Monoclonal antibody of immunoglobulin A and application thereof

Abstract

The invention provides a monoclonal antibody of immunoglobulin A. The monoclonal antibody is a monoclonal antibody of specific immunoglobulin A secreted by a mouse anti-human immunoglobulin A monoclonal antibody hybridoma cell line 5F5A4F3 strain, can be used for quantitatively detecting the concentration of RBD-IgA generated by stimulation of a specific antigen, and also can be used for quantitatively detecting the concentration of the immunoglobulin A. The immunoglobulin A monoclonal antibody can be used for detecting the immunoglobulin A monoclonal antibody, can also be used for quantitatively detecting the concentration of total IgA generated by mucosal immunity, and is good in linearity, high in sensitivity, strong in specificity, suitable for evaluating the immune effect of vaccines, and also suitable for screening and monitoring the IgA antibody level in large-scale people.

Description

Technical Field

[0001] The present invention relates to the field of immunology and diagnostic reagents, and in particular to a monoclonal antibody of immunoglobulin A, which is classified and named as mouse anti-human immunoglobulin A monoclonal antibody. The present invention also relates to a hybridoma cell line secreting the monoclonal antibody of immunoglobulin A, and the application of the monoclonal antibody of immunoglobulin A in IgA detection. Background Art

[0002] Since the outbreak of the novel coronavirus, respiratory virus co-infection has occurred from time to time. As a result, global vaccine research and development has entered a rapid development stage, among which new progress has been made in the research on the immune protection mechanism of nasal spray or inhaled respiratory mucosal immunization vaccines, and significant breakthroughs have been made in vaccine research and development. Existing literature has disclosed that: by nasal inoculation, natural viral infection is simulated, secretory immunoglobulin A (SIgA) is induced locally in the nasal mucosa, first establishing the first effective defense barrier between the body and the external environment, and then stimulating cellular immunity and antibody response; the multi-dimensional protective immune factors in the local respiratory tract have better spatial distance advantages than peripheral immune factors, and can respond to viral infections more promptly; compared with injection vaccines, although the humoral immune response rate and neutralizing antibody level produced by nasal attenuated influenza vaccine are significantly lower than those of inactivated influenza vaccine (Inactivated influenza virus, IIV), the nasal inoculation route is more likely to produce natural immunity similar to that caused by infection, and ultimately show relatively higher protection derived from mucosal immunity; nasal spray vaccines can induce multi-dimensional broad-spectrum protective immune responses, including: cellular immunity, mucosal immunity, innate immunity and trained immunity; the nasal spray live attenuated influenza vaccine (live-attenuated influenza vaccine) of Flumist Company in the United States virus, LAIV) can produce good immunogenicity in children; after vaccination with nasal spray LAIV, the IgA produced by the nasal mucosa plays an important role in resisting influenza virus infection; on the 31st day after vaccination with Changchun Baike Biotechnology Co., Ltd.'s freeze-dried nasal spray LAIV, the vaccine recipients whose IgA antibody titers corresponding to H3N2 and B types increased by 2 times accounted for 38.75% and 31.05% of the vaccinated population, respectively, which was significantly higher than the positive conversion rate calculated based on HI (hemagglutination inhibition) antibodies. However, among the vaccine recipients whose IgA antibody titers increased by 2 times, only 3%~23% of the serum samples had an increase of more than 4 times in HI antibody titers. It can be seen from this that vaccines administered by injection mainly induce the production of serum immunoglobulin G (IgG), while vaccines administered by nasal administration can simultaneously induce the production of serum IgG and nasal mucosal immunoglobulin A (IgA), as well as possible induced cellular immune responses, which can provide the body with more extensive and lasting immune protection. In particular, the level of SIgA produced by the nasal mucosa is closely related to the protective efficacy of the vaccine. Therefore, the mucosal IgA level can be used as an important monitoring indicator for evaluating the immune effect of the vaccine.

[0003] Vaccines administered through the nasal route have the advantages of being non-invasive and easy to accept, and are particularly suitable for children and other groups. Common vaccination methods include nasal spray, inhalation, and other respiratory routes. In addition, the unique advantages of the nasal route in defending against infection by external pathogens through the mucosal immune system are also reflected in the following: the immune cells contained in the mucosal tissue account for 80% of all immune cells and have a wide coverage; the mucosal immune system has the regulation of specific T cells and cytokines, and a large amount of SIgA is produced by differentiated plasma cells to stimulate local immune protection. However, there is currently a lack of antibodies with high titers and strong specificity for mucosal IgA. Therefore, it is very necessary to develop a specific IgA and a rapid, simple, quantitative, sensitive, accurate, and specific mucosal IgA detection method.

[0004] The enzyme linked immunosorbent assay (ELISA) has the advantages of low cost, high sensitivity, strong specificity and good repeatability. It can be used to detect trace amounts of specific proteins in body fluids and can be applied to mucosal IgA detection. The basis and key to the research of the ELISA method is to screen a highly specific antigen or antibody protein, and adsorb it to a solid phase carrier while maintaining the activity of the protein to form an antigen or antibody complex containing an enzyme as a marker. In addition, there are technical problems such as sample collection when combining ELISA with IgA detection: on the one hand, the body produces 60 to 66 mg / kg of IgA every day, which exceeds the total production of all antibodies and can be renewed every 8 hours. Due to the high frequency of secretion and excretion of IgA, IgA may be unevenly distributed in nasal mucosal samples, and the sampling techniques of sampling personnel vary greatly, making the collection process difficult; on the other hand, IgA sampling in nasal mucosa generally adopts nasal washing method, swab or sponge sampling method, nasal scraping method or nasal absorption method, and the sample collection process uses physiological saline, culture medium or PBS Solution (including FBS and EDTA), etc., leads to the complexity of the matrix of the collected nasal swab samples; on the other hand, IgA has sequence similarities with other antibody types such as IgM and IgG, and may have non-specific reactions during detection, and IgA detection has high requirements for the affinity and titer of anti-IgA protein; therefore, it is necessary to develop a high-affinity, high-titer, high-specificity, and anti-interference anti-human IgA monoclonal antibody on the basis of overcoming these technical problems, and on this basis, establish a rapid, simple, efficient, quantitative mucosal IgA ELISA detection method with high sensitivity, accuracy and specificity. Summary of the invention

[0005] The object of the present invention is to provide a monoclonal antibody of immunoglobulin A and a hybridoma cell line secreting the monoclonal antibody of immunoglobulin A.

[0006] The present invention also aims to provide a method, a kit and application thereof for detecting immunoglobulin A with high sensitivity and high specificity based on the monoclonal antibody of immunoglobulin A.

[0007] The present invention also aims to overcome the influence of sampling on immunoglobulin A detection.

[0008] To achieve the above object, the present invention provides the following technical solutions: In the first aspect, the present invention provides a monoclonal antibody of immunoglobulin A, a monoclonal antibody of specific immunoglobulin A secreted by the mouse anti-human immunoglobulin A monoclonal antibody hybridoma cell line 5F5A4F3, recorded as anti-IgA-McAb, the deposit number of the mouse anti-human immunoglobulin A monoclonal antibody hybridoma cell line 5F5A4F3 is CGMCC No.46012, the deposit unit is the General Microbiology Center of the China Microbiological Culture Collection Administration, the deposit unit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is July 2, 2024. The classification name of 5F5A4F3 is Mouse anti-human immunoglobulin A monoclonal antibody hybridoma cell line, and the Latin name of the classification name is Mouse anti-humanimmunoglobulin A monoclonal antibody hybridoma cell line.

[0009] According to a preferred embodiment of the present invention, the monoclonal antibody of immunoglobulin A can specifically recognize and bind to the heavy chain constant region of secretory immunoglobulin A.

[0010] In a second aspect, the present invention provides a hybridoma cell line for secreting monoclonal antibodies that produce immunoglobulin A. The preservation number of the hybridoma cell line is CGMCC No.46012, the preservation address is the General Microbiology Center of China National Culture Collection Administration, and the preservation date is July 2, 2024.

[0011] In a third aspect, the present invention provides an enzyme-linked immunosorbent assay method, which uses a monoclonal antibody of immunoglobulin A for detection; this detection method does not belong to a method for diagnosing and / or treating a disease.

