Detection method of porcine acute diarrhea syndrome coronavirus neutralizing antibody

Abstract

The invention relates to the technical field of virus detection, in particular to a method for detecting a neutralizing antibody of a porcine acute diarrhea syndrome coronavirus. The detection method comprises the steps of neutralization reaction, cell adsorption, cell cleaning, cell culture and antibody titer detection. The cleaning comprises the following steps: after the cell adsorption is carried out for 1-2 hours, discarding supernate, and cleaning the cells. Research finds that in the stage of adsorbing cells by viruses, serum has a neutralization effect on pancreatin in a virus diluent so as to inhibit cytopathy, so that the influence of the serum and pancreatin action on cytopathy is eliminated through cleaning after the cell adsorption stage; the detection limit of the porcine acute diarrhea syndrome coronavirus neutralizing antibody can be obviously reduced, and the detection accuracy is improved.

Description

Technical Field

[0001] The invention relates to the technical field of virus detection, and in particular to a method for detecting neutralizing antibodies of porcine acute diarrhea syndrome coronavirus. Background Art

[0002] Vaccination plays an important role in the prevention and control of viral infectious diseases. Effective vaccines can quickly induce high-titer neutralizing antibodies in the body, and the level of neutralizing antibodies is one of the key indicators to measure the immune protection effect of vaccines. In order to meet the needs of rapid vaccine development, large-scale clinical trial testing, and immunogenicity testing of animal populations after vaccination, it is urgent to establish a highly accurate, simple and fast virus neutralizing antibody evaluation method.

[0003] Traditional methods for detecting virus neutralizing antibodies include plaque inhibition assay and cytopathic assay based on live viruses. With the development of technology, more detection methods have been proposed, such as detection methods of recombinant live viruses containing reporter genes and neutralizing antibody detection methods based on pseudoviruses, but these methods have their own limitations or require the intervention of instruments and equipment, which are relatively complicated. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a method for detecting neutralizing antibodies of porcine acute diarrhea syndrome coronavirus.

[0005] The present invention found in the research process that in the detection process of neutralizing antibodies of porcine acute diarrhea syndrome coronavirus, after the virus adsorption stage, the serum has a certain neutralizing effect on the pancreatic enzyme in the virus diluent, which causes the pathological process to be inhibited after the cell is infected with the virus, and it is difficult to achieve accurate detection of neutralizing antibodies of porcine acute diarrhea syndrome coronavirus (or greatly improve the detection limit). Therefore, the present invention adds a virus adsorption process to the cell neutralization process, and the cells are normally diseased after washing, thereby achieving accurate detection of neutralizing antibodies of porcine acute diarrhea syndrome coronavirus. This technical improvement is difficult for technicians to think of. On the one hand, technicians have not found that the effect between serum / pancreatic enzymes will affect cell pathological changes. On the other hand, technicians usually think that washing may affect the process of virus adsorption of cells and thus affect the cell pathological process. However, the present invention has been experimentally verified to find that after adsorption, even if the supernatant is discarded by washing 2 to 3 times, the cells can still develop pathological changes normally. These technical discoveries have laid a theoretical foundation for the technical solution provided by the present invention.

[0006] In a first aspect, the present invention provides a method for detecting neutralizing antibodies of porcine acute diarrhea syndrome coronavirus, comprising: Neutralization reaction, cell adsorption, cell washing, cell culture and antibody titer detection; The cleaning comprises: After the cell adsorption was allowed to proceed for 1 to 2 hours, the supernatant was discarded and the cells were washed.

[0007] The detection method of the present invention is suitable for disease diagnosis or treatment. In addition to disease diagnosis and treatment, the detection method can also be used to evaluate the effectiveness of porcine acute diarrhea syndrome coronavirus vaccines. By comparing the neutralizing antibody levels of vaccinated pigs and unvaccinated pigs, it can be determined whether the vaccine can induce sufficient immune response to fight porcine acute diarrhea syndrome coronavirus. In addition, the detection method can also be used for pig herd immune status detection, helping farmers and veterinarians to assess the immune status of pigs and understand whether re-vaccination or other measures are needed to improve immunity. The detection method can also be used for scientific research related to porcine acute diarrhea syndrome coronavirus, such as studying the variation of porcine acute diarrhea syndrome coronavirus.

[0008] Furthermore, the washing comprises: washing the cells until there is no residue in the supernatant after the cell adsorption stage; preferably, the following method is adopted: washing the cells 2 to 4 times with a buffer solution or a culture medium.

[0009] Furthermore, the buffer is PBS buffer or the culture medium is one of DMEM.

