Screening method for raw plasma containing high-titer anti-hcmv neutralizing antibodies and method for detecting anti-hcmv neutralizing antibody titer

Abstract

The application discloses a screening method of raw blood plasma containing high-titer anti-HCMV neutralizing antibodies and a detection method of anti-HCMV neutralizing antibody titer, and belongs to the technical field of blood products. The screening method and the detection method provided by the application are both based on a fluorescence immunodot test, and the fluorescence immunodot test is performed on a HCMV virus diluent which can be exactly neutralized by anti-HCMV immunoglobulin national standard containing a known amount of anti-HCMV neutralizing antibodies and a to-be-detected blood plasma sample diluted according to a determined multiple or a to-be-detected sample diluted in a series of gradients. The method provided by the application can obtain a result in about 48 hours, and the result data is collected and analyzed by an immunodot analyzer, without naked-eye observation, so that the determination of the result is more objective and accurate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of blood product detection, and particularly relates to a screening method of raw plasma containing high-titer anti-HCMV (human cytomegalovirus) neutralizing antibodies and a detection method of the titer of anti-HCMV neutralizing antibodies in a sample, and especially relates to a rapid, objective and accurate screening method of raw plasma containing high-titer anti-HCMV neutralizing antibodies for preparing cytomegalovirus immunoglobulin and a rapid, objective and accurate detection method of the titer of anti-HCMV neutralizing antibodies in a sample (such as a blood product, raw plasma or a combination thereof). BACKGROUND

[0002] Human cytomegalovirus (HCMV) is a DNA virus of the β genus of the Herpesviridae family, and is extremely common in the human population, and most of the infections are asymptomatic latent infections. However, for immunocompromised populations (such as bone marrow and solid organ transplant recipients, HIV-infected persons and tumor patients), HCMV infection can cause serious HCMV clinical complications (such as pneumonia, hepatitis, nephritis, etc.), and can also lead to organ transplant failure; primary or recurrent HCMV infection in pregnant women can cause intrauterine infection or perinatal infection in newborns, and can cause serious consequences such as miscarriage, stillbirth, fetal malformation, developmental retardation, etc. Anti-viral drugs combined with cytomegalovirus-specific human immunoglobulin (CMVIG) are widely used in foreign clinics to prevent and treat postoperative HCMV infection and related diseases in solid organ transplant patients.

[0003] As an effective means for preventing and treating HCMV infection, there are CMVIG-related products on the market abroad, but there are currently no products on the market in China. CMVIG is an immunoglobulin preparation prepared by mixing healthy human plasma containing high-titer anti-HCMV neutralizing antibodies obtained by a certain screening method and the corresponding screening standard, and using the Cohn low-temperature ethanol method and virus inactivation process. Therefore, the establishment of a screening method for raw plasma containing high-titer anti-HCMV neutralizing antibodies is a prerequisite and guarantee for the preparation of CMVIG.

[0004] Currently, the detection methods for anti-HCMV antibody titer level generally include ELISA method, microcytopathic neutralization test method and rapid micro-neutralization test method. The antigen-antibody reaction of the ELISA method cannot reflect the true level of the anti-HCMV neutralizing antibody titer in raw plasma and other samples, and thus is not suitable for the detection of anti-HCMV neutralizing antibody titer in raw plasma and other samples and the screening of healthy human plasma containing high-titer anti-HCMV neutralizing antibodies (Yang Chun et al., Establishment and application of rapid micro-detection method for human cytomegalovirus neutralizing antibodies, Chinese Journal of Transfusion, February 2012, Vol. 25, No. 2, hereinafter referred to as Document 1). The microcytopathic neutralization test method is a relatively intuitive antibody neutralization titer determination method, but the experimental period is relatively long (see Document 1), and the results usually cannot be determined until 7-10 days later (for example, CN103554252A, hereinafter referred to as Document 2). The rapid micro-neutralization test method (see Document 1) can greatly shorten the experimental period (the detection results can be obtained in about 48 h) compared with the microcytopathic neutralization test method, but the determination of the results of the two test methods is based on the observation of cytopathic effect under a microscope, and the professional ability and experience of the experimenters are required to be high, and the subjectivity is relatively strong. SUMMARY

[0005] In view of one or more of the problems existing in the prior art, one aspect of the present application provides a screening method for raw plasma containing high-titer anti-HCMV neutralizing antibodies, which comprises the following steps:

[0006] S1: Prepare a portion of HCMV virus liquid (for example, the titer of the HCMV virus liquid is 4000-6000 TCID 50 / 0.1 mL of HCMV virus liquid), which can detect the expression of HCMV gB glycoprotein by fluorescence immunodot assay after infecting MRC-5 cells for 48 h, and the HCMV virus liquid can be exactly neutralized by an equal volume of anti-HCMV immunoglobulin national standard containing a known amount of anti-HCMV neutralizing antibodies (for example, about 25 U / mL);

[0007] S2: According to the known amount of anti-HCMV neutralizing antibodies that can exactly neutralize the HCMV virus liquid in step S1 and the screening standard for raw plasma containing high-titer anti-HCMV neutralizing antibodies, the dilution multiple of the to-be-tested plasma sample is determined, and all to-be-tested plasma samples are diluted according to the determined dilution multiple to obtain to-be-tested plasma sample dilutions;

[0008] S3: Mix the to-be-tested plasma sample obtained in step S2 with an equal volume of HCMV virus liquid thoroughly to obtain a virus-sample mixture, and place it in an incubator for incubation; at the same time, set a virus control with only the HCMV virus liquid and a blank control with only the culture solution;

[0009] S4: Take the virus-sample mixture solution after the end of step S3, the virus control and the blank control, and add MRC-5 cell suspension (1-2x10 4 cells per well) to obtain a culture system, and culture in an incubator for 48±2h;

[0010] S5: After the end of step S4, the culture system is subjected to the following operations: removal of the culture solution in the culture system, first PBS rinsing, fixation, second PBS rinsing, permeation, third PBS rinsing, blocking with blocking solution and incubation at room temperature to obtain an incubation system;

[0011] S6: Add anti-HCMV gB glycoprotein antibody as specific primary antibody to the incubation system of step S5, and after incubation and rinsing, add the corresponding secondary antibody with fluorescent labeling, and again perform incubation and rinsing to obtain the observation plate of the plasma sample to be tested;

[0012] S7: Place the observation plate of the plasma sample to be tested obtained in step S6 in an immunospot analyzer for counting. When a large number of fluorescent spots appear in the virus control well and the number of fluorescent spots in the blank control well does not exceed 20 (i.e. ≤20), the experiment is valid. The sample detection result determination standard is: if the number of fluorescent spots in the detection well corresponding to the plasma sample to be tested does not exceed 20, preferably no fluorescent spots appear, then the sample meets the plasma screening standard and is raw plasma containing high-titer anti-HCMV neutralizing antibodies; if the number of fluorescent spots in the detection well corresponding to the plasma sample to be tested is more than 20 (>20), then the sample does not meet the plasma screening standard and is raw plasma not containing high-titer anti-HCMV neutralizing antibodies.

[0013] In the above method, the fixed conditions in step S4 are: 0.18%-0.22% glutaraldehyde fixation for 50-70min.

[0014] In the above method, the specific operation of step S3 is: incubation in a 37±1℃, 5% CO2 incubator for 50-70min.

[0015] In the above method, the MRC-5 cell suspension in step S4 is obtained by: taking MRC-5 cells in the logarithmic growth phase, performing routine digestion using trypsin, adjusting the cell density to 1-2x10 5 cells / mL using MEM culture solution containing 5% fetal bovine serum to obtain the MRC-5 cell suspension; and / or the culture conditions in the incubator in step S4 are 37±1℃, 5% CO2.

[0016] In the method, the culture system in step S5 is subjected to the following operations: removing the culture solution in the culture system; PBS rinsing for 3 times; adding 0.18%-0.22% glutaraldehyde for fixation for 50-70 min; PBS rinsing for 3 times; adding 0.1% Triton-X 100 for permeation for 15 min, and PBS rinsing for 3 times; adding 200 μL blocking solution (containing 1% BSA in PBS) per well for blocking, and incubating at room temperature for 60 min.

[0017] In the method, the specific operation of step S6 is: adding anti-HCMV gB glycoprotein antibody as specific primary antibody to the incubation system of step S5, and incubating at 37°C for 120 min, and PBS rinsing for 3 times; adding fluorescently labeled corresponding secondary antibody, and incubating at 37°C in dark for 30 min, and PBS rinsing for 3 times, to obtain the observation plate of the to-be-tested plasma sample.

[0018] In the method, step S7 specifically uses Immunospot professional analysis software to perform fluorescent spot counting analysis on the ELISPOT image obtained by the immunospot analyzer.

