Monoclonal antibodies to immunoglobulin a and their use

Abstract

The application provides a monoclonal antibody of immunoglobulin A and application thereof, the monoclonal antibody is a specific monoclonal antibody of immunoglobulin A secreted by a mouse anti-human immunoglobulin A monoclonal antibody hybridoma cell line 5F5A4F3 strain, can be used for not only quantitative detection of the concentration of RBD-IgA generated by specific antigen stimulation, but also quantitative detection of the concentration of total IgA generated by mucosal immunity, and the ELISA method constructed based on the monoclonal antibody of immunoglobulin A is good in linearity, high in sensitivity and strong in specificity, is suitable for being used for evaluating the immunization effect of a vaccine, and is also suitable for being used for screening and monitoring the antibody level of IgA in a large-scale population.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology and diagnostic reagents, in particular to a monoclonal antibody of immunoglobulin A, classified as a monoclonal antibody of murine anti-human immunoglobulin A, and 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

[0002] Since the outbreak of COVID-19, co-infection of respiratory viruses has occurred from time to time. Subsequently, global vaccine research and development has entered a stage of rapid development, and the study of the immune protection mechanism of nasal spray or inhalation respiratory mucosal immunity vaccines has made new progress and achieved significant breakthroughs in vaccine development. It has been reported that by nasal inoculation, the natural infection of the virus is simulated, and the secretory immunoglobulin A (SIgA) is induced in the nasal mucosa to establish the first effective defense barrier between the body and the external environment, and then to stimulate cellular immunity and antibody response; the local multidimensional protective immune factors in the respiratory tract have better spatial distance advantage than peripheral immune factors, and can respond to viral infection more timely; compared with injection of vaccines, although the humoral immune response rate and neutralizing antibody level of nasal influenza attenuated live vaccine are significantly lower than those of inactivated influenza virus (IIV), but the nasal inoculation route is more likely to produce natural immunity similar to infection, and ultimately shows relatively higher protection derived from mucosal immunity; nasal spray vaccine can induce multidimensional broad-spectrum protective immune response, including cellular immunity, mucosal immunity, innate immunity and trained immunity; the nasal spray live-attenuated influenza virus (LAIV) of Flumist company in the United States can produce good immunogenicity in children; after vaccination with nasal spray LAIV, IgA produced in the nasal mucosa plays an important role in resisting influenza virus infection; on the 31st day after vaccination with freeze-dried nasal spray LAIV of Changchun Btk Biological Technology Co., Ltd., the proportion of vaccinees with 2-fold increase in H3N2 and B type IgA antibody titers was 38.75% and 31.05%, respectively, which was significantly higher than the positive conversion rate calculated based on HI (hemagglutination inhibition) antibody, however, only 3%~23% of serum samples with 2-fold increase in IgA antibody titers had more than 4-fold increase in HI antibody titers. Therefore, injection route vaccines mainly induce the production of serum immunoglobulin G (IgG), while nasal inoculation vaccines can induce the production of serum IgG and nasal mucosal immunoglobulin A (IgA), as well as possible cellular immune response, which can provide more extensive and durable immune protection for the body, especially the level of SIgA produced by the nasal mucosa is closely related to the protective efficacy of the vaccine, so the level of mucosal IgA can be used as an important monitoring indicator for evaluating the immune effect of the vaccine.

[0003] Vaccines via nasal inoculation route have the advantages of non-invasiveness and easy acceptance, and are especially suitable for children and other groups. Common inoculation methods include nasal spray, inhalation and other respiratory routes. In addition, the unique advantages of the mucosal immune system in defending against external pathogen infection also include that the immune cells contained in the mucosal tissue account for 80% of all immune cells, and the mucosal immune system has specific T cells and cytokine regulation, 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 titer and high specificity for mucosal IgA. Therefore, it is necessary to develop a specific IgA and a rapid, simple and efficient mucosal IgA detection method that can be quantified and has high sensitivity, accuracy and specificity.

[0004] The enzyme-linked immunosorbent assay (ELISA) has the advantages of low cost, high sensitivity, strong specificity and good repeatability, and can be used to detect specific proteins in body fluids. The ELISA method can be used in mucosal IgA detection. The basis and key of the ELISA method are to screen a specific antigen or antibody protein, and to adsorb the protein to a solid carrier under the premise of maintaining the activity of the protein, to form an antigen or antibody complex containing an enzyme as a label. In addition, there are technical problems such as sample collection when combining ELISA with IgA detection. On the one hand, the body produces 60-66 mg / kg of IgA per day, which is more than the total amount of all antibodies, and can be updated once every 8 hours. Due to the high frequency of IgA secretion and excretion, the distribution of IgA in the nasal mucosa sample may be uneven, and the sampling personnel have large differences in sampling techniques. On the other hand, the nasal mucosa IgA sampling generally uses nasal washing, swab or sponge sampling, scraping the nose or nasal absorption method. The sample collection process uses physiological saline, culture medium or PBS solution (containing FBS and EDTA), etc., resulting in a complex matrix of the collected nasal swab sample. In addition, IgA has sequence similarity with IgM, IgG and other antibody types, which may have non-specific reactions during detection. 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 to establish a rapid, simple and efficient mucosal IgA ELISA detection method that can be quantified and has high sensitivity, accuracy and specificity. SUMMARY

[0005] The present application provides a monoclonal antibody of immunoglobulin A, and a hybridoma cell line secreting the monoclonal antibody of immunoglobulin A.

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

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

[0008] To achieve the above-mentioned purposes, the present application provides the following technical solutions.

[0009] In a first aspect, the present application provides a monoclonal antibody of immunoglobulin A, which is a specific monoclonal antibody of immunoglobulin A secreted by a mouse anti-human immunoglobulin A monoclonal antibody hybridoma cell line 5F5A4F3 strain, and is denoted as anti-IgA-McAb. The preservation number of the mouse anti-human immunoglobulin A monoclonal antibody hybridoma cell line 5F5A4F3 is CGMCC No. 46012, the preservation unit is China General Microbiological Culture Collection Center, the preservation unit address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation 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-human immunoglobulin A monoclonal antibody hybridoma cell line.

[0010] According to a preferred embodiment of the present application, the monoclonal antibody of immunoglobulin A can specifically recognize and bind to the heavy chain constant region of secreted immunoglobulin A.

[0011] In a second aspect, the present application provides a hybridoma cell line for secreting a monoclonal antibody of immunoglobulin A, and the preservation number of the hybridoma cell line is CGMCC No. 46012, the preservation address is China General Microbiological Culture Collection Center, and the preservation date is July 2, 2024.

[0012] In a third aspect, the present application provides an enzyme-linked immunosorbent assay method, which uses a monoclonal antibody of immunoglobulin A for detection. The detection method is not a method for diagnosing and / or treating diseases.

[0013] According to a preferred embodiment of the present application, the detection step is: 1) after the sample to be tested is diluted in proportion with the sample diluent, it is added to the corresponding well of the enzyme-labeled plate; 2) it is washed with the washing solution; 3) the enzyme-labeled antibody is added, and incubated at 35-39°C for 50-70 minutes; 4) it is washed with the washing solution; 5) the color developing agent is added, and color development is carried out in a constant-temperature incubator at 35-39°C for 10-20 minutes in the dark, and then the termination solution is added to terminate, and the color change process of the positive result of the enzyme-labeled well is blue first and then yellow; and 6) the detection result is output by the enzyme-labeled instrument, and compared with the cutoff value to determine the positive result or the negative result.

[0014] According to a preferred embodiment of the present application, the enzyme-linked immunosorbent assay detection method adopts the double-antibody sandwich ELISA method, is used for detecting specific immunoglobulin A generated by specific antigen stimulation, and obtains the quantitative result of specific immunoglobulin A.

[0015] Preferably, the enzyme-linked immunosorbent assay detection method is used for detecting novel coronavirus RBD-IgA.

