Preparation method and application of high-throughput FAMA virus plate

Abstract

The present invention provides a preparation method and application of a high-throughput FAMA virus plate, which belongs to the field of biotechnology. By using a cell culture plate to prepare a high-throughput FAMA virus plate, the technical parameters such as the formula of the fixing solution and the blocking solution are optimized. A high-throughput FAMA virus plate prepared from a 96-well cell plate can detect 7 samples at the same time, increasing the detection throughput by 7 times, simplifying the detection process, shortening the detection time from 4 hours to 2.5 hours, and improving the timeliness of the FAMA method for detecting VZV antibodies; the present invention also provides a method for detecting the uniformity of a high-throughput FAMA virus plate, which is conducive to establishing and improving the quality control standards for the production, manufacturing and application of high-throughput FAMA virus plates. The present invention provides a high-throughput FAMA detection platform, which improves the automation level of the preparation and detection of high-throughput FAMA virus plates, and is conducive to the promotion and application of large-scale VZV antibody automated detection.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and relates to a preparation method and application of a high-throughput FAMA virus plate, and specifically relates to a high-throughput FAMA virus plate prepared from a cell culture plate and comprising FAMA positive antigen wells and FAMA negative control wells, wherein one high-throughput FAMA virus plate can realize simultaneous detection of multiple samples to be tested; the invention also relates to a detection method for the uniformity of the FAMA virus plate, which is used for evaluating the uniformity of the high-throughput FAMA virus plate. Background Art

[0002] Varicella-zoster virus (VZV) is a human alpha herpes virus that can cause chickenpox and / or herpes zoster. Vaccination with varicella vaccine and / or herpes zoster vaccine is a preferred preventive measure. VZV antibodies are widely used in the evaluation of varicella vaccine immunogenicity and are one of the important tools for assessing individual immune status and epidemiological surveys.

[0003] There are many methods for detecting VZV antibodies, including the FAMA method, enzyme-linked immunosorbent assay, complement fixation test, immunoadhesion hemagglutination test, radioimmunoassay, neutralization test, time-resolved fluorescence immunoassay, chemiluminescence immunoassay, lateral flow immunochromatography test, luciferase immunoprecipitation system, etc. The FAMA method is the gold standard for VZV antibody detection and is widely used in clinical serum testing of varicella vaccines at home and abroad. The FAMA method has a good correlation with the susceptibility and protection of clinical varicella. According to the US CDC report, the FAMA titer of the subject 16 weeks after vaccination is >1:4 and is associated with disease protection. In clinical practice, a serum titer increase of 4 times or more is considered positive. Merck's follow-up results 10 years after vaccination showed that the incidence of varicella in subjects with a FAMA titer ≤8 6 weeks after vaccination was 4 times that of subjects with a FAMA titer ≥64. A clinical trial by GSK also showed that high FAMA titers will produce higher immune efficacy. Up to now, the FAMA method is still the mainstream method for clinical VZV antibody detection of varicella vaccine. However, the traditional FAMA method is to prepare FAMA antigen slices through glass slides and 12-well glass slides. The process of preparing FAMA antigen slices from glass slides and the process of using FAMA antigen slices for VZV antibody detection all present a series of problems such as labor-intensive detection, small preparation batches, inconvenient storage, inability to achieve automation, and low detection throughput. The detection efficiency is low, and when testing a large number of clinical samples, it will also lead to queuing for testing. In addition, FAMA antigen slices lack unified quality control standards and quality control methods, and the prepared antigen slices have large differences between wells, slices, and batches, which seriously affects the detection efficiency and stability of VZV antibodies. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a high-throughput membrane antigen fluorescent antibody (FAMA) method for detecting varicella zoster virus antibody virus plates, which overcomes the problems of traditional FAMA antigen plate preparation being manual and labor-intensive and having low throughput.

[0005] The present invention also aims to provide a method for detecting the uniformity of a FAMA virus plate, thereby improving the quality control standard of the FAMA virus plate.

[0006] The present invention also aims to provide a high-throughput FAMA detection platform, which improves the automation level of FAMA detection and solves the timeliness problem of large-scale VZV antibody detection.

[0007] In a first aspect, the present invention provides a method for preparing a high-throughput FAMA virus plate, which is prepared by coating a cell culture plate with diseased cells infected with VZV, and is used to detect VZV antibodies, with diseased cells as coating antigens and uninfected susceptible cells as negative controls of coating antigens; the preparation steps include: 1) using VZV to infect susceptible cells and then culturing the virus; 2) until the pathological rate of susceptible cells reaches an appropriate pathological stage, using trypsin digestion, terminating the digestion and preparing a cell suspension; 3) adjusting the cell suspension to a certain cell density, adding it to the corresponding wells of a cell culture plate to the plating density, and standing it for sedimentation; 4) using a fixing solution to fix for 10 to 30 minutes, discarding the fixing solution, and washing; 5) using a blocking solution to block, discarding the blocking solution to dry and then freezing.

[0008] According to a preferred embodiment of the present invention, in step 3), a multi-channel micropipette or an automatic sample loader is used to add the cell suspension into the corresponding wells of the cell culture plate, and the suspension is allowed to settle at room temperature.

[0009] According to a preferred embodiment of the present invention, in step 3), after static sedimentation, the supernatant can be discarded.

[0010] According to a preferred embodiment of the present invention, in the method for preparing a high-throughput FAMA virus plate, the susceptible cells include MRC-5 cells; the cell culture plate includes a 96-well cell plate; the suitable pathological stage is a pathological rate of 60% to 80%; the plating density is 10,000 to 30,000 cells / well; the fixing solution includes 1% to 4% formaldehyde or paraformaldehyde solution; the blocking solution includes 5% to 12% animal serum, and the blocking time is 6 to 16 hours.

[0011] According to a preferred embodiment of the present invention, in the method for preparing a high-throughput FAMA virus plate, the suitable pathological stage is a pathological rate of 70%; the fixing liquid is a 2% formaldehyde solution, fixed for 20 min; the blocking liquid is 10% animal serum. Among them, the animal serum includes goat serum, fetal bovine serum, horse serum, etc., preferably goat serum.

