A stabilizing protective agent, a cell antigen dry sheet, and a preparation method and application of the dry sheet

Abstract

The application belongs to the technical field of biology, and discloses a stabilizing protective agent, a cell antigen dry sheet, and a preparation method and application of the dry sheet. The stabilizing protective agent comprises 0.4% glycerol in volume fraction, 0.1% gelatin in mass volume fraction, 0.1 mM EDTA, 0.03% trehalose in mass volume fraction, and 100 muM resveratrol. The stabilizing protective agent is used to prepare the cell antigen dry sheet and is applied in CBA detection. The stabilizing protective agent has good permeability, penetrates into the cell interior through the cell membrane, provides protection inside and outside the cell, has moisturizing and antioxidant properties, can prevent cell deformation and cell fixation layer from falling off, well maintains the stability and reliability of antigen protein, and the prepared cell antigen dry sheet can be stored for a long time without damaging the antigenicity of the membrane signal index. When a positive sample is detected, obvious green fluorescent cells can be seen, and red fluorescent quenching does not occur, so that the co-localization of red fluorescence and green fluorescence can be observed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a stabilizing protective agent, a cell antigen dry sheet, and a preparation method and application of the dry sheet. BACKGROUND

[0002] At present, in a commercial CBA detection reagent kit, a cell antigen sheet is generally a wet sheet, that is, after cell antigens in a 96-well plate are fixed by 4% paraformaldehyde, 200 μL PBS is added to each well as a cell protective agent for sealing and preservation. Therefore, the cell antigen wet sheet for CBA detection has the characteristics of simple operation, low cost, and superior performance. However, the cell antigen wet sheet for CBA detection also has some shortcomings: (1) after the protective liquid is added, the stability of the cell antigen sheet is poor, and the cells attached to the bottom of the well will slowly fall off; (2) the protective liquid is easy to introduce contaminants and contaminate the antigen sheet. Therefore, we use a cell antigen dry sheet for detection, but the cell antigen dry sheet needs to solve the problems of cell deformation, moisturizing, antioxidation, and maintaining antigen stability. SUMMARY

[0003] In order to solve at least one of the above problems, the present application provides a stabilizing protective agent, a cell antigen dry sheet, and a preparation method and application of the dry sheet. The stabilizing protective agent takes into account moisturizing and antioxidation, can prevent cell deformation and prevent the fixed layer from falling off, and the prepared cell antigen dry sheet can better maintain the long-term stability of antigen proteins, and is convenient for application in CBA detection.

[0004] In order to achieve the above purpose, the present application adopts the following technical means:

[0005] The first aspect of the present application provides a stabilizing protective agent for a cell antigen dry sheet, which comprises 0.4% glycerol by volume fraction, 0.1% gelatin by mass volume fraction, 0.1 mM EDTA, 0.03% trehalose by mass volume fraction, and 100 μM resveratrol.

[0006] The second aspect of the present application provides the application of the stabilizing protective agent for the cell antigen dry sheet as described in the first aspect in the preparation of a detection reagent kit.

[0007] The third aspect of the present application provides a preparation method of a cell antigen dry sheet, comprising the following steps:

[0008] S1, using target cells to perform plating to obtain a cell plate;

[0009] S2, washing the cell plate with cells grown thereon with PBS for 2-3 times;

[0010] S3, fixing with 4% paraformaldehyde for 25-30 min, and then washing with PBS for 2-3 times;

[0011] S4, after adding the stabilizing protective agent of the first aspect, drying in a 37℃ drying oven for 2-3 h, to obtain a cell antigen dry sheet.

[0012] In some embodiments of the present application, the target cell is capable of specifically overexpressing an antigen by integrating an antigen gene, and the antigen includes one of CASPR2, AQP4, MOG, AQP1, Flotillin1 / 2, PLP1, NMDAR, AMPAR1, AMPAR2, GABABR, DPPX, IgLON5, GlyRα1, D2R, mGluR5, mGluR1, Neurexin-3α, GABAARα1, GABAARβ3, GABAARγ2, GluK2, CaVα2δ, KCTD16, RGS8, ganglionic AChR, SEZ6L2, MuSk, LRP4, ATP1A3, mGluR2, mGluR8, mGluR3, NF155, NF140, NF186, CNTN1, CNTN2, CASPR1, MAG, PLA2R, THSD7A.

[0013] The fourth aspect of the present application provides a cell antigen dry sheet, which is prepared by the method of the third aspect.

[0014] The fifth aspect of the present application provides the use of the cell antigen dry sheet of the fourth aspect in the preparation of a CBA detection kit.

[0015] The sixth aspect of the present application provides a detection kit, which comprises the stabilizing protective agent of the first aspect or the cell antigen dry sheet of the fourth aspect.

[0016] In some embodiments of the present application, the kit further comprises a PBS buffer, HEK293T cells, a cell culture microplate, a blocking solution, and a fluorescent secondary antibody, wherein the blocking solution is a serum of the species in which the fluorescent secondary antibody is located.

[0017] In some embodiments of the present application, the HEK293T cells specifically overexpress an antigen, which includes one of CASPR2, AQP4, MOG, AQP1, Flotillin1 / 2, PLP1, NMDAR, AMPAR1, AMPAR2, GABABR, DPPX, IgLON5, GlyRα1, D2R, mGluR5, mGluR1, Neurexin-3α, GABAARα1, GABAARβ3, GABAARγ2, GluK2, CaVα2δ, KCTD16, RGS8, ganglionic AChR, SEZ6L2, MuSk, LRP4, ATP1A3, mGluR2, mGluR8, mGluR3, NF155, NF140, NF186, CNTN1, CNTN2, CASPR1, MAG, PLA2R, THSD7A, and a red fluorescent protein on the antigen.

