Antigen stabilizer, its application and kit

Abstract

The present invention relates to the field of biotechnology, and more particularly to antigen stabilizers, their applications, and kits. The present invention provides antigen stabilizers, combination reagents, and their applications, as well as an in vitro diagnostic kit. The present invention overcomes the shortcomings of the prior art of respiratory syncytial virus, such as poor heat resistance during storage and large batch-to-batch variability. Furthermore, the antigen stabilizers and combination reagents provided by the technical solutions of the present invention can be widely used in RSV antibody detection reagents, providing enhanced reagent stability and specificity, effectively extending the shelf life of the reagents.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an antigen stabilizer, application thereof, and a kit. Background Art

[0002] RSV was first isolated from a chimpanzee with a cold in 1956, and in 1957 the virus was isolated from the respiratory tract of a child with pneumonia using cell culture technology.

[0003] Respiratory syncytial virus (RSV) is a filamentous, enveloped, negative-sense, single-stranded RNA virus belonging to the genus Orthopneumovirus of the family Pneumoviridae. RSV replicates briefly in epithelial cells of the nasopharynx and upper respiratory tract before spreading to the small bronchioles or alveoli of the lower respiratory tract. The host immune response to RSV infection increases mucus production and inflammation, leading to airway narrowing and potentially causing bronchiolitis in young children and acute respiratory illness in the elderly or those with underlying chronic diseases.

[0004] Due to the lipid envelope structure of respiratory syncytial virus (RSV), it is sensitive to ether or other lipid solvents. RSV is very sensitive to changes in the environment. After exposure to 55°C for 5 minutes, only 10% of RSV still maintains infectious activity; after 48 hours at room temperature, RSV's infectious activity drops to 10%; after 7 days at 4°C, RSV only retains 1% infectious activity. After each freeze-thaw cycle, the titer of RSV's infectious activity decreases by approximately 90%. Chloroform and detergents (such as 0.1% sodium deoxycholate, sodium dodecyl sulfate (SDS), and Triton X-100) can quickly inactivate RSV. RSV's ability to survive in the environment depends in part on the drying time and environmental humidity. Quick freezing of specimens in ethanol or dry ice and adding stabilizers (such as glycerol or sucrose) to the freezing solution can enhance the long-term storage of RSV.

[0005] RSV is very unstable and requires special handling. Clinical specimens for culture should be transported within a cold chain and promptly inoculated into sensitive cell lines. Sensitivity decreases after 1 hour at 37°C, and viral viability is completely lost if the virus is frozen and thawed and then slowly frozen to -20°C. Specimens should be inoculated into culture medium within 4 hours of collection. Specimens that cannot be processed promptly should be snap-frozen in an ethanol and dry ice bath. Adding sucrose or glycerol to the cryopreservation medium can increase the recovery rate of RSV after cryopreservation.

[0006] In vitro cell culture is an effective method for amplifying RSV; however, the unstable nature of RSV increases the difficulty of in vitro cell culture of RSV.

[0007] For primary culture, human aneuploid cells, such as HEP-2, HeLa, and A549 cells, are often the first choice for viral amplification. Other cell lines, such as human kidney cells, monkey kidney cells, and diploid fibroblasts, can also be used for culture.

[0008] Regarding the timing of virus harvest, cell lines may develop characteristic syncytial cytopathic effects 3–7 days after culture. However, the extent of syncytial formation depends on the cell type, confluency of the monolayer, the virus strain, and the multiplicity of infection (MOI). Consequently, the timing of RSV harvest using conventional culture methods can vary widely. This variability is partially due to: 1. decreased viral viability; 2. the need for experienced technicians; and 3. different cell lines used in different laboratories.

[0009] There are two main types of processing techniques after collecting the virus: collecting cultured cells and collecting culture supernatant.

