CN111579789B - Method for rapidly detecting varicella virus titer by using fluorescence method - Google Patents
Method for rapidly detecting varicella virus titer by using fluorescence method Download PDFInfo
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- CN111579789B CN111579789B CN202010515720.7A CN202010515720A CN111579789B CN 111579789 B CN111579789 B CN 111579789B CN 202010515720 A CN202010515720 A CN 202010515720A CN 111579789 B CN111579789 B CN 111579789B
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Abstract
The invention provides an antibody for detecting varicella-zoster virus (VZV) virus titer and a related detection method, which comprises the steps of inoculating diploid cells into a 96-well plate for culture, then infecting and inoculating a sample with unknown virus titer, adding the antibody specifically combined with the VZV virus and a FITC-labeled secondary antibody after culture, carrying out fluorescent staining, and observing the number of infected lesions under a mirror to obtain the virus titer of the sample. The method provided by the invention has the advantages of strong specificity, sensitivity, quickness and easiness in observation, can obtain the same detection accuracy as that of the traditional plaque method, and can obviously shorten the detection time.
Description
The technical field is as follows:
the invention belongs to the technical field of biological detection, and particularly relates to varicella zoster virus titer detection and a method for quickly detecting varicella zoster virus titer at high flux by using a fluorescence immunoassay method.
Background art:
varicella-zoster virus (VZV), belonging to the genus human herpesvirus, human herpesvirus type 3. It was first isolated from the herpes solution of varicella patients in 1971 by the Japanese scholar Gaoqiao (Taka-hashiM). Subsequent studies show that VZV belongs to herpesvirus alphasubfamily, is a double-stranded DNA virus, consists of 124.9kb, has the diameter of 150-200 nm, contains 71 genomes, and encodes 67 proteins, but the virus has only one serotype and has neurotrophism. VZV has no animal reservoir host, and humans are the only natural host. The skin is the major target organ for the virus. In humans, initial infection with VZV causes chickenpox, and subsequently VZV becomes latent in the ganglia, and when the body's immunity declines, reactivation causes shingles.
VZV infection presents two distinct clinical symptoms. The primary infection typically occurs in children and is manifested as chickenpox, after which the virus enters the dorsal root ganglion and becomes latent, and in the event of immune decline or immunosuppression, the virus may be reactivated and manifest as herpes zoster (Zerboni, L. et al, 2014.NatRev Microbiol12(3): 197-210). Herpes zoster is often accompanied with postherpetic neuralgia, which is painful and can last for years, thus greatly affecting the life quality of patients. The incidence of herpes zoster and postherpetic neuralgia increases with age, which imposes a large social burden on the aging society.
The current leading preventive measure against varicella zoster virus infection is the injection of vaccines. Varicella live attenuated vaccines for varicella prevention, recombinant subunit herpes zoster vaccines and live attenuated herpes zoster vaccines for shingles prevention (Brisson, M.etal,2003.J Med Virol 70suppl 1: S31-37; Oxman, N.etal,2005.N Med (352): 2271-. Although both vaccines and systemic antivirals have brought about significant improvements, the disease persists. The identification and production of novel antibodies and antibody fragments that more effectively detect and block infection and reproduction of VZVs in populations is of particular importance for establishing improved treatments for the treatment and/or prevention of such infectious diseases.
For the vaccine industry, the detection of the vaccine titer of the varicella attenuated live vaccine is very important. The plaque method is a conventional method for measuring the titer of the varicella virus currently used in China, and the simple process is as follows: cells were first seeded in 6-well plates, typically 2 × 105-4x105Each well is inoculated with 3 ml; 37 ℃ and 5% CO2Culturing for 3 days under the condition; inoculating viruses; 37 ℃ and 5% CO2Culturing for 7 days under the condition; finally, staining with Coomassie brilliant blue, and manually counting the number of plaques in each well.
The titer detection method by the plaque method has the titration compound pore number of 2, and has the inaccuracy on the method caused by the insufficient compound pore number; the detection time of the whole process of the plaque method is 10 days, and the experimental result cannot be obtained in a short time; the plaque etching method requires an experimenter to count the plaques by naked eyes, and human errors are difficult to control. Therefore, a method for rapidly and accurately detecting the titer of the varicella-zoster virus is urgently needed.
