a red strip appears. The complex continues to chromatograph upward and is captured by the quality control line (C line) antibody (sheep anti-mouse IgG antibody) coated on the nitrocellulose membrane, and a red strip appears. When the content of the analyte in the sample is lower than the minimum detection limit, the detection line does not develop color. The respiratory syncytial virus, adenovirus, and mycoplasma pneumonia antigen detection kit provided detects oropharyngeal swab samples, adds samples once, and reports the test results of three indicators simultaneously within 15 minutes.

[0031] 2. The present application provides a colloidal gold prepared by a seed growth method, and in the preparation process, hexadecyltrimethylammonium bromide and polyethylene glycol 20000 are used as stabilizers. The colloidal gold obtained by this method can not only simultaneously label Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1 and syncytial virus monoclonal antibody 1, so that the formed antibody conjugate is used to prepare a gold label pad, and a detection kit for simultaneous diagnosis of respiratory syncytial virus, adenovirus and Mycoplasma pneumoniae can be obtained; and the colloidal gold prepared in the present application has a suitable particle size, good uniformity, high sensitivity and good specificity.

[0032] 3. In the labeling process, the present application adds sodium lauryl polyoxyethylene ether sulfate, which is weakly alkaline in aqueous solution, which helps to further maintain the weakly alkaline state of the labeling environment and reduce the dead gold aggregation phenomenon caused by the change of the isoelectric point of the labeled antibody due to the change of pH value; secondly, AES has good dispersibility and antistatic effect, and the colloidal gold particles, labeled antibodies, and gold-labeled conjugates can be evenly distributed in the reaction system, reducing the large difference in sensitivity of different items caused by the difference in coupling efficiency under the same pH conditions during the colloidal gold labeling of multiple antibodies due to the different isoelectric points of antibodies, and even the occurrence of dead gold aggregation.

[0033] 4. The present application can form an antibody conjugate with one labeling, and only one gold label pad is needed in the test strip, thus reducing the production cost of the test kit.

[0034] 5. This application uses colloidal gold as a carrier, which is convenient and quick to detect. The result can be interpreted without the help of relevant instruments and equipment, and it has higher sensitivity than the traditional colloidal gold preparation process.

[0035] 6. Adding dithiothreitol or TCEP hydrochloride as a blocking agent to the lysate of the kit of the present application can improve the specificity of the product. DETAILED DESCRIPTION

[0036] The present application is further described in detail below with reference to the embodiments.

[0037] Unless otherwise specified, the relevant raw materials in this example can be obtained from the market, and the preparation of relevant reagents is shown in Table 1.

[0038] Table 1 Table of Reagent Components Related to the Examples

[0039]

[0040]

[0041] Example

[0042] The embodiment of the present application first provides an immunogold for combined detection of multiple virus antigens, the raw materials of which include colloidal gold, Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, syncytial virus monoclonal antibody 1, and sodium lauryl alcohol polyoxyethylene ether sulfate.

[0043] Wherein, colloidal gold simultaneously labels Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 to form an antibody conjugate.

[0044] The colloidal gold is prepared by a seed growth method, and during the preparation of the colloidal gold, a mixture of hexadecyltrimethylammonium bromide and polyethylene glycol 20000 is added as a stabilizer during the gold seed growth process.

[0045] Specifically, the preparation method of colloidal gold comprises the following steps:

[0046] S1: Add ultrapure water to the chloroauric acid solution, heat and stir under reflux until boiling, and add sodium citrate solution; the mass ratio of chloroauric acid to sodium citrate in the mixed solution is 1:(1.6-2), and the concentration of chloroauric acid is 0.19-0.2g / L; after the solution turns wine red, continue to heat and reflux for 10-15min, then stop heating, and wait for the solution to cool naturally to room temperature to obtain a small particle gold seed solution;

[0047] S2: Add ultrapure water to the chloroauric acid solution, at which time the chloroauric acid concentration in the mixed solution is 0.09-0.1 g / L, and then add the gold seed solution, the ratio of the mass amount of chloroauric acid (g) to the volume amount of gold seed solution (L) in S2 is 1:(0.1-0.2); add a stabilizer, heat to 70-90°C, add a reducing agent to react, and naturally cool to room temperature to obtain colloidal gold.

[0048] Furthermore, in step S2, the stabilizer is a mixture of hexadecyltrimethylammonium bromide and polyethylene glycol 20000, the mass ratio of hexadecyltrimethylammonium bromide to polyethylene glycol 20000 is 1:(0.8-1.2), and the mass ratio of hexadecyltrimethylammonium bromide to chloroauric acid in S2 is (80-120):1.

[0049] Further, in step S2, a certain amount of reducing agent is added every 9-11 minutes for 54-66 minutes, and specifically, a certain amount of reducing agent is added every 10 minutes for 60 minutes.

[0050] Further, the reducing agent is a mixture of hydroxylamine hydrochloride and sodium citrate or a mixture of p-phenylenediamine phenol and sodium citrate. In the mixture of hydroxylamine hydrochloride and sodium citrate, the ratio of the amount of chloroauric acid (g), the amount of hydroxylamine hydrochloride (mmol), and the amount of sodium citrate (g) in S2 is 1: (0.6-0.8): (0.15-0.2). In the mixture of p-phenylenediamine phenol and sodium citrate, the ratio of the amount of chloroauric acid (g), the amount of p-phenylenediamine phenol (mol), and the amount of sodium citrate (g) in S2 is 1: (0.0045-0.006): (0.15-0.2).

[0051] Specifically, the reducing agent is prepared in advance, and the reducing agent is specifically a solution containing 40mM hydroxylamine hydrochloride and 10g / L sodium citrate or a solution containing 0.3M p-phenylenediamine phenol and 10g / L sodium citrate; the ratio of the mass amount of chloroauric acid (g) in S2 to the volume amount of the reducing agent each time (L) is 1: (0.015-0.02).

[0052] The present application also provides a method for preparing immunogold for joint detection of multiple virus antigens, comprising the following steps: taking a certain amount of colloidal gold in a beaker, adding sodium lauryl polyoxyethylene ether sulfate, the ratio of the mass amount (g) of sodium lauryl polyoxyethylene ether sulfate to the volume amount (ml) of colloidal gold is (0.01-0.05):1; adjusting the pH value to 7.2-7.8, adding 1 Mycoplasma pneumoniae monoclonal antibody, 1 adenovirus monoclonal antibody, and 1 syncytial virus monoclonal antibody to the colloidal gold, the ratio of the mass amount (ug) of each antibody to the volume amount (ml) of colloidal gold is (50-75):(4-6), and stirring at room temperature for 8-15 minutes;

[0053] After centrifugation, the supernatant was discarded, and a blocking solution was added for blocking, and the volume ratio of colloidal gold to the blocking solution was (10-20): 1; after centrifugation, the supernatant was discarded, and the preservation solution was added for re-dissolution to obtain colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 conjugate. Specifically, the centrifugation conditions for the two times were: temperature 4°C, speed 8000-10000rpm, 15min), and blocking solution blocking for 20min.

