Blocking agent for cell immunofluorescence detection by slide method and use method of blocking agent

Abstract

The invention discloses a blocking agent for cell immunofluorescence detection by a slide method and a use method of the blocking agent. The blocking agent is a homologous no-load cell lysis solution without transfection plasmids, and the protein concentration is greater than or equal to 30mg / mL. The blocking agent is completely the same as cell cytoplasm and cell membrane components of cells to be detected except for plasmids-expressed antigens, when in use, the blocking agent is pre-mixed with a blood cleaning sample in advance, interfering antibodies except target antibodies in serum can be purified, and meanwhile, due to the fact that no antigen protein expressed after plasmid transfection exists, the blocking agent can be used for detecting the cells to be detected. And the protein concentration is equivalent to the globulin concentration in serum, so that the target antibody cannot be lost in the serum, and the slide method CBA detection sensitivity can be greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, in particular to a blocking agent for slide method cell immunofluorescence detection and a use method thereof. BACKGROUND

[0002] Cellular indirect immunofluorescence method (CBA) is the gold standard for detection of neural immune system autoantibodies. The recombinant protein overexpressed by plasmid transfection 293T cells or Hela cells is the full-length conformational antigen, which is superior to ELISA and immunoblotting methods. However, in the actual detection process, there are unavoidable experimental defects affecting the accuracy of the results, including interference from complex and highly concentrated globulin in serum, which results in high specificity and limited sensitivity of the method, only about 75%. In order to improve the sensitivity of the detection method, some laboratories use well plates, and some use slides. It is difficult to establish a unified detection standard. The same patient sample may provide completely different test results in different laboratories, which interferes with the clinician's correct diagnosis of the disease when the gold standard method is applied in the clinic, and even causes the patient to lose the opportunity for early diagnosis and treatment, affects the prognosis of the disease, and greatly reduces the quality of life of the patient.

[0003] Among them, compared with L-CBA and well plate method, the slide method has the advantages of saving samples, high throughput, and can be made into commercial kits; it also has unavoidable disadvantages. The detection sensitivity is lower than that of L-CBA method due to the fixing, drying or freeze-drying operation in the preparation of the slide process, resulting in frequent missed detection of some items such as low titer AQP4 and MOG. How to improve the sensitivity of the slide method has become the core technology that the industry urgently needs to solve.

[0004] Any link in the slide production process has been standardized by the process and cannot be changed. Under this premise, how to remove the high concentration of interfering substances in the serum sample has become the only breakthrough to solve the strong fluorescence background, improve the specific binding of the overexpressed antigen and the sample primary antibody, and the primary antibody and the fluorescent secondary antibody, and thus improve the detection sensitivity of the slide method. In the entire immune reaction system, the commonly used method in the industry to remove interfering antibodies in serum is to use commercial blocking agents. Due to the different compositions of blocking agents from different manufacturers, there are two drawbacks in the use process. First, the addition of unknown substances to the reaction system may affect the antigen-antibody reaction in the system, which is an uncertain factor. Second, the blocking agent has batch differences, which makes it difficult to accurately explore the standardized use conditions, and may lead to false negative or false positive results. Moreover, the existing blocking agent is relatively expensive. Therefore, it is urgent to improve the existing blocking agent. SUMMARY

[0005] In order to solve the problems existing in the prior art, the present application uses the homologous empty cell lysate corresponding to the cells on the slide, which is not contained in the transfection plasmid, and is treated by the commercial cell lysate to obtain the empty cell lysate which is completely the same as the cell cytoplasm and cell membrane on the slide except the plasmid expressed antigen, and the empty cell lysate is used as a blocking agent for the slide method CBA detection to improve the sensitivity and greatly reduce the cost.

[0006] The present application is implemented by the following technical solutions: A blocking agent for slide method cell immunofluorescence detection, the blocking agent is a homologous empty cell lysate not containing a transfection plasmid, and the protein concentration of the blocking agent is greater than or equal to 30 mg / mL. The blocking agent is completely the same as the cell cytoplasm and cell membrane of the cell to be detected except the plasmid expressed antigen, and when used, it is premixed with the serum sample, which can purify the interfering antibodies in the serum except the target antibody, and since there is no antigen protein expressed after plasmid transfection in the blocking agent and the protein concentration is equivalent to the globulin concentration in the serum, the target antibody in the serum will not be lost, so the detection sensitivity can be improved.

