CN107831306B - H7 subtype avian influenza virus double-antibody sandwich ELISA kit and detection method thereof - Google Patents
H7 subtype avian influenza virus double-antibody sandwich ELISA kit and detection method thereof Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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Abstract
The invention discloses an H7 subtype avian influenza virus double-antibody sandwich ELISA kit and a detection method thereof. The kit comprises an ELISA plate coated with a monoclonal antibody against H7 subtype avian influenza virus, wherein the monoclonal antibody is secreted and generated by a hybridoma cell strain 1H 11. The monoclonal antibody has high titer, good sensitivity and best self-sandwich effect, so the invention selects the 1H11 monoclonal antibody as the coating monoclonal antibody, and the 1H11-HRP as the enzyme-labeled secondary antibody to establish the double-antibody sandwich ELISA method. The method has the advantages of rapidness, stability, high specificity and high sensitivity, and can be used for rapidly detecting whether a sample contains H7 subtype avian influenza virus; the method is suitable for the mass detection of whether the poultry farm is infected with the H7 subtype avian influenza virus.
Description
Technical Field
The invention belongs to the technical field of pathogen detection, and particularly relates to an H7 subtype avian influenza virus double-antibody sandwich ELISA kit and a detection method thereof.
Background
Avian Influenza (AI) is an avian syndrome caused by infection with influenza a virus (AIV), ranging from infections that do not present clinical symptoms, respiratory diseases, systemic diseases with a drop in egg production to mortality rates approaching 100%. Avian influenza is classified into nonpathogenic avian influenza, low-pathogenic avian influenza, moderately pathogenic avian influenza and highly pathogenic avian influenza, and highly pathogenic avian influenza belongs to a class a disease in the OIE classification.
So far, H5 and H7 subtypes belong to highly pathogenic avian influenza, which not only causes huge economic loss to the poultry industry, but also threatens the public health safety of human beings. In recent years, the H7 subtype avian influenza virus has caused millions of poultry to die, the spread range is wide, and the outbreak subtype is many. The H7N7 virus was isolated from chickens in Australia as in 1976 and 1985, and H7N4 was isolated in 1997; the H7N3 virus was isolated from chickens by pakistan 1994; H7N1 virus was isolated from turkeys in Italy in 1999-2000; chile's discovery H7N3 in 2002; outbreak of H7N7 in the netherlands, germany, belgium 2003; canada in 2004 developed H7N 3; 2007; korea occurrence of H7N8, UK occurrence of H7N2, Italy occurrence of H7N3, etc.; in 2008, danish genesis H7N1, german genesis H7N3, norwegian genesis H7NX, japanese genesis H7N6, and czech genesis H7N 9. Therefore, the H7 subtype avian influenza virus has attracted global attention. In 3 months in 2013, a novel A-type avian influenza virus (H7N9) is detected for the first time in China, 135 people are infected and 45 people die after 12 days in 8 months in 2013, epidemic situation spreads very quickly, and high death rate is caused. 2016-2017, during winter and spring handover, the fifth H7 subtype avian influenza outbreak peak appears in China, the epidemic situation is more serious than before, the outbreak time is early, the outbreak rate is high, and the virus has the phenomena of drug resistance mutation and virulence increase. Therefore, an effective and rapid detection method is urgently needed to be established, the threat of the disease to the public health safety of human beings is reduced, and the loss of the disease to the breeding industry and the national economy is reduced.
The monoclonal antibody plays an important role in the diagnosis, prevention and treatment of avian influenza virus. The monoclonal antibody is a single antibody generated by fusing spleen lymphocytes and myeloma cells, is directed against a single epitope, has high titer and strong specificity, and has been widely applied to diagnosis and treatment of diseases based on the characteristics.
In the antibody detection method, ELISA and AGP can detect specific antibodies, and HI is used for subtype detection and has lower sensitivity than ELISA. Therefore, it is required to provide an effective detection means with simplicity, rapidness, high sensitivity and good specificity for detecting H7 subtype avian influenza virus in a large number of samples.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a double-antibody sandwich ELISA kit for H7 subtype avian influenza virus, which is convenient for more simply and rapidly detecting H7 subtype avian influenza virus in a large number of samples.
Another object of the present invention is to provide a method for detecting the above-mentioned kit.
The above object of the present invention is achieved by the following means.
The hybridoma cell strain 1H11 is applied to H7 subtype avian influenza virus double-antibody sandwich ELISA detection, the hybridoma cell strain 1H11 is preserved in China center for type culture Collection, and the preservation number is CCTCC NO: c2017112; and (3) classification and naming: hybridoma cell 1H11, address: wuhan university in Wuhan, China; the preservation time is 7 months and 18 days in 2017.