[0010] Methods for detecting respiratory syncytial virus infection include nucleic acid testing, antigen detection, serological specific antibody testing, and virus isolation and culture. A positive serum-specific IgM antibody test is crucial for etiological diagnosis. Currently, methods for detecting respiratory syncytial virus (RSV) infection in China include electron microscopy, culture identification, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), agglutination, and fluorescent immunoassays. A four-component capture kit is used to detect IgM antibodies in serum / plasma. The kit includes magnetic microparticles conjugated to anti-human IgM antibodies, a RSV antigen solution, a horseradish peroxidase-labeled anti-RSV antibody solution (enzyme conjugate solution), and a sample diluent. The RSV antigen solution is prepared from naturally cultured inactivated RSV antigen and a buffer solution. However, different batches of the kit exhibit poor specificity and high background signal when testing clinical samples, primarily due to differences in antigen batches.

[0011] The existing antigen stabilizer formula cannot serve as a carrier or attachment protection for the virus, so its stability is poor. It cannot provide a buffer medium for the virus under heat conditions, causing the virus to become ineffective, and therefore it cannot play a stabilizing and protective role. Summary of the Invention

[0012] In view of this, the present invention provides an antigen stabilizer, its application, and a kit. The present invention provides an antigen stabilizer and a combination reagent and its application in an in vitro diagnostic kit. The kit of the present invention is simple and easy to prepare. Due to the thermal stability and improved specificity of its reagent components, it will not easily denature during storage and testing, resulting in product failure. Therefore, it is beneficial to ensure the accuracy of test results and effectively improve the problem of inter-batch variability of naturally cultured RSV virus antigens.

[0013] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0014] The present invention provides an antigen stabilizer, which comprises a buffer, a protein protectant, a stabilizer, a biocompatible polymer and a biopreservative;

[0015] The buffer comprises PBS buffer; the protein protective agent comprises bovine serum albumin; the stabilizer comprises sucrose; the biocompatible polymer comprises poloxamer; and the biological preservative comprises Proclin 300.

[0016] In some specific embodiments, the concentration of the PBS buffer is 0.02 to 0.1 mol / L, and the pH thereof is 6.5 to 8.0; and / or

[0017] The concentration of the bovine serum albumin is 5 to 50 g / L; and / or

[0018] The concentration of the sucrose is 10 to 50 g / L; and / or

[0019] The concentration of the poloxamer is 1 to 50 g / L; preferably, the molecular weight of the poloxamer is not less than 8000; and / or

[0020] The concentration of Proclin300 is 0.1-1 g / L.

[0021] Preferably, in some specific embodiments of the present invention, the concentration of bovine serum albumin is 10 g / L.

[0022] Preferably, in some specific embodiments of the present invention, the sucrose concentration is 15 g / L.

[0023] Preferably, in some specific embodiments of the present invention, the molecular weight of the poloxamer is 12600-14600, and its concentration is 1 g / L.

[0024] Preferably, in some specific embodiments of the present invention, the concentration of Proclin300 is 1 g / L.

[0025] The present invention also provides a combined reagent comprising the antigen stabilizer and an enzyme conjugate solution.

[0026] The enzyme conjugate solution includes sodium chloride.

[0027] The concentration of the sodium chloride is 0.15 mol / L to 1.0 mol / L; and / or

[0028] The concentration of the PBS buffer is 0.02 to 0.1 mol / L, and the pH thereof is 6.5 to 8.0; and / or

[0029] The concentration of the bovine serum albumin is 5 to 50 g / L; and / or

[0030] The concentration of the sucrose is 10 to 50 g / L; and / or

[0031] The concentration of the poloxamer is 1 to 50 g / L; preferably, the molecular weight of the poloxamer is not less than 8000; and / or

[0032] The concentration of Proclin300 is 0.1-1 g / L.

[0033] Preferably, in some specific embodiments of the present invention, the sodium chloride is not less than 0.3 mol / L.

[0034] Preferably, in some specific embodiments of the present invention, the concentration of bovine serum albumin is 10 g / L.

[0035] Preferably, in some specific embodiments of the present invention, the sucrose concentration is 15 g / L.

[0036] Preferably, in some specific embodiments of the present invention, the molecular weight of the poloxamer is 12600-14600, and its concentration is 1 g / L.

