A dry sheet stabilizer, cytoplasmic antigen dry sheets, their preparation method and application

Abstract

This invention belongs to the field of biotechnology and discloses a dry film stabilizer, cytoplasmic antigen dry films, their preparation methods, and applications. The dry film stabilizer includes polyethylene glycol, trehalose, BSA, melatonin, and Tween-20. Cytoplasmic antigen dry films are prepared using this stabilizer and applied in CBA detection. This dry film stabilizer has good permeability, penetrating the cell membrane to enter the cell interior, providing protection both inside and outside the cell. It also possesses moisturizing and antioxidant properties, preventing cell deformation and shedding of the cell fixation layer, effectively maintaining the stability and reliability of intracellular antigen proteins. The prepared cytoplasmic antigen dry films can be stored long-term without damaging the antigenicity of cytoplasmic signal indicators. When detecting positive samples, strong and numerous green filamentous fluorescence signals are observed in the cytoplasm, with consistent green and red fluorescence intensities. Software synthesis reveals co-localization of green and red antigen fluorescence.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a dry sheet stabilizer, cytoplasmic antigen dry sheets, their preparation methods, and applications. Background Technology

[0002] Currently, in commercially available CBA assay kits, cell antigen slides are generally wet slides. This means that after the cell antigen slides in a 96-well plate are fixed with 4% paraformaldehyde, 200 μL of PBS is added to each well as a cell protectant for sealing and preservation. Therefore, wet slides for CBA (Cell-based assay) detection are simple to operate, low in cost, and have superior performance. However, wet slides for CBA detection also have some drawbacks: (1) After adding the protective solution, the cell antigen slides have poor stability and cannot be preserved for a long time; the cells attached to the bottom of the wells will gradually detach. (2) The protective solution easily introduces contaminants, contaminating the antigen slides. Therefore, we use dry cell antigen slides for detection. However, dry cell antigen slides require solutions to problems such as cell deformation, moisture retention, fixation layer detachment, antioxidant properties, and maintaining antigen stability. Summary of the Invention

[0003] To address at least one of the aforementioned problems, this invention provides a dry sheet stabilizer, cytoplasmic antigen dry sheets, their preparation method, and applications. The dry sheet stabilizer combines moisturizing and antioxidant properties, preventing cell deformation and fixation layer detachment. The prepared cytoplasmic antigen dry sheets can better maintain the long-term stability of antigen proteins in the cytoplasm, facilitating their application in CBA detection.

[0004] To achieve the above objectives, the present invention employs the following technical means:

[0005] A first aspect of the present invention provides a dry tablet stabilizer comprising 0.8% by weight / volume of polyethylene glycol, 0.01% by weight / volume of trehalose, 5% by weight / volume of BSA, 2 mM melatonin, and 0.05% by volume of Tween-20.

[0006] A second aspect of the invention provides the use of the dry sheet stabilizer as described in the first aspect in the preparation of a test kit.

[0007] A third aspect of the present invention provides a method for preparing dried cytoplasmic antigen slices, comprising the following steps:

[0008] S1. The target cells are plated to obtain cell plates;

[0009] S2. Wash the cell plate with grown cells 2-3 times with PBS;

[0010] S3. Fix with 4% paraformaldehyde for 25-30 min, then wash with PBS 2-3 times.

[0011] S4. After adding the dry sheet stabilizer described in the first aspect, dry in a drying oven at 37°C for 2-3 hours to obtain cytoplasmic antigen dry sheets.

[0012] In some embodiments of the present invention, the engineered cells are enabled to specifically overexpress the target antigen by integrating the target antigen gene. In some specific embodiments of the present invention, the target antigen is the antigen corresponding to GFAP.

[0013] A fourth aspect of the present invention provides a cytoplasmic antigen dry sheet, which is prepared by the method described in the third aspect.

[0014] The fifth aspect of the present invention provides the use of the cytoplasmic antigen dry strips described in the fourth aspect in the preparation of a CBA detection kit.

[0015] A sixth aspect of the present invention provides a detection kit comprising the dry sheet stabilizer described in the first aspect or the cytoplasmic antigen dry sheet described in the fourth aspect.

[0016] In some embodiments of the present invention, the kit further comprises PBS buffer, engineered 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. In some embodiments of the present invention, the engineered cells are HEK293T cells.

[0017] In some embodiments of the present invention, the engineered cells are enabled to specifically overexpress the target antigen by integrating the target antigen gene, wherein the target antigen is the antigen corresponding to GFAP and the target antigen is a fluorescent protein that emits red light.

[0018] A seventh aspect of the present invention provides a method of using the test kit described in the sixth aspect, comprising the following steps:

[0019] S1. Engineered cells with integrated antigen genes are plated to obtain cell plates;

[0020] S2. Wash the cell plate with grown cells 2-3 times with PBS;

[0021] S3. Fix with 4% paraformaldehyde for 25-30 min, then wash with PBS 2-3 times.

[0022] S4. After adding the dry sheet stabilizer described in the first aspect, dry in a drying oven at 37°C for 2-3 hours to obtain cytoplasmic antigen dry sheets;

[0023] S5. Add a permeabilizing agent to the dried cytoplasmic antigen slide, incubate at room temperature for 10-12 min, discard the liquid, wash with buffer and discard the liquid again.

