CN111198265A - Combined rapid detection method for various poultry antibodies - Google Patents
Combined rapid detection method for various poultry antibodies Download PDFInfo
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- CN111198265A CN111198265A CN201811383056.4A CN201811383056A CN111198265A CN 111198265 A CN111198265 A CN 111198265A CN 201811383056 A CN201811383056 A CN 201811383056A CN 111198265 A CN111198265 A CN 111198265A
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Abstract
The invention belongs to the technical field of antibody detection, and provides a combined rapid detection method for various avian antibodies, which comprises the following steps: step 1: adding 0.1-0.2mL of diluent into all the holes of the 1 st-10 th row of the original reaction plate; step 2: adding 0.1-0.2mL of blood serum into each hole of the 1 st column in the step 1; and step 3: sucking 0.1-0.2mL of the solution from each hole in the 1 st row of the step 2 to the hole in the 2 nd row; and 4, step 4: transferring the blood clear liquid in the step 3 into corresponding reaction holes of ND, H9, H5 and H7 reaction plates according to 0.02-0.04mL per hole respectively; and 5: adding 0.02-0.04mL of diluent into each hole of the 11 th row of each reaction plate in the step 4 respectively; step 6: adding 0.02-0.04mL of ND, H9, H5 and H7 virus antigen liquid into each hole of the 1 st row to the 11 th row of each reaction plate in the step 5; and 7: adding 0.02-0.04mL of chicken erythrocyte suspension into each hole of each reaction plate in the step 6, and judging the antibody titer of ND, H9, H5 and H7; meanwhile, the invention has the characteristic of simultaneously detecting various antibodies by a single person.
Description
Technical Field
The invention belongs to the technical field of antibody detection, and particularly relates to a combined rapid detection method for multiple avian antibodies.
Background
Some viruses contain hemagglutinin on their surface, which binds to mucin receptors on the surface of chicken erythrocytes, causing agglutination of the erythrocytes, known as agglutination (HA). This agglutination phenomenon can be suppressed by specific immune sera, called Hemagglutination Inhibition (HI), and the known viruses can be used to check the content of the corresponding antibodies in the sera examined by the HA-HI assay. The test is suitable for detecting the antibody titer of Newcastle disease, avian influenza and egg drop syndrome in serum.
However, the existing HA/HI method can only detect one avian disease antibody, and generally, a poultry farm needs to simultaneously detect multiple antibodies such as newcastle disease, avian influenza, egg drop syndrome and the like, and the traditional HA/HI method needs repeated tests, so that the efficiency is low, and multiple dilutions can cause artificial detection errors.
Therefore, in order to overcome the defects, the invention urgently needs to provide a combined rapid detection method for multiple avian antibodies.
Disclosure of Invention
The invention aims to provide a method for quickly detecting multiple poultry antibodies in a combined manner, so as to at least solve the problems of heavy repeated sample adding work and poor accuracy of detection technicians in the prior art.
The invention provides a combined rapid detection method for various avian antibodies, which comprises the following technical scheme:
a method for rapidly detecting multiple avian antibodies in combination comprises the following steps of 1: adding 0.1-0.2mL of diluent into all the holes of the 1 st-10 th row of the original reaction plate; step 2: adding 0.1-0.2mL of blood serum into each hole of the 1 st column in the step 1; and step 3: sucking 0.1-0.2mL of the solution from each hole in the 1 st row of the step 2 to each hole in the 2 nd row; and 4, step 4: transferring the blood serum of the step 3 into corresponding holes of ND, H9, H5 and H7 reaction plates according to 0.02-0.04mL per hole respectively; and 5: adding 0.02-0.04mL of diluent into each hole of the 11 th row of each reaction plate in the step 4 respectively; step 6: adding 0.02-0.04mL of ND, H9, H5 and H7 virus antigen liquid into each hole of the 1 st row to the 11 th row of each reaction plate in the step 5; and 7: 0.02-0.04mL of chicken red blood cell suspension was added to each well of each reaction plate of step 6, and the antibody titers of ND, H9, H5, and H7 were determined.