[0007] Further, the blocking agent is prepared by the following method: After diluting the cell lysate, the prepared empty cell is added, and the cell is lysed at 4 DEG C or on ice, after the lysis is completed, the supernatant is obtained by centrifugation to obtain the empty cell lysate; Or, the cell lysate is added to the prepared empty cell, and the cell is lysed at 4 DEG C or on ice, and then diluted after the lysis is completed, and the supernatant is obtained by centrifugation to obtain the empty cell lysate.

[0008] That is, the lysate can be diluted first and then lysed, or lysed and then diluted, and the dilution solution is usually PBS solution, etc., so that the protein concentration of the obtained lysate is more appropriate.

[0009] Further, the prepared empty cell is obtained by centrifuging the cultured empty cell to remove the cell culture solution, then adding PBS and mixing, and then centrifuging again, and discarding the supernatant; wherein the centrifugal rate is 2000-5000 rpm, and the time is 20-30 min.

[0010] Further, the number of the empty cell is 2*10 5 ~5*10 6 per 20 μL of the cell lysate. The cell lysate can be various strength lysates, such as weak strength lysate, medium strength lysate, and strong lysate, which are selected according to the thickness of the cell membrane, the number, etc. For example, in some specific embodiments, for HEK 293T empty cells, 1*10 6Preferably, the lysis solution is of medium or high strength, and 20 μL of the lysis solution is used for 2 x 10 5 Preferably, the lysis solution is of medium or high strength, and 20 μL of the lysis solution is used for 2 x 10

[0011] Further, the dilution ratio is 2-10, preferably 3-8, and more preferably 5. The dilution ratio can be the dilution ratio of the lysis solution before lysis or the dilution ratio after lysis. PBS buffer is usually used for dilution.

[0012] Further, the lysis time is 10-15 min at 4°C or on ice, the centrifugation rate is 20,000-25,000 rpm, and the centrifugation time is 10-20 min.

[0013] Further, the homologous empty cells are any of HEK 293T empty cells, Hela empty cells, Hep-2 empty cells, CHO empty cells, COS empty cells, HepG2 empty cells, MCF-7 empty cells, A549 empty cells, and Vero empty cells.

[0014] Another aspect of the present application provides the use of the blocking agent in a slide method cell immunofluorescence detection. The use of the blocking agent in the slide method cell immunofluorescence detection includes the following steps: Preparation of a monoclonal antibody serum, mixing the blocking agent, incubation at 37°C, dilution with a diluent, centrifugation, and collection of the supernatant. Alternatively, preparation of a monoclonal antibody serum, mixing the blocking agent, dilution with a diluent, incubation at 37°C, and centrifugation to collect the supernatant.

[0015] Further, the monoclonal antibody serum: blocking agent: diluent = 1:0.5-1:8-10 by volume. In a preferred embodiment, the monoclonal antibody serum: blocking agent: diluent = 1:1:8 by volume. The diluent is usually PBS buffer or the like.

[0016] Further, the incubation time is 15-3 min at 37°C, the centrifugation rate is 20,000-25,000 rpm, and the centrifugation time is 10-15 min.

[0017] Further, the monoclonal antibody serum can be an AQP4 monoclonal antibody serum, a MOG monoclonal antibody serum, an LGI 1 monoclonal antibody serum, or an antinuclear antibody serum.

[0018] Compared with the prior art, the present application has the following advantages: 1) The blocking agent of the present application is a homologous empty cell lysate without transfection plasmid, wherein, in addition to the antigen expressed by the plasmid, the cytoplasmic and membrane components of the cells to be detected are completely the same, and the protein concentration is greater than or equal to 30 mg / mL, and the globulin concentration in the serum is consistent. When used, the serum sample is pre-mixed with the blocking agent, the interfering antibodies in the serum are adsorbed and removed, and only the target antibodies to be detected are left. Since there is no antigen protein expressed after plasmid transfection in the blocking agent of the present application, the target antibodies in the serum will not be lost, which can purify the serum, reduce the steric hindrance affecting the specific binding of the Fc segment of the primary antibody and the secondary antibody, and greatly improve the sensitivity of the slide method reagent detection.