[0024] S6. Add the sample to be tested to the detection well of the cytoplasmic antigen dry strip, shake gently to mix, and incubate at 37°C.

[0025] S7. After incubation, remove and discard the liquid in the detection well, wash thoroughly with PBS buffer, and then discard.

[0026] S8. 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.

[0027] S9. Repeat the washing steps of S7, 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 in the cytoplasm under a fluorescence microscope and take pictures.

[0028] S10. Observe whether the two fluorescences co-localize and obtain the test results.

[0029] In some embodiments of the present invention, the permeabilizer is 0.25% Triton X-100.

[0030] In some embodiments of the present invention, the incubation time for the above-mentioned incubation step is 30-40 min, preferably 35 min. In some embodiments, the cleaning step involves removing and discarding the liquid in the detection wells after incubation, washing twice with PBS buffer while gently shaking the plate during the washing process, and then placing it on a shaker for a third time for 5 min.

[0031] Furthermore, in some embodiments of the present invention, if the test result is positive and titer verification is required, the sample is diluted according to the ratio and the experimental process of steps S5 to S10 is repeated.

[0032] In some embodiments of the present invention, if the sample to be tested is a serum sample, it needs to be diluted with a blocking solution; if it is a cerebrospinal fluid sample, the undiluted solution is used. In some embodiments of the present invention, the serum sample is diluted 20 times or more with the blocking solution.

[0033] Beneficial effects of the present invention

[0034] Compared with existing technologies, the present invention has the following beneficial effects: The dry sheet stabilizer for cytoplasmic antigen dry sheets for CBA detection provided by the present invention 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 protectant has moisturizing and antioxidant properties, and can also prevent cell deformation and cell fixation layer shedding, and can well maintain the stability and reliability of antigen proteins in the cytoplasm. The cytoplasmic antigen dry sheets prepared using this dry sheet stabilizer can be stored for a long time without damaging the antigenicity of the target cytoplasmic signal. The dry sheet stabilizer requires 0.8% polyethylene glycol, 0.01% trehalose, 5% BSA, 2mM melatonin, and 0.05% Tween-20 by mutual synergy to achieve the best effect: when detecting positive samples, the green filamentous fluorescence signal in the cytoplasm is strong and numerous, and the green and red fluorescence intensities are consistent. After software synthesis, the co-localization of green fluorescence and red antigen fluorescence can be observed. Attached Figure Description

[0035] Figure 1 The following graph shows the fluorescence detection results of positive and negative samples obtained from wet mounts in Example 1 (comparative scheme).

[0036] Figure 2 The following diagram shows the fluorescence detection results of positive and negative samples in Example 1.

[0037] Figure 3 The following diagram shows the fluorescence detection results of positive and negative samples in Example 2.

[0038] Figure 4 The following diagram shows the fluorescence detection results of positive and negative samples in Example 3.

[0039] Figure 5 The following diagram shows the fluorescence detection results of positive and negative samples in Example 4.

[0040] Figure 6 The following diagram shows the fluorescence detection results of positive and negative samples in Example 5.

[0041] Figure 7 The following diagram shows the fluorescence detection results of positive and negative samples in Example 6.

[0042] Figure 8 The following diagram shows the fluorescence detection results of positive and negative samples in Example 7.

[0043] Figure 9 The following diagram shows the fluorescence detection results of positive and negative samples in Example 8.

[0044] Figure 10 The following diagram shows the fluorescence detection results of positive and negative samples in Example 9.

[0045] Figure 11 The following diagram shows the fluorescence detection results of positive and negative samples in Example 10.

[0046] Figure 12 The following diagram shows the fluorescence detection results of positive and negative samples in Example 11.

[0047] Figure 13 The following graph shows the fluorescence detection results of positive and negative samples in Example 12.

[0048] Figure 14 The following diagram shows the fluorescence detection results of positive and negative samples in Example 13.

[0049] Figure 15 The following diagram shows the fluorescence detection results of positive and negative samples in Example 14.

[0050] Figure 16 The following diagram shows the fluorescence detection results of positive and negative samples in Example 15.

[0051] Figure 17 The following diagram shows the fluorescence detection results of positive and negative samples in Example 16.

[0052] Figure 18 The following graph shows the fluorescence detection results of positive and negative samples in Example 17.

[0053] Figure 19 The following diagram shows the fluorescence detection results of positive and negative samples in Example 18.

[0054] Figure 20 The following graph shows the fluorescence detection results of positive and negative samples in Example 19.

[0055] Figure 21 The following graph shows the fluorescence detection results of positive and negative samples in Example 20.

[0056] Figure 22 The following diagram shows the fluorescence detection results of positive and negative samples in Example 21.