[0037] Preferably, in some specific embodiments of the present invention, the concentration of Proclin300 is 1 g / L.

[0038] The present invention also provides the use of any of the following items in the preparation of a product for improving the stability of viral antigens:

[0039] (I), the antigen stabilizer; and / or

[0040] (II), the combined reagent;

[0041] Such viruses include respiratory syncytial virus.

[0042] The present invention also provides the use of any of the following items in the preparation of a product for improving the specificity of viral antigen detection:

[0043] (I), the antigen stabilizer; and / or

[0044] (II), the combined reagent;

[0045] Such viruses include respiratory syncytial virus.

[0046] The present invention also provides the use of any of the following items in the preparation of an in vitro diagnostic kit:

[0047] (I), the antigen stabilizer; and / or

[0048] (II), the combined reagent.

[0049] The present invention also provides a kit comprising any of the following items, and an acceptable adjuvant or carrier:

[0050] (I), the antigen stabilizer; and / or

[0051] (II), the combined reagent.

[0052] The present invention also provides a method for improving the stability of viral antigens and / or improving the specificity of viral antigen detection, wherein the viral antigens are detected based on any of the following:

[0053] (I), the antigen stabilizer; and / or

[0054] (II), the combined reagent;

[0055] Such viruses include respiratory syncytial virus.

[0056] The present invention has achieved the following beneficial effects including but not limited to:

[0057] The stability of respiratory syncytial virus antigens, mainly thermal stability, is improved; the components in the antigen stabilizer act synergistically with each other, thereby enabling long-term storage; and the invention can be widely used in in vitro diagnostic reagents for RSV antibody detection, having a better stabilizing effect on the reagents and effectively extending the shelf life of the reagents.

[0058] Adding NaCl to the antigen solution (up to 1.0 mol / L in the experiment) improved specificity and effectively reduced background signal values, but it also caused the thermal stability of the antigen solution to deteriorate. Adding 0.15 mol / L NaCl or not adding any NaCl did not resolve the specificity and background issues. Adding 0.3 mol / L NaCl to the enzyme conjugate solution also achieved the same effect and did not affect the thermal stability of the antigen solution.

[0059] The kit of the present invention is simple and easy to prepare. Due to the thermal stability and enhanced specificity of its reagent components, it will not easily denature during storage and testing, resulting in product failure. Therefore, it is beneficial to ensure the accuracy of the test results and effectively improve the problem of batch-to-batch differences in naturally cultured RSV virus antigens. DETAILED DESCRIPTION

[0060] The present invention discloses an antigen stabilizer and its application and kit. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0061] The object of the present invention is to provide a method for improving the thermal stability of respiratory syncytial virus.

[0062] The present invention also aims to provide a RSV antibody test kit having heat stability prepared by the above method.

[0063] To achieve the above-mentioned purpose of the invention, the present invention realizes an RSV antibody detection kit antigen stabilizer through the following technical scheme, which comprises a buffer, a protein protectant, a stabilizer, a biocompatible polymer, and a biological preservative.

[0064] As a preferred antigen stabilizer, the specific ingredients include: PBS buffer with a concentration of 0.02-0.1 mol / L and a pH of 6.5-8.0; bovine serum albumin with a concentration of 5 g / L-50 g / L as a protein protectant; sucrose with a concentration of 10 g / L-50 g / L as a stabilizer; poloxamer with a concentration of 1.0 g / L-50 g / L as a biocompatible polymer; and Proclin 300 with a concentration of 0.1 g / L-1 g / L as a biological preservative.

[0065] There are no biocompatible polymers in the existing technical formulas, or most of the biocompatible polymers are low molecular weight biocompatible polymers; they cannot act as carriers or attachment protection for viruses, and therefore have poor stability. Under heat conditions, they cannot provide a buffer medium for viruses, resulting in the ineffectiveness of the viruses, and therefore cannot play a stabilizing and protective role.