[0018] The seventh aspect of the present application provides a method for using the detection kit of the sixth aspect, comprising the following steps:

[0019] S1, using HEK293T cells integrated with antigen genes for plating to obtain a cell plate;

[0020] S2, washing the cell plate with cells grown with PBS for 2-3 times;

[0021] S3, fixing with 4% paraformaldehyde for 25-30 min and then washing with PBS for 2-3 times;

[0022] S4, adding the stabilizing protective agent of the first aspect and drying in a 37°C drying box for 2-3 h to obtain a cell antigen dry sheet;

[0023] S5, adding a sample to be tested to the detection hole of the cell antigen dry sheet, mixing gently, and incubating at 37°C;

[0024] S6, after incubation, removing the liquid in the detection hole, and washing completely with PBS buffer and then discarding;

[0025] S7, adding a fluorescent secondary antibody diluted with a blocking solution to the detection hole, mixing gently, and incubating at 37°C in the dark;

[0026] S8, repeating the washing step of S6, discarding the liquid in the hole, adding PBS buffer to the detection hole to cover the cells, and then observing the green fluorescence and red fluorescence of the cells under a fluorescence microscope and taking a photo;

[0027] S9, observing whether the two kinds of fluorescence are co-localized and obtaining a test result.

[0028] In some embodiments, the incubation time of the above-mentioned incubation step is 30-40 min, preferably 35 min. In some embodiments, the washing step is to remove the liquid in the detection hole after incubation, wash twice with PBS buffer, gently shake the hole plate during the washing process, and place on a shaker for 5 min for the third time.

[0029] Further, in some embodiments, if the test result is positive, and if titer verification is required, the sample is diluted in proportion, and the experimental process of steps S5 to S9 is repeated.

[0030] In some embodiments of the present application, if the sample to be tested is a serum sample, a blocking solution is used for dilution; if it is a cerebrospinal fluid sample, a stock solution is used. In some embodiments of the present application, the serum sample is diluted 20 times or more using a blocking solution.

[0031] Advantages of the present application

[0032] Compared with the prior art, the present application has the following advantages: the stabilizing protective agent for CBA detection cell antigen dry sheet provided by the present application has good permeability, can penetrate the cell membrane and enter the cell interior, thereby providing protection inside and outside the cell; at the same time, the protective agent has moisturizing and antioxidant properties, can also prevent cell deformation and cell fixation layer shedding, and can well maintain the stability and reliability of the antigen protein. The cell antigen dry sheet prepared by using the stabilizing protective agent can be stored for a long time without damaging the antigenicity of the membrane signal target index. The stabilizing protective agent needs 0.4% glycerol by volume fraction, 0.1% gelatin by mass volume fraction, 0.1 mM EDTA, 0.03% trehalose by mass volume fraction, and 100 μM resveratrol to synergize to achieve the best effect: when the detection sample is a positive sample, obvious green fluorescent cells can be seen, and there is no red fluorescent quenching. Using a fluorescence microscope, it can be observed that the red fluorescent and green fluorescent are co-localized. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 A fluorescence detection result diagram of a positive sample and a negative sample obtained by the wet sheet of Example 1 is shown;

[0034] Figure 2 A fluorescence detection result diagram of a positive sample and a negative sample obtained by the wet sheet of Example 1 is shown;

[0035] Figure 3 A fluorescence detection result diagram of a positive sample and a negative sample obtained by the wet sheet of Example 1 is shown;

[0036] Figure 4 A fluorescence detection result diagram of a positive sample and a negative sample obtained by the wet sheet of Example 1 is shown;

[0037] Figure 5 FIG. 8 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 8;

[0038] Figure 6 FIG. 9 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 9;

[0039] Figure 7 FIG. 10 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 10;

[0040] Figure 8 FIG. 11 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 11;

[0041] Figure 9 FIG. 12 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 12;

[0042] Figure 10 FIG. 13 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 13;

[0043] Figure 11 FIG. 14 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 14;

[0044] Figure 12 FIG. 15 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 15;

[0045] Figure 13 FIG. 16 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 16;

[0046] Figure 14 FIG. 17 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 17;

[0047] Figure 15 FIG. 18 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 18;

[0048] Figure 16 FIG. 19 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 19;

[0049] Figure 17 FIG. 20 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 20;

[0050] Figure 18 FIG. 21 shows a plot of the fluorescence detection results for positive and negative samples in the scheme of Example 21;

[0051] Figure 19Fluorescence detection results for positive and negative samples from Example 18 are shown in the graphs below.

[0052] Figure 20 Fluorescence detection results for positive and negative samples from Example 19 are shown in the graphs below.

[0053] Figure 21 Fluorescence detection results for positive and negative samples from Example 20 are shown in the graphs below.

[0054] Figure 22 Fluorescence detection results for positive and negative samples from Example 21 are shown in the graphs below.

[0055] Figure 23 Fluorescence detection results for positive and negative samples from Example 22 after 1 week of accelerated aging of dry film and control wet film are shown in the graphs below.

[0056] Figure 24 Fluorescence detection results for positive and negative samples from Example 22 after 2 weeks of accelerated aging of dry film and control wet film are shown in the graphs below.

