Cell adherence promoting agent, method for realizing rapid cell planking by using cell adherence promoting agent and application of cell adherence promoting agent

Abstract

The invention discloses a cell adherence promoting agent, a method for realizing rapid cell planking by using the cell adherence promoting agent and application of the cell adherence promoting agent. The rapid planking method comprises the following steps: pretreating a cell pore plate by using the adherence promoting agent, adding target cells obtained by resuscitation and resuspension into the pretreated cell pore plate, and incubating in a water bath at 35-37 DEG C for 1-1.2 hours to finish planking; the adhesion promoting agent is sericin. By adopting the method, the adherence rate of the target cell GABABR-HEK293T can reach 80% or above, even a pore plate treated by 1mu g / mL sericin is adopted, the cell does not fall off, the complete adherence is achieved, the total time is about 2.5 hours when the method is used for CBA detection, the time is consumed by 121 hours compared with the conventional CBA detection experiment process, the time is saved by nearly 5 days, and the labor and time cost is greatly reduced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a cell adhesion promoting agent, a method for realizing rapid cell plating using the same, and an application thereof. Background Art

[0002] HEK293T cells are one of the most widely used derivatives of HEK293 cells. They are created by integrating a temperature-sensitive SV40T antigen mutant into the original HEK293 cell genome. Due to its high transfection efficiency, easy culture and amplification, rapid growth, and strong adaptability, it is often used in the expression of exogenous genes. However, it has poor adhesion and is easy to fall off, which is not conducive to experimental operations.

[0003] Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system and plays a key role in regulating neuronal activity. Gamma-aminobutyric acid type B receptors (GABABR) are widely distributed in the central nervous system, including the cerebral cortex, hippocampus, cerebellum and thalamus. GABABR belongs to inhibitory synaptic proteins in neurons and plays an important role in neurotransmitter transmission and synaptic plasticity.

[0004] Patients with anti-GABABR encephalitis usually present subacutely, with an age range of 18 to 85 years, most commonly in the 60s to 70s, and there are also reports of 3-year-old children with no gender difference in onset. Most of the clinical manifestations of anti-GABABR encephalitis are similar to those of limbic encephalitis, with epileptic seizures, memory loss, mental disorders, and impaired consciousness as the main manifestations. Detection of GABABR antibodies is a specific indicator for diagnosing anti-GABABR encephalitis, and the simple, rapid, and intuitive immunofluorescence method is often used for autoantibody detection. Summary of the invention

[0005] In order to solve at least one of the above problems, the present invention provides a cell adhesion promoter, a method for achieving rapid cell plating using the same, and an application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides the use of sericin in preparing a cell adhesion promoting agent.

[0007] In some embodiments of the present invention, the concentration of sericin is 0.1-10 μg / mL. In a preferred embodiment of the present invention, the concentration of sericin is 1 μg / mL.

[0008] The second aspect of the present invention provides a method for rapid cell plating, wherein the cell plate is pretreated with an adhesion promoter, and the target cells are plated at a rate of (2-3)×10 5The inoculation amount of cells / well is added to the pretreated cell well plate, and the plate is incubated in a 35-37° C. water bath for 1-1.2 h to complete the plating; the adhesion promoting agent is sericin.

[0009] In some embodiments of the present invention, the pretreatment method is: cover the bottom of the well with an adhesion promoter solution, incubate in a 35-37° C. saturated humidity incubator for 1-1.2 h, wash with sterile saline and then air-dry.

[0010] In some embodiments of the present invention, the concentration of the sericin is 0.1-10 μg / mL. In a preferred embodiment of the present invention, the concentration of the sericin is 1 μg / mL.

[0011] The third aspect of the present invention provides a cell plate prepared according to the method described in the first aspect. In some specific embodiments of the present invention, the target cells are GABABR-HEK293T cells.

[0012] The fourth aspect of the present invention provides the use of the cell plate prepared according to the method described in the first aspect in preparing a cell immunofluorescence detection kit.

[0013] The fifth aspect of the present invention provides an anti-GABABR encephalitis detection kit, comprising the cell adhesion promoter described in the first aspect or the GABABR-HEK293T cell plate prepared by the method described in the second aspect.