[0010] Further, the neutralization reaction comprises: The serum sample to be tested is diluted according to a predetermined ratio to obtain reaction systems with different dilution ratios; porcine acute diarrhea syndrome coronavirus is added to all reaction systems; and the reaction systems are neutralized at 37° C. and 5% CO2 for 1 to 2 hours.

[0011] Furthermore, the virus titer for neutralization of porcine acute diarrhea syndrome coronavirus is 200 TCID 50 / 0.1mL.

[0012] Furthermore, the cell adsorption comprises: mixing the neutralized sample with the 96-well plate cells that have grown a monolayer and discarded the supernatant, and then adsorbing for 1 to 2 hours.

[0013] Furthermore, the cells are Vero cells.

[0014] Furthermore, the cell culture includes: adding the washed cells to a virus culture medium (DMEM+12.5 μg / mL trypsin), and culturing at 37° C. and 5% CO 2 .

[0015] Furthermore, the antibody titer detection includes: The pathological changes of the cells are observed during the culture process, and the titer of the neutralizing antibodies against porcine acute diarrhea syndrome coronavirus in the serum sample to be tested is determined according to the pathological changes.

[0016] In a second aspect, the present invention provides application of the detection method in evaluating the effectiveness of porcine acute diarrhea syndrome coronavirus vaccine.

[0017] The present invention further provides application of the detection method in detecting the immune status of pig herds.

[0018] The present invention has the following beneficial effects: The present invention provides a method for detecting neutralizing antibodies of porcine acute diarrhea syndrome coronavirus. By washing after cell adsorption, the inhibitory effect of serum / pancreatic enzyme interaction on cytopathic effect is avoided, and effective detection of porcine acute diarrhea syndrome coronavirus (with a lower detection limit) is achieved, and the detection accuracy is improved, which has important application value in the relevant fields of porcine acute diarrhea syndrome coronavirus detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 It is the specific pathological condition that occurs 24 hours after the adsorption group, positive control group and negative control group provided in Example 2 of the present invention are exposed to the virus. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1 This example verifies the effect of serum and pancreatic enzyme mixture on neutralizing antibodies of porcine acute diarrhea syndrome coronavirus (SADS-CoV) (derived from seed virus; provided by Jinyu Baoling Biopharmaceutical Co., Ltd., pathogen isolation platform), including the following experimental process: 1. Inactivation of the serum to be tested: Inactivate the SADS-CoV negative serum (Gibco's fetal bovine serum), and then place it in a water bath at 56°C for 30 minutes.

[0023] 2. Dilution of serum after inactivation: dilute the SADS-CoV negative serum sample 2-fold with maintenance solution.

[0024] 3. Virus dilution: Dilute the SADS-CoV virus to 200 TCID using a maintenance solution containing pancreatin (purchased from Gibco). 50 / 0.1mL, respectively configured according to the following system: Well A: The predetermined serum dilution ratio is 1:2, and the added neutralization virus (SADS-CoV virus) contains 12.5 μg / mL pancreatic enzyme.

[0025] Well B: The predetermined serum dilution ratio is 1:4, and the added neutralization virus (SADS-CoV virus) contains 12.5 μg / mL trypsin.

[0026] Well C: The predetermined serum dilution ratio is 1:8, and the added neutralization virus (SADS-CoV virus) contains 12.5 μg / mL trypsin.

[0027] Well D: The predetermined serum dilution ratio is 1:16, and the added neutralization virus (SADS-CoV virus) contains 12.5 μg / mL trypsin.

[0028] Well E: The predetermined serum dilution ratio is 1:32, and the added neutralization virus (SADS-CoV virus) contains 12.5 μg / mL trypsin.

[0029] Well F: The predetermined serum dilution ratio is 1:64, and the added neutralization virus (SADS-CoV virus) contains 12.5 μg / mL trypsin.

[0030] Well G: The predetermined serum dilution ratio is 1:128, and the added neutralization virus (SADS-CoV virus) contains 12.5 μg / mL trypsin.

[0031] Well H: The predetermined serum dilution ratio is 1:256, and the added neutralization virus (SADS-CoV virus) contains 12.5 μg / mL trypsin.

[0032] 4. Neutralization: Mix the neutralizing toxin with the diluted SADS-CoV negative serum sample in equal amounts (50 μL: 50 μL), add them to a 96-well plate according to the predetermined ratio in step 3, make 2 replicate wells for each serum dilution, place in a 37°C, 5% CO2 incubator, and neutralize for 1 hour.

