CN108303529B - Immune colloidal gold test strip for detecting avian influenza virus H7 subtype and preparation method thereof - Google Patents
Immune colloidal gold test strip for detecting avian influenza virus H7 subtype and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an immune colloidal gold test strip for detecting avian influenza virus H7 subtype and a preparation method thereof, belonging to the technical field of animal disease diagnosis. The test strip consists of a sample pad, a colloidal gold combination pad sprayed with a monoclonal antibody 1H11(CCTCC NO: C2017112) colloidal gold mark for resisting avian influenza virus H7 subtype, and a nitrocellulose membrane respectively coated with a monoclonal antibody 6B3(CCTCC NO: C2017204) for resisting avian influenza virus H7 subtype and a goat anti-mouse IgG polyclonal antibody on a detection line and a quality control line, wherein the sample pad, the colloidal gold combination pad, the nitrocellulose membrane and the water absorption pad are sequentially adhered to a PVC bottom plate. The main principle of the immune colloidal gold test strip for detecting the avian influenza virus H7 subtype is that the antigen is detected by a double-antibody sandwich, and the immune colloidal gold test strip has high sensitivity and specificity. Compared with the common PCR and ELISA antigen detection technology in laboratories, the method is simpler, more convenient and faster, does not need special personnel and detection equipment, and can be widely applied to the basement layer.
Description
Technical Field
The invention belongs to the technical field of animal disease diagnosis, and particularly relates to an immune colloidal gold test strip for detecting avian influenza virus H7 subtype and a preparation method thereof.
Background
Avian influenza is a highly contagious disease caused by Avian Influenza Virus (AIV) and infected by poultry and wild poultry, and has the characteristics of strong infectivity, high transmission speed, easy antigen variation and the like. According to different antigenicity of Hemagglutinin (HA) and Neuraminidase (NA) of the avian influenza virus, 15 specific HA and 9 specific NA which are found at present are respectively named as H1-H15 and N1-N9, and more than 100 subtypes of avian influenza virus can be formed between different HA and NA. AIV subtype H7 has 9 NA subtypes, among which H7N3, H7N7, H7N1, etc. are highly pathogenic to birds. The H7 subtype of avian influenza has emerged in australia, england, germany and other countries over the past decades. In 2003, H7N2 cases are separated from chicken bodies for the first time in China, and in 2013, death cases of human infection with H7N9 subtype avian influenza virus are reported for the first time in China. 2016-2017 shows a fifth H7 subtype avian influenza outbreak peak in China during winter and spring handover, and the epidemic situation is more serious than before, and is represented by early onset time and high onset rate, and the virus has the phenomena of drug resistance mutation and increased virulence. Aiming at the current situation of H7 subtype avian influenza epidemic, the office of Ministry of agriculture at the beginning of 6 months in 2017 firstly carries out H7N9 immunization work in two broad areas, and provides strong technical support for carrying out avian influenza prevention and control work in two broad areas and nationwide efficiently. In view of the great challenge brought to the health of poultry breeding industry and human beings by the outbreak of avian influenza, a rapid and visual pathogen detection method is established, and the development of poultry breeding industry and public health safety can be greatly promoted by early discovery, early elimination and early immunity.
The Immune colloidal gold technology (Immune colloidal gold technology) is a novel Immune labeling technology which applies colloidal gold as a tracer marker to antigen and antibody. The technology is a novel rapid and simple diagnostic technology developed on the basis of technologies such as McAb, ELISA and the like. The principle is that colloidal gold is used as a tracing marker, a microporous membrane is used as a solid phase carrier, known antigen or antibody is coated, after a sample to be detected is added, the antigen or antibody in the sample is combined with the antigen or antibody on the membrane through the osmosis action of the microporous membrane, and then the red visible result is formed through the reaction of the antigen or antibody and the colloidal gold marker.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides an immune colloidal gold test strip for detecting avian influenza virus H7 subtype and a preparation method thereof.