[0014] Beneficial Effects of the Invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a cell adhesion promoter, a method and application of using the same to achieve rapid cell plating, and the cell well plate treated with the adhesion promoter has a density of (2-3)×10 5 Cells were added to each well and incubated in a 35-37℃ water bath for 1-1.2 h to complete the plating. The plated cells were arranged evenly, without overlapping, and were stretched and oval in shape. Not only could the GABABR-HEK293T cell adhesion rate be maintained at more than 80%, but even with the well plate treated with 1μg / mL sericin, the cells did not fall off and were completely adhered to the wall. The improved plating method was used for CBA detection. The operation steps in the detection method were simple and convenient, and the detection cycle was short. The total time was saved to 2.5 h, which saved nearly 5 days compared with the conventional CBA detection experiment process that took 121 h, greatly shortening the detection cycle, which was conducive to accelerating the progress of related research, saving time costs and reducing manpower consumption.

[0015] In the scheme of the present invention, the cell well plate is arranged according to (2-3)×10 5Cells are added to each well for incubation, and the number of cells just fills the well. The cell adhesion rate will not be affected by too few cells; nor will the effect of CBA be affected by too many or too few cells: if there are too many adherent cells on the cell plate, the cell density is too high, and the cell spacing is shortened, it may cause interference and overlap of the detected fluorescence signals. If the density is too high, the cells will compete for limited nutrients and growth space, causing some cells to grow poorly or die, and reducing the cell reactivity and activity during CBA detection. At the same time, too high cell concentration may also increase the nonspecific binding of the detection reagent, bringing additional signal interference; if there are too few cells, the detection signal will be weak and the heterogeneity of individual cells will increase, affecting the entire detection result. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The bright field microscopy results of the cell adhesion effect obtained in Example 1 of the present invention are shown; Figure 2 The bright field microscopy results of the cell adhesion effect obtained in Example 2 of the present invention are shown; Figure 3 The bright field microscopy results of the cell adhesion effect obtained in Example 3 of the present invention are shown; Figure 4 The bright field microscopy results of the cell adhesion effect obtained in Example 4 of the present invention are shown; Figure 5 The bright field microscopy results of the cell adhesion effect obtained in Example 5 of the present invention are shown; Figure 6 The bright field microscopy results of the cell adhesion effect obtained in Comparative Example 1 of the present invention are shown; Figure 7 The time consumption comparison results of the experimental process of Example 1 of the present invention and Comparative Example 1 are shown; Figure 8 The result diagram showing the CBA detection performance comparison between the preferred scheme in Example 4 of the present invention and the conventional method in Comparative Example 1 is shown. DETAILED DESCRIPTION

[0017] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0018] 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 the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0019] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0020] Example 1 Study on Poly-L-Lysine as Adhesion Promoter In this example, poly-L-lysine was used as an adhesion promoter to investigate the adhesion of GABABR-HEK293T cells.

[0021] 1. Principle Poly-L-lysine is a non-specific cell adhesion factor that promotes the adhesion of cells to solid matrices by enhancing the electrostatic interaction between negatively charged ions on the cell membrane surface and the culture surface.

[0022] 2. Preparation of adhesion promoter The poly-L-lysine powder was first re-dissolved with sterile deionized water, and the gelatin solution was filtered and sterilized using a 0.22 μm filter. It was then diluted with sterile deionized water to obtain 8 concentration gradients of 0 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, and 1 mg / mL, respectively. The number of cells before acetone treatment was used as the initial cell amount for comparison with the results after the experiment.

[0023] 3. Cell plate pretreatment Add 50 μL of adhesion promoters with different concentration gradients to the reaction wells, tap the well plate to ensure that the solution covers the bottom of the wells, incubate in a 37°C saturated humidity incubator for 1 h, add 150 μL of sterile saline to wash three times, and dry for later use.

[0024] 4. Cell recovery (1) Thawing: Preheat the DMEM culture medium and water bath to 37°C. Quickly take out a tube of GABABR-HEK293T cells from the liquid nitrogen tank and quickly place it in the water bath (quickly check whether the lid is tightened before placing it in the water bath. Do not immerse the lid in water). Shake it quickly to melt it quickly until only a small ice core remains.

[0025] (2) Resuspension: Aspirate the cells in the tube and add 1 mL of DMEM to complete the culture. Gently pipette to mix.

[0026] (3) Centrifugation: Centrifuge at 1500 rpm for 5 min.

[0027] (4) Resuspend: Carefully remove the supernatant, add 1 mL of DMEM and mix by gently pipetting.