[0033] 5. Inoculation of cells: Wash the 96-well plate 2-3 times with PBS or serum-free medium and culture the VERO cells for 48 hours to grow a monolayer. Take out the samples from the 96-well plate in step 4 and add them to the washed 96-well plate with VERO cells that have grown a monolayer for 48 hours. Set up a regression group with four dilutions, namely (100, 10, 1, 0.1) TCID 50 / 0.1mL, set 8 wells for each dilution, place in a 37℃, 5% CO2 incubator for 3-5 days, observe and record the cytopathic effect (CPE) every day.

[0034] 6. Result determination: Use an inverted microscope to observe whether CPE appears in the cell wells, count the number of serum lesion wells to be tested, and calculate the serum neutralizing antibody titer according to the Reed-Muench method. The conditions for the test to be established are: negative serum should show CPE in all wells, normal cell controls should not show CPE, and the cell lesion in the regression group is 100TCID 50 / 0.1mL hole lesion hole is 8 / 8, 10TCID 50 / 0.1mL hole lesion hole 8 / 8, 1TCID 50 / 0.1mL hole lesion hole 4 / 8, 0.1TCID 50 / 0.1mL hole lesion hole 0 / 8.

[0035] 7. The results are shown in Table 1 below. It can be seen from the results that the presence of pancreatic enzymes affects the level of cytopathic effect after viral infection, which leads to the negative serum showing no cytopathic effect in the test wells. Therefore, this method is prone to false negative results and cannot be used for neutralizing antibody detection.

[0036] Table 1 Statistical results of lesions in different experimental groups

[0037] Example 2 The present invention further verifies whether the SADS-CoV virus can still cause pathological changes after being adsorbed on adherent cells and then discarded. The specific steps are as follows: 1. Inoculation cells: Use T75 adherent cell culture flasks to culture Vero cells for 24 to 48 hours until they grow into a monolayer (culture medium volume 30 mL), discard the supernatant, and wash 2 to 3 times with PBS or serum-free DMEM medium.

[0038] 2. Virus inoculation scheme: For the adsorption virus group, add the virus titer of 10 to the cells described in step 1 at a ratio of 1% of the cell culture volume. 6 ~10 7 TCID 50 / 0.1mL of virus (the volume of the virus solution is 300μL), adsorb for 1 hour to 2 hours (1 hour in this embodiment), wash 2 to 3 times with PBS or serum-free culture medium, and then supplement with 30mL of DMEM culture medium containing 12.5g / mL trypsin.

[0039] The positive control group added a 10% toxin to the cells in step 1 at a ratio of 1% of the cell culture volume. 6 ~10 7 TCID 50 / 0.1ml of virus, supplement with 30mL of DMEM culture medium containing 12.5g / mL trypsin.

[0040] In the negative control group, the cells described in step 1 were directly supplemented with 30 ml of DMEM culture medium containing 12.5 g / mL trypsin.

[0041] All the above were placed in a 37°C, 5% CO2 incubator for 1 to 3 days, and the cytopathic effect (CPE) was observed and recorded daily.

[0042] 3. Result determination: Use an inverted microscope to observe whether the cells have CPE. Both the adsorption group and the positive control group have CPE, while the negative control group does not have CPE and the cell status is normal, which is significantly different from the positive control.

[0043] 4. Result analysis: The results are as follows Figure 1 As shown, both the adsorption group and the positive control group developed specific lesions 24 hours after infection, while the negative control group did not develop any lesions. This experiment shows that cells treated with SADS-CoV virus adsorption can still produce specific lesions after being washed twice.

[0044] Example 3 The present invention further provides a method for detecting the toxicity of SADS-CoV by adsorption method, comprising the following process: 1. 96-well plate: Inoculate the Vero adherent cell suspension into a 96-well plate for 24-48 hours to grow into a full monolayer. The culture medium is DMEM (Gbico) + 10% FBS (Gbico).

[0045] 2. Dilute the virus: dilute the virus by 10-fold gradient dilution method, dilute (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 ), DMEM medium (Gibco) was used as dilution medium.

[0046] 3. Wash cells: Wash the cells grown in a monolayer on the 96-well plate in step 1 above 2 to 3 times with PBS or DMEM medium.

[0047] 4. Virus adsorption: Take 100 μL of each dilution of the SADS-CoV virus diluted in step 2 and add it to the 96-well cell plate washed in step 3 above. Make 8 duplicate wells for each dilution. Adsorb for 1-2 hours and discard the supernatant. Wash and discard the supernatant using the method described in step 3. Add 100 μL of virus culture solution (DMEM solution (Gibco) containing 12.5 μg of trypsin (Gibco)) to each well, and culture in a carbon dioxide incubator at 37°C and 5% CO2. Observe and record the cytopathic effect every day.

