CN103941005B - Detect the colloidal gold strip of H7 subtype avian influenza virus - Google Patents

Abstract

The invention discloses a colloidal gold test strip for detecting H7 subtype avian influenza virus. A colloidal gold immunochromatographic test strip for detecting H7 subtype avian influenza virus provided by the invention comprises a support plate and a sample pad placed thereon, a coupling pad perfused with a gold-labeled antibody, and a detection line T and the NC membrane and absorbent pad of quality control line C; the gold-labeled antibody is colloidal gold-labeled H7 subtype avian influenza virus monoclonal antibody. In the present invention, NC membranes and absorbent pads labeled with sample pads, coupling pads, detection lines and quality control lines are assembled on the support plate according to the following process, wherein the coupling pads are perfused with H7 gold-labeled monoclonal antibody, detection lines and quality control lines. Lines consisted of H7 polyclonal antibody and rabbit anti-mouse IgG spray membrane. The test strip of the present invention has the following advantages: simple operation, good specificity, high sensitivity, and reliable diagnosis result; it is suitable for on-site detection of H7 subtype avian influenza virus infection, especially suitable for on-site rapid detection of grassroots units or individuals.

Description

Colloidal gold test strip for detecting H7 subtype avian influenza virus

technical field

The invention relates to the field of biotechnology, in particular to a colloidal gold test strip for detecting H7 subtype avian influenza virus.

Background technique

Since 2002, up to 247 cases of human infection with H7 subtype avian influenza virus (AIVH7) have been reported worldwide (Netherlands, Italy, Canada, the United States, the United Kingdom and other countries). According to statistics from the Chinese Center for Disease Control and Prevention, from March 31, 2013 to August 15, 2013, a total of 134 cases were reported in 10 provinces including Shanghai, Beijing, Jiangsu, Zhejiang, Anhui, Fujian, Jiangxi, Shandong, Henan, and Hunan. Among the cases of H7N9 subtype avian influenza virus (AIVs) human infection, 1 case was confirmed in Taiwan, of which 45 died. The H7 subtype avian influenza virus not only affects the healthy development of the poultry industry, but also seriously threatens human life and health. In order to effectively control the spread of H7 subtype avian influenza virus, scholars from various countries are working on developing various methods for detecting H7 subtype avian influenza virus.

At present, the main methods for detecting AIV include virus isolation and identification, enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), nucleic acid sequence-dependent amplification (NASBA), and loop-mediated isothermal amplification ( LAMP) and the GeXP method, etc. Colloidal gold immunochromatography assay (gold-immunochromatography assay, GICA) is an immunological analysis technique developed in recent years that uses colloidal gold as a marker to qualitatively analyze biological macromolecules in samples through antigen-antibody immunological reactions. The application of colloidal gold as an immunological marker in immunological analysis began in 1971. Since the first AIDS antibody percolation detection reagent developed with colloidal gold as a marker in 1989 was born, the rapid detection technology of immunocolloidal gold has been widely used in various fields of life medical diagnosis. has been widely used. There is no report on the immunocolloidal gold test strip for rapid detection of H7 subtype avian influenza virus at present.

Contents of the invention

One object of the present invention is to provide a colloidal gold immunochromatographic test strip for detecting H7 subtype avian influenza virus.

The test strip provided by the present invention includes a support plate and a sample pad placed thereon, a coupling pad perfused with a gold-labeled antibody, an NC membrane containing a detection line T and a quality control line C, and an absorbent pad;

The gold-labeled antibody is colloidal gold-labeled H7 subtype avian influenza virus monoclonal antibody.

Of the above test strips,

The particle size of the colloidal gold is 15-25nm.

Of the above test strips,

The detection line T is formed by the polyclonal antibody of H7 subtype avian influenza virus;

The quality control line C is formed by mouse-derived anti-IgG antibody.

Of the above test strips,

The mouse-derived anti-IgG antibody is rabbit anti-mouse IgG;

The above-mentioned polyclonal antibody to H7 subtype avian influenza virus is specifically a polyclonal antibody to H7N2 subtype avian influenza virus.

Another object of the present invention is to provide a method for preparing the above test strip.

The method provided by the invention comprises the steps of:

1) First mix the colloidal gold solution and the H7 subtype avian influenza virus monoclonal antibody, react, collect the solid reactant, and then block the solid reactant with BSA to obtain the gold-labeled antibody;

After the blocking in the step 1), the following steps are further included: collecting the precipitate of the blocked product and redissolving it to obtain a gold-labeled antibody.