[0054] The present application also provides a kit for combined detection of multiple virus antigens, which mainly includes a test strip and a lysis solution. The lysis solution includes a blocking agent, which is dithiothreitol or TCEP hydrochloride, and specifically, the concentration of the blocking agent is 0.1-0.5 g / L.

[0055] The test strip includes a base plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorbent pad. The sample pad, the gold label pad, the nitrocellulose membrane and the water absorbent pad are overlapped and pasted on the base plate in sequence according to the chromatography direction. The nitrocellulose membrane is coated with a quality control line and a detection line. The quality control line is coated with sheep anti-mouse IgG. The detection lines include detection line T1, detection line T2 and detection line T3. Detection line T1 is coated with Mycoplasma pneumoniae monoclonal antibody 2, detection line T2 is coated with adenovirus monoclonal antibody 2, and detection line T3 is coated with syncytial virus monoclonal antibody 2.

[0056] The preparation method of the gold-labeled pad is as follows: dilute the antibody conjugate 20 times, soak the blank pad, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0057] The sample pad treatment method is as follows: soak the blank pad in the sample pad treatment solution, take it out and dry it at 37°C.

[0058] The preparation method of the nitrocellulose membrane is as follows: dilute 2 Mycoplasma pneumoniae monoclonal antibodies, 2 adenovirus monoclonal antibodies, 2 syncytial virus monoclonal antibodies, and goat anti-mouse IgG to corresponding concentrations with coating solution, use a film scribing instrument to coat corresponding positions on the nitrocellulose membrane in sequence, and dry at 45°C for 2 hours.

[0059] The following is an explanation through specific embodiments.

[0060] Example 1

[0061] This embodiment provides an immunogold for combined detection of multiple virus antigens. The immunogold is an antibody conjugate formed by simultaneously labeling Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 with colloidal gold.

[0062] Specifically, colloidal gold is prepared by a seed growth method, and the preparation method of colloidal gold comprises the following steps:

[0063] S1: Add 2mL of 10g / L chloroauric acid solution to 100mL of ultrapure water, heat and stir under reflux until boiling, add 4mL of 10g / L sodium citrate solution, the mass ratio of chloroauric acid to sodium citrate in the mixture is 1:2, and the concentration of chloroauric acid is 0.2g / L; after the solution turns wine red, continue to heat and reflux for 15min, then stop heating, wait for the solution to cool naturally to room temperature, and obtain a small particle gold seed solution;

[0064] S2: Add 1 mL of 10 g / L chloroauric acid solution to 100 mL of ultrapure water. At this time, the concentration of chloroauric acid in the mixed solution is 0.01 g / L. Then add 1 mL of gold seed solution. The ratio of the mass amount of chloroauric acid (g) to the volume amount of gold seed solution (L) in S2 is 1:0.1. Add stabilizer, heat to 90°C, add a certain amount of reducing agent every 10 minutes, continue for 1 hour, and naturally cool to room temperature to obtain colloidal gold.

[0065] In the above preparation process, the stabilizer is a mixture of 0.8 g of hexadecyltrimethylammonium bromide and 0.8 g of polyethylene glycol 20000. The mass ratio of hexadecyltrimethylammonium bromide to polyethylene glycol 20000 is 1:1, and the mass ratio of hexadecyltrimethylammonium bromide to chloroauric acid in S2 is 80:1.

[0066] The reducing agent is a solution containing 40 mM hydroxylamine hydrochloride and 10 g / L sodium citrate. Every ten minutes, 150 uL of the reducing agent is added; that is, the ratio of the mass amount of chloroauric acid (g) to the volume amount of the reducing agent each time (L) is 1:0.015.

[0067] The present application also provides a method for preparing immunogold for combined detection of multiple virus antigens, comprising the following steps: taking 10 mL of colloidal gold in a beaker, adding 0.1 g of sodium lauryl polyoxyethylene ether sulfate and mixing evenly, adjusting the pH value to 7.5 with 0.2 M potassium carbonate solution, adding 100 ug of Mycoplasma pneumoniae monoclonal antibody 1, 100 ug of adenovirus monoclonal antibody 1, and 100 ug of syncytial virus monoclonal antibody 1, and stirring at room temperature for reaction for 10 minutes; the ratio of the mass amount (ug) of each antibody to the volume amount (ml) of colloidal gold is (50):(5).

[0068] After centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of blocking solution was added for blocking for 20 min, with the volume ratio of colloidal gold to blocking solution being 10:1; after centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of preservation solution was added for reconstitution to obtain colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 conjugates.

[0069] The present application also provides a kit for combined detection of multiple virus antigens, mainly comprising a test strip and a lysate. The test strip comprises a base plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water-absorbing pad, the sample pad, the gold label pad, the nitrocellulose membrane and the water-absorbing pad are sequentially overlapped and pasted on the base plate in the direction of chromatography, the nitrocellulose membrane is coated with a quality control line and a detection line, the quality control line is coated with sheep anti-mouse IgG, the detection line comprises a detection line T1, a detection line T2, and a detection line T3, the detection line T1 is coated with a Mycoplasma pneumoniae monoclonal antibody 2, the detection line T2 is coated with an adenovirus monoclonal antibody 2, and the detection line T3 is coated with a syncytial virus monoclonal antibody 2.

[0070] The preparation method of the gold-labeled pad is as follows: dilute the antibody conjugate 20 times, soak the blank pad, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0071] The sample pad treatment method is as follows: soak the blank pad in the sample pad treatment solution, take it out and dry it at 37°C.

[0072] The preparation method of the nitrocellulose membrane is as follows: the original monoclonal antibody 2 is diluted to 1 mg / mL with the coating solution, and coated at the detection line T1 position on the nitrocellulose membrane with a film stripper at a parameter of 1uL / cm;

[0073] Adenovirus monoclonal antibody 2 was diluted to 1 mg / mL with coating solution and coated at the detection line T2 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0074] The syncytial virus monoclonal antibody 2 was diluted to 1 mg / mL with the coating solution and coated at the detection line T3 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0075] Dilute goat anti-mouse IgG to 1 mg / mL with coating solution and coat it at the quality control line position on the nitrocellulose membrane with a film stripper at a parameter of 1uL / cm.

[0076] The coated membrane was placed in a 45°C oven and dried for 2 hours.

