Preparation method and application of cell protection solution and HPV E6 / E7 quality control tablet

Abstract

The present invention discloses a preparation method and application of a cell protective solution and an HPV E6 / E7 quality control tablet. The cell protective solution comprises polysaccharide, polyethylene glycol, anhydrous ethanol, a nonionic surfactant, and betaine. The addition of polysaccharide, surfactant, and betaine improves the ability of the cell protective solution to protect protein activity, and the optimization of polyethylene glycol improves the ability of the cell protective solution to maintain cell structure. When applied to the quality control tablet, the cell protective solution can withstand a certain high temperature during the preparation process, thereby extending the shelf life of the quality control tablet.

Description

Technical Field

[0001] The present invention relates to the technical field of protein control tablets, and in particular to a preparation method and application of a cell protection solution and an HPV E6 / E7 quality control tablet. Background Art

[0002] Cervical cancer is the most common gynecological malignancy, and persistent infection with high-risk human papillomavirus is closely associated with the development of cervical cancer. The circular HPV genome includes two types of coding gene regions: seven early region genes (E1, E2, E4, E5, E6, E7, and E8) and two late regions (L1 and L2). L1 and L2 encode viral capsid proteins, which play a role in coordination and recognition during the self-assembly of viral particles and invasion of target cells. Among the protein genes encoding the early region (E region), the oncogenic proteins encoded by the E6 and E7 genes are important factors in the development of cervical epithelial carcinogenesis. Therefore, the detection of HPV E6E7 protein expression is of great significance in reducing the incidence of cervical cancer and precancerous lesions.

[0003] Currently, the HPV E6 / E7 protein immunocytochemical staining reagent urgently needs a quality control plate for the HPV E6 / E7 protein. The quality control plate for the HPV E6 / E7 protein immunocytochemical staining reagent requires that (1) the quality control plate can be stored under specific conditions for more than three months, and the quality control plate within the validity period can still accurately control the experimental results in the experiment when it is unpacked and used; (2) the quality control plate processed by a specific preparation method can still accurately control the experimental results in the experiment when it is used; (3) the raw material source of the quality control plate is stable and sufficient.

[0004] The quality control plate of HPV E6 / E7 protein prepared according to the existing technology, after simple air drying and sealing, can no longer produce normal quality control experimental results after three days of storage. Therefore, it is necessary to improve the existing quality control plate technology. Summary of the Invention

[0005] The purpose of the present invention is to provide a cell protection solution that provides quality control for HPV E6 / E7 protein immunocytochemical staining reagents and prolongs the validity period of HPV E6 / E7 quality control films.

[0006] To achieve the above object, the scheme of the present invention is as follows:

[0007] A cell protection solution comprises, by mass percentage, 0.1-1% trehalose, 1-10% polyethylene glycol, 30-70% anhydrous ethanol, 0.01-1% nonionic surfactant, 0.01-1% betaine and the balance water; the molecular weight of the polyethylene glycol is 2000-5000.

[0008] Furthermore, the invention comprises, by mass percentage, 0.5% trehalose, 5% polyethylene glycol, 50% anhydrous ethanol, 0.05% nonionic surfactant, 0.05% betaine and the balance water.

[0009] Furthermore, the molecular weight of the polyethylene glycol is 3500.

[0010] Furthermore, the ionic surfactant is at least one of Triton-100, Tween-20, and Tween-80.

[0011] Another object of the present invention is to provide the use of the above-mentioned cell protection solution in the preparation of HPV E6 / E7 quality control tablets. On the one hand, it can withstand a certain high temperature during the preparation process, and on the other hand, it can maintain the protein activity of HPV E6 / E7 for a long time, thereby extending the shelf life of the quality control tablets.

[0012] Another object of the present invention is to provide a method for preparing an HPV E6 / E7 quality control sheet, comprising the following steps:

[0013] 1) Centrifuge the HPV E6 / E7 cell suspension, remove the supernatant, add buffer, resuspend the cells, and allow to settle;

[0014] 2) After sedimentation is complete, remove excess liquid and wash the slide outside the cell area;

[0015] 3) adding the cell protection solution of claim 1 and immersing the tube, reacting at room temperature for 30 minutes, and then drying the tube at 37-65°C without removing the tube;

[0016] 4) After removing the sleeve, air-dry the slide at 37-65°C and vacuum-seal to obtain the HPV E6 / E7 quality control slide.