[0028] 5. Cell incubation According to 2×10 5 The inoculum of 10 cells / well was added to 96-well plates treated with different concentrations of adhesion promoters and incubated in a 37°C water bath for 1 h.

[0029] 6. Cell Fixation (1) PBS washing: Discard the cell incubation solution and gently add 200 μL PBS buffer to the reaction wells for washing once.

[0030] (2) Acetone fixation: Add 50 μL of pre-cooled acetone and fix at -20°C for 5 min.

[0031] (3) PBS washing: Discard the fixative solution and add 200 μL PBS buffer to the reaction wells for washing three times.

[0032] 7. Microscopic examination The fixed 96-well plate was placed under a microscope to observe the adhesion effect of 8 concentration gradients. The bright field photos are shown in the figure. Figure 1 shown.

[0033] The results showed that compared with the initial cell amount, after acetone fixation, all the cells in the control group without poly-L-lysine were detached, while the cells in the poly-L-lysine-treated wells still remained, about 20-50%. This shows that poly-L-lysine has a promoting effect on cell adhesion, but the effect is not obvious. In the different concentration gradients of 0.01-1 mg / mL, the wells treated with 0.01-0.05 mg / mL poly-L-lysine had the worst effect, with only 20% of cells remaining, while the wells treated with 0.2-1 mg / mL poly-L-lysine had a slightly better effect, with 40% of cells remaining, and the best effect was the wells treated with 0.1 mg / mL poly-L-lysine, with 50% of cells remaining.

[0034] Example 2 Study of gelatin as an adhesion promoter The experimental steps were the same as those in Example 1, except that gelatin was selected as an adhesion promoter to investigate the adhesion of GABABR-HEK293T cells; 1. Principle Gelatin is a product of the extracellular matrix protein collagen that has undergone irreversible denaturation and still maintains some of its structure and functional properties, and can promote cell adhesion and extension.

[0035] 2. Preparation of adhesion promoter Gelatin powder was dissolved in sterile deionized water and placed in a 56°C water bath for full dissolution. The gelatin solution was sterilized by filtration using a 0.22 μm filter, and then diluted with sterile deionized water to obtain 8 concentration gradients of 0 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, and 10 mg / mL, respectively. The number of cells before acetone treatment was used as the initial cell amount for comparison with the results after the experiment.

[0036] The fixed 96-well plate was placed under a microscope to observe the adhesion effect of 8 concentration gradients. The bright field photos are shown in the figure. Figure 2 shown.

[0037] The results showed that compared with the initial cell amount, after acetone fixation, all the cells in the control group without gelatin were detached, while more cells remained in the wells treated with gelatin, with 50-80% remaining. This shows that gelatin has a good promoting effect on cell adhesion. In the different concentration gradients of 0.1-10 mg / mL, the wells treated with 0.1-0.5 mg / mL gelatin had the worst effect, with only 50% of cells remaining, while the wells treated with 5 mg / mL and 10 mg / mL gelatin had a slightly better effect, with 60% of cells remaining, and the best effect was the wells treated with 1 mg / mL and 2 mg / mL gelatin, with 80% of cells remaining.

[0038] Example 3 Study on carboxymethyl chitosan as an adhesion promoter The experimental steps were the same as those in Example 1, except that carboxymethyl chitosan was selected as an adhesion promoter to investigate the adhesion of GABABR-HEK293T cells; 1. Principle Carboxymethyl chitosan is one of the advanced derivatives of chitosan, which is obtained by reacting chitosan with chloroacetic acid under alkaline conditions. It has good water solubility, film-forming properties, moisturizing and water retention, antibacterial properties and strong heavy metal chelation, and can enhance the adhesion and proliferation of osteoblasts, fibroblasts, keratinocytes and endothelial cells.

[0039] 2. Preparation of adhesion promoter The carboxymethyl chitosan powder was dissolved in sterile deionized water and placed in a 37°C water bath for full dissolution. The carboxymethyl chitosan solution was filtered and sterilized using a 0.22 μm filter, and then diluted with sterile deionized water to obtain 8 concentration gradients of 0 μg / mL, 0.01 μg / mL, 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and 1 μg / mL, and the number of cells before acetone treatment was used as the initial cell amount for comparison with the results after the experiment.

[0040] The fixed 96-well plate was placed under a microscope to observe the adhesion effect of 8 concentration gradients. The bright field photos are shown in the figure. Figure 3 shown.