The application of the monoclonal antibody 1H11 in double-antibody sandwich ELISA detection of H7 subtype avian influenza virus, wherein the monoclonal antibody 1H11 is secreted and generated by the hybridoma cell strain 1H11 of claim 1.
The hybridoma cell strain 1H11 or the monoclonal antibody 1H11 is applied to preparation of a H7 subtype avian influenza virus double-antibody sandwich ELISA kit.
The invention utilizes H7 subtype avian influenza antigen to immunize a BALB/C mouse to prepare an anti-H7 subtype AIV monoclonal antibody, utilizes an ELISA serum antibody detection technology with high sensitivity and good specificity to screen positive hybridomas, establishes an avian influenza virus H7 subtype double-antibody sandwich ELISA detection kit and a detection method, and provides an effective means for simply, conveniently and quickly detecting H7 subtype avian influenza virus in a large number of samples.
Specifically, preparation of monoclonal antibody: an H7 subtype hemagglutination inhibition test antigen (H7 subtype hemagglutination inhibition test antigen purchased from Harveaceae) is used for immunizing female BALB/c mice with the age of 6-8 weeks, after the antibody titer reaches more than 104 three days, splenocytes of the mice and SP2/0 myeloma cells are subjected to cell fusion by a PEG method, an indirect ELISA method and the hemagglutination inhibition method are used for simultaneously detecting antibodies and screening positive hybridoma cells, and ascites monoclonal antibodies prepared by the hybridoma cells are purified by an octanoic acid-ammonium sulfate method.
A double-antibody sandwich ELISA kit for H7 subtype avian influenza virus comprises an ELISA plate coated with a monoclonal antibody against H7 subtype avian influenza virus, wherein the monoclonal antibody is the monoclonal antibody 1H 11.
The monoclonal antibody solution is marked by HRP by a sodium periodate method, and the double-antibody sandwich effect of the monoclonal antibody solution marked by HRP is verified by an ELISA method, and the result proves that the sandwich effect of the 1H11-HRP and 1H11 monoclonal antibodies is optimal.
Preferably, the coating amount of the monoclonal antibody is 7 mug/mL.
Preferably, the kit further comprises: enzyme-labeled secondary antibody resisting H7 subtype avian influenza virus, confining liquid, diluent and washing liquid; the enzyme-labeled secondary antibody is a monoclonal antibody 1H11 marked by HRP.
Preferably, the dilution of the enzyme-labeled secondary antibody is 1: 1600.
Preferably, the blocking solution is 10mM phosphate buffer pH7.2 containing 5g/L BSA; the diluent is 10mM PBS buffer solution with pH 7.2; the washing solution is 0.01M PBS solution added with 0.05 percent of Tween-20.
The method for detecting H7 subtype avian influenza virus by using the kit is carried out according to a double-antibody sandwich ELISA detection method, and comprises the following steps:
s1, coating an enzyme label plate of a monoclonal antibody 1H11 of anti-H7 subtype avian influenza virus, 100 mu L per well, and standing overnight at 4 ℃; adding washing liquid into each hole, washing the plate for multiple times, and drying by beating;
s2, adding sealing liquid into each hole, adding washing liquid into each hole after sealing, washing the plate for multiple times, and drying by beating;
s3, adding 100 mu L/hole of a sample to be detected, incubating at room temperature, washing the plate for multiple times, and drying by patting;
s4, diluting the enzyme-labeled secondary antibody by using a diluent according to the ratio of 1: 1600 times, adding the diluted enzyme-labeled secondary antibody into an enzyme-labeled plate according to the ratio of 100 mu L/hole, incubating at room temperature, washing the plate for multiple times, and patting the plate dry;
s5, adding a color development liquid into each hole, stopping the reaction after color development in a dark place at room temperature, and measuring the OD450nm value; when OD450nm is more than 0.2, the sample is judged to be positive, namely the sample to be detected contains H7 subtype avian influenza virus; when OD450nm is lower than 0.18, the sample to be detected is judged to be negative, namely the sample to be detected does not contain H7 subtype avian influenza virus; when OD450nm is between 0.18-0.2, it is judged to be suspected and re-detected.
The invention firstly uses a serological method to detect the H7 subtype avian influenza virus. Compared with the prior art, the invention is more suitable for the large-scale pathogen detection of the basic layer, and is a simple, convenient, rapid and specific double-antibody sandwich ELISA method.
