Antigen plate for detecting varicella-zoster virus antibody as well as preparation method and application of antigen plate

Abstract

The invention relates to the technical field of biological detection, and particularly discloses an antigen plate for detecting a varicella-zoster virus antibody as well as a preparation method and application of the antigen plate. The preparation method of the antigen plate for detecting the chicken pox-herpes zoster virus antibody specifically comprises the following steps in sequence: infecting MRC-5 cells by using a chicken pox-herpes zoster virus Oka strain, inoculating the MRC-5 cells into a cell culture plate in a mixed planting manner, culturing for 60-84 hours, fixing by using a fixing solution, protecting by using a protecting solution, washing and drying to obtain the antigen plate for detecting the chicken pox-herpes zoster virus antibody. When the antigen plate disclosed by the invention is used for detecting the chicken pox-herpes zoster virus antibody, relatively high sensitivity and repeatability are achieved, and the preservation stability of the antigen plate is relatively good.

Description

Technical Field

[0001] The present application relates to the technical field of biological detection, and in particular to an antigen plate for detecting varicella-zoster virus antibodies and a preparation method and application thereof. Background Art

[0002] Currently, the commonly used methods for detecting varicella-zoster virus antibodies include FAMA method, ELISA method, neutralization test and lactoball assay. The ELISA method is convenient and fast and suitable for large-scale sample testing, but commercial ELISA kits may have false positive or false negative results. They are unreliable when evaluating serum after varicella vaccination and cannot well reflect the protectiveness of antibodies. The neutralization test has low sensitivity, long cycle, and complex operation, and is not suitable for high-throughput detection. The lactoball assay has room for improvement and needs further verification. The FAMA method is currently the internationally recognized gold standard for varicella-zoster virus antibody detection. It has the characteristics of high sensitivity and strong specificity. Its principle is: cells infected with varicella virus are used as antigens, serum to be tested is used as primary antibody, and the corresponding fluorescently labeled IgG is used as secondary antibody. The antigen-antibody complex formed is mainly located on the surface of infected cells, and a unique ring-shaped membrane fluorescence can be seen under a fluorescence microscope.

[0003] In the traditional FAMA method, cells infected with varicella virus need to be digested with trypsin, prepared into a cell suspension and then added to a glass slide for the preparation of an antigen plate. This operation process is difficult to control and will inevitably result in uneven cell dispersion, which will affect the determination of the results. In addition, FAMA testing requires experienced technicians to determine the results under a fluorescence microscope, which not only involves subjective judgment, but also limits the high throughput of the test. Currently, there is an urgent need for a method that can quickly and accurately detect the titer of varicella-zoster virus antibodies. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides an antigen plate for detecting varicella-zoster virus antibodies and a preparation method and application thereof.

[0005] The present application provides a method for preparing an antigen plate for detecting varicella-zoster virus antibodies, which specifically comprises the following steps in sequence: The MRC-5 cells were infected with the varicella-zoster virus Oka strain, seeded into a cell culture plate in a mixed manner, cultured for 60 to 84 hours, fixed with a fixative, protected with a protective solution, washed and dried to obtain the obtained cells.

[0006] Preferably, the parameter conditions for the virus Oka strain to infect MRC-5 cells are: the MRC-5 cell density is 1.5×10 5 / ml~2.5×10 5 / ml; virus concentration is 10000PFU / ml~20000PFU / ml.

[0007] In a specific embodiment, the MRC-5 cell density is 1.5×10 5 / ml, 2.0×10 5 / ml, 2.5×10 5 / ml; virus concentrations are 10000PFU / ml, 15000PFU / ml, and 20000PFU / ml.

[0008] In some specific embodiments, the MRC-5 cell density is 1.5×10 5 / ml~2.0×10 5 / ml, 2.0×10 5 / ml~2.5×10 5 / ml; virus concentration is 10000PFU / ml~15000PFU / ml, 15000PFU / ml~20000PFU / ml.

[0009] Preferably, the fixative conditions are: fixation at 2-8°C for 25-35 min, or fixation at -25--15°C for 15-25 min; the fixative consists of components with the following concentrations: cold acetone 75-85 v / v%, arginine 2-8 w / v%, and the dissolving liquid used is water.