[0019] Another aspect of the present application provides a method for detecting anti-HCMV neutralizing antibodies in a sample, which comprises the following steps:

[0020] T1: preparing a portion of HCMV virus liquid (virus titer can be known), which can detect the expression of HCMV gB glycoprotein by fluorescent immunospot test after infecting MRC-5 cells for 48 h, and the HCMV virus liquid can be exactly neutralized by using an equal volume of anti-HCMV immunoglobulin national standard containing a known amount of anti-HCMV neutralizing antibodies;

[0021] T2: performing a series of gradient dilutions on the to-be-tested sample, to obtain a series of gradient-diluted to-be-tested samples;

[0022] T3: mixing the series of gradient-diluted to-be-tested samples obtained in step T2 with an equal volume of HCMV virus liquid respectively, to obtain virus-sample mixed liquids, and placing the virus-sample mixed liquids in an incubator for incubation; meanwhile, setting a virus control with only the HCMV virus liquid and a blank control with only the culture solution;

[0023] T4: taking the virus-sample mixed liquid, the virus control and the blank control after the incubation in step T3 is completed, and adding MRC-5 cell suspension (1-2×10 4 cells per well) to the virus-sample mixed liquid, the virus control and the blank control, to obtain culture systems, and culturing the culture systems in an incubator for 48±2 h;

[0024] T5: After the end of step T4 culture, the culture system is subjected to the following operations: removing the culture solution in the culture system, first PBS rinsing, fixing, second PBS rinsing, permeation, third PBS rinsing, blocking with blocking solution, and room temperature incubation, to obtain an incubation system;

[0025] T6: Anti-HCMV gB glycoprotein antibody is added to the incubation system of step T5 as specific primary antibody, after incubation and rinsing, the corresponding secondary antibody with fluorescent label is added, and incubation and rinsing are performed again, to obtain an observation plate of serially gradient-diluted samples to be tested;

[0026] T7: The observation plate of serially gradient-diluted samples to be tested obtained in step T6 is placed in an immunospot analyzer for counting. When a large number of fluorescent spots appear in the virus control well, and the number of fluorescent spots appearing in the blank control well is not more than 20, the experiment is valid. The sample detection result determination standard is as follows: the wells with 0-20 (0, exactly no) fluorescent spots appearing in the sample detection well are recorded, the highest dilution multiple of the sample well corresponding to the sample to be tested is determined, and the titer of anti-HCMV neutralizing antibody in the sample is calculated in combination with the known amount of anti-HCMV neutralizing antibody.

[0027] In the above method, the sample can include blood products, raw plasma, or a combination thereof.

[0028] In the above method, the observation plate can be a 96-well plate or an ELISPOT plate, and different wells in the plate can be the same sample or different samples.

[0029] The screening method and the detection method of the anti-HCMV neutralizing antibody titer in the sample (e.g., blood products, raw plasma or a combination thereof) provided based on the above technical scheme are both based on the principle of fluorescent immunospot test. The screening method provided utilizes the HCMV virus liquid which can be exactly neutralized by the anti-HCMV immunoglobulin national standard containing a known amount of anti-HCMV neutralizing antibody to perform the fluorescent immunospot test with the plasma sample to be detected diluted according to a determined multiple. The detection method of the anti-HCMV neutralizing antibody titer provided utilizes the HCMV virus liquid which can be exactly neutralized by the anti-HCMV immunoglobulin national standard containing a known amount of anti-HCMV neutralizing antibody to perform the fluorescent immunospot test with the sample to be detected diluted in a series of gradient. The whole screening process or detection process only needs about 48 hours to screen the raw plasma containing high-titer anti-HCMV neutralizing antibody or detect the anti-HCMV neutralizing antibody titer in the sample, and the result data are collected and analyzed by the immunospot analyzer without the need of naked eye observation, and the determination of the result is more objective and accurate. Therefore, the method provided by the present application has the advantages of rapidness (about 48 hours) and objectivity and accuracy compared with the rapid micro-neutralization test method provided by the above document 1 and the micro-cellular pathogenic neutralization test method provided by the above document 2, and is more suitable for screening the raw plasma containing high-titer anti-HCMV neutralizing antibody or detecting the anti-HCMV neutralizing antibody titer in the sample. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The fluorescent image observed when the fixed conditions for the fluorescent immunospot test are optimized.

[0031] Figure 2 The fluorescent spot image collected and obtained by the immunospot analyzer when the raw plasma containing high-titer anti-HCMV neutralizing antibody is screened according to one embodiment of the present application.