[0057] Figure 23 The following graphs show the fluorescence detection results of positive and negative samples obtained after one week of accelerated aging of dry film and wet film in Example 22 and the control scheme;

[0058] Figure 24The graph shows the fluorescence detection results of positive and negative samples obtained after two weeks of accelerated aging of dry film and wet film in Example 22 and the control scheme;

[0059] Figure 25 The graph shows the fluorescence detection results of positive and negative samples obtained after three weeks of accelerated aging of dry film and wet film in Example 22 and the control scheme;

[0060] Figure 26 The graph shows the fluorescence detection results of positive and negative samples obtained after 4 weeks of accelerated aging of dry film and wet film in Example 22 and the control scheme;

[0061] Figure 27 The graph shows the fluorescence detection results of positive and negative samples obtained after 5 weeks of accelerated aging of dry film and wet film in Example 22 and the control scheme;

[0062] Figure 28 The diagram shows the fluorescence detection results of positive and negative samples obtained after 6 weeks of accelerated aging of dry film and wet film in Example 22 and the control scheme. Detailed Implementation

[0063] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0064] 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 invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.

[0065] The technical solution of this application will be further explained in detail below, taking the GFAP antigen as an example and in conjunction with specific embodiments.

[0066] Example 1

[0067] I. Preparation of Cell Plates

[0068] 1. Cell washing: After culturing HEK293T cells with integrated GFAP antigen gene for 48 h, discard the old culture supernatant and add an appropriate amount of physiological saline to wash the cells 1-2 times;

[0069] 2. Cell digestion: Add an appropriate amount of 0.25% trypsin-EDTA solution to the culture dish, gently shake the culture dish to allow the trypsin to fully contact the cells, digest at room temperature, and observe the cell digestion under a microscope. If most of the cells become round and detach, quickly return to the operating table, aspirate the trypsin, and add 2 mL of stop culture medium to terminate the digestion.

[0070] 3. Cell centrifugation: Gently pipette the cells until they are completely detached, then transfer them to a 2 mL centrifuge tube. Centrifuge at 1400 rpm for 6 min, remove the supernatant, add 1 mL of complete culture medium, and gently mix.

[0071] 4. Cell plating: Adjust cell density to 3 × 10⁶ cells / year using complete culture medium. 6 Cells / mL, add 100 μL of cell suspension to each well of a 96-well plate, and incubate at 37°C and 5% CO2 for 12–16 h.

[0072] II. Preparation of Cytoplasmic Antigen Slides

[0073] 1. Wash the 96-well cell plate filled with HEK293T cells (integrated with the GFAP antigen gene) twice with PBS;

[0074] 2. Fix with 4% paraformaldehyde for 30 min;

[0075] 3. Wash twice with PBS;

[0076] 4. Add 50 μL of dry tablet stabilizer: 0.1% w / v polyethylene glycol, and dry in a drying oven at 37℃ for 3 h.

[0077] III. Performance Testing

[0078] (1) Principle of testing

[0079] The detection principle of this embodiment is indirect immunofluorescence. The antibody corresponding to GFAP in the sample forms an antigen-antibody complex with the specific overexpressed antigen expressed on HEK293T cells in the reagent. This antigen carries mCherry fluorescent protein (red fluorescence). Subsequently, the human antibody bound to the antigen is labeled with goat anti-human fluorescent secondary antibody (green fluorescence). Finally, the presence of co-localization between red and green fluorescence is observed using a fluorescence microscope. If co-localization exists, it indicates that the sample contains the antibody; otherwise, the corresponding antibody is not present in the sample.

[0080] (2) Test method

[0081] Preparation:

[0082] 1. Preparation of PBS buffer: Dilute 10 times the amount of PBS buffer with pure water or distilled water.

[0083] 2. Preparation of blocking solution: Dilute goat serum 10 times with PBS buffer;

[0084] 3. Samples: If the sample is serum, dilute it at least 20 times with blocking solution; if the sample is cerebrospinal fluid, use the undiluted solution.

[0085] 4. Fluorescent secondary antibody: Dilute 1500 times with blocking solution.

[0086] Inspection steps:

[0087] 1. Permeability testing: Add 80 µL of 0.25% Triton X-100 to the detection wells of the cytoplasmic antigen dry slide, incubate at room temperature for 10 min, discard the liquid in the wells, then add 200 µL of 1×PBS buffer to wash once, and discard the liquid in the wells.

[0088] 2. Sample addition: Add 80 µL of diluted sample to the detection well, gently shake to mix, and incubate at 37°C for 35 min;

[0089] 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.

[0090] 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.

[0091] 5. Cleaning: Repeat step 3;

[0092] 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 cell fluorescence under a fluorescence microscope and take pictures.

[0093] 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.

[0094] (3) Detection indicators

[0095] GFAP index of cytoplasmic signaling.

[0096] (4) Comparison of schemes

[0097] The preparation and testing methods for wet slides of cytoplasmic signaling GFAP index are as follows:

[0098] A. Fixation method

[0099] 1. Prepare 4% paraformaldehyde solution using PBS;

[0100] 2. Wash the 96-well cell culture microplates filled with HEK293T cells (integrated with the GFAP antigen gene) twice with PBS;

[0101] 3. Fix with 4% paraformaldehyde for 30 min;

[0102] 4. Wash twice with PBS;

[0103] 5. Add 200 μL of PBS as a cell protectant and store under vacuum;

[0104] B. Inspection steps:

[0105] 1. Pre-wash: Remove the cell culture microplate and discard the pre-filled liquid in the wells. Add 200 µL of 1×PBS buffer and wash once, then discard the liquid in the wells again.