[0057] Figure 25 Fluorescence detection results for positive and negative samples from Example 22 after 3 weeks of accelerated aging of dry film and control wet film are shown in the graphs below.

[0058] Figure 26 Fluorescence detection results for positive and negative samples from Example 22 after 4 weeks of accelerated aging of dry film and control wet film are shown in the graphs below.

[0059] Figure 27 Fluorescence detection results for positive and negative samples from Example 22 after 5 weeks of accelerated aging of dry film and control wet film are shown in the graphs below.

[0060] Figure 28 Fluorescence detection results for positive and negative samples from Example 22 after 6 weeks of accelerated aging of dry film and control wet film are shown in the graphs below. DETAILED DESCRIPTION

[0061] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present application, and are not intended to limit the scope of what the inventors regard as their application. One skilled in the art will recognize that the examples that are disclosed herein represent techniques that can be used in the practice of the present application, and thus are to be considered within the scope of the application. Of course, those of ordinary skill in the art will forwardly appreciate that the specific embodiments disclosed herein can be easily modified and various modifications can be made without departing from the spirit or scope of the present application.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the materials described herein will be referred to by the names given in the references cited herein. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the following claims.

[0063] In embodiments of the application, the target cells are made to specifically overexpress an antigen by integrating an antigen gene, the antigen including one of CASPR2, AQP4, MOG, AQP1, Flotillin1 / 2, PLP1, NMDAR, AMPAR1, AMPAR2, GABABR, DPPX, IgLON5, GlyRα1, D2R, mGluR5, mGluR1, Neurexin-3α, GABAARα1, GABAARβ3, GABAARγ2, GluK2, CaVα2δ, KCTD16, RGS8, ganglionic AChR, SEZ6L2, MuSk, LRP4, ATP1A3, mGluR2, mGluR8, mGluR3, NF155, NF140, NF186, CNTN1, CNTN2, CASPR1, MAG, PLA2R, THSD7A.

[0064] The technical solutions of the present application are further described in detail below with the CASPR2 antigen as an example in combination with the specific embodiments.

[0065] Embodiment 1

[0066] I. Preparation of cell plate

[0067] 1. Cell washing: After culturing the HEK293T cells integrated with the CASPR2 antigen gene for 48 h, the old culture supernatant was poured out, and the cells were washed with an appropriate amount of physiological saline for 1-2 times;

[0068] 2. Cell digestion: An appropriate amount of 0.25% trypsin-EDTA solution was added to the culture dish, the culture dish was gently shaken to make the trypsin fully contact the cells, and the cells were digested at room temperature. The cell digestion was observed under a microscope. If most of the cells were rounded and detached, the cells were quickly taken back to the operation table, and 2 mL of the termination medium was added to terminate the digestion after the trypsin was removed;

[0069] 3. Cell centrifugation: The cells were gently blown with a pipette, and after complete detachment, they were aspirated into a 2 mL centrifuge tube and centrifuged at 1400 rpm for 6 min. The supernatant was removed, and 1 mL of complete medium was added and gently blown evenly;

[0070] 4. Cell plating: The cell density was adjusted to 3×106 The HEK293T cells (integrating CASPR2 antigen gene) in the 96-well cell plate are washed twice with PBS.

[0071] II. Preparation of cell antigen dry sheet

[0072] 1. The 96-well cell plate covered with HEK293T cells (integrating CASPR2 antigen gene) is washed twice with PBS;

[0073] 2. Fixed with 4% paraformaldehyde for 30 min;

[0074] 3. Washed twice with PBS;

[0075] 4. Add 50 μL stabilizing protective agent: 0.05% v / v glycerol, and dry in a 37°C drying oven for 3 h.

[0076] III. Performance test

[0077] (1) Test principle

[0078] The detection principle of this embodiment is indirect immunofluorescence. The antibody corresponding to CASPR2 in the sample forms an antigen-antibody complex with the specific overexpressed antigen expressed on the HEK293T cells in the reagent, and this antigen has mCherry fluorescent protein (red fluorescence). Then, the human secondary antibody bound to the antigen is labeled with goat anti-human fluorescent secondary antibody (green fluorescence), and finally, the presence or absence of co-localization of red fluorescence and green fluorescence is observed using a fluorescence microscope. If co-localization exists, it indicates that the sample contains the antibody, otherwise the sample does not contain the corresponding antibody.

[0079] (2) Test method

[0080] Preparation work:

[0081] 1. Preparation of PBS buffer: dilute 10 times of PBS buffer with pure water or distilled water;

[0082] 2. Blocking solution preparation: dilute goat serum with PBS buffer by 10 times;

[0083] 3. Sample: if it is a serum sample, dilute it with blocking solution by at least 20 times; if it is a cerebrospinal fluid sample, use the original solution;

[0084] 4. Fluorescent secondary antibody: dilute it with blocking solution by 1500 times.

[0085] Test steps:

[0086] 1. Add sample: add 80 μL of diluted sample to the detection well, mix gently, and incubate at 37°C for 35 min;

[0087] 2. Wash: After incubation, remove the liquid in the detection hole, and add 200 μL of PBS buffer for washing twice. Shake the hole slightly during the washing process. Place the hole on the shaker for the third time, and wash for 5 min;

[0088] 3. Add fluorescent secondary antibody: Discard the liquid in the hole, and add 80 μL of diluted fluorescent secondary antibody to the detection hole. Mix gently, and incubate at 37°C for 35 min in the dark;

[0089] 4. Wash: Repeat step 3;

[0090] 5. Observe: Discard the liquid in the hole, and add 100 μL of PBS buffer to the sample hole to cover the cells. Then, observe the red fluorescence of the cells under a fluorescence microscope, and take a photo;

[0091] 6. Observe whether the two types of fluorescence are co-localized to obtain the test result. If a positive result needs to be verified for titer, dilute the sample according to the ratio, and repeat steps 1 to 7.