[0032] Figure 3 The fluorescent spot image collected and obtained by the immunospot analyzer when the anti-HCMV neutralizing antibody titer level in the sample is detected according to one embodiment of the present application. DETAILED DESCRIPTION

[0033] In view of the defects of the prior art that the method for detecting the titer level of anti-HCMV neutralizing antibody in raw plasma sample and the method for screening or detecting raw plasma containing high-titer anti-HCMV neutralizing antibody are time-consuming and the result determination is subjective, the present application provides a screening method for raw plasma containing high-titer anti-HCMV neutralizing antibody and a method for detecting the titer level of anti-HCMV neutralizing antibody in a sample based on a fluorescent immunospot test, and the result data is collected and analyzed by an immunospot analyzer, so the method is more objective and accurate than the microcytopathic neutralization test or the fast micro-neutralization test method for observing cytopathic changes with the naked eye, and the method provided by the present application can obtain results in about 48 hours, and can achieve the same fast effect as the fast micro-neutralization test method.

[0034] The content of the present application will be described in detail below in combination with specific examples and drawings.

[0035] In the following, only some exemplary embodiments are simply described, the embodiments of each serial number are helpful for reading, and each can be independently implemented and implemented in association to constitute a strong support for the content of the present application. As can be recognized by those skilled in the art, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present application. Therefore, the drawings and the description are considered to be essentially exemplary rather than limiting.

[0036] The methods used in the following examples are all conventional methods unless otherwise specified, and the specific steps can be referred to: Molecular Cloning Experiment Guide (Molecular Cloning: A Laboratory Manual, Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0037] The obtaining path of various biological materials described in the examples is only provided as a path for experimental obtaining to achieve the specific disclosed purpose, and should not be a limitation on the source of biological materials of the present application. In fact, the source of the biological materials used is extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the examples.

[0038] Example 1: Determination of fixation conditions in fluorescent immunospot test

[0039] This example uses HCMV virus liquid to infect MRC-5 cells, and uses 4% paraformaldehyde, 0.1% glutaraldehyde plus 1% formaldehyde, 0.2% glutaraldehyde, and 1% formaldehyde as fixing liquids (all of which are commonly used in the art) to fix the cells, respectively. By comparing the expression of HCMV gB glycoprotein under different conditions, the optimal fixing conditions for the fluorescent immunospot assay for anti-HCMV neutralizing antibodies in the present application are selected, which specifically include the following steps.

[0040] (1) Take HCMV virus liquid (measured virus titer is 10 6.85 TCID 50 / 0.1mL, AD169 strain, China Institute for Control of Drug and Food), dilute 64 times with MEM culture solution (Gibico) to obtain HCMV virus diluent. Add 50 μL of HCMV virus diluent to each well of a 96-well culture plate, and add 50 μL of MEM culture solution. At the same time, set up a blank control with only culture solution.

[0041] (2) Take MRC-5 cells in logarithmic growth phase (purchased from ATCC), and perform routine digestion with trypsin. Adjust the cell density to 2×10 5 cells / mL with MEM culture solution containing 5% fetal bovine serum to obtain a cell suspension. Add 100 μL of the cell suspension to each well of the 96-well culture plate in step (2), i.e., 2×10 4 cells per well. Place the 96-well culture plate in a 37°C, 5% CO2 incubator for 48 h.

[0042] (3) After 48 h of culture, remove the culture solution in the wells, and rinse with PBS for 3 times. Fix for 5 min with 0.1% glutaraldehyde plus 1% formaldehyde, for 15 min with 4% paraformaldehyde, for 60 min with 0.2% glutaraldehyde, and for 60 min with 1% formaldehyde, respectively. After the fixation, rinse with PBS for 3 times. Add blocking solution (PBS containing 1% BSA) to block, and incubate at room temperature for 60 min. Rinse with PBS for 3 times. Add 100 μL of specific primary antibody (purified anti-HCMV gB glycoprotein antibody (mouse monoclonal antibody, 1:800 dilution), purchased from abcam) to each well, and incubate at 37°C for 120 min. Rinse with PBS for 3 times. Add 100 μL of fluorescently labeled corresponding secondary antibody (FITC-labeled goat anti-mouse IgG, 1:500 dilution, purchased from Biyun Tian Company) to each well, and incubate at 37°C for 30 min in the dark. Rinse with PBS for 3 times. After the last rinsing, discard the PBS.

[0043] (4) Place the 96-well culture plate under a fluorescence microscope to observe the expression of HCMV gB glycoprotein under different fixing conditions.