[0012] According to a preferred embodiment of the present invention, the detection steps are: 1) the sample to be tested is diluted with a sample diluent in proportion and added to the corresponding well of the ELISA plate; 2) washed with a washing solution; 3) the enzyme-labeled antibody is added and incubated at 35-39°C for 50-70 minutes; 4) washed with a washing solution; 5) the color developer is added and the color is developed in a constant temperature incubator at 35-39°C in the dark for 10-20 minutes, and then the stop solution is added to stop. The color change process of the positive result of the ELISA well is first blue and then yellow; 6) the ELISA instrument outputs the test result, which is compared with the cutoff value to determine the positive result or negative result.

[0013] According to a preferred embodiment of the present invention, the enzyme-linked immunosorbent assay method adopts a double antibody sandwich ELISA method to detect specific immunoglobulin A produced by specific antigen stimulation to obtain a quantitative result of specific immunoglobulin A.

[0014] Preferably, the enzyme-linked immunosorbent assay method is used to detect the new coronavirus RBD-IgA.

[0015] According to a preferred embodiment of the present invention, the enzyme-linked immunosorbent assay method adopts a double antibody sandwich ELISA method for universal detection of all immunoglobulins A produced by mucosal immunity to obtain a quantitative result of total immunoglobulin A.

[0016] In a fourth aspect, the present invention provides an enzyme-linked immunosorbent assay kit, comprising an enzyme-labeled antibody and a coated antibody, wherein the enzyme-labeled antibody is prepared from the above-mentioned monoclonal antibody of immunoglobulin A and is used to detect specific immunoglobulin A.

[0017] According to a preferred embodiment of the present invention, the ELISA kit uses the above-mentioned ELISA method to perform quantitative detection on the sample to be tested, and the kit uses a monoclonal antibody of immunoglobulin A.

[0018] According to a preferred embodiment of the present invention, the enzyme-linked immunosorbent assay kit is used to detect the new coronavirus RBD-IgA.

[0019] According to a preferred embodiment of the present invention, the components of the enzyme-linked immunosorbent assay kit include an enzyme-labeled plate, an enzyme-labeled antibody, a negative control, a positive control, a sample diluent, and an expanded sponge swab for collecting nasopharyngeal swab samples.

[0020] Preferably, the enzyme-labeled antibody is a monoclonal antibody of immunoglobulin A labeled with HRP.

[0021] In a fifth aspect, the present invention provides a method for detecting the relative content of immunoglobulin A, by calculating the ratio of the quantitative result of specific immunoglobulin A obtained by detecting the specific immunoglobulin A produced by specific antigen stimulation and the quantitative result of total immunoglobulin A obtained by the universal detection of all immunoglobulins A produced by mucosal immunity to obtain the relative content of immunoglobulin A.

[0022] In a sixth aspect, the present invention provides the above-mentioned monoclonal antibodies of immunoglobulin A, hybridoma cell lines for secreting and producing monoclonal antibodies of immunoglobulin A, enzyme-linked immunosorbent assay methods based on monoclonal antibodies of immunoglobulin A, enzyme-linked immunosorbent assay kits based on monoclonal antibodies of immunoglobulin A, and the use of a method for detecting the relative content of immunoglobulin A based on monoclonal antibodies of immunoglobulin A in detecting the content of immunoglobulin A.

[0023] In a seventh aspect, the present invention provides the above-mentioned monoclonal antibody of immunoglobulin A, and the use of a hybridoma cell line that secretes and produces the monoclonal antibody of immunoglobulin A in the evaluation of vaccine immune effects.

[0024] According to a preferred embodiment of the present invention, the application in the evaluation of vaccine immune effect is achieved by calculating the ratio of specific immunoglobulin A produced by specific antigen stimulation to all immunoglobulin A produced by mucosal immunity.

[0025] Preferably, the specific antigen is the new coronavirus and the specific immunoglobulin A is RBD-IgA.

[0026] Beneficial effects of the present invention: The invention provides a monoclonal antibody of immunoglobulin A. The monoclonal antibody of immunoglobulin A is prepared by using collected human serum samples as raw materials, and a human IgA protein with a purity of 90% obtained by PEG precipitation, Protein G and Protein L purification is used as an immunogen. Through multiple screening, a preferred positive hybridoma cell strain capable of secreting the monoclonal antibody of immunoglobulin A of the invention is screened from 6 monoclonal positive hybridoma cell strains, and the monoclonal antibody of immunoglobulin A (anti-IgA-McAb) that is expanded and cultured is obtained by preparing ascites and purifying the protein. Through titer and affinity detection, identification of specificity and epitope, and identification of binding segments, the titer of the preferred monoclonal antibody of immunoglobulin A of the invention is 2.35 million, and the affinity detected by high salt elution is 72%. Moreover, the monoclonal antibody of immunoglobulin A is a linear epitope antibody, which can specifically recognize and bind to the heavy chain constant region of secretory immunoglobulin A, and can be combined with an enzyme-linked immunosorbent assay method for quantitative detection of immunoglobulin A.

[0027] Based on the final preferred monoclonal antibody of immunoglobulin A No. 2, the present invention constructs a novel coronavirus RBD-IgA ELISA detection platform, including a novel coronavirus RBD-IgA ELISA detection method, a novel coronavirus RBD-IgA ELISA detection kit and related applications thereof. It can be used to quantitatively detect the content of novel coronavirus RBD-IgA in the sample to be tested, especially the XBB strain novel coronavirus. By obtaining the presence and level of RBD-IgA, it can be used to assist in the analysis of the immune status after novel coronavirus infection. It can also be combined with antibody detection such as IgM and IgG to evaluate the immunogenicity and effectiveness of the vaccine, or for screening and monitoring of RBD-IgA levels in a large population. In addition, potential infected persons can be found and corresponding prevention and control measures can be taken. The novel coronavirus RBD-IgA ELISA test can also be used to detect the level of RBD-specific antibodies at different time points, in different populations and after different vaccinations, which can provide a deep understanding of the immune response rules and influencing factors of the novel coronavirus, and provide a scientific basis for disease prevention and control and vaccine development.

[0028] High-quality and highly specific antibodies are the key to establishing ELISA detection of total immunoglobulin A. Based on the final preferred monoclonal antibody of immunoglobulin A No. 2, the present invention establishes a universal ELISA detection method and a kit for immunoglobulin A, which has high sensitivity and specificity for the detection of total immunoglobulin A, good accuracy, high precision, and good repeatability. The quantitative detection range of all immunoglobulins A is 0.75-12 ng / mL, R 2 >0.99, the test results have good linearity and high credibility. The quantitative detection of total immunoglobulin A can not only be used to evaluate the body's immune status and monitor abnormal immune system reactions caused by diseases related to IgA abnormalities, but also be used for in-depth research on the function of immunoglobulin A. In addition, based on the detection of specific immunoglobulin A produced by specific antigen stimulation and the detection of total immunoglobulin A produced by mucosal immunity, the ratio of the two can be used more objectively to evaluate the immune effect of vaccines, thereby overcoming the errors caused by sampling factors.