[0019] 2) The use cost of the blocking agent of the present application is less than one percent of that of the commercial blocking agent, which can greatly reduce the detection cost under the premise of improving the result accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The detection results of the Hela cell blocking agent; Figure 2 The detection results of the HEK 293T cell blocking agent; Figure 3 The detection results of the blocking agent of the present application and the serum with different pre-mixing proportions are compared. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and examples.

[0022] In the present application, the homologous empty cell (without transfection plasmid) corresponding to the cell on the slide is treated by the commercial cell lysate to obtain the empty cell lysate which is completely the same as the cytoplasmic and membrane components of the cell on the slide except for the antigen expressed by the plasmid, and by optimizing the experimental conditions, the protein concentration of ≥30 g / L can be obtained at one time, which is equivalent to the globulin concentration in the serum. The empty cell lysate is used as a blocking agent, and before detection, the patient's serum sample is pre-mixed with the blocking agent and incubated at 37°C, which can adsorb and remove the interfering antibodies in the serum, leaving only the target antibodies to be detected. Since there is no antigen protein expressed after plasmid transfection in the blocking agent of the present application, the target antibodies in the serum will not be lost, which can purify the serum, remove the interfering antibodies, and reduce the steric hindrance affecting the specific binding of the Fc segment of the primary antibody and the secondary antibody, greatly improving the sensitivity of the slide method reagent detection. Moreover, the use cost of the blocking agent of the present application is less than one percent of that of the commercial blocking agent, which can greatly reduce the detection cost under the premise of improving the result accuracy.

[0023] The principle of the blocking agent of this invention is to premix serum samples with empty vector cell lysates expressing antigens after plasmid transfection, thus purifying the serum of interfering antibodies other than the target antibody. Therefore, for different detection items, corresponding climbing cells must be selected to obtain the corresponding empty vector cell lysates as blocking agents. For example, for detection items using 293T cells, empty vector 293T cells must be used to prepare the blocking agent; for detection items using HeLa cells, empty vector HeLa cells must be used to prepare the blocking agent, and so on. In addition to 293T cells and HeLa cells, it can also be used with Hep-2 cells, CHO cells, COS cells, HepG2 cells, MCF-7 cells, A549 cells, Vero cells, etc.

[0024] The blocking agent of the present invention is applicable to the detection of a variety of antibodies, including but not limited to AQP4, MOG, LGI 1 monoclonal antibody or antinuclear antibody.

[0025] The preparation and application of the inhibitor of the present invention are illustrated below using 293T cells and HeLa cells as examples. The weak, medium, and strong lysis buffers used in the following examples were all from Shanghai Beyotime Biotechnology Co., Ltd.

[0026] Example 1: Exploration of the preparation method of the blocking agent

[0027] To obtain the optimal concentration of lysed protein, three methods were designed: repeated freeze-thaw cycles, pre-dilution of the lysis buffer, and post-dilution of the lysis buffer. For different cell types, a weak, medium, and strong lysis buffer were used to lyse a fixed number of cells. For HEK 293T empty vector cells, the cell number was 1.0 × 10⁻⁶ cells. 6 Lysis was performed using medium-strength and weak-strength lysis buffers, respectively; for HeLa empty vector cells, the cell fixation quantity was 2.0 × 10⁶ cells / year. 5 They were pyrolyzed using strong pyrolysis buffer and medium-strength pyrolysis buffer, respectively.

[0028] The specific experimental method is as follows:

[0029] (1) Repeated freeze-thaw method A fixed number of empty vector cells were added to 1 ml of PBS and mixed well. The cells were then placed at room temperature and at -80°C for 30 minutes each. After repeated freeze-thaw cycles three times, the supernatant was collected by centrifugation for protein quantification.

[0030] (2) Pre-diluted lysis buffer Transfer a fixed number of cultured empty vector cells to a 2 ml EP tube, centrifuge at 3000 rpm / min for 25 minutes, discard the cell culture medium, add 1 ml PBS, mix by pipetting, centrifuge again at 3000 rpm / min for 25 minutes, discard the supernatant and keep the cell pellet for later use. Add 20 μl of lysis buffer to 80 μl of PBS, then add 100 μl of the diluted lysis buffer to the prepared cell pellet. Mix well and incubate at 4°C for 15 minutes to lyse the cells and obtain empty cell lysate.