[0017] Furthermore, in step 3), the drying is carried out at 45-50° C. for 30 minutes without removing the casing, and in step 4), the casing is removed and the slices are air-dried at 45-50° C. for 2.5 hours.

[0018] Another object of the present invention is to provide an HPV E6 / E7 quality control sheet obtained by the above-mentioned preparation method, wherein the quality control sheet can maintain the protein activity of HPV E6 / E7.

[0019] Another object of the present invention is to provide the use of the above-mentioned quality control sheet in HPV E6 / E7 protein immunocytochemical staining, which can accurately control the experimental results.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The cell protection solution provided by the present invention improves the ability of the cell protection solution to protect protein activity by adding polysaccharides, surfactants, and betaine. Combined with polyethylene glycol, it improves the ability of the cell protection solution to maintain cell structure and high temperature resistance. It has good prospects for application in quality control films.

[0022] 2. The preparation method of the HPV E6 / E7 quality control sheet provided by the present invention can obtain reliable quality control sheets by combining only two simple drying methods. It is simple, fast and has strong scalability.

[0023] 3. The HPV E6 / E7 quality control sheet provided by the present invention can significantly extend the shelf life compared with the existing technology, and has ideal quality control value and economic value when applied to HPV E6 / E7 protein immunocytochemical staining. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] An embodiment of the present invention provides an HPV E6 / E7 cell protective solution comprising trehalose, polyethylene glycol, anhydrous ethanol, a nonionic surfactant, betaine, and pure water.

[0026] Wherein: the mass percentage of trehalose is 0.1-1%, preferably 0.5%.

[0027] The mass percentage of polyethylene glycol is 1-10%, preferably 5%; the molecular weight is 2000-5000, preferably 3500.

[0028] The mass percentage of anhydrous ethanol is 30-70%, preferably 50%.

[0029] The nonionic surfactant is at least one of Triton 100, Tween 20, and Tween 80, preferably Tween 20, with a mass ratio of 0.01 to 1%, preferably 0.05%.

[0030] The betaine is betaine citrate or lauryl betaine, preferably betaine citrate, with a mass ratio of 0.01-1%, preferably 0.05%.

[0031] The synergistic effect of polyethylene glycol, trehalose, and betaine can reduce protein degradation in positive quality control samples during the preparation of quality control samples. Therefore, this cell protection solution can enhance protein protection, reduce protein degradation, improve the ability to maintain cell structure, and extend the shelf life of the quality control samples.

[0032] In another embodiment, a method for preparing a quality control sheet for HPV E6 / E7 is proposed. The quality control sheet treated with the cell protection solution described above can withstand a certain high temperature during the subsequent drying process, and does not affect the binding ability of the protein and antibody reagent during the use of the quality control sheet, nor the quality control ability of the quality control sheet.

[0033] The specific steps are as follows:

[0034] 1. Equipment Preparation

[0035] Open the drying box in advance, set the temperature inside the drying box at 30°C, and put desiccant in the drying box to control the humidity within 50%.

[0036] 2. Centrifugation

[0037] Take an appropriate amount of suspended cells (positive cells or negative cells) preserved in cell preservation solution and place them in a 12 mL conical tube. Centrifuge at 400 × g for 5 minutes. After centrifugation, remove the supernatant.

[0038] 3. Settlement

[0039] After centrifugation, remove the supernatant and resuspend the cells in secondary buffer (1 slide / 500 μL secondary buffer). Transfer 500 μL of the suspension to a 10 mm cannula, mix the cells using the cross-hatch technique, and allow them to settle for 10 minutes. While the cells are settling, label the slides with (E6+, E6-, E7+, E7-) and the date of slide preparation.