The buffer used for the above blocking is 20mmol/L sodium borate aqueous solution containing 1% BSA by mass percentage;

The buffer used for the dissolution is a 20 mmol/L sodium borate aqueous solution containing 1% BSA and 0.1% sodium azide.

2) Prepare the coupling pad perfused with the gold-labeled antibody and the NC membrane containing the detection line T and the quality control line C;

The coupling pad infused with the gold-labeled antibody is obtained by pouring the gold-labeled antibody onto glass fiber cotton and drying it; the above-mentioned drying is vacuum freeze-drying; the glass fiber cotton used for infusion of the gold-labeled antibody In order to put the glass fiber cotton into the PBS treatment solution containing 5% BSA, 2% sucrose, 0.8% NaCl and 0.05% NaN ₃ for 20 minutes, and dry it at a constant temperature of 37 ° C to obtain.

The NC membrane containing the detection line T and the quality control line C is that the H7 subtype avian influenza virus polyclonal antibody and the mouse anti-IgG antibody are respectively spotted on the NC membrane to form an NC membrane containing the detection line T and the quality control line C. membrane;

3) Assemble the NC membrane, the sample pad, the coupling pad and the water-absorbing pad to a support plate to obtain a colloidal gold immunochromatographic test strip.

In the above method,

In step 1), the feeding volume ratio of the colloidal gold solution and the H7 subtype avian influenza virus monoclonal antibody is 200:1;

The concentration of the H7 subtype avian influenza virus monoclonal antibody is 1mg/mL;

The particle diameter of the colloidal gold in the colloidal gold solution is 15-25nm;

The reaction was stirred at 25°C for 30min;

The pH value of described colloidal gold solution is 9.0;

In step 2), the mouse-derived anti-IgG antibody is rabbit anti-mouse IgG.

In the above method,

The colloidal gold solution is prepared according to the following method: the tetrachloroauric acid aqueous solution and the sodium citrate aqueous solution are mixed, and react to obtain the colloidal gold solution; the mass ratio of the sodium citrate and the tetrachloroauric acid is 5:1;

The reaction temperature of the tetrachloroauric acid aqueous solution is 97 DEG C;

The reactions were A and B in sequence: A was reacted at 97°C for 10 minutes; B was stirred at 25°C for 10 minutes.

In the above method,

The H7 subtype avian influenza virus polyclonal antibody is the H7N2 subtype avian influenza virus polyclonal antibody.

The application of the above-mentioned colloidal gold immunochromatographic test strips in the preparation of kits for detecting or assisting in the detection of H7 subtype avian influenza virus is also within the protection scope of the present invention.

The experiment of the present invention proves that the present invention uses sample pads, coupling pads, NC membranes marked with detection lines and quality control lines, and absorption pads to be assembled on the support plate according to the following process, wherein the coupling pads are perfused with H7 gold-labeled monoclonal antibodies , The detection line and the quality control line are composed of H7 polyclonal antibody and rabbit anti-mouse IgG spray film. The test strip of the present invention has the following advantages: simple operation, good specificity, high sensitivity, and reliable diagnostic results; it is suitable for on-site detection of H7 subtype avian influenza virus infection, especially suitable for on-site rapid detection of grassroots units or individuals.

Description of drawings

Figure 1 is a schematic diagram of the test strip structure

Figure 2 is the judgment of the test strip results

Figure 3 shows the sensor sensitivity test results

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

H7N2 subtype avian influenza virus (Duck/HK/47/76H7N2), H5N1 subtype avian influenza virus (Chicken/Guangxi/1/04H5N1), H1N3 subtype avian influenza virus (Duck/HK/717/79-d1H1N3), H9N2亚型禽流感病毒（Duck/Guangxi/1/00H9N2）均记载在：AmultiplexRT-PCRfordetectionoftypeAinfluenzavirusanddifferentiationofavianH5,H7,andH9hemagglutininsubtypes；ZhixunXie,Yao-shanPang,JiaboLiu,XianwenDeng,XiaofeiTang,JianhuaSun,MazharI.Khan；MolecularandCellularProbes20(2006)245 –249; the public can all be obtained from the Veterinary Research Institute of Guangxi Zhuang Autonomous Region.

NDV strain GX1/00 is recorded in the literature: Chen Anli, Xie Zhixun, Zhou Chenyu, et al. Establishment of a detection method for the identification of strong and weak Newcastle disease virus LAMP strains[J]. Chinese Veterinary Science, 2011,41(09):917-922 ; Publicly available from Guangxi Zhuang Autonomous Region Veterinary Research Institute.