In order to achieve the purpose, the invention adopts the technical scheme that: an immune colloidal gold test strip for detecting avian influenza virus H7 subtype comprises a PVC base plate, wherein a sample pad and a water absorption pad are respectively arranged at two ends of the PVC base plate, and the sample pad provides a position for adding a sample to be detected; the detection line coated with the avian influenza virus H7 subtype-resistant monoclonal antibody 6B3 and the quality control line coated with the goat anti-mouse IgG polyclonal antibody are sequentially arranged on the nitrocellulose membrane, and the monoclonal antibody 1H11 is secreted by a hybridoma cell strain 1H 11; the monoclonal antibody 6B3 is secreted by a hybridoma cell strain 6B3, the hybridoma cell strains 1H11 and 6B3 are preserved in the China Center for Type Culture Collection (CCTCC) of Wuhan university, and the cell preservation numbers are as follows in sequence: CCTCC NO: c2017112 and C2017204, wherein the preservation dates are 7 and 18 in 2017 and 10 and 17 in 2017 respectively.
The invention also provides a preparation method of the immune colloidal gold test strip for detecting the avian influenza virus H7 subtype, which comprises the following steps:
1) preparing colloidal gold;
2) labeling the monoclonal antibody 1H11 with the colloidal gold prepared in the step 1) to obtain a colloidal gold-labeled monoclonal antibody 1H 11;
3) preparing a colloidal gold bonding pad;
4) respectively spraying monoclonal antibody 6B3 and goat anti-mouse IgG polyclonal antibody on the positions of a nitrocellulose membrane detection line and a quality control line, and drying for later use;
5) treating the sample pad: soaking absorbent paper in PB buffer solution for 30min, drying at 37 deg.C, and cutting into strips with width of 1.6 cm; obtaining a processed sample pad; the formula of the buffer solution is Na2HPO4 & 12H2O 3.58.58 g, NaH2PO4 & 2H2O1.56g, and the volume of the injection water is fixed to 1000 mL; and sequentially sticking the treated sample pad, the colloidal gold combined pad sprayed with the monoclonal antibody 1H 11-colloidal gold labeled for resisting avian influenza virus H7 subtype, the nitrocellulose membrane sprayed with the monoclonal antibody 6B3 detection line for resisting avian influenza virus H7 subtype and the goat anti-mouse IgG multi-antibody quality control line and the water absorption pad on a hard PVC bottom plate, and cutting into strips of 1.6mm for later use.
Further, the specific steps of step 1) are as follows: adding 100mL of water for injection into a siliconized bottle, adding 1mL of chloroauric acid, heating to boil with medium fire, adding 1.8mL of 1.05% trisodium citrate solution at one time, continuing heating until the color of the solution is stable wine red, naturally cooling at room temperature, and finally diluting to 100mL with double distilled water to obtain the colloidal gold solution.
Further, the specific steps of step 2) are as follows: adjusting the pH value of the colloidal gold obtained in the step 1) to 9.0 by using 0.2mol/L potassium carbonate, adding the monoclonal antibody 1H11, wherein the monoclonal antibody labeling amount of each 1mL of the colloidal gold solution is 8.0ug, slowly stirring for 45min to obtain a monoclonal antibody-colloidal gold marker for resisting the H7 subtype of avian influenza virus, and adding 0.5mL of 5% PEG20000 solution under stirring for sealing for 30 min. The labeled colloidal gold solution was centrifuged at 8000rpm for 30min and then resuspended in 10mM Tris-HCl, pH9.0.
Further, the specific steps of step 3) are as follows:
a) preparing a gold pad treatment solution: 10mM Tris-HCl, 0.45% PVP-40, 0.45% Tween-20, 0.5% SDS-L + SDS-F and 3% trehalose, stirring and mixing uniformly, and filtering by a 0.22uM filter;
b) soaking the glass cellulose membrane in the treatment liquid obtained in the step a) for 30min, and drying at 37 ℃. Spraying the colloidal gold-labeled anti-avian influenza virus H7 subtype monoclonal antibody 1H11 prepared in the step 2) on the treated colloidal gold conjugate pad, wherein the spraying amount is 10uL/cm, and drying at 37 ℃.