[0106] 2. Permeability testing: Add 80 µL of 0.25% Triton X-100 to the wells of the cell culture microplate, incubate at room temperature for 10 min, discard the liquid in the wells, then add 200 µL of 1×PBS buffer to wash once, and discard the liquid in the wells again.

[0107] 3. Sample addition: Add 80 µL of diluted serum to the test well. Gently shake to mix, and incubate at 37°C for 35 min;

[0108] 4. Washing: After incubation, remove the plate, discard the liquid in the wells, and add 200 µL of 1×PBS buffer to wash twice. Gently shake the plate during the washing process. For the third wash, place the plate on a shaker and wash for 5 min.

[0109] 5. 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.

[0110] 6. Cleaning: Repeat step 4;

[0111] 7. Observation: Discard the liquid in the well, add 100 µL of 1×PBS buffer to the sample well to cover the cells, and then observe the cell fluorescence under a fluorescence microscope and take pictures.

[0112] 8. 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 8.

[0113] (5) Comparison of test samples

[0114] Both positive and negative samples were clinical samples, and were validated using the human anti-glial fibrillary acidic protein antibody (Anti-GFAP) enzyme-linked immunosorbent assay kit (catalog number SB-EH3694) from Shanghai Shenger Biotechnology Co., Ltd.

[0115] (6) Result interpretation

[0116] Criteria for interpreting negative and positive samples:

[0117] Negative: No green filamentous fluorescence was observed in the cytoplasm under the green fluorescence channel, or no overlap between the green filamentous fluorescence and the red antigen fluorescence was observed after software synthesis.

[0118] Positive: Under the green fluorescence channel, green filamentous fluorescence can be seen in the cytoplasm, and it can be observed to overlap with the red antigen fluorescence after software synthesis.

[0119] 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.

[0120] 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.

[0121] The results showed that GFAP cell antigen dry slides treated with 0.1% w / v polyethylene glycol exhibited slightly shrunken cell morphology. Under the red fluorescence channel, the background antigen showed weak red fluorescence, and under the green fluorescence channel, the green filamentous fluorescence signal in the cytoplasm of positive samples was weak and few in number. Compared with GFAP cell antigen wet slides, the effect was worse. Therefore, 0.1% w / v polyethylene glycol cannot be used to prepare GFAP cell antigen dry slides because the polyethylene glycol concentration is too low to form a protective film on its surface, resulting in damage to the protein structure.

[0122] Example 2

[0123] The method is the same as in Example 1, except that the dry sheet stabilizer is 0.2% w / v polyethylene glycol.

[0124] 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 3 As shown.

[0125] The results showed that GFAP cell antigen dry slides treated with 0.2% w / v polyethylene glycol had normal cell morphology. Under the red fluorescence channel, the background antigen showed weak red fluorescence, and under the green fluorescence channel, the green filamentous fluorescence signal in the cytoplasm of positive samples was weak and few in number. Compared with GFAP cell antigen wet slides, the effect was worse. Therefore, 0.2% w / v polyethylene glycol cannot be used to prepare GFAP cell antigen dry slides because the polyethylene glycol concentration is too low to form a protective film on the surface, resulting in damage to the protein structure.

[0126] Example 3

[0127] The method is the same as in Example 1, except that the dry sheet stabilizer is 0.5% w / v polyethylene glycol.

[0128] 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 4 As shown.

[0129] The results showed that GFAP cell antigen dry slides treated with 0.5% w / v polyethylene glycol had normal cell morphology. Under the red fluorescence channel, the background antigen showed moderate to strong red fluorescence. Under the green fluorescence channel, the positive samples showed weak green filamentous fluorescence signals in the cytoplasm, but the number of cells was large. Compared with GFAP cell antigen wet slides, the effect was slightly worse. Therefore, 0.5% w / v polyethylene glycol cannot be used to prepare GFAP cell antigen dry slides because the polyethylene glycol concentration is too low and cannot form a protective film on its surface, resulting in damage to the protein structure.

[0130] Example 4

[0131] The method is the same as in Example 1, except that the dry sheet stabilizer is 0.8% w / v polyethylene glycol.

[0132] 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 5 As shown.

[0133] The results showed that the GFAP cell antigen dry slides treated with 0.8% w / v polyethylene glycol had normal cell morphology. Under the red fluorescence channel, the background antigen showed strong red fluorescence, and under the green fluorescence channel, the positive samples showed strong green filamentous fluorescence signals in the cytoplasm and a large number of cells. The effect was similar to that of the GFAP cell antigen wet slides. Therefore, 0.8% w / v polyethylene glycol can be used as the basis for the preparation of GFAP cell antigen dry slides.

[0134] Based on this, we will design experiments to introduce chemical substances with properties such as moisturizing, preventing cell deformation, preventing fixation layer shedding, maintaining antigen protein stability, and anti-oxidation, in order to obtain a dry sheet stabilizer with performance close to or equal to that of wet sheet and which is universal.