The IBV strain Massachusetts41 is recorded in the literature: Xie Zhiqin, Xie Zhixun, Lu Huagang. et al. Cloning and sequence analysis of the S1 gene of 30 standard strains and isolates of chicken infectious bronchitis virus [J]. Southwest Agricultural Journal, 2008, 21(6) : 1733-1736; publicly available from the Veterinary Research Institute of Guangxi Zhuang Autonomous Region.

Among them, the virus content of H7N2 subtype avian influenza virus (Duck/HK/47/76H7N2) is 2 ⁷ HAunit/50ul;

H1 subtype avian influenza virus content is 2 ⁸ HAunit/50ul

H5 subtype avian influenza virus content is 2 ⁹ HAunit/50ul

H9 subtype avian influenza virus content is 2 ⁹ HAunit/50ul

The virus content of NDV strain GX1/00 is 2 ⁷ HAunit/50ul

The virus content of IBV strain Massachusetts 41 was 10 ^5.7 EID ₅₀ /ml.

Embodiment 1, the preparation of colloidal gold immunochromatography test strip

1. Preparation of colloidal gold solution

Accurately pipette 1mL of ₁ % HAuCl4 solution into a 150mL Erlenmeyer flask, add water to a total volume of about 95mL to obtain tetrachloroauric acid aqueous solution; heat the water bath to 97°C, add 5.0mL of 10g/L sodium citrate aqueous solution under stirring conditions, Mix well to obtain a mixture (mass ratio of HAuC1 ₄ and sodium citrate 1:5); then heat the mixture in a 97°C water bath for 10 minutes (stirring), then stir at room temperature (25°C) for 10 minutes, and cool naturally Finally, the colloidal gold solution was obtained, and the volume was fixed to a 100mL volumetric flask with water.

The particle size of colloidal gold is 15-25nm.

2. Preparation of gold-labeled antibody

Adjust the colloidal gold solution to pH 9.0 with 0.1mol/L K ₂ CO ₃ solution;

Add 50 μL of H7 avian influenza virus monoclonal antibody with a concentration of 1 mg/mL to 10 ml of colloidal gold solution with a pH value of 9.0, stir for 30 min at room temperature at 25 ° C, centrifuge at 5200 g for 20 min, discard the supernatant to collect the precipitate; 1% BSA in 20mmol/L sodium borate aqueous solution, stir at 25°C for 30min to seal, centrifuge at 9000g for 20min, discard the supernatant to collect the precipitate; In sodium borate aqueous solution, restore the gold-labeled antibody to 1/10 of its original volume, store it in a 4°C refrigerator for later use, and obtain the gold-labeled antibody.

H7 subtype avian influenza virus monoclonal antibody was purchased from abcam company, product code: ab82458, product link website: http://www.abcam.cn/Influenza-A-Virus-Hemagglutinin-H7-antibody-1H11-ab82458-references .html.

3. Development of colloidal gold immunochromatographic test strips

H7 subtype avian influenza virus polyclonal antibody is positive serum of H7 subtype avian influenza chicken: purchased from Harbin Veken Biotechnology Development Company, identified as positive by hemagglutination inhibition test method, the concentration is 0.2mg/mL.

The test strip is mainly composed of NC membrane (NC-a101Millipore135, pore size 8μm, 2.5cm×30cm, with backing, Millipore), sample pad (BT50, thickness 0.52mm, water absorption 53mg/cm2, Shanghai Gold Standard Biotechnology Co., Ltd. ), coupling pad (SB08, thickness 0.33mm, water absorption 63.1mg/cm2, Shanghai Gold Standard Biotechnology Co., Ltd.), water absorption pad (CH27, thickness 0.69mm, water absorption speed 65.8s/4cm, water absorption 62.5mg/cm2 , Shanghai Gold Standard Biotechnology Co., Ltd.) and the support board (PVC rubber board) and other five parts.

Put the NC membrane on the BIODOTXYZ spraying system (XYZ3200) platform to flatten and compact, put 200μL 0.2mg/mL H7 subtype avian influenza virus polyclonal antibody in pool A, put 200μL 1mg/mL rabbit anti-mouse IgG in pool B, and turn on the machine Afterwards, the H7 subtype avian influenza virus polyclonal antibody and rabbit anti-mouse IgG (product number BA1048, Boster Biotech) were spotted on the NC membrane respectively to form a test line (T) and a quality control line (C). After drying naturally at room temperature at 25°C, soak it in the blocking solution (1%BSA in PBS buffer solution, pH=7.4) for 30min, dry at 37°C, add desiccant, and seal it at 4°C to obtain the detection line ( T) and quality control line (C) NC membrane;