[0066] The biocompatible polymer Poloxamer, whose trade name is Pluronic, is a new type of high molecular weight non-ionic triblock copolymer with a molecular formula of HO·(C2H4O) m (C3H6O) nH, composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), has the properties of a biocompatible polymer due to its amphiphilic structure. It is used as an emulsifier and stabilizer, exhibiting stable physical properties and the ability to withstand autoclaving and freezing. It can also be used as a solubilizer, absorption enhancer, sustained-release material, and solid dispersant.

[0067] In the antigen stabilizer for the RSV antibody detection kit of the present invention, poloxamer can bind to the hydrophobic region of the virus. After the binding, its external hydrophilic end forms a hydration film, which protects the virus. At the same time, due to its large molecular weight, it acts as a carrier for the virus after binding and has better thermal stability.

[0068] The preparation method of the antigen stabilizer of the RSV antibody detection kit provided by the present invention:

[0069] (1) preparing the PBS buffer according to the component contents to obtain solution 1;

[0070] (2) The protein protectant, stabilizer, biocompatible polymer, and biopreservative are measured according to their component contents and added to solution 1, and mixed to obtain solution 2; thus, the antigen stabilizer is obtained.

[0071] On the other hand, the present invention also provides a detection kit comprising an antibody prepared by the above method and having thermal stability.

[0072] In the kit, the respiratory syncytial virus antigen solution R2 exists as a separate component and plays a role of cross-linking in antibody detection. The kit can be used for qualitative detection of respiratory syncytial virus antibodies, and the type of detection antibody depends on the coated anti-human antibody.

[0073] Preferably, the above-mentioned antigen stabilizer is selected as the diluent of the above-mentioned antigen solution R2.

[0074] The other components of the kit are all related ingredients generally known in the industry; including anti-human antibody-coupled magnetic particles R1, horseradish peroxidase-labeled anti-respiratory syncytial virus antibody R3, sample diluent R4, chemiluminescent substrate A solution R5 and chemiluminescent substrate B solution R6.

[0075] The preparation method of the enzyme conjugate solution (horseradish peroxidase-labeled anti-respiratory syncytial virus antibody R3) of the RSV antibody detection kit provided by the present invention is as follows:

[0076] (1) 6.038 g of tris(hydroxymethyl)aminomethane (Tris) and 0.15-1.0 mol of sodium chloride were taken according to the component content, and the above components were fully dissolved and mixed in 1 L of purified water. The pH value of the solution was adjusted to pH 7.4 with 6 mol / L hydrochloric acid to obtain Solution 1;

[0077] (2) Calf serum 203.430 g, Proclin 3001 g, Bronidox preservative 0.199 g, aminopyrine (ADP protein protectant) 0.997 g, and casein 1.994 g were weighed and added to solution 1 according to the component content, and mixed to obtain solution 2; thus, the enzyme conjugate dilution solution was prepared;

[0078] (3) Adding the enzyme conjugate active material to solution 2 at a volume ratio of 1:8000 to prepare solution 3; that is, preparing an enzyme conjugate solution.

[0079] Compared with the existing technology, the RSV antibody detection kit antigen stabilizer provided by the present invention has the following beneficial effects:

[0080] The stability of respiratory syncytial virus antigens, mainly thermal stability, is improved; the components in the antigen stabilizer act synergistically with each other, thereby enabling long-term storage; and the invention can be widely used in in vitro diagnostic reagents for RSV antibody detection, having a better stabilizing effect on the reagents and effectively extending the shelf life of the reagents.

[0081] Adding NaCl to the antigen solution (up to 1.0 mol / L in the experiment) improved specificity and effectively reduced background signal values, but it also caused the antigen solution's thermal stability to deteriorate. Adding 0.15 mol / L NaCl (or no NaCl) did not resolve the specificity and background issues. Adding 0.3 mol / L NaCl to the enzyme conjugate solution also achieved the same effect and did not affect the antigen solution's thermal stability.