[0135] Example 5

[0136] The method is the same as in Example 1, except that the dry sheet stabilizer is 1% w / v polyethylene glycol.

[0137] 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 6 As shown.

[0138] The results showed that GFAP cell antigen dry slides treated with 1% w / v polyethylene glycol had normal cell morphology. Under the red fluorescence channel, the red fluorescence intensity of the background antigen was weak. Under the green fluorescence channel, the green filamentous fluorescence signal in the cytoplasm of positive samples was weak and few in number. Compared with GFAP cell antigen wet slides, the effect was worse. Therefore, 1% w / v polyethylene glycol cannot be used to prepare GFAP cell antigen dry slides because the polyethylene glycol concentration is too high, which may produce excessive osmotic pressure shock and toxicity to cells.

[0139] Example 6

[0140] The method is the same as in Example 1, except that the dry sheet stabilizer is 2% w / v polyethylene glycol.

[0141] 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 7 As shown.

[0142] The results showed that GFAP cell antigen dry slides treated with 2% w / v polyethylene glycol exhibited slightly shrunken cell morphology. Under the red fluorescence channel, the red fluorescence intensity of the background antigen was weak. Under the green fluorescence channel, the green filamentous fluorescence signal in the cytoplasm of positive samples was weak and few in number. Compared with GFAP cell antigen wet slides, the effect was worse. Therefore, 2% w / v polyethylene glycol cannot be used for the preparation of GFAP cell antigen dry slides because the polyethylene glycol concentration is too high, which may produce excessive osmotic pressure shock and toxicity to the cells.

[0143] From the above Figures 1 to 7 The results show that the cell antigen dry slides prepared with 0.8% w / v polyethylene glycol as a dry slide stabilizer exhibit the best detection performance for the GFAP index.

[0144] Depend on Figure 5It can be seen that the GFAP cell antigen dry slides prepared by using 0.8% w / v polyethylene glycol as a dry slide stabilizer still have certain differences compared with the GFAP cell antigen wet slides: the green filamentous fluorescence signal in the cytoplasm and the red fluorescence signal of the background antigen in the dry slides are not ideal, and the background of the dry slides is stronger than that of the wet slides.

[0145] Therefore, we still need to find a stabilizer formulation based on 0.8% w / v polyethylene glycol that can not only maintain the stability of the antigen protein and resist oxidation from air, but also eliminate the background caused by the dried sheet.

[0146] Example 7

[0147] The method was the same as in Example 4, except that the dry sheet stabilizer was 0.8% w / v polyethylene glycol, 1% w / v BSA, 1mM melatonin, and 0.05% v / v Tween-20. GFAP cell antigen wet sheets were used as the control group.

[0148] 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 8 As shown.

[0149] The results showed that the cells in the GFAP cell antigen dry slides treated with this protocol had normal cell morphology. Under the red fluorescence channel, the background antigen exhibited weak red fluorescence, while under the green fluorescence channel, the cell background was strong. In positive samples, the green filamentous fluorescence signal in the cytoplasm was weak and few in number, resulting in poorer performance compared to GFAP cell antigen wet slides. Therefore, this protocol cannot be used for the preparation of GFAP cell antigen dry slides.

[0150] Example 8

[0151] The method was the same as in Example 1, except that the dry sheet stabilizer was 0.8% w / v polyethylene glycol, 2% w / v BSA, 2mM melatonin, and 0.08% v / v Tween-20. GFAP cell antigen wet sheets were used as the control group.

[0152] 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 9 As shown.

[0153] The results showed that the GFAP cell antigen dry slides treated with this protocol exhibited normal cell morphology. Under the red fluorescence channel, the background antigen showed weak red fluorescence, while under the green fluorescence channel, the cell background was weak. In positive samples, the green filamentous fluorescence signal in the cytoplasm was weak and few in number, resulting in poorer performance compared to GFAP cell antigen wet slides. Therefore, this protocol cannot be used for the preparation of GFAP cell antigen dry slides.

[0154] Example 9

[0155] The method was the same as in Example 1, except that the dry sheet stabilizer was 0.8% w / v polyethylene glycol, 5% w / v BSA, 3mM melatonin, and 0.1% v / v Tween-20. GFAP cell antigen wet sheets were used as the control group.

[0156] 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.

[0157] The results showed that the GFAP cell antigen dry slides treated with this method had shrunken cell morphology, weak red fluorescence of the background antigen under the red fluorescence channel, weak cell background under the green fluorescence channel, and weak and fewer green filamentous fluorescence signals in the cytoplasm of positive samples. Compared with GFAP cell antigen wet slides, the effect was poor. Therefore, this method cannot be used for the preparation of GFAP cell antigen dry slides.

[0158] Example 10

[0159] The method was the same as in Example 1, except that: 0.8% w / v polyethylene glycol, 0.01% w / v trehalose, 1 mM melatonin, and 0.08% v / v Tween-20 were used. GFAP cell antigen wet mounts were used as the control group.

[0160] 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.