[0029] The present invention combines the screened monoclonal antibody with high affinity, high titer, strong anti-interference ability, and specific recognition and binding to immunoglobulin A with the ELISA detection method with high sensitivity, strong specificity, and good repeatability. By optimizing the reaction conditions, a high-sensitivity and high-specificity detection method for specific immunoglobulin A and a universal detection method for all immunoglobulin A produced by mucosal immunity are established. The detection cost is low, the throughput is high, and the operation is simple. Moreover, by calculating the ratio of the two detection results, the relative content of immunoglobulin A can also be obtained, which can effectively overcome the error caused by the sampling method and can be used to more objectively evaluate the immune effect of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure is a result of the identification of the recognition specificity and epitope of the immunoglobulin A monoclonal antibody of the present invention. The superscripts in the figure represent different loaded proteins, wherein 1 is human IgG, 2 is human IgA, and 3 is human IgM; M is a protein marker; the subscripts in the figure represent the 6 monoclonal antibodies Ab1 to Ab6 added in the WB experiment; Figure 2 The SDS-PAGE electrophoresis comparison diagram of immunoglobulin A of the present invention before and after being cleaved by pepsin, in the superscript of the figure, M is a protein marker, 1 is human IgA protein, and 2 is a protein sample after human IgA protease cleavage reaction; Figure 3 The results of WB detection of the immunoglobulin A monoclonal antibody of the present invention and human IgA protein before and after enzyme cleavage reaction are shown in the figure. In the superscript of the figure, M is a protein marker, 1 is a human IgA protein, and 2 is a protein sample after the human IgA protein is cleaved by enzyme cleavage reaction; the subscript of the figure indicates that the monoclonal antibodies to be tested added in the WB experiment are Ab2 and Ab5 respectively; Figure 4 The results of Dot Blot screening of the enzyme-labeled antibody of the present invention are shown in Figure 1. A is the enzyme-labeled antibody of the immunoglobulin IgA monoclonal antibody Ab2 of the present invention, and B is the goat anti-human-IgA enzyme-labeled antibody of the third-party control. The top of the figure represents the sample concentrations of 5 concentration gradients of human immunoglobulins. The vertical lines ①, ②, and ③ respectively represent different types of human immunoglobulins: human IgA, human IgM, and human IgG. Figure 5 This is a standard curve diagram of the universal ELISA test of immunoglobulin A of the present invention: the abscissa is the concentration of total IgA, and the ordinate is the OD value of the corresponding ELISA test. DETAILED DESCRIPTION

[0031] Exemplary embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. Unless otherwise specified, the same or similar terms represent the same concepts, such as monoclonal antibody = single antibody, polyclonal antibody = polyclonal antibody, IgA = immunoglobulin A, SIgA = secretory immunoglobulin A, anti-IgA-McAb = IgA-McAb = monoclonal antibody to immunoglobulin A, Ab = antibody, etc.

[0032] 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.

[0033] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified. Among them, male Balb / c mice were purchased from the China Food and Drug Administration; human serum samples were from Beijing Wantai Biological Pharmaceutical Co., Ltd.; goat anti-mouse IgG-HRP (horseradish peroxidase, referred to as HRP) was homemade by Beijing Wantai Biological Pharmaceutical Co., Ltd.; XBB.1.5RBD recombinant protein (Cat. No.: 40592-V08H146) was purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.; and the third-party control reagent of goat anti-human-IgA enzyme-labeled antibody was purchased from Wuhan Aokebotai Biotechnology Co., Ltd. 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.

[0034] 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 Laboratory Guide" (3rd edition) by J. Sambrook, or in accordance with the reagent and product instructions. The object of the test - the sample to be tested, is an inanimate sample preserved in vitro; the direct purpose of the test is to determine in vitro whether the sample to be tested contains specific IgA or total IgA and to quantitatively determine the concentration; the process, results or related applications of the test do not include the diagnosis and treatment of the disease, and the diagnosis results of the disease or the health status cannot be directly obtained based on the test results. 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.

[0035] The present invention uses the collected human serum samples as the preparation raw materials, and uses PEG precipitation, Protein G and Protein The purified human IgA protein with a purity of 90% was used as an immunogen. Balb / c mice were immunized with the purified human IgA protein using Freund's complete adjuvant and incomplete adjuvant. Mice with a serum titer of about one million were selected by the indirect ELISA method. The spleen cells were fused with myeloma cells sp2 / 0 at a cell ratio of 1:5. The positive hybridoma cell lines that could secrete high-specificity and high-affinity anti-IgA were preliminarily screened by the indirect ELISA method, HAT pressure method and WB method. After three rounds of limiting dilution method, the positive hybridoma cell lines were monoclonalized to obtain 6 monoclonal positive hybridoma cell lines. These 6 positive hybridoma cell lines were frozen to establish a cell bank. At the same time, sensitized Balb / c mice were inoculated to prepare ascites for large-scale culture of monoclonal antibodies. After protein purification of the collected ascites, 6 corresponding mouse anti-human immunoglobulin A monoclonal antibodies (anti-IgA-McAb) were obtained, and the titer detection and performance evaluation were carried out respectively. The specific evaluation contents included: using WB method and Dot The antibody reactivity and specificity of the six monoclonal antibodies were preliminarily evaluated by the Blot method, and the monoclonal antibody titer, affinity, etc. were then tested by the indirect ELISA method. Finally, through a specific reaction pairing experiment with different types of human immunoglobulins, a monoclonal antibody specific for human mucosal IgA with high titer, high affinity, high specificity, and no cross-reaction with IgG and IgM was screened from the six monoclonal antibodies. This is the preferred monoclonal antibody specific for immunoglobulin A (anti-IgA-McAb).

[0036] The present invention is based on a preferred hybridoma cell line that can secrete a monoclonal antibody (anti-IgA-McAb) of specific immunoglobulin A and the monoclonal antibody (Ab2) of immunoglobulin A secreted by it, fixes the RBD recombinant protein on a solid phase carrier, and optimizes the enzyme-labeled antibody HRP-Ab2 through a pairing experiment. The sample to be tested can be a nasal swab sample, thereby constructing an ELISA detection method for XBB strain new coronavirus RBD-IgA, which is used to detect the content of XBB strain new coronavirus RBD-IgA in human nasal mucosal samples. The detection method can quantitatively obtain the antibody concentration of RBD-IgA. The quantitative detection method has a sensitivity of 0.16U / mL and a specificity of 100%, and has strong resistance to matrix interference. The XBB strain new coronavirus RBD-IgA indirect ELISA quantitative detection kit constructed by the detection method has performance indicators such as linear range, specificity, accuracy, precision, and stability that meet the requirements, and can be used to evaluate the mucosal immune response after immunization with a nasal spray new coronavirus vaccine. In addition, an ELISA detection method for total immunoglobulin A produced by mucosal immunity is constructed based on HRP-Ab2 as an enzyme-labeled antibody and sheep anti-IgA polyclonal antibody as a coating antibody. It can analyze the change trend of IgA in clinical samples at different periods and can be used as a quality control method for total IgA detection. The relative content of immunoglobulin A can also be obtained by calculating the ratio of specific IgA to total IgA, which can effectively avoid the influence of sampling factors and more objectively evaluate the immune effect of the vaccine. The specific embodiments of the present invention are described in detail below, and the specific contents are as follows: Example 1. Preparation and screening of positive hybridoma cells The preparation and screening of positive hybridoma cells include the preparation of human IgA protein immunogen, animal immunization, cell fusion and screening of positive hybridoma cell lines.

[0037] Preparation of immunogen: human serum sample was used as raw material, PEG precipitation method was adopted, 7% of the prepared PEG precipitation mother solution was added, mixed and then allowed to stand; protein purification step was carried out in two steps, Protein G purification and Protein L purification.

[0038] Protein G purification: Take the supernatant after the above precipitation, dilute it appropriately with purified water, and then filter it through a 0.45µm filter membrane to obtain a filtrate; column treatment step: rinse the column with 1×PBS equilibration buffer until the pH value, conductivity, UV280 absorbance and other parameters reach a stable baseline for standby use; sample loading purification process: pump the filtered filtrate into the Protein G column at a flow rate of 3mL / min, and start collecting the penetration liquid when the UV280 absorbance rises to 50mAU; after the sample loading is completed, continue to collect until the UV280 absorbance drops to 50mAU and stop collecting; rinse the column again with the equilibration solution until all parameters return to the baseline stable state; elution step: elute the column with 0.05M glycine eluent to obtain the first eluate.

[0039] Protein L purification: including column treatment, sample purification, elution and collection steps; column treatment step: use 1×PBS equilibrium buffer to rinse the Protein L column until the pH value, conductivity and UV280 absorbance parameters reach a stable baseline and then standby; sample purification process: pump the first eluent into the Protein L column at a flow rate of 3mL / min In the L column, the change of UV280 absorbance is continuously monitored; after the sample is loaded, the column is rinsed with the balance solution until the UV280 absorbance returns to the baseline stable state to ensure that there is no residual sample in the column; elution step: the column is eluted with 0.05M glycine eluent, and during the elution process, the change of UV280 absorbance is closely monitored to identify the appearance of the elution peak; when the UV280 absorbance rises to 50mAU, the eluent is collected, and the collection is continued until the UV280 absorbance drops to 50mAU, and the collection is stopped to ensure that the complete elution peak is collected. The second eluent is the target protein - human IgA protein. The column is rinsed with the balance solution again until the UV280 absorbance returns to the baseline stable state, and it is ready for the next purification operation.