[0041] The results showed that compared with the initial cell amount, after acetone fixation, all the cells in the well plate without carboxymethyl chitosan in the control group fell off, while the cells in the well plate treated with carboxymethyl chitosan still remained, but very little, with 10-40% remaining. This shows that carboxymethyl chitosan has a promoting effect on cell adhesion, but the effect is not obvious. In the different concentration gradients of 0.01-1 μg / mL, the best effect was the well plate treated with 0.1 μg / mL carboxymethyl chitosan, with 40% of cells remaining, and the effects of the other concentrations were poor, with only 10-20% of cells remaining.

[0042] Example 4 Study on Sericin as Adhesion Promoter The experimental steps were the same as those in Example 1, except that sericin was selected as an adhesion promoter to investigate the adhesion of GABABR-HEK293T cells; 1. Principle Sericin is derived from natural silkworm cocoons and has good biocompatibility and a series of unique biological properties. It is a natural biomaterial with excellent performance. The unique amino acid composition and structural properties of sericin give it good water solubility, cell adhesion and proliferation activity, in situ fluorescence, antioxidant activity, and tyrosinase inhibition activity.

[0043] 2. Preparation of adhesion promoter The sericin powder was dissolved in sterile deionized water, and the sericin solution was filtered and sterilized using a 0.22 μm filter. Then, it was diluted with sterile deionized water to obtain 8 concentration gradients, namely 0 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, and 10 μg / mL. The cell number before acetone treatment was taken as the initial cell amount and compared with the results after the experiment.

[0044] The fixed 96-well plate was placed under a microscope to observe the adhesion effect of 8 concentration gradients. The bright field photos are shown in the figure. Figure 2 shown.

[0045] The results showed that compared with the initial cell amount, after acetone fixation, all the cells in the well plates without sericin added in the control group fell off, while most of the cells in the well plates treated with sericin adhered to the wall, with 80%-100% of the cells. This shows that sericin significantly promotes cell adhesion. In the different concentration gradients of 0.1-10 μg / mL, the well plates treated with 0.1-0.5 μg / mL gelatin had a slightly worse effect, with 80% of the cells remaining, while the well plates treated with 2-10 μg / mL gelatin had a slightly better effect, with 90% of the cells remaining. The best effect was the well plate treated with 1 μg / mL sericin, with no cell shedding and complete adherence.

[0046] Example 5 Study on Sodium Carboxymethyl Cellulose as Adhesion Promoter The experimental steps were the same as those in Example 1, except that sodium carboxymethyl cellulose was selected as an adhesion promoter to investigate the adhesion of GABABR-HEK293T cells; 1. Principle Sodium carboxymethyl cellulose is an anionic water-soluble polymer compound made from natural cellulose through chemical modification. Natural cellulose is its main raw material, and carboxymethyl groups are introduced through chemical reactions to give it good water solubility and unique physical and chemical properties. This modified cellulose not only has the excellent properties of natural cellulose, but also increases its solubility, stability and thickening properties.

[0047] 2. Preparation of adhesion promoter Sodium carboxymethyl cellulose powder was dissolved in sterile deionized water and placed in a 37°C water bath for full dissolution. The sodium carboxymethyl cellulose solution was sterilized by filtration using a 0.22 μm filter, and then diluted with sterile deionized water to obtain 8 concentration gradients of 0 μg / mL, 0.01 μg / mL, 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and 1 μg / mL, and the number of cells before acetone treatment was used as the initial cell amount for comparison with the results after the experiment.

[0048] The fixed 96-well plate was placed under a microscope to observe the adhesion effect of 8 concentration gradients. The bright field photos are shown in the figure. Figure 5 shown.

[0049] The results showed that compared with the initial cell amount, after acetone fixation, all the cells in the wells without sodium carboxymethylcellulose in the control group fell off, while the cells in the wells treated with sodium carboxymethylcellulose still remained, but very little, with 20-40% remaining. This shows that sodium carboxymethylcellulose has a promoting effect on cell adhesion, but the effect is not obvious. In the different concentration gradients of 0.01-1 μg / mL, the best effect was the well plate treated with 0.2 μg / mL sodium carboxymethylcellulose, with 40% of cells remaining, and the other concentrations had poor effects, with only 20-30% of cells remaining.

