A method for screening for canine distemper virus microneutralizing antibodies

Abstract

This invention discloses a method for screening trace amounts of neutralizing antibodies against canine distemper virus (CDV). The method involves neutralizing the sample with live canine distemper virus, then inoculating it into Vero-Slam cells, using fluorescent lesions as the criterion. Detecting neutralizing antibodies using the true conformation of the virus ensures the highest degree of accuracy in detecting neutralizing antibodies. CDV-positive neutralizing serum (neutralizing titer 1:708) was serially diluted twofold to multiple concentration groups as the sample to be tested. The detection rate and limit of detection of CDV neutralizing activity of the method for each dilution of serum were determined. Results showed that serum with a CDV neutralizing titer of 1:708 diluted twofold... 12 The neutralizing activity after doubling is the lowest detection limit of the method of this invention, with a detection rate of 63.54 ± 4.774%. The method of this invention uses few reagents, none of which are expensive, resulting in low cost. This invention provides a new technical means for screening trace neutralizing antibodies against canine distemper virus.

Description

TECHNICAL FIELD

[0001] The present application relates to a screening method of canine distemper virus neutralizing antibody, in particular to a screening method of canine distemper virus micro-neutralizing antibody. The present application belongs to the field of biotechnology. BACKGROUND

[0002] Canine distemper (CD) is a highly contagious disease caused by infection of canine distemper virus (CDV) in dogs. Among various diseases suffered by dogs, CD ranks first in terms of infection intensity, severity of disease, and mortality rate.

[0003] Neutralizing antibody is the corresponding antibody produced when pathogenic microorganisms invade the body. When pathogenic microorganisms invade cells, they need to rely on specific molecules expressed by the pathogen to bind to receptors on the cell surface in order to infect the cell and further proliferate. Neutralizing antibody is a certain antibody produced by B lymphocytes, which can bind to the antigen on the surface of pathogenic microorganisms, thereby preventing the pathogenic microorganisms from adhering to the target cell receptors and preventing invasion of the cell. Therefore, compared with general antibodies, neutralizing antibodies can neutralize viruses, making the viruses lose activity and thus be phagocytosed by cells, and have a stronger protective effect on the body.

[0004] In "The establishment of canine distemper pseudovirus system and its application in neutralizing antibody detection method, Ma Zhihua, master's degree thesis", the author established a canine distemper virus neutralizing antibody detection method based on pseudovirus technology. Pseudovirus refers to a retrovirus that can integrate the envelope glycoprotein of another different virus, thereby forming an envelope with exogenous virus, while the genome remains the same as the original retrovirus. Due to its safe characteristics, it is often used to simulate high-risk viruses. However, this method has the following disadvantages: 1. Canine distemper virus is not a high-risk virus and can be used in P2 laboratory operations. Therefore, there is no need to use pseudovirus simulation instead. 2. Canine distemper pseudovirus is only expressed on the slow virus backbone of the H and F antigen proteins of canine distemper virus, and these two antigen proteins undergo conformational changes, which do not exist in the original virus's natural conformation. Therefore, this method will miss some real neutralizing antibodies, resulting in false negatives; it will also misdiagnose some false neutralizing antibodies, resulting in false positives. 3. The canine distemper pseudovirus neutralizing antibody detection method is only compared with the traditional neutralizing antibody level detection method. It is proved that this method can replace the traditional neutralizing antibody detection method, but it has not tested the detection ability of the method for trace neutralizing antibodies (i.e. the minimum detection limit and its detection rate). Therefore, it cannot be used for trace neutralizing antibody detection at present. 4. High cost: the cost of preparing canine distemper pseudovirus is high, and the yield is low. SUMMARY

[0005] The purpose of the present application is to establish a canine distemper virus (CDV) trace neutralizing antibody detection method suitable for screening samples containing trace CDV neutralizing activity in high-throughput micro-volume samples.

[0006] In order to achieve the above-mentioned purpose, the following technical means are adopted in the present application:

[0007] The method of the present application is an indirect immunofluorescence detection method (IFA) based on Vero-slam cell line and canine distemper virus (CDV-OR12 strain), by repeatedly groping for the fixed neutralizing virus amount. The method of the present application uses canine distemper virus with fixed neutralizing virus amount to mix with the sample to be tested in equal volume, neutralize at 37℃ for 1 hour, then inoculate the prepared 96-well plate cells (Vero-slam cells), and perform indirect immunofluorescence experiment after 72 hours. The commercial canine distemper antibody CDV-NP (VRMD) is selected as the primary antibody. Through repeated groping, the fixed neutralizing virus amount is set to 4TCID 50 / 0.1ml. Again, the CDV positive neutralizing serum (neutralization titer is 1:708) is serially diluted by 2 times to form multiple concentration groups as the samples to be detected, so as to detect the detection rate of CDV neutralization activity of the method of the application to each dilution serum and the lowest detection limit.