Further, the specific steps of step 4) are as follows:
A. preparing a coating solution: 0.2M Tris-HCl, 1% Tween-20 and 0.5% X-100, stirring and mixing uniformly, and filtering by a 0.22uM filter;
B. respectively diluting the monoclonal antibody 6B3 resisting avian influenza virus H7 subtype and the goat anti-mouse IgG polyclonal antibody with the coating solution, spraying the diluted antibodies on a nitrocellulose membrane as a detection line and a quality control line, wherein the concentrations of the antibodies are 1mg/mL and 0.5mg/mL respectively, the spraying amount is 1uL/cm, the distance between the detection line and the quality control line is 5mm, and drying the antibodies at 37 ℃ for later use.
Compared with the prior art, the invention has the beneficial effects that:
1) the avian influenza virus H7 subtype monoclonal antibodies 1H11 and 6B3 obtained by the invention have good effect of detecting avian influenza virus H7 subtype antigen through sandwich ELISA verification, and the use of the monoclonal antibodies improves the specificity and sensitivity of detection results.
2) Two strains of hybridoma cells 1H11 and 6B3 with better biological characteristics are screened from the obtained hybridoma cells secreting the avian influenza virus H7 subtype-resistant monoclonal antibody, the HI titers of hemagglutination inhibition of the avian influenza virus H7 subtype after ascites prepared by the hybridoma cells is purified respectively reach 18log2 and 16log2, and the ELISA titer reaches 4 × 105 and 3 × 105.
3) The test strip is suitable for rapid detection of avian influenza virus H7 subtype, and has high sensitivity and strong specificity;
4) the test strip does not need any instrument and equipment, is convenient to carry and has low detection cost;
5) the test strip is convenient to operate, does not need to be operated by professional personnel, and saves manpower;
6) the test strip is convenient to store, has stable property, and has consistent detection results when stored at normal temperature for 12 months and at 4 ℃.
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FIG. 1 is a schematic structural diagram of an immune colloidal gold test strip for detecting avian influenza virus subtype H7 according to the present invention;
FIG. 2 is a schematic diagram of the sensitivity test in example 5 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: hemagglutination inhibition detection of ascites of cell strain on avian influenza virus H7 subtype
Taking female BALB/c mice more than 8 weeks old, injecting sterilized paraffin 0.5 mL/mouse intraperitoneally, carrying out amplification culture on hybridoma cell strains 1H11 and 6B3 obtained in the laboratory after 7 days, collecting well-grown hybridoma cell strains, centrifuging for 10min at 1000r/min, discarding supernatant, suspending cell precipitates in serum-free culture solution, adjusting cell density, and injecting 0.5mL of cell suspension (about 5 multiplied by 105 cells/mouse) into the abdominal cavity of each mouse. After 7-10 days, the abdomen of the mouse is obviously enlarged, and ascites is collected. Ascites titers were determined by indirect ELISA and HI assay, and the results are shown in Table 1. Purifying ascites by two-step precipitation with octanoic acid-ammonium sulfate, centrifuging at 4 deg.C at 10000rpm for 15min, collecting supernatant, and filtering with microporous membrane to remove clot and fat drop. Adding 4 times volume of sodium acetate buffer (pH4.5) into ascites, adding 20 μ L of octanoic acid into each ml of ascites under stirring, centrifuging at 4 deg.C and 10000rpm for 30min after 30min, and adjusting pH of the supernatant to 7.4 with sodium hydroxide solution. Adding saturated ammonium sulfate with volume less than 40% of ascites volume under stirring for 30min, centrifuging at 4 deg.C at 10000rpm for 15min, and removing supernatant. The pellet was resuspended in 1/2 volumes of 10mM Tris-HCl (pH9.0) and dialyzed against the same Tris-HCl buffer in dialysis bags, with the dialysis solution being changed every 4h for 3 total changes. The concentrations of purified monoclonal antibody 1H11 and 6B3 were 11.2 mg/ml and 6.8mg/ml, respectively. ELISA verifies that the obtained monoclonal antibody only has specific reaction with the avian influenza virus H7 subtype, but has no cross reaction with other viruses such as H5 subtype avian influenza virus, H9 subtype avian influenza virus, infectious bursal disease virus, infectious bronchitis virus, Marek's virus and Newcastle disease virus, and shows that the monoclonal antibody has good specificity.