[0016] According to a preferred embodiment of the present application, the enzyme-linked immunosorbent assay detection method adopts the double-antibody sandwich ELISA method, is used for detecting all immunoglobulin A generated by mucosal immunity, and obtains the quantitative result of total immunoglobulin A.

[0017] In a fourth aspect, the present application provides an enzyme-linked immunosorbent assay detection kit containing an enzyme-labeled antibody and a coating antibody, wherein the enzyme-labeled antibody is prepared from the above-mentioned monoclonal antibody of immunoglobulin A, and is used for detecting specific immunoglobulin A.

[0018] According to a preferred embodiment of the present application, the enzyme-linked immunosorbent assay detection kit adopts the above-mentioned enzyme-linked immunosorbent assay detection method to quantitatively detect the sample to be tested, and the monoclonal antibody of immunoglobulin A is used in the kit.

[0019] According to a preferred embodiment of the present application, the enzyme-linked immunosorbent assay detection kit is used for detecting novel coronavirus RBD-IgA.

[0020] According to a preferred embodiment of the present application, the components of the enzyme-linked immunosorbent assay detection kit include an enzyme-labeled plate, an enzyme-labeled antibody, a negative control, a positive control, a sample diluent, and an inflatable sponge swab for collecting a nasopharyngeal swab sample.

[0021] Preferably, the enzyme-labeled antibody is a monoclonal antibody of immunoglobulin A labeled with HRP.

[0022] In a fifth aspect, the present application provides a method for detecting the relative content of immunoglobulin A, which comprises: obtaining a specific immunoglobulin A by detecting the specific immunoglobulin A produced by stimulation of the specific antigen; obtaining a total immunoglobulin A by detecting all the immunoglobulin A produced by mucosal immunity; and calculating the ratio of the specific immunoglobulin A to the total immunoglobulin A to obtain the relative content of the immunoglobulin A.

[0023] In a sixth aspect, the present application provides a monoclonal antibody of the immunoglobulin A, a hybridoma cell line for secreting the monoclonal antibody of the immunoglobulin A, an enzyme-linked immunosorbent assay method based on the monoclonal antibody of the immunoglobulin A, an enzyme-linked immunosorbent assay kit based on the monoclonal antibody of the immunoglobulin A, and an application of a method for detecting the relative content of the immunoglobulin A based on the monoclonal antibody of the immunoglobulin A in the detection of the content of the immunoglobulin A.

[0024] In a seventh aspect, the present application provides the monoclonal antibody of the immunoglobulin A and the application of the hybridoma cell line for secreting the monoclonal antibody of the immunoglobulin A in the evaluation of the immune effect of a vaccine.

[0025] According to a preferred embodiment of the present application, the application in the evaluation of the immune effect of a vaccine is achieved by calculating the ratio of the specific immunoglobulin A produced by stimulation of the specific antigen to all the immunoglobulin A produced by mucosal immunity.

[0026] Preferably, the specific antigen is a novel coronavirus, and the specific immunoglobulin A is RBD-IgA.

[0027] The present application has the following beneficial effects:

[0028] The present application provides a monoclonal antibody of the immunoglobulin A, which is prepared from a human serum sample, and the human IgA protein with a purity of 90% obtained after PEG precipitation, Protein G and Protein L purification is used as an immunogen. Through repeated screening, the present application selects a positive hybridoma cell line capable of secreting the monoclonal antibody of the immunoglobulin A from 6 positive hybridoma cell lines, and obtains the monoclonal antibody of the immunoglobulin A (anti-IgA-McAb) by preparing ascites and protein purification. Through titer and affinity detection, specificity and epitope identification, and identification of the binding segment, the titer of the preferred monoclonal antibody of the immunoglobulin A of the present application is 2.35 million, the affinity of the high-salt elution detection is 72%, and the monoclonal antibody of the immunoglobulin A is a linear epitope antibody, which can specifically recognize and combine with the heavy chain constant region of the secreted immunoglobulin A, and can be combined with the enzyme-linked immunosorbent assay method for quantitative detection of the immunoglobulin A.

[0029] Based on the final preferred No. 2 immunoglobulin A monoclonal antibody, the application constructs a new coronavirus RBD-IgA ELISA detection platform, including a new coronavirus RBD-IgA ELISA detection method, a new coronavirus RBD-IgA ELISA kit and related applications thereof. It can be used for quantitative detection of the content of new coronavirus RBD-IgA in the sample to be tested, especially XBB strain of new coronavirus. The presence and level of RBD-IgA can be used to assist in analyzing the immune status after new coronavirus infection, and can also be combined with IgM and IgG antibody detection to evaluate the immunogenicity and effectiveness of vaccines, or used for screening and monitoring of RBD-IgA levels in large population. In addition, to find potential infected persons and take corresponding prevention and control measures. The new coronavirus RBD-IgA ELISA detection can also be used to detect the level of RBD-specific antibody at different time points, different populations and after different vaccination, which can further understand the immune response rules and influencing factors of new coronavirus, and provide scientific basis for disease prevention and control and vaccine development.

[0030] High-quality and high-specificity antibodies are the key to establish ELISA detection of total immunoglobulin A. Based on the final preferred No. 2 immunoglobulin A monoclonal antibody, the application establishes an immunoglobulin A universal ELISA detection method and kit, which has high sensitivity and specificity for detection of total immunoglobulin A, good accuracy, high precision, good repeatability, and the quantitative detection range of all immunoglobulin A is 0.75-12 ng / mL, R 2 >0.99, the linearity of the detection result is good, and the reliability is high. The quantitative detection of total immunoglobulin A can not only be used for evaluating the immune status of the body, monitoring the abnormal immune system response 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, according to the ratio of the two, it can be more objectively used for evaluation of the immune effect of vaccines, thereby overcoming the error caused by sampling factors.

[0031] The application combines the screened monoclonal antibody with high affinity, high titer, strong anti-interference ability, specific recognition and combination of immunoglobulin A, and an ELISA detection method with high sensitivity, strong specificity and good repeatability, to establish a specific detection method for immunoglobulin A with high sensitivity and high specificity, and a universal detection method for all immunoglobulin A produced by mucosal immunity, which has low detection cost, high throughput, simple operation, and can obtain the relative content of immunoglobulin A by calculating the ratio of the two detection results, effectively overcome the error caused by the sampling method, and be more objectively used for evaluating the immunization effect of the vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a recognition specificity and epitope identification result diagram of the immunoglobulin A monoclonal antibody of the application, and the different protein samples are indicated on the picture, wherein 1 is human IgG, 2 is human IgA, and 3 is human IgM; M is a protein marker; and the six monoclonal antibodies Ab1 to Ab6 added in the WB experiment are indicated below the picture.

[0033] Figure 2 It is an SDS-PAGE electrophoresis comparison diagram of the immunoglobulin A before and after the enzyme cutting reaction of pepsin, and in the upper mark of the picture, M is a protein marker, 1 is a human IgA protein, and 2 is a protein sample after the enzyme cutting reaction of human IgA protein;

[0034] Figure 3 It is a WB detection result diagram of the immunoglobulin A monoclonal antibody and human IgA protein before and after the enzyme cutting reaction, and in the upper mark of the picture, M is a protein marker, 1 is a human IgA protein, and 2 is a protein sample after the enzyme cutting reaction of human IgA protein; and the added monoclonal antibodies for detection in the WB experiment are Ab2 and Ab5, respectively, which are indicated below the picture.

[0035] Figure 4 It is a Dot Blot screening result of the enzyme-labeled antibody, A is the enzyme-labeled antibody of the immunoglobulin IgA monoclonal antibody Ab2 of the application, and B is a third-party control goat anti-human IgA enzyme-labeled antibody; the sample concentration of human immunoglobulin with five concentration gradients is represented on the top of the diagram; and the vertical ①, ② and ③ represent different types of human immunoglobulin: human IgA, human IgM and human IgG.