[0010] In a specific embodiment, the fixative conditions are: fixation at 2°C for 25 min, fixation at 4°C for 25 min, fixation at 8°C for 25 min, fixation at 2°C for 30 min, fixation at 4°C for 30 min, fixation at 8°C for 30 min, fixation at 2°C for 35 min, fixation at 4°C for 35 min, fixation at 8°C for 35 min, fixation at -25°C for 15 min, fixation at -20°C for 15 min, fixation at -15°C for 15 min, fixation at -25°C for 20 min, fixation at -20°C for 20 min, fixation at -15°C for 20 min, fixation at -25°C for 25 min, fixation at -20°C for 25 min, and fixation at -15°C for 25 min.

[0011] In a specific embodiment, in the fixing solution, the concentration (v / v) of the cold acetone can be: 75%, 80%, 85%; the concentration (w / v) of the arginine can be: 2%, 5%, 8%, and the dissolving solution used is water.

[0012] Preferably, the protection conditions of the protective solution are: protection at 6-14°C for 90-150 min; the protective solution is composed of the following concentration components: 1.5-2.5 v / v% glycerol, 0.5-1.5 w / v% carbohydrates, 0.05-0.15 w / v% oils and fats), 0.02-0.12 w / v% aluminum glycolate, and the dissolving solution used is PBS buffer with a pH of 7.4.

[0013] After experimental analysis, the applicant found that the protective liquid composed of the above-mentioned raw material components can further improve the comprehensive performance of the antigen plate by protecting the fixed antigen plate. Glycerol, sugar substances, oils and aluminum glycolate in the protective liquid are used as protective agents, each of which has a unique role, and at the same time they cooperate with each other to provide comprehensive protection for the antigen plate. Among them, glycerol has good hygroscopicity and moisture retention, which can effectively prevent the antigen plate from losing water in a dry environment and causing antigen inactivation; glycerol can also enhance the stability of the antigen plate and extend its service life. Sugar substances mainly play the role of stabilizing and protecting antigens; by combining with antigens, sugar substances can form a protective film to reduce the damage of external factors to antigens; in addition, sugar substances can also provide energy to support the physiological activities of antigens during storage. Oils and fats are mainly used as lubricants and sealants in the antigen plate protective agent, which can reduce the friction between the antigen plates and prevent scratches or damage during transportation and use; at the same time, oils and fats can also effectively seal the micropores on the antigen plate to prevent the invasion of harmful substances such as moisture and oxygen, thereby protecting the integrity and activity of the antigen. Aluminum glyoxylate has excellent antioxidant and preservative properties; it can remove free radicals and harmful substances generated during the storage of antigen plates, preventing the inactivation of antigens due to oxidation; at the same time, aluminum glyoxylate can also inhibit the growth and reproduction of microorganisms, ensuring the long-term sterile storage of antigen plates.

[0014] In a specific embodiment, the protection conditions of the protective solution can be: 6°C protection for 90 min, 6°C protection for 120 min, 6°C protection for 150 min, 10°C protection for 90 min, 10°C protection for 120 min, 10°C protection for 150 min, 14°C protection for 90 min, 14°C protection for 120 min, and 14°C protection for 150 min.

[0015] Furthermore, the protective solution is composed of the following components in concentrations: 1.7-2.3 v / v% glycerol, 0.7-1.3 w / v% sugars, 0.08-0.12 w / v% oils and fats, and 0.04-0.1 w / v% aluminum glycolate.

[0016] In a specific embodiment, in the protective solution, the concentration of the glycerol (v / v) can be 1.5%, 1.7%, 2.0%, 2.3%, or 2.5%; the concentration of the carbohydrate (w / v) can be 0.5%, 0.7%, 1%, 1.3%, or 1.5%; the concentration of the oil (w / v) can be 0.05%, 0.08%, 0.1%, 0.12%, or 0.15%; the concentration of aluminum glycolate (w / v) can be 0.02%, 0.04%, 0.07%, 0.1%, or 0.12%.

[0017] Preferably, the oil is selected from one or more of caprylic / capric triglyceride, diisostearyl malate, polyglyceryl-2 triisostearate, phytosteryl oleate, ethylhexyl methoxycinnamate, petrolatum, lecithin, hydrogenated lecithin, oleyl alcohol, triglyceride, ethylhexyl palmitate, avocado esters, hexyl laurate, dicaprylyl carbonate, propyl heptyl ester, sesame oil, tea seed oil, shark liver oil, almond oil, and olive oil.