[0040] 4. Cleaning

[0041] After sedimentation, remove excess liquid in the cannula and wash once with an elbow wash bottle filled with 50% alcohol, avoiding the cell area to prevent washing away the cells on the slide.

[0042] 5. Cell protection solution

[0043] Shake off excess 50% alcohol in the cannula, add about 40 μL of protective solution to immerse the cells, mix the protective solution using the cross method, and react at room temperature for 30 minutes.

[0044] 6. Drying

[0045] After the cell protection solution has reacted for 30 minutes, place the slide upright and horizontally in a drying oven at 37-65°C without removing the sleeve and dry for 30 minutes before removing the sleeve (to ensure that the protection solution forms a hydrogel film in the cell area and the liquid does not overflow from the cell area). Place the slide on a drying plate and dry at 37-65°C for 2.5 hours. If a few slides are not completely dry after the drying time is over, the drying time can be appropriately extended by 30 minutes to 1 hour.

[0046] 7. Vacuum seal

[0047] After drying, place the slides into a 5-slide box, place several packets of desiccant in the slide box containing cells, seal with a sealed bag vacuum sealer, and store at 2-8°C.

[0048] In particular, the drying method in step 6 is preferably: drying at 40-50°C for 30 minutes without removing the casing, then removing the casing and placing it on a drying plate at 40-50°C for 2.5 hours, which can achieve better results.

[0049] Example 1

[0050] Trehalose concentration screening

[0051] A cell protection solution containing trehalose at concentrations of 0 g / mL, 0.3 g / mL, 0.5 g / mL, and 0.8 g / mL, 50% by volume of anhydrous ethanol, 0.05% by volume of Tween-20, and 1% by weight of PEG-6000 was prepared. HPV E6 / E7 quality control plates were prepared according to the above method. The HPV E6 / E7 protein immunocytochemical staining kit was used for the experiment and compared with freshly settled positive cells. The results are shown in Table 1.

[0052] Table 1:

[0053]

[0054]

[0055] Among them, the results of 0.5 g / mL trehalose were closer to those of fresh cells, so 0.5 g / mL was selected.

[0056] Note: + represents the degree of color development; - represents half of the degree of color development; N represents no color development (the same below).

[0057] Example 2

[0058] Screening of polyethylene glycol molecular weight

[0059] A cell protection solution containing 1% PEG (molecular weights of 2000, 3500, and 6000), 0.5 g / mL trehalose, 50% by volume anhydrous ethanol, 0.05% by volume Tween-20, and 1% by weight PEG-6000 was prepared. HPV E6 / E7 quality control plates were prepared according to the above method. The HPV E6 / E7 protein immunocytochemical staining kit was used for the assay and compared with freshly settled positive cells. The results are shown in Table 2.

[0060] Table 2:

[0061]

[0062]

[0063] Among them, the results of PEG3500 were closer to those of fresh cells, so PEG3500 was selected.

[0064] Example 3

[0065] Screening of polyethylene glycol concentration

[0066] Prepare a cell protection solution with polyethylene glycol 3500 concentrations of 0%, 1%, 5%, and 10%, trehalose concentration of 0.5 g / mL, Tween-20 volume ratio of 0.05%, and anhydrous ethanol volume ratio of 50%, and prepare HPV E6 / E7 quality control slices according to the above preparation method.

[0067] The HPV E6 / E7 protein immunocytochemical staining kit was used for the experiment and compared with freshly precipitated positive cells. The results are shown in Table 3.

[0068] Table 3:

[0069]

[0070]

[0071] Among them, the results of 5% PEG3500 were closer to those of fresh cells, so 5% PEG3500 was selected.

[0072] Example 4

[0073] Prepare the following formulas of cell protection solution L1 (Table 4) and cell protection solution L2 (Table 5) to prepare HPV E6 / E7 quality control plates.

[0074] The HPV E6 / E7 protein immunocytochemical staining kit was used for the experiment. For comparison with freshly precipitated positive cells, HPV E6 / E7 quality control slides were stored at 2-8°C for 1 day and at 37°C for 7 days. The results are shown in Table 6.