Table 1: results of two ascites HI and ELISA titer detections
| Cell line | HI | Potency of the |
| 1H11 | ||
| 218 | 1∶409600 |
| |
216 | 1∶320000 |
Example 2: preparation of avian influenza virus H7 subtype test strip
(1) Preparation of colloidal gold
Adding 100mL of water for injection into a siliconized conical flask, adding 1mL of chloroauric acid, placing the mixture into a microwave oven, heating the mixture to boiling with medium fire, adding 1.8mL of 1.05% trisodium citrate solution at one time, continuing heating until the solution is in a stable wine red color, naturally cooling the solution at room temperature, and finally adding double distilled water to a constant volume of 100mL to obtain the colloidal gold solution.
(2) Preparation of colloidal gold-labeled monoclonal antibody 1H11
And (2) regulating the pH value of the colloidal gold obtained in the step (1) to 9.0 by using 0.2mol/L potassium carbonate, adding the monoclonal antibody 1H11, wherein the monoclonal antibody labeling amount of each 1mL of the colloidal gold solution is 8.0ug, slowly stirring for 45min to obtain a monoclonal antibody-colloidal gold marker for resisting the H7 subtype of avian influenza virus, and adding 0.5mL of 5% PEG20000 solution under stirring for sealing for 30 min. The labeled colloidal gold solution was centrifuged at 8000rpm for 30min and then resuspended in 10mM Tris-HCl, pH9.0.
(3) Preparation of colloidal gold bonding pad
a) Preparing a gold pad treatment solution: 10mM Tris-HCl, 0.45% PVP-40, 0.45% Tween-20, 0.5% SDS-L + SDS-F and 3% trehalose, stirring and mixing uniformly, and filtering by a 0.22uM filter;
b) soaking the glass cellulose membrane in the treatment liquid obtained in the step a) for 30min, and drying in a blast drying oven at 37 ℃. And (3) spraying the colloidal gold-labeled anti-avian influenza virus H7 subtype monoclonal antibody 1H11 prepared in the step (2) on the treated colloidal gold conjugate pad, wherein the spraying amount is 10uL/cm, and drying in a 37 ℃ forced air drying oven.
(4) Spraying detection line and quality control line
1. Preparing a coating solution: 0.2M Tris-HCl, 1% Tween-20 and 0.5% X-100, stirring and mixing uniformly, and filtering by a 0.22uM filter;
2. respectively diluting the monoclonal antibody 6B3 resisting avian influenza virus H7 subtype and the goat anti-mouse IgG polyclonal antibody with the coating solution, spraying the diluted antibodies on a nitrocellulose membrane as a detection line and a quality control line, wherein the concentrations of the antibodies are 1mg/mL and 0.5mg/mL respectively, the spraying amount is 1uL/cm, the distance between the detection line and the quality control line is 5mm, and drying the antibodies in a blast drying oven at 37 ℃ for later use.
(5) Sample pad handling
Soaking absorbent paper in PB buffer solution for 30min, drying in a blast drying oven at 37 deg.C, and cutting into strips with width of 1.6 cm; obtaining a processed sample pad; the formula of the buffer solution comprises Na2HPO4 & 12H2O 3.58.58 g, NaH2PO4 & 2H2O1.56g and water for injection with constant volume of 1000 mL.