[0077] Test strip assembly: overlap and paste the sample pad, gold label pad, nitrocellulose membrane and absorbent pad on the PVC base plate in sequence according to the chromatography direction, and cut them into test strips with a width of 3 mm.

[0078] Example 2

[0079] This embodiment provides an immunogold for combined detection of multiple virus antigens. The immunogold is an antibody conjugate formed by simultaneously labeling Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 with colloidal gold.

[0080] Specifically, colloidal gold is prepared by a seed growth method, and the preparation method of colloidal gold comprises the following steps:

[0081] S1: Add 2mL of 10g / L chloroauric acid solution to 100mL of ultrapure water, heat and stir under reflux until boiling, add 4mL of 10g / L sodium citrate solution, the mass ratio of chloroauric acid to sodium citrate in the mixture is 1:2, and the concentration of chloroauric acid is 0.2g / L; after the solution turns wine red, continue to heat and reflux for 15min, then stop heating, wait for the solution to cool naturally to room temperature, and obtain a small particle gold seed solution;

[0082] S2: Add 1 mL of 10 g / L chloroauric acid solution to 100 mL of ultrapure water, at which point the chloroauric acid concentration in the mixture is 0.1 g / L; then add 1 mL of gold seed solution, the ratio of the mass amount of chloroauric acid (g) to the volume amount of gold seed solution (L) in S2 is 1:0.1; add a stabilizer, heat to 90°C, add a certain amount of reducing agent every 10 minutes, continue for 1 hour, and naturally cool to room temperature to obtain colloidal gold.

[0083] In the above preparation process, the stabilizer is a mixture of 0.8 g of hexadecyltrimethylammonium bromide and 0.8 g of polyethylene glycol 20000. The mass ratio of hexadecyltrimethylammonium bromide to polyethylene glycol 20000 is 1:1, and the mass ratio of hexadecyltrimethylammonium bromide to chloroauric acid in S2 is 80:1.

[0084] The reducing agent is a solution containing 0.3M p-phenylenediamine phenol and 10g / L sodium citrate. Every ten minutes, 150uL of the reducing agent is added; that is, the ratio of the mass amount of chloroauric acid (g) to the volume amount of the reducing agent each time (L) is 1:0.015.

[0085] The present application also provides a method for preparing immunogold for combined detection of multiple virus antigens, comprising the following steps: taking 10 mL of colloidal gold in a beaker, adding 0.1 g of sodium lauryl polyoxyethylene ether sulfate and mixing evenly, adjusting the pH value to 7.5 with 0.2 M potassium carbonate solution, adding 150 ug of Mycoplasma pneumoniae monoclonal antibody 1, 150 ug of adenovirus monoclonal antibody 1, and 150 ug of syncytial virus monoclonal antibody 1, and stirring at room temperature for reaction for 10 minutes; the ratio of the mass amount (ug) of each antibody to the volume amount (ml) of colloidal gold is (75):(5).

[0086] After centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, 1 mL of blocking solution was added for blocking for 20 min, and after centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of preservation solution was added for reconstitution to obtain colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 conjugates.

[0087] The present application also provides a kit for combined detection of multiple virus antigens, mainly comprising a test strip and a lysate. The test strip comprises a base plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water-absorbing pad, the sample pad, the gold label pad, the nitrocellulose membrane and the water-absorbing pad are sequentially overlapped and pasted on the base plate in the direction of chromatography, the nitrocellulose membrane is coated with a quality control line and a detection line, the quality control line is coated with sheep anti-mouse IgG, the detection line comprises a detection line T1, a detection line T2, and a detection line T3, the detection line T1 is coated with a Mycoplasma pneumoniae monoclonal antibody 2, the detection line T2 is coated with an adenovirus monoclonal antibody 2, and the detection line T3 is coated with a syncytial virus monoclonal antibody 2.

[0088] The preparation method of the gold-labeled pad is as follows: dilute the antibody conjugate 20 times, soak the blank pad, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0089] The sample pad treatment method is as follows: soak the blank pad in the sample pad treatment solution, take it out and dry it at 37°C.

[0090] The preparation method of the nitrocellulose membrane is as follows: the Mycoplasma pneumoniae monoclonal antibody 2 is diluted to 0.8 mg / mL with a coating solution, and coated at the detection line T1 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0091] Adenovirus monoclonal antibody 2 was diluted to 0.8 mg / mL with coating solution and coated at the detection line T2 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0092] The syncytial virus monoclonal antibody 2 was diluted to 0.8 mg / mL with the coating solution and coated at the detection line T3 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0093] Dilute goat anti-mouse IgG with coating solution to 1 mg / mL, and coat it at the quality control line position on the nitrocellulose membrane with a film stripper at a parameter of 1uL / cm.

[0094] Test strip assembly: overlap and paste the sample pad, gold label pad, nitrocellulose membrane and absorbent pad on the PVC base plate in sequence according to the chromatography direction, and cut them into test strips with a width of 3 mm.

[0095] Example 3

[0096] This embodiment provides an immunogold for combined detection of multiple virus antigens. The immunogold is an antibody conjugate formed by simultaneously labeling Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 with colloidal gold.

[0097] Specifically, colloidal gold is prepared by a seed growth method, and the preparation method of colloidal gold comprises the following steps:

[0098] S1: Add 2mL of 10g / L chloroauric acid solution to 100mL of ultrapure water, heat and stir under reflux until boiling, add 4mL of 10g / L sodium citrate solution, the mass ratio of chloroauric acid to sodium citrate in the mixture is 1:2, and the concentration of chloroauric acid is 0.2g / L; after the solution turns wine red, continue to heat and reflux for 15min, then stop heating, wait for the solution to cool naturally to room temperature, and obtain a small particle gold seed solution;

[0099] S2: Add 1 mL of 10 g / L chloroauric acid solution to 100 mL of ultrapure water. At this time, the concentration of chloroauric acid in the mixed solution is 0.1 g / L. Then add 1 mL of gold seed solution. The ratio of the mass amount of chloroauric acid (g) to the volume amount of gold seed solution (L) in S2 is 1:0.1. Add stabilizer, heat to 90°C, add a certain amount of reducing agent every 10 minutes, continue for 1 hour, and naturally cool to room temperature to obtain colloidal gold.

[0100] In the above preparation process, the stabilizer is a mixture of 1.2 g of hexadecyltrimethylammonium bromide and 1.2 g of polyethylene glycol 20000. That is, the mass ratio of hexadecyltrimethylammonium bromide to polyethylene glycol 20000 is 1:1, and the mass ratio of hexadecyltrimethylammonium bromide to chloroauric acid in S2 is 120:1.