[0012] According to a preferred embodiment of the present invention, in the method for preparing a high-throughput FAMA virus plate, the plating density is 15,000 per well.

[0013] In a second aspect, the present invention provides a high-throughput FAMA virus plate, which is prepared by the preparation method of the high-throughput FAMA virus plate of the first aspect.

[0014] In a third aspect, the present invention provides a method for detecting the uniformity of a FAMA virus plate, wherein the detection object is the high-throughput FAMA virus plate of the second aspect, and the steps include: 1) staining with a nuclear staining reagent; 2) discarding the nuclear staining reagent, washing and patting dry; 3) adding a glycerol solution and counting cells using a high-throughput microscope; 4) calculating CV values ​​based on the cell counting results within and / or between plates; 5) analyzing the uniformity within and / or between plates of the high-throughput FAMA virus plate based on the CV value; wherein, if the CV value is less than 20%, the uniformity within and / or between plates of the high-throughput FAMA virus plate is good.

[0015] According to a preferred embodiment of the present invention, in step 1), dyeing is performed at room temperature.

[0016] According to a preferred embodiment of the present invention, in step 3), the concentration of the glycerol solution is 65% to 75%, preferably 70%.

[0017] According to a preferred embodiment of the present invention, in the method for detecting the uniformity of the FAMA virus plate, the nuclear staining reagent includes one or two of Hoechest, DAIP or Evans blue, which are diluted at 1:500 to 1:2000 before use; the staining time does not exceed 30 minutes.

[0018] According to a preferred embodiment of the present invention, in the method for detecting the uniformity of the FAMA virus plate, when counting cells, a high-throughput microscope is aimed at the central field of view of the sampling well on the high-throughput FAMA virus plate, and at least one field of view under the same magnification is selected for cell counting, and the cell number is taken as the number of cells in the well.

[0019] Further preferably, no less than 10% of the total number of wells in each high-throughput FAMA virus plate are selected for cell counting.

[0020] In a fourth aspect, the present invention provides a kit comprising a VZV antibody positive quality control product and the high-throughput FAMA virus plate of the second aspect, for detecting VZV antibodies.

[0021] According to a preferred embodiment of the present invention, the titer of the VZV antibody positive quality control product is 64±2.

[0022] In a fifth aspect, the present invention provides a method for preparing a high-throughput FAMA virus plate, a high-throughput FAMA virus plate, a method for detecting the uniformity of a FAMA virus plate, or a kit, and their use in the preparation of products related to VZV antibody detection.

[0023] Beneficial effects of the present invention:

[0024] The present invention provides a method for preparing a high-throughput FAMA virus plate for detecting VZV antibodies. The plate is prepared using a porous cell culture plate, which can not only realize high-throughput detection of multiple samples to be tested at the same time and improve the detection throughput, but also the detection results are highly consistent with the FAMA antigen sheet prepared by the traditional slide glass, and the specificity is good, the sensitivity is high, and the stability is good. In particular, the high-throughput automated detection is realized by using upgraded experimental equipment such as an automatic sampler or a liquid dispenser, so as to solve the problems of manual and labor-intensiveness that cannot be overcome in the subsequent use of the FAMA antigen sheet and the relatively low detection throughput. The high-throughput FAMA virus plate provided by the present invention, in combination with a high-throughput detection system and an automated analysis system, improves the automation level of preparation and detection, and is expected to be applied in large-scale detection such as sample screening, VZV antibody group detection, etc. more quickly, more efficiently and more deeply. Compared with the traditional FAMA antigen slide, the high-throughput FAMA virus plate provided by the present invention has a good detection result compliance rate and significantly improved detection timeliness.

[0025] The high-throughput FAMA virus plate provided by the present invention improves the detection flux and automation level of FAMA detection. Common FAMA antigen sheets are made by coating VZV antigens artificially and in small batches on 12-well slides. During the preparation process and the detection process, each hole of the FAMA antigen sheet needs to be manually operated separately. There are serious labor-intensive characteristics, and it is impossible to perform operations such as automated film making and detection, and it is impossible to meet the detection needs of large quantities, stability, rapidity and efficiency. The high-throughput FAMA virus plate prepared by the present invention is used for detection, and 7 samples to be tested can be detected at the same time, and the detection flux is increased by 7 times. At the same time, the detection process is simplified, and the instrument automation plate washing is realized, and the detection time is shortened from 4 hours to 2.5 hours. And common FAMA antigen sheets all use 12-well slides, which are limited by the low flux of the slides, and the preparation and detection process are cumbersome, which limits the increase of FAMA antigen sheet uniformity quality control and detection scale.

[0026] In the traditional FAMA antigen sheet preparation process, the uniformity of the cell suspension added to each well has a decisive influence on the accuracy of the FAMA test results, but there is a lack of objective and effective quality control methods for FAMA antigen sheets. The present invention provides a method for detecting the uniformity of a FAMA virus plate, which can be used for the quality control of the uniformity within and between plates of a FAMA virus plate to ensure its stable and uniform performance. The uniformity detection method is to stain the diseased cells fixed on a high-throughput FAMA virus plate with a nuclear staining reagent, count the cells in the wells with a high-throughput microscope, calculate the CV values ​​within and between plates, and the uniformity detection results of the high-throughput FAMA virus plate show that the CV values ​​within and between plates are less than 20%, and the uniformity is good. It can be seen that not only the preparation method of the high-throughput FAMA virus plate is stable and available, but also the uniformity detection method is suitable for the quality control of the preparation and application of high-throughput FAMA virus plates.

[0027] In the preparation method of the high-throughput FAMA virus plate, susceptible cells are infected with VZV, and diseased cells are used as coating antigens. The susceptible cells are preferably MRC-5 cells. When the virus is cultured to the preferred mid-range disease rate, the virus is harvested, and the cell suspension is prepared into a certain density after trypsin digestion, termination, and resuspension. The cell suspension is added to the preferred 96-well cell plate wells to the preferred plating density of 10,000 to 30,000 / well with a pipette, fixed with a preferred fixative, washed, and then blocked with a preferred blocking solution containing animal serum, and the blocking solution is discarded and dried and frozen. In the preparation method of the high-throughput FAMA virus plate, specific technical parameters such as the disease rate, the type of fixative, and the type of blocking solution are screened, and the mid-range disease rate of 60% to 80% is selected as the optimal disease rate, 2% to 4% formaldehyde is the available fixative, and 5% to 12% animal serum is the blocking solution; after the blocking treatment, complex preparation steps such as vacuum sealing are no longer required, which simplifies the production procedure and preparation cost. Moreover, the preparation method of the high-throughput FAMA virus plate has controllable uniformity and stable performance. In particular, VZV-infected cells are fixed on a 96-well cell plate, replacing the traditional 12-well FAMA antigen plate, improving the labor-intensiveness, small preparation batches, inconvenient storage and other problems in the preparation process, increasing throughput, and realizing automated preparation and detection.