[0044] The results are as follows Figure 1 As shown, A represents the observation results after fixation with 0.1% glutaraldehyde and 1% formaldehyde for 5 min; B represents the observation results after fixation with 4% paraformaldehyde for 15 min; C represents the observation results after fixation with 0.2% glutaraldehyde for 60 min; D represents the observation results after fixation with 1% formaldehyde for 60 min; and E represents the observation results of the blank control. Figure 1 As can be seen, no fluorescence was observed in the blank control well (E); when fixed with 0.2% glutaraldehyde for 60 min (C), a large amount of green fluorescence was observed under a fluorescence microscope; while no fluorescence was observed when using the other fixation conditions (A, B, D). This indicates that when using 0.2% glutaraldehyde for 60 min as the fixation condition, the expression of HCMV gB glycoprotein can be detected, while the expression of HCMV gB glycoprotein cannot be detected when using other conventional fixatives (e.g., 0.1% glutaraldehyde plus 1% formaldehyde, 4% paraformaldehyde, or 1% formaldehyde). Therefore, in the screening or detection system for anti-HCMV neutralizing antibodies based on fluorescence immunospot assay established in this invention, 0.2% glutaraldehyde fixation for 60 min can be used as the fixation condition for the experiment.

[0045] In this embodiment, the inventors also tested the following fixation conditions: 0.2% glutaraldehyde fixation for 50 min, 0.2% glutaraldehyde fixation for 70 min, 0.18% glutaraldehyde fixation for 60 min, and 0.22% glutaraldehyde fixation for 60 min. The results showed that the expression of HCMV gB glycoprotein could be detected under all of the above fixation conditions, and therefore all can be used as fixation conditions based on the fluorescent immunospot assay established in this invention. These conditions will not be elaborated further here. Based on the results of the above embodiments, in the screening or detection system for anti-HCMV neutralizing antibodies based on the fluorescent immunospot assay established in this invention, 0.18%-0.22% glutaraldehyde can be used as the fixative, and the fixation time can be 50-70 min.

[0046] Example 2: Screening method for plasma samples containing high-titer anti-HCMV neutralizing antibodies

[0047] This embodiment is based on the principle of fluorescent immunospot assay and the fixation conditions determined in Example 1 above, using a titer of 5000 TCID. 50 The process involves using 0.1 mL of HCMV virus solution and an anti-HCMV immunoglobulin standard (purchased from the China National Institutes for Food and Drug Control) containing 25 U / mL of anti-HCMV neutralizing antibodies to screen for plasma containing high-titer anti-HCMV neutralizing antibodies (titer higher than 200 U / mL). The specific steps include:

[0048] (1) Prepare a titer of 5000 TCID50 / 0.1 mL of HCMV virus liquid, which can detect the expression of HCMV gB glycoprotein by fluorescence immunodot test after infecting MRC-5 cells for 48 h, can be neutralized by the national standard of anti-HCMV immunoglobulin containing 25 U / mL of anti-HCMV neutralizing antibody.

[0049] (2) The plasma sample containing high-titer anti-HCMV neutralizing antibody (titer higher than 200 U / mL) is screened, and the dilution multiple of the plasma sample is calculated according to the formula: plasma dilution multiple = high-titer plasma screening standard (200 U / mL) / anti-HCMV neutralizing antibody amount (25 U / mL) that can just neutralize the HCMV virus liquid, which is 8 times.

[0050] (3) Three plasma samples to be tested (provided by Shenzhen Weiguang Bioproducts Co., Ltd., numbered 1, 2, and 3, respectively) are subjected to complement inactivation at 56°C water bath for 30 min. The plasma sample after complement inactivation is diluted 8 times with MEM culture solution to obtain the diluted plasma sample to be tested. 50 μL of the diluted plasma sample to be tested and an equal volume of HCMV virus liquid (5000 TCID 50 / 0.1 mL) are added to the wells of a 96-well culture plate and mixed well, and then incubated in a 37°C, 5% CO2 incubator for 60 min. At the same time, a virus control with only HCMV virus liquid and a blank control with only culture solution are set.

[0051] (4) Logarithmic growth phase MRC-5 cells are routinely digested with trypsin, and the cell density is adjusted to 2×10 5 / mL using MEM culture solution containing 5% fetal bovine serum as a cell suspension. After the incubation in step (3) is completed, 100 μL of the cell suspension is added to the virus-plasma mixture wells, virus control wells, and blank control wells, i.e., 2×10 4 / mL of cells per well. The 96-well culture plate is incubated in a 37°C, 5% CO2 incubator for 48 h.

[0052] (5) After culturing for 48 h, aspirate the culture medium from the wells and wash three times with PBS. Add 0.2% glutaraldehyde for fixation for 60 min, and wash three times with PBS. Add 0.1% Triton-X 100 for permeation for 15 min, and wash three times with PBS. Add blocking buffer (PBS containing 1% BSA) to the wells for blocking, incubate at room temperature for 60 min, and wash three times with PBS. Add 100 μL of specific primary antibody (purified anti-HCMV gB glycoprotein antibody, 1:800 dilution) to each well, incubate at 37 °C for 120 min, and wash three times with PBS. Add 100 μL of the corresponding fluorescently labeled secondary antibody (FITC-labeled goat anti-mouse IgG, 1:500 dilution) to each well, incubate at 37 °C in the dark for 30 min, and wash three times with PBS. After the last wash, discard the PBS.