[0092] (3) Detection index

[0093] Membrane signal CASPR2 index.

[0094] (4) Comparison scheme

[0095] The preparation and test method of the membrane signal CASPR2 index are as follows:

[0096] A. Fixing method

[0097] 1. Prepare 4% paraformaldehyde with PBS;

[0098] 2. Wash the 96-well cell culture microplate covered with HEK293T cells (integrated CASPR2 antigen gene) with PBS twice;

[0099] 3. Fix with 4% paraformaldehyde for 30 min;

[0100] 4. Wash with PBS twice;

[0101] 5. Add 200 μL of PBS as a cell protectant, and vacuum seal;

[0102] B. Test steps:

[0103] 1. Pre-wash: Take out the cell culture microplate, discard the reserved liquid in the hole. Add 200 μL of PBS buffer for washing once, and discard the liquid in the hole again;

[0104] 2. Add sample: Add 80 μL of diluted serum to the detection hole. Mix gently, and incubate at 37°C for 35 min;

[0105] 3. Washing: After incubation, remove the plate, discard the liquid in the wells, and wash twice with 200 µL PBS buffer. Gently shake the plate during the washing process. For the third wash, place the plate on a shaker and wash for 5 min.

[0106] 4. Add fluorescent secondary antibody: Discard the liquid in the well, add 80 µL of diluted fluorescent secondary antibody to the detection well, shake gently to mix, and incubate at 37°C in the dark for 35 min.

[0107] 5. Cleaning: Repeat step 3;

[0108] 6. Observation: Discard the liquid in the well, add 100 µL of PBS buffer to the sample well to cover the cells, and then observe the red fluorescence of the cells under a fluorescence microscope and take pictures.

[0109] 7. Observe whether the two fluorescences co-localize to obtain the test result. If a positive result requires titer verification, dilute the sample proportionally and repeat steps 1 to 7.

[0110] (5) Comparison of test samples

[0111] Both positive and negative samples were clinical samples, and were validated using the human anti-contactin-associated protein 2 IgG antibody (Caspr2-IgG) ELISA kit (Catalog No. BY-HS 13702) from Shanghai Senxingyan Biotechnology Co., Ltd.

[0112] (6) Result interpretation

[0113] Criteria for interpreting negative and positive samples:

[0114] Negative: No green fluorescent cells were observed in the green fluorescence channel or no overlap of green fluorescent cells with red fluorescence was observed after software synthesis.

[0115] Positive: Green fluorescent cells are visible under the green fluorescent channel, and can be observed to overlap with the red antigen fluorescence after software synthesis.

[0116] Dry slide performance evaluation criteria: If the dry slides of cell antigens exhibit the same performance as the wet slides of cell antigens, regardless of whether the sample is negative or positive, it indicates that the performance of the dry slide stabilizer is ideal.

[0117] The test results of the comparison scheme are as follows: Figure 1 As shown, the test results of the implementation plan are as follows: Figure 2 As shown.

[0118] The results showed that when the test sample was positive, faint green and red fluorescent cells were visible; compared with CASPR2 cell antigen wet mount ( Figure 1The green and red fluorescence intensity of the positive sample is too weak compared to the CASPR2 cell antigen wet slice, so 0.05% v / v glycerol cannot be used for the preparation of the CASPR2 cell antigen dry slice. The reason is that when the glycerol concentration is too low, the hydration layer on the surface of the protein is lost with the evaporation of the solvent or the drying of the environment, which leads to the destruction of the antigen protein structure, and then leads to the reduction of effective antigens.

[0119] Example 2

[0120] The method is the same as example 1, except that the stabilizing protective agent is 0.1% v / v glycerol.

[0121] The cell fluorescence is observed under a fluorescence microscope, and the test results are obtained by observing whether the two kinds of fluorescence are co-located. The results are shown in Table 2. Figure 3

[0122] The results show that when the test sample is a positive sample, weak green and red fluorescent cells can be seen; compared to the CASPR2 cell antigen wet slice ( Figure 1 ), the green and red fluorescence intensity of the positive sample is too weak, so 0.1% v / v glycerol cannot be used for the preparation of the CASPR2 cell antigen dry slice. The reason is that when the glycerol concentration is too low, the hydration layer on the surface of the protein is lost with the evaporation of the solvent or the drying of the environment, which leads to the destruction of the antigen protein structure, and then leads to the reduction of effective antigens.

[0123] Example 3

[0124] The method is the same as example 1, except that the stabilizing protective agent is 0.2% v / v glycerol.

[0125] The cell fluorescence is observed under a fluorescence microscope, and the test results are obtained by observing whether the two kinds of fluorescence are co-located. The results are shown in Table 3. Figure 4

[0126] The results show that when the test sample is a positive sample, medium-strong green and red fluorescent cells can be seen; compared to the CASPR2 cell antigen wet slice ( Figure 1 ), the green and red fluorescence intensity of the positive sample is slightly weak, so 0.2% v / v glycerol cannot be used for the preparation of the CASPR2 cell antigen dry slice. The reason is that when the glycerol concentration is too low, the hydration layer on the surface of the protein is lost with the evaporation of the solvent or the drying of the environment, which leads to the destruction of the antigen protein structure, and then leads to the reduction of effective antigens.