[0082] The kit of the present invention is simple and easy to prepare. Due to the thermal stability and enhanced specificity of its reagent components, it will not easily denature during storage and testing, resulting in product failure. Therefore, it is beneficial to ensure the accuracy of the test results and effectively improve the problem of batch-to-batch differences in naturally cultured RSV virus antigens.

[0083] The bovine serum albumin used in the present invention is produced by Zhengzhou Yimenuo Biotechnology Co., Ltd.; sucrose is produced by Beijing Yili Fine Chemicals Co., Ltd.; poloxamer is produced by Aladdin; Proclin 300 is produced by SIGMA; Tween 20 is produced by Beijing Yili Fine Chemicals Co., Ltd.; PVA is produced by SIGMA; PVP is produced by SIGMA; and PEG is produced by SIGMA.

[0084] The kit used in the present invention is a respiratory syncytial virus IgM antibody detection kit (magnetic particle chemiluminescence method); registration certificate number: National Medical Device Registration No. 20203400075; product batch number: 20220426; specification: 100 tests / box.

[0085] Unless otherwise specified, the antigen stabilizer provided by the present invention and its application as well as the raw materials and reagents used in the kit can all be purchased from the market.

[0086] The present invention will be further described below in conjunction with the embodiments:

[0087] Preparation Example 1 Preparation of 0.1% Poloxamer Antigen Solution

[0088] Components of the RSV antibody detection kit: Reagent 1: magnetic particle suspension containing magnetic particles coupled with anti-human IgM antibodies; Reagent 2: antigen solution: antigen solution containing respiratory syncytial virus; Reagent 3: enzyme conjugate solution containing horseradish peroxidase-labeled anti-respiratory syncytial virus antibodies; Reagent 4: sample diluent, a buffer containing protein.

[0089] 1. Preparation of 0.1% poloxamer antigen stabilizer

[0090] (1) Weigh 0.59 g of sodium dihydrogen phosphate, 5.8 g of disodium hydrogen phosphate, and 8.766 g of sodium chloride according to their respective content, and fully dissolve and mix the above components in 1 L of purified water to obtain Solution 1;

[0091] (2) 10 g of bovine serum albumin, 15 g of sucrose, 300 1 g / L of Proclin, and 12600 1 g of poloxamer were weighed according to their component contents, and added to solution 1, and mixed to obtain solution 2; thus, an antigen stabilizer containing 0.1% poloxamer was prepared.

[0092] 2. Preparation of 0.1% poloxamer antigen solution

[0093] Live material manufacturer and number: Manufacturer ATCC (full name: American Type Culture Collection), strain number: VR-26.

[0094] Viral antigen preparation process:

[0095] (1) Cell preparation: cell recovery, adherent subculture, and scale-up to a 10-layer cell factory;

[0096] (2) Virus preparation: virus inoculation, virus-to-virus ratio of 1:5, virus-carrying passage, cell factory expansion to 40 layers, sampling and testing, and virus harvesting;

[0097] (3) Virus inactivation and inactivation verification: inactivation with β-propiolactone and blind transmission after inactivation;

[0098] (4) Virus concentration: The virus liquid is concentrated through a virus liquid concentration pipeline system (concentration ratio is 1:10) to obtain respiratory syncytial virus antigen.

[0099] (5) A 0.1% poloxamer antigen solution was prepared according to a volume ratio of respiratory syncytial virus antigen to 0.1% poloxamer antigen stabilizer = 1:15.

[0100] Preparation Example 2 Preparation of enzyme conjugate solution

[0101] (1) 6.038 g of tris(hydroxymethyl)aminomethane (Tris) and 0.15, 0.3, 0.5, and 1 mol of sodium chloride were taken according to their component contents, and the above components were fully dissolved and mixed in 1 L of purified water. The pH value of the solution was adjusted to pH 7.4 with 6 mol / L hydrochloric acid to obtain solution 1 containing different concentrations of sodium chloride;

[0102] (2) Calf serum 203.430 g, Proclin 3001 g, Bronidox preservative 0.199 g, aminopyrine (ADP protein protectant) 0.997 g, and casein 1.994 g were weighed and added to solution 1 according to the component content, and mixed to obtain solution 2; thus, enzyme conjugate dilutions containing different concentrations of sodium chloride were prepared;

[0103] (3) Adding the enzyme conjugate active material to solution 2 at a volume ratio of 1:8000 to prepare solution 3; that is, enzyme conjugate solutions containing different concentrations of sodium chloride were prepared.