Cut the glass fiber cotton into thin strips of 10 mm, put them into the PBS treatment solution containing 5% BSA, 2% sucrose, 0.8% NaCl and 0.05% NaN ₃ for 20 minutes, and dry them at a constant temperature of 37 ° C, and then put the gold standard prepared by the above 2 The antibody is perfused on the treated glass fiber cotton, and vacuum freeze-dried for 4 hours, which is the coupling pad;

The sample pad should be treated with PBS containing ₂ % BSA, 1% sucrose, 0.5% sodium borate and 0.1% NaN3, and then dried at 37°C for use;

On the support plate, assemble the NC membrane, sample pad, coupling pad, water-absorbing pad, etc. according to the following process (as shown in Figure 1): the bottom surface of the NC membrane is pasted on the support plate, and the two ends of the NC membrane Paste a coupling pad and a water-absorbent pad on the top, and paste a sample pad on the other end of the glass fiber membrane, with a lamination width of 2mm. Make 3.5mm wide test strips with a cutting machine, store them in an airtight container with a desiccant to obtain colloidal gold immunochromatographic test strips.

The detection principle of the test strip: the test strip is designed according to the principle of "antibody-antigen-antibody" sandwich immunochromatography. After dropping the sample, under the capillary action of the NC membrane, the sample solution migrates upwards, and when it reaches the coupling pad, the gold-labeled antibody will be dissolved. When the sample is positive for the H7 subtype avian influenza virus, the H7 subtype avian influenza virus will combine with the gold-labeled antibody and migrate upward together to reach the detection line T where the H7 subtype avian influenza virus polyclonal antibody is immobilized. The "H7 subtype avian influenza virus polyclonal antibody-H7 subtype avian influenza virus-gold-labeled antibody" complex finally forms a red precipitation line, while the unfixed gold-labeled antibody continues to ascend to the rabbit anti-mouse IgG to form "Gold-labeled antibody-rabbit anti-mouse IgG" complex forms a red precipitation line, which is quality control line C. When the sample is negative for H7 subtype avian influenza virus, the gold-labeled antibody dissolves in the sample and migrates upwards together, and when it reaches the detection line T where the H7 subtype avian influenza virus polyclonal antibody is immobilized, it cannot be fixed, and the detection line T There is no red band (the unfixed gold-labeled antibody continues to ascend to the rabbit anti-mouse IgG, forming a "gold-labeled antibody-rabbit anti-mouse IgG" complex, forming a red precipitation line, which is the quality control line C. The negative and positive results of the test strips are shown in Figure 2.

Embodiment 2, the specificity and sensitivity detection of colloidal gold immunochromatography test strip

1. Specific detection

The following inactivated avian influenza viruses were obtained by inactivating the corresponding subtype of avian influenza virus strains by shaking with 0.1% formaldehyde at 37°C for 4 hours.

Inactivated H7N2 subtype avian influenza virus (positive control, H7), inactivated H5N1 subtype avian influenza virus (H5), inactivated H1N3 subtype avian influenza virus (H1), inactivated H9N2 subtype avian influenza virus (H9 ), Newcastle disease virus (NDV) GX1/00 and infectious bronchitis virus (IBV) Massachusetts41 are samples to be tested, and are detected with the colloidal gold immunochromatography test strip prepared by embodiment 1, and the detection method is as follows: The test sample was diluted 10 times with PBS solution (pH=7.4, containing 0.8% NaCl), that is, 10 μL of the sample to be tested was added to 90 μL PBS solution, mixed evenly, and dropped into the sample hole (S) of the test strip, and allowed to move along the test strip. The paper strip spreads freely, and the results are observed within 15 minutes.

Results: Inactivated H7N2 subtype avian influenza virus appeared two red reaction bands at the position of T line and C line at the same time, and the result was judged as positive; inactivated H5N1 subtype avian influenza virus (H5), H1N3 subtype avian influenza virus, H9N2 Subtype avian influenza virus, Newcastle disease virus and infectious bronchitis virus only had a red reaction band at the C line, and the results were judged as negative.

2. Sensitivity detection

SPF chicken cloacal cotton test paper sample (healthy chicken) was used as a negative control, and the H7N2 subtype avian influenza virus (Duck/HK/47/76H7N2) was diluted 10 times, 50 times, 100 times, 200 times, 300 times with PBS buffer solution , 400 times, 500 times, 600 times, 700 times, detect according to the method of the following steps with the colloidal gold immunochromatography test strip prepared by embodiment 1.