[0040] Animal immunization: The immunized animals are male Balb / c mice (18-22 g), the immunogen is human IgA protein, and the immunization procedure is three immunizations. In the first immunization, the immunogen is fully emulsified with an equal volume of Freund's complete adjuvant until it does not spread, and the final concentration of the immunogen is 100µg / mL. The immunization route is multiple subcutaneous injections on the back of the mouse, the immunization dose is 20µg / mouse, the immunization volume is 0.2 mL / mouse, and 6 Balb / c male mice are injected and immunized in each group at the same time. In the second immunization, two weeks after the first immunization, the immunogen is fully emulsified with an equal volume of Freund's incomplete adjuvant until it does not spread, and the same final concentration of the immunogen, immunization dose, immunization volume, and immunization route as the first immunization are used, that is, 100µg / mL, 20µg / mouse, 0.2mL / mouse, and multiple subcutaneous injections on the back of the mouse. In the third immunization, two weeks after the second immunization, the immunogen is fully emulsified with an equal volume of Freund's incomplete adjuvant until it does not spread even when dripped. The same final concentration of the immunogen, immunization dose, immunization volume, and immunization route as the first immunization are used again, i.e., 100µg / mL, 20µg / mouse, 0.2mL / mouse, and multiple subcutaneous injections on the back of the mouse. Blood is collected two weeks after the third immunization, and the antibody titer produced after immunization is detected by indirect ELISA. Mice with an antibody titer of about 1:1000000 can continue to be used for subsequent impact immunization and cell fusion. In the impact immunization, the immunogen immunization dose is 20 µg / mouse, and 10mM phosphate buffer is added to dilute it to an immunization volume of 100 µL / mouse. The immunization route is injection into the mouse tail vein, and spleen cells are taken 3-4 days later for cell fusion.

[0041] Cell fusion: Prepare spleen cells and SP2 / 0 myeloma cells for fusion in advance, mix them in a 50 mL fusion tube, collect the cells by centrifugation, and aspirate the supernatant as much as possible to avoid affecting the concentration of polyethylene glycol 1500. Rotate the centrifuge tube, slowly add 1 mL of 50% PEG1500 (pH 8.0) preheated to 37°C along the tube wall, collect the cells by centrifugation, add an appropriate amount of HAT medium to resuspend the cells, and inoculate the cell suspension in batches of 11 96-well cell plates with feeder cells at 100 µL / well, and continue to culture for 10 to 14 days before screening positive hybridoma cells.

[0042] The steps of screening positive hybridoma cells are as follows: continue to culture for 10-14 days after cell fusion, aspirate the culture supernatant of the fused cells in the 96-well cell plate, load 50 µL / well into the 96-well detection plate coated with the above-mentioned human IgA protein (coating concentration is 0.2 µg / mL), add 50 µL / well of sample diluent, incubate in a 37°C water bath for 60 minutes, and wash 5 times; dilute goat anti-mouse IgG-HRP by 5000 times, add 100 µL / well into the 96-well detection plate, incubate in a 37°C water bath for 45 minutes, and wash 5 times; add TMB colorimetric solution for 15 minutes, add stop solution (2M H2SO4) to terminate the color reaction, and use 450 / 630 dual wavelength to measure the absorbance OD, with OD ≥ 0.2 as the criterion for positive hybridoma cells, and combined with the growth of fused cells in the cell culture plate, the 21 positive hybridoma cell lines initially screened were subjected to three monoclonal screenings using the limiting dilution method, and 6 hybridoma cells that can secrete and produce specific IgA monoclonal antibodies were obtained. These 6 cloned positive hybridoma cell lines were cultured on a large scale to establish a cell bank, and then frozen for seed preservation.

[0043] At the same time, the culture supernatants of these 6 positive hybridoma cells were used for the preparation and identification of ascites containing anti-IgA-McAb. These 6 positive hybridoma cells were named cell line 1 to cell line 6, among which cell line 2 was the mouse anti-human immunoglobulin A monoclonal antibody hybridoma cell line 5F5A4F3.

[0044] Example 2: Identification of anti-IgA-McAb The preparation and identification process of ascites containing anti-IgA-McAb includes preparation of ascites, purification of monoclonal antibody, titer and affinity detection, identification of specificity and epitope, and identification of binding segment.

[0045] Preparation of ascites: Take the 6 positive hybridoma cells collected after the above expansion culture, wash them twice with RPMI-1640 medium, and add 5×10 5 ~1×10 6 The injection volume of 10 ...

[0046] The purification of monoclonal antibodies includes crude purification and fine purification. First, the saturated ammonium sulfate crude purification step includes: first diluting the collected ascites (containing Ab1~Ab6) by 3~5 times with 10mM phosphate buffer, then adding an equal volume of saturated ammonium sulfate for precipitation, and then adding 10mM phosphate buffer again after precipitation for re-dissolution. Secondly, Mabselect Protein A affinity purification method was used for purification. The purification steps included: first treating the affinity column with binding buffer (including 0.05 M Tris, 0.5 M NaCl) and elution buffer (including 0.05 M glycine, 0.5 M NaCl), and then balancing to neutrality with 10 mM phosphate buffer, and then taking the crude and purified monoclonal antibodies (Ab1~Ab6) re-dissolved in the above 10 mM phosphate buffer and loading them onto the column respectively. After loading, the column was rinsed with 10 mM phosphate buffer until the OD280 value of the flow-through returned to the baseline, and then eluted with 0.05 M glycine-hydrochloric acid solution (pH3.0), and the solution of the entire elution peak was collected. The eluate was the purified monoclonal antibody (Ab1~Ab6) for further performance testing and identification of the monoclonal antibody.

[0047] Titer and affinity detection: Take the above-mentioned purified 6 monoclonal antibodies (Ab1~Ab6) as the monoclonal antibodies to be tested, and use indirect enzyme-linked immunosorbent assay and high salt elution method to detect the antibody titer and affinity of these 6 monoclonal antibodies respectively; in the high salt elution method, the high concentration of salt can be selected from urea or urea, preferably urea. The specific operation steps are: take the monoclonal antibodies to be tested and dilute them to the same protein concentration with 1×PBS. Dilute the antibodies to be tested with the same protein concentration 100 times as the starting sample detection concentration, and then dilute them in a 5-fold series to prepare a total of 8 concentration gradients. The 8 concentrations of dilutions are added to the corresponding wells of the microplate (96-well detection plate coated with human IgA protein at a concentration of 0.2 µg / mL) at a dosage of 100 µL / well, and each concentration is repeated 2 wells, of which 1 well is used as an experimental well and 1 well is used as a control well; after the addition of the sample, incubate at 37°C for 60 minutes. Washing plate: The amount of washing solution is 300µL / well. Wash the whole plate once with 1×PBST, soak and stand at room temperature for 3 minutes after adding washing solution. Wash the experimental wells with 1×PBST containing 4M urea for the second washing solution, and wash the control wells with 1×PBST for the second washing solution. Then wash the whole plate with 1×PBST washing solution once and try to dry it. Add 50µL / well of color development solution A and B respectively, place in a 37℃ constant temperature incubator to develop color for 15 minutes away from light; add 50µL / well of stop solution to stop the color development reaction; use 450 / 630 dual wavelength to measure the absorbance OD value of each well. The test result of the control group is the titer of the monoclonal antibody, and the titer value is at least 400,000; the test result of the experimental well is to select the OD value of about 1.0 after urea elution and divide it by the test OD value of the control group sample with the same dilution multiple. The percentage calculated is the relative affinity of the monoclonal antibody. The titer and affinity test results of these 6 monoclonal antibodies are shown in Table 1.