[0104] Comparative Example 1 Preparation of 0.1% TW20 antigen solution

[0105] In the preparation of reagent 2 in this comparative example, the preparation process and the amounts of components added were the same as those in 1-2 of Preparation Example 1, except that the poloxamer molecular weight of 12600 was replaced with TW20 to prepare an antigen solution containing 0.1% TW20.

[0106] Comparative Example 2 Preparation of 0.1% PVA80 antigen solution

[0107] In the preparation of reagent 2 in this comparative example, the preparation process and the amounts of components added were the same as those in 1-2 of Preparation Example 1, except that the poloxamer with a molecular weight of 12600 was replaced with PVA80 to prepare an antigen solution containing 0.1% PVA80.

[0108] Comparative Example 3 Preparation of 0.1% PVA203 antigen solution

[0109] In the preparation of reagent 2 in this comparative example, the preparation process and the amounts of components added were the same as those in 1-2 of Preparation Example 1, except that the poloxamer with a molecular weight of 12600 was replaced with PVA203 to prepare an antigen solution containing 0.1% PVA203.

[0110] Comparative Example 4 Preparation of 0.1% PVP (K30) antigen solution

[0111] In the preparation of reagent 2 in this comparative example, the preparation process and the amounts of components added were the same as those in 1-2 of Preparation Example 1, except that the poloxamer with a molecular weight of 12600 was replaced with PVP (K30), thereby obtaining an antigen solution containing 0.1% PVP (K30).

[0112] Comparative Example 5 Preparation of 0.1% PEG2000 antigen solution

[0113] In the preparation of reagent 2 in this comparative example, the preparation process and the amount of components added were the same as those in 1-2 of Preparation Example 1, except that the poloxamer molecular weight of 12600 was replaced with PEG2000 to prepare an antigen solution containing 0.1% PEG2000.

[0114] Comparative Example 6 Preparation of 0.1% PEG4000 antigen solution

[0115] In the preparation of reagent 2 in this comparative example, the preparation process and the amounts of components added were the same as those in 1-2 of Preparation Example 1, except that the poloxamer molecular weight of 12600 was replaced with PEG4000 to prepare an antigen solution containing 0.1% PEG4000.

[0116] Comparative Example 7 Preparation of Antigen Solution Containing 0.1% PEG20000

[0117] In the preparation of reagent 2 in this comparative example, the preparation process and the amounts of components added were the same as those in 1-2 of Preparation Example 1, except that the poloxamer molecular weight of 12600 was replaced with PEG 20000 to obtain an antigen solution containing 0.1% PEG 20000.

[0118] Comparative Example 8 Preparation of Antigen Solution Containing 0.1% PEG100000

[0119] In the preparation of reagent 2 in this comparative example, the preparation process and the amounts of components added were the same as those in 1-2 of Preparation Example 1, except that the poloxamer molecular weight of 12600 was replaced with PEG 100000 to obtain an antigen solution containing 0.1% PEG 100000.

[0120] Example 1 Stability and repeatability test

[0121] The kit used in the present invention is a respiratory syncytial virus IgM antibody detection kit (magnetic particle chemiluminescence method); registration certificate number: National Medical Device Registration No. 20203400075; product batch number: 20220426; specification: 100 tests / box.