The results are shown in Figure 3, 10 times, 50 times, 100 times, 200 times, 300 times, 400 times, two red reaction bands appeared at the position of T line and C line at the same time, the result was judged as positive, 500 times, 600 times, The 700-fold sample only had a red reaction band at the position of the C line, and the result was judged as negative. The results showed that the sensitivity of the test strip was 1:400.

Therefore, use the above-mentioned test strip to detect or assist in detecting whether the sample to be tested contains H7 subtype avian influenza virus,

If two red reaction bands appear at both the detection line T and the quality control line C, the result is determined to be positive, and the sample to be tested contains or is a candidate to contain H7 subtype avian influenza virus;

If there is only one red reaction band at the quality control line C, the result is judged as negative; the sample to be tested does not contain or the candidate does not contain H7 subtype avian influenza virus;

In other cases, the test strips are invalid.

3. Check against conventional methods

Using Dot-ELYSA detection, the specific method is to dilute H7N2 subtype avian influenza virus (Duck/HK/47/76H7N2) by 10 times, 50 times, 100 times, 200 times, 300 times, 400 times, 500 times with PBS buffer solution , 600 times, and 700 times, respectively, take 5 μL samples on the smooth surface of nitrocellulose membrane (NC membrane, pore size 0.45 μm, product of Zhejiang Huangyan People’s Chemical Factory), dry at 37°C, seal in 5% skimmed milk for 1 h, take out NC membrane, washed 3 times with PBST, 10 min each time; add 1:200 diluted H7 monoclonal antibody and incubate at 37°C for 1 h, after washing 3 times with PBST, add 1:200 diluted HPR-labeled rabbit anti-mouse IgG, 37°C Incubate for 1 h, wash 3 times with PBST, develop color in DAB for 15 min, wash NC membrane with PBST, and those with brown spots on the membrane are judged as positive.

The results showed that brown spots appeared at 10 times, 50 times, 100 times, 200 times, and 300 times, but no brown spots appeared in samples with 400 times, 500 times, 600 times, and 700 times. The detection sensitivity of Dot-ELYSA was 1:300.

Therefore, the method of the present invention has high sensitivity.

Claims (2)

1., for the preparation of the method detecting H7 subtype avian influenza virus colloidal gold immuno-chromatography test paper strip, comprise the steps:

1) first by colloidal gold solution and the mixing of H7 subtype avian influenza virus monoclonal antibody, reaction, collects solid reactant, then is closed through BSA by described solid reactant, namely obtain golden labeling antibody;

2) preparation is perfused with the coupling pad of golden labeling antibody and the NC film containing detection line T and nature controlling line C;

The described coupling pad being perfused with golden labeling antibody for described golden labeling antibody to be filled on glass fibre cotton, dry, obtain coupling pad;

The described NC film containing detection line T and nature controlling line C is that H7 subtype avian influenza virus polyclonal antibody and rabbit against murine lgG are distinguished fixed fire on NC film, forms the NC film containing detection line T and nature controlling line C;

3) described NC film, sample pad, described coupling pad and adsorptive pads are assembled into support plate, obtain colloidal gold immuno-chromatography test paper strip;

Step 1) in, the reinforced volume ratio of described colloidal gold solution and described H7 subtype avian influenza virus monoclonal antibody is 200:1;

The concentration of described H7 subtype avian influenza virus monoclonal antibody is 1mg/mL;

The particle diameter of the collaurum in described colloidal gold solution is 15-25nm;

Described reaction is 25 DEG C and stirs 30min;

The pH value of described colloidal gold solution is 9.0;

Described colloidal gold solution is prepared as follows: tetra chlorauric acid aqueous solution and sodium citrate aqueous solution mixing, and reaction, obtains colloidal gold solution; The mass ratio of described sodium citrate and described tetra chlorauric acid is 5:1;

The temperature of reaction of described tetra chlorauric acid aqueous solution is 97 DEG C;

It is 97 DEG C of reaction 10min that described reaction is followed successively by A, B:A; B is 25 DEG C and stirs 10min;

Step 2) described in drying be vacuum freeze-drying; For pouring into the glass fibre cotton of golden labeling antibody for put into containing 5%BSA by glass fibre cotton, 2% sucrose, 0.8%NaCl and 0.05%NaN ₃pBS treating fluid in 20min, 37 DEG C of constant temperature dryings, obtain;

Step 3) in, for being assembled into the sample pad of described support plate for using containing 2%BSA by sample pad, 1% sucrose, 0.5% sodium borate and 0.1%NaN ₃the process of PBS treating fluid, 37 DEG C of dryings, obtain.

2. method according to claim 1, is characterized in that:

Described H7 subtype avian influenza virus polyclonal antibody is H7N2 subtype avian influenza virus polyclonal antibody.