[0161] The results showed that the GFAP cell antigen dry slides treated with this method had normal cell morphology. Under the red fluorescence channel, the background antigen showed medium to strong red fluorescence, while under the green fluorescence channel, the cell background was strong. In the positive samples, the green filamentous fluorescence signal in the cytoplasm was medium to strong and numerous. Compared with GFAP cell antigen wet slides, the effect was slightly worse. Therefore, this method cannot be used for the preparation of GFAP cell antigen dry slides.

[0162] Example 11

[0163] The method was the same as in Example 1, except that the dry sheet stabilizer consisted of 0.8% w / v polyethylene glycol, 0.01% w / v trehalose, 1% w / v BSA, and 0.1% v / v Tween-20. GFAP cell antigen wet sheets served as the control group.

[0164] 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.

[0165] The results showed that the GFAP cell antigen dry slides treated with this method had normal cell morphology. Under the red fluorescence channel, the background antigen red fluorescence was weak, and under the green fluorescence channel, the cell background was strong. In the positive samples, the green filamentous fluorescence signal in the cytoplasm was weak and the number was small. Compared with GFAP cell antigen wet slides, the effect was poor. Therefore, this method cannot be used for the preparation of GFAP cell antigen dry slides.

[0166] Example 12

[0167] The method was the same as in Example 1, except that the dry sheet stabilizer consisted of 0.8% w / v polyethylene glycol, 0.01% w / v trehalose, 2% w / v BSA, and 3mM melatonin. GFAP cell antigen wet sheets served as the control group.

[0168] 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 13 As shown.

[0169] The results showed that the GFAP cell antigen dry slides treated with this method had normal cell morphology. Under the red fluorescence channel, the background antigen showed medium to strong red fluorescence, while under the green fluorescence channel, the cell background was weak. In the positive samples, the green filamentous fluorescence signal in the cytoplasm was medium to strong and numerous. Compared with GFAP cell antigen wet slides, the effect was slightly worse. Therefore, this method cannot be used for the preparation of GFAP cell antigen dry slides.

[0170] Example 13

[0171] The method was the same as in Example 1, except that the dry sheet stabilizer consisted of 0.8% w / v polyethylene glycol, 0.01% w / v trehalose, 5% w / v BSA, 2 mM melatonin, and 0.05% v / v Tween-20. GFAP cell antigen wet sheets served as the control group.

[0172] 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 14 As shown.

[0173] The results showed that the GFAP cell antigen dry slides treated with this method had normal cell morphology, strong red fluorescence of the background antigen under the red fluorescence channel, and weak cell background under the green fluorescence channel. In the positive samples, the green filamentous fluorescence signal in the cytoplasm was strong and numerous. The effect was basically the same as that of GFAP cell antigen wet slides. Therefore, this method can be used to prepare GFAP cell antigen dry slides.

[0174] Example 14

[0175] The method was the same as in Example 1, except that the dry sheet stabilizer was 0.8% w / v polyethylene glycol, 0.2% w / v trehalose, 2 mM melatonin, and 0.1% v / v Tween-20. GFAP cell antigen wet sheets were used as the control group.

[0176] 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 15 As shown.

[0177] The results showed that the GFAP cell antigen dry slides treated with this method had normal cell morphology. Under the red fluorescence channel, the background antigen showed medium to strong red fluorescence, while under the green fluorescence channel, the cell background was strong. In the positive samples, the green filamentous fluorescence signal in the cytoplasm was medium to strong and numerous. Compared with GFAP cell antigen wet slides, the effect was slightly worse. Therefore, this method cannot be used for the preparation of GFAP cell antigen dry slides.

[0178] Example 15

[0179] The method was the same as in Example 1, except that the dry sheet stabilizer consisted of 0.8% w / v polyethylene glycol, 0.2% w / v trehalose, 1% w / v BSA, 3mM melatonin, and 0.08% v / v Tween-20. GFAP cell antigen wet sheets served as the control group.

[0180] 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 16 As shown.

[0181] The results showed that the GFAP cell antigen dry slides treated with this method had normal cell morphology. Under the red fluorescence channel, the background antigen red fluorescence was weak, and under the green fluorescence channel, the cell background was strong. In the positive samples, the green filamentous fluorescence signal in the cytoplasm was weak and the number was small. Compared with GFAP cell antigen wet slides, the effect was poor. Therefore, this method cannot be used for the preparation of GFAP cell antigen dry slides.

[0182] Example 16

[0183] The method was the same as in Example 1, except that the dry sheet stabilizer consisted of 0.8% w / v polyethylene glycol, 0.2% w / v trehalose, 2% w / v BSA, and 0.05% v / v Tween-20. GFAP cell antigen wet sheets served as the control group.

[0184] 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 17 As shown.

[0185] The results showed that the GFAP cell antigen dry slides treated with this method had normal cell morphology. Under the red fluorescence channel, the background antigen showed medium to strong red fluorescence, while under the green fluorescence channel, the cell background was weak. In the positive samples, the green filamentous fluorescence signal in the cytoplasm was medium to strong and numerous. Compared with GFAP cell antigen wet slides, the effect was slightly worse. Therefore, this method cannot be used for the preparation of GFAP cell antigen dry slides.