[0075] Table 4:

[0076]

[0077]

[0078] Table 5:

[0079]

[0080] Table 6:

[0081]

[0082] It can be seen that the positive quality control strips prepared with Cell Protection Solution L1, which contains trehalose and polyethylene glycol, produce results closer to those of fresh cells. Therefore, the addition of trehalose and polyethylene glycol is essential. Furthermore, the quality control strips prepared with Cell Protection Solution L1 maintain results close to those of fresh cells even when stored at 37°C for 7 days under accelerated conditions. This significantly extends the shelf life of the quality control strips, offering a significant advantage over existing quality control strips, which only last for 3 days.

[0083] Example 5

[0084] Prepare cell protection solution L1 and quality control plates according to Table 4. The drying methods are as follows:

[0085] A. After removing the cannula, place it directly into a 37°C drying oven and dry for 1 hour.

[0086] B. After removing the cannula, place it directly into a 37°C drying oven and dry for 3 hours.

[0087] C. Dry at 37°C for 30 minutes without removing the cannula, then remove the cannula and dry for 2.5 hours.

[0088] The quality control plates obtained under the three drying methods were tested using the HPV E6 / E7 protein immunocytochemical staining kit and compared with freshly precipitated positive cells. The results are shown in Table 7.

[0089] Table 7:

[0090]

[0091] The cells in group B showed signs of rupture, so method C was selected as the best drying method.

[0092] Example 6

[0093] Prepare cell protection solutions L1 and L2 according to Table 4 and Table 5, respectively, and prepare quality control plates. The drying methods are as follows:

[0094] A. Dry at 37°C for 30 minutes without removing the cannula, then remove the cannula and dry for 2.5 hours.

[0095] B. Without removing the casing, dry at 50℃ for 30 minutes, then remove the casing and dry for 2.5 hours.

[0096] C. Without removing the sleeve, dry at 65℃ for 30 minutes, then remove the sleeve and dry for 2.5 hours.

[0097] The quality control slides obtained under the three drying methods were subjected to experiments using the HPV E6 / E7 protein immunocytochemical staining kit and compared with freshly precipitated positive cells. The results are shown in Table 8.

[0098] Table 8:

[0099]

[0100] As can be seen from Table 8, the staining results of group B using cell protection solution L1 were more stable. Therefore, the final drying method selected was to dry the cells at 50°C for 30 minutes without removing the tube, then remove the tube and dry for 2.5 hours.

[0101] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A cell protection solution, characterized in that: Calculated by mass percentage, the composition includes 0.5% trehalose, 5% polyethylene glycol, 50% anhydrous ethanol, 0.05% Tween-20, 0.05% betaine and the balance water; the molecular weight of the polyethylene glycol is 2000-5000.

2. The cell protection solution according to claim 1, characterized in that The molecular weight of the polyethylene glycol is 3500.

3. Use of the cell protection solution according to claim 1 or 2 in the preparation of HPV E6 / E7 quality control slices.

4. A method for preparing an HPV E6 / E7 quality control sheet, characterized in that: The steps include: 1) Centrifuge the HPV E6 / E7 cell suspension, remove the supernatant, add buffer, resuspend the cells, and allow to settle. 2) After sedimentation is complete, remove excess liquid and wash the slide outside the cell area; 3) adding the cell protection solution of claim 1, immersing the tube, reacting at room temperature for 30 minutes, and then drying at 37-65°C without removing the tube; 4) After removing the sleeve, air-dry the slide at 37-65°C and vacuum-seal to obtain the HPV E6 / E7 quality control slide.

5. The preparation method according to claim 4, characterized in that In step 3), the slices are dried at 45-50° C. for 30 minutes without removing the sleeve. In step 4), the slices are dried at 45-50° C. for 2.5 hours after removing the sleeve.

6. The HPV E6 / E7 quality control sheet obtained by the preparation method according to claim 4 or 5, characterized in that: It can maintain the protein activity of HPVE6 / E7 and extend its validity period.

7. Use of the quality control sheet according to claim 6 in immunocytochemical staining of HPV E6 / E7 proteins.

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