[0031] (3) Post-dilution of lysis buffer Transfer a fixed number of cultured empty vector cells to a 2 ml EP tube, centrifuge at 3000 rpm / min for 25 minutes, discard the cell culture medium, add 1 ml PBS, mix by pipetting, centrifuge again at 3000 rpm / min for 25 minutes, discard the supernatant and keep the cell pellet for later use. First, add 20 μl of pure lysis buffer to a fixed amount of cell pellet, mix well, and place in a 4°C refrigerator for 15 minutes. Then, take out the lysed cell solution, add 80 μl of PBS, mix by pipetting, and centrifuge for later use.

[0032] The specific experimental groups are shown in Table 1. Protein detection was performed on the corresponding samples. Each treatment method was tested 3 times, and the average value was taken.

[0033] The protein concentration data of the lysate obtained by different treatment methods are shown in Table 1.

[0034] Table 1. Test results of protein concentration in blocker samples obtained by different treatment methods (unit: mg / ml)

[0035] As shown in Table 1, the number of 293T cells was 1×10⁻⁶. 7 There were 2 × 10⁶ HeLa cells. 6 After treatment with the lysis buffer, the protein concentration required to be greater than 30 mg / ml was achieved; however, the protein concentration obtained by repeated freeze-thaw cycles was significantly lower than that obtained by the lysis buffer method. For 293T cells, the highest protein concentration was obtained by diluting the cells 5-fold with a moderate lysis buffer, and therefore this method was used as the lysis method for 293T cells in subsequent experiments. For HeLa cells, repeated freeze-thaw cycles could not obtain the ideal protein concentration, and the protein concentration obtained by diluting the cells 5-fold with a moderate lysis buffer was second only to that obtained by the strong lysis buffer.

[0036] Example 2: Use of blocking agents

[0037] The lysis buffers obtained in the above experiments were used as blocking agents for CBA detection and compared with positive samples without the addition of blocking agents.

[0038] (1) HeLa cells: The samples from Example 1, which underwent repeated freeze-thaw cycles (1-2-5), 5-fold pre-dilution with lysis buffer (1-2-1), and 5-fold strong pre-dilution with lysis buffer (1-2-3), were premixed 1:10 with NMDAR positive serum. Specifically, 20 μl of positive serum was mixed with 20 μl of the blocking agent obtained from each treatment method. 180 μl of PBS was added to each tube to dilute the positive serum 1:10. After mixing, the tubes were incubated at 37°C for 20 minutes, centrifuged at 22000 rpm / min for 15 minutes, and 90 μl of the supernatant was collected as the detection sample. This sample was added to a pre-prepared NMDAR cell slide and detected according to the CBA method standard operating procedure. The results were compared with positive samples without the blocking agent. The fluorescence images are shown below. Figure 1 As shown.

[0039] (2) 293T cells: The samples from Example 1, which underwent repeated freeze-thaw cycles (1-1-5), were diluted 5-fold in the lysis buffer before dilution (1-1-3), and were diluted 5-fold in the lysis buffer after dilution (1-1-4). These samples were premixed with MOG 1:10 positive serum at a 1:1 ratio (20 μl of positive serum plus 20 μl of the blocking agent obtained from different treatments). 180 μl of PBS was added to each tube to dilute the positive serum 1:10. After mixing, the tubes were incubated at 37°C for 20 minutes, centrifuged at 22000 rpm / min for 15 minutes, and 90 μl of the supernatant was collected as the detection sample. This sample was added to a pre-prepared MOG cell slide and detected according to the CBA method standard operating procedure. The results were compared with positive samples without the blocking agent. The fluorescence images are shown below. Figure 2 As shown.