[0028] During the application process, the high-throughput FAMA virus plate further verified its better performance indicators. In terms of specificity, 10 negative sera with clinical background were selected, and the results of the high-throughput FAMA virus plate and the FAMA antigen sheet were completely consistent, that is, the specificity of the high-throughput FAMA virus plate met the requirements; in terms of sensitivity, the WHO VZV immunoglobulin international standard was used as the VZV antibody standard for sensitivity experiments, and the results showed that the sensitivity was 24.4 mIU / mL, which was equivalent to the classic FAMA antigen sheet method; in terms of stability, the stability of the test results of the high-throughput FAMA virus plate after repeated freezing and thawing 3 times, 5 times and room temperature placement for 4h and 6h was verified. The same VZV antibody positive quality control product was used for detection, and the titer difference was within ±2 times, indicating that the high-throughput FAMA virus plate of the present invention has good stability; in terms of compliance rate, the titers of 15 positive sera were all within ±2 times, and all 10 negative sera were negative, which was completely consistent with the results of the FAMA antigen sheet, showing that the detection compliance rate of this method with the classic FAMA antigen sheet method was 100%.

[0029] Therefore, the present invention provides a high-throughput FAMA detection platform, including an optimized high-throughput FAMA virus plate preparation method, a FAMA virus plate homogeneity detection method, high-throughput FAMA virus plate related detection reagents and their corresponding quality control standards. Among them, the high-throughput FAMA virus plate for detecting VZV antibodies can improve the detection throughput of the FAMA antigen sheet prepared by the current membrane antigen fluorescent antibody method, and can be used for epidemiological surveys of varicella and / or herpes zoster, immunogenicity detection after varicella vaccine and / or herpes zoster vaccine immunization, and monitoring of the status of herd immunity of varicella / herpes zoster virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The cell status diagram of the present invention at different lesion rates, A is a low-grade lesion rate (30%-50%), B is a medium-grade lesion rate (60%-80%), and C is a high-grade lesion rate (90% and above);

[0031] Figure 2 The figure is a test result diagram of the same sample by the high-throughput FAMA virus plate prepared by harvesting cells at different lesion rates of the present invention. The horizontal direction is: different dilution multiples of the same sample, "-" is the FAMA negative control, and "NTC" is the blank control without adding the sample; the vertical direction is: A is the low-grade lesion rate (30%~50%); B is the medium-grade lesion rate (60%~80%);

[0032] Figure 3 The results of the cell dispersion degree after the samples were placed on the plates at different plating densities in the present invention are shown in Figure 1. A represents a plating density of 10,000 cells / well; B represents a plating density of 15,000 cells / well; C represents a plating density of 20,000 cells / well; and D represents a plating density of 30,000 cells / well.

[0033] Figure 4 The figure is a test result diagram of the high-throughput FAMA virus plate prepared with different fixatives of the present invention for the same sample, the horizontal direction is: different dilution multiples of the same sample, "-" is the FAMA negative control; the vertical direction is different fixatives: A is 4% formaldehyde solution; B is 2% formaldehyde solution; C is 4% paraformaldehyde solution;

[0034] Figure 5 The finished product image of the high-throughput FAMA virus plate prepared after the preparation conditions of the high-throughput FAMA virus plate of the present invention are optimized and the local microscopic magnified image of the cell wells;

[0035] Figure 6 This is a staining effect diagram of the Hoechest nuclear staining reagent used in the high-throughput FAMA virus plate uniformity detection method of the present invention;

[0036] Figure 7 This is a diagram of the specific detection results of the high-throughput FAMA virus plate and the comparison reagent of the present invention. In the figure, "+" represents a positive test result, "-" represents a negative test result, A is a high-throughput FAMA virus plate, and B is a comparison reagent, i.e., a traditional FAMA antigen sheet; white arrows indicate typical positive signals, and blue arrows indicate typical negative signals. DETAILED DESCRIPTION

[0037] Exemplary embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein the same or similar concepts represent the same concepts.

[0038] In the detailed description below, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. In the detailed description below, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. The following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0039] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified. Among them, trypsin was purchased from Gibco Life Technologies; the virus used in the present invention is the varicella Oka strain virus working seed batch (referred to as VZV-Oka strain) prepared by Beijing Wantai Biological Pharmaceutical Co., Ltd.; the varicella-zoster virus IgG antibody standard (referred to as VZV antibody standard) is the WHO VZV immunoglobulin IgG international standard, with a concentration of 50 IU / mL, purchased from NIBSC; the VZV antibody positive quality control product is the VZV IgG antibody internal quality control product, which is self-made by Beijing Wantai Biological Pharmaceutical Co., Ltd.; Nissui MEM medium is purchased from Nissui Pharmaceutical Co., Ltd.; MRC-5 cells are purchased from the European Certified Cell Culture Collection Center ECACC. However, it is obvious that one or more embodiments can be implemented without these specific details. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0040] The molecular biology experimental methods not specifically described in the following embodiments are all carried out with reference to the specific methods listed in the book "Molecular Cloning Experiment Guide" (3rd edition) by J. Sambrook, or in accordance with the instructions for reagents and products. The object of the present invention, the sample to be tested, is a biological sample that has been inactivated and stored in vitro, such as serum; the direct purpose of the test is to determine the content of VZV antibodies in the sample to be tested in vitro; the process, results or related applications of the test do not include the diagnosis and treatment of the disease, and it is impossible to directly obtain the diagnosis results of the disease or the health status based on the test results. It is well known in the art that the detection of VZV antibodies can be used for the clinical immunogenicity evaluation of varicella vaccine or herpes zoster vaccine, for epidemiological surveys of varicella and herpes zoster, or for monitoring the immunity of the population to varicella and herpes zoster viruses. Therefore, the present invention does not belong to the diagnosis and treatment of diseases, and meets the basic requirements of the Patent Law for patent protection objects.