[0036] Figure 5 It is a standard curve diagram of the universal ELISA detection of immunoglobulin A: the horizontal coordinate is the concentration of total IgA, and the vertical coordinate is the OD value of the corresponding ELISA detection. DETAILED DESCRIPTION

[0037] The following detailed description of the application will be made with reference to the accompanying drawings, in which like numerals designate like elements in all of the figures, and in which the exemplary embodiments of the application are described in sufficient detail to provide a thorough understanding of the embodiments of the application being disclosed. Where specifically recited, the same or similar designations will be used to represent the same or similar concepts, such as monoclonal antibody = mAb, polyclonal antibody = pAb, IgA = immunoglobulin A, SIgA = secretory immunoglobulin A, anti-IgA-McAb = IgA-McAb = monoclonal antibody to immunoglobulin A, Ab = antibody, etc.

[0038] In the following detailed description of the application, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the application being disclosed. In the following detailed description of the application, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the application being disclosed. The following examples are intended to be illustrative only and are not to be construed as limiting the scope of the application.

[0039] In the following detailed description of the application, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the application being disclosed. In the following detailed description of the application, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the application being disclosed. The following examples are intended to be illustrative only and are not to be construed as limiting the scope of the application.

[0040] In the following detailed description of the application, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the application being disclosed. In the following detailed description of the application, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the application being disclosed. The following examples are intended to be illustrative only and are not to be construed as limiting the scope of the application.

[0041] The present application takes human serum samples as raw materials, and uses human IgA protein with a purity of 90% obtained by PEG precipitation, Protein G and Protein L purification as immunogen. The purified human IgA protein is immunized to Balb / c mice using Freund's complete adjuvant and incomplete adjuvant. Mice with serum titers of about one million are selected by indirect ELISA method, and their spleen cells are fused with myeloma cells sp2 / 0 at a cell ratio of 1:5. Positive hybridoma cell lines secreting high specificity and high affinity anti-IgA are screened by indirect ELISA method, HAT pressure method and WB method. After three rounds of limited dilution, six strains of monoclonal positive hybridoma cell lines are obtained. The six strains of positive hybridoma cell lines are frozen to establish a cell bank, and are inoculated into sensitized Balb / c mice to prepare ascites for large-scale culture of monoclonal antibodies. After protein purification of the collected ascites, six strains of monoclonal antibodies (anti-IgA-McAb) against human immunoglobulin A are obtained. The titers and performance of the monoclonal antibodies are detected and evaluated, respectively. The specific evaluation content includes: the preliminary evaluation of the antibody reactivity and specificity of the six monoclonal antibodies by WB method and Dot Blot method, the detection of the monoclonal antibody titer and affinity by indirect ELISA method, and the specific reaction pairing experiment of different types of human immunoglobulin to screen one strain of monoclonal antibody with high titer, high affinity, high specificity and no cross reaction with IgG and IgM from the six monoclonal antibodies, which is the preferred specific monoclonal antibody of immunoglobulin A (anti-IgA-McAb).

[0042] The application is based on a preferred monoclonal antibody (anti-IgA-McAb) secreting hybridoma cell line capable of secreting specific immunoglobulin A and the monoclonal antibody (Ab2) of immunoglobulin A secreted by the hybridoma cell line, the RBD recombinant protein is fixed on a solid carrier, the enzyme-labeled antibody HRP-Ab2 is selected by pairing experiment, and the sample to be detected can be a nasal swab sample, thereby constructing an ELISA detection method for RBD-IgA of the XBB strain of the novel coronavirus, which is used for detecting the content of RBD-IgA of the XBB strain of the novel coronavirus in human nasal mucosa samples. The detection method can quantitatively obtain the antibody concentration of RBD-IgA, the sensitivity of the quantitative detection method is 0.16 U / mL, the specificity is 100%, the anti-matrix interference is strong, the XBB strain of the novel coronavirus RBD-IgA indirect ELISA quantitative detection kit constructed by the detection method meets the requirements in linear range, specificity, specificity, accuracy, precision, stability and other performance indicators, and can be used to evaluate the mucosal immune response after nasal spray novel coronavirus vaccine immunization. In addition, based on HRP-Ab2 as the enzyme-labeled antibody and goat anti-IgA polyclonal antibody as the coating antibody, an ELISA detection method for total immunoglobulin A produced by mucosal immunity is constructed, which can analyze the change trend of IgA in different periods of clinical samples, can be used as a quality control method for total IgA detection, and can obtain the relative content of immunoglobulin A 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 application are described in detail as follows:

[0043] Example 1, preparation and screening of positive hybridoma cells

[0044] The preparation and screening of positive hybridoma cells include preparation of human IgA protein immunogen, animal immunization, cell fusion and screening of positive hybridoma cell lines.

[0045] Preparation of immunogen: human serum samples were used as raw materials, PEG precipitation method was used, 7% PEG precipitation mother liquor was added and mixed, and then static setting was carried out; the protein purification steps were carried out in the order of Protein G purification and Protein L purification.

[0046] Protein G purification: The supernatant after the above precipitation was appropriately diluted with purified water, and then filtered through a 0.45 μm filter to obtain a filtrate; column treatment step: the column was washed with 1xPBS equilibration buffer until the pH value, conductivity and UV280 absorbance parameters reached a stable baseline and were ready for use; sample loading purification process: the filtered filtrate was pumped into the Protein G column at a flow rate of 3 mL / min, and when the UV280 absorbance rose to 50 mAU, the breakthrough liquid was collected; after sample loading was completed, collection was continued until the UV280 absorbance dropped to 50 mAU, and collection was stopped; the column was again washed with the equilibration liquid until the parameters returned to a stable baseline state; elution step: the column was eluted with 0.05M glycine eluent to obtain the first eluate.

[0047] Protein L purification: including column treatment, sample loading purification, elution and collection steps; wherein, the column treatment step: the Protein L column was washed with 1xPBS equilibration buffer until the pH value, conductivity and UV280 absorbance parameters reached a stable baseline and were ready for use; sample loading purification process: the first eluate was pumped into the Protein L column at a flow rate of 3 mL / min, and the change in UV280 absorbance was continuously monitored; after sample loading was completed, the column was washed with the equilibration liquid until the UV280 absorbance returned to a stable baseline state to ensure that there was no residual sample in the column; elution step: the column was eluted with 0.05M glycine eluent, and during the elution process, the change in UV280 absorbance was closely monitored to identify the appearance of the elution peak; when the UV280 absorbance rose to 50 mAU, the eluate was collected, and the collection was continued until the UV280 absorbance dropped to 50 mAU, and the collection was stopped, ensuring that the complete elution peak was collected, and the second eluate, i.e., the target protein, human IgA protein, was obtained. The column was again washed with the equilibration liquid until the UV280 absorbance returned to a stable baseline state, ready for the next purification operation.

[0048] 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 to form a drop that does not spread, and the final concentration of the immunogen is 100 μg / mL. The immunization route is subcutaneous injection at multiple points on the back of the mouse, the immunization dose is 20 μg per mouse, and the immunization volume is 0.2 mL per mouse. Six Balb / c male mice are injected simultaneously in each group. In the second immunization, two weeks after the first immunization, the immunogen is fully emulsified with an equal volume of Freund's incomplete adjuvant to form a drop that does not spread, and the same final concentration of the immunogen, immunization dose, immunization volume, and immunization route as in the first immunization are used, i.e. 100 μg / mL, 20 μg per mouse, 0.2 mL per mouse, and subcutaneous injection at multiple points 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 to form a drop that does not spread, and the same final concentration of the immunogen, immunization dose, immunization volume, and immunization route as in the first immunization are used again, i.e. 100 μg / mL, 20 μg per mouse, 0.2 mL per mouse, and subcutaneous injection at multiple points on the back of the mouse. Two weeks after the third immunization, blood is collected, and the antibody titer produced after immunization is detected by indirect ELISA. Mice with an antibody titer of about 1:1,000,000 can be used for subsequent challenge immunization and cell fusion. In the challenge immunization, the immunization dose of the immunogen is 20 μg per mouse, which is diluted with 10 mM phosphate buffer to an immunization volume of 100 μL per mouse. The immunization route is intravenous injection into the tail vein of the mouse, and the spleen cells are collected 3-4 days later for cell fusion.