[0018] Preferably, the oil is composed of a mixture of polyglyceryl-2 triisostearate and hydrogenated lecithin in a weight ratio of 0.5-1.5:0.5-1.5.

[0019] Preferably, the sugar substance is selected from one or more of polysucrose, trehalose, dextran and maltose.

[0020] In a second aspect, the present application provides an antigen plate for detecting varicella-zoster virus antibodies, which is prepared using the above-mentioned preparation method.

[0021] In a third aspect, the present application provides a method for detecting varicella-zoster virus antibodies, using the above-mentioned antigen plate for detection; specifically comprising the following steps in sequence: Antibody detection: Add the serially diluted serum to be tested to the antigen plate, 20-40 μl / well, 3-6 replicate wells for each dilution; incubate at 37°C for 50-70 min, rinse with PBS for 3 times, 180-220 μl / well, and air-dry; add fluorescent secondary antibody to the detection plate, 20-40 μl / well, and incubate at 37°C for 50-70 min; after the incubation, rinse with PBS for 3 times, 180-220 μl / well, spin dry, and place on an enzyme reader to detect fluorescence; then determine the result.

[0022] Preferably, the specific steps of the result determination are: taking the average value of the fluorescence intensity of the negative control wells + 3SD as the CUT OFF value, if the fluorescence intensity of the test well is greater than the CUT OFF value, it is determined to be positive, otherwise it is negative; the highest dilution at which a positive result occurs is taken as the determination endpoint.

[0023] In summary, the technical solution of this application has the following effects: The present application adopts the Oka strain of varicella-zoster virus as the infectious strain, and selects MRC-5 cells as the cell base to prepare the antigen plate. Compared with Vero cells, the antigen plate applied to the detection method of varicella-zoster virus antibodies has higher sensitivity and repeatability; compared with the group without adding protective liquid, the storage stability of the antigen plate is better.

[0024] The antigen plate preparation method provided in the present application simplifies the preparation steps, omits the preparation process of the antigen sheet in the traditional method, directly inoculates the MRC-5 cells and the varicella virus into a 96-well culture plate in a mixed manner for culture, and is fixed with a fixing solution, protected with a protective solution, and dried to make an antigen plate, which can be directly used for the detection of varicella antibodies and can be stored at -20°C or 2-8°C for 1 year.

[0025] The method for detecting varicella-zoster virus antibodies provided in the present application sets up duplicate wells in the experiment, which significantly improves the accuracy of antibody titer detection; secondly, the method uses an ELISA instrument to read the fluorescence intensity signal, realizes automation, reduces labor intensity and experimental human errors, and meets the requirements of GMP data integrity; the method has the advantages of rapidity, strong specificity, accuracy, high throughput, and automation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are microscopic morphology images of MRC-5 cells during culture in Example 1 (a - culture day 1; b - culture day 2; c - culture day 3).

[0027] Figure 2 These are microscopic morphology images of MRC-5 cells during culture in Example 2 (a - culture day 1; b - culture day 2; c - culture day 3).

[0028] Figure 3 These are microscopic morphology images of MRC-5 cells during culture in Example 3 (a - culture day 1; b - culture day 2; c - culture day 3). DETAILED DESCRIPTION

[0029] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in the present application. Example

[0030] Example 1 Example 1 provides an antigen plate for detecting varicella-zoster virus antibodies.

[0031] The specific steps of the method for preparing the antigen plate for varicella-zoster virus antibody detection in this embodiment are as follows: Virus solution dilution: Dilute varicella-zoster virus to 15,000 PFU / ml with 6% bovine serum L-15 culture medium and set aside.

[0032] Preparation of MRC-5 cell suspension: Take MRC-5 cells with good morphology, discard the liquid in the culture bottle, add trypsin digestion solution, 10 ml / bottle, wash twice, and digest to a density of 2.0×10 5 Cell suspension of 100 μl / ml was added to a 96-well plate, 200 μl / well, and cultured at 37°C and 5% carbon dioxide for 3 days. During this period, the plate was observed under a microscope every day to select the appropriate cell seeding density. The morphology after microscopic observation is shown in the figure below. Figure 1 As shown (a-culture day 1; b-culture day 2; c-culture day 3), it can be observed that the cells are fully grown and in good growth condition.