[0127] Example 4

[0128] The method is the same as example 1, except that the stabilizing protective agent is 0.3% v / v glycerol.

[0129] ​​The fluorescence of the cells was observed under a fluorescence microscope, and the test results were obtained by observing whether the two kinds of fluorescence were co-localized. The results are shown in Table 1. Figure 5

[0130] The results show that when the detection sample is a positive sample, strong green and red fluorescent cells appear; compared with the CASPR2 cell antigen wet sheet Figure 1 , the green and red fluorescence intensity of the positive sample is not ideal, so 0.3% v / v glycerol cannot be used for the preparation of the CASPR2 cell antigen dry sheet. The reason is that when the concentration of glycerol is too low, the hydration layer on the surface of the protein is lost with the evaporation of the solvent or the drying of the environment, thereby causing the destruction of the non-antigen protein structure and then leading to the reduction of the effective antigen.

[0131] Example 5

[0132] The method is the same as that in Example 1, except that the stabilizing protective agent is 0.4% v / v glycerol.

[0133] The fluorescence of the cells was observed under a fluorescence microscope, and the test results were obtained by observing whether the two kinds of fluorescence were co-localized. The results are shown in Table 1. Figure 6

[0134] The results show that when the detection sample is a positive sample, strong green and red fluorescent cells appear; compared with the CASPR2 cell antigen wet sheet Figure 1 , the green and red fluorescence intensity of the positive sample is not ideal, so 0.3% v / v glycerol cannot be used for the preparation of the CASPR2 cell antigen dry sheet. The reason is that when the concentration of glycerol is too low, the hydration layer on the surface of the protein is lost with the evaporation of the solvent or the drying of the environment, thereby causing the destruction of the non-antigen protein structure and then leading to the reduction of the effective antigen.

[0135] Example 6

[0136] The method is the same as that in Example 1, except that the stabilizing protective agent is 0.5% v / v glycerol.

[0137] The fluorescence of the cells was observed under a fluorescence microscope, and the test results were obtained by observing whether the two kinds of fluorescence were co-localized. The results are shown in Table 1. Figure 7

[0138] The results show that when the detection sample is a positive sample, strong green and red fluorescent cells appear; compared with the CASPR2 cell antigen wet sheet Figure 1 ​​​green and red fluorescence intensity of the positive sample was slightly weaker, so 0.5% v / v glycerol could not be used for the preparation of the CASPR2 cell antigen dry sheet. The reason is that when the concentration of glycerol is too high, it will significantly increase the viscosity of the solution, limiting the conformational dynamics of protein molecules (such as the flexible swing of antigen epitopes). Some antigen epitopes need to be exposed by dynamic conformation to be recognized by antibodies. The increase in viscosity will inhibit this process, resulting in reduced accessibility of antigen epitopes, leading to decreased antigen-antibody binding efficiency and weakened detection signal.

[0139] From the above Figure 1 to Figure 6 results, it can be seen that the CASPR2 index cell antigen dry sheet prepared by using 0.4% v / v glycerol as a dry sheet stabilizer exhibits the best detection performance. From Figure 6 , it can be seen that the CASPR2 index cell antigen dry sheet prepared by using 0.4% v / v glycerol as a dry sheet stabilizer still has some gap compared with the CASPR2 cell antigen wet sheet.

[0140] Example 7

[0141] The method is the same as that of Example 1, except that the stabilizing and protecting agent is 0.4% v / v glycerol, 0.1 mM EDTA, 0.01% w / v trehalose, and 50 μM resveratrol. The CASPR2 cell antigen wet sheet is used as the control group.

[0142] The fluorescence of the cells is observed under a fluorescence microscope, and photographs are taken to observe whether the two types of fluorescence are co-localized to obtain the test results. The results are shown in Figure 8 .

[0143] The results show that when the detection sample is a positive sample, weak green and red fluorescent cells can be seen; compared with the CASPR2 cell antigen wet sheet ( Figure 1 ), the green and red fluorescence intensity of the positive sample is too weak, so this scheme cannot be used for the preparation of the CASPR2 cell antigen dry sheet.

[0144] Example 8

[0145] The method is the same as that of Example 1, except that the stabilizing and protecting agent is 0.4% v / v glycerol, 0.2 mM EDTA, 0.02% w / v trehalose, and 100 μM resveratrol. The CASPR2 cell antigen wet sheet is used as the control group.

[0146] The fluorescence of the cells is observed under a fluorescence microscope, and photographs are taken to observe whether the two types of fluorescence are co-localized to obtain the test results. The results are shown in Figure 9 .

[0147] The results show that when the detection sample is a positive sample, weak green and red fluorescent cells can be seen; compared with the CASPR2 cell antigen wet sheet (Figure 1 In comparison, the green and red fluorescence intensity of the positive samples is too weak, so this method cannot be used to prepare CASPR2 cell antigen dry slides.

[0148] Example 9

[0149] The method was the same as in Example 1, except that the stabilizers were 0.4% v / v glycerol, 0.3 mM EDTA, 0.03% w / v trehalose, and 150 μM resveratrol. CASPR2 cell antigen wet mounts were used as the control group.

[0150] Cell fluorescence was observed under a fluorescence microscope, photographed, and the co-localization of the two fluorescence types was determined to obtain the test results. The results are as follows: Figure 10 As shown.