[0122] Reagent 2 in the kit was replaced with the 0.1% poloxamer antigen solution obtained in Preparation Example 1, the 0.1% TW20 antigen solution obtained in Comparative Examples 1 to 8, the 0.1% PVA80 antigen solution, the 0.1% PVA203 antigen solution, the 0.1% PVP (K30) antigen solution, the 0.1% PEG2000 antigen solution, the 0.1% PEG4000 antigen solution, the 0.1% PEG20000 antigen solution, and the 0.1% PEG100000 antigen solution. The finished kit was then placed in a laboratory constant temperature incubator set to 37°C, and accelerated for 3 days, 7 days, and 10 days. It was placed in the positive order, taken out at the time point, and the clinical samples were tested together with the unaccelerated assessment reagents under the same conditions. The kit is used in conjunction with instruments such as the AutoLumo A2000 Plus, a fully automatic chemiluminescence analyzer produced by Zhengzhou Antu Bioengineering Co., Ltd., to realize automated detection of samples, provide the signal value of the test sample for the day, detect the luminescence value based on the magnetic particle platform and through HRP enzymatic chemiluminescence, and calculate the ratio of the test sample signal value to that of the unaccelerated assessment reagent.

[0123] The repeatability of the sample signal value was assessed using unaccelerated reagents.

[0124] The stability test comparison data are shown in Tables 1 to 3:

[0125] Table 1 37℃ heat accelerated stability test on the 3rd day

[0126]

[0127]

[0128] Table 2 37℃ heat accelerated stability test on the 7th day

[0129] Preparation Example 1 Comparative Example 1 Antigen diluent addition 0.1% poloxamer 0.1% TW20 condition Day 7 / Day 0 Day 7 / Day 0 Sample 1 96％ 52％ Sample 2 94％ 48％ Sample 3 96％ 51％ Sample 4 97％ 37％ Total ratio 96％ 47％

[0130] Table 3 37℃ heat accelerated stability test on the 10th day

[0131] Preparation Example 1 Comparative Example 1 Antigen diluent addition 0.1% poloxamer 0.1% TW20 condition Day 10 / Day 0 Day 10 / Day 0 Sample 1 93％ 23％ Sample 2 96％ 18％ Sample 3 97％ 22％ Sample 4 98％ 20％ Total ratio 96％ 21％

[0132] The results showed that the average signal value ratio of the antigen solution added with poloxamer after thermal acceleration for 3, 7, and 10 days was stable at above 95%, while the signal value ratio of the comparative reagent with other components added after thermal acceleration was significantly worse than the poloxamer average and was below 87%; and the signal value ratio of Tween 20 was on a downward trend as the thermal acceleration was prolonged, showing instability.

[0133] The repeatability test results are shown in Table 4:

[0134] Table 4 Repeatability test

[0135] Preparation Example 1 Comparative Example 1 Antigen diluent addition 0.1% poloxamer 0.1% TW20 Repeatability 1.34％ 1.84％

[0136] The results showed that the repeatability of the detection sample signal value was also improved by 0.5% after adding poloxamer.

[0137] Example 2 Specificity Detection

[0138] The kit used in the present invention is a respiratory syncytial virus IgM antibody detection kit (magnetic particle chemiluminescence method); registration certificate number: National Medical Device Registration No. 20203400075; product batch number: 20220426; specification: 100 tests / box.

[0139] 1. Sodium chloride was added to the 0.1% poloxamer antigen solution prepared in Preparation Example 1 to obtain 0.1% poloxamer antigen solutions containing 0, 0.5, and 1 mol / L sodium chloride. Reagent 2 in the kit was replaced with 0.1% poloxamer antigen solutions containing 0, 0.5, and 1 mol / L sodium chloride, respectively. The signal value level of the background sample and the signal value of the positive sample on the 10th day of 37°C heat acceleration were tested. The background signal value level test results are shown in Table 5, and the signal value of the positive sample on the 10th day of 37°C heat acceleration is shown in Table 6:

[0140] Table 5 Background signal level

[0141]

[0142]

[0143] Table 6 Signal values of positive samples at 37℃ thermal acceleration on the 10th day

[0144]

[0145] The results showed that adding NaCl to the antigen solution (up to 1.0 mol / L in the experiment) improved specificity and effectively reduced background signal values, with the average background signal dropping from over 1 million to the tens of thousands. However, adding NaCl to the antigen diluent from 0.15 mol / L to 1.0 mol / L decreased the thermal stability of the antigen solution from 22% to 42%, resulting in a decrease in thermal stability. Adding 0.15 mol / L or no NaCl did not resolve the specificity and background issues.