[0049] Cell fusion: prepare the spleen cells and SP2 / 0 myeloma cells for fusion in advance, mix them in a 50 mL fusion tube, centrifuge to collect the cells, 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, centrifuge to collect the cells, add an appropriate amount of HAT medium to resuspend the cells, and inoculate the cell suspension in 100 μL per well into 11 96-well cell plates coated with feeder cells. Continue to culture for 10-14 days for screening of positive hybridoma cells.

[0050] The steps for screening positive hybridoma cells are as follows: after cell fusion, continue to culture for 10-14 days, aspirate the culture supernatant of the fusion cells in the 96-well cell plate, load 50 μL / well into the 96-well detection plate coated with the above human IgA protein (coating concentration is 0.2 μg / mL), add 50 μL / well of sample diluent, incubate at 37°C for 60 minutes, wash 5 times; dilute the goat anti-mouse IgG-HRP by 5000 times, add 100 μL / well to the 96-well detection plate, incubate at 37°C for 45 minutes, wash 5 times; add TMB color developing solution for color development for 15 minutes, add stop solution (2M H2SO4) to terminate the color development reaction, use 450 / 630 dual-wavelength to determine the absorbance OD, and take OD≥0.2 as the judgment standard for positive hybridoma cells, and combine the growth of the fusion cells in the cell culture plate to preliminarily screen 21 positive hybridoma cell strains. The 21 positive hybridoma cell strains are screened by limited dilution method for 3 times of monoclonalization screening, and 6 hybridoma cell strains capable of secreting specific IgA monoclonal antibodies are obtained. The 6 cloned positive hybridoma cell strains are scaled up for culture to establish a cell bank, and are cryopreserved.

[0051] Meanwhile, the culture supernatant of the 6 positive hybridoma cell strains is used for preparation and identification of anti-IgA-McAb ascites, and the 6 positive hybridoma cell strains are named as cell strain 1 to cell strain 6, wherein the cell strain 2 is a mouse anti-human immunoglobulin A monoclonal antibody hybridoma cell line 5F5A4F3 strain.

[0052] Example 2, identification of anti-IgA-McAb

[0053] The preparation and identification process of anti-IgA-McAb ascites includes preparation of ascites, purification of monoclonal antibodies, detection of titer and affinity, identification of specificity and epitope, and identification of binding segments.

[0054] Preparation of ascites: take the 6 positive hybridoma cell strains collected after the above scaling-up culture, wash with RPMI-1640 medium for 2 times, inoculate into the abdominal cavity of the sensitized mouse at an injection amount of 5×10 5 ~1×10 6 The mouse is used to collect the ascites in the abdominal cavity with a sterile syringe every 1-2 days, and the ascites collected in the abdominal cavity of the mouse contains the expanded immunoglobulin A monoclonal antibody (anti-IgA-McAb), which is stored at -20°C for standby use after labeling and sub-packaging. The monoclonal antibodies secreted by the cell strain 1 to the cell strain 6 correspond to anti-IgA-McAb 1 to anti-IgA-McAb 6 (abbreviated as Ab1 to Ab6), and the monoclonal antibody secreted by the 5F5A4F3 hybridoma cell line corresponds to anti-IgA-McAb 2 (abbreviated as Ab2).

[0055] The purification of the monoclonal antibodies includes crude purification and fine purification. First, the crude purification step by saturated ammonium sulfate includes: the collected ascites (containing Ab1 to Ab6) is diluted 3 to 5 times with 10 mM phosphate buffer, then an equal volume of saturated ammonium sulfate is added for precipitation, and after precipitation, the 10 mM phosphate buffer is added again for redissolution. Second, the fine purification is performed by using Mabselect Protein A affinity purification method, and the fine purification step includes: the affinity column is treated with a binding buffer (including 0.05 M Tris, 0.5 M NaCl) and an elution buffer (including 0.05 M glycine, 0.5 M NaCl), then the column is equilibrated to neutral with 10 mM phosphate buffer, then the crude purified monoclonal antibodies (Ab1 to Ab6) redissolved in the 10 mM phosphate buffer are loaded on the column, after the loading is completed, the column is washed with 10 mM phosphate buffer until the OD280 value of the flow-through returns to the baseline, then the elution is performed with 0.05 M glycine-hydrochloric acid solution (pH 3.0), and the solution of the whole elution peak is collected, which is the fine purified monoclonal antibodies (Ab1 to Ab6) and is further subjected to performance detection and identification of the monoclonal antibodies.

[0056] Titer and affinity detection: Take the above purified 6 monoclonal antibodies (Ab1-Ab6) as the tested monoclonal antibodies, and detect the antibody titer and affinity of the 6 monoclonal antibodies by indirect enzyme-linked immunoassay and high-salt elution method respectively. In the high-salt elution method, the high-concentration salt can be selected from hydrogen peroxide or urea, and urea is preferred. The specific operation steps are as follows: take the tested monoclonal antibody and dilute it with 1xPBS to the same protein concentration. Dilute the tested antibody with the same protein concentration by 100 times as the starting loading detection concentration, and then dilute it by 5 times according to the series, a total of 8 concentration gradients are prepared, 8 concentrations of diluents are added to the corresponding holes of the micro-hole plate (0.2 μg / mL concentration coated with human IgA protein 96-hole detection plate) with a dosage of 100 μL / hole, each concentration is repeated 2 holes, of which 1 hole is used as an experimental hole and 1 hole is used as a control hole; after the addition is completed, it is incubated at 37°C for 60 minutes. Wash the plate: the amount of washing solution is 300 μL / hole, first wash the plate with 1xPBST for 1 time, after adding the washing solution, soak at room temperature for 3 minutes, the experimental hole is washed twice with 1xPBST containing 4M urea, and the control hole is washed twice with 1xPBST, then wash the plate with 1xPBST for 1 time, and try to dry it. Add color developing solution A and B each 50 μL / hole, and place it in a 37°C constant temperature incubator to develop color for 15 minutes in the dark; add the stop solution according to 50 μL / hole to terminate the color developing reaction; use 450 / 630 dual-wavelength to measure the absorbance OD value of each hole. The detection result of the control group is the titer of the monoclonal antibody, and the titer value is at least 400,000; the detection result of the experimental hole is selected, and the OD value of 1.0 after urea elution is divided by the detection OD value of the control sample with the same dilution multiple, and the percentage calculated is the relative affinity of the monoclonal antibody. The titer and affinity detection results of the 6 monoclonal antibodies are shown in Table 1.

[0057] Table 1 Titer and affinity detection results

[0058] ;

[0059] Specificity and epitope identification: Take human IgA, human IgG, and human IgM proteins, respectively, and dilute them to 0.2 mg / mL, 10 μL per hole, and perform SDS-PAGE electrophoresis. Subsequently, semi-dry transfer membrane method is used, and the membrane is transferred at 1600mA current for 10 minutes. Dilute the purified monoclonal antibody to an appropriate concentration (5 μg / mL) for WB detection analysis. Through at least 3 repeated experiments, the results prove that the 6 purified monoclonal antibodies can specifically bind to IgA and develop color, and there is no cross reaction with IgG and IgM, as shown in Figure 1 Table 2, it can be seen that the 6 monoclonal antibodies can specifically recognize IgA. In addition, through WB detection, it is preliminarily confirmed that the 6 monoclonal antibodies are linear epitope antibodies that can specifically recognize IgA.