[0048] 5. Observation and result determination: Specific lesions appeared on the second day after the test, and the results were determined on the third day after the test. The sample's toxicity was 10 5 TCID 50 / 0.1ml.

[0049] Example 4 The present invention provides a method for detecting SADS-CoV virus neutralizing antibodies by an adsorption method, comprising the following process: 1. 96-well plate: Inoculate the Vero adherent cell suspension into a 96-well plate and allow it to grow into a monolayer for 24-48 hours. The culture medium is DMEM (Gbico) + 10% FBS (Gbico).

[0050] 2. Neutralization: Dilute the virus obtained in Example 3 to 200 TCID with DMEM medium. 50 / 0.1mL for future use.

[0051] 3. Wash cells: Wash the 96-well plate Vero cells grown in monolayer in step 1 above 2-3 times with PBS or DMEM medium (wash twice in this example).

[0052] 4. Dilute serum: dilute the serum to be tested in a 96-well plate using DMEM medium at a 2-fold ratio to the 12th well (2 1 , 2 2 , 2 3 , 2 4 , 2 5 , 2 6 , 2 7 , 2 8 , 2 9 , 2 10 , 2 11 , 2 12 ), make 2 replicate wells for each sample, and the volume of each well is 50μL.

[0053] 5. Neutralization: Add 50 μL of the neutralizing agent diluted in step 2 to each well of the serum diluted in step 4 above, and incubate in a carbon dioxide incubator for 1 hour at 37°C and 5% CO2.

[0054] 6. Adsorption: Place the neutralized sample in step 5 above into the 96-well plate cells washed in step 3 above, adsorb for 1-2 hours, discard, wash with PBS, and add virus culture medium (DMEM medium containing 12.5 μg / mL trypsin).

[0055] 7. Culture and observation: Culture the cells in a carbon dioxide incubator at 37°C, 5% CO2. Observe and record the cytopathic effects daily. Calculate the neutralizing antibody titer. The results are shown in Table 2 below.

[0056] Table 2 Neutralizing antibody test results

[0057] Note: The positive samples are the positive serum samples collected from piglets after virus challenge but before autopsy, and the negative serum samples are the serum samples collected from piglets before virus challenge.

[0058] The results of Example 4 above show that the method is feasible. The method solves the problem of non-pathological changes caused by the inhibitory effect of serum on trypsin in the virus dilution solution during cell neutralizing antibody detection. The detection limit of the method is consistent with the conventional cell neutralization method. The method has the advantages of simple operation and high repeatability. Therefore, the method can be applied to experiments such as screening negative experimental animals and evaluating vaccine efficacy tests.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting neutralizing antibodies of porcine acute diarrhea syndrome coronavirus, characterized in that: include: Neutralization reaction, cell adsorption, cell washing, cell culture and antibody titer detection; The cleaning comprises: After the cell adsorption was allowed to proceed for 1 to 2 hours, the supernatant was discarded and the cells were washed.

2. The detection method according to claim 1, characterized in that: The washing comprises: washing the cells until there is no residue in the supernatant after the cell adsorption stage; preferably, the following method is adopted: washing the cells 2 to 4 times with a buffer solution or a culture medium.

3. The detection method according to claim 1 or 2, characterized in that: The neutralization reaction comprises: The serum sample to be tested is diluted according to a predetermined ratio to obtain reaction systems with different dilution ratios; porcine acute diarrhea syndrome coronavirus is added to all reaction systems; and the reaction systems are neutralized at 37° C. and 5% CO2 for 1 to 2 hours.

4. The detection method according to claim 3, characterized in that: The virus titer for neutralization of porcine acute diarrhea syndrome coronavirus is 200TCID 50 / 0.1mL.

5. The detection method according to any one of claims 1 to 4, characterized in that: The cell adsorption comprises: mixing the neutralized sample with cells and then adsorbing for 1 to 2 hours.

6. The detection method according to claim 5, characterized in that: The cells are Vero cells.

7. The detection method according to any one of claims 1 to 6, characterized in that: The cell culture comprises: adding the washed cells into a culture medium and culturing them under the conditions of 37° C. and 5% CO 2 .

8. The detection method according to any one of claims 1 to 7, characterized in that The antibody titer detection comprises: The pathological changes of the cells are observed during the culture process, and the titer of the neutralizing antibodies against porcine acute diarrhea syndrome coronavirus in the serum sample to be tested is determined according to the pathological changes.

9. Use of the detection method according to any one of claims 1 to 8 in the effectiveness evaluation of porcine acute diarrhea syndrome coronavirus vaccine.

10. Use of the detection method according to any one of claims 1 to 8 in detecting the immune status of pig herds.

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