[0050] From the above experimental results, it can be seen that the cell coating solution composed of different adhesion promoters has different adhesion promoting effects on GABABR-HEK293T. Among them, 1 μg / mL sericin has the most obvious effect, and GABABR-HEK293T cells are completely attached to the wall; secondly, gelatin has a better effect, and 80% of GABABR-HEK293T cells adhere to the well plate after 1 mg / mL and 2 mg / mL gelatin treatment; poly-L-lysine has a general effect, and only 50% of GABABR-HEK293T cells adhere to the wall at the optimal concentration; carboxymethyl chitosan and sodium carboxymethyl cellulose have poor effects, and only 40% of GABABR-HEK293T cells adhere to the wall at the optimal concentration.

[0051] Comparative Example 1 Conventional method 1. Cell recovery (1) Thawing: Preheat the DMEM culture medium and water bath to 37°C. Quickly take out a tube of GABABR-HEK293T cells from the liquid nitrogen tank and quickly place it in the water bath (quickly check whether the lid is tightened before placing it in the water bath. Do not immerse the lid in water). Shake it quickly to melt it quickly until only a small ice core remains.

[0052] (2) Resuspension: Aspirate the cells in the tube and add 1 mL of DMEM to complete the culture. Gently pipette to mix.

[0053] (3) Centrifugation: Centrifuge at 1500 rpm for 5 min.

[0054] (4) Resuspend: Carefully remove the supernatant, add 1 mL of DMEM and mix by gently pipetting.

[0055] 2. Cell culture Pipette 500 μL of cell suspension into a 6 cm culture dish with 4 mL of preheated complete culture medium, shake the dish to evenly distribute the cells, place it in an incubator, and culture it at 37°C, 5% CO2 for 48 h.

[0056] 3. Cell Digestion (1) Cell washing: When the cell confluence in the culture dish reaches 80-90% under a microscope, remove the old supernatant culture medium and gently add an appropriate amount of saline (enough to cover the cells) to rinse the cells 1-2 times.

[0057] (2) Cell digestion: Add 1 mL of trypsin (0.25% Trypsin-EDTA) to the culture dish, gently shake the 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 fall off, quickly take them back to the operating table, aspirate the trypsin, and add 1-2 mL of stop medium to terminate the digestion.

[0058] (3) Cell centrifugation: Use a pipette to gently blow the cells to completely dislodge and mix them, then pipette them into a 2 mL centrifuge tube. Centrifuge at 1400 rpm for 5 min, aspirate the supernatant, add 1 mL of complete culture medium, and gently blow to mix.

[0059] 4. Cell culture Add the cell suspension to a new 10 cm culture dish at a subculture ratio of 1:2, then add 15 mL of DMEM complete medium, shake the dish to evenly distribute the cells, place it in an incubator, and culture it at 37°C, 5% CO2 for 48 h.

[0060] 5. Cell plating After digesting the 10 cm culture dish with trypsin, the cells were counted using a cell counter at a rate of (0.5-1)×10 5 Cells / well were inoculated into a 96-well plate. 6. Cell culture The 96-well plate was incubated at 37°C and 5% CO2 for 24 h.

[0061] 7. Cell Fixation (1) PBS washing: Discard the cell incubation solution and gently add 200 μL PBS buffer to the reaction wells for washing once.

[0062] (2) Acetone fixation: Add 50 μL of pre-cooled acetone and fix at -20°C for 5 min.

[0063] (3) PBS washing: Discard the fixative solution and add 200 μL PBS buffer to the reaction wells for washing three times.

[0064] 8. Microscopic examination The fixed cell wells were placed under a microscope to observe the adhesion effect. The bright field photos are shown in the figure. Figure 6 shown.

[0065] The results showed that when cells were attached to the wall using conventional methods and fixed with acetone, the cells were observed under bright field and were evenly distributed, stretched and oval in shape, and completely attached to the wall.

[0066] The operation process of Examples 1-5 is compared with that of Comparative Example 1. Figure 7 .

[0067] The results showed that pretreatment of the cell wells with adhesion promoters could speed up cell plating and fixation, shortening the time from the original 121 h to 2.5 h, greatly saving time.

[0068] Example 6 Anti-GABABR encephalitis detection using GABABR-293T cells Using cell immunofluorescence detection, the antibody corresponding to GABABR in the sample and the specific overexpressed antigen expressed on HEK293T cells in the reagent formed an antigen-antibody complex. This antigen carries mcherry fluorescent protein (red fluorescence). Subsequently, goat anti-human fluorescent secondary antibody (green fluorescence) is used to label the human secondary antibody bound to the antigen. Finally, a fluorescence microscope is used to observe whether there is co-localization of green fluorescence and red fluorescence. If there is co-localization, it means that the sample contains the antibody, otherwise the corresponding antibody does not exist in the sample.