(6) Assembly of test strips
And sequentially sticking the treated sample pad, the colloidal gold combined pad sprayed with the monoclonal antibody 1H 11-colloidal gold labeled for resisting avian influenza virus H7 subtype, the nitrocellulose membrane sprayed with the monoclonal antibody 6B3 detection line for resisting avian influenza virus H7 subtype and the goat anti-mouse IgG multi-antibody quality control line and the water absorption pad on a hard PVC bottom plate, and cutting into strips of 1.6mm for later use.
Example 3: application of H7 subtype avian influenza test strip
1) Collection of secretion swab samples: the swab stained with the secretion was placed in 500. mu.L PBS (containing 2000 units/mL penicillin and streptomycin) overnight at 4 ℃, centrifuged at 10000r/min for 5min, and the supernatant was collected (25% glycerol was added to PBS if long-term storage was required).
2) And (3) detection: diluting a sample by using normal saline at a ratio of 1:10, dripping 40 mu L of the diluted sample on the test strip, standing for 15min, and observing the result. Simultaneously, 40 mu L of physiological saline and 1: the positive antigen diluted by 50 is dripped on a test strip to be used as a negative control and a positive control. The positive control is avian influenza virus H7 subtype hemagglutination inhibition test antigen purchased from Harbin Vitaceae biotechnology development company.
3) And (4) judging a result: and (3) a positive result shows that the quality control line and the detection line both present red bands, which indicates that the avian influenza virus H7 subtype exists. Negative results: and the detection result shows no red strip, and the quality control line shows a red strip, which indicates that no avian influenza virus H7 subtype exists in the sample. And if the detection line and the quality control line do not have red strips or only the detection line has the red strips, the product is invalid.
Example 4: specificity test of avian influenza virus H7 subtype test strip
Clinically collected newcastle disease virus positive samples, adenovirus positive samples, bursal disease virus positive samples, infectious bronchitis virus positive samples and normal saline are respectively spotted with avian influenza virus H5, H7 and H9 hemagglutination inhibition test antigens purchased from Harbin Vitaceae biotechnology development companies for detection. Only the test strip quality control line and the detection line which are dropwise added with the avian influenza virus H7 subtype antigen present red strips at the same time, and other test strips only present red quality control lines, which shows that the test strip has good specificity.
Example 5: susceptibility test of avian influenza virus H7 subtype test strip
After an antigen with a hemagglutination inhibition valence HA of 29 purchased from Harbin Vitaceae biotechnology development company is diluted by physiological saline from a 1: 2-fold ratio, 40 mu L of virus liquid is dripped into each test strip, and the result shows that the test strip can still detect the antigen as positive when the antigen is diluted to 128-fold, which indicates that the test strip HAs good sensitivity.
Example 6: stability test of avian influenza virus H7 subtype test strip
The 3 batches of test strips are respectively placed at normal temperature and 4 ℃, taken out every 1 month and simultaneously used for detecting 10 avian influenza virus H7 subtype positive samples and 10 avian influenza virus H7 subtype negative samples. The results show that: the test result is stable after being placed at room temperature and 4 ℃ for 6 months.