[0060] Identification of IgA segment bound by monoclonal antibody: According to the results of WB detection, 2 strains of monoclonal antibodies (Ab2, Ab5) with specific binding, high reactivity and stable repeated experimental results were selected as verification objects, and WB experiment was further used to analyze the specific binding segment of the 2 strains of monoclonal antibodies to IgA. Take human IgA protein, first use pepsin for enzyme reaction, and take the sample before and after enzyme reaction respectively, dilute to 1 mg / mL, and load 10 μL per well for SDS-PAGE electrophoresis. Subsequently, semi-dry transfer membrane method was used, and the membrane was transferred at 1600 mA current for 10 minutes. The purified monoclonal antibody was diluted to an appropriate concentration (5 μg / mL), and the binding of the monoclonal antibody to human IgA before and after enzyme reaction was analyzed by WB experiment. The results of SDS-PAGE electrophoresis of human IgA protein before and after enzyme reaction are shown in Figure 2 The results of WB experiment of monoclonal antibody binding to human IgA protein before and after enzyme reaction are shown in Figure 3 .

[0061] Human IgA can be divided into IgA1 and IgA2 two subtypes in structure, the amino acid sequences of the 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, especially lacks cysteine residues required for disulfide bond formation, so the heavy chain and light chain are not connected by disulfide bond, but by non-covalent bond. This structural difference leads to different sensitivity 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 and enzymatically cleaved by pepsin, while IgA2 cannot be recognized and enzymatically 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 multimer structure; each SIgA molecule contains a J chain and a secretory piece, and the J chain connects two or more IgA monomers through multiple disulfide bonds and other interactions, while the secretory piece enhances the structural stability of SIgA, so that it can further resist enzymatic degradation by pepsin. Combined with the fact that the two subtypes of IgA have different sensitivities to pepsin, it can be seen that the two subtypes of IgA have different structures and functions, and the two subtypes of IgA have different sensitivities to pepsin. Figures 2-3 It can be seen that the two strains of monoclonal antibodies (Ab2, Ab5) prepared and preferably obtained in the present application can not only specifically recognize IgA, but also specifically recognize and bind to the heavy chain (α chain), and do not react with the variable region (Fab segment) remaining after pepsin enzyme cleavage. Therefore, it is speculated that the two strains of monoclonal antibodies (Ab2, Ab5) bind to the constant region of the heavy chain (α chain) of IgA, so the two strains of monoclonal antibodies (Ab2, Ab5) can be used for the analysis and application of SIgA produced by nasal mucosa.

[0062] Example 3, application 1: establishment of a new coronavirus RBD-IgA antibody ELISA detection method

[0063] The receptor binding domain (RBD) of the novel coronavirus XBB strain is a specific research object for the application of the present application. The RBD recombinant protein of the novel coronavirus XBB strain is used as the coating protein of the enzyme-labeled plate; the nasopharyngeal sample with a concentration of 1000 U / mL is used as the sample to be tested;

[0064] Coating concentration optimization: the RBD recombinant protein was diluted to 2 μg / mL, 1 μg / mL and 0.5 μg / mL respectively with carbonate buffer, and the enzyme-labeled plates were coated respectively; the nasopharyngeal sample was diluted by sample diluent and then added for ELISA reaction to screen the protein coating concentration of the enzyme-labeled plate, and the screening standard was that the coating concentration with low background and relatively high P / N value in the ELISA detection result was relatively optimal, and the detection result is shown in Table 2.

[0065] Table 2 Optimization results of coating concentration

[0066] ;

[0067] From Table 2, it can be seen that 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.

[0068] Enzyme-labeled antibody optimization: the first step is to screen the enzyme-labeled antibody, and the second step is to label and verify the performance of the enzyme-labeled antibody. The key to screening the enzyme-labeled antibody lies in specificity. The first screening method is to detect the reaction specificity of the enzyme-labeled antibody to different types of human immunoglobulin by Dot-blot method, and the third party control selects goat anti-human IgA enzyme-labeled antibody. The specific process includes: the initial concentration of purified human IgA, human IgM and human IgG is diluted to 50 μg / mL, 4 times of dilution with 5 gradients, 2 μL is taken and loaded on the PVDF membrane, and 5% skim milk is used for blocking; the enzyme-labeled antibody is diluted with 5% skim milk at a ratio of 1:1000, and then incubated with the spotted PVDF membrane. The Dot-blot detection result is as follows: Figure 4As shown: from the dose comparison, the minimum dose of Ab2 and the third party control reacting with human IgA is 12.5 μg / mL, and there is no significant difference; but from the specificity, only Ab2 has a very strong specific binding reaction with human IgA protein, which is consistent with the identification result above, and the third party control also has a non-specific reaction with human IgG, which 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 screened mouse anti-human immunoglobulin A monoclonal antibodies Ab2 and Ab5 are labeled with peroxidase dismutase (HRP) to prepare enzyme-labeled antibodies (two enzyme-labeled antibodies are referred to as HRP-Ab2 and HRP-Ab5, respectively), and the enzyme-labeled antibodies are compared with the third party control reagent of the goat anti-human IgA enzyme-labeled antibody; preparation of the test sample: the nasopharyngeal sample (concentration: 1000 U / mL) and the serum sample (concentration: 1000 U / mL) are diluted with the sample diluent, and then 2-fold gradient dilution is performed to obtain the test sample; ELISA reaction is performed, and the detection differences of the three enzyme-labeled antibodies (HRP-Ab2, HRP-Ab5 and the third party control) for the serum sample and the nasopharyngeal sample are compared, and the enzyme-labeled antibody with high detection result sensitivity and good dilution linearity is preferred, and the detection results are shown in Table 3. The detection sensitivity results of the three enzyme-labeled antibodies for the nasopharyngeal sample and the serum sample are shown in Table 4.

[0069] Table 3 Enzyme-labeled antibody optimization detection results

[0070]

[0071] Table 4 Sensitivity detection results of three enzyme-labeled antibodies

[0072] ;

[0073] From the results of Tables 3-4, it can be seen that when the enzyme-labeled antibody is HRP-Ab2 and the third party control, the detection sensitivity of RBD-IgA in the nasopharyngeal sample and the serum sample is higher than that of HRP-Ab5. Although the detection sensitivity (0.08) of the enzyme-labeled antibody as the third party control is slightly higher than that (0.16) of HRP-Ab2 in the detection results of the serum sample, the difference is not obvious. It is speculated that when the enzyme-labeled antibody is HRP-Ab2 and the third party control, SIgA in the form of a dimer in the nasal mucosa can be sensitively detected, and IgA mainly in the form of a monomer in the serum can also be sensitively detected, and the detection sensitivity of the two forms of IgA is relatively high, so Ab2 can be used as a preferred enzyme-labeled antibody, and further optimization of the use concentration can be continued.

[0074] Optimization of the use concentration of enzyme-labeled antibody: Taking HRP-labeled Ab2 (HRP-Ab2) as the preferred, the use concentration was optimized and screened, and the enzyme-labeled antibody was diluted by 1:500, 1:1000 and 1:2000. The nasopharyngeal sample (initial concentration: 1000 U / mL) was diluted by 2 times to prepare the test sample, and the ELISA reaction was carried out. The detection results of the enzyme-labeled antibody diluted by 3 times were compared, and the screening standard was that the use concentration of the enzyme-labeled antibody with low background and relatively high P / N value in the ELISA detection result was the relatively optimal concentration. The detection results are shown in Table 5.

[0075] Table 5 Optimization of the use concentration of enzyme-labeled antibody

[0076] ;

[0077] From Table 5, it can be seen that when the use concentration of the enzyme-labeled antibody is diluted by 1:1000, the P / N value is the highest (P / N=45.5), so the preferred use concentration of the enzyme-labeled reagent is 1:1000. Although the P / N value corresponding to the use concentration of 1:1000 is the highest, but from the specific data, the P / N value corresponding to the use concentration of 1:1000 and 1:2000 is not much different. From the perspective of detection cost, 1:1000-1:2000 is relatively optimal, and the detection cost is relatively lower when the use concentration is 1:2000.