[0048] Table 1 Titer and affinity test results ; Identification of specificity and epitopes: Take three proteins, human IgA, human IgG, and human IgM, dilute them to 0.2 mg / mL respectively, load 10 μL into each well, and perform SDS-PAGE electrophoresis. Subsequently, the semi-dry transfer method was used to transfer the membrane at a current of 1600 mA for 10 minutes. The purified monoclonal antibody was diluted to an appropriate concentration (5 μg / mL) for WB detection and analysis. After at least 3 repeated experiments, the results showed that the 6 purified monoclonal antibodies were able to specifically bind to IgA and develop color, and had no cross-reaction with IgG and IgM. Figure 1 As shown, it can be seen that these 6 monoclonal antibodies can specifically recognize IgA. In addition, WB detection preliminarily confirmed that these 6 monoclonal antibodies are linear epitope antibodies that can specifically recognize IgA.

[0049] Identification of monoclonal antibody binding to IgA segment: According to the WB test results, two monoclonal antibodies (Ab2 and Ab5) with specific binding, high reaction activity and stable repeated experimental results were selected as verification objects, and WB experiments were used to further analyze the specific binding segments of these two monoclonal antibodies to IgA. Take human IgA protein, first use pepsin for enzymatic reaction, take samples before and after enzymatic reaction and dilute to 1 mg / mL, load 10 μL into each well, and perform SDS-PAGE electrophoresis. Subsequently, the semi-dry transfer method was used to transfer the membrane at a current of 1600mA for 10 minutes. The purified monoclonal antibody was diluted to an appropriate concentration (5μg / mL), and the binding of the monoclonal antibody to human IgA before and after the enzymatic reaction was analyzed by WB experiment. The SDS-PAGE electrophoresis results of human IgA protein before and after the enzymatic reaction are shown in the figure. Figure 2 As shown, the heavy chain (α chain) of IgA is about 56KD, and the light chain is about 25KD; the WB experimental results of the monoclonal antibody binding to human IgA protein before and after enzyme cleavage reaction are shown in Figure 3 shown.

[0050] Human IgA can be divided into two subtypes, IgA1 and IgA2, from a structural perspective. The amino acid sequences of these two subtypes are not much different in the constant region of the heavy chain, but there are significant differences in the hinge region. The hinge region of IgA2 is shorter than that of IgA1 and has different functions. In particular, it lacks the cysteine ​​residues required for the formation of disulfide bonds. Therefore, its heavy chain and light chain are not connected by disulfide bonds, but by non-covalent bonds. This structural difference leads to different sensitivities of the two subtypes of IgA to pepsin. The hinge region (J chain) of IgA1 contains multiple sites that can be recognized by pepsin, so it can be recognized by pepsin and undergo enzymatic cleavage, while IgA2 will not be recognized and cleaved by pepsin. The proportion of IgA2 in secretory immunoglobulin A (SIgA) is usually higher than that of IgA1, and SIgA is mostly a dimer or polymer structure; each SIgA molecule contains a J chain and a secretory piece. The J chain connects two or more IgA monomers to each other through multiple disulfide bonds and other interactions, while the secretory piece enhances the structural stability of SIgA, enabling it to further resist enzymatic degradation by pepsin. Figure 2-Figure 3 It can be seen that the two monoclonal antibodies (Ab2, Ab5) prepared and preferably obtained by the present invention can not only specifically recognize IgA, but also can specifically recognize and bind to the heavy chain (α chain), and do not react with the variable region (Fab segment) remaining after pepsin cleavage. Therefore, it is speculated that the epitope bound by these two monoclonal antibodies (Ab2, Ab5) is the constant region of the heavy chain (α chain) of IgA, so these two monoclonal antibodies (Ab2, Ab5) can continue to be used for the analysis and application of SIgA produced by nasal mucosa.

[0051] Example 3, Application 1: Establishment of ELISA detection method for novel coronavirus RBD-IgA antibody The receptor binding domain (RBD) of the novel coronavirus XBB strain is a specific research object of the present invention. The RBD recombinant protein of the novel coronavirus XBB strain is used as the coating protein of the ELISA plate; a nasopharyngeal sample with a concentration of 1000U / mL is taken as the sample to be tested; Optimization of coating concentration: The RBD recombinant protein was diluted to 3 concentrations of 2 μg / mL, 1 μg / mL, and 0.5 μg / mL with carbonate buffer, and coated on the ELISA plate respectively; the nasopharyngeal samples were diluted in multiples with sample diluent and then loaded for ELISA reaction to screen the protein coating concentration of the ELISA plate. The screening criteria were that the coating concentration with low background and relatively high bias ratio (P / N value) in the ELISA test results was relatively optimal. The test results are shown in Table 2.

[0052] Table 2 Optimization results of coating concentration ; As can be seen from Table 2, when the coating concentration of the RBD recombinant protein is 2 μg / mL, the P / N value (P / N=82.0) is the highest, so the preferred coating concentration of the RBD recombinant protein is 2 μg / mL.

[0053] Optimization of enzyme-labeled antibodies: The first step is to screen enzyme-labeled antibodies, and the second step is to label and verify the performance of enzyme-labeled antibodies. The key to screening enzyme-labeled antibodies lies in specificity. The first step of the screening method is to use the Dot-blot method to detect the specificity of the reaction of enzyme-labeled antibodies to different types of human immunoglobulins. The third-party control selects goat anti-human-IgA enzyme-labeled antibodies. The specific process includes: diluting the initial concentration of purified human IgA, human IgM, and human IgG to 50μg / mL, diluting 5 gradients at a 4-fold ratio, taking 2μL and loading it onto the PVDF membrane, and blocking it with 5% skim milk; diluting the enzyme-labeled antibody with 5% skim milk at a ratio of 1:1000 and incubating the spotted PVDF membrane. The Dot-blot test results are as follows: Figure 4As shown: in terms of dosage, the lowest dosage of Ab2 and the third-party control that reacted with human IgA was 12.5 μg / mL, with no significant difference; however, in terms of specificity, only Ab2 had a very strong specific binding reaction to human IgA protein, which was consistent with the identification results above, while the third-party control also had a non-specific reaction with human IgG. This shows that Ab2 has good specificity for human IgA and can be used as a relatively preferred enzyme-labeled antibody. The second step is quantitative screening by ELISA: first, the mouse anti-human immunoglobulin A monoclonal antibodies Ab2 and Ab5 were labeled with superoxide dismutase (HRP) to prepare enzyme-labeled antibodies (the two enzyme-labeled antibodies are referred to as HRP-Ab2 and HRP-Ab5, respectively). The enzyme-labeled antibodies were simultaneously compared and verified with the third-party control reagent of goat anti-human-IgA enzyme-labeled antibodies; preparation of the samples to be tested: using sample diluent to appropriately dilute the nasopharyngeal samples (concentration: 1000U / mL) and serum samples (concentration: 1000U / mL), and then dilute them in a 2-fold gradient as samples to be tested; ELISA reaction was performed to compare the detection differences of the three enzyme-labeled antibodies (HRP-Ab2, HRP-Ab5 and third-party control) on serum samples and nasopharyngeal samples, respectively. The screening criteria were that the enzyme-labeled antibodies with high detection sensitivity and good dilution linearity were preferred, and the detection results were summarized in Table 3. The detection sensitivity results of the three enzyme-labeled antibodies for nasopharyngeal samples and serum samples are summarized in Table 4.

[0054] Table 3 Optimization results of enzyme-labeled antibody detection

[0055] Table 4 Comparison of sensitivity test results of three enzyme-labeled antibodies ; From the results in Tables 3 and 4, it can be seen that when the enzyme-labeled antibody is HRP-Ab2 and the third-party control, the sensitivity of detecting RBD-IgA in nasopharyngeal samples and serum samples is higher than that of HRP-Ab5. Although in the test results of serum samples, the detection sensitivity (0.08) of the enzyme-labeled antibody as a third-party control is slightly higher than that of HRP-Ab2 (0.16), the difference between the two is not obvious. It is speculated that when the enzyme-labeled antibody is HRP-Ab2 and the third-party control, it can sensitively detect SIgA in the form of dimers in the nasal mucosa, and can also sensitively detect IgA mainly in the form of monomers in the serum, and the detection sensitivity of these two forms of IgA is relatively high, so Ab2 can be used as the preferred enzyme-labeled antibody, and further optimization of the concentration can be continued in the future.