[0008] Specifically, the method for screening the micro neutralizing antibody of canine distemper virus comprises the following steps:

[0009] (1) Before inoculating the Vero-Slam cells, the cell counting is performed, and the cell concentration is diluted to 1.5×10 5 / ml, and then transferred to a sterile sample groove for fully mixing, and finally inoculated into a 96-well plate by a row gun at 100ul / well; within 24 hours, the cells can be attacked when the cell coverage area reaches more than 90%;

[0010] (2) The CDV virus liquid containing 2% v / v serum is serially diluted by 10 times to form 4TCID50 / 0.1ml, and mixed with the samples to be detected in equal volume, and neutralized at 37°C for 1h; after the neutralization is completed, the 96-well plate Vero-Slam cells obtained in step (1) are washed once with 150ul PBS / well, and then each sample to be detected is inoculated into 1 well at 100ul, and incubated at 37°C for 72h under the condition of 5% CO2, so as to obtain the 96-well plate Vero-Slam cells to be detected;

[0011] (3) Indirect immunofluorescence experiment observation (IFA)

[0012] The IFA experiment is performed after the samples to be detected are inoculated and neutralized for 72h, and the specific steps are as follows:

[0013] 1) The 96-well plate Vero-Slam cells to be detected are washed twice with PBS at pH 7.2, 150ul / well, and rested for 3min;

[0014] 2) Fixation: 100ul / well of fixing solution is added, and the resting action is performed for 20min; PBS is washed for 3 times, 150ul / well, and rested for 3min;

[0015] 3) Blocking: 100ul / well of 1% w / v BSA is added, and the action is performed at 37°C for 1h;

[0016] 4) Primary antibody incubation: the canine distemper virus monoclonal antibody CDV-NP is diluted to 1ug / ml, 50ul / well, and the action is performed at 37°C for 1.5h;

[0017] 5) PBS is washed for 3 times, 100ul / well, and rested for 3min;

[0018] 6) Second antibody incubation: rabbit anti-mouse antibody was diluted at 1:500, 50ul / well, 37℃ for 1h; PBS was used for washing 3 times, 100ul / well, 3min;

[0019] 7) Nucleus staining: DAPI staining solution (E607303, Sangon Biotech) was diluted at 1:60, 50ul / well, 15min at room temperature in dark; PBS was used for washing 2 times, 100ul / well, 3min;

[0020] 8) Observation under fluorescence microscope, judgment standard:

[0021] The absence of positive fluorescence in the well was used as the positive standard of neutralization activity, and the presence of positive fluorescence in the well was used as the negative standard of neutralization activity.

[0022] Preferably, the canine distemper virus is CDV-OR12 strain.

[0023] Preferably, the fixing solution is prepared by mixing ethanol and acetone at a volume ratio of 3:2.

[0024] Preferably, the method is not used for the diagnosis and treatment of diseases.

[0025] Preferably, the method is used for screening antibodies with neutralization activity, and the antibodies with neutralization activity can be used for preparing medicines for preventing or treating diseases caused by canine distemper virus infection.

[0026] Compared with the prior art, the present application has the following advantages:

[0027] 1. Real neutralizing antibody detection:

[0028] After the method of the present application uses the live canine distemper virus to neutralize the sample to be detected, the sample is inoculated on vero-slam cells (canine distemper virus sensitive cells), and fluorescence plaques are used as the judgment standard. The real conformation of the virus is used to detect neutralizing antibodies, which can most effectively ensure the authenticity of the detected neutralizing antibodies.