[0146] 2. Sodium chloride was added to the enzyme conjugate solution prepared in Preparation Example 2 to obtain enzyme conjugate solutions containing 0.15, 0.3, 0.5, and 1 mol / L sodium chloride. Reagent 3 in the kit was replaced with enzyme conjugate solutions containing 0.15, 0.3, 0.5, and 1 mol / L sodium chloride. The sample signal values were detected, and the results are shown in Table 7:

[0147] Table 7 Sample signal values

[0148] Enzyme conjugate solution 0.15mol / L 0.3mol / L 0.5mol / L 1.0 mol / L Positive clinical 1 824,997 897,406 829,509 646,699 Positive clinical 2 903,427 854,187 771,085 550,570 Positive clinical 3 1,289,049 1,180,621 1,090,707 882,046 Background sample 1 12,166 10,037 8,918 6,723 Background sample 2 6,565 4,531 4,309 3,668 Background sample 3 278,358 32,865 20,738 23,052 Background sample 4 206,057 12,961 9,798 11,367 Background sample 5 2,322,707 159,404 92,407 76,930 Background sample 6 1,949,612 298,169 158,849 165,493 Background signal mean 795,911 86,328 49,170 47,872

[0149] Result analysis: Adding 0.3 mol / L NaCL to the enzyme conjugate solution can also achieve the same background reduction effect, with the average background signal below 100,000, and does not affect the stability of the antigen.

[0150] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An antigen stabilizer, characterized in that The antigen stabilizer includes a buffer, a protein protectant, a stabilizer, a biocompatible polymer and a biopreservative; The buffer is PBS buffer; the protein protective agent is bovine serum albumin; the stabilizer is sucrose; the biocompatible polymer is poloxamer; and the biological preservative is Proclin 300. The concentration of the PBS buffer is 0.02-0.1 mol / L, and the pH thereof is 6.5-8.0; and The concentration of the bovine serum albumin is 5 to 50 g / L; and The concentration of the sucrose is 10-50 g / L; and The concentration of the poloxamer is 1 to 50 g / L; the molecular weight of the poloxamer is not less than 8000; and The concentration of Proclin300 is 0.1-1 g / L; The antigen is respiratory syncytial virus.

2. A combination reagent, characterized in that: It comprises the antigen stabilizer and enzyme conjugate solution as claimed in claim 1; The enzyme conjugate solution includes sodium chloride; The concentration of sodium chloride is 0.3 mol / L to 1.0 mol / L; and The concentration of the PBS buffer is 0.02-0.1 mol / L, and the pH thereof is 6.5-8.0; and The concentration of the bovine serum albumin is 5 to 50 g / L; and The concentration of the sucrose is 10-50 g / L; and The concentration of the poloxamer is 1 to 50 g / L; the molecular weight of the poloxamer is not less than 8000; and / or The concentration of Proclin300 is 0.1~1g / L.

3. Use of any of the following in the preparation of products that improve the stability of viral antigens: (I) The antigen stabilizer according to claim 1; and / or (II), the combined reagent according to claim 2; The virus is respiratory syncytial virus.

4. Use of any of the following in the preparation of products that improve the specificity of viral antigen detection: (I) The antigen stabilizer according to claim 1; and / or (II), the combined reagent according to claim 2; The virus is respiratory syncytial virus.

5. Use of any of the following in the preparation of an in vitro diagnostic kit for respiratory syncytial virus: (I) The antigen stabilizer according to claim 1; and / or (II) The combined reagent according to claim 2.

6. A respiratory syncytial virus diagnostic kit, characterized in that: Include any of the following, and acceptable adjuvants or carriers: (I) The antigen stabilizer according to claim 1; and / or (II) The combined reagent according to claim 2.

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