[0040] from Figure 1 and Figure 2 As can be seen from the fluorescence images, no specific fluorescence signal was detected in any of the positive sera without the blocking agent. For both 293T cells and HeLa cells, when detecting the corresponding items, the lysis buffer with a 5-fold dilution maximizes the amplification of the target antibody's fluorescence signal and minimizes background fluorescence. For HeLa cells, although the protein concentration obtained by treating empty vector cells with a strong lysis buffer is the highest, the background fluorescence is also the strongest under the same detection conditions. Therefore, it can be seen that for cell numbers of 1×10⁻⁶, the lysis buffer is more effective. 7 and 2×10 6 The most effective inhibitor can be prepared from 293T cells and HeLa cells by first diluting the medium lysis buffer 5-fold.

[0041] Example 3: The effect of the blocking agent of the present invention on the detection sensitivity of monoclonal antibodies 3.1 Theoretical Basis for the Application of Blocking Agents in Monoclonal Antibody Plate Detection

[0042] The well plate method is a recognized method with sensitivity that meets clinical testing needs. To date, there are no reports on the extreme sensitivity of the well plate method. In order to prove the existence of interfering antibodies in the serum matrix, this experiment was designed to demonstrate the existence of the serum matrix effect and to obtain data on the extreme sensitivity of the well plate method, thus providing a theoretical basis for the application of the blocking agent of this invention.

[0043] Different concentrations of AQP4 and MOG monoclonal antibody-BSA and monoclonal antibody serum were prepared using 5% BSA and 10% negative serum as diluents, respectively. The concentrations of the two monoclonal antibodies ranged from 0.04 μg / ml to 1 μg / ml. The well plate reagents were prepared in advance and used within 3 days. The results were interpreted using a Nikon inverted microscope according to the detection procedures of well plate method and slide method (without blocking agent). The experimental data are shown in Tables 3 and 4 below.

[0044] Table 3. Extreme sensitivity results for monoclonal antibody detection in 5% BSA matrix.

[0045] Table 4. Extreme sensitivity results of monoclonal antibody detection in 10% negative serum.

[0046] Experimental conclusion: As can be seen from Tables 3 and 4: For monoclonal antibodies in a 5% BSA matrix: using the plate method, the extreme sensitivity for AQP4 and MOG was 0.06 μg / ml; using the slide method, the extreme sensitivity for AQP4 was 0.08 μg / ml, and the extreme sensitivity for MOG was 0.2 μg / ml. For monoclonal antibody-negative serum: the extreme sensitivity of AQP4 and MOG was 0.8 μg / ml using the plate method; neither of the two methods reached the lower limit of sensitivity using the slide method.

[0047] It is evident that, regardless of whether it is the plate method or the slide method, the sensitivity of the monoclonal antibody serum matrix solution simulating real samples decreases significantly, indicating that the matrix effect of serum is the core factor affecting experimental sensitivity.

[0048] Monoclonal antibody-negative serum can simulate real samples and quantitatively detect the extreme sensitivity of the plate method and slide method. The above results confirm the extreme sensitivity of the plate method for detecting AQP4 and MOG, proving the existence of the matrix effect of serum, and providing reliable theoretical data for the application of the blocking agent of the present invention.

[0049] 3.2 Optimization of the usage conditions of the blocking agent of the present invention

[0050] This experiment investigated the effect of different dilution methods on the blocking agent of the present invention—using three different premixing methods, the specific experimental schemes are as follows: 1) Premix serum and blocking agent 1:1, that is, add 20 μl of blocking agent to 20 μl of serum, then add 160 μl of PBS, incubate in 37℃ for 20 minutes, take out, mix well, centrifuge at 22000 rpm / min for 15 minutes, and take the supernatant for detection. 2) Premix serum and blocking agent 1:1, that is, add 20 μl of blocking agent to 20 μl of serum, incubate in 37℃ for 20 minutes, take it out, add 160 μl of PBS, mix well, centrifuge at 22000 rpm / min for 15 minutes, and take the supernatant for detection; 3) Premix serum and blocking agent at a ratio of 1:0.5, i.e., add 10 μl of blocking agent to 20 μl of serum, then add 170 μl of PBS, mix well, and incubate at 37°C for 10 minutes. Remove, mix well, centrifuge at 22000 rpm / min for 15 minutes, and take the supernatant for detection.