[0101] The reducing agent is a solution containing 40 mM hydroxylamine hydrochloride and 10 g / L sodium citrate, and the amount of reducing agent added is 200 uL every ten minutes. That is, the ratio of the mass amount of chloroauric acid in S2 (g) to the volume amount of reducing agent each time (L) is 1:0.02.

[0102] The present application also provides a method for preparing immunogold for joint detection of multiple virus antigens, comprising the following steps: taking 10 mL of colloidal gold in a beaker, adding 0.5 g of sodium lauryl polyoxyethylene ether sulfate and mixing, adjusting the pH value to 7.5 with 0.2 M potassium carbonate solution, adding 125 ug of Mycoplasma pneumoniae monoclonal antibody 1, 125 ug of adenovirus monoclonal antibody 1, and 125 ug of syncytial virus monoclonal antibody 1, the ratio of the mass amount (ug) of each antibody to the volume amount (ml) of colloidal gold is 62.5:5, and stirring at room temperature for 10 minutes;

[0103] After centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, 1 mL of blocking solution was added for blocking for 20 min, and after centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of preservation solution was added for reconstitution to obtain colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 conjugates.

[0104] The present application also provides a kit for combined detection of multiple virus antigens, mainly comprising a test strip and a lysate. The test strip comprises a base plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water-absorbing pad, the sample pad, the gold label pad, the nitrocellulose membrane and the water-absorbing pad are sequentially overlapped and pasted on the base plate in the direction of chromatography, the nitrocellulose membrane is coated with a quality control line and a detection line, the quality control line is coated with sheep anti-mouse IgG, the detection line comprises a detection line T1, a detection line T2, and a detection line T3, the detection line T1 is coated with a Mycoplasma pneumoniae monoclonal antibody 2, the detection line T2 is coated with an adenovirus monoclonal antibody 2, and the detection line T3 is coated with a syncytial virus monoclonal antibody 2.

[0105] The preparation method of the gold-labeled pad is as follows: dilute the antibody conjugate 20 times, soak the blank pad, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0106] The sample pad treatment method is as follows: soak the blank pad in the sample pad treatment solution, take it out and dry it at 37°C.

[0107] The preparation method of the nitrocellulose membrane is as follows: the Mycoplasma pneumoniae monoclonal antibody 2 is diluted to 1 mg / mL with a coating solution, and coated at the detection line T1 position on the nitrocellulose membrane with a film stripper at a parameter of 1uL / cm;

[0108] Adenovirus monoclonal antibody 2 was diluted to 1 mg / mL with coating solution and coated at the detection line T2 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0109] The syncytial virus monoclonal antibody 2 was diluted to 1 mg / mL with the coating solution and coated at the detection line T3 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0110] Dilute goat anti-mouse IgG to 1 mg / mL with coating solution and coat it at the quality control line position on the nitrocellulose membrane with a film stripper at a parameter of 1uL / cm.

[0111] The coated membrane was placed in a 45°C oven and dried for 2 hours.

[0112] Test strip assembly: overlap and paste the sample pad, gold label pad, nitrocellulose membrane and absorbent pad on the PVC base plate in sequence according to the chromatography direction, and cut them into test strips with a width of 3 mm.

[0113] Example 4

[0114] This embodiment provides an immunogold for combined detection of multiple virus antigens. The immunogold is an antibody conjugate formed by simultaneously labeling Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 with colloidal gold.

[0115] Specifically, colloidal gold is prepared by a seed growth method, and the preparation method of colloidal gold comprises the following steps:

[0116] S1: Add 2mL of 10g / L chloroauric acid solution to 100mL of ultrapure water, heat and stir under reflux until boiling, add 4mL of 10g / L sodium citrate solution, the mass ratio of chloroauric acid to sodium citrate in the mixture is 1:2, and the concentration of chloroauric acid is 0.2g / L; after the solution turns wine red, continue to heat and reflux for 15min, then stop heating, wait for the solution to cool naturally to room temperature, and obtain a small particle gold seed solution;

[0117] S2: Add 1 mL of 10 g / L chloroauric acid solution to 100 mL of ultrapure water. At this time, the concentration of chloroauric acid in the mixed solution is 0.1 g / L. Then add 1 mL of gold seed solution. The ratio of the mass amount of chloroauric acid (g) to the volume amount of gold seed solution (L) in S2 is 1:0.1. Add stabilizer, heat to 90°C, add a certain amount of reducing agent every 10 minutes, continue for 1 hour, and naturally cool to room temperature to obtain colloidal gold.

[0118] In the above preparation process, the stabilizer is 1.2 g of hexadecyltrimethylammonium bromide and 1.2 g of polyethylene glycol 20000. The mass ratio of hexadecyltrimethylammonium bromide to polyethylene glycol 20000 is 1:1, and the mass ratio of hexadecyltrimethylammonium bromide to chloroauric acid in S2 is 120:1.

[0119] The reducing agent is a solution containing 0.3M p-phenylenediamine phenol and 10g / L sodium citrate, and the amount of reducing agent added is 200uL every ten minutes. The ratio of the mass amount of chloroauric acid (g) in S2 to the volume amount of reducing agent (L) each time is 1:0.02.

[0120] The present application also provides a method for preparing immunogold for joint detection of multiple virus antigens, comprising the following steps: taking 10 mL of colloidal gold in a beaker, adding 0.5 g of sodium lauryl polyoxyethylene ether sulfate and mixing, adjusting the pH value to 7.5 with 0.2 M potassium carbonate solution, adding 125 ug of Mycoplasma pneumoniae monoclonal antibody 1, 125 ug of adenovirus monoclonal antibody 1, and 125 ug of syncytial virus monoclonal antibody 1, the ratio of the mass amount (ug) of each antibody to the volume amount (ml) of colloidal gold is 62.5:5, and stirring at room temperature for 10 minutes;

[0121] After centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, 1 mL of blocking solution was added for blocking for 20 min, and after centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of preservation solution was added for reconstitution to obtain colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 conjugates.

[0122] The present application also provides a kit for combined detection of multiple virus antigens, mainly comprising a test strip and a lysate. The test strip comprises a base plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water-absorbing pad, the sample pad, the gold label pad, the nitrocellulose membrane and the water-absorbing pad are sequentially overlapped and pasted on the base plate in the direction of chromatography, the nitrocellulose membrane is coated with a quality control line and a detection line, the quality control line is coated with sheep anti-mouse IgG, the detection line comprises a detection line T1, a detection line T2, and a detection line T3, the detection line T1 is coated with a Mycoplasma pneumoniae monoclonal antibody 2, the detection line T2 is coated with an adenovirus monoclonal antibody 2, and the detection line T3 is coated with a syncytial virus monoclonal antibody 2.