[0078] Optimization of the loading ratio of the nasal swab sample: 15 negative and positive nasal swab samples were taken, and each sample was diluted by 1:2, 1:4 and 1:8 to prepare the initial detection concentration of the nasal swab test sample. The novel coronavirus RBD-IgA ELISA detection method established by the above-mentioned condition optimization was detected, and the initial detection concentration of the nasal swab sample with relatively optimal specificity and sensitivity was screened. The detection results are shown in Table 6.

[0079] Table 6 Optimization of the initial detection concentration of the nasal swab sample

[0080] ;

[0081] From table 6, it can be seen that: if the initial detection concentration is 1:2, the specific detection result is relatively low, that is, false positive results caused by non-specific reactions occur, and it is speculated that different sampling matrices at 1:2 may interfere with the detection results; if the initial detection concentration is 1:8, the sensitivity detection result is relatively low, that is, false negative results occur for samples with low IgA concentration, and it is speculated that the high dilution ratio of the sample at 1:8 leads to low detection sensitivity; therefore, relatively speaking, when the initial detection concentration is 1:4, the sensitivity and specificity of the detection result are relatively high, and the coincidence degree is the best, which can be used as the preferred initial sample detection concentration.

[0082] Negative sample cutoff value: the new coronavirus RBD-IgA ELISA detection method established after the above condition optimization, and the above optimized initial detection concentration (1:4) of the nasal swab sample are used as the preferred new coronavirus RBD-IgA ELISA detection method of the application, 50 nasal swab samples collected from people without a history of new coronavirus infection are selected as negative samples to be detected, the 50 negative samples are detected to verify the cutoff value of the detection method, the detection result is calculated according to the formula (cutoff=2.1x mean of negative sample detection A value), if the mean of negative sample detection A value is less than 0.05, 0.05 is calculated, and the final detection result is shown in table 7.

[0083] Table 7 cutoff value

[0084] ;

[0085] From the detection results shown in table 7, it can be seen that the mean of negative sample detection A value is 0.026, which is less than 0.05, and 0.05 is calculated, Cutoff=2.1x0.05=0.105, so the cutoff value is 0.105.

[0086] The reaction principle of the new coronavirus RBD-IgA ELISA detection method of the application is a double antibody sandwich ELISA method, the optimized reaction conditions in the detection method include using 2 μg / mL RBD recombinant protein as the preferred coating concentration, preparing enzyme-labeled Ab2 (HRP-Ab2) using HRP-labeled Ab2, using a concentration diluted according to 1:1000, the cutoff value of the ELISA detection method is 0.105, the initial detection concentration of the sample is preferably diluted by 1:4, the new coronavirus RBD-IgA ELISA detection kit is prepared according to the above reaction conditions, the use mode of the kit is improved according to the preferred ELISA reaction conditions, and thus a new coronavirus RBD-IgA ELISA detection platform is established. The specific detection steps of the new coronavirus RBD-IgA ELISA detection include:

[0087] 1) The sample to be tested is diluted with sample diluent according to the ratio of sample to be tested: sample diluent = 1:4, then added to the corresponding wells of the enzyme-labeled plate, and the RBD recombinant protein coated on the enzyme-labeled plate specifically recognizes and binds to the RBD-IgA in the sample to form an "RBD recombinant protein-RBD-IgA" complex;

[0088] 2) Wash with washing solution to wash away the substances on the enzyme-labeled plate that are not specifically combined with the RBD recombinant protein;

[0089] 3) Add enzyme-labeled antibody and incubate at 37°C for 60 minutes to form an "RBD recombinant protein-RBD-IgA-HRP-Ab2" complex after specific recognition and combination;

[0090] 4) Wash with washing solution to wash away the enzyme-labeled antibody McAb-HRP that has not been specifically combined on the enzyme-labeled plate;

[0091] 5) Add color developing agent and incubate in a 37°C constant temperature incubator for 15 minutes in the dark, and the enzyme-labeled wells on the enzyme-labeled plate that form a complex react with the color developing agent to first turn blue, then turn yellow after adding the stop solution;

[0092] 6) The enzyme-labeled instrument outputs the detection results: first, if the detection value is higher than 0.105, it is determined as a positive result, indicating that RBD-IgA exists in the sample to be tested; if the detection value is lower than 0.105, it is determined as a negative result, indicating that RBD-IgA does not exist or exists in extremely low amount in the sample to be tested.

[0093] Further, by measuring the absorbance values of different concentrations of standard samples, a standard curve is drawn to establish the quantitative relationship between RBD-IgA concentration and absorbance value; according to the absorbance value of the sample to be tested and the Cutoff value, 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 during the detection process that the setting of the Cutoff value has a high false positive rate or false negative rate, the Cutoff value needs to be re-determined.

[0094] The components of the novel coronavirus RBD-IgA ELISA detection kit include: RBD recombinant protein coated enzyme-labeled plate, enzyme-labeled antibody, negative control, positive control, sample diluent, washing solution, color developing solution, stop solution, inflatable sponge swab for nasopharyngeal swab sample collection, and instruction manual; it can also include novel coronavirus XBB strain IgA standard, with a concentration in the range of 0.31-5 U / mL. The novel coronavirus RBD-IgA ELISA detection kit prepared according to the above method is subjected to performance evaluation, and the content of performance evaluation includes linear range, specificity, specificity, repeatability, precision, accuracy and stability, etc. The verification results of performance evaluation are as follows.

[0095] Linear range: Take nasopharyngeal sample (1000 U / mL), dilute it to 10 U / mL as the initial detection concentration of the sample to be tested, dilute it by 2 times, a total of 7 concentration gradients, detect respectively, repeat 3 times for each concentration, draw the standard curve according to the detection result data, get the linear regression equation and the regression coefficient (R 2 ), determine the linear range of quantitative detection. The data of the above detection results are shown in Table 8, and the linear regression equation of the standard curve is shown in Table 9.

[0096] Table 8 Linear range detection data

[0097]

[0098] Table 9 Linear regression equation

[0099] ;

[0100] From the detection results shown in Tables 8-9, it can be seen that when the concentration range of the sample to be tested is between 0.31-5 U / mL, the linear correlation of the detection method is relatively optimal, R 2 =0.9951. If the concentration of the novel coronavirus RBD-IgA is higher than 5 U / mL, in order to improve the detection accuracy, the sample to be tested can be diluted and rechecked.

[0101] Specificity: Take positive nasopharyngeal sample as the main sample, dilute the positive nasopharyngeal sample with 3 volumes of negative nasopharyngeal sample and sample diluent respectively, dilute the positive nasopharyngeal sample according to the ratio of 3:1, and take the diluted mixture as the sample to be tested. The mixture sample with a ratio of negative nasopharyngeal sample: positive nasopharyngeal sample = 3:1 is taken as the experimental group, and the mixture sample with a ratio of sample diluent: positive nasopharyngeal sample = 3:1 is taken as the control group. After detection, draw the standard curve, calculate the recovery rate, and the recovery rate = experimental group / control group x 100%, use the recovery rate to evaluate the specificity of the method, and the detection results of the recovery rate are shown in Table 10.

[0102] Table 10 Recovery rate

[0103] ;

[0104] From Table 10, it can be seen that the detection results of the positive nasopharyngeal sample diluted with negative nasopharyngeal sample and sample diluent are very similar, and the recovery rate is 100%, so the interference of the sample matrix liquid to the detection system is relatively small, and the specificity is good.

[0105] Specificity: The nasopharyngeal samples or oropharyngeal samples collected from the positive confirmed patients infected with 4 respiratory viruses (rhinovirus, influenza A, influenza B and metapneumovirus) similar to the symptoms of the new coronavirus were used as the test samples, and the new coronavirus RBD-IgA ELISA detection was performed to confirm the detection specificity of the method, and the detection results are shown in Table 11.