Preferably, the dilution of the sample to be tested in step S3 is 1: 8.
Preferably, the incubation time in steps S3 and S4 is 30 min.
More preferably, the method for detecting comprises the following steps:
s1, coating, namely coating a 96-hole enzyme label plate with the monoclonal antibody 1H11 solution with the concentration of 7 mu g/mL according to 100 mu L/hole, and standing overnight at 4 ℃; adding washing liquid into the coated enzyme label plate according to 300 mu L/hole, washing for 4-5 times, each time for 5min, and patting dry;
s2, sealing, namely adding sealing liquid into the ELISA plate which is dried in the S1 manner according to 200 mu L/hole for sealing, incubating for 2h at 37 ℃, adding 300 mu L/hole washing liquid, washing for 3-5 times, each time for 5min, and drying in the manner of drying;
s3, sample adding, namely diluting a sample to be detected according to the ratio of 1: 8, adding 100 mu L/hole of the sample into an enzyme label plate which is dried by beating after being sealed, placing the mixture at room temperature for incubation for 30min, washing for 4-5 times, and drying by beating for 5min each time;
s4, enzyme-labeled antibody, namely diluting an enzyme-labeled secondary antibody with a diluent according to the volume ratio of 1: 1600, adding an enzyme-labeled plate according to 100 mu L/hole, incubating at room temperature for 30min, washing for 4-5 times, washing for 5min each time, and patting dry;
s5, TMB color development, namely adding TMB color development liquid with the volume of 100 mu L per hole, performing color development for 10min in a dark place at room temperature, then adding 50 mu L0.5M H2SO4 stop solution per hole to stop color development reaction, and reading the value of the reaction liquid OD450nm of each hole on an enzyme-labeling instrument; determination of negative and positive cut-off values:
according to the formula: obtaining a negative and positive critical value, namely the average value of negative sample OD450 +3SD (standard deviation); when OD450nm is more than 0.2, the sample to be detected can be judged to be positive, namely the sample to be detected contains H7 subtype avian influenza virus; when the OD450nm is between 0.18 and 0.2, the result is judged to be suspected, and the detection is carried out again according to the step 6; and when the OD450nm is lower than 0.18, judging the sample to be detected to be negative, namely the sample to be detected does not contain H7 subtype avian influenza virus.
Compared with the prior art, the invention has the beneficial effects that: the H7 subtype avian influenza virus sandwich ELISA detection method established by the invention has the advantages of rapidness, stability, high specificity and high sensitivity, and can be used for rapidly detecting whether a sample contains H7 subtype avian influenza virus; the method is suitable for the mass detection of whether the poultry farm is infected with the H7 subtype avian influenza virus.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The reagents and components used in the invention are as follows:
coating buffer solution: 1 Xcarbonate buffer (pH 9.6): dissolving Na2CO30.2756g and NaHCO30.6216g in distilled water, diluting to 1000mL, adjusting pH to 9.6, and storing at 4 deg.C;
PBS buffer: NaCl 4.25g, NaH2PO4 & 2H2O 0.178.178 g and Na2HPO4 & 12H2O 1.386g, which are dissolved by distilled water and have a constant volume of 500mL and a pH value of 7.2-7.4;
sealing liquid: 5g BSA was dissolved in 1000mL of the dilution and stored at 4 ℃ for a short period and-20 ℃ for a long period.
TMB color development liquid:
color developing substrate liquid A: dissolving 3,3',5, 5' -tetramethylbenzidine in DMSO to make the final concentration of 3,3',5, 5' -tetramethylbenzidine L0mg/mL, adding 10% by volume of glycerol, and adding L-hemisarcosine hydrochloride to make the final concentration of 0.6mg/mL to obtain chromogenic substrate solution A;
the chromogenic substrate solution B: dissolving 0.1 percent by weight of citric acid, 0.2 percent by weight of disodium hydrogen phosphate, 0.02 percent by weight of sodium sulfite, 0.003 percent by weight of EDTA and 0.02 percent by weight of urea hydrogen peroxide in ddH2O, and adjusting the pH value to 5.3 to prepare the chromogenic substrate liquid B; storing at 4 ℃ in the dark.
When in use, the chromogenic substrate liquid A is diluted by 50 times with the chromogenic substrate liquid B and is mixed uniformly, and then the chromogenic substrate liquid A is prepared for use;
0.5M H2SO4 stop solution: adding 27.2mL of concentrated H2SO4 into distilled water, metering to 1L, mixing uniformly, cooling, and storing at room temperature.
The process of the present invention will be further described below by taking specific examples of the conditions for carrying out the process.