[0056] Optimization of enzyme-labeled antibody concentration: HRP-labeled Ab2 (HRP-Ab2) was selected as the preferred concentration for optimization screening. The enzyme-labeled antibody was diluted with enzyme-labeled antibody diluent at three ratios of 1:500, 1:1000, and 1:2000. The nasopharyngeal samples (initial concentration: 1000U / mL) were gradiently diluted at a ratio of 2 times with sample diluent and used as test samples for ELISA reaction. The detection results of enzyme-labeled antibodies diluted at three ratios were compared. The screening criteria were that the concentration of enzyme-labeled antibodies with low background and relatively high partial ratio (P / N value) in the ELISA test results was the relatively optimal concentration. The test results are shown in Table 5.

[0057] Table 5 Optimization test results of enzyme-labeled antibody concentration ; As can be seen from Table 5: When the enzyme-labeled antibody is diluted at a ratio of 1:1000, the P / N value in the test result is the highest (P / N=45.5), so the preferred concentration of the enzyme-labeled reagent is determined to be 1:1000. Although the P / N value corresponding to the concentration of 1:1000 is the highest, it can be seen from the specific data that the P / N values ​​corresponding to the two dilution ratios of 1:1000 and 1:2000 are not much different. From the perspective of test cost, 1:1000 to 1:2000 are relatively better, and the test cost is relatively lower when the concentration is 1:2000.

[0058] Optimization of the loading ratio of nasal swab samples: 15 negative and 15 positive nasal swab samples were taken, and each sample was diluted according to three ratios of 1:2, 1:4 and 1:8 as the starting detection concentration of the nasal swab samples to be tested. The new coronavirus RBD-IgA ELISA detection method established after the above conditions were optimized was tested to screen the starting detection concentration of the nasal swab samples with the best specificity and sensitivity. The test results are shown in Table 6.

[0059] Table 6 Optimization of the initial detection concentration of nasal swab samples ; It can be seen from Table 6 that: if the initial detection concentration is 1:2, the specificity detection result is relatively low, that is, a false positive result caused by a nonspecific reaction occurs. It is speculated that at 1:2, different sampling matrices may interfere with the detection result to a certain extent; if the initial detection concentration is 1:8, the sensitivity detection result is relatively low, that is, samples with low IgA concentrations are missed. It is speculated that at 1:8, the detection sensitivity is reduced due to the large dilution ratio of the sample. Therefore, relatively speaking, when the initial detection concentration is 1:4, the sensitivity and specificity of the test result are relatively high, and the compliance is the best, which can be used as the preferred starting sample detection concentration.

[0060] Calculation of cutoff value for negative samples: The novel coronavirus RBD-IgA ELISA detection method established after the above-mentioned conditions were optimized, and the above-mentioned optimized initial detection concentration of nasal swab samples (1:4) was used as the preferred novel coronavirus RBD-IgA ELISA detection method of the present invention. 50 nasal swab samples collected from patients with no history of novel coronavirus infection were selected as negative samples to be tested. These 50 negative samples were tested to verify the cutoff value of the detection method. The test results were calculated according to the formula (cutoff = 2.1 × the mean of the A values ​​of the negative sample tests). If the mean of the A values ​​of the negative sample tests were all less than 0.05, they were calculated as 0.05. The final test results are shown in Table 7.

[0061] Table 7 Cutoff values ; From the test results shown in Table 7, it can be seen that the mean A value of the negative sample test is 0.026, which is less than 0.05. Calculated based on 0.05, Cutoff=2.1×0.05=0.105, so the cutoff value is determined to be 0.105.

[0062] The reaction principle of the novel coronavirus RBD-IgA ELISA detection method of the present invention is the double antibody sandwich ELISA method. The optimized reaction conditions in the detection method include using 2μg / mL RBD recombinant protein as the preferred coating concentration, preparing enzyme-labeled antibody (HRP-Ab2) with HRP-labeled Ab2, using a concentration of 1:1000 dilution, the cutoff value of the ELISA detection method = 0.105, and the initial detection concentration of the sample is preferably 1:4 dilution. According to the above reaction conditions, a novel coronavirus RBD-IgA ELISA detection kit is prepared, and the use of the kit is improved according to the preferred ELISA reaction conditions, thereby establishing a novel coronavirus RBD-IgA ELISA detection platform. Among them, the specific detection steps of the novel coronavirus RBD-IgA ELISA detection include: 1) The sample to be tested is diluted with sample diluent at a ratio of 1:4, and then added to the corresponding well of the ELISA plate. The RBD recombinant protein coated on the ELISA plate specifically recognizes and binds to the RBD-IgA in the sample to form a "RBD recombinant protein-RBD-IgA" complex; 2) Wash with washing solution to remove substances on the ELISA plate that are not specifically bound to the RBD recombinant protein; 3) Add enzyme-labeled antibody and incubate at 37°C for 60 minutes to form a "RBD recombinant protein-RBD-IgA-HRP-Ab2" complex after recognition and specific binding; 4) Wash with washing solution to remove the enzyme-labeled antibody McAb-HRP that has not yet specifically bound to the ELISA plate; 5) Add the colorimetric reagent and develop the color in a 37°C constant temperature incubator for 15 minutes away from light. The enzyme-labeled wells on the ELISA plate that form a complex will react with the colorimetric reagent and turn blue first. Then add the stop solution to stop the reaction, and the blue enzyme-labeled wells will turn yellow. 6) The ELISA instrument outputs the test results: First, if the test value is higher than 0.105, it is judged as a positive result, indicating that RBD-IgA exists in the test sample; if the test value is lower than 0.105, it is judged as a negative result, indicating that RBD-IgA does not exist or exists in very low amounts in the test sample.

[0063] Further, by measuring the absorbance values ​​of standards of different concentrations, a standard curve is drawn to establish a quantitative relationship between RBD-IgA concentration and absorbance value; according to the absorbance value and Cutoff value of the sample to be tested, the corresponding RBD-IgA concentration can be found on the standard curve, thereby realizing the ELISA quantitative detection of RBD-IgA. If it is found that the false positive rate or false negative rate of the Cutoff value setting is too high during the detection process, the Cutoff value needs to be re-determined.

[0064] The components of the novel coronavirus RBD-IgA ELISA test kit include: RBD recombinant protein-coated ELISA plate, ELISA antibody, negative control, positive control, sample diluent, washing solution, colorimetric solution, stop solution, expanded sponge swab for nasopharyngeal swab sample collection and instructions; it can also include novel coronavirus XBB strain IgA standard with a concentration in the range of 0.31 to 5 U / mL. The novel coronavirus RBD-IgA ELISA test kit prepared according to the above method was evaluated for performance, including linear range, specificity, repeatability, precision, accuracy and stability, etc. The verification results of the performance evaluation are as follows.

[0065] Linear range: Take nasopharyngeal samples (1000U / mL) and dilute them to 10U / mL as the starting detection concentration of the sample to be tested. Perform multiple dilutions according to the 2-fold dilution ratio, dilute 7 concentration gradients in total, and test them separately. Repeat the test for 3 times for each concentration. According to the test result data, use the concentration as the horizontal axis and the detection OD value as the vertical axis to draw the standard curve, and obtain the linear regression equation and regression coefficient (R 2 ), determine the linear range of quantitative detection. The data of the above test results are shown in Table 8, and the linear regression equation of the standard curve is shown in Table 9.

[0066] Table 8 Linear range test data

[0067] Table 9 Linear regression equation ; From the test results shown in Tables 8 and 9, it can be seen that when the concentration range of the sample to be tested is between 0.31 and 5 U / mL, the linear correlation of this detection method is relatively optimal, R 2 =0.9951. If the concentration of the novel coronavirus RBD-IgA is higher than 5 U / mL, the sample to be tested can be appropriately diluted for retesting in order to improve the accuracy of the test.