The method for the combined rapid detection of the various avian antibodies is further preferably as follows: and 3, sequentially diluting the solution to 10 th row of wells in a multiple ratio, sucking 0.1-0.2mL of the solution from the 10 th row of wells, and discarding the solution.
The method for the combined rapid detection of the various avian antibodies is further preferably as follows: the volume fraction of the chicken red blood cell suspension in the step 7 is 1%.
The method for the combined rapid detection of the various avian antibodies is further preferably as follows: in the step 6, 4 units of ND, H9, H5 and H7 virus antigen solution 0.02-0.04mL is added into each well of the 1 st to 11 th rows of each reaction plate in the step 5.
The method for the combined rapid detection of the various avian antibodies is further preferably as follows: step 1 was performed using an 8-channel micropipette.
The method for the combined rapid detection of the various avian antibodies is further preferably as follows: step 2 is pipetting by using a single-channel micropipette.
The method for the combined rapid detection of the various avian antibodies is further preferably as follows: step 7 uses a 12-channel micropipette for pipetting.
The method for the combined rapid detection of the various avian antibodies is further preferably as follows: and step 5, adding 0.04-0.08mL of diluent into each hole of the 12 th row of each reaction plate in the step 4.
The method for the combined rapid detection of the various avian antibodies is further preferably as follows: the diluent is any one of normal saline or PBS.
The method for the combined rapid detection of the various avian antibodies is further preferably as follows: the original, ND, H9, H5 and H7 reaction plates are all 96-well reaction plates.
Analysis shows that compared with the prior art, the invention has the advantages and beneficial effects that:
the invention provides a combined rapid detection method for multiple avian antibodies, which can realize the simultaneous operation and detection of 4-6 avian influenza and Newcastle disease antibodies by a single person, improves the working efficiency by 200 percent, and has good judgment result stability, high accuracy, simple operation and low cost.
Drawings
FIG. 1 is a schematic diagram of the principle of the method for the combined rapid detection of multiple avian antibodies according to the present invention;
in the figure: 1-original reaction plate; 2-ND reaction plates; 3-H9 reaction plate; 4-H5 reaction plate; 5-H7 reaction plate.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
The invention provides a combined rapid detection method for multiple avian antibodies, which selects 8 samples and takes Newcastle disease ND, avian influenza H9, H5 and H7 as examples.
Example 1
Step 1: 0.125mL of physiological saline was added to the 1 st to 10 th wells of the 96-well original reaction plate 1 by using an 8-channel micropipette.
Step 2: using a single-channel micropipette to respectively suck 0.125mL of blood serum to be detected to each hole of the 1 st row of the original reaction plate 1, repeatedly blowing and beating for 5 times, and uniformly mixing.
And step 3: sucking 0.125mL from each hole of the 1 st row of the original reaction plate 1 by using an 8-channel micropipette, repeatedly blowing and beating for 5 times, uniformly mixing, sequentially diluting to the 10 th row of holes in a multiple ratio manner, sucking 0.125mL from the 10 th row of holes, and discarding.
And 4, step 4: as shown in fig. 1, the diluted blood serum to be tested in step 3 was transferred to the corresponding wells of the 96-well ND reaction plate 2, H9 reaction plate 3, H5 reaction plate 4 and H7 reaction plate 5 at a volume of 0.025mL per well.
And 5: 0.025mL of physiological saline was added to the 11 th well of each reaction plate of step 4, and 0.05mL of physiological saline was added to the 12 th well of each reaction plate of step 4.
Step 6: to the 1 st to 11 th wells of each reaction plate of step 5, 0.025mL of 4 units of ND, H9, H5, and H7 virus antigen solution was added, shaken for 1 minute, and allowed to stand at room temperature for 30 minutes.