[0151] The results showed that when the sample was positive, medium-strong green and red fluorescent cells were visible; compared with the CASPR2 cell antigen wet mount ( Figure 1 Compared to the positive sample, the green and red fluorescence intensity of the positive sample is slightly weaker, so this method cannot be used to prepare CASPR2 cell antigen dry slides.

[0152] Example 10

[0153] The method was the same as in Example 1, except that the stabilizers were 0.4% v / v glycerol, 0.05% w / v gelatin, 0.01% w / v trehalose, and 100 μM resveratrol. CASPR2 cell antigen wet mounts were used as the control group.

[0154] Cell fluorescence was observed under a fluorescence microscope, photographed, and the co-localization of the two fluorescence types was determined to obtain the test results. The results are as follows: Figure 11 As shown.

[0155] The results showed that when the sample was positive, slightly weaker green and red fluorescent cells were visible; compared with CASPR2 cell antigen wet mount ( Figure 1 In comparison, the green and red fluorescence intensity of the positive samples is too weak, so this method cannot be used to prepare CASPR2 cell antigen dry slides.

[0156] Example 11

[0157] The method was the same as in Example 1, except that the stabilizers were 0.4% v / v glycerol, 0.05% w / v gelatin, 0.1 mM EDTA, and 150 μM resveratrol. CASPR2 cell antigen wet mounts were used as the control group.

[0158] Cell fluorescence was observed under a fluorescence microscope, photographed, and the co-localization of the two fluorescence types was determined to obtain the test results. The results are as follows: Figure 12 As shown.

[0159] Results show: when the detection sample is a positive sample, the green and red fluorescence cells of medium intensity can be seen; compared with the CASPR2 cell antigen wet film ( Figure 1 ), the green and red fluorescence intensity of the positive sample is too weak, so this scheme cannot be used for the preparation of the CASPR2 cell antigen dry film.

[0160] Example 12

[0161] The method is the same as that of Example 1, except that the stabilizing protective agent is 0.4% v / v glycerol, 0.05% w / v gelatin, 0.2 mM EDTA, and 0.03% w / v trehalose. The CASPR2 cell antigen wet film is used as the control group.

[0162] Observe the cell fluorescence under the fluorescence microscope, take pictures, and observe whether the two kinds of fluorescence are co-localized to obtain the test results. The results are shown in Figure 13 .

[0163] Results show: when the detection sample is a positive sample, the green and red fluorescence cells of medium intensity can be seen; compared with the CASPR2 cell antigen wet film ( Figure 1 ), the green and red fluorescence intensity of the positive sample is too weak, so this scheme cannot be used for the preparation of the CASPR2 cell antigen dry film.

[0164] Example 13

[0165] The method is the same as that of Example 1, except that the stabilizing protective agent is 0.4% v / v glycerol, 0.05% w / v gelatin, 0.3 mM EDTA, 0.02% w / v trehalose, and 50 μM resveratrol. The CASPR2 cell antigen wet film is used as the control group.

[0166] Observe the cell fluorescence under the fluorescence microscope, take pictures, and observe whether the two kinds of fluorescence are co-localized to obtain the test results. The results are shown in Figure 14 .

[0167] Results show: when the detection sample is a positive sample, the green and red fluorescence cells of medium intensity can be seen; compared with the CASPR2 cell antigen wet film ( Figure 1 ), the green and red fluorescence intensity of the positive sample is too weak, so this scheme cannot be used for the preparation of the CASPR2 cell antigen dry film.

[0168] Example 14

[0169] The method is the same as that of Example 1, except that the stabilizing protective agent is 0.4% v / v glycerol, 0.1% w / v gelatin, 0.02% w / v trehalose, and 150 μM resveratrol. The CASPR2 cell antigen wet film is used as the control group.

[0170] The fluorescence of the cells was observed under a fluorescence microscope, and the test results were obtained by observing whether the two kinds of fluorescence were co-localized. The results are shown in Table 1. Figure 15

[0171] The results show that when the detection sample is a positive sample, the strongest green and red fluorescent cells appear; compared with the CASPR2 cell antigen wet slice Figure 1 , the green and red fluorescence intensity of the positive sample is weaker, so this scheme cannot be used for the preparation of the CASPR2 cell antigen dry slice.

[0172] Example 15

[0173] The method is the same as that in Example 1, except that the stabilizing protective agent is 0.4% v / v glycerol, 0.1% w / v gelatin, 0.1 mM EDTA, 0.03% w / v trehalose, and 100 μM resveratrol. The CASPR2 cell antigen wet slice is used as the control group.

[0174] The fluorescence of the cells was observed under a fluorescence microscope, and the test results were obtained by observing whether the two kinds of fluorescence were co-localized. The results are shown in Table 1. Figure 16

[0175] The results show that when the detection sample is a positive sample, the strongest green and red fluorescent cells appear; compared with the CASPR2 cell antigen wet slice Figure 1 , the green and red fluorescence intensity of the positive sample is stronger, so this scheme can be used for the preparation of the CASPR2 cell antigen dry slice.

[0176] Example 16

[0177] The method is the same as that in Example 1, except that the stabilizing protective agent is 0.4% v / v glycerol, 0.1% w / v gelatin, 0.2 mM EDTA, and 50 μM resveratrol. The CASPR2 cell antigen wet slice is used as the control group.