[0041] The present invention provides a method for preparing a high-throughput FAMA virus plate by replacing a glass slide with a cell culture plate. By optimizing key preparation conditions and improving the detection method of the uniformity of the FAMA virus plate, a FAMA detection platform capable of high-throughput rapid detection of VZV antibodies is established. The FAMA detection platform provided by the present invention has the advantages of good specificity, high sensitivity and good stability while ensuring a 100% conformity rate with the detection results of the traditional FAMA antigen sheet. The present invention significantly improves the detection flux and detection time efficiency of FAMA. The specific embodiments of the present invention are described in detail below, and the specific contents are as follows:

[0042] Example 1: High-throughput FAMA virus plate preparation method

[0043] The traditional FAMA method is to prepare FAMA antigen slides on glass slides, 12-well glass slides or 12-well tissue in situ glass slides. During the preparation process, VZV strains with higher virus titers (such as VZV Oka strain or VZV84-7 strain) are selected to infect susceptible cells at an MOI of 0.01-0.05. Susceptible cells are preferably 2BS cells, Vero E6 cells, etc. After the virus effectively replicates to the peak of replication, it is harvested and prepared into a cell suspension. The density of plating on the slide is 1.2-1.8×10 5 / mL, or up to 2.5-3.5×10 5 / mL, add 20-25μL or 10μL to each well when loading the sample. Use a fixative containing cold acetone to fix the sample, wash and dry it, and then use a protective solution containing multiple sugar components (equivalent to a blocking solution) to improve the stability of the FAMA antigen slice and extend its shelf life. After the protective solution is dried, the FAMA antigen slice also needs to be vacuum-sealed to prevent contamination from the external environment and degradation of the antigen.

[0044] However, the present invention creatively selects a cell culture plate (preferably a flat-bottom 96-well cell plate) to prepare a high-throughput FAMA virus plate, and the specific preparation steps are as follows:

[0045] 1) MRC-5 cells were used as VZV susceptible cells. When the MRC-5 cells of passage M36 to M38 grew to a thin monolayer, VZV-Oka strain virus was inoculated into the cell culture flask of MRC-5 cells at an MOI of 0.005 to 0.02. The virus culture medium was MEM medium containing 2% serum, 0.03% glutamine, and pH adjusted to about 7.3 with 5.6% sodium bicarbonate. The virus was cultured in a 35°C ± 1°C, 5% carbon dioxide incubator for about 3 days to ensure that VZV could effectively infect MRC-5 cells.

[0046] 2) After VZV infects MRC-5 cells, continue to culture until the pathological rate of MRC-5 cells reaches a certain level, digest them with 0.25% trypsin in turn, and terminate the digestion with cell resuspension solution. The pathological rate is divided into three levels from low to high: low (30% to 50%), medium (60% to 80%), and high (90% and above); the cell states at the three different pathological rates are as follows Figure 1 As shown;

[0047] 3) Use cell resuspension solution (formula is 10% serum, 0.03% glutamine, and MEM medium with pH adjusted to about 7.3 with 5.6% sodium bicarbonate) to adjust the diseased cells to cell suspensions after different plating densities, and use a pipette or automatic sampler (loading error is less than 1 μL) to inoculate the cell suspension into a cell culture plate, preferably a 96-well cell plate, with a loading volume of 50-150 μL / well (preferably 100 μL / well); discard the supernatant after standing at room temperature. The standing time is suitable for the infected MRC-5 cells to stably adhere to the bottom of the cell plate, and the standing time at room temperature is preferably 1.5 hours.

[0048] 4) Fix the plate with a fixative at room temperature for 10 to 30 minutes, discard the fixative, wash the plate with an automated plate washer to remove unattached cells and impurities, and pat dry after washing; the fixative is a 1% to 4% formaldehyde / paraformaldehyde solution.

[0049] 5) Blocking with blocking solution, discarding the blocking solution and air drying; wherein the blocking solution is selected from different formulas containing various ingredients such as proteins, sugars, alcohols, etc., and the staining effect, specificity and sensitivity of the high-throughput FAMA virus plate blocked with different blocking solutions in the detection are verified respectively.

[0050] 6) After sealing, the high-throughput FAMA virus plate is ready for quality stability testing. The remaining finished products can be stored at -20°C.

[0051] During the preparation of the high-throughput FAMA virus plate, the buffer used for washing was PBST buffer.

[0052] Example 2: Optimization of preparation conditions for high-throughput FAMA virus plates

[0053] 1. Optimal lesion rate at the peak of lesions

[0054] VZV was inoculated into MRC-5 cells at an MOI of 0.01, and MRC-5 cells reached the peak of pathology after about 3 days. The progression of cell pathology was observed, and the cells were harvested at 3 different pathology rates and the same technical parameters were used to prepare high-throughput FAMA virus plates. During the preparation process, it was found that when the pathology rate was low and medium, the lesions were bright and typical, and the digested cell suspension had good adhesion to the bottom of the cell plate after plating. However, when the pathology rate was high, the lesions were unstable and a small amount of melting spots would appear. After the digested cell suspension was plating, the adhesion to the bottom of the cell plate was poor, which was not conducive to subsequent data processing and observation. The same sample was tested using a high-throughput FAMA virus plate prepared after harvesting cells at 3 different pathology rates. The test results are as follows Figure 2As shown: when the lesion rate is from low to high, no positive signal appears in the negative control in the test results, and the specificity is good; when the lesion rate is medium, not only is the fluorescence intensity slightly higher than the low, but the detection titer of the high-throughput FAMA virus plate prepared by medium-grade harvest is 512, while the detection titer of the high-throughput FAMA virus plate prepared by low-grade harvest is 256; considering comprehensively, in the preparation process of high-throughput FAMA virus plate, the lesion rate at the peak of lesions is preferably medium (i.e., lesion rate 60% to 80%), which not only has better adhesion ability during the preparation process, but also better subsequent lesion staining effect and higher final detection sensitivity.