[0106] Table 11 Specificity

[0107] ;

[0108] From Table 11, it can be seen that the results of the new coronavirus RBD-IgA ELISA detection of 17 test samples from 4 respiratory virus infections were all negative, and the new coronavirus RBD-IgA ELISA detection method of the present application does not have cross-reaction with other 4 respiratory viruses, and the detection specificity meets the requirements.

[0109] Repeatability, accuracy and precision: The new coronavirus XBB strain IgA standard was diluted with the kit sample diluent to 3 concentrations: high value (3 U / mL), medium value (1.5 U / mL) and low value (0.75 U / mL). Each concentration of the test sample was determined by repeating 8 times in the same experiment to evaluate repeatability and accuracy. The samples of different concentrations were repeatedly loaded 8 times by three different experimenters at different times to determine the precision of the system. The performance evaluation of the reagent requires that the CV is less than 15% and the accuracy is greater than 85%. The performance evaluation results of the new coronavirus RBD-IgA ELISA detection kit established by the present application are shown in Table 12. From Table 12, it can be seen that the repeatability and precision CV values of the system are all less than 15%, the accuracy recovery rate is between 85%-115%, and the requirements are met.

[0110] Table 12 Repeatability, precision and accuracy test results

[0111] ;

[0112] Stability: Comparative heat accelerated stability and low temperature stability, the new coronavirus RBD-IgA ELISA detection kit prepared by the application was placed at 37℃ constant temperature condition for 6 days, and the new coronavirus RBD-IgA ELISA detection kit placed in 2-8℃ low temperature storage environment was used as control. Preparation of test sample: take nasopharyngeal sample (1000U / mL), dilute to 10U / mL as the starting detection concentration of test sample, dilute by 2 times dilution ratio, a total of 6 concentration gradients. Comparative analysis of detection results before and after 37℃ heat acceleration of the kit, focusing on the change of detection OD value and recovery rate. The stability test results are shown in Table 13. From Table 13, it can be seen that after the kit is placed at 37℃ for 6 days, compared with the 2-8℃ control group, the OD value recovery rate is between 80%-120%, the background value (BLK) is less than 0.05, and the heat accelerated stability meets the requirements.

[0113] Table 13 Stability test results

[0114] ;

[0115] In summary: In this study, a new coronavirus RBD-IgA ELISA detection platform for XBB strain of new coronavirus was established, and a new coronavirus RBD-IgA ELISA detection kit was prepared therefrom. Through the performance evaluation of the kit, it can be seen that the kit can specifically and quantitatively detect the content of XBB strain of new coronavirus RBD-IgA in mucosal samples, and the performance of the linear range, specificity, specificity and stability of the kit meets the needs.

[0116] Example 4, application 2: establishment of immunoglobulin A universal ELISA detection method

[0117] Based on the 2 strains of mouse anti-human immunoglobulin A monoclonal antibodies Ab2 and Ab5 obtained by the application, a universal ELISA detection platform for immunoglobulin A was constructed, and the matching screening of coating antibody / enzyme-labeled antibody and the coating concentration in the detection system were optimized, and finally a double antibody sandwich IgA universal ELISA detection system was established. The specific content is as follows.

[0118] Two goat anti-IgA polyclonal antibodies (goat polyclonal antibody 1 and goat polyclonal antibody 2) were used as coating antibodies, and HRP-labeled Ab2 (HRP-Ab2) and a commercially available enzyme-labeled antibody were used as enzyme-labeled antibodies for paired experiments for screening and optimizing the coating antibodies. The screening criteria were goat polyclonal antibodies with good specificity and appropriate sensitivity. The coating antibodies were diluted to 2 μg / mL with a phosphate buffer, and the enzyme-labeled antibodies were diluted 1000 times with an enzyme-labeled diluent. The samples to be tested were IgA, which was diluted to six concentration gradients (10000 ng / mL, 2500 ng / mL, 625 ng / mL, 156 ng / mL, 39 ng / mL and 10 ng / mL) with a sample diluent. The swab preservation solution and the sample diluent were used as negative controls to verify the anti-interference of the sample matrix. The specificity of the detection was verified by using 10 μg / mL human IgM antibody as a sample to be tested. The detection results of the ELISA detection were as follows:

[0119] Table 14. Pairing screening results

[0120] ;

[0121] As can be seen from Table 14, among the two goat polyclonal antibodies used as coating antibodies, goat polyclonal antibody 1 had relatively strong non-specific reactions with the swab preservation solution, the sample diluent and human IgM. Although goat polyclonal antibody 2 also had some non-specific reactions, the sensitivity of goat polyclonal antibody 2 for detecting IgA (10 ng / mL) was 1000 times higher than that for detecting IgM (10 μg / mL). The comparison of the enzyme-labeled antibodies showed that the detection OD value of the commercially available enzyme-labeled antibody was about 2 times higher than that of HRP-Ab2. However, the detection OD values of the commercially available enzyme-labeled antibody with the swab preservation solution, the sample diluent and the human IgM sample were significantly higher than those of HRP-Ab2 as the enzyme-labeled antibody. Therefore, after comprehensive evaluation, goat anti-IgA polyclonal antibody 2 (goat polyclonal antibody 2) was used as the coating antibody, and HRP-Ab2 prepared from the preferred monoclonal antibody of the application was used as the enzyme-labeled antibody for pairing, which was used for universal ELISA detection of immunoglobulin A (IgA), which can also be referred to as ELISA detection of total IgA.

[0122] Coating concentration optimization: The screened sheep polyclonal antibody 2 was used as the coating antibody, and was diluted to 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 in phosphate buffer solution, respectively, to obtain a total of 6 concentration gradients. IgA was diluted with the sample diluent to obtain the sample to be detected, and was diluted by 6 concentration gradients at a ratio of 1:2, i.e. 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 ELISA detection result data bias and linearity were comprehensively analyzed to determine the optimal coating concentration. The detection results are shown in Table 15.

[0123] Table 15 Coating concentration

[0124] ;

[0125] As shown in Table 15, the OD values of the negative control were all less than 0.05 at the 6 coating concentrations (1.78-57 μg / mL) of the coating antibody sheep polyclonal antibody 2. The standard curve drawn therefrom had an R 2 > 0.98, but when the coating concentration was greater than 28.5 μg / mL, the OD values of the positive sample rapidly entered the plateau period. Therefore, the coating concentration of sheep polyclonal antibody 2 was preferably 28.5 μg / mL.

[0126] According to the preferred sheep polyclonal antibody 2 as the coating antibody and the preferred coating concentration of 28.5 μg / mL, an immunoglobulin A universal ELISA plate was prepared, and was used together with HRP-Ab2 as the preferred enzyme-labeled antibody, together with washing solution, color developing solution, termination solution and the like 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 the linear range and the specificity. The performance evaluation results are as follows.

[0127] Linear range: A nasopharyngeal sample (1000 U / mL) was diluted to 24 ng / mL with the sample diluent to obtain a standard for the immunoglobulin A universal ELISA detection kit. The sample to be detected was diluted by 5 concentration gradients at a ratio of 1:2 based on the standard, and was subjected to immunoglobulin A universal ELISA detection. Each concentration was detected twice. According to the detection results, a standard curve was drawn with the concentration as the abscissa and the average OD value as the ordinate. The regression equation and the regression coefficient (R 2), for investigating the linear range of the immunoglobulin A universal ELISA detection method. The results are shown in Table 16, and it can be seen that when the concentration of the sample to be detected is in the range of 0.75-12 ng / mL, the linearity of the detection OD value is relatively optimal, R2 is 0.9991, and the standard curve of the immunoglobulin A universal ELISA detection kit is shown in Figure 5

[0128] Table 16 Linear range of immunoglobulin A universal ELISA detection method ;

[0129] Specificity: The samples to be detected were human IgA (25 ng / mL), human IgG (100 ng / mL), human IgM (100 ng / mL), and swab preservation solution (diluted according to the ratio of swab preservation solution: sample diluent = 1:4), and the immunoglobulin A universal ELISA detection was performed on the four samples to be detected to investigate the specificity of the detection method, and the detection results are shown in Table 17. It can be seen that the detection result of human IgA is positive, and the detection results of human IgG, human IgM and swab preservation solution are all negative, that is, no specific immunoglobulin A (IgA) is detected, that is, the immunoglobulin A universal ELISA detection method constructed by the present application has no cross reaction with human IgG, human IgM, etc., and can specifically detect and recognize immunoglobulin A (IgA), and the detection specificity is good.