And 7: 0.025mL of a chicken erythrocyte suspension with the volume fraction of 1% (added after fully shaking the erythrocyte suspension) is added into each hole by a 12-channel micropipette, the mixture is shaken for 1 minute, and is kept stand for 30 minutes at room temperature, and the antibody titers ND, H9, H5 and H7 are judged, wherein the judgment result is shown in a table 1, and the antibody titer corresponding to the dilution degree is shown in a table 2.
TABLE 1 antibody Titer results (unit: log2)
TABLE 2 dilution versus antibody drop table (log2)
Example 2
Step 1: PBS0.125mL was added to the 1 st to 10 th wells of the original 96-well reaction plate 1 using an 8-well micropipette.
Step 2: using a single-channel micropipette to respectively suck 0.125mL of blood serum to be detected to each hole of the 1 st row of the original reaction plate 1, repeatedly blowing and beating for 5 times, and uniformly mixing.
And step 3: sucking 0.125mL from each hole of the 1 st row of the original reaction plate 1 by using an 8-channel micropipette, repeatedly blowing and beating for 5 times, uniformly mixing, sequentially diluting to the 10 th row of holes in a multiple ratio manner, sucking 0.125mL from the 10 th row of holes, and discarding.
And 4, step 4: as shown in fig. 1, the diluted blood serum to be tested in step 3 was transferred to the corresponding wells of the 96-well ND reaction plate 2, H9 reaction plate 3, H5 reaction plate 4 and H7 reaction plate 5 at a volume of 0.025mL per well.
And 5: 0.025mLPBS was added to the 11 th well of each reaction plate of step 4, and 0.05mLPBS was added to the 12 th well of each reaction plate of step 4.
Step 6: to the 1 st to 11 th wells of each reaction plate of step 5, 0.025mL of 4 units of ND, H9, H5, and H7 virus antigen solution was added, shaken for 1 minute, and allowed to stand at room temperature for 30 minutes.
And 7: 0.025mL of a 1% volume fraction chicken erythrocyte suspension (added after fully shaking the erythrocyte suspension) is added into each hole by a 12-channel micropipette, the mixture is shaken for 1 minute, and is kept stand for 30 minutes at room temperature, and the antibody titer of ND, H9, H5 and H7 is judged.
Example 3
Step 1: 0.1mL of physiological saline was added to the 1 st to 10 th wells of the 96-well original reaction plate 1 by using an 8-channel micropipette.
Step 2: using a single-channel micropipette to respectively suck 0.1mL of blood serum to be detected to each hole of the 1 st row of the original reaction plate 1, repeatedly blowing and beating for 5 times, and uniformly mixing.
And step 3: sucking 0.1mL of the sample from each 1 st row of wells of the original reaction plate 1 by using an 8-channel micropipette, repeatedly blowing and beating the sample for 5 times, uniformly mixing the sample into the 2 nd row of wells, sequentially diluting the sample in multiple proportions to the 10 th row of wells, sucking 0.1mL of the sample from the 10 th row of wells, and discarding the sample.
And 4, step 4: as shown in fig. 1, the diluted blood serum to be tested in step 3 was transferred to the corresponding wells of the 96-well ND reaction plate 2, H9 reaction plate 3, H5 reaction plate 4 and H7 reaction plate 5 at a volume of 0.02mL per well.
And 5: 0.02mL of physiological saline was added to the 11 th well of each reaction plate of step 4, and 0.04mL of physiological saline was added to the 12 th well of each reaction plate of step 4.
Step 6: to the 1 st to 11 th wells of each reaction plate of step 5, 0.02mL of 4 units of ND, H9, H5, and H7 virus antigen solution was added, shaken for 1 minute, and allowed to stand at room temperature for 30 minutes.
And 7: 0.02mL of a chicken erythrocyte suspension with the volume fraction of 1% (added after fully shaking the erythrocyte suspension) is added into each hole by a 12-channel micropipette, the mixture is shaken for 1 minute, and is kept stand for 30 minutes at room temperature, and the antibody titer of ND, H9, H5 and H7 is judged.