[0069] The experimental steps are as follows: 1. Sample addition: Add 80 μL of sample (GABABR positive patient serum and negative serum, 1:20 dilution) into the test well. Shake gently to mix, and incubate at 37°C for 35 min.

[0070] 2. Washing: After incubation, remove the wells, discard the liquid in the test wells, and add 200 µL PBS buffer to wash twice. Slightly shake the wells during the washing process. Place the wells on a shaker for the third time and wash for 5 min.

[0071] 3. Add fluorescent secondary antibody: discard the liquid in the well, add 80 µL fluorescent secondary antibody (1:1500 dilution, purchased from abcam) to the detection well, shake gently to mix, and incubate at 37℃ in the dark for 35 min.

[0072] 4. Cleaning: Repeat step 3.

[0073] 5. Observation: Discard the liquid in the wells, add 100 µL of PBS buffer to the sample wells to cover the cells, then observe the green fluorescence of the cells under a fluorescence microscope and take pictures.

[0074] The photo results are as follows Figure 8 shown.

[0075] The results showed that after cell immunofluorescence, green fluorescent cells were visible in the green fluorescence channel of positive patient samples, and overlap with red antigen fluorescence could be observed after software synthesis; in negative patient samples, no green fluorescent cells were seen in the green fluorescence channel or no overlap between green fluorescent cells and red fluorescence was observed after software synthesis. The test results of cells obtained by the new method and cells obtained by the conventional method were consistent, and both positive and negative were synergistic. Moreover, compared with the conventional method, the green fluorescence signal of positive cells in the new method was stronger, and the background of negative cells was cleaner. This shows that GABABR-293T cells plated on the well plate treated with sericin can be used for the detection of anti-GABABR encephalitis, and the positive fluorescence signal is more obvious.

[0076] Example 7 Synergy Test Using the conventional method of Comparative Example 1 and the method corresponding to the preferred embodiment in Example 4, 15 healthy human sera and 15 GABABR positive serum samples were tested for anti-GABABR encephalitis.

[0077] The test results are shown in Table 1 below.

[0078] Table 1. Test results of sample synergy between conventional and new methods

[0079] Note: Positive (1:100) is the patient's antibody titer, which is the test result. The larger the titer value, the higher the antibody concentration in the body.

[0080] The results showed that the results of negative and positive samples using the conventional method and the new method were synergistic, with a synergy rate of 100%. Among the 15 positive samples, 10 samples had consistent antibody titers detected by the two methods, accounting for 67%. The 5 inconsistent samples, accounting for 33%, all had higher antibody titers using the new method than the conventional method. In summary, the sensitivity of the new method is higher than that of the conventional method.

[0081] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. Application of sericin in the preparation of cell adhesion promoters.

2. The use according to claim 1, characterized in that: The concentration of sericin in the cell adhesion promoting agent is 0.1-10 μg / mL.

3. A method for rapid cell plating, characterized in that: The cell wells were pre-treated with an adhesion promoter and the target cells were plated at (2-3)×10 5 The inoculation amount of cells / well is added to the pretreated cell well plate, and the plate is incubated in a 35-37° C. water bath for 1-1.2 h to complete the plating; the adhesion promoting agent is sericin.

4. The method according to claim 3, characterized in that: The pretreatment method is: make the adhesion promoter solution cover the bottom of the cell well plate, place it in a saturated humidity incubator at 35-37° C. for 1-1.2 h, wash it with sterile saline and then dry it.

5. The method according to claim 3, characterized in that: The concentration of sericin in the cell adhesion promoting agent is 0.1-10 μg / mL.

6. The method according to claim 3, characterized in that: The target cells are GABABR-HEK293T cells.

7. A cell plate prepared according to the method according to any one of claims 3 to 6.

8. Use of the cell plate according to claim 7 in preparing a cell immunofluorescence detection kit.

9. An anti-GABABR encephalitis detection kit, characterized in that: The invention comprises the cell adhesion promoting agent according to claims 1-2.

10. An anti-GABABR encephalitis detection kit, characterized in that: The invention comprises a GABABR-HEK293T cell plate prepared according to the method of claim 6.

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