[0029] 2. Micro neutralizing antibody sample screening ability: sensitivity and stability

[0030] A CDV positive neutralizing serum (neutralization titer of 1:708) was serially diluted by 2 times to form multiple concentration groups as samples to be detected, and the detection rate of CDV neutralization activity of each dilution serum and the lowest detection limit were detected by the method of the present application. The results show that: the detection rate of the positive neutralizing serum (neutralization titer of 1:708) diluted by 2 times is 93.75±3.608%; the detection rate of the serum dilution group diluted by 2 times is 88.54±4.774%; and the detection rate of the serum dilution group diluted by 2 times is 88.54±4.774%. 10 11 The detection rate of the positive neutralizing serum (neutralization titer of 1:708) diluted by 2 times is 93.75±3.608%; the detection rate of the serum dilution group diluted by 2 times is 88.54±4.774%; and the detection rate of the serum dilution group diluted by 2 times is 88.54±4.774%.​12 The detection rate of the 2-fold serum dilution group was 63.54±4.774%; the detection rate of the 2 13 The detection rate of the 2-fold serum dilution group was 46.88±6.250%; the detection rate of the 2 14 The detection rate of the 2-fold serum dilution group was 14.58±6.505%; the detection rate of the 2 15 The detection rate of the 2-fold serum dilution group was 7.29±1.804%. In this study, the detection rate greater than 60% was considered as effective detection. Therefore, the neutralization activity of the serum diluted 2 12 times in the CDV was the lowest detection limit of the method of the present application, and the detection rate was 63.54±4.774%.

[0031] 3. Low cost

[0032] The method of the present application uses fewer reagents, and there is no expensive reagent. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is a technical roadmap;

[0034] Figure 2 is a canine distemper virus fluorescence CPE and conventional CPE observation chart;

[0035] A~D are inoculated with 10 3 fold diluted virus liquid, E~H are inoculated with 10 4 fold diluted virus liquid, and I~L are inoculated with DMEM instead of virus liquid; the fluorescence indicated by the white arrow is positive fluorescence CPE, and the corresponding H chart does not find conventional CPE, proving that the method is more sensitive than the conventional neutralizing antibody detection method;

[0036] Figure 3 is the neutralization activity detection rate result of the 2 10 ~2 15 fold serum dilution group. DETAILED DESCRIPTION

[0037] The advantages and characteristics of the present application will become clearer with the description. However, these examples are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements all fall within the protection scope of the present application.

[0038] Example 1: A screening method for canine distemper virus micro-neutralizing antibody

[0039] The technical roadmap is shown in Figure 1 , and specifically includes the following steps:

[0040] (1) CDV positive neutralizing serum (neutralization titer 1:708) was serially diluted by 2 times for 2 times as the sample to be tested 10

[0041] (2) Cell counting was performed before inoculation of Vero-Slam cells, and the cell concentration was diluted to 1.5 x 10 5 / ml, and then transferred to a sterile sample addition groove for thorough mixing, and finally inoculated into a 96-well plate at 100ul / well by using a row gun; within 24 hours, the cells were attacked when the coverage area reached more than 90%;

[0042] (3) CDV virus solution containing 2% v / v serum was serially diluted by 10 times to 4 TCID50 / 0.1ml, and mixed with the sample to be tested in equal volume, and neutralized at 37°C for 1h; after the neutralization was completed, the 96-well plate Vero-Slam cells obtained in step (2) were washed once with 150ul PBS / well, and each sample to be tested was inoculated at 100ul / well, and incubated at 37°C for 72h under 5% CO2 conditions to obtain the 96-well plate Vero-Slam cells to be tested;

[0043] (4) Indirect immunofluorescence test (IFA)

[0044] IFA test was performed after the sample to be tested was inoculated and neutralized for 72h, and the specific steps were as follows:

[0045] 1) The 96-well plate Vero-Slam cells to be tested were washed twice with PBS at pH 7.2: 150ul / well, and rested for 3min;

[0046] 2) Fixation: 100ul / well of fixing solution (ethanol and acetone were prepared in a volume ratio of 3:2) was added, and the effect was 20min; PBS was washed 3 times: 150ul / well, and rested for 3min;

[0047] 3) Blocking: 100ul / well of 1% w / v BSA was added, and incubated at 37°C for 1h;

[0048] 4) Primary antibody incubation: canine distemper virus monoclonal antibody CDV-NP was diluted to 1ug / ml, 50ul / well, and incubated at 37°C for 1.5h;

[0049] 5) PBS was washed 3 times: 100ul / well, and rested for 3min;

[0050] 6) Secondary antibody incubation: rabbit anti-mouse antibody (SAB3701081, sigma) was diluted at 1:500, 50ul / well, and incubated at 37°C for 1h; PBS was washed 3 times in the dark: 100ul / well, and rested for 3min;

[0051] ​7) Nuclear staining: Dilute DAPI staining solution (E607303, Sangon Biotech) 1:60, 50ul / well, incubate at room temperature in the dark for 15min; wash twice with PBS in the dark: 100ul / well, stand for 3min.