Example 4
Step 1: 0.2mL of physiological saline was added to the 1 st to 10 th wells of the 96-well original reaction plate 1 by using an 8-channel micropipette.
Step 2: using a single-channel micropipette to respectively suck 0.2mL of blood serum to be detected to each hole of the 1 st row of the original reaction plate 1, repeatedly blowing and beating for 5 times, and uniformly mixing.
And step 3: sucking 0.2mL of the sample from each 1 st row of wells of the original reaction plate 1 by using an 8-channel micropipette, repeatedly blowing and beating the sample for 5 times, uniformly mixing the sample into the 2 nd row of wells, sequentially diluting the sample in multiple proportions to the 10 th row of wells, sucking 0.2mL of the sample from the 10 th row of wells, and discarding the sample.
And 4, step 4: as shown in fig. 1, the diluted blood serum to be tested in step 3 was transferred to the corresponding wells of the 96-well ND reaction plate 2, H9 reaction plate 3, H5 reaction plate 4 and H7 reaction plate 5 at a volume of 0.04mL per well.
And 5: 0.04mL of physiological saline was added to the 11 th well of each reaction plate of step 4, and 0.08mL of physiological saline was added to the 12 th well of each reaction plate of step 4.
Step 6: to the 1 st to 11 th wells of each reaction plate of step 5, 0.04mL of 4 units of ND, H9, H5, and H7 virus antigen solution was added, shaken for 1 minute, and allowed to stand at room temperature for 30 minutes.
And 7: 0.04mL of a suspension of chicken red blood cells with a volume fraction of 1% (added after fully shaking the suspension of red blood cells) was added to each well with a 12-channel micropipette, shaken for 1 minute, and left standing at room temperature for 30 minutes to determine the antibody titers of ND, H9, H5 and H7.
Analysis shows that compared with the prior art, the invention has the advantages and beneficial effects that:
the invention provides a combined rapid detection method for multiple avian antibodies, which can simultaneously detect 4-6 avian influenza and newcastle disease antibodies by one person and improve the working efficiency by 200 percent.
Comparative example
Step 1: 0.025mL of physiological saline was added to each well of the 1 st to 12 th rows of the 96-well reaction plate by a micropipette.
Step 2: sucking 0.025mL of blood serum to be detected in each hole of the 1 st row by using a micropipettor, repeatedly blowing and beating for 4-5 times, uniformly mixing, sucking out 0.025mL to the 2 nd row of holes, sequentially diluting to the 11 th row of holes in multiple proportion, and discarding 0.025 mL; no serum was added to well 12 for antigen control.
And step 3: 0.025mL4 units of antigen were added to each of the wells in columns 1-12.
And 4, step 4: and (3) placing the reaction plate in the step (3) on an oscillator, shaking for 1 minute, and standing for 30 minutes at room temperature.
And 5: 0.025mL of a 1% volume fraction suspension of chicken red blood cells was added to each of the wells in columns 1-12 using a micropipette.
Step 6: placing the reaction plate obtained in the step 5 on an oscillator, and oscillating for 1 minute; the antibody titer was judged after 30 minutes of standing at room temperature.
And 7: the steps 1-6 are repeated for the avian influenza H9/H7/H5.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for rapidly detecting multiple avian antibodies in a combined manner is characterized by comprising the following steps:
step 1: adding 0.1-0.2mL of diluent into all the holes of the 1 st-10 th row of the original reaction plate;
step 2: adding 0.1-0.2mL of blood serum into each hole of the 1 st column in the step 1;
and step 3: sucking 0.1-0.2mL of the solution from each hole in the 1 st row of the step 2 to the hole in the 2 nd row;
and 4, step 4: transferring the blood serum of the step 3 into corresponding holes of ND, H9, H5 and H7 reaction plates according to 0.02-0.04mL per hole respectively;
and 5: adding 0.02-0.04mL of diluent into each hole of the 11 th row of each reaction plate in the step 4 respectively;
step 6: adding 0.02-0.04mL of ND, H9, H5 and H7 virus antigen liquid into each hole of the 1 st row to the 11 th row of each reaction plate in the step 5;
and 7: 0.02-0.04mL of chicken red blood cell suspension was added to each well of each reaction plate of step 6, and the antibody titers of ND, H9, H5, and H7 were determined.