Example 1 preparation of monoclonal antibody against subtype H7 avian influenza Virus
1) Animal immunization
Taking a bottle of H7 standard antigen purchased from Harveaceae, resuspending the antigen with 500 mu L of normal saline, fully emulsifying the antigen with equivalent volume of Freund's complete adjuvant, and injecting 200 mu L of BALB/c mice of 8 weeks old by subcutaneous points; 2 weeks, 4 weeks later, the antigen emulsified in Freund's incomplete adjuvant was immunized once more each time in the same manner; after 6 weeks, the same dose of antigen is taken for intraperitoneal injection without adjuvant; after 4 days, fusion was carried out.
2) Cell fusion
Preparation of myeloma cells: SP2/0 cells with good growth state were selected, the supernatant was discarded when the density reached 75% of the bottom of the flask, and after washing once with incomplete DMEM medium, the cells were gently purged with 10mL of incomplete DMEM medium.
Preparation of splenic lymphocytes: taking a mouse 4 days after the boosting immunity, removing an eyeball and taking blood as positive serum; dislocation of cervical vertebra to kill mouse, placing in 75% alcohol for 10min, placing onto a dissecting plate with abdomen facing upwards and fixed in a super clean bench; aseptically opening the abdominal cavity, taking out the spleen, washing with incomplete DMEM medium, and removing excessive connective tissues and fat; the spleen was then transferred to another dish containing incomplete DMEM medium. Spleen was ground on a cell mesh using a grinding rod, and spleen cell suspension was collected.
Preparation of feeder cells: feeder cells can be prepared the evening before fusion as follows: taking a mature and healthy ICR mouse, picking eyeballs, collecting blood as negative serum, dislocating cervical vertebra and killing; placing in 75% alcohol for 10min, fixing limbs, cutting skin to expose peritoneum, and wiping peritoneum with alcohol cotton ball; 10mL of HAT medium was carefully injected into the abdominal cavity by aspiration with a 10mL syringe, 12# needle, gently massaging the abdomen with an alcohol cotton ball, withdrawing the intra-abdominal fluid, injecting into a prepared container, counting and diluting, and then adding to a 96-well cell culture plate.
Fusing: the spleen cells and myeloma cells prepared above were mixed in a 50mL fusion tube, centrifuged at 1000g for 10min, and the supernatant was discarded. Placing the fusion tube in the palm, and lightly rubbing the bottom to mix the two cells; slowly adding 1mL of preheated PEG1500 into the fusion tube in a water bath at 37 ℃ within 45s, and gently shaking up while adding; then slowly and quickly dripping 30mL of incomplete DMEM medium preheated at 37 ℃ within 90s to terminate the reaction, standing in a water bath at 37 ℃ for 10min, and centrifuging at 1000g for 10 min; discarding the supernatant, using 60mL HAT culture medium to gently suspend and precipitate cells, subpackaging the cells in 96-well cell culture plates paved with feeder cells, and then placing the culture plates in a 37 ℃ 6% CO2 incubator for culture; after 5d, half-changing the culture solution by using a fresh HAT culture medium; after 10 days, replacing HAT culture medium with preheated HT culture medium; observing the growth of the hybridoma cells, and sucking 100 mu L of cell supernatant for antibody detection when the cell culture supernatant turns yellow or clones are distributed to more than 1/10 of the area of the bottom of the hole.
3) Screening of hybridoma cells
The positive clones were screened by indirect ELISA and HI assay using H7 standard antigen from Harveaceae as the detection antigen.
Determining the coating concentration of a detection antigen (the antigen used in the embodiment is H7 standard antigen purchased from Harvey as the detection antigen), performing gradient dilution on the standard antigen by using a coating buffer solution, performing dilution on each line, coating an ELISA plate by using 100 mu L/hole, and coating for 2H at 37 ℃; washing with washing solution for 3 times, 5min each time, and drying; adding 10% PBS diluted calf serum 200 μ L/well, sealing at 4 deg.C overnight, washing 3 times; adding 100 mu L/hole of positive serum diluted in gradient, incubating for 1h at 37 ℃, and washing for 3 times; adding 50 mu L/hole goat anti-mouse HRP-IgG with working concentration (diluted 1: 10000), incubating at 37 ℃ for 1h, and washing 3 times; adding 50 mu L/hole of TMB color development liquid, and acting at 37 ℃ for 10-15 min; the reaction was stopped by adding 2M H2SO 450. mu.L/well. The OD450nm reading was determined. Coating the enzyme label plate with the antigen concentration of 1: 100 determined according to the square matrix, and storing at-20 ℃ for later use. ELISA was performed to detect the hybridoma cell supernatants as described above. The serum of the immune mouse is diluted according to the concentration determined by the square matrix and is used as a positive control during screening, the serum of the mouse which is used as a feeder cell is used as a negative control, and meanwhile, a blank zero-adjusting hole is arranged. HI assay screening method: the method screens positive hybridoma cells for cloning aiming at avian influenza virus H7 subtype hemagglutinin. The positive wells thus detected were subcloned (hybridoma cells positive for ELISA detection were cloned by limiting dilution) until the antibody secreted from the cell line stably reacted specifically with avian influenza virus subtype H7. 3 positive hybridoma cell lines were obtained by 3 subcloning and named 1H11, 5D7 and 50G 2.