First month detection results:
third month test results
Test results in the sixth month
Example 7: comparison of avian influenza virus H7 subtype test strip with HA test
60 cloaca swabs of chickens are detected by using two methods, namely an avian influenza virus H7 subtype antigen detection test strip and an HA test, wherein 30 parts are stored in a P3-grade laboratory of the national emphasis laboratory of agricultural microbiology of university of agriculture in Huazhong, and 30 parts are clinically collected cloaca swabs. And comparing the test results, and calculating the coincidence rate. The positive coincidence rate is 100%, the negative coincidence rate is 96.67%, and the total coincidence rate is 98.33%, which shows that the test strip HAs good coincidence rate with HA tests, and is suitable for popularization and application in large-scale detection of a base layer.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. An immune colloidal gold test strip for detecting avian influenza virus H7 subtype comprises a PVC base plate, wherein a sample pad and a water absorption pad are respectively arranged at two ends of the PVC base plate, and the sample pad provides a position for adding a sample to be detected; PVC bottom plate middle part is provided with the nitrocellulose membrane, be provided with the colloidal gold between nitrocellulose membrane and the sample pad and combine the pad, the nitrocellulose membrane is connected its characterized in that with the pad that absorbs water: the colloidal gold combined pad is composed of a glass cellulose membrane adsorbed with a colloidal gold-labeled anti-avian influenza virus H7 subtype monoclonal antibody 1H11, a detection line coated with an anti-avian influenza virus H7 subtype monoclonal antibody 6B3 and a quality control line coated with a goat anti-mouse IgG polyclonal antibody are sequentially arranged on the nitrocellulose membrane, the monoclonal antibody 1H11 is secreted by a hybridoma cell strain 1H11, the monoclonal antibody 6B3 is secreted by a hybridoma cell strain 6B3, the hybridoma cell strains 1H11 and 6B3 are preserved in the China Committee culture Collection of university in Wuhan, and the cell preservation numbers are sequentially as follows: CCTCC NO: c2017112 and C2017204, with the preservation dates of 7 and 18 in 2017 and 10 and 17 in 2017, respectively.
2. The method for preparing the immune colloidal gold test strip for detecting the avian influenza virus H7 subtype of claim 1, which is characterized in that: further comprising the steps of:
1) preparing colloidal gold;
2) labeling the monoclonal antibody 1H11 with the colloidal gold prepared in the step 1) to obtain a colloidal gold-labeled monoclonal antibody 1H 11;
3) preparing a colloidal gold bonding pad;
4) respectively spraying monoclonal antibody 6B3 and goat anti-mouse IgG polyclonal antibody on the positions of a nitrocellulose membrane detection line and a quality control line, and drying for later use;
5) treating the sample pad: soaking absorbent paper in PB buffer solution for 30min, drying at 37 ℃, and cutting into strips with the width of 1.6cm for later use to obtain a processed sample pad; the above buffer solution is Na2HPO4·12H2O 3.58g，NaH2PO4·2H2O1.56g, and the volume of the injection water is up to 1000 mL; and sequentially sticking the treated sample pad, the colloidal gold combined pad sprayed with the monoclonal antibody 1H11 colloidal gold mark resisting avian influenza virus H7 subtype, the nitrocellulose membrane sprayed with the monoclonal antibody 6B3 detecting line resisting avian influenza virus H7 subtype and the goat anti-mouse IgG multi-antibody quality control line and the water absorption pad on a hard PVC bottom plate, and cutting into strips of 1.6mm for later use.
3. The method for preparing the immune colloidal gold test strip for detecting the avian influenza virus H7 subtype according to claim 2, which is characterized in that: the specific steps of the step 1) are as follows: adding 100mL of water for injection into a siliconized bottle, adding 1mL of chloroauric acid, heating to boil with medium fire, adding 1.8mL of 1.05% trisodium citrate solution at one time, continuing heating until the color of the solution is stable wine red, naturally cooling at room temperature, and finally diluting to 100mL with double distilled water to obtain the colloidal gold solution.
4. The method for preparing the immune colloidal gold test strip for detecting the avian influenza virus H7 subtype according to claim 2, which is characterized in that: the specific steps of the step 2) are as follows: adjusting the pH value of the colloidal gold obtained in the step 1) to 9.0 by using 0.2mol/L potassium carbonate, adding a monoclonal antibody 1H11, wherein the monoclonal antibody labeling amount of each 1mL of the colloidal gold solution is 8.0ug, slowly stirring for 45min to obtain a monoclonal antibody-colloidal gold marker for resisting avian influenza virus H7 subtype, and adding 0.5mL of 5% PEG20000 solution under stirring for sealing for 30 min; the labeled colloidal gold solution was centrifuged at 8000rpm for 30min and then resuspended in 10mM Tris-HCl, pH9.0.