[0033] The prepared MRC-5 cell suspension was added to a 96-well plate, 200 μl per well; the diluted virus suspension was added to a 96-well plate, 50 μl per well, and cultured at 37°C and 5% carbon dioxide for 72 h.

[0034] Fixation with fixative solution: After the incubation, discard the liquid in the 96-well plate, add fixative solution, 50 μl / well, and fix at 4°C for 30 minutes; after fixation, discard the fixative solution and wait for evaporation and drying; the preparation method of the fixative solution is: prepare it by mixing 80 ml of cold acetone, 5 g of arginine, and 20 ml of water; Protection with protection solution: add protection solution, 50μl / well, and protect at 10℃ for 120min; discard the protection solution, then wash once with PBS buffer solution at pH=7.4, and wait for evaporation and drying; the preparation method of protection solution is as follows: take 20ml of glycerol, 10g of polysucrose, 1g of oil (composed of a mixture of polyglycerol-2 triisostearate and hydrogenated lecithin in a weight ratio of 1.5:1), 0.7g of aluminum glycolate, make up to 1000ml with PBS buffer solution (pH 7.4), mix and set aside.

[0035] Storage: Seal with sealing film, mark and store at 2-8℃ for later use.

[0036] Embodiments 2 to 5 Examples 2 to 5 each provide an antigen plate for detecting varicella-zoster virus antibodies.

[0037] The difference between the above embodiment and embodiment 1 is that the parameter conditions for the virus Oka strain to infect MRC-5 cells are different, as shown below.

[0038] In Example 2: the MRC-5 cell density is 1.5×10 5 / ml; the morphology after microscopic observation is as follows Figure 2As shown (a-culture day 1; b-culture day 2; c-culture day 3), it can be observed that the cells are fully grown and in good growth condition.

[0039] In Example 3: the MRC-5 cell density is 2.5×10 5 / ml; the morphology after microscopic observation is as follows Figure 3 As shown (a-1st day of culture; b-2nd day of culture; c-3rd day of culture), it can be observed that on the 2nd day of culture, the cells began to aggregate and fall off.

[0040] In Example 4: the virus concentration is 10000 PFU / ml.

[0041] In Example 5: the virus concentration is 20000 PFU / ml.

[0042] The remaining parameters and conditions in the above embodiment are the same as those in embodiment 1.

[0043] Embodiments 6 to 10 Examples 6 to 10 respectively provide an antigen plate for detecting varicella-zoster virus antibodies.

[0044] The difference between the above embodiment and embodiment 1 is that the parameter conditions of the fixation liquid are different, as shown below.

[0045] In Example 6: the fixing liquid is cold acetone.

[0046] In Example 7: The preparation method of the stationary phase is as follows: take 80 ml of cold acetone and 5 g of glycine, and dilute to 100 ml with water.

[0047] In Example 8: The preparation method of the stationary phase is as follows: take 70 ml of cold acetone and 10 g of arginine, and dilute to 100 ml with water.

[0048] In Example 9: The preparation method of the stationary phase is as follows: take 75 ml of cold acetone and 8 g of arginine, and dilute to 100 ml with water.

[0049] In Example 10: The preparation method of the stationary phase is as follows: 85 ml of cold acetone and 2 g of arginine are taken and the volume is made up to 100 ml with water.

[0050] The remaining parameters and conditions in the above embodiment are the same as those in embodiment 1.

[0051] Examples 11 to 18 Examples 11 to 18 each provide an antigen plate for detecting varicella-zoster virus antibodies.

[0052] The difference between the above embodiment and embodiment 1 is that the parameter conditions of the protection liquid are different, as shown below.

[0053] In Example 11: The preparation method of the protective solution is as follows: take 20 ml of glycerol and 10 g of polysucrose, dilute to 1000 ml with PBS buffer (pH 7.4), mix well and set aside.

[0054] In Example 12: The preparation method of the protective solution is as follows: take 30 ml of glycerol, 2 g of polysucrose, 0.3 g of oil (composed of a mixture of polyglycerol-2 triisostearate and hydrogenated lecithin in a weight ratio of 1.5:1), and 1.5 g of aluminum glycolate, dilute to 1000 ml with PBS buffer (pH 7.4), mix well and set aside.