[0055] 2. Planking density

[0056] The cell suspension is made by adjusting the diseased cells to the plating density by cell resuspension, and adding them to the cell culture plate using a pipette or automatic sampler, preferably a 96-well cell plate, and the loading volume per well is preferably 100 μL / well. The same technical parameters are subsequently used to prepare a high-throughput FAMA virus plate; among them, the plating density is set in 4 gradients within the range of 10,000 / well to 30,000 / well to screen the optimal plating density, and the screening assessment indicators are the degree of cell dispersion after plating, the staining effect after detection, and the sensitivity of titer detection. The degree of cell dispersion requires that the cells are neither too stacked nor too sparse; the staining effect after detection requires that the staining of the lesions is clearly discernible; the detection sensitivity requires that the test results of the sample titer are not reduced.

[0057] Screening results of plating density: The results of cell dispersion after stabilization at 4 different plating densities are as follows Figure 3 As shown in Table 1, when the plating density is 10,000 cells / well, the cells are relatively sparse after dispersion; when the plating density is 20,000 cells / well and 30,000 cells / well, a very small amount of stacking can be seen after the cells are dispersed; and when the plating density is 15,000 cells / well, the degree of cell dispersion is more appropriate. The high-throughput FAMA virus plates prepared with different plating densities were tested and verified by testing two samples separately and found that different plating densities had little effect on the staining effect, the staining effect after detection was clear and discernible, and the detection titer of the same sample was not reduced, as shown in Table 1, that is, there was no significant difference in the sensitivity of the detection results for the different plating densities of the four gradients, and all of them could meet the requirements for detection sensitivity. Taking all factors into consideration, in the preparation of high-throughput FAMA virus plates, the preferred cell density for plating density is 15,000 cells / well.

[0058] Table 1. Test results of different plating densities

[0059]

[0060] 3. Fixative

[0061] FAMA detection also belongs to the field of immunofluorescence detection. Different fixative formulations will have a significant impact on the morphology, structure and subsequent immunofluorescence reaction of cell surface antigens after cell fixation. Traditional FAMA antigen plates often use cold acetone as a fixative. Due to the strong penetrating power of acetone, it is often used as a strong dehydrating agent and can quickly fix cells. However, the fixation process may cause damage to antigenic substances in cells due to excessive dehydration, thereby affecting subsequent immunofluorescence experiments. The present invention screens formaldehyde or paraformaldehyde solutions with a concentration of 1% to 4% as fixatives to investigate the fixation effect on high-throughput FAMA virus plates and the impact on the sensitivity of subsequent immunofluorescence detection; in terms of fixation effect, formaldehyde or paraformaldehyde solutions at a concentration of 1% to 4% can complete the fixation of cells in the cell suspension within 30 minutes; the high-throughput FAMA virus plates prepared using 1% to 4% formaldehyde / paraformaldehyde solutions as fixatives do not show significant differences in the test results for the same sample, and the test results of 3 representative fixative formulations are as follows. Figure 4 As shown in the figure: There is no significant difference in the sensitivity of the detection results of the high-throughput FAMA virus plate prepared with different formulas of fixatives, and all can meet the requirements for detection sensitivity. The lower the concentration of formaldehyde or paraformaldehyde solution, the longer the fixation time; the higher the concentration of formaldehyde or paraformaldehyde solution, the stronger its irritation is, which is not conducive to subsequent cleaning; therefore, considering the fixation time and irritation, the fixative formula preferably uses a lower concentration of 2% formaldehyde solution.

[0062] 4. Blocking solution

[0063] In order to extend the shelf life of the antigen slice and improve the detection stability, the traditional FAMA antigen slice is often treated with a protective solution containing sugar components before vacuum sealing, and the preparation process is relatively cumbersome. In order to screen out a new sealing solution that has both protective functions and does not require vacuum sealing, the present invention verifies the treatment effects of the sealing solutions prepared by various formulas such as proteins, sugars, and alcohols by testing the same sample, and focuses on the subsequent cell staining effect, detection specificity and sensitivity of the high-throughput FAMA virus plate prepared with different sealing solutions. The formulas of different sealing solutions have no effect on subsequent cell staining, and the stained cell morphology is complete and clearly distinguishable. The test results of the high-throughput FAMA virus plate prepared by different sealing solution formulas for the same sample are shown in Table 2. The sealing solution component containing animal peptone showed non-specific binding phenomenon in the subsequent detection process, and the detection specificity was poor; while the sealing solution containing only 5% to 12% animal serum had a relatively high sensitivity in subsequent detection.

[0064] Table 2. Test results of different blocking solutions

[0065]

[0066] Taking into account the components of different blocking solutions and their blocking effects, the preferred formula of the blocking solution is a relatively single component 5% to 12% animal serum, with a preferred concentration of 10%. The animal serum is preferably goat serum.

[0067] According to the layer-by-layer optimization and verification experiments of the preparation conditions of the high-throughput FAMA virus plate, the key optimal conditions of the high-throughput FAMA virus plate of the present invention are: the optimal lesion rate of virus harvest in the cell suspension is mid-range (60% to 80%), and the cell suspension plating density is preferably 1.5×10 5 / mL, the loading volume is 100 μL / well, and the cell density after loading is 15,000 / well. After standing for about 1.5 hours, discard the supernatant, add a fixative containing 2% formaldehyde at 100 μL / well, the fixation time is preferably 20 min, discard the fixative, and after washing, preferably use a blocking solution containing 10% goat serum for overnight blocking treatment. The next day, discard the blocking solution and dry it. The finished high-throughput FAMA virus plate does not need to be vacuum-sealed and can be directly frozen. The finished cell plate prepared by optimizing the above conditions is as follows Figure 5 shown.