[0053] (6) Place the 96-well culture plate in an immunospot analyzer for counting. Analyze the ELISPOT images using Immunospot professional analysis software. The experiment is considered successful when a large number of fluorescent spots appear in the virus control wells and the number of fluorescent spots in the blank control wells does not exceed 20. Sample detection result judgment criteria: If the number of fluorescent spots in the detection wells corresponding to the plasma sample to be tested does not exceed 20, then the sample meets the plasma screening criteria and is raw plasma containing high-titer anti-HCMV neutralizing antibodies; if the number of fluorescent spots in the detection wells corresponding to the plasma sample to be tested is more than 20, then the sample does not meet the plasma screening criteria and is raw plasma without high-titer anti-HCMV neutralizing antibodies.

[0054] The results are as follows Figure 2 As shown, A represents the fluorescent spot image of the blank control, B represents the fluorescent spot image of the virus control, and C and E represent the fluorescent spot images of plasma samples 1, 2, and 3, respectively. The numbers in the figure (e.g., 2, 370, 8, 12, 353) indicate the number of fluorescent spots analyzed using Immunospot professional analysis software on the ELISPOT images. Figure 2It can be seen that a large number of fluorescent spots (370) appeared in the virus control hole (B), and only a small amount of fluorescent spots (2, not more than 20) appeared in the blank control hole (A), and the experiment was established. The number of fluorescent spots appearing in the detection hole (C) corresponding to the first plasma sample and the detection hole (D) corresponding to the second plasma sample in the 3 plasma samples was 8 and 12 respectively, both of which were not more than 20, indicating that the anti-HCMV neutralizing antibody titer of the 2 plasma samples diluted 8 times was higher than 25 U / mL, that is, the anti-HCMV neutralizing antibody titer in the 2 plasma samples was higher than 200 U / mL, which was the plasma containing high-titer anti-HCMV neutralizing antibody; while the number of fluorescent spots appearing in the detection hole (E) corresponding to the third plasma sample was 353, indicating that the anti-HCMV neutralizing antibody titer of the plasma sample diluted 8 times was lower than 25 U / mL, that is, the anti-HCMV neutralizing antibody titer in the plasma sample was lower than 200 U / mL, which did not meet the blood screening standard.

[0055] Example 3: Detection method of anti-HCMV neutralizing antibody titer in plasma sample

[0056] This example is based on the principle of fluorescent immunospot test and the fixed conditions determined in the above example 1, using HCMV virus liquid with a titer of 5000 TCID 50 / 0.1 mL and anti-HCMV immunoglobulin standard containing anti-HCMV neutralizing antibody with a neutralizing amount of 25 U / mL, the anti-HCMV neutralizing antibody titer of the plasma sample to be tested is detected, which specifically includes the following steps.

[0057] (1) Prepare HCMV virus liquid with a titer of 5000 TCID 50 / 0.1 mL, which can detect the expression of HCMV gB glycoprotein by fluorescent immunospot test after infecting MRC-5 cells for 48 h, and can be neutralized by anti-HCMV immunoglobulin national standard containing anti-HCMV neutralizing antibody with a neutralizing amount of 25 U / mL.

[0058] (2) The plasma sample (provided by Shenzhen Weiguang Bioproducts Co., Ltd.) was subjected to complement inactivation at 56℃ water bath for 30 min. The complement-inactivated plasma sample was gradiently diluted with MEM culture solution to obtain 8-fold, 16-fold, 32-fold, 64-fold, 128-fold and 256-fold diluted plasma sample diluents to be tested. 50 μL of each dilution of the plasma sample diluent to be tested was added to the wells of a 96-well culture plate in an equal volume of HCMV virus liquid (5000 TCID 50 / 0.1 mL) and mixed well, and then incubated in a 37℃, 5% CO2 incubator for 60 min. At the same time, virus control with only HCMV virus liquid and blank control with only culture solution were set.

[0059] (3) MRC-5 cells in the logarithmic growth phase were routinely digested with trypsin and the cell density was adjusted to 2 × 10⁶ cells using MEM culture medium containing 5% fetal bovine serum. 5 Cells / mL were used as cell suspension. After the above step (2) was completed, 100 μL of cell suspension was added to the virus-plasma mixture wells, virus control wells, and blank control wells, respectively, so that the cell volume per well was 2 × 10⁶ cells / mL. 4 Each 96-well culture plate was placed in a 37°C, 5% CO2 incubator and incubated for 48 hours.