[0055] In Example 13: The preparation method of the protective solution is as follows: 20 ml of glycerol, 10 g of polysucrose, 1 g of oil (composed of a mixture of polyglycerol-2 triisostearate and phytosteryl oleate in a weight ratio of 1.5:1), and 0.7 g of aluminum glycolate, and the mixture is fixed to 1000 ml with PBS buffer (pH 7.4), mixed and set aside.

[0056] In Example 14: The preparation method of the protective solution is as follows: take 20 ml of glycerol, 10 g of polysucrose, 1 g of oil (composed of a mixture of hexyl laurate and hydrogenated lecithin in a weight ratio of 1.5:1), and 0.7 g of aluminum glycolate, dilute to 1000 ml with PBS buffer (pH 7.4), mix well and set aside.

[0057] In Example 15: The preparation method of the protective solution is as follows: take 20 ml of glycerol, 10 g of polysucrose, 1 g of oil (composed of a mixture of polyglycerol-2 triisostearate and hydrogenated lecithin in a weight ratio of 0.2:2), and 0.7 g of aluminum glycolate, dilute to 1000 ml with PBS buffer (pH 7.4), mix well and set aside.

[0058] In Example 16: The preparation method of the protective solution is as follows: take 20 ml of glycerol, 10 g of polysucrose, 1 g of oil (composed of a mixture of polyglycerol-2 triisostearate and hydrogenated lecithin in a weight ratio of 2:0.3), and 0.7 g of aluminum glycolate, dilute to 1000 ml with PBS buffer (pH 7.4), mix well and set aside.

[0059] In Example 17: The preparation method of the protective solution is as follows: take 20 ml of glycerol, 10 g of polysucrose, 1 g of oil (composed of a mixture of polyglycerol-2 triisostearate and hydrogenated lecithin in a weight ratio of 0.5:1.5), and 0.7 g of aluminum glycolate, dilute to 1000 ml with PBS buffer (pH 7.4), mix well and set aside.

[0060] In Example 18: The preparation method of the protective solution is as follows: take 20 ml of glycerol, 10 g of polysucrose, 1 g of oil (composed of a mixture of polyglycerol-2 triisostearate and hydrogenated lecithin in a weight ratio of 1.5:0.5), and 0.7 g of aluminum glycolate, make up to 1000 ml with PBS buffer (pH 7.4), mix well and set aside.

[0061] The remaining parameters and conditions in the above embodiment are the same as those in embodiment 1.

[0062] Comparative Example Comparative Example 1 This comparative example provides an antigen plate for detecting varicella-zoster virus antibodies.

[0063] In the preparation method of the antigen plate for varicella-zoster virus antibody detection in this comparative example, Vero cells are used instead of MRC-5 cells.

[0064] The remaining parameters in this comparative example are the same as those in Example 1.

[0065] Comparative Example 2 This comparative example provides an antigen plate for detecting varicella-zoster virus antibodies.

[0066] In the preparation method of the antigen plate for varicella-zoster virus antibody detection in this comparative example, no protective liquid is added for protection.

[0067] The remaining parameters in this comparative example are the same as those in Example 1.

[0068] Performance testing Add the doubly diluted serum to be tested to the antigen plate prepared in the embodiment or comparative example, 96-well plate, 30μl / well, 4 wells for each sample. Place the test plate in a 37°C incubator and incubate for 60 minutes to fully neutralize the virus and serum. After the incubation is completed, wash it 3 times with PBS, 200μl / well, 5 minutes each time, and dry it. Dilute the FITC-labeled IgG 200 times with PBS (pH7.4) to obtain the working concentration of secondary antibody, add it to the test plate, 30μl / well, and incubate it in a 37°C incubator for 60 minutes. After the incubation is completed, wash it 3 times with PBS, 200μl / well, 5 minutes each time, dry it, and read the fluorescence on the microplate reader (excitation wavelength 485, emission wavelength 535); then determine the result.