The inventor applies the sensitivity and the specificity of a fluorescence immunoassay method to the detection of virus titer and develops a method for detecting the titer of varicella zoster virus. The fluorescence method is a method for combining specificity of antigen-antibody combination and high sensitivity of the fluorescence method and is applied to the field of virus titer detection. Firstly, 4-8 multiple holes can be set in the experiment, so that the accuracy of titer detection is obviously improved; secondly, the whole experimental process of the method takes 5 days, compared with the 10 days in the whole process of the plaque etching method, the experimental time is reduced by half; the method can automatically read and count the plate on the fluorescent enzyme-linked immunosorbent spot analyzer, realizes automation, reduces labor cost and reduces labor intensity and artificial errors of experiments; furthermore, the automatic plate reading and counting of the method meet the requirements of GMP data integrity. The method has the advantages of rapidness, specificity, accuracy, high flux, capability of automatic counting and the like, and is expected to become an alternative (substitute) method for detecting the titer of the varicella zoster virus. A comparison of the two methods is shown in the table below.
Comparison of plaque assay with fluorescence assay
The inventor mutates an existing antibody aiming at VZV envelope protein gE to obtain a mutated antibody in the process of researching a VZV virus-related antibody, and unexpectedly finds that the mutated antibody is particularly suitable for detecting the titer of the VZV virus, and particularly can obtain the accuracy equivalent to that of the traditional plaque method when being used for detecting by a fluorescence method, but the detection time is obviously shortened.
Accordingly, the present application provides a fluorescent detection method for detecting varicella-zoster virus titer; the method is rapid and accurate in detection and convenient for industrial application.
Disclosure of Invention
The invention provides a novel method for detecting varicella-zoster virus, which utilizes the characteristic of antigen-antibody specific binding and combines the advantage of high sensitivity of fluorescent signal detection, and the method is quicker and more accurate in detection. And the instrument reading plate can be used, so that the artificial judgment error is reduced.
Specifically, the invention relates to a fluorescence detection method for detecting varicella-zoster virus titer, which is characterized by comprising the following steps of:
(1) inoculating the diploid cells into a 96-well plate, and culturing in a carbon dioxide incubator;
(2) diluting a virus sample to be detected, inoculating the diluted virus sample to the 96-well plate, supplementing a maintenance liquid, and continuously culturing in a carbon dioxide incubator;
(3) taking out the 96-well plate cultured in the step (2), removing the culture solution, fixing the plate by using a fixing solution, and adding a primary antibody for incubation, wherein the primary antibody is a specific VZV monoclonal antibody;
(4) washing the 96-well plate incubated by the primary antibody in the step (3), and adding a fluorescence-labeled secondary antibody for continuous incubation, wherein the secondary antibody is an anti-species antibody;
(5) and (4) washing the 96-well plate incubated by the second antibody in the step (4), detecting by using a fluorescence quantitative detector, and calculating the virus titer of the sample.
The diploid cell is an optional diploid cell sensitive to varicella-zoster virus, and is preferably a human embryonic lung diploid cell 2BS cell strain, or an MRC-5 cell strain, or a KMB-17 cell strain; more preferably, the human embryonic lung diploid cell 2BS cell line is selected.
The viruses detected by the invention are varicella-zoster viruses, including attenuated varicella-zoster viruses and non-attenuated varicella-zoster viruses.
The specific VZV antibody is a polyclonal antibody and a monoclonal antibody aiming at VZV and glycoprotein E (VZV-gE), glycoprotein B (VZV-gB) and glycoprotein H (VZV-gH).
The secondary antibody is an anti-species antibody marked by fluorescein, and comprises an anti-mouse (mouse), an anti-mouse (rats), an anti-rabbit and an anti-human anti-species antibody.
The number of cell inoculation in the step (1) of the present invention is 2X105-5×105The culture time is 20-48 hours, the culture condition is 35-40 ℃, the carbon dioxide concentration is 4-6%.
The culture conditions in the step (2) of the invention are 35-40 ℃, the concentration of carbon dioxide is 4-6%, and the culture time is 48-96 hours.
Further, the optimization condition is that the inoculation number of the cells in the step (1) is 2 multiplied by 105-4×105The culture time is 24 hours, the culture condition is 37 ℃, the carbon dioxide concentration is 5 percent;
further, the conditions are optimized in the step (2) that the culture conditions are 37 ℃, the carbon dioxide concentration is 5% and the culture time is 72 hours.
Diluting in the step (2) of the invention by using a diluent, and inoculating 50 microliters of the diluent into each hole, wherein each dilution is used for inoculating 4-8 holes; the diluent is as follows: PBS + 50% sucrose + 10% sodium glutamate + 10% fetal bovine serum, or MEM + 2% newborn bovine serum + 1% glutamine + 2% NaHCO3。
The dilution in the step (2) of the present invention is a dilution containing 2 to 50PFU per 50. mu.l of virus.