[0060] (4) After culturing for 48 h, aspirate the culture medium from the wells and wash three times with PBS. Add 0.2% glutaraldehyde for fixation for 60 min, and wash three times with PBS. Add 0.1% Triton-X 100 for permeation for 15 min, and wash three times with PBS. Add blocking buffer (PBS containing 1% BSA) to the wells for blocking, incubate at room temperature for 60 min, and wash three times with PBS. Add 100 μL of specific primary antibody (purified anti-HCMV gB glycoprotein antibody, 1:800 dilution) to each well, incubate at 37 °C for 120 min, and wash three times with PBS. Add 100 μL of the corresponding fluorescently labeled secondary antibody (FITC-labeled goat anti-mouse IgG, 1:500 dilution) to each well, incubate at 37 °C in the dark for 30 min, and wash three times with PBS. After the last wash, discard the PBS.

[0061] (5) Place the 96-well culture plate in an immunospot analyzer for counting. Analyze the ELISPOT images using Immunospot professional analysis software. The experiment is considered successful when a large number of fluorescent spots appear in the virus control wells and the number of fluorescent spots in the blank control wells does not exceed 0. Sample detection result judgment criteria: Record the wells with no more than 20 fluorescent spots in the sample detection wells, determine the highest dilution factor of the sample corresponding to no more than 20 fluorescent spots in the test sample wells, and calculate the titer of the anti-HCMV neutralizing antibody in the sample based on the known amount of anti-HCMV neutralizing antibody (25 U / mL).

[0062] The results are as follows Figure 3 As shown, A represents the fluorescent spot image of the blank control, B represents the fluorescent spot image of the virus control, and CH represent the fluorescent spot images of plasma samples diluted 8-fold, 16-fold, 32-fold, 64-fold, 128-fold, and 256-fold, respectively. The numbers in the figure (e.g., 1, 336, 1, 1, 98, 176, 259, 291) indicate the number of fluorescent spots analyzed using Immunospot professional analysis software on the ELISPOT images. Figure 3It can be seen that a large number of fluorescent spots (336) appear in the virus control hole (B), and only a small number of fluorescent spots (1, not more than 20) appear in the blank control hole (A), and the experiment is established. The detection hole (C) corresponding to the 8-fold diluted plasma sample and the detection hole (D) corresponding to the 16-fold diluted plasma sample each have only one spot, not more than 20; the detection hole (E) corresponding to the 32-fold diluted plasma sample, the detection hole (F) corresponding to the 64-fold diluted plasma sample, the detection hole (G) corresponding to the 128-fold diluted plasma sample, and the detection hole (H) corresponding to the 256-fold diluted plasma sample have 98, 176, 259 and 291 fluorescent spots respectively, all of which are much more than 20. This result shows that the plasma sample diluted by 16 times can still neutralize 5000 TCID 50 / 0.1 mL HCMV virus liquid, the amount of anti-HCMV neutralizing antibody in the plasma sample diluted by 16 times is higher than 25 U / mL. Combined with the amount of anti-HCMV neutralizing antibody in the national standard of anti-HCMV immunoglobulin that can exactly neutralize the HCMV virus liquid (25 U / mL), it is calculated that the titer of the anti-HCMV neutralizing antibody in the plasma sample detected in this example is 320 U / mL.

[0063] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A screening method of raw plasma containing high-titer anti-HCMV neutralizing antibodies, comprising the following steps: S1: preparing a dilution of HCMV virus which is just neutralized by an equal volume of anti-HCMV immunoglobulin national standard containing a known amount of anti-HCMV neutralizing antibodies; S2: determining the dilution factor of the raw plasma to be screened according to the known amount of anti-HCMV neutralizing antibodies and the screening standard of the titer of raw plasma containing high-titer anti-HCMV neutralizing antibodies, diluting all raw plasma to be screened according to the determined dilution factor to obtain a sample to be tested; S3: mixing the sample to be tested obtained in step S2 with an equal volume of the dilution of HCMV virus to obtain a virus-sample mixture, and incubating in an incubator; S4: taking the virus-sample mixture after the incubation in step S3, and adding an MRC-5 cell suspension to obtain a culture system, and culturing in an incubator for 48±2h; S5: after the culture in step S4, the culture system is subjected to the following operations: removing the culture solution in the culture system, first PBS rinsing, fixing, second PBS rinsing, permeation, third PBS rinsing, blocking with blocking solution, and incubation at room temperature to obtain an incubation system; S6: adding an anti-HCMV gB glycoprotein antibody as a specific primary antibody to the incubation system in step S5, and after incubation and rinsing, adding a corresponding secondary antibody labeled with fluorescence, and again incubating and rinsing to obtain a sample to be tested observation plate; S7: placing the sample to be tested observation plate obtained in step S6 in an immunospot analyzer for counting, and the raw plasma corresponding to the wells in which the number of fluorescent spots in the plate is not more than 20 is the raw plasma containing high-titer anti-HCMV neutralizing antibodies.