[0051] The results obtained are as follows Figure 3 As shown in the image, the fluorescence image corresponding to premixing method 1) shows that a 1:1 premix of serum and inhibitor (i.e., 20 μl serum plus 20 μl of preferred antibiotic, followed by 160 μl PBS) and incubation at 37°C for 20 minutes maximizes the fluorescence signal and reduces the fluorescence background. While premixing method 2) is not significantly different from 1), the results show a weaker fluorescence signal intensity compared to premixing method 1). This is likely because the 40 μl sample, when incubated at 37°C, causes partial degradation and inactivation of the protein, thus reducing the detection sensitivity. Therefore, premixing method 1) was used in subsequent experiments.

[0052] 3.3 Confirmation of the method of using the blocking agent of the present invention

[0053] 3.3.1 293T empty cell blocker 1. To compare the sensitivity differences between the slide detection methods prepared with different blocking agents and those without blocking agents, and to quantify the detection sensitivity after serum treatment with blocking agents, AQP4 and MOG monoclonal antibodies were added to fresh negative serum to simulate the real serum matrix. The results of detecting different concentrations of monoclonal antibodies on slides of the same batch were compared. A fluorescence interpretation result of 1:10 was considered positive.

[0054] 2. Prepare monoclonal antibody sera of different concentrations, from highest to lowest: 0.5 μg / ml, 0.1 μg / ml, 0.06 μg / ml, 0.03 μg / ml, and 0.01 μg / ml. Add 20 μl of each concentration of monoclonal antibody serum to 2 ml EP tubes, add 20 μl of the blocking agent prepared from 293T empty vector cells to each tube, mix well by pipetting, and incubate at 37°C for 20 minutes. After incubation, centrifuge at 22000 rpm / min for 15 minutes, and transfer 40 μl of the supernatant to a new EP tube (avoiding aspirating the precipitate at the bottom of the tube). Add 160 μl of PBS, mix well, and use as a 1:10 dilution of monoclonal antibody serum for later use. 3. Following the standard operating procedure of the CBA method, 90 μl of pretreated monoclonal antibody serum was added to a cell slide overexpressing AQP4 and MOG antigens for indirect immunofluorescence detection. The specific fluorescence signal on the 293T cell membrane was read under a fluorescence microscope using a 488 nm filter. 4. The interpretation results of parallel detections after different concentrations of monoclonal antibody serum were treated with blocking agents obtained by different treatment methods are shown in Tables 5 and 6 below.

[0055] Table 5 Comparison results of simulated AQP4 positive serum experiments

[0056] Table 6. Comparison results of simulated MOG positive serum experiments

[0057] 3.3.2 Method of using HeLa empty cell blocker: 1. To compare the sensitivity differences between the slide detection methods prepared with different blocking agents and those without blocking agents, and to quantify the detection sensitivity after serum treatment with blocking agents, LGI 1 monoclonal antibody was added to fresh negative serum to simulate the real serum matrix. The results of different concentrations of monoclonal antibody were compared on slides of the same batch. A fluorescence interpretation result of 1:10 was considered positive. 2. Prepare LGI1 monoclonal antibody serum at different concentrations, from highest to lowest: 0.5 μg / ml, 0.1 μg / ml, 0.06 μg / ml, 0.03 μg / ml, and 0.01 μg / ml. Add 20 μl of each concentration of monoclonal antibody serum to 2 ml EP tubes, add 20 μl of the blocking agent prepared from HeLa empty vector cells to each tube, mix well by pipetting, and incubate at 37°C for 20 minutes. After incubation, centrifuge at 22000 rpm / min for 15 minutes, and transfer 40 μl of the supernatant to a new EP tube (avoiding aspirating the precipitate at the bottom of the tube). Add 160 μl of PBS, mix well, and use as a 1:10 diluted monoclonal antibody serum for later use. 3. Following the standard operating procedure of the CBA method, 90 μl of pretreated monoclonal antibody serum was added to a cell slide overexpressing LGI1 antigen for indirect immunofluorescence detection. The specific fluorescence signal on the HeLa cell membrane was read under a fluorescence microscope using a 488 nm filter. 4. The interpretation results of parallel detections after treating the blocking agents obtained from different concentrations of monoclonal antibody serum using different treatment methods are shown in Table 7 below. Table 7 Comparison results of simulated LGI1 positive serum experiments