2. The method for rapidly detecting the combination of the antibodies of various birds according to claim 1, which is characterized in that: and 3, sequentially diluting the solution to 10 th row of holes in a multiple ratio, sucking 0.1-0.2mL of the solution from the 10 th row of holes, and discarding the solution.
3. The method for rapidly detecting the combination of the antibodies of various birds according to claim 1, which is characterized in that: the volume fraction of the chicken red blood cell suspension in the step 7 is 1%.
4. The method for rapidly detecting the combination of the antibodies of various birds according to claim 1, which is characterized in that: in the step 6, 4 units of ND, H9, H5 and H7 virus antigen solution 0.02-0.04mL is added into each well of the 1 st to 11 th rows of each reaction plate in the step 5.
5. The method for the combined rapid detection of various avian antibodies according to one of claims 1 to 4, characterized in that: step 1 was performed using an 8-channel micropipette.
6. The method for rapidly detecting the combination of the antibodies of various birds according to claim 5, which is characterized in that: step 2 is pipetting by using a single-channel micropipette.
7. The method for rapidly detecting the combination of the antibodies of various birds according to claim 6, which is characterized in that: step 7 uses a 12-channel micropipette for pipetting.
8. The method for the combined rapid detection of multiple avian antibodies according to claim 7, which is characterized in that: and 5, adding 0.04-0.08ml of diluent into each hole of the 12 th row of each reaction plate in the step 4.
9. The method for the combined rapid detection of multiple avian antibodies according to claim 8, which is characterized in that: the diluent is any one of normal saline or PBS.
10. The method for the combined rapid detection of multiple avian antibodies according to claim 9, which is characterized in that: the original, ND, H9, H5 and H7 reaction plates are all 96-well reaction plates.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4059491A (en) * | 1976-02-02 | 1977-11-22 | Takeda Chemical Industries, Ltd. | Diluents for rubella virus hemagglutination-inhibition test |
| US20110097705A1 (en) * | 2009-10-20 | 2011-04-28 | Vaxdesign Corp. | Surface-assisted hemagglutination and hemagglutination inhibition assays |
| CN104730259A (en) * | 2015-03-12 | 2015-06-24 | 青岛易邦生物工程有限公司 | Method for detecting hemagglutination inhibition antibody of chicken infectious bronchitis |
| CN107091935A (en) * | 2017-04-20 | 2017-08-25 | 陈凡 | Automate General layout Plan of blood clotting Inhibition test work station and application thereof |
| CN108088995A (en) * | 2017-12-05 | 2018-05-29 | 广西凤翔集团股份有限公司 | Bird flu hemagglutination-inhibition test detection method |
-
2018
- 2018-11-20 CN CN201811383056.4A patent/CN111198265A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4059491A (en) * | 1976-02-02 | 1977-11-22 | Takeda Chemical Industries, Ltd. | Diluents for rubella virus hemagglutination-inhibition test |
| US20110097705A1 (en) * | 2009-10-20 | 2011-04-28 | Vaxdesign Corp. | Surface-assisted hemagglutination and hemagglutination inhibition assays |
| CN104730259A (en) * | 2015-03-12 | 2015-06-24 | 青岛易邦生物工程有限公司 | Method for detecting hemagglutination inhibition antibody of chicken infectious bronchitis |
| CN107091935A (en) * | 2017-04-20 | 2017-08-25 | 陈凡 | Automate General layout Plan of blood clotting Inhibition test work station and application thereof |
| CN108088995A (en) * | 2017-12-05 | 2018-05-29 | 广西凤翔集团股份有限公司 | Bird flu hemagglutination-inhibition test detection method |
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