[0068] Specificity: The positive nasopharyngeal samples were used as the main test samples. Three times the volume of negative nasopharyngeal samples and sample diluent were used to dilute the positive nasopharyngeal samples in a ratio of 3:1. The diluted mixture was used as the test sample. The mixed sample of negative nasopharyngeal samples: positive nasopharyngeal samples = 3:1 was used as the experimental group, and the mixed sample of sample diluent: positive nasopharyngeal samples = 3:1 was used as the control group. After the test, the standard curve was drawn and the recovery rate was calculated. The recovery rate = experimental group / control group × 100%. The specificity of the method was evaluated by the recovery rate. The test results of the recovery rate are shown in Table 10.

[0069] Table 10 Recovery rate ; As can be seen from Table 10: the test results of using negative nasopharyngeal samples and sample diluent to dilute positive nasopharyngeal samples are very similar, and the recovery rate is 100%. Therefore, the interference of the sample matrix liquid on the detection system is relatively small, and the specificity is good.

[0070] Specificity: Nasopharyngeal or oropharyngeal specimens collected from confirmed patients infected with four respiratory viruses (rhinovirus, influenza A, influenza B, and metapneumovirus) with symptoms similar to those of the novel coronavirus were used as test samples. The novel coronavirus RBD-IgA ELISA test was performed to confirm the detection specificity of the method. The test results are shown in Table 11.

[0071] Table 11 Specificity ; It can be seen from Table 11 that the results of the new coronavirus RBD-IgA ELISA test for 17 samples infected with four respiratory viruses were all negative. It can be seen that the new coronavirus RBD-IgA ELISA detection method described in the present invention does not have a cross reaction to the other four respiratory viruses, and the detection specificity meets the requirements.

[0072] Repeatability, accuracy and precision: The new coronavirus XBB strain IgA standard was diluted to three concentrations with the kit sample diluent: high value (3U / mL), medium value (1.5U / mL), and low value (0.75U / mL). Each concentration of the sample to be tested was repeated 8 times in the same experiment to evaluate the repeatability and accuracy; three different experimenters repeated the sample loading 8 times at different times to measure the precision of the system. The assessment requirements for the performance evaluation of the reagent were CV <15% and accuracy>85%. The performance evaluation results of the new coronavirus RBD-IgA ELISA detection kit established by the present invention are shown in Table 12. It can be seen from Table 12 that the same experimenter tested the high value, medium value, and low value samples in the same experiment and different experimenters tested them at different times. The repeatability and precision CV values ​​of the system were all <15%, and the accuracy recovery rate was between 85%-115%, which met the requirements.

[0073] Table 12 Repeatability, precision and accuracy test results ; Stability: Comparing the thermal acceleration stability and low temperature stability, the novel coronavirus RBD-IgA ELISA detection kit prepared by the present invention was placed at a constant temperature of 37°C for 6 days, and the novel coronavirus RBD-IgA ELISA detection kit placed in a low temperature storage environment of 2-8°C was used as a control. Preparation of the sample to be tested: Take a nasopharyngeal sample (1000U / mL), dilute it to 10U / mL as the starting detection concentration of the sample to be tested, and dilute it in multiples according to a 2-fold dilution ratio, and dilute a total of 6 concentration gradients. The test results before and after the 37°C thermal acceleration of the kit were compared and analyzed, focusing on the changes in the detection OD value and the recovery rate. The stability test results are shown in Table 13. It can be seen from Table 13 that after the kit was placed at 37°C for 6 days, the OD value recovery rate was between 80%-120% compared with the 2-8°C control group, and the background value (BLK) was less than 0.05, and the thermal acceleration stability met the requirements.

[0074] Table 13 Stability test results ; In summary: This study established a novel coronavirus RBD-IgA ELISA detection platform for the XBB strain of novel coronavirus, and prepared a novel coronavirus RBD-IgA ELISA detection kit based on it. The performance evaluation of the kit showed that the kit can specifically and quantitatively detect the content of XBB strain novel coronavirus RBD-IgA in mucosal samples, and the linear range, specificity, specificity and stability of the kit all meet the requirements.

[0075] Example 4, Application 2: Establishment of a universal ELISA detection method for immunoglobulin A Based on the two strains of mouse anti-human immunoglobulin A monoclonal antibodies Ab2 and Ab5 preferably obtained by the present invention, a universal ELISA detection platform for immunoglobulin A is constructed. The detection system focuses on optimizing the pairing screening of coating antibody / enzyme-labeled antibody and the coating concentration, and finally a double-antibody sandwich IgA universal ELISA detection system is established. The specific contents are as follows.

[0076] Two sheep anti-IgA polyclonal antibodies (sheep polyclonal antibody 1 and sheep polyclonal antibody 2) were used as coating antibodies, and HRP-labeled Ab2 (HRP-Ab2) and purchased enzyme-labeled antibodies were used as enzyme-labeled antibodies for pairing experiments for optimization and screening of coating antibodies. The screening criteria were sheep polyclonal antibodies with good specificity and appropriate sensitivity. The coating antibody was diluted to 2 μg / mL with phosphate buffer, and the enzyme-labeled plate was coated at 100 μL / well. The enzyme-labeled antibody was diluted 1000 times with enzyme-labeled diluent and set aside for use. The sample to be tested was IgA, and the sample diluent was used to dilute 6 concentration gradients, namely 10000 ng / mL, 2500 ng / mL, 625 ng / mL, 156 ng / mL, 39 ng / mL and 10 ng / mL. The swab preservation solution and sample diluent were used as negative controls to verify the anti-interference of the sample matrix. In parallel, 10 μg / mL human IgM antibody was used as the sample to be tested to verify the specificity of the test. The test results of ELISA test are as follows: Table 14 Paired screening results ; As can be seen from Table 14: of the two sheep polyclonal antibodies used as coating antibodies, sheep polyclonal antibody 1 has relatively strong non-specific reactions to swab preservation solution, sample diluent and human IgM, and although sheep polyclonal antibody 2 also has certain non-specific reactions, the sensitivity of sheep polyclonal antibody 2 to detect IgA (10ng / mL) is 1000 times higher than that of detecting IgM (10μg / mL). The comparison of enzyme-labeled antibodies shows that the detection OD value of the purchased enzyme-labeled antibody is about 2 times higher than that of HRP-Ab2, but the detection OD values ​​of the purchased enzyme-labeled antibody with swab preservation solution, sample diluent and human IgM samples are significantly higher than the detection OD value of HRP-Ab2 as an enzyme-labeled antibody. Therefore, after comprehensive evaluation, sheep anti-IgA polyclonal antibody 2 (sheep polyclonal antibody 2) was used as the coating antibody, and HRP-Ab2 prepared from the preferred monoclonal antibody of the present invention was paired as the enzyme-labeled antibody for universal ELISA detection of immunoglobulin A (IgA), which can also be called ELISA detection for total IgA.

[0077] Optimization of coating concentration: The screened sheep polyclonal antibody 2 was used as the coating antibody and diluted with phosphate buffer to 6 concentration gradients of 57.0 μg / mL, 28.5 μg / mL, 14.25 μg / mL, 7.13 μg / mL, 3.56 μg / mL and 1.78 μg / mL respectively; IgA was diluted with sample diluent as the sample to be tested and diluted in 6 concentration gradients, namely 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.13 ng / mL and 1.57 ng / mL. The sample diluent was used as the negative control. The data bias and linearity of the ELISA test results were comprehensively analyzed to determine the optimal coating concentration. The test results are shown in Table 15.

[0078] Table 15 Coating concentration ; As can be seen from Table 15, within the 6 coating concentrations (1.78-57 μg / mL) of the coated antibody sheep polyclonal antibody 2, the OD values ​​of the negative control were all less than 0.05, and the standard curve R 2 However, when the coating concentration was greater than 28.5 μg / mL, the detection OD value of the positive sample quickly entered the plateau phase, so the coating concentration of sheep polyclonal antibody 2 was preferably 28.5 μg / mL.