[0068] 5. Contrast reagent

[0069] Preparation of traditional FAMA antigen slices as comparison reagents: According to the cell suspension prepared with the preferred pathological rate in the preparation process of the high-throughput FAMA virus plate of the present invention, the cell suspension is loaded onto a classic 12-well glass slide using a pipette. The loading volume of the 12-well glass slide is 20 μL / well. The cell suspension is evenly applied to the surface of each microwell by drawing circles and the plating density of each microwell is about 7,000 / well. Since the storage time of the cell suspension at room temperature is limited, the loading process requires the participation of multiple people. The loaded slide is first left at room temperature for 30 minutes, then gently placed horizontally on the slide rack, and transferred to a constant temperature water tank at 25°C and left to stand for 30 minutes. Then, the preferred fixing solution for preparing high-throughput FAMA virus plates of the present invention is fixed for about 15 minutes, the fixing solution is discarded, and the plate is placed in a fume hood for about 15 minutes to dry, and then washed 3 times and left to stand, and left to dry naturally overnight; the next day, the preferred blocking solution for preparing high-throughput FAMA virus plates of the present invention is used to block at room temperature for 120 minutes, then washed twice, and finally washed once with purified water, and left to stand to dry to obtain the FAMA antigen sheet, which is stored at -60°C for standby use. During the preparation process, the smearing and cleaning operations of the 12 wells on the FAMA antigen sheet need to be performed manually, such as manually drawing circles for each well; the sedimentation process requires the FAMA antigen sheet to be placed on the slide rack one by one, and the cleaning process requires manual gentle shaking of the beaker.

[0070] Example 3: Method for detecting uniformity of FAMA virus plate

[0071] The degree of standardization of the preparation and detection of traditional FAMA antigen slices is relatively low, and there will be certain differences in the test results between FAMA antigen slices prepared by different manufacturers. Due to the lack of objective and effective methods to evaluate the quality of FAMA antigen slices, the FAMA-based test kits lack technical standards such as performance and results, and their consistency is affected. Therefore, no FAMA-based detection products have been approved for marketing at home and abroad. More stringent detection methods and quality control standards are urgently needed to ensure the uniformity, accuracy and comparability of test results of similar products.

[0072] The present invention provides a method for detecting the uniformity of a FAMA virus plate, which is used for quality control of the FAMA virus plate by detecting the uniformity within and / or between plates, and the detection steps are: first, a nuclear staining reagent is used to stain the prepared high-throughput FAMA virus plate product at room temperature, the nuclear staining reagent can be Hoechest, DAIP or Evans blue, the nuclear staining reagent is diluted at a ratio of 1:1000 and added to each well of the high-throughput FAMA virus plate, and the nuclear staining reagent is discarded after staining for a certain time (for example, Evans blue staining for 30 min), and an automatic plate washer is used for automatic cleaning, and after cleaning, it is patted dry, and then a 70% glycerol solution is added to count cells using a high-throughput microscope. Calculate the CV value according to the cell counting results within and / or between plates, and then analyze and judge the uniformity within and / or between plates of the high-throughput FAMA virus plate according to the CV value. Among them, in the central field of view of each well of the high-throughput FAMA virus plate, a field of view under the same magnification (such as 4 times) is selected for cell counting as the number of cells in the well; each high-throughput FAMA virus plate selects cell wells of no less than 10% of the total number of wells for observation and cell counting. The CV values ​​calculated between different wells in the same high-throughput FAMA virus plate are used to determine the intra-plate uniformity of the high-throughput FAMA virus plate; the CV values ​​calculated between different high-throughput FAMA virus plates are used to determine the inter-plate uniformity of different high-throughput FAMA virus plates. Three high-throughput FAMA virus plates prepared from the same batch were sampled. According to the above-mentioned high-throughput FAMA virus plate uniformity detection method, the nuclear staining reagent is preferably Hoechest, and the intra-plate and inter-plate uniformity detections are respectively carried out. The staining effect of the nuclear staining reagent is shown in FIG. Figure 6 The uniformity test results of the three high-throughput FAMA virus plates are shown in Table 3. It can be seen that the CV values ​​within and between the three high-throughput FAMA virus plates sampled were less than 20%, and the uniformity was good.

[0073] Table 3 High-throughput FAMA virus plate uniformity test results

[0074]

[0075] Example 4: Application and evaluation of high-throughput FAMA virus plates

[0076] The present invention provides a high-throughput FAMA detection platform, including an optimized preparation method of a high-throughput FAMA virus plate, a method for detecting the uniformity of a FAMA virus plate, high-throughput FAMA virus plate-related detection reagents and their corresponding quality control standards. According to the above preparation method, a high-throughput FAMA virus plate was trial-produced, and the following detection applications and performance analysis were performed.

[0077] 1. FAMA detection steps

[0078] 1) High-throughput FAMA virus panel detection steps:

[0079] FAMA plate equilibration at room temperature: remove the high-throughput FAMA virus plate from the ≤-15°C condition and equilibrate at room temperature for 30 min;

[0080] Sample dilution: First, dilute the inactivated serum sample 4-fold in the dilution plate, and then dilute it 2-fold in 12 steps;

[0081] Sample loading: Use a pipette to transfer the serially diluted serum samples to the FAMA plate, with a sample volume of 100 μL / well;

[0082] Incubation: Place in a 37°C incubator for 60 min;

[0083] Wash the plate: Wash the plate with a plate washer for 2 min / plate, and pat dry;

[0084] Add enzyme: dilute goat anti-human IgG fluorescent labeled antibody with FAMA enzyme label diluent at a dilution ratio of 1:400, load 100 μL / well, and avoid light from this step on;

[0085] Incubation: Place in a 37°C incubator for 30 min;

[0086] Nuclear staining: discard the enzyme-labeled antibody, pat dry, add nuclear staining reagent (1:1000), 100 μL / well, and incubate at room temperature for 5 minutes;

[0087] Wash the plate: Wash the plate with a plate washer for 2 min / plate, and pat dry;

[0088] Microscopic examination: Add 100 μL of 70% glycerol solution to each well and examine under a microscope.