[0186] Example 17

[0187] The method was the same as in Example 1, except that the dry sheet stabilizer consisted of 0.8% w / v polyethylene glycol, 0.2% w / v trehalose, 5% w / v BSA, and 1 mM melatonin. GFAP cell antigen wet sheets served as the control group.

[0188] 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 18 As shown.

[0189] The results showed that the GFAP cell antigen dry slides treated with this method had normal cell morphology. Under the red fluorescence channel, the background antigen red fluorescence was weak, and under the green fluorescence channel, the cell background was weak. In the positive samples, the green filamentous fluorescence signal in the cytoplasm was weak and the number was small. Compared with GFAP cell antigen wet slides, the effect was poor. Therefore, this method cannot be used for the preparation of GFAP cell antigen dry slides.

[0190] Example 18

[0191] The method was the same as in Example 1, except that the dry sheet stabilizer was 0.8% w / v polyethylene glycol, 0.3% w / v trehalose, 3mM melatonin, and 0.05% v / v Tween-20. GFAP cell antigen wet sheets were used as the control group.

[0192] 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 19 As shown.

[0193] The results showed that the GFAP cell antigen dry slides treated with this method had shrunken cell morphology, weak red fluorescence of the background antigen under the red fluorescence channel, and strong cell background under the green fluorescence channel. In the positive samples, the green filamentous fluorescence signal in the cytoplasm was weak and few in number. Compared with GFAP cell antigen wet slides, the effect was poor. Therefore, this method cannot be used for the preparation of GFAP cell antigen dry slides.

[0194] Example 19

[0195] The method was the same as in Example 1, except that the dry sheet stabilizer consisted of 0.8% w / v polyethylene glycol, 0.3% w / v trehalose, 1% w / v BSA, and 2 mM melatonin. GFAP cell antigen wet sheets served as the control group.

[0196] 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 20 As shown.

[0197] The results showed that the GFAP cell antigen dry slides treated with this method had normal cell morphology. Under the red fluorescence channel, the background antigen showed medium to strong red fluorescence, while under the green fluorescence channel, the cell background was strong. In the positive samples, the green filamentous fluorescence signal in the cytoplasm was medium to strong and numerous. Compared with GFAP cell antigen wet slides, the effect was slightly worse. Therefore, this method cannot be used for the preparation of GFAP cell antigen dry slides.

[0198] Example 20

[0199] The method was the same as in Example 1, except that the dry sheet stabilizer consisted of 0.8% w / v polyethylene glycol, 0.3% w / v trehalose, 2% w / v BSA, 1 mM melatonin, and 0.1% v / v Tween-20. GFAP cell antigen wet sheets served as the control group.

[0200] 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 21 As shown.

[0201] The results showed that the GFAP cell antigen dry slides treated with this method had normal cell morphology. Under the red fluorescence channel, the background antigen showed medium to strong red fluorescence, while under the green fluorescence channel, the cell background was weak. In the positive samples, the green filamentous fluorescence signal in the cytoplasm was medium to strong and numerous. Compared with GFAP cell antigen wet slides, the effect was slightly worse. Therefore, this method cannot be used for the preparation of GFAP cell antigen dry slides.

[0202] Example 21

[0203] The method was the same as in Example 1, except that the dry sheet stabilizer consisted of 0.8% w / v polyethylene glycol, 0.3% w / v trehalose, 5% w / v BSA, and 0.08% v / v Tween-20. GFAP cell antigen wet sheets served as the control group.

[0204] 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 22 As shown.

[0205] The results showed that the GFAP cell antigen dry slides treated with this method had normal cell morphology. Under the red fluorescence channel, the background antigen showed medium to strong red fluorescence, while under the green fluorescence channel, the cell background was weak. In the positive samples, the green filamentous fluorescence signal in the cytoplasm was medium to strong and numerous. Compared with GFAP cell antigen wet slides, the effect was slightly worse. Therefore, this method cannot be used for the preparation of GFAP cell antigen dry slides.

[0206] Example 22

[0207] In the above scheme, when the dry sheet stabilizer is 0.8% w / v polyethylene glycol, 0.01% w / v trehalose, 5% w / v BSA, 2mM melatonin, and 0.05% v / v Tween-20, it can be used to prepare GFAP cell antigen dry sheets. To further determine the stability and reliability of this scheme under long-term use, we conducted accelerated stability tests. The accelerated stability test design is based on the assumption that the chemical reactions involved in material deterioration follow the Arrhenius reaction rate function.

[0208] The Arrhenius reaction rate function is as follows:

[0209]

[0210] Where k 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, and A is the pre-exponential factor (constant).

[0211] Based on the above formula, we can calculate the biological / chemical fields ( =2) The aging time, that is, 6 weeks (42 days) of treatment at 37°C is equivalent to about 413 days of storage at 4°C.

[0212] Our ideal shelf life is one year. If Example 13 still exhibits performance superior to or equal to that of the corresponding wet tablets within a 6-week aging period, then our dry tablet stabilizer can be considered successful.