[0079] According to the preferred sheep polyclonal antibody 2 as the coating antibody, 28.5 μg / mL was used as the preferred coating concentration to prepare an immunoglobulin A universal ELISA plate, and HRP-Ab2 was used as the preferred enzyme-labeled antibody, supplemented with washing solution, color development solution, stop solution, etc. to form an immunoglobulin A universal ELISA detection kit. The performance of the ELISA plate, the kit and the ELISA detection method was evaluated. The performance evaluation mainly included: linear range and specificity. The results of the performance evaluation are as follows.

[0080] Linear range: Take nasopharyngeal samples (1000U / mL) and dilute them to 24ng / mL with sample diluent as the standard of immunoglobulin A universal ELISA detection kit. The samples to be tested are based on the standard and diluted with sample diluent at a 2-fold dilution ratio. A total of 5 concentration gradients are diluted and tested by immunoglobulin A universal ELISA. Each concentration is tested twice. According to the test results, the concentration is used as the horizontal axis and the average OD value of the test is used as the vertical axis to draw the standard curve, and the regression equation and regression coefficient (R 2), used to investigate the linear range of the immunoglobulin A universal ELISA detection method. The results are shown in Table 16. It can be seen that when the concentration of the sample to be tested is in the range of 0.75-12 ng / mL, the linearity of the detection OD value is relatively optimal, with an R2 of 0.9991. The standard curve of the immunoglobulin A universal ELISA detection kit is shown in Figure 5 As shown, the linearity of the standard curve is good.

[0081] Table 16 Linear range of immunoglobulin A universal ELISA detection method ; Specificity: The samples to be tested were human IgA (25 ng / mL), human IgG (100 ng / mL), human IgM (100 ng / mL) and swab preservation solution (diluted at a ratio of swab preservation solution: sample diluent = 1:4). The four samples to be tested were respectively subjected to immunoglobulin A universal ELISA test to investigate the specificity of the detection method. The test results are shown in Table 17, which shows that the test result for human IgA was positive, and the test results for the three samples to be tested, namely human IgG, human IgM and swab preservation solution, were all negative, that is, no specific immunoglobulin A (IgA) was detected. That is, the immunoglobulin A universal ELISA detection method constructed by the present invention has no cross-reaction to human IgG, human IgM, etc., and can specifically detect and identify immunoglobulin A (IgA), and the detection specificity is good.

[0082] Table 17 Specificity of the universal ELISA method for immunoglobulin A

[0083] Testing clinical samples: 30 clinical samples with a history of nasal spray new coronavirus vaccine were taken. First, the new coronavirus RBD-IgA ELISA detection method constructed by the present invention was used for detection. According to the quantitative detection results of RBD-IgA concentration, the samples were divided into three groups of high value, medium value and low value, with 10 samples in each group. Then, the universal immunoglobulin A ELISA detection kit constructed by the present invention was used to detect 30 samples in three groups to obtain the quantitative detection results of total immunoglobulin A. The test results are summarized in Table 18. The quantitative concentration results of total immunoglobulin A of individual samples are inconsistent with the three groups of concentration gradients divided based on RBD-IgA concentration. The difference in the quantitative detection results of total immunoglobulin A is large, with the highest value of 213.07μg / mL and the lowest value of 1.08μg / mL. The difference between these two extreme concentrations is nearly 200 times. The large difference may be due to the relatively unstable mucosal sampling, which may result in large differences between different people or different sampling times. Therefore, by calculating the ratio of specific IgA to total IgA to obtain the relative content of immunoglobulin A, we can effectively avoid the influence of sampling factors and more objectively evaluate the immune effect of the vaccine.

[0084] Table 18 Clinical sample test results

[0085] After excluding the outliers, the ratio of the quantitative test results of 29 clinical samples obtained by the two detection methods was further compared and analyzed, that is, the ratio of the detection value of the new coronavirus RBD-IgA to the detection value of total IgA. The comparative analysis is shown in Table 19.

[0086] Table 19 Analysis of quantitative results of RBD-IgA and total IgA ; In Table 19, ratio = RBD-IgA (U / mL) / total IgA (μg / mL) × 100, when the detection concentration of RBD-IgA is less than 4U / mL, it is calculated as 1U / mL. The detection method of the relative content of immunoglobulin A established by the screened monoclonal antibody of the present invention can effectively avoid the difference in sampling methods.

[0087] Table 20 Correlation analysis between the two detection methods

[0088] The present invention further analyzed the correlation between the detection concentration of RBD-IgA and the detection concentration of total IgA obtained by the two detection methods through Pearson correlation (see Table 20). The results showed that the correlation coefficient values ​​of the quantitative concentration of RBD-IgA and the quantitative concentration of total IgA were both 1, indicating that the results of the novel coronavirus RBD-IgA detection alone or the immunoglobulin A universal ELISA detection alone constructed by the present invention were linearly positively correlated when the sampling method standard was stable. Moreover, the Pearson correlation analysis showed that the correlation value between the RBD-IgA test results and the total IgA test results was <0.001 (at the 0.01 two-tailed level), that is, the correlation between the two was significant. Although a small number of samples among the 30 clinical samples had inhibitor interference, which made the correlation between the two IgA test results poor, the Pearson correlation analysis showed that overall, there was a certain regularity in the relative content of specific immunoglobulin A (such as RBD-IgA concentration) in the total IgA concentration in the immune population after vaccination, and the correlation was significant. Therefore, the specific IgA proportion method can be used to analyze the immune effect, thereby further avoiding the errors caused by sampling, and used to more objectively evaluate the immune effect of the vaccine. At the same time, if combined with other test data, it is more conducive to a comprehensive evaluation of the immune protection effect.

[0089] 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 monoclonal antibody of immunoglobulin A, characterized in that A monoclonal antibody of specific immunoglobulin A secreted by the mouse anti-human immunoglobulin A monoclonal antibody hybridoma cell line 5F5A4F3. The preservation number of the mouse anti-human immunoglobulin A monoclonal antibody hybridoma cell line 5F5A4F3 is CGMCC No.46012, the preservation address is the General Microbiology Center of China National Microbiological Culture Collection Administration, and the preservation date is July 2, 2024.

2. A hybridoma cell line, characterized in that Used to secrete a monoclonal antibody that produces the immunoglobulin A described in claim 1, the hybridoma cell line has a preservation number of CGMCC No.46012, the preservation address is the General Microbiology Center of the China Microbiological Culture Collection Administration, and the preservation date is July 2, 2024.

3. An enzyme-linked immunosorbent assay method, characterized in that: The monoclonal antibody of immunoglobulin A according to claim 1 is used for detection.

4. The enzyme-linked immunosorbent assay method according to claim 3, characterized in that: The double antibody sandwich ELISA method is used to detect the specific immunoglobulin A produced by specific antigen stimulation to obtain the quantitative results of specific immunoglobulin A.

5. The enzyme-linked immunosorbent assay method according to claim 3, characterized in that: The double antibody sandwich ELISA method is used for universal detection of all immunoglobulin A produced by mucosal immunity to obtain quantitative results of total immunoglobulin A.

6. The enzyme-linked immunosorbent assay method according to claim 4, characterized in that: Used to detect RBD-IgA of the new coronavirus.

7. An enzyme-linked immunosorbent assay kit, characterized in that: It contains an enzyme-labeled antibody and a coated antibody, wherein the enzyme-labeled antibody is prepared from the monoclonal antibody of the immunoglobulin A according to claim 1 and is used to detect specific immunoglobulin A.

8. A method for detecting the relative content of immunoglobulin A, characterized in that: The quantitative result of specific immunoglobulin A obtained by the enzyme-linked immunosorbent assay method described in claim 4 and the quantitative result of total immunoglobulin A obtained by the enzyme-linked immunosorbent assay method described in claim 5 are calculated as a ratio of the two quantitative results to obtain the relative content of immunoglobulin A.

9. Use of the monoclonal antibody of immunoglobulin A according to claim 1, the hybridoma cell line according to claim 2, the enzyme-linked immunosorbent assay method according to any one of claims 3 to 6, the enzyme-linked immunosorbent assay kit according to claim 7, or the method for detecting the relative content of immunoglobulin A according to claim 8 in detecting the content of immunoglobulin A.

10. Use of the monoclonal antibody of immunoglobulin A according to claim 1 or the hybridoma cell line according to claim 2 in evaluating the immune effect of vaccines.

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