[0130] Table 17 Specificity of immunoglobulin A universal ELISA detection method

[0131] ​Detection of clinical samples: 30 clinical samples with a history of nasal spray new coronavirus vaccination were taken. First, the new coronavirus RBD-IgA ELISA detection method constructed by the application was used for detection, and 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, each group having 10 samples. Then, the immunoglobulin A universal ELISA detection kit constructed by the application was used to detect the 30 samples in the three groups, and the quantitative detection results of total immunoglobulin A were obtained. The detection results are shown in Table 18. The quantitative concentration results of total immunoglobulin A of individual samples are inconsistent with the three concentration gradients divided based on RBD-IgA concentration. The quantitative detection results of total immunoglobulin A have large differences, the highest value is 213.07 μg / mL, and the lowest value is 1.08 μg / mL, and the difference between the two extreme concentrations is nearly 200 times. The large difference may be because the mucosal sampling is less stable, and there is a large difference between different people or different sampling times. Therefore, by calculating the ratio of specific IgA to total IgA, the relative content of immunoglobulin A can be obtained, which can effectively avoid the influence of sampling factors and more objectively evaluate the immune effect of the vaccine.

[0132] Table 18 Detection results of clinical samples

[0133]

[0134] Abnormal value analysis of total immunoglobulin A quantitative detection results, after excluding abnormal values, further comparative analysis of the ratio of the quantitative detection results of 29 clinical samples obtained by two detection methods, that is, the ratio of the detection values of new coronavirus RBD-IgA and total IgA, is shown in Table 19.

[0135] Table 19 Analysis of RBD-IgA and total IgA quantitative results

[0136] ;

[0137] In Table 19, the ratio = RBD-IgA (U / mL) / total IgA (μg / mL) x 100, when the detection concentration of RBD-IgA is less than 4 U / mL, it is calculated according to 1 U / mL. The detection method of relative content of immunoglobulin A established by the monoclonal antibody screened by the application can effectively avoid the difference of sampling methods.

[0138] Table 20 Correlation analysis of two detection methods

[0139]

[0140] The present application further analyzes 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), and the correlation coefficient values of the quantitative concentration of RBD-IgA and the quantitative concentration of total IgA are both 1, which can indicate that the results of the novel coronavirus RBD-IgA detection or the general ELISA detection of immunoglobulin A constructed by the present application are linearly positively correlated under the condition that the sampling method is stable. Moreover, the correlation value between the RBD-IgA detection result and the total IgA detection result is <0.001 (attributed to 0.01 two-tailed level) through Pearson correlation analysis, that is, the correlation between the two is significant. Although a small amount of samples in the 30 clinical samples exist inhibitor interference phenomenon, the correlation between the two IgA detection results is poor, but the Pearson correlation analysis shows that the relative content of specific immunoglobulin A (such as RBD-IgA concentration) in the total IgA concentration in the immune population after vaccination generally has certain regularity, and the correlation is significant, so the specific IgA proportion method can be used to analyze the immune effect, so as to further avoid the error caused by sampling, and be used to more objectively evaluate the vaccine immune effect. At the same time, if combined with other detection data, it is more conducive to comprehensively evaluate the immune protection effect.

[0141] The specific embodiments of the present application have been described in detail, so that those skilled in the art will easily understand. However, all the descriptions have been disclosed, and different modifications or substitutions can be made to those details, and these changes are all within the protection scope of the present application. The whole scope of the present application is given by the appended claims and any equivalents thereof.

Claims

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

2. A hybridoma cell line, characterized in that, The hybridoma cell line for secreting the immunoglobulin A monoclonal antibody in claim 1, the preservation number of the hybridoma cell line is CGMCC No. 46012, the preservation address is China General Microbiological Culture Collection Center, and the preservation date is July 2, 2024.

3. A novel coronavirus RBD-IgA antibody ELISA detection method, characterized in that, The bis-antibody sandwich ELISA detection method using the immunoglobulin A monoclonal antibody in claim 1 is used for detecting novel coronavirus RBD-IgA to obtain a quantitative result of the novel coronavirus RBD-IgA; The bis-antibody sandwich ELISA detection method uses the RBD recombinant protein of the novel coronavirus XBB strain as the coating protein of the enzyme-labeled plate, uses the immunoglobulin A in claim 1 as the enzyme-labeled antibody, and uses the novel coronavirus RBD-IgA in the sample as the capture antibody; the sample is a nasopharyngeal sample or a serum sample; The detection method does not include a disease diagnosis and treatment method.

4. An immunoglobulin A universal ELISA detection method, characterized in that, The bis-antibody sandwich ELISA detection method using the immunoglobulin A monoclonal antibody in claim 1 is used for detecting all immunoglobulin A produced by mucosal immunity to obtain a quantitative result of total immunoglobulin A; The bis-antibody sandwich ELISA detection method uses a goat anti-IgA polyclonal antibody as the coating antibody and uses the immunoglobulin A in claim 1 as the enzyme-labeled antibody; the sample is a nasopharyngeal sample; The detection method does not include a disease diagnosis and treatment method.

5. A novel coronavirus RBD-IgA antibody ELISA detection kit, characterized in that, The bis-antibody sandwich ELISA detection method using the immunoglobulin A monoclonal antibody in claim 1 is used for detecting all immunoglobulin A produced by mucosal immunity to obtain a quantitative result of total immunoglobulin A; 6. An immunoglobulin A universal ELISA test kit, characterized in that, The bis-antibody sandwich ELISA detection method uses a goat anti-IgA polyclonal antibody as the coating antibody and uses the immunoglobulin A in claim 1 as the enzyme-labeled antibody; the sample is a nasopharyngeal sample; 7. A method for detecting the relative amount of immunoglobulin A, characterized by, The bis-antibody sandwich ELISA detection method using the immunoglobulin A monoclonal antibody in claim 1 is used for detecting all immunoglobulin A produced by mucosal immunity to obtain a quantitative result of total immunoglobulin A; The bis-antibody sandwich ELISA detection method uses a goat anti-IgA polyclonal antibody as the coating antibody and uses the immunoglobulin A in claim 1 as the enzyme-labeled antibody; the sample is a nasopharyngeal sample; The detection method does not include disease diagnosis and treatment methods.

8. The application of the monoclonal antibody of immunoglobulin A in claim 1, the hybridoma cell line in claim 2, the universal ELISA detection method of immunoglobulin A in claim 4, or the universal ELISA detection kit of immunoglobulin A in claim 6 in the detection of total content of immunoglobulin A. The application does not include disease diagnosis and treatment methods.

9. The application of the monoclonal antibody of immunoglobulin A in claim 1, the hybridoma cell line in claim 2, the novel coronavirus RBD-IgA antibody ELISA detection method in claim 3, the universal ELISA detection method of immunoglobulin A in claim 4, the ELISA detection kit in any one of claims 5-6, or the detection method of relative content of immunoglobulin A in claim 7 in the evaluation of immune effect of vaccine; the vaccine is a novel coronavirus vaccine. The application does not include disease diagnosis and treatment methods.

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