TABLE 1 hybridoma cell supernatant titer assay results
Example 2 preparation and potency assay of ascites
Taking 10-week-old BALB/c mice, and injecting sterilized liquid paraffin into the abdominal cavity, wherein each mouse is 0.5 mL; hybridoma cells in logarithmic growth phase diluted with PBS, 5 × 105 cells/cell, were intraperitoneally injected 1 week later; when the abdomen of the mouse is obviously raised, collecting ascites from the abdominal cavity by using a No. 16 needle, centrifuging for 10min at 2500g, removing adipose tissues, sucking supernatant, and storing at-70 ℃ for later use.
Ascites titer determination: the ascites titer was measured by two methods, indirect ELISA and HI assay, with the following results:
TABLE 2 determination of ascites titer of monoclonal antibodies
| Cell line | HI potency | ELISA potency |
| 1H11 | 218 | 1∶409600 |
| 5D7 | 217 | 1∶102400 |
| 50G2 | 216 | 1∶204800 |
Subclass identification: the cell lines of 3 strains were all IgG1 and the results are shown in Table 3, which were identified by using a Sigma monoclonal antibody subclass identification kit (Ig61, IgG2a, IgG2b, IgG3, IgM, IgA) according to the method described in the specification.
TABLE 3 monoclonal antibody subclass identification results
| Monoclonal antibody cell line name | 1H11 | 5D7 | 50G2 |
| Subclass of | 1gG1 | IgG1 | IgG1 |
EXAMPLE 3 purification of monoclonal antibodies
The prepared monoclonal antibody (monoclonal antibody) ascites is purified by a Protein G affinity chromatography method, and the Protein G purification method comprises the following steps: the container containing Protein G affinity resin was gently inverted several times to mix the resin for complete suspension. The appropriate amount of Protein G resin was loaded onto the column and 5mL of binding buffer was added to equilibrate the column. The monoclonal antibody ascites fluid is diluted with the same or more volume of binding buffer and loaded. After loading, the column was washed with 10ml of binding buffer. After the binding buffer had run off, the antibody was eluted with 10mL of elution buffer and the eluted product was collected. And detecting the pH value of the eluent in real time in the elution process, and neutralizing the eluent by using 1M Tris-Cl (pH 8.5) in time to ensure that the pH value of the eluent containing the elution product is 7.4. The eluate is put into a dialysis bag, dialyzed overnight at 4 ℃ using 0.01M Tris-Cl buffer (pH7.4), and the solution is changed 2 to 3 times in the middle. The concentration of the purified monoclonal antibody and the ascites titer are shown in Table 4.
TABLE 4 determination of concentration and potency of monoclonal antibody after purification of ascites fluid
| Monoclonal antibody cell line name | 1H11 | 5D7 | 50G2 |
| Concentration after purification (mg/mL) | 11.2 | 7.6 | 8.0 |
| HI potency of purified ascites | 218 | 214 | 216 |
| ELISA potency of purified ascites | 1∶409600 | 1∶102400 | 1∶204800 |
The 3 monoclonal antibodies have higher potency and better sensitivity.
Example 4 monoclonal antibody HRP enzyme label and Sandwich ELISA establishment
1) Preparation of enzyme-labeled antibody
10mg of HRP was dissolved in 2ml of water for injection to give a brownish red color, 1ml of 0.06M/L NaIO4 was added to give a greenish brown color at 4 ℃ for 30min, 1ml of 0.16M/L ethylene glycol was added to terminate the reaction, the solution was protected from light at room temperature for 30min to give a brownish yellow color, and 1ml of the 1H11 antibody purified in example 3, 1.5ml of the 5D7 antibody, 1.2ml of the 5062 antibody, 0.05M, pH 9.5, and dialyzed at CB at 4 ℃ overnight. Sucking out, adding 5mg/ml NaBH40.4ml, adding equal volume saturated ammonium sulfate at 4 deg.C for 30min and 10000r/min for 5min, then using 2ml 20mM pH7.4 PBS to make heavy suspension dialysis, harvesting enzyme-labeled antibody 4.5ml, adding equal volume glycerol, and storing at-20 deg.C for standby.