[0089] 2) FAMA antigen detection steps:

[0090] Thawing of FAMA slices: Take out FAMA slices from the condition of ≤-60℃, place each slice flat on a horizontal slide rack, and thaw at room temperature for 60 min;

[0091] Sample dilution: First, dilute the inactivated sample 4-fold in the first well of the dilution plate, and then dilute it 2-fold, for a total of 10 gradients;

[0092] Sample loading: Use a pipette to load the sample into each well, spread it evenly in each well, and the sample loading volume is 20 μL / well;

[0093] Incubation: Place in a 37°C constant temperature water tank and incubate for 30 min;

[0094] Washing: Take the slides out of the horizontal slide rack, place them upright on the vertical slide rack, gently place them in a beaker, and shake them manually 3 times, 5 minutes each time. After each change of solution, let them stand for 5 minutes. Place the washed slides on absorbent paper, leave them at room temperature for 30 minutes, and air dry;

[0095] Add enzyme: Place the dried slides on a horizontal slide rack, dilute goat anti-human IgG fluorescent-labeled antibody at a ratio of 1:400, and add 1% Evans blue solution at a ratio of 1:100. Load the slides one by one using a single-channel pipette at 20 μL / well;

[0096] Incubation: Place in a 37°C incubator for 30 min;

[0097] Washing: Take the slides out of the horizontal slide rack, discard the enzyme-labeled antibody, and place them upright on the vertical slide rack. Gently place them in a beaker and wash them manually 3 times, 5 minutes each time. After each change of solution, let them stand for 5 minutes. Place the washed slides on absorbent paper, leave them at room temperature for 30 minutes, and air dry.

[0098] Microscopic examination: Add 70 μL of 70% glycerol solution to each slide, cover with a coverslip, and wait for examination under a microscope.

[0099] Comparison of detection timeliness: When using FAMA antigen slices as comparison reagents for detection, similar to its preparation process, it needs to be completed manually and cannot be automated. Moreover, one FAMA antigen slice can only be used to detect one sample to be tested. The detection time for each FAMA antigen slice is about 4 hours. In contrast, the high-throughput FAMA virus plate of the present invention is preferably prepared from a 96-well cell culture plate, and an eight-channel pipette or an automatic sampler can be used in the preparation and detection processes, and high-end automated equipment such as automatic robotic arms can be upgraded to complete loading, pipetting, plate washing and other tasks; for example, during detection, after the original solution of the sample to be tested is added to the corresponding wells respectively, the automatic sampler can be used to transfer and dilute the samples to be tested in the first row / row in the subsequent detection wells in sequence to complete the multiple dilution operation; for example, the automatic plate washer can be used for fully automatic plate washing for cleaning, which gets rid of the excessive dependence of FAMA antigen sheets on manual operation and greatly improves the timeliness of detection; in addition, one high-throughput FAMA virus plate can detect at least 7 samples to be tested at the same time, and the detection time of each high-throughput FAMA virus plate from loading, dilution to detection is about 2.5 hours.

[0100] 2. Timeliness and specificity of high-throughput FAMA virus panel qualitative detection

[0101] Ten clinically confirmed negative sera and 15 positive sera were selected as the test samples, totaling 25 samples, which were tested in parallel using the high-throughput FAMA virus plate of the present invention and the FAMA antigen piece of the comparison reagent to verify the specificity of the high-throughput FAMA virus plate.

[0102] Timeliness comparison: There are 25 samples to be tested in the specificity verification experiment, which requires 25 FAMA antigen sheets. If one tester performs the detection operation in sequence and continuously, it takes about 6 hours to consider the crossover time in the actual operation. If these 25 samples are tested using a high-throughput FAMA virus plate, less than 4 high-throughput FAMA virus plates are required. If one tester also performs the detection operation on the 4 high-throughput FAMA virus plates in sequence, in actual operation, since many operations of the high-throughput FAMA virus plate can be automated, the simultaneous detection operation of 4 high-throughput FAMA virus plates can be realized, which can be completed in only about 2.5 hours. Compared with the FAMA antigen sheet, the high-throughput FAMA virus plate of the present invention greatly improves the detection speed, detection flux and detection timeliness of VZV antibodies. It can be reasonably expected that when the number of samples to be tested is larger, the use of high-throughput FAMA virus plates for detection will show stronger timeliness. Therefore, the high-throughput FAMA virus plates of the present invention are very suitable for high-throughput, high-efficiency and automated detection of large-scale clinical samples.

[0103] The parallel detection results of the high-throughput FAMA virus plate and the FAMA antigen plate were analyzed and interpreted using the same microscope: the appearance of a typical complete, bright ring was a positive signal, while a negative signal was an opaque sphere, such as Figure 7 As shown in the figure, the morphology of positive and negative signals detected by the high-throughput FAMA virus plate and the FAMA antigen piece is basically the same. The results of the specific experiment are: 10 negative sera are all negative, without typical positive signals; 15 positive sera have typical positive signals and are judged as positive; the results of the interpretation on the high-throughput FAMA virus plate of the virus-uninfected negative are all negative; that is, the qualitative detection results of the high-throughput FAMA virus plate and the FAMA antigen piece are consistent, and the negative coincidence rate, positive coincidence rate and total coincidence rate are all 100%. The specific results are shown in Table 4.

[0104] Table 4 Comparison of detection specificity of the two methods

[0105]

[0106] 3. Detection sensitivity of high-throughput FAMA virus panels

[0107] The high-throughput FAMA virus plate and FAMA antigen sheet were taken respectively, and the VZV antibody standard was used as the sample to be tested to compare the detection sensitivity of the two methods. The test was repeated 3 times. The first well of the experiment was diluted with the VZV antibody standard at a ratio of 1:4 and then added. The subsequent 2-fold gradient dilution was performed in sequence, that is, the dilutions of the VZV antibody standard were 1:4, 1:8, 1:16, 1:32...1:2048, 1:4096, and the corresponding concentrations of the VZV antibody standard were 12500 mIU / mL, 6250 mIU / mL, 3125 mIU / mL, 1562.5 mIU / mL...24.4 mIU / mL, 12.2 mIU / mL. The high-throughput FAMA virus plate can determine the titer of the test to be 2048, which is equivalent to 24.4 mIU / mL of the VZV antibody standard; the parallel FAMA antigen sheet can also determine the titer of the test to be 2048, and the specific sensitivity test results are shown in Table 5. Therefore, the high-throughput FAMA virus plate of the present invention and the FAMA antigen sheet have the same determination test results, that is, the sensitivity of the high-throughput FAMA virus plate is not inferior to that of the FAMA antigen sheet.