[0123] The preparation method of the gold-labeled pad is as follows: dilute the antibody conjugate 20 times, soak the blank pad, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0124] The sample pad treatment method is as follows: soak the blank pad in the sample pad treatment solution, take it out and dry it at 37°C.

[0125] The preparation method of the nitrocellulose membrane is as follows: the Mycoplasma pneumoniae monoclonal antibody 2 is diluted to 0.8 mg / mL with a coating solution, and coated at the detection line T1 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0126] Adenovirus monoclonal antibody 2 was diluted to 0.8 mg / mL with coating solution and coated at the detection line T2 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0127] The syncytial virus monoclonal antibody 2 was diluted to 0.8 mg / mL with the coating solution and coated at the detection line T3 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0128] Dilute goat anti-mouse IgG to 1 mg / mL with coating solution and coat it at the quality control line position on the nitrocellulose membrane with a film stripper at a parameter of 1uL / cm.

[0129] The coated membrane was placed in a 45°C oven and dried for 2 hours.

[0130] Test strip assembly: overlap and paste the sample pad, gold label pad, nitrocellulose membrane and absorbent pad on the PVC base plate in sequence according to the chromatography direction, and cut them into test strips with a width of 3 mm.

[0131] Example 5

[0132] This embodiment provides an immunogold for combined detection of multiple virus antigens. The immunogold is an antibody conjugate formed by simultaneously labeling Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 with colloidal gold.

[0133] Specifically, colloidal gold is prepared by a seed growth method, and the preparation method of colloidal gold comprises the following steps:

[0134] S1: Add 2mL of 10g / L chloroauric acid solution to 100mL of ultrapure water, heat and stir under reflux until boiling, add 4mL of 10g / L sodium citrate solution, the mass ratio of chloroauric acid to sodium citrate in the mixture is 1:2, and the concentration of chloroauric acid is 0.2g / L; after the solution turns wine red, continue to heat and reflux for 15min, then stop heating, wait for the solution to cool naturally to room temperature, and obtain a small particle gold seed solution;

[0135] S2: Add 1 mL of 10 g / L chloroauric acid solution to 100 mL of ultrapure water. At this time, the concentration of chloroauric acid in the mixed solution is 0.01 g / L. Then add 2 mL of gold seed solution. The ratio of the mass amount of chloroauric acid (g) to the volume amount of gold seed solution (L) in S2 is 1:0.2. Add stabilizer, heat to 90°C, add a certain amount of reducing agent every 10 minutes, continue for 1 hour, and naturally cool to room temperature to obtain colloidal gold.

[0136] In the above preparation process, the stabilizer is a mixture of 1.0 g of hexadecyltrimethylammonium bromide and 0.8 g of polyethylene glycol 20000. The mass ratio of hexadecyltrimethylammonium bromide to polyethylene glycol 20000 is 1:0.8, and the mass ratio of hexadecyltrimethylammonium bromide to chloroauric acid in S2 is 80:1.

[0137] The reducing agent is a solution containing 40 mM hydroxylamine hydrochloride and 10 g / L sodium citrate. Every ten minutes, 150 uL of the reducing agent is added; that is, the ratio of the mass amount of chloroauric acid (g) to the volume amount of the reducing agent each time (L) is 1:0.015.

[0138] The embodiment of the present application also provides a method for preparing immunogold for joint detection of multiple virus antigens, comprising the following steps: taking 8 mL of colloidal gold in a beaker, adding 0.2 g of sodium lauryl polyoxyethylene ether sulfate and mixing, adjusting the pH value to 7.5 with 0.2 M potassium carbonate solution, adding 100 ug of Mycoplasma pneumoniae monoclonal antibody 1, 100 ug of adenovirus monoclonal antibody 1, and 100 ug of syncytial virus monoclonal antibody 1, and stirring at room temperature for 10 minutes; the ratio of the mass amount (ug) of each antibody to the volume amount (ml) of colloidal gold is 50:4.

[0139] After centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 0.4 mL of blocking solution was added for blocking for 20 min, with the volume ratio of colloidal gold to blocking solution being 20:1; after centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of preservation solution was added for reconstitution to obtain colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 conjugates.

[0140] The present application also provides a kit for combined detection of multiple virus antigens, mainly comprising a test strip and a lysate. The test strip comprises a base plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water-absorbing pad, the sample pad, the gold label pad, the nitrocellulose membrane and the water-absorbing pad are sequentially overlapped and pasted on the base plate in the direction of chromatography, the nitrocellulose membrane is coated with a quality control line and a detection line, the quality control line is coated with sheep anti-mouse IgG, the detection line comprises a detection line T1, a detection line T2, and a detection line T3, the detection line T1 is coated with a Mycoplasma pneumoniae monoclonal antibody 2, the detection line T2 is coated with an adenovirus monoclonal antibody 2, and the detection line T3 is coated with a syncytial virus monoclonal antibody 2.

[0141] The preparation method of the gold-labeled pad is as follows: dilute the antibody conjugate 20 times, soak the blank pad, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0142] The sample pad treatment method is as follows: soak the blank pad in the sample pad treatment solution, take it out and dry it at 37°C.

[0143] The preparation method of the nitrocellulose membrane is as follows: the original monoclonal antibody 2 is diluted to 1 mg / mL with the coating solution, and coated at the detection line T1 position on the nitrocellulose membrane with a film stripper at a parameter of 1uL / cm;

[0144] Adenovirus monoclonal antibody 2 was diluted to 1 mg / mL with coating solution and coated at the detection line T2 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0145] The syncytial virus monoclonal antibody 2 was diluted to 1 mg / mL with the coating solution and coated at the detection line T3 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0146] Dilute goat anti-mouse IgG to 1 mg / mL with coating solution and coat it at the quality control line position on the nitrocellulose membrane with a film stripper at a parameter of 1uL / cm.

[0147] The coated membrane was placed in a 45°C oven and dried for 2 hours.

[0148] Test strip assembly: overlap and paste the sample pad, gold label pad, nitrocellulose membrane and absorbent pad on the PVC base plate in sequence according to the chromatography direction, and cut them into test strips with a width of 3 mm.

[0149] Example 6

[0150] This embodiment provides an immunogold for combined detection of multiple virus antigens. The immunogold is an antibody conjugate formed by simultaneously labeling Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 with colloidal gold.