2) Double antibody sandwich ELISA preliminary test
The 3 monoclonal antibodies purified in example 3 were diluted to 10. mu.g/ml, 100. mu.l of each well was coated with an ELISA plate, 100. mu.l of each well, and the antigen concentration of H7 was fixed (1: 10 fold dilution) and cross-arrayed with labeled HRP-1H11, HRP-5D7, and HRP-50G2, respectively, to verify the sandwich ELISA effect. The results show that 1H11 has the best sandwich effect by itself, and the OD450 value is as high as 2.68. The results are detailed in Table 5. Therefore, 1H11 monoclonal antibody is selected as coating monoclonal antibody, and 1H11-HRP is selected as enzyme-labeled secondary antibody to establish a double-antibody sandwich ELISA method.
Table 53 monoclonal antibody sandwich ELISA effect preliminary verification
3) Double antibody sandwich ELISA condition optimization
a.1H11 monoclonal antibody coating concentration and optimization of 1H11-HRP secondary antibody concentration
The optimal coating concentration and secondary antibody concentration were determined by square matrix titration. 1H11 mAb stock was diluted 1: 50 fold with coating buffer and then diluted 1: 6400 fold, 100. mu.l was added to each well in ELISA plates and coated overnight at 4 ℃. Then the liquid was spun off, blocked with 200. mu.l of 0.5% BSA per well, blocked at 37 ℃ for 2h, washed 3 times and patted dry. 100 μ l H7 standard antigen (diluted 1: 10 times) and PBS control were added to each well of a 96-well microplate, reacted at room temperature for 30min, washed 5 times, and patted dry. Then 1H11-HRP secondary antibody is diluted by PBS 1: 100 times, and then diluted by the ratio of left to right to 1: 3200 times, each hole is 100 mul, the reaction is carried out for 30min at room temperature, and the washing is carried out for 5 times and the swatter is dried. Substrate was added and the reaction was stopped by developing at room temperature for 15min and then 50. mu.l of stop solution was added to each well and the OD450 value was read and the results are detailed in tables 6 and 7.
TABLE 6 detection results of OD450 values by square matrix titration
TABLE 7 Square matrix titration P/N values
As shown in Table 7, the highest P/N value was found to be 15.70 when the coating dilution of the 1H11 monoclonal antibody was 1: 1600 and the dilution of the 1H11-HRP secondary antibody was 1: 1600. Therefore, the dilution of the monoclonal antibody coating is determined to be 1: 1600, namely 7 mu g/ml, and the dilution of the enzyme-labeled secondary antibody is 1: 1600.
b. Antigen dilution optimization
The 1H11 mAb coating buffer was diluted 1: 1600 times to coat the ELISA plates, 100. mu.l of each well was added to the ELISA plates and coated overnight at 4 ℃. Then the liquid was spun off, blocked with 200. mu.l of 0.5% BSA per well, blocked at 37 ℃ for 2h, washed 3 times and patted dry. Then H7 standard antigen was diluted in multiple ratios, reacted at room temperature for 30min, washed 5 times, and patted dry. Then, the secondary 1H11-HRP antibody was diluted 1: 1600 times with PBS, 100. mu.l of the secondary antibody was added to each well and reacted at room temperature for 30min, washed 5 times, and patted dry. Substrate was added and the reaction was stopped by developing at room temperature for 15min and then 50. mu.l of stop solution was added to each well and the OD450 value was read. The results show that the P/N value is highest when the antigen is diluted 1: 8 fold, so that the antigen dilution is selected to be 1: 8. See table 8 for details.