[0052] 8) Observation under a fluorescence microscope, judgment criteria:

[0053] The absence of positive fluorescence in the wells was considered a positive standard for neutralizing activity, while the presence of positive fluorescence in the wells was considered a negative standard for neutralizing activity.

[0054] (5) The detection rate using this method was 93.75 ± 3.608%.

[0055] Example 2:

[0056] CDV-positive neutralizing serum (neutralizing titer 1:708) was serially diluted 2-fold. 11 The sample was taken as a multiple of the sample to be tested, and the remaining steps were the same as in Example 1. The detection rate using this method was 88.54 ± 4.774%.

[0057] Example 3:

[0058] CDV-positive neutralizing serum (neutralizing titer 1:708) was serially diluted 2-fold. 12 The sample was taken as a multiple of the sample to be tested, and the remaining steps were the same as in Example 1. The detection rate using this method was 63.54 ± 4.774%.

[0059] Example 4:

[0060] CDV-positive neutralizing serum (neutralizing titer 1:708) was serially diluted 2-fold. 13 The sample was taken as a multiple of the sample to be tested, and the remaining steps were the same as in Example 1. The detection rate using this method was 46.88±6.250%.

[0061] Example 5:

[0062] Serum from animals infected with CDV was serially diluted 2-fold. 10 Using the sample as the test sample, the remaining steps are the same as in Example 1, and the neutralizing antibody animal serum can be screened to obtain the neutralizing antibody. The neutralizing antibody can be further prepared into a vaccine or drug according to conventional methods in the art.

Claims

1. A method for screening for micro-neutralizing antibodies to canine distemper virus, characterized in that, The method comprises the following steps: (1) The canine distemper virus positive neutralization serum with neutralization titer of 1:708 was serially diluted by 2 times for 2 times as the sample to be tested. 10 -2 12 times as the sample to be tested. (2) Cell counting was performed before inoculation of Vero-Slam cells, and the cell concentration was diluted to 1.5 x 10 5 / ml, then transferred to a sterile sample tank for thorough mixing, and finally inoculated into a 96-well plate at 100 μl / well using a dispensing gun; within 24 hours, the cells were infected when the cell coverage area reached more than 90%; (3) The CDV-OR12 strain virus solution was serially diluted by 10 times gradient for 4 TCID 50 / 0.1ml, respectively mixed with the same volume of the sample to be detected, neutralized at 37°C for 1h, and then the 96-well plate Vero-Slam cells obtained in step (2) were washed once with 150μl PBS per well, and then each sample neutralization solution was inoculated into 1 well at 100μl, incubated at 37°C for 72h under 5% CO2, to obtain the 96-well plate Vero-Slam cells to be detected; (4) indirect immunofluorescence experiment observation The indirect immunofluorescence experiment is carried out after 72 hours of neutralization and inoculation of the sample to be detected, and the specific steps are as follows: 1) The Vero-Slam cells in the 96-well plate to be detected are washed twice with pH 7.2 PBS: 150ul / well, and stand for 3 minutes; 2) Fixation: 100ul / well of fixing solution is added, and stand for 20 minutes; PBS is washed for 3 times: 150ul / well, and stand for 3 minutes; 3) Blocking: 100ul / well of 1% w / v BSA is added, and stand for 1 hour at 37℃; 4) Primary antibody incubation: the canine distemper virus monoclonal antibody CDV-NP is diluted to 1ug / ml, 50ul / well, and stand for 1.5 hours at 37℃; 5) PBS is washed for 3 times: 100ul / well, and stand for 3 minutes; 6) Secondary antibody incubation: the rabbit anti-mouse antibody is diluted at 1:500, 50ul / well, and stand for 1 hour at 37℃; PBS is washed for 3 times in the dark: 100ul / well, and stand for 3 minutes; 7) Nucleus staining: DAPI staining solution is diluted at 1:60, 50ul / well, and stand for 15 minutes in the dark at room temperature; PBS is washed for 2 times in the dark: 100ul / well, and stand for 3 minutes; 8) Observation under a fluorescence microscope, and the judgment standard is: The absence of positive fluorescence in the well is the positive standard of neutralization activity, and the presence of positive fluorescence in the well is the negative standard of neutralization activity.

2. The screening method of claim 1, wherein, The fixing solution is prepared by ethanol and acetone at a volume ratio of 3:

2.

3. The screening method of claim 1, wherein, The method is not used for the diagnosis and treatment of diseases.

4. The screening method of claim 1, wherein, The method is used for screening antibodies with neutralization activity, and the antibodies with neutralization activity are used for preparing medicines for preventing or treating diseases caused by canine distemper virus infection.

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