[0151] Specifically, colloidal gold is prepared by a seed growth method, and the preparation method of colloidal gold comprises the following steps:

[0152] S1: Add 2mL of 10g / L chloroauric acid solution to 100mL of ultrapure water, heat and stir under reflux until boiling, add 4mL of 10g / L sodium citrate solution, the mass ratio of chloroauric acid to sodium citrate in the mixture is 1:2, and the concentration of chloroauric acid is 0.2g / L; after the solution turns wine red, continue to heat and reflux for 15min, then stop heating, wait for the solution to cool naturally to room temperature, and obtain a small particle gold seed solution;

[0153] S2: Add 1 mL of 10 g / L chloroauric acid solution to 100 mL of ultrapure water. At this time, the concentration of chloroauric acid in the mixed solution is 0.01 g / L. Then add 1 mL of gold seed solution. The ratio of the mass amount of chloroauric acid (g) to the volume amount of gold seed solution (L) in S2 is 1:0.1. Add stabilizer, heat to 90°C, add a certain amount of reducing agent every 10 minutes, continue for 1 hour, and naturally cool to room temperature to obtain colloidal gold.

[0154] In the above preparation process, the stabilizer is a mixture of 1.0 g of hexadecyltrimethylammonium bromide and 1.2 g of polyethylene glycol 20000. The mass ratio of hexadecyltrimethylammonium bromide to polyethylene glycol 20000 is 1:1.2, and the mass ratio of hexadecyltrimethylammonium bromide to chloroauric acid in S2 is 80:1.

[0155] The reducing agent is a solution containing 40 mM hydroxylamine hydrochloride and 10 g / L sodium citrate. Every ten minutes, 150 uL of the reducing agent is added; that is, the ratio of the mass amount of chloroauric acid (g) to the volume amount of the reducing agent each time (L) is 1:0.015.

[0156] The embodiment of the present application also provides a method for preparing immunogold for joint detection of multiple virus antigens, comprising the following steps: taking 12 mL of colloidal gold in a beaker, adding 0.3 g of sodium lauryl polyoxyethylene ether sulfate and mixing, adjusting the pH value to 7.5 with 0.2 M potassium carbonate solution, adding 150 ug of Mycoplasma pneumoniae monoclonal antibody 1, 150 ug of adenovirus monoclonal antibody 1, and 150 ug of syncytial virus monoclonal antibody 1, stirring at room temperature for 10 minutes; the ratio of the mass dosage (ug) of each antibody to the volume dosage (ml) of colloidal gold is 75:6.

[0157] After centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of blocking solution was added for blocking for 20 min, with the volume ratio of colloidal gold to blocking solution being 12:1; after centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of preservation solution was added for reconstitution to obtain colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 conjugates.

[0158] The present application also provides a kit for combined detection of multiple virus antigens, mainly comprising a test strip and a lysate. The test strip comprises a base plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water-absorbing pad, the sample pad, the gold label pad, the nitrocellulose membrane and the water-absorbing pad are sequentially overlapped and pasted on the base plate in the direction of chromatography, the nitrocellulose membrane is coated with a quality control line and a detection line, the quality control line is coated with sheep anti-mouse IgG, the detection line comprises a detection line T1, a detection line T2, and a detection line T3, the detection line T1 is coated with a Mycoplasma pneumoniae monoclonal antibody 2, the detection line T2 is coated with an adenovirus monoclonal antibody 2, and the detection line T3 is coated with a syncytial virus monoclonal antibody 2.

[0159] The preparation method of the gold-labeled pad is as follows: dilute the antibody conjugate 20 times, soak the blank pad, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0160] The sample pad treatment method is as follows: soak the blank pad in the sample pad treatment solution, take it out and dry it at 37°C.

[0161] The preparation method of the nitrocellulose membrane is as follows: the original monoclonal antibody 2 is diluted to 1 mg / mL with the coating solution, and coated at the detection line T1 position on the nitrocellulose membrane with a film stripper at a parameter of 1uL / cm;

[0162] Adenovirus monoclonal antibody 2 was diluted to 1 mg / mL with coating solution and coated at the detection line T2 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0163] The syncytial virus monoclonal antibody 2 was diluted to 1 mg / mL with the coating solution and coated at the detection line T3 position on the nitrocellulose membrane with a film stripper at a parameter of 1 uL / cm;

[0164] Dilute goat anti-mouse IgG to 1 mg / mL with coating solution and coat it at the quality control line position on the nitrocellulose membrane with a film stripper at a parameter of 1uL / cm.

[0165] The coated membrane was placed in a 45°C oven and dried for 2 hours.

[0166] Test strip assembly: overlap and paste the sample pad, gold label pad, nitrocellulose membrane and absorbent pad on the PVC base plate in sequence according to the chromatography direction, and cut them into test strips with a width of 3 mm.

[0167] Comparative Example

[0168] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that the conventional process for preparing colloidal gold is adopted, and the preparation method of colloidal gold is as follows:

[0169] Add 2mL of 10g / L chloroauric acid solution to 100mL of ultrapure water, heat and stir under reflux until boiling, add 4mL of 10g / L sodium citrate solution, and continue heating and reflux for 15min after the solution turns wine red, then stop heating and wait for the solution to cool naturally to room temperature to obtain colloidal gold.

[0170] Comparative Example 2: The difference between Comparative Example 2 and Example 2 is that no stabilizer is added in the colloidal gold preparation process.

[0171] Comparative Example 3: The difference between Comparative Example 3 and Example 3 is that the lysate does not contain dithiothreitol.

[0172] Comparative Example 4: The difference between Comparative Example 4 and Example 1 is that the stabilizer is only 0.8 g of hexadecyltrimethylammonium bromide, and the mass ratio of hexadecyltrimethylammonium bromide to chloroauric acid in S2 is 80:1.

[0173] Comparative Example 5: The difference between Comparative Example 5 and Example 1 is that the stabilizer is only 0.8 g of polyethylene glycol 20000, and the mass ratio of polyethylene glycol 20000 to chloroauric acid in S2 is 80:1.

[0174] Comparative Example 6: The difference between Comparative Example 6 and Example 1 is that the immunogold raw material does not contain sodium lauryl alcohol polyoxyethylene ether sulfate. Colloidal gold is used to label Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1, respectively, and the labeling process is as follows:

[0175] Take 10 mL of colloidal gold solution in a beaker, adjust the pH value to 7.5 with 0.2 M potassium carbonate solution, add 100 ug of Mycoplasma pneumoniae monoclonal antibody 1, and stir at room temperature for 10 minutes; the ratio of the mass amount of each antibody (ug) to the volume amount of colloidal gold solution (ml) is (50):(5).

[0176] After centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of blocking solution was added for blocking for 20 min, with the volume ratio of colloidal gold solution to blocking solution being 10:1; after centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of preservation solution was added for reconstitution to obtain colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody 1 conjugate.

[0177] Take 10 mL of colloidal gold solution in a beaker, adjust the pH value to 7.2 with 0.2 M potassium carbonate solution, add 100 ug of adenovirus monoclonal antibody 1, and stir at room temperature for 10 minutes; the ratio of the mass amount (ug) of each antibody to the volume amount (ml) of colloidal gold solution is (50):(5).