[0178] The fluorescence of the cells was observed under a fluorescence microscope, and the test results were obtained by observing whether the two kinds of fluorescence were co-localized. The results are shown in Table 1. Figure 17

[0179] The results show that when the detection sample is a positive sample, the strongest green and red fluorescent cells appear; compared with the CASPR2 cell antigen wet slice Figure 1 , the green and red fluorescence intensity of the positive sample is weaker, so this scheme cannot be used for the preparation of the CASPR2 cell antigen dry slice.

[0180] Example 17

[0181] ​​​The method is the same as example 1, except that the stabilizing protectant is 0.4% v / v glycerol, 0.1% w / v gelatin, 0.3 mM EDTA, 0.01% w / v trehalose. The CASPR2 cell antigen wet slice is used as a control group.

[0182] The fluorescence of the cells is observed under a fluorescence microscope, and photographs are taken. The test results are obtained by observing whether the two kinds of fluorescence are co-localized. The results are shown in Table 1. Figure 18

[0183] The results show that when the detection sample is a positive sample, strong green and red fluorescent cells appear; compared with the CASPR2 cell antigen wet slice, Figure 1 the green and red fluorescence intensity of the positive sample is slightly weaker, so this scheme cannot be used for the preparation of the CASPR2 cell antigen dry slice.

[0184] Example 18

[0185] The method is the same as example 1, except that the stabilizing protectant is 0.4% v / v glycerol, 0.15% w / v gelatin, 0.03% w / v trehalose, 50 μM resveratrol. The CASPR2 cell antigen wet slice is used as a control group.

[0186] The fluorescence of the cells is observed under a fluorescence microscope, and photographs are taken. The test results are obtained by observing whether the two kinds of fluorescence are co-localized. The results are shown in Table 1. Figure 19

[0187] The results show that when the detection sample is a positive sample, strong green and red fluorescent cells appear; compared with the CASPR2 cell antigen wet slice, Figure 1 the green and red fluorescence intensity of the positive sample is slightly weaker, so this scheme cannot be used for the preparation of the CASPR2 cell antigen dry slice.

[0188] Example 19

[0189] The method is the same as example 1, except that the stabilizing protectant is 0.4% v / v glycerol, 0.15% w / v gelatin, 0.1 mM EDTA, 0.02% w / v trehalose. The CASPR2 cell antigen wet slice is used as a control group.

[0190] The fluorescence of the cells is observed under a fluorescence microscope, and photographs are taken. The test results are obtained by observing whether the two kinds of fluorescence are co-localized. The results are shown in Table 1. Figure 20

[0191] The results show that when the detection sample is a positive sample, strong green and red fluorescent cells appear; compared with the CASPR2 cell antigen wet slice, Figure 1 ​​​The green and red fluorescence intensity of the positive sample is slightly weaker than that of the CASPR2 cell antigen wet slice, so this scheme cannot be used for the preparation of the CASPR2 cell antigen dry slice.

[0192] Example 20

[0193] The method is the same as that in Example 1, except that the stabilizing protective agent is 0.4% v / v glycerol, 0.15% w / v gelatin, 0.2 mM EDTA, 0.01% w / v trehalose, and 150 μM resveratrol. The CASPR2 cell antigen wet slice is used as a control group.

[0194] The fluorescence of the cells is observed under a fluorescence microscope, and a photograph is taken. Whether the two types of fluorescence are co-localized is observed to obtain the test result. The results are shown in Table 2. Figure 21

[0195] The results show that when the detection sample is a positive sample, strong green and red fluorescent cells appear; compared with the CASPR2 cell antigen wet slice, Figure 1 the green and red fluorescence intensity of the positive sample is too weak, so this scheme cannot be used for the preparation of the CASPR2 cell antigen dry slice.

[0196] Example 21

[0197] The method is the same as that in Example 1, except that the stabilizing protective agent is 0.4% v / v glycerol, 0.15% w / v gelatin, 0.3 mM EDTA, 100 μM resveratrol. The CASPR2 cell antigen wet slice is used as a control group.

[0198] The fluorescence of the cells is observed under a fluorescence microscope, and a photograph is taken. Whether the two types of fluorescence are co-localized is observed to obtain the test result. The results are shown in Table 2. Figure 22

[0199] The results show that when the detection sample is a positive sample, strong green and red fluorescent cells appear; compared with the CASPR2 cell antigen wet slice, Figure 1 the green and red fluorescence intensity of the positive sample is not ideal, so this scheme cannot be used for the preparation of the CASPR2 cell antigen dry slice.

[0200] Example 22

[0201] ​​In the above scheme, when the stabilizing protectant is 0.4% v / v glycerol, 0.1% w / v gelatin, 0.1 mM EDTA, 0.03% w / v trehalose, and 100 μM resveratrol, it can be used for the preparation of CASPR2 cell antigen dry sheet. To further determine the stability and reliability of this scheme in long-term use scenarios, we carried out an accelerated stability test. The accelerated stability test is based on the assumption that the chemical reactions involved in the deterioration of the material follow the Arrhenius reaction rate function.

[0202] The Arrhenius reaction rate function is as follows:

[0203]

[0204] is the reaction rate constant, is the activation energy (J / mol), R is the gas constant (8.314 J / (mol·K)), T is the absolute temperature, A is the pre-exponential factor (constant)

[0205] According to the above formula, we can calculate the aging time of biological / chemical field (k = 2), that is, 6 weeks (42 days) at 37°C is equivalent to about 413 days of storage at 4°C.