5. The method for preparing the immune colloidal gold test strip for detecting the avian influenza virus H7 subtype according to claim 2, which is characterized in that: the specific steps of the step 3) are as follows:
a) preparing a gold pad treatment solution: 10mM Tris-HCl, 0.45% PVP-40, 0.45% Tween-20, 0.5% SDS-L + SDS-F and 3% trehalose, stirring and mixing uniformly, and filtering by a 0.22uM filter;
b) soaking a glass cellulose membrane in the treatment liquid obtained in the step a) for 30min, drying at 37 ℃, spraying the colloidal gold-labeled anti-avian influenza virus H7 subtype monoclonal antibody 1H11 prepared in the step 2) on a treated colloidal gold conjugate pad, wherein the spraying amount is 10uL/cm, and drying at 37 ℃.
6. The method for preparing the immune colloidal gold test strip for detecting the avian influenza virus H7 subtype according to claim 2, which is characterized in that: the specific steps of the step 4) are as follows:
A. preparing a coating solution: 0.2M Tris-HCl, 1% Tween-20 and 0.5% X-100, stirring and mixing uniformly, and filtering by a 0.22uM filter;
B. and (2) respectively diluting the monoclonal antibody 6B3 resisting avian influenza virus H7 subtype and the goat anti-mouse IgG polyclonal antibody with the coating solution, spraying the diluted antibodies on a nitrocellulose membrane to serve as a detection line and a quality control line, wherein the concentrations of the antibodies are 1mg/mL and 0.5mg/mL respectively, the spraying amount is 1uL/cm, the distance between the detection line and the quality control line is 5mm, and drying the antibodies at 37 ℃ for later use.
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| CN111551745B (en) * | 2020-05-15 | 2023-04-07 | 安徽中起生物科技有限公司 | Avian influenza virus H7N9 subtype N protein IgY antibody detection colloidal gold test paper and method |
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|---|---|---|---|---|
| CN103805571A (en) * | 2014-02-19 | 2014-05-21 | 西安天星生物药业股份有限公司 | Preparation method of ascitic-type avian influenza monoclonal antibody and hybridoma cell strain as well as avian influenza immunodetection kit |
| CN204731247U (en) * | 2013-06-28 | 2015-10-28 | 广州万孚生物技术股份有限公司 | H7 subtype avian influenza virus colloidal gold strip |
| CN107475203A (en) * | 2017-07-19 | 2017-12-15 | 武汉科前生物股份有限公司 | A kind of H7 avian influenza virus monoclonal antibody and application |
-
2018
- 2018-01-23 CN CN201810063609.1A patent/CN108303529B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN204731247U (en) * | 2013-06-28 | 2015-10-28 | 广州万孚生物技术股份有限公司 | H7 subtype avian influenza virus colloidal gold strip |
| CN103805571A (en) * | 2014-02-19 | 2014-05-21 | 西安天星生物药业股份有限公司 | Preparation method of ascitic-type avian influenza monoclonal antibody and hybridoma cell strain as well as avian influenza immunodetection kit |
| CN107475203A (en) * | 2017-07-19 | 2017-12-15 | 武汉科前生物股份有限公司 | A kind of H7 avian influenza virus monoclonal antibody and application |
Non-Patent Citations (2)
| Title |
|---|
| H7亚型禽流感病毒单克隆抗体的制备及胶体金免疫层析试纸条的研制;刘晓燕;《中国优秀硕士学位论文全文数据库 农业科技辑》;20160815;摘要 * |
| 猪流感病毒胶体金方法的建立及应用;明晓;《中国优秀硕士学位论文全文数据库 农业科技辑》;20150315;第14-48页 * |
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