[0058] 3.3.3 Experimental Conclusions: 1. Compared to untreated monoclonal antibody serum, the 293T empty vector cell blocking agent obtained from a 5-fold diluted moderate lysis buffer showed the best results, increasing the sensitivity of AQP4 and MOG detection by slide assay from 2.5 μg / ml to 0.1 μg / ml (25-fold); the HeLa blocking agent increased the sensitivity of LGI 1 detection by slide assay from 0.5 μg / ml to 0.03 μg / ml (16-17-fold); both the 293T blocking agent and the HeLa blocking agent significantly improved the sensitivity of slide assay. 2. The data above show that after treatment with the blocking agent, the extreme sensitivity of HeLa slides for detecting LGI 1 is 0.03 μg / ml, and the extreme sensitivity of 293T cell slides for detecting AQP4 and MOG is 0.1 μg / ml. In summary, serum treatment with the blocking agent increases the extreme sensitivity of slide detection of AQP4 and MOG from 0.8 μg / ml to 0.1 μg / ml. This means that using the blocking agent makes the sensitivity of slide detection of AQP4 and MOG 8 times higher than that of the well plate method. The slide method can completely replace and far surpass the well plate method, becoming a powerful tool for detecting demyelinating events.

[0059] It is evident that the use of the blocking agent in this invention can greatly improve the sensitivity of CBA detection by slide method, overturning the previous literature reports on the false negative rate of 20%-40% for AQP4 and MOG, enabling early diagnosis and treatment of diseases, and effectively solving the pain point of timely and accurate diagnosis and treatment in clinical practice, thus showing good application prospects.

[0060] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A blocking agent for slide-based immunofluorescence assay, characterized in that, The blocking agent is a homologous empty vector cell lysate without transfection plasmid, with a protein concentration ≥30mg / mL.

2. The blocking agent according to claim 1, characterized in that, The blocking agent is prepared by the following method: Dilute the cell lysis buffer and add it to the prepared empty cell vector. Incubate at 4°C or on ice for lysis. After lysis, centrifuge and collect the supernatant. Alternatively, add the cell lysis buffer to the prepared empty cell vector and lyse at 4°C or on ice. After lysis, dilute and centrifuge to collect the supernatant.

3. The blocking agent according to claim 2, characterized in that, The prepared empty vector cells are obtained by centrifuging the cultured empty vector cells to remove the cell culture medium, then adding PBS, mixing by pipetting, centrifuging again, and discarding the supernatant; wherein the centrifugation rate is 2000~5000 rpm and the time is 20~30 min.

4. The blocking agent according to claim 2, characterized in that, The ratio of the number of empty cells to the amount of cell lysis buffer was 2 × 10⁻⁶. 5 ~5×10 6 Quantity: 20 μL.

5. The blocking agent according to claim 2, characterized in that, The dilution factor is 2 to 10 times.

6. The blocking agent according to claim 2, characterized in that, The pyrolysis time at 4℃ or on ice is 10~15 min, the centrifugation rate is 20000~25000 rpm, and the time is 10~20 min.

7. The blocking agent according to claim 1, characterized in that, The homologous empty vector cells are any one of HEK 293T empty vector cells, Hela empty vector cells, Hep-2 empty vector cells, CHO empty vector cells, COS empty vector cells, HepG2 empty vector cells, MCF-7 empty vector cells, A549 empty vector cells, and Vero empty vector cells.

8. The method of using the blocking agent as described in any one of claims 1-7 in slide immunofluorescence assay, characterized in that, Includes the following steps: Prepare monoclonal antibody serum, add the blocking agent as described in any one of claims 1-7 and mix well. After incubation at 37°C, add diluent to dilute and centrifuge to collect the supernatant. Alternatively, prepare monoclonal antibody serum by adding the blocking agent as described in any one of claims 1-7 and mixing well; then dilute with diluent, incubate at 37°C, and finally centrifuge to collect the supernatant.

9. The method of use according to claim 8, characterized in that, According to the volume ratio, the monoclonal antibody serum:blocker:diluent = 1:0.5-1:8-10.

10. The method of use according to claim 8, characterized in that, The incubation time at 37℃ is 15-30 min, the centrifugation rate is 20000~25000 rpm, and the time is 10~15 min.

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