Further, the optimization condition is that in the step (2), the virus quantity is 5-30PFU per 50 μ l.
The formula of the maintenance liquid in the step (2) of the invention is as follows: MEM + 5% newborn calf serum + 1% glutamine, 150. mu.l of maintenance medium was added to each well.
The fixing solution in the step (3) is 90% ethanol and 10% formaldehyde; the fixed solution is 100-300 microliter of the fixed solution per hole, and the fixed solution is placed at room temperature for 10-30 minutes.
Further, the optimization conditions are that the fixing solution in the step (3) is 90% ethanol and 10% formaldehyde; the fixing process is optimized by adding 100 microliter of fixing liquid into each hole, and the fixing condition is that the mixture is placed for 15 minutes at room temperature.
The primary antibody incubation conditions in step (3) of the invention are as follows: incubating at 37 deg.C or 25 deg.C or room temperature for 0.5-2 hr; or incubating for 18-24 hours at 2-8 ℃.
Further, the optimal conditions are that the incubation temperature is 37 ℃ and the incubation time is 1 h.
The secondary antibody incubation conditions in the step (4) of the invention are as follows: incubating at 37 deg.C, 25 deg.C or room temperature for 0.5-2 hr or 2-8 deg.C for 18-24 hr.
Further, the optimal conditions are that the incubation temperature is 37 ℃ and the incubation time is 0.5 h.
The washing liquid used in the washing process in the steps (3) and (4) is PBST washing liquid, and the formula of the washing liquid is as follows: PBS + 0.5-2 ‰ Twwen 20; the number of washing times was 2-5.
Further, the optimized condition is that the washing solution is PBS +1 ‰ Twwen20, and the washing times are 3 times.
The inventor of the present application found that although the fluorescence method is superior to the plaque method in many aspects, the detection result is still unstable, and found that the detection result is unstable due to factors such as unstable binding ability of the existing specific VZV antibody to the antigen, and the like, and it is presumed that the binding of the antibody to the antigen VZV in the cultured monolayer cells is affected by the cell environment, so that the binding is unstable, and the detection result is unstable. In contrast, the inventors of the present invention obtained the mutant anti-VZV antibody of the present invention by random mutation, which has improved antigen binding properties compared to the wild type, and in experiments, it was shown that the mutant anti-VZV antibody can be stably bound to VZV, and when used in the detection method of the present invention, the detection accuracy comparable to that of the conventional plaque method can be obtained, but the detection time can be significantly reduced, and a surprising technical effect can be obtained. The inventors of the present application selected an anti-VZV antibody disclosed in application No. CN201510884880.8 as a prototype (defined as a wild-type antibody) in the study, and performed mutation screening to obtain a human IgG-type antibody in which the constant region is a human IgG constant region. The light chain variable region sequence of the antibody is shown as SEQ ID No: 3, the variable region sequence of the antibody heavy chain is shown as SEQ ID No: 4, respectively.
Compared with the existing virus titer detection method, the method of the invention mainly has the following advantages:
(1) the detection speed is high, and compared with the traditional plaque method which needs 10 days, the detection method only needs 5 days.
(2) The detection flux is large, the plate can be read by a machine, the automation degree is high, and the labor intensity of manual judgment is obviously reduced;
(3) the method is simple and quick to operate, has high detection accuracy, and can obtain detection accuracy equivalent to that of the traditional plaque method.
(4) The specificity is strong.
Drawings
FIG. 1: the invention discloses a single-hole detection map of a 96-hole plate in a fluorescence detection method;
FIG. 2: in the fluorescence detection method, a fluorescence detector is used for detecting the detection image of the 96-well plate.
Detailed Description
The following examples further illustrate the present invention but should not be construed as limiting the invention and modifications or alterations to the methods, procedures and conditions of the present invention may be made without departing from the spirit and substance of the invention.
The most key technology for detecting the titer of the varicella-zoster virus by a fluorescence method is the selection of virus dilution times and fluorescence staining, and the accurate titer of the virus can be obtained only by selecting a proper virus dilution range and a correct fluorescence staining method.