2. The screening method according to claim 1, wherein in step S7, the raw plasma corresponding to the wells in which no fluorescent spots appear in the plate is the raw plasma containing high-titer anti-HCMV neutralizing antibodies.

3. The screening method according to claim 1, wherein the fixing condition in step S5 is 0.18%-0.22% glutaraldehyde for 50-70min.

4. The screening method according to any one of claims 1-3, wherein the specific operation of incubating in an incubator in step S3 is incubating in a 37±1℃, 5% CO2 incubator for 0.9-1.1h.

5. The screening method according to any one of claims 1-3, wherein the MRC-5 cell suspension in step S4 is obtained by the following methods: taking MRC-5 cells in logarithmic growth phase, performing conventional digestion using trypsin, and adjusting the cell density using MEM culture solution containing 5% fetal bovine serum to obtain the MRC-5 cell suspension; and / or the culture system in step S4 is obtained by mixing the virus-sample mixture with an equal volume of the MRC-5 cell suspension; and / or the culture condition in the incubator in step S4 is 37±1℃, 5% CO2. ​ ​ ​ ​ ​ ​ ​ ​ ​ 6. The screening method according to any one of claims 1-3, wherein in step S5, the culture system is subjected to the following operations: removing the culture solution in the culture system, PBS rinsing for 3 times; adding 0.18%-0.2% glutaraldehyde for fixation for 50-70 min; rinsing with PBS for 3 times; adding 0.1% Triton-X 100 for permeation for 15 min, rinsing with PBS for 3 times; adding 200 μL / well blocking solution for blocking, and incubating at room temperature for 60 min.

7. The screening method according to any one of claims 1-3, wherein in step S6, the following operations are performed: adding anti-HCMV gB glycoprotein antibody as specific primary antibody to the incubation system of step S5, incubating at 37°C for 120 min, rinsing with PBS for 3 times; adding fluorescently labeled corresponding secondary antibody, incubating at 37°C in the dark for 30 min, rinsing with PBS for 3 times, and obtaining the observation plate of the plasma sample to be detected.

8. The screening method according to any one of claims 1-3, wherein in step S7, the ELISPOT image obtained by the immunospot analyzer is subjected to fluorescent spot counting analysis using Immunospot analysis software.

9. A method for detecting anti-HCMV neutralizing antibodies in a sample, comprising the following steps: T1: preparing an HCMV virus diluent which is just neutralized with an equal volume of anti-HCMV immunoglobulin national standard containing a known amount of anti-HCMV neutralizing antibodies; T2: gradient diluting the sample to obtain a series of gradient-diluted samples to be detected; T3: mixing the series of gradient-diluted samples to be detected obtained in step T2 with an equal volume of the HCMV virus diluent respectively to obtain virus-sample mixtures, and placing in an incubator for incubation; T4: taking the virus-sample mixture after the incubation in step T3 is completed, and adding MRC-5 cell suspension thereto to obtain a culture system, and culturing in an incubator for 48±2 h; T5: after the culture in step T4 is completed, the culture system is subjected to the following operations: removing the culture solution in the culture system, first PBS rinsing, fixation, second PBS rinsing, permeation, third PBS rinsing, blocking with blocking solution, and incubation at room temperature to obtain an incubation system; T6: adding anti-HCMV gB glycoprotein antibody as specific primary antibody to the incubation system of step T5, after incubation and rinsing, adding fluorescently labeled corresponding secondary antibody, and again performing incubation and rinsing to obtain the observation plate of the series of gradient-diluted samples to be detected; T7: placing the observation plate of the series of gradient-diluted samples to be detected obtained in step T6 in an immunospot analyzer for counting, recording the highest dilution fold of the sample corresponding to the well in which the number of fluorescent spots is 0-20, and calculating the titer of anti-HCMV neutralizing antibodies in the sample in combination with the known amount of anti-HCMV neutralizing antibodies.

10. The detection method according to claim 9, wherein the sample comprises blood products, raw plasma, or a combination thereof.

11. The method according to claim 1 or 9, wherein the observation plate is a 96-well plate or an ELISPOT plate, and different wells in the plate are the same sample or different samples.

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