TABLE 8 dilution optimization of antigen
| 1H11 | PBS | |
| 1∶1 | 1.78 | 0.08 |
| 1∶2 | 1.67 | 0.07 |
| 1∶4 | 1.98 | 0.09 |
| 1∶8 | 2.21 | 0.06 |
| 1∶16 | 1.02 | 0.09 |
| 1∶32 | 0.76 | 0.07 |
| 1∶64 | 0.57 | 0.08 |
| 1∶128 | 0.34 | 0.09 |
c. Optimization of optimal reaction conditions
Optimization of the reaction time is carried out on the basis of steps a and b. And (c) taking the coated ELISA (the coating and sealing method is the same as the step b), adding the H7 standard antigen diluted by 1: 8 times by using PBS, standing and incubating for 15min, 30min, 45min and 60min at room temperature respectively, then washing for 5 times, and patting to dry. Then 1H11-HRP secondary antibody diluted by 1: 1600 times is added, and the mixture is respectively kept stand and incubated for 15min, 30min, 45min and 60min at room temperature. Washed 5 times and patted dry. Substrate was added and the reaction was stopped by developing at room temperature for 15min, then 50. mu.l of stop solution was added to each well, and OD450 values were read and P/N values were calculated. According to the result, considering convenience, the incubation time of H7 standard antigen and the incubation time of secondary antibody are both selected to be 30min finally.
TABLE 9 optimal reaction time optimization of antigen with secondary antibody
d. Experiment of specificity
The specificity test is carried out on avian influenza virus H7 subtype hemagglutination inhibition test antigen (H7-AIAg), avian influenza virus H1 subtype hemagglutination inhibition test antigen (H1-AIAg), avian influenza virus H5 subtype hemagglutination inhibition test antigen (H5-AIAg), avian influenza virus H9 subtype hemagglutination inhibition test antigen (H9-AIAg), Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), egg laying descent syndrome virus (EDS-76) and Infectious Bursal Disease Virus (IBDV) by using the method established by the invention, and the method only reacts positively with the H7 subtype avian influenza virus (namely the H7 subtype hemagglutination inhibition test antigen), which shows that the method has good specificity. The detailed results are shown in Table 10.
TABLE 10 results of specificity experiments
| Antigens | H7 | H1 | H5 | H9 | NDV | IBV | EDS | IBDV | PBS control |
| OD450 | 2.30 | 0.05 | 0.04 | 0.05 | 0.07 | 0.06 | 0.07 | 0.04 | 0.04 |
Determination of positive and negative cutoff values
32 chicken serum samples which are detected as H7 subtype avian influenza virus negative by PCR are detected, meanwhile, standard positive control and negative control are set, and OD450 values are repeatedly detected for multiple times, so that the negative and positive critical value judgment standard of the method is finally determined as follows:
table 1132 negative sample test results
According to the formula: negative-positive cut-off value was obtained by averaging OD450 of negative samples +3SD (standard deviation). And when the OD450nm is less than 0.20, the result is positive, namely the sample to be detected contains H7 subtype avian influenza virus. When the OD450nm is between 0.18 and 0.2, the result is judged to be suspected, and the detection is carried out again according to the step 6; and when the OD450nm is lower than 0.18, judging the sample to be detected to be negative, namely the sample to be detected does not contain H7 subtype avian influenza virus.
Example 5 kit detection procedure
S1, coating, namely coating a 96-hole enzyme label plate with the monoclonal antibody 1H11 solution with the concentration of 7 mu g/mL according to 100 mu L/hole, and standing overnight at 4 ℃; adding washing liquid into the coated enzyme label plate according to 300 mu L/hole, washing for 4-5 times, each time for 5min, and patting dry;
s2, sealing, namely adding sealing liquid into the ELISA plate which is dried in the S1 manner according to 200 mu L/hole for sealing, incubating for 2h at 37 ℃, adding 300 mu L/hole washing liquid, washing for 3-5 times, each time for 5min, and drying in the manner of drying;
s3, sample adding, namely diluting a sample to be detected according to the ratio of 1: 8, adding 100 mu L/hole of the sample into an enzyme label plate which is dried by beating after being sealed, placing the mixture at room temperature for incubation for 30min, washing for 4-5 times, and drying by beating for 5min each time;
s4, enzyme-labeled antibody, namely diluting an enzyme-labeled secondary antibody with a diluent according to the volume ratio of 1: 1600, adding an enzyme-labeled plate according to 100 mu L/hole, incubating at room temperature for 30min, washing for 4-5 times, washing for 5min each time, and patting dry;
s5, TMB color development, namely adding TMB color development liquid with the volume of 100 mu L per hole, performing color development for 10min in a dark place at room temperature, then adding 50 mu L0.5M H2SO4 stop solution per hole to stop color development reaction, and reading the value of the reaction liquid OD450nm of each hole on an enzyme-labeling instrument; determination of negative and positive cut-off values:
according to the formula: obtaining a negative and positive critical value, namely the average value of negative sample OD450 +3SD (standard deviation); when OD450nm is more than 0.2, the sample to be detected can be judged to be positive, namely the sample to be detected contains H7 subtype avian influenza virus; when the OD450nm is between 0.18 and 0.2, the result is judged to be suspected, and the detection is carried out again according to the step 6; and when the OD450nm is lower than 0.18, judging the sample to be detected to be negative, namely the sample to be detected does not contain H7 subtype avian influenza virus.