1. Test materials
(1) Cell matrix: diploid cells such as MRC-5 cells, 2BS cells, KMB-17 cells, etc.;
(2) cell culture solution: MEM + 10% newborn bovine serum + 1% glutamine + 2% NaHCO3；
(3) Virus diluent: MEM + 2% newborn bovine serum + 1% glutamine + 2% NaHCO3Or PBS + 50% sucrose + 10% sodium glutamate +10 fetal bovine serum;
(4) virus maintenance liquid: MEM + 5% newborn bovine serum + 1% glutamine;
(5) fixing liquid: 90% ethanol + 10% formaldehyde;
(6) anti-varicella-zoster virus antibody
(7) Fluorescence labeling secondary antibody (such as goat anti-mouse FITC-IgG)
(8) Washing liquid: PBS + 1% Tween20
2. Fluorescence detection method
First step cell culture: diploid cells (MRC-5 cells or 2BS cells or KMB-17 cells) were counted at 2.0-2.5X 1050.1ml of growth medium MEM containing 10% newborn calf serum, 1% glutamine, and NaHCO was inoculated into 96-well plate per ml3Adjusting the pH value to about 7.2, and culturing at 37 ℃ in a 5% carbon dioxide incubator for 24 hours.
The second step is to inoculate the virus sample: diluting a virus sample to be detected to 5-30PFU/50 mu l by using a diluent, inoculating the diluted virus sample to the monolayer cells of the 96-well plate in the step one, wherein each hole is 50 mu l, each diluted sample is inoculated to 4-8 holes, each hole is supplemented with 150 mu l of maintenance solution, and the diluted virus sample is cultured in a 5% carbon dioxide incubator at 37 ℃ for 72 hours.
Thirdly, cell fixation: the old solution of 96-well plate is discarded, 200. mu.l of cell fixative is added to each well, the solution is fixed for 15 minutes at room temperature, the fixative is discarded, and the plate is washed 3 times with PBST.
Fourth step dyeing: adding 100 mu l of the anti-poxvirus antibody with proper concentration into each well, incubating for 1 hour at 37 ℃ and 5% carbon dioxide, washing the plate for 3 times by PBST, adding 100 mu l of fluorescence labeled antibody (secondary antibody) with proper concentration into each well, incubating for 40 minutes at 37 ℃ and 5% carbon dioxide, and washing the plate for 3 times by PBST.
And fifthly, reading the plate: and (4) observing the number of the plaques in each hole under a fluorescence microscope, or detecting the result by using a fluorescence counter to calculate the virus titer. The results are shown in FIGS. 1 and 2.
As can be seen from FIG. 1, the detection result of the fluorescence detection method of the present invention is visually clear and distinguishable, and the influence of human misjudgment factors can be effectively reduced. In fig. 2, the detection result can be automatically detected quickly and accurately by combining with a fluorescence detection instrument, so that misjudgment is remarkably reduced.
3. Comparison experiment of fluorescence detection method and traditional plaque etching method
4 groups of detection comparison experiments are set, each group is respectively detected by the fluorescence detection method and the traditional plaque method, the fluorescence detection method respectively selects the wild type anti-VZV antibody and the mutant type anti-VZV antibody to carry out detection, and the detection results are shown in Table 1.
Table 1: comparison of fluorescence detection method with traditional plaque method
As can be seen from the results in Table 1, the fluorescence detection method of the present invention can effectively reduce the detection time (only 5 days), which is much faster than the detection time of 10 days in the conventional plaque method, and significantly improves the detection efficiency; and the comparison result shows that the mutant antibody of the invention can obtain the experimental result equivalent to the traditional plaque method; the detection result of the wild type antibody group obviously has a certain problem of insufficient stability, and the result fluctuation is obvious.
4. The invention discloses a method for evaluating the repeatability of detection results of a fluorescence detection method
The samples from each set were evaluated in a multi-well assay (3 wells) including comparison of wild type versus mutant types, and the results are shown in the table.
Table 2 (1): results of repeated titer determination of samples by fluorescence method (mutant type): (Limitable range 0.25log PFU/ml)
| Sample (I) | Sample 1 | Sample 2 | Sample 3 | Sample No. 4 |
| Hole 1 | 4.36 | 4.46 | 3.74 | 4.15 |
| Hole 2 | 4.28 | 4.51 | 3.90 | 4.09 |
| Hole 3 | 4.33 | 4.39 | 3.82 | 4.18 |
Table 2 (2): results of fluorescent sample replicate titer determination (wild type): (Limitable range 0.25log PFU/ml)
| Sample (I) | Sample 1 | Sample 2 | Sample 3 | Sample No. 4 |
| Hole 1 | 4.38 | 4.21 | 3.22 | 3.78 |
| Hole 2 | 4.01 | 4.58 | 3.76 | 4.22 |
| Hole 3 | 3.89 | 4.08 | 3.46 | 4.01 |
As can be seen from the results in Table 2(1), the results of multiple detections of the same sample by the antibody and the fluorescence detection method are very similar, and the error is small.