[0178] After centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of blocking solution was added for blocking for 20 min, with the volume ratio of colloidal gold solution to blocking solution being 10:1; after centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of preservation solution was added for reconstitution to obtain a colloidal gold-labeled adenovirus monoclonal antibody 1 conjugate.

[0179] Take 10 mL of colloidal gold solution in a beaker, adjust the pH value to 7.8 with 0.2 M potassium carbonate solution, add 100 ug of syncytial virus monoclonal antibody 1, and stir at room temperature for 10 minutes; the ratio of the mass amount (ug) of each antibody to the volume amount (ml) of colloidal gold solution is (50):(5).

[0180] After centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of blocking solution was added for blocking for 20 min, with the volume ratio of colloidal gold solution to blocking solution being 10:1; after centrifugation (temperature 4°C, speed 8500 rpm, 15 min), the supernatant was discarded, and 1 mL of preservation solution was added for reconstitution to obtain a colloidal gold-labeled syncytial virus monoclonal antibody 1 conjugate.

[0181] The preparation method of the gold-labeled pad is as follows: colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody 1 conjugate, colloidal gold-labeled adenovirus monoclonal antibody 1 conjugate, and colloidal gold-labeled adenovirus monoclonal antibody 1 conjugate are mixed at a volume ratio of 1:1:1, the mixed conjugate is diluted 7 times and then soaked in a blank gasket, and then taken out and dried at 37°C to obtain a gold-labeled pad.

[0182] Comparative Example 7

[0183] The difference between Comparative Example 7 and Example 1 is that: a method for preparing immunogold for joint detection of multiple virus antigens, comprising the following steps: taking 10 mL of colloidal gold in a beaker, adjusting the pH value to 7.5 with 0.2 M potassium carbonate solution, adding 100 ug of Mycoplasma pneumoniae monoclonal antibody 1, 100 ug of adenovirus monoclonal antibody 1, and 100 ug of syncytial virus monoclonal antibody 1, stirring at room temperature for 10 minutes; the ratio of the mass amount (ug) of each antibody to the volume amount (ml) of colloidal gold is (50): (5). Coagulation occurs during the labeling process.

[0184] Performance Testing

[0185] 1. Minimum detection limit test

[0186] The syncytial virus-positive samples (the samples confirmed as positive by commercially available fluorescent PCR methodology reagents) were diluted 10 times in a gradient manner to obtain 6 gradient samples, L1-L6. The 6 samples were tested, and the test results are shown in Table 1 below.

[0187] Adenovirus-positive samples (samples confirmed as positive by commercially available fluorescent PCR methodology reagents) were diluted 10 times to obtain 6 gradient samples, L7-L12. The 6 samples were tested, and the test results are shown in Table 2 below.

[0188] The Mycoplasma pneumoniae positive samples (the samples confirmed as positive by commercially available fluorescent PCR methodology reagents) were diluted 10 times to obtain 6 gradient samples, L13-L18. The 6 samples were tested, and the test results are shown in Table 3 below.

[0189] Table 1 Results of the minimum detection limit of syncytial virus antigen

[0190]

[0191]

[0192] Note: "+" indicates positive, "-" indicates negative

[0193] Table 2 Results of minimum detection limit of adenovirus antigen

[0194] Detection object L7 L8 L9 L10 L11 L12 Example 1 + + + + - - Example 2 + + + + - - Example 3 + + + + - - Example 4 + + + + - - Example 5 + + + + - - Example 6 + + + + - - Comparative Example 1 + + - - - - Comparative Example 2 + + + - - - Comparative Example 3 + + + + - - Comparative Example 4 + + + - - - Comparative Example 5 + + + - - - Comparative Example 6 + + + + - -

[0195] Note: "+" indicates positive, "-" indicates negative

[0196] Table 3 Results of the minimum detection limit of Mycoplasma pneumoniae

[0197] Detection object L13 L14 L15 L16 L17 L18 Example 1 + + + + + - Example 2 + + + + + - Example 3 + + + + + - Example 4 + + + + + - Example 5 + + + + + - Example 6 + + + + + - Comparative Example 1 + + + - - - Comparative Example 2 + + + + - - Comparative Example 3 + + + + + - Comparative Example 4 + + + + - - Comparative Example 5 + + + + - - Comparative Example 6 + + + + + -

[0198] Note: "+" indicates positive, "-" indicates negative

[0199] From Table 1, Table 2, and Table 3, it can be seen that the sensitivity of Examples 1-6 is 100 times higher than that of Comparative Example 1, which is consistent with Comparative Example 3, indicating that the colloidal gold prepared by the seed growth method in this application effectively improves the sensitivity of the product;

[0200] The sensitivity of Examples 1-6 is 10 times higher than that of Comparative Examples 2, 4, and 5, which is consistent with Comparative Example 3, indicating that the colloidal gold prepared by using hexadecyltrimethylammonium bromide and polyethylene glycol 20000 as stabilizers in this application can improve the sensitivity of the product.

[0201] The sensitivity of Examples 1-6 is consistent with that of Comparative Example 6, indicating that the method of adding sodium lauryl polyoxyethylene ether sulfate to colloidal gold and labeling three antibodies at the same time is feasible, the sensitivity is not affected, and the labeling efficiency can be improved. In addition, during the labeling process of Comparative Example 7, coagulation occurred, indicating that the addition of sodium lauryl polyoxyethylene ether sulfate can reduce the dead gold coagulation phenomenon caused by the change of the isoelectric point of the labeled antibody due to the change of pH value, and at the same time reduce the large difference in sensitivity of different items caused by the difference in coupling efficiency under the same pH condition due to the different isoelectric points of antibodies in the process of colloidal gold labeling of multiple antibodies, and even the occurrence of dead gold coagulation phenomenon.

[0202] 2. Specificity detection

[0203] Oropharyngeal swab samples were collected from 400 subjects who were confirmed to be negative for syncytial virus, adenovirus, and Mycoplasma pneumoniae by fluorescent PCR methodology, and then processed in corresponding lysis buffers for testing. The test results are shown in Table 4 below.

[0204] Table 4 Specificity test results of reagents prepared in Example 1-4

[0205]

[0206] From the comparison in Table 4, it can be seen that the specificity of Examples 1-4 is better than that of Comparative Example 1, indicating that the colloidal gold prepared by the seed growth method used in this application effectively improves the specificity of the product;

[0207] The specificity of Examples 1-4 is better than that of Comparative Examples 2, 4 and 5, indicating that the colloidal gold method prepared by using hexadecyltrimethylammonium bromide and polyethylene glycol 20000 as stabilizers in the present application can improve the specificity of the product;

[0208] The specificity of Examples 1-4 is better than that of Comparative Example 3, indicating that the addition of blocking agent dithiothreitol or TCEP hydrochloride in the lysate of the present application can improve the specificity of the product.