The H7 subtype avian influenza virus sandwich ELISA detection method established by the invention has the advantages of rapidness, stability, high specificity and high sensitivity, and whether the sample contains the H7 subtype avian influenza virus can be rapidly detected only by collecting tracheal or cloacal secretion or other samples containing the H7 subtype avian influenza virus; the method is suitable for the mass detection of whether the poultry farm is infected with the H7 subtype avian influenza virus.
The implementation of the present invention has been described in detail, however, the present invention is not limited to the specific details of the above-described embodiments, and various simple modifications can be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
Claims (3)
1. A double-antibody sandwich ELISA kit for H7 subtype avian influenza virus, which is characterized by comprising: the kit comprises an enzyme label plate coated with a monoclonal antibody 1H11 for resisting H7 subtype avian influenza virus, an enzyme-labeled secondary antibody for resisting H7 subtype avian influenza virus, a sealing solution, a diluent and a washing solution; the monoclonal antibody 1H11 is secreted and produced by a hybridoma cell strain 1H11, the hybridoma cell strain 1H11 is preserved in China center for type culture Collection, and the preservation number is CCTCC NO: c2017112; the enzyme-labeled secondary antibody is a monoclonal antibody 1H11 marked by HRP.
2. The double-antibody sandwich ELISA kit for H7 subtype avian influenza virus according to claim 1, wherein the coating amount of the monoclonal antibody is 7 μ g/mL.
3. The double-antibody sandwich ELISA kit for H7 subtype avian influenza virus according to claim 1 or 2, wherein the blocking solution is 10mM phosphate buffer pH7.2 containing 5g/L BSA; the diluent is 10mM PBS buffer solution with pH7.2; the washing solution is 0.01M PBS solution added with 0.05 percent of Tween-20.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009035412A1 (en) * | 2007-09-13 | 2009-03-19 | Temasek Life Sciences Laboratory Limited | Monoclonal antibodies specific to hemagglutinin from influenza virus h5-subtype and uses thereof |
| CN101553732A (en) * | 2006-08-31 | 2009-10-07 | 大阪府 | Rapid diagnosis method specific to avian influenza virus |
| CN103266088A (en) * | 2013-04-15 | 2013-08-28 | 北京世纪元亨动物防疫技术有限公司 | H7 subtype avian influenza virus monoclonal antibody of and kit |
| CN104312979A (en) * | 2014-09-30 | 2015-01-28 | 江苏省农业科学院 | Anti-H7 subtype avian influenza virus monoclonal antibody and application thereof |
| CN106771181A (en) * | 2016-11-24 | 2017-05-31 | 剑药（苏州）生物技术有限公司 | A kind of bird flu H7N9 hemagglutinin HA antigens detect ELISA kits and detection method |
-
2017
- 2017-10-10 CN CN201710935599.1A patent/CN107831306B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101553732A (en) * | 2006-08-31 | 2009-10-07 | 大阪府 | Rapid diagnosis method specific to avian influenza virus |
| WO2009035412A1 (en) * | 2007-09-13 | 2009-03-19 | Temasek Life Sciences Laboratory Limited | Monoclonal antibodies specific to hemagglutinin from influenza virus h5-subtype and uses thereof |
| CN103266088A (en) * | 2013-04-15 | 2013-08-28 | 北京世纪元亨动物防疫技术有限公司 | H7 subtype avian influenza virus monoclonal antibody of and kit |
| CN104312979A (en) * | 2014-09-30 | 2015-01-28 | 江苏省农业科学院 | Anti-H7 subtype avian influenza virus monoclonal antibody and application thereof |
| CN106771181A (en) * | 2016-11-24 | 2017-05-31 | 剑药（苏州）生物技术有限公司 | A kind of bird flu H7N9 hemagglutinin HA antigens detect ELISA kits and detection method |
Non-Patent Citations (2)
| Title |
|---|
| H7亚型禽流感病毒单克隆抗体的制备及ELISA诊断方法的建立;王桂芹;《中国优秀博硕士学位论文全文数据库（硕士）农业科技辑》;20070215(第02期);正文第17-33页 * |
| 禽流感病毒H5、H7和H9亚型单克隆抗体的制备及鉴定;贾强;《中国优秀博硕士学位论文全文数据库（硕士）农业科技辑 》;20050915(第05期);正文第10-25页 * |
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