[0069] The specific steps for determining the results are as follows: the mean value of the fluorescence intensity of the negative control wells + 3SD is taken as the CUT OFF value. If the fluorescence intensity of the test well is greater than the CUT OFF value, it is determined to be positive, otherwise it is negative; all 4 wells are positive, indicated by "++++"; 3 wells are positive, indicated by "+++"; 2 wells are positive, indicated by "++"; 1 well is positive, indicated by "+"; "++" is the end point, that is, the antibody titer unit, as shown in Table 1 for the calculation method of antibody titer. The highest dilution with a positive result is taken as the determination end point.

[0070] Table 1 Antibody titer calculation method result Antibody titer calculation method ++++ 1: (highest dilution multiple of positive × 3 / 2) +++ 1: (highest dilution multiple of positive × 5 / 4) ++ 1: The highest dilution multiple of positive + 1: (highest dilution multiple of positive × 7 / 8) (1) Specificity experiment The antigen plate freshly prepared in Example 1 was used, and mouse post-immune serum (test group), mouse pre-immune negative serum (negative control group), and rat post-immune serum (interference serum control group) were used as test samples, and detected with FITC-goat anti-mouse IgG. At the same time, normal MRC-5 cells were used as controls (cell control group).

[0071] Acceptable criteria: The result of mouse post-immunization serum (test group) should be positive; the result of mouse pre-immunization negative serum (negative control group) should be negative; the result of rat post-immunization serum (interference serum control group) should be negative; the result of cell control group should be negative.

[0072] The results showed that the test group was positive, the negative control group was negative, the interference serum control group was negative, and the interference serum had no interference with the mouse serum test, and the cell control group was negative. The test results met the acceptable standards, indicating that the method has good specificity.

[0073] (2) Sensitivity test The antigen plates freshly prepared in the embodiments and comparative examples were used to measure the positive reference serum to be tested (mouse immunization serum) in multiple dilution ratios, and the gradient dilutions were performed at 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1024 times, respectively. The positive or negative results of the samples to be tested were determined, and the minimum detection limit was detected to examine the sensitivity.

[0074] Test results: as shown in Table 2.

[0075] Table 2 Sensitivity test results in the embodiments and comparative examples By analyzing the test results in Table 2, it can be seen that when the gradient diluted positive reference serum to be tested is tested using the antigen plate provided by the present application, the antibody titer of the serum is 1:224-1:384; in contrast, when the gradient diluted serum to be tested is tested using the antigen plate of Comparative Example 1, the antibody titer of the serum is 1:48.

[0076] The above test results show that the antigen plate provided in the present application has a high sensitivity when used to detect varicella-zoster virus antibodies.

[0077] (3) Repeatability experiment The freshly prepared antigen plates of the embodiment and comparative example 2 were used to measure the mouse immunization serum, and the fluorescence values ​​of 1:16, 1:64, and 1:256 of each embodiment were counted, and the RSD of the fluorescence values ​​was calculated. The results are shown in Table 3. The RSD of the antibody titer in each embodiment and comparative example 2 and the ratio of the maximum value to the minimum value were counted, and the results are shown in Table 4.

[0078] Table 3 Repeatability test results of the embodiment - fluorescence value Table 4 Repeatability test results of the embodiments and comparative examples - antibody titer By analyzing the test results in Table 3, it can be seen that when the gradient diluted post-immunization serum is tested using the antigen plate provided by the present application, the RSD of the fluorescence value result is small. By the test results in Table 4, it can be seen that when the post-immunization serum is tested using the antigen plate provided by the present application, the RSD of the antibody titer is less than 30%, and the ratio of the maximum value to the minimum value is less than 2.

[0079] The above test results show that when the antigen plate provided in the present application is used to detect varicella-zoster virus antibodies, the detection method has good repeatability and stability.

[0080] (4) Storage stability of antigen plate The antigen plates prepared in Example 1 and Comparative Example 2 (storage conditions were -20°C for 6 months, -20°C for 12 months, 2-8°C for 6 months, and 2-8°C for 12 months) were used to measure the mouse immunization serum, 6 times in parallel, and the average antibody titer was calculated; at the same time, the freshly prepared antigen plate was used as the control group to calculate the storage stability of the antigen plate; the calculation formula was: antigen plate storage stability = average antibody titer result after storage for a period of time / average antibody titer result of freshly prepared × 100%; the closer the stability result is to 100%, the better the storage stability of the antigen plate; the results are shown in Table 5.