[0213] The accelerated stability test process is as follows:

[0214] (1) Cell antigen dry sheets and contrast reagent cell antigen wet sheets were prepared by vacuum sealing with 0.8% w / v polyethylene glycol, 0.01% w / v trehalose, 5% w / v BSA, 2mM melatonin and 0.05% v / v Tween-20 as dry sheet stabilizers;

[0215] (2) The prepared material was placed in a constant temperature chamber at 37°C for accelerated aging test (lasting 6 weeks);

[0216] (3) Test once a week. After each test, the remaining reagents need to be vacuum sealed.

[0217] Results of accelerated aging from week 1 to week 6 are as follows Figures 23 to 28 As shown.

[0218] The results showed that during the accelerated aging period from week 1 to week 3, cell morphology was normal, background antigen showed strong red fluorescence under the red fluorescence channel, and weak cell background under the green fluorescence channel. In positive samples, the green filamentous fluorescence signal in the cytoplasm was strong and numerous. Compared with GFAP cell antigen wet mounts, the green and red fluorescence intensities of positive samples were consistent, and the reliability and stability of the protocol were equivalent to those of GFAP cell antigen wet mounts.

[0219] During the accelerated aging period from week 4 to week 6, cell morphology was normal. Under the red fluorescence channel, the background antigen exhibited strong red fluorescence, while under the green fluorescence channel, the cellular background was weak. In positive samples, the cytoplasmic green filamentous fluorescence signal was strong and abundant. Compared with GFAP cell antigen wet mounts, the green and red fluorescence intensities of positive samples were stronger, and the reliability and stability of the protocol were superior to those of GFAP cell antigen wet mounts.

[0220] Therefore, the optimal solution selected in this application has better reliability and long-term stability than GFAP cell antigen wet mount.

[0221] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. Use of a dry sheet stabilizer in the preparation of a cytoplasmic antigen dry sheet, characterized in that: The dry slice stabilizer comprises 0.8% polyethylene glycol by mass volume, 0.01% trehalose by mass volume, 5% BSA by mass volume, 2mM melatonin, and 0.05% Tween-20 by volume.

2. A method of preparing a cytoplasmic antigen dry sheet, characterized by, The method comprises the following steps: S1, using the engineering cell to plate to obtain a cell plate; S2, washing the cell plate with PBS for 2-3 times; S3, fixing with 4% paraformaldehyde for 25-30 min and then washing with PBS for 2-3 times; S4, adding a dry slice stabilizer and drying in a 37℃ drying box for 2-3 h to obtain the cytoplasmic antigen dry slice; the dry slice stabilizer comprises 0.8% polyethylene glycol by mass volume, 0.01% trehalose by mass volume, 5% BSA by mass volume, 2mM melatonin, and 0.05% Tween-20 by volume.

3. The method for preparing a dried cytoplasmic antigen slide according to claim 2, characterized in that, The engineering cell can specifically overexpress the target antigen by integrating the target antigen gene.

4. A cytoplasmic antigen dry sheet, characterized by, The cytoplasmic antigen dry slice is prepared by the method in any one of claims 2-3.

5. The cytoplasmic antigen dry slice in claim 4 is used in the preparation of a CBA detection kit.

6. A CBA assay kit characterized in that: The CBA detection kit comprises the cytoplasmic antigen dry slice in claim 4.

7. The CBA assay kit of claim 6, wherein: The kit further comprises PBS buffer, engineering cell, cell culture microplate, blocking solution, and fluorescent secondary antibody, wherein the blocking solution is serum of the species in which the fluorescent secondary antibody is located.

8. The CBA assay kit of claim 7, wherein: The engineering cell can specifically overexpress the target antigen by integrating the target antigen gene.

9. The method of using the CBA assay kit of claim 8, characterized in that, The method is a method for non-disease diagnosis purposes, comprising the following steps: S1, using the engineering cell integrated with the target antigen gene to plate to obtain a cell plate; S2, washing the cell plate with PBS for 2-3 times; S3, fixing with 4% paraformaldehyde for 25-30 min and then washing with PBS for 2-3 times; S4, adding a dry slice stabilizer and drying in a 37℃ drying box for 2-3 h to obtain the cytoplasmic antigen dry slice; S5, adding a permeabilization agent to the cytoplasmic antigen dry slice, incubating at room temperature for 10-12 min, discarding the liquid and then washing with buffer once and discarding the liquid; S6, adding the sample to be tested to the detection well of the cytoplasmic antigen dry slice, mixing gently, and incubating at 37℃; S7, after incubation, discarding the liquid in the detection well, washing completely with PBS buffer and discarding; S8, adding the fluorescent secondary antibody diluted with the blocking solution to the detection well, mixing gently, and incubating at 37℃ in the dark; S9, repeating the washing step S7, discarding the liquid in the well, adding PBS buffer to the detection well to cover the cells, and then observing the green fluorescence and red fluorescence in the cytoplasm under a fluorescence microscope and taking a photo; S10, observing whether the two kinds of fluorescence are co-localized and obtaining a test result; The dry slice stabilizer comprises 0.8% polyethylene glycol by mass volume, 0.01% trehalose by mass volume, 5% BSA by mass volume, 2mM melatonin, and 0.05% Tween-20 by volume.

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