[0206] The accelerated stability test process is as follows:

[0207] (1) Prepare cell antigen dry sheets with 0.4% v / v glycerol, 0.1% w / v gelatin, 0.1 mM EDTA, 0.03% w / v trehalose, and 100 μM resveratrol as stabilizing protectants, and vacuum seal the cell antigen wet sheets of the comparative reagent;

[0208] (2) Place the prepared materials in a constant temperature oven at 37°C for accelerated aging test (for 6 weeks);

[0209] (3) Test once a week, and after each test, the remaining reagent needs to be vacuum sealed.

[0210] The results of the first week to the sixth week of accelerated aging are shown in Figure 23 to Figure 28

[0211] The results show that during the first week to the sixth week of accelerated aging, when the test sample is a positive sample, the strongest green and red fluorescent cells appear; compared with the CASPR2 cell antigen wet sheet, the green and red fluorescent intensity of the positive sample is stronger, so the reliability and stability of the optimal scheme selected by the present application are better than that of the CASPR2 cell antigen wet sheet.​​

[0212] All documents referred to in the present application are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to readers of the application from the teaching provided.

Claims

1. The application of a stabilizer in the preparation of dried cell antigen slides, characterized in that: The stabilizer comprises 0.4% glycerol (volume fraction), 0.1% gelatin (mass volume fraction), 0.1mM EDTA, 0.03% trehalose (mass volume fraction), and 100μM resveratrol.

2. A method for preparing dried cell antigen slices, characterized in that, Includes the following steps: S1. The target cells are plated to obtain cell plates; S2. Wash the cell plate with grown cells 2-3 times with PBS; S3. Fix with 4% paraformaldehyde for 25-30 min, then wash with PBS 2-3 times. S4. After adding the stabilizer, dry in a 37°C drying oven for 2-3 hours to obtain the cell antigen dry sheet; The stabilizer comprises 0.4% glycerol (volume fraction), 0.1% gelatin (mass volume fraction), 0.1mM EDTA, 0.03% trehalose (mass volume fraction), and 100μM resveratrol.

3. The method for preparing a cell antigen dried slide according to claim 2, characterized in that, The target cells are enabled to specifically overexpress antigens by integrating antigen genes, wherein the antigens include one of the following: CASPR2, AQP4, MOG, AQP1, Flotillin1 / 2, PLP1, NMDAR, AMPAR1, AMPAR2, GABABR, DPPX, IgLON5, GlyRα1, D2R, mGluR5, mGluR1, Neurexin-3α, GABAARα1, GABAARβ3, GABAARγ2, GluK2, CaVα2δ, KCTD16, RGS8, ganglionic AChR, SEZ6L2, MuSk, LRP4, ATP1A3, mGluR2, mGluR8, mGluR3, NF155, NF140, NF186, CNTN1, CNTN2, CASPR1, MAG, PLA2R, and THSD7A.

4. A dried cell antigen slide, characterized in that, The cell antigen dry slices were prepared using the method described in any one of claims 2-3.

5. The use of the cell antigen dry slides as described in claim 4 in the preparation of the CBA detection kit.

6. A CBA test kit, characterized in that: The CBA test kit contains the cell antigen dry slide as described in claim 4.

7. The CBA test kit according to claim 6, characterized in that: The kit also includes PBS buffer, HEK293T cells, cell culture microplates, blocking solution, and fluorescent secondary antibody, wherein the blocking solution is serum of the species for which the fluorescent secondary antibody is present.

8. The CBA test kit according to claim 7, characterized in that: The antigen specifically overexpressed on HEK293T cells includes one of the following antigens: CASPR2, AQP4, MOG, AQP1, Flotillin1 / 2, PLP1, NMDAR, AMPAR1, AMPAR2, GABABR, DPPX, IgLON5, GlyRα1, D2R, mGluR5, mGluR1, Neurexin-3α, GABAARα1, GABAARβ3, GABAARγ2, GluK2, CaVα2δ, KCTD16, RGS8, ganglionic AChR, SEZ6L2, MuSk, LRP4, ATP1A3, mGluR2, mGluR8, mGluR3, NF155, NF140, NF186, CNTN1, CNTN2, CASPR1, MAG, PLA2R, and THSD7A.

9. The method of using the CBA test kit according to claim 8 for non-diagnostic purposes, characterized in that, Includes the following steps: S1. HEK293T cells with integrated antigen genes were plated to obtain cell plates; S2. Wash the cell plate with grown cells 2-3 times with PBS; S3. Fix with 4% paraformaldehyde for 25-30 min, then wash with PBS 2-3 times. S4. After adding the stabilizer, dry in a 37℃ drying oven for 2-3 hours to obtain cell antigen dry sheets; S5. Add the sample to be tested to the detection well of the cell antigen dry strip, shake gently to mix, and incubate at 37°C. S6. After incubation, remove and discard the liquid in the detection well, wash thoroughly with PBS buffer, and then discard. S7. Add the fluorescent secondary antibody diluted with the blocking solution to the detection well, shake gently to mix, and incubate at 37°C in the dark. S8. Repeat the washing steps of S6, discard the liquid in the well, add PBS buffer to the detection well until it covers the cells, and then observe the green and red fluorescence of the cells under a fluorescence microscope and take pictures. S9. Observe whether the two fluorescences co-localize and obtain the test results; The stabilizer comprises 0.4% glycerol (volume fraction), 0.1% gelatin (mass volume fraction), 0.1mM EDTA, 0.03% trehalose (mass volume fraction), and 100μM resveratrol.

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