[0209] 3. Positive sample detection

[0210] Oropharyngeal swab samples were collected from 100 patients with syncytial virus-positive confirmed by fluorescent PCR methodology, 100 patients with adenovirus-positive confirmed by fluorescent PCR methodology, and 100 patients with Mycoplasma pneumoniae-positive confirmed by fluorescent PCR methodology, and tested after treatment with corresponding lysis buffer.

[0211] Table 5 Detection results of syncytial virus positive samples

[0212]

[0213] Table 6 Adenovirus positive sample test results

[0214]

[0215] Table 7 Mycoplasma pneumoniae positive sample test results

[0216]

[0217] As can be seen from the comparison of Tables 5-7, the positive detection rates of Examples 1-6 of the present application are higher than those of Comparative Example 1, Comparative Example 2, Comparative Example 4, and Comparative Example 5, indicating that the respiratory syncytial virus, adenovirus, and Mycoplasma pneumoniae antigen detection reagents prepared in the present application can meet clinical needs.

[0218] The positive detection rates of Examples 1-6 are consistent with those of Comparative Example 6, indicating that the method of adding sodium lauryl polyoxyethylene ether sulfate to colloidal gold and simultaneously labeling three antibodies is feasible, the positive detection rates are not affected, and the labeling efficiency can be improved.

[0219] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. An immunogold for combined detection of multiple viral antigens, characterized in that: The raw materials of the immune gold include colloidal gold, Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, syncytial virus monoclonal antibody 1, and lauryl alcohol polyoxyethylene ether sulfate sodium salt; The colloidal gold simultaneously labels Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 to form an antibody conjugate, and the ratio of the mass amount (g) of the sodium lauryl polyoxyethylene ether sulfate to the volume amount (ml) of the colloidal gold is (0.01-0.05): 1; The colloidal gold is prepared by a seed growth method, and the seed growth method uses hexadecyltrimethylammonium bromide and polyethylene glycol 20000 as stabilizers, and the mass ratio of the hexadecyltrimethylammonium bromide to the polyethylene glycol 20000 is 1:(0.8-1.2).

2. The immunogold for combined detection of multiple viral antigens according to claim 1, characterized in that: The preparation method of colloidal gold comprises the following steps: S1: Add ultrapure water to the chloroauric acid solution, heat and stir under reflux until boiling, and add sodium citrate solution; the mass ratio of chloroauric acid to sodium citrate in the mixed solution is 1:(1.6-2), and the concentration of chloroauric acid is 0.19-0.2g / L; after the solution turns into wine red, continue heating and reflux for 10-15min, then stop heating, and wait for the solution to cool naturally to room temperature to obtain a small particle gold seed solution; S2: Add ultrapure water to the chloroauric acid solution, at which time the chloroauric acid concentration in the mixed solution is 0.09-0.1 g / L, and then add the gold seed solution, the ratio of the mass amount of chloroauric acid (g) to the volume amount of gold seed solution (L) in S2 is 1:(0.1-0.2); add a stabilizer, heat to 70-90°C, add a reducing agent to react, and naturally cool to room temperature to obtain colloidal gold.

3. The immunogold for combined detection of multiple viral antigens according to claim 1, characterized in that: In the stabilizer, the mass ratio of hexadecyltrimethylammonium bromide to chloroauric acid in S2 is (80-120):

1.

4. The immunogold for combined detection of multiple viral antigens according to claim 1, characterized in that: In the step S2, a certain amount of reducing agent is added every 9-11 minutes for 54-66 minutes.

5. The immunogold for combined detection of multiple viral antigens according to claim 4, characterized in that: The reducing agent is a mixture of hydroxylamine hydrochloride and sodium citrate or a mixture of p-phenylenediamine phenol and sodium citrate.

6. The immunogold for combined detection of multiple viral antigens according to claim 5, characterized in that: The reducing agent is a solution containing 40 mM hydroxylamine hydrochloride and 10 g / L sodium citrate or a solution containing 0.3 M p-phenylenediamine phenol and 10 g / L sodium citrate; the ratio of the mass amount (g) of chloroauric acid in S2 to the volume amount (L) of the reducing agent each time is 1: (0.015-0.02).

7. A method for preparing immunogold for combined detection of multiple viral antigens, characterized in that: The following steps are involved: Take a certain amount of colloidal gold in a beaker, add sodium lauryl polyoxyethylene ether sulfate; adjust the pH value to 7.2-7.8, add 1 monoclonal antibody to Mycoplasma pneumoniae, 1 monoclonal antibody to adenovirus, and 1 monoclonal antibody to syncytial virus into the colloidal gold, the ratio of the mass amount (ug) of each antibody to the volume amount (ml) of colloidal gold is (50-75): (4-6), and react at room temperature with stirring for 8-15 minutes; After centrifugation, the supernatant is discarded, and blocking solution is added for blocking, the volume ratio of colloidal gold to blocking solution is (10-20):1; after centrifugation, the supernatant is discarded, and preservation solution is added for re-dissolution to obtain colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody 1, adenovirus monoclonal antibody 1, and syncytial virus monoclonal antibody 1 conjugate, namely immunogold.

8. A gold label pad, characterized in that: The antibody conjugate according to any one of claims 1 to 6 is diluted and soaked in a blank pad, and then dried to obtain a gold-labeled pad.

9. A test strip for combined detection of multiple virus antigens, characterized in that: The invention comprises a bottom plate, a sample pad, a gold label pad as claimed in claim 8, a nitrocellulose membrane and a water absorbent pad, wherein the sample pad, the gold label pad, the nitrocellulose membrane and the water absorbent pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction, the nitrocellulose membrane is coated with a quality control line and a detection line, the quality control line is coated with sheep anti-mouse IgG, the detection lines comprise detection line T1, detection line T2 and detection line T3, the detection line T1 is coated with a Mycoplasma pneumoniae monoclonal antibody 2, the detection line T2 is coated with an adenovirus monoclonal antibody 2, and the detection line T3 is coated with a syncytial virus monoclonal antibody 2.

10. A kit for combined detection of multiple viral antigens, characterized in that: The method comprises the test strip and lysis solution as described in claim 8.

11. A kit for combined detection of multiple viral antigens according to claim 10, characterized in that: The lysate comprises a blocking agent, and the blocking agent is dithiothreitol or TCEP hydrochloride.

12. A kit for combined detection of multiple viral antigens according to claim 11, characterized in that: In the lysate, the concentration of the blocking agent is 0.1-0.5 g / L.

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