As is clear from the results of table 2(2), although the detection result of the wild-type antibody can detect the virus titer of the sample well, the detection result fluctuates greatly, and a certain error is caused in the detection result, and it is assumed that the wild-type antibody is affected by the detection environment and the like, and the binding stability thereof is not sufficient.
That is to say, the antibody and the detection method of the invention can obviously shorten the VZV virus titer detection time, and the detection result is accurate and reliable and the operation is simple.
Sequence listing
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Claims (9)
1. A fluorescence detection method for detecting varicella-zoster virus titer, which is characterized by comprising the following steps:
(1) inoculating the diploid cells into a 96-well plate, and culturing in a carbon dioxide incubator;
(2) diluting a virus sample to be detected, inoculating the diluted virus sample to the 96-well plate, supplementing a maintenance liquid, and continuously culturing in a carbon dioxide incubator;
(3) taking out the 96-well plate cultured in the step (2), removing the culture solution, fixing the plate by using a fixing solution, and adding a primary antibody for incubation, wherein the primary antibody is a specific VZV monoclonal antibody;
(4) washing the 96-well plate incubated by the primary antibody in the step (3), and adding a fluorescence-labeled secondary antibody for continuous incubation, wherein the secondary antibody is an anti-species antibody;
(5) washing the 96-well plate incubated by the second antibody in the step (4), detecting by using a fluorescence quantitative detector, and calculating the virus titer of the sample;
wherein, the specificity VZV monoclonal antibody is human IgG monoclonal antibody, and the variable region sequence of the monoclonal antibody light chain is shown in SEQ ID No: 3, the sequence of the single-antibody heavy chain variable region is shown as SEQ ID No: 4, respectively.
2. The fluorescence detection method for detecting the titer of varicella-zoster virus according to claim 1, wherein the diploid cells are diploid cells sensitive to varicella-zoster virus and are human embryonic lung diploid cell 2BS cell strains, MRC-5 cell strains or KMB-17 cell strains;
the secondary antibody is an anti-species antibody marked by fluorescein, and comprises an anti-human anti-species antibody.
3. The fluorescence detection method for detecting varicella-zoster virus titer according to any one of claims 1-2, wherein the number of inoculated cells in step (1) is 2x105-5×105The culture time is 20-48 hours, the culture condition is 35-40 ℃, the carbon dioxide concentration is 4-6%; the culture conditions in the step (2) are 35-40 ℃ and 4-6% of carbon dioxide concentration, and the culture is carried out for 60-84 hours.
4. The fluorescence detection method for detecting varicella-zoster virus titer according to claim 3, wherein the inoculation number of the cells in the step (1) is 2x105-4×105The culture time is 24 hours, the culture condition is 37 ℃, the carbon dioxide concentration is 5 percent;
the culture conditions in the step (2) are 37 ℃ and 5% of carbon dioxide concentration, and the culture is carried out for 72 hours.
5. The fluorescence detection method for detecting varicella-zoster virus titer according to claim 4, wherein the dilution in step (2) is dilution with a diluent, 50 μ l of the diluted solution is inoculated to each well, and 4-8 wells are inoculated to each dilution; the diluent is as follows: PBS + 50% sucrose + 10% sodium glutamate + 10% fetal bovine serum, or MEM + 2% newborn bovine serum + 1% glutamine + 2% NaHCO3。
6. The fluorescence detection method for detecting varicella-zoster virus titer according to claim 5, characterized in that the virus count is 2-50PFU per 50 μ l.
7. The fluorescence detection method for detecting varicella-zoster virus titer according to claim 1, wherein the maintenance fluid in the step (2) is prepared from the following formula: MEM + 5% newborn calf serum + 1% glutamine, 150 microliters of maintenance liquid is added to each well;
the fixing solution in the step (3) is 90% ethanol and 10% formaldehyde; the fixation was performed by adding 100-300. mu.l of the fixative per well and standing at room temperature for 10-30 minutes.
8. The fluorescence detection method for detecting varicella-zoster virus titer according to claim 7, wherein the fixing solution is 90% ethanol + 10% formaldehyde; the fixation was performed by adding 100. mu.l of fixative to each well and allowing to stand at room temperature for 15 minutes.
9. The fluorescence detection method for detecting varicella-zoster virus titer according to claim 1, wherein the incubation conditions in steps (3) and (4) are as follows: incubating at 37 deg.C or room temperature for 0.5-2 hr; or incubating for 18-24 hours at 2-8 ℃;
the washing solution used for washing in the steps (4) and (5) is PBST washing solution, and the formula of the washing solution is as follows: PBS + 0.5-2% Tween20, and washing for 2-5 times.
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