[0108] Table 5 Sensitivity test results

[0109]

[0110] 4. High-throughput FAMA virus panel detection stability

[0111] The VZV antibody positive quality control product was used as the sample to be tested, wherein the titer of the VZV antibody positive quality control product on the FAMA antigen sheet was 64±2. After the high-throughput FAMA virus plate was repeatedly frozen and thawed 3 times, 5 times and placed at room temperature for 4h and 6h, the titer of the VZV antibody positive quality control product after gradient dilution was detected. The test results are shown in Table 6. It can be seen that after the high-throughput FAMA virus plate was repeatedly frozen and thawed 3 times and placed at room temperature for 4h and 6h, the test results were consistent with the test results of the high-throughput FAMA virus plate that was not frozen and thawed and placed at room temperature. The titer of the VZV antibody positive quality control product was 64. After the high-throughput FAMA virus plate was repeatedly frozen and thawed 5 times, the titer of the VZV antibody positive quality control product was 32, which was slightly decreased, but still within the range of ±2 times the titer ratio, indicating good stability.

[0112] Table 6 Stability test results

[0113]

[0114] 5. The consistency rate between the high-throughput FAMA virus panel and the FAMA antigen plate test results

[0115] Further analysis of the determination of the specific antibody titers of 25 clinical samples by the high-throughput FAMA virus plate and FAMA antigen sheet is shown in Table 7. It can be seen that the ratio (B / A) of the determination results of the titers of 15 positive serum samples by the high-throughput FAMA virus plate and FAMA antigen sheet is mostly 1, the maximum is no more than 2, and the minimum is not less than 0.5, that is, the ratio is within ±2 times; the determination results of the titers of 10 negative serum samples by the high-throughput FAMA virus plate and FAMA antigen sheet are all negative, and the ratio (B / A) is 1. It is generally believed that the ratio of the detection titer of the test method to be evaluated to the detection titer of the FAMA antigen sheet as the gold standard needs to be within ±2 times for positive and more than 90% for negative. The determination result of the test method to be evaluated is within the acceptable range of VZV antibody titer detection, meets the requirements of conventional detection standards, and has good specificity and high positive and negative detection conformity. Therefore, the titer determination results of the high-throughput FAMA virus plate and the FAMA antigen sheet have a high overall compliance rate. The high-throughput FAMA virus plate of the present invention meets the requirements of the detection standard, has good specificity, sensitivity and compliance rate, and can be applied to actual VZV antibody detection.

[0116] Table 7 Correspondence test results

[0117]

[0118] The specific embodiments of the present invention have been described in detail so that those skilled in the art will be easily understood. However, according to all the descriptions disclosed, different modifications or replacements can be made to those details, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for preparing a high-throughput FAMA virus plate, characterized in that: The high-throughput FAMA virus plate is prepared by coating a cell culture plate with diseased cells infected by VZV, wherein the cell culture plate comprises a 96-well cell plate; it is used to detect VZV antibodies, wherein the diseased cells are used as coating antigens, and uninfected susceptible cells are used as negative controls of coating antigens; the preparation steps include: 1) After infecting susceptible cells with VZV, virus culture is performed; the susceptible cells are MRC-5 cells; 2) When the pathological rate of susceptible cells reaches an appropriate pathological stage, trypsin digestion is performed and the cell suspension is prepared after terminating the digestion; 3) After adjusting the cell suspension to a certain cell density, add the cell suspension to the corresponding wells of the cell culture plate to the plating density, and let it settle; the plating density is 10,000 to 30,000 cells / well; 4) Fixing with a fixative for 10 to 30 minutes, discarding the fixative, and washing; the fixative includes 1% to 4% formaldehyde or paraformaldehyde solution; 5) Block with blocking solution, discard the blocking solution, dry it and freeze it.

2. The method for preparing a high-throughput FAMA virus plate according to claim 1, characterized in that: The suitable pathological stage is a pathological rate of 60% to 80%; the blocking solution includes 5% to 12% animal serum, and the blocking time is 6 to 16 hours.

3. The method for preparing a high-throughput FAMA virus plate according to claim 2, characterized in that: The appropriate pathological stage is a pathological rate of 70%; the plating density is 15,000 per well; the fixing solution is 2% formaldehyde solution, fixed for 20 minutes; and the blocking solution is 10% animal serum.

4. A high-throughput FAMA virus plate, characterized in that: The high-throughput FAMA virus plate is prepared by the preparation method of the high-throughput FAMA virus plate described in any one of claims 1 to 3.

5. A method for detecting the homogeneity of a FAMA virus plate, characterized in that: The detection object is the high-throughput FAMA virus plate according to claim 4, and the steps include: 1) Stain with nuclear staining reagent; 2) Discard the nuclear staining reagent, wash and pat dry; 3) After adding glycerol solution, cell counting was performed using a high-throughput microscope; 4) Calculate the CV value based on the cell counting results within the plate and / or between plates; 5) analyzing the intra-plate and / or inter-plate homogeneity of the high-throughput FAMA virus plate based on the CV value; Among them, if the CV value is less than 20%, the high-throughput FAMA virus plate has good intra-plate and / or inter-plate uniformity.

6. The method for detecting the homogeneity of the FAMA virus plate according to claim 5, characterized in that: The nuclear staining reagent includes one or two of Hoechest, DAIP or Evans blue, which are used after being diluted at 1:500-1:2000; and the staining time does not exceed 30 minutes.

7. The method for detecting the homogeneity of the FAMA virus plate according to claim 5, characterized in that: When performing the cell counting, a high-throughput microscope is aimed at the central field of view of the sampling wells on the high-throughput FAMA virus plate, and at least one field of view under the same magnification is selected for cell counting. For each high-throughput FAMA virus plate, cell wells of no less than 10% of the total number of wells are selected for cell counting.

8. A kit, characterized in that: The invention comprises a VZV antibody positive quality control product and the high-throughput FAMA virus plate described in claim 4, and is used for detecting VZV antibodies.

9. The kit according to claim 8, characterized in that: The titer of the VZV antibody positive quality control product was 64±2.

10. Use of the high-throughput FAMA virus plate according to claim 4, or the kit according to any one of claims 8 to 9, in the preparation of products related to VZV antibody detection.

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