[0081] Table 5 Stability test results of antigen plates in Examples and Comparative Examples By analyzing the test results in Table 5, it can be seen that after the antigen plate provided by the present application is stored under different conditions, the stability of the antibody titer results when testing serum is good. The above test results show that the antigen plate prepared using the technical solution provided by the present application has good storage stability.

[0082] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing an antigen plate for detecting varicella-zoster virus antibodies, characterized in that: Specifically, the following steps are performed in sequence: MRC-5 cells were infected with the varicella-zoster virus Oka strain, seeded into a cell culture plate in a mixed manner, cultured for 60-84 hours, fixed with a fixative, protected with a protective solution, washed and dried to obtain the obtained cells.

2. The method for preparing the antigen plate for varicella-zoster virus antibody detection according to claim 1, characterized in that: The parameter conditions for the virus Oka strain to infect MRC-5 cells are: the MRC-5 cell density is 1.5×10 5 / ml~2.5×10 5 / ml; virus concentration is 10000PFU / ml~20000PFU / ml.

3. The method for preparing the antigen plate for varicella-zoster virus antibody detection according to claim 1, characterized in that: The fixation conditions of the fixative are: fixation at 2-8°C for 25-35 min, or fixation at -25--15°C for 15-25 min; the fixative is composed of components with the following concentrations: cold acetone 75-85 v / v%, arginine 2-8 w / v%, and the solvent used is water.

4. The method for preparing the antigen plate for varicella-zoster virus antibody detection according to claim 1, characterized in that: The protection conditions of the protection solution are: protection at 6~14℃ for 90~150min; the protection solution is composed of the following components in the following concentrations: 1.5~2.5v / v% glycerol, 0.5~1.5w / v% sugar, 0.05~0.15w / v oil, 0.02~0.12w / v% aluminum glycolate, and the dissolving solution used is PBS buffer with a pH of 7.

4.

5. The method for preparing the antigen plate for varicella-zoster virus antibody detection according to claim 4, characterized in that: The oil is selected from one or more of caprylic / capric triglyceride, diisostearyl malate, polyglyceryl-2 triisostearate, phytosteryl oleate, ethylhexyl methoxycinnamate, petrolatum, lecithin, hydrogenated lecithin, oleyl alcohol, triglyceride, ethylhexyl palmitate, avocado esters, hexyl laurate, dicaprylyl carbonate, propyl heptyl ester, sesame oil, tea seed oil, shark liver oil, almond oil, and olive oil.

6. The method for preparing the antigen plate for detecting varicella-zoster virus antibodies according to claim 5, characterized in that: The oil is composed of a mixture of polyglycerol-2 triisostearate and hydrogenated lecithin in a weight ratio of 0.5-1.5:0.5-1.

5.

7. The method for preparing the antigen plate for detecting varicella-zoster virus antibodies according to claim 4, characterized in that: The sugar substance is selected from one or more of polysucrose, trehalose, dextran and maltose.

8. An antigen plate for detecting varicella-zoster virus antibodies, characterized in that: The method is prepared by the method according to any one of claims 1 to 7.

9. A method for detecting varicella-zoster virus antibodies, characterized in that: The method of using the antigen plate according to claim 8 for detection comprises the following steps: Antibody detection: add the serum to be tested in multiple dilutions to the antigen plate, 20-40 μl / well, 4 replicate wells for each dilution; Incubate at 37°C for 50-70 min, wash three times with PBS, 180-220 μl / well, and air dry; Add fluorescent secondary antibody to the detection plate, 20~40μl / well, incubate at 37℃ for 50~70min; after incubation, wash 3 times with PBS, 180~220μl / well, spin dry, and place in an enzyme reader to detect fluorescence; then determine the results.

10. The method for detecting varicella-zoster virus antibodies according to claim 9, characterized in that: The specific steps of the result determination are as follows: taking the average value of the fluorescence intensity of the negative control wells + 3SD as the CUT OFF value, if the fluorescence intensity of the test well is greater than the CUT OFF value, it is determined to be positive, otherwise it is negative; if all 4 wells are positive, it is indicated by "++++"; if 3 wells are positive, it is indicated by "+++"; if 2 wells are positive, it is indicated by "++"; if 1 well is positive, it is indicated by "+"; "++" is the end point, that is, the antibody titer unit; the highest dilution with a positive result is used as the determination end point.

Images