Compositions for cell lysis and uses thereof, kits

Abstract

The application provides a composition for cell lysis and application and kit thereof, and relates to the technical field of biology. The composition comprises, in terms of mass fraction, 0.5-5 parts of serum albumin, 1-10 parts of Triton X-100, 0.1-1 parts of ethylenediaminetetraacetate and 0.05-0.2 parts of surfactant S-22. The composition can effectively lyse epithelial cells and improve the detection effect of analyte in the lysis product of the epithelial cells.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a composition for cell lysis and its application, as well as a kit. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Molecular biology detection methods facilitate the rapid detection of pathogens causing respiratory diseases. These methods primarily target cell lysis products, such as proteins, DNA, and RNA within cells or on cell membranes. Therefore, cell lysis is often required as a pretreatment step in most detection methods. Cell lysis buffer is a solution used to disrupt cell and nuclear membranes, extracting intracellular proteins and other molecules. The function of the cell lysis buffer significantly impacts the detection results.

[0004] In in vitro diagnostics, diagnostic samples often include epithelial cells, such as throat and nasal swabs. While various cell lysis buffers exist, such as RIPA and IP lysis buffers, the main difference between these buffers lies in their lysis intensity on cells or tissues. A cell lysis buffer specifically designed for a particular cell type, such as epithelial cells, is lacking. Thorough cell lysis helps obtain more target molecules during detection, improving diagnostic results. Therefore, a cell lysis buffer that can effectively lyse epithelial cells is urgently needed.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a composition for cell lysis and its application to improve the lysis effect of epithelial cells.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] In a first aspect, a composition for cell lysis is provided, comprising, by weight parts: 0.5 to 5 parts serum albumin, 1 to 10 parts Triton X-100, 0.1 to 1 part ethylenediaminetetraacetic acid (EDTA), and 0.05 to 0.2 parts surfactant S-22.

[0009] In a second aspect, the use of the composition of the first aspect for cell lysis is provided in the lysis of epithelial cells for non-diagnostic and therapeutic purposes, or in the preparation of a kit for detecting epithelial cell lysis products.

[0010] Thirdly, a detection kit is provided that comprises the cell lysis composition of the first aspect.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The cell lysis composition provided by this invention comprises serum albumin, Triton X-100, ethylenediaminetetraacetic acid (EDTA), and surfactant S-22. The chemicals used in this composition are readily available, simple to prepare, and require no complex processes, making the technical solution relatively easy to implement. This composition facilitates the lysis of epithelial cells, allowing for a more complete release of the cell contents into the detection system, increasing the content and concentration of the target analyte in a fixed-volume detection system, and enabling the target analyte to be more fully recognized by the detection reagent. This composition also helps release housekeeping proteins from epithelial cells, increasing the signal value of housekeeping proteins when used as an internal control.

[0013] In the preferred embodiment, immunochromatography is used to detect the target. During the immunochromatographic process, the target can be retained as much as possible by the antibodies coated on the immunochromatographic test strip, improving the reading signal and thus enhancing detection sensitivity. Simultaneously, this composition effectively eliminates background on the immunochromatographic test strip, improves the chromatographic membrane surface, and enhances readings, making the results clearer and easier to obtain. Attached Figure Description

[0014] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. (The actual experiment used three parallel samples and averaged them, but the accompanying drawings only provide one sample for a clearer comparison of the membrane surfaces.)

[0015] Figure 1 The images show test strips obtained by lysing samples using lysis buffer 3 and lysis buffer 4 in Example 2, and then testing them with a novel coronavirus latex immunochromatographic test strip. The left side shows lysis buffer 3, and the right side shows lysis buffer 4.

[0016] Figure 2 The images show the test strips after the samples were lysed using lysis buffer 3 and lysis buffer 4 in Example 2, and then detected using the novel coronavirus fluorescent immunochromatographic test strip after fluorescence excitation and color development. The left side shows lysis buffer 4, and the right side shows lysis buffer 3. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] In this document, "prepared from" and "comprising" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0019] In this document, the conjunction "composed of" excludes any unspecified elements, steps, or components. If used in a claim, this phrase will make the claim closed, meaning it does not contain any materials other than those described, except for conventional impurities associated with them.

[0020] In this document, when a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1–5” is disclosed, the described range should be interpreted as including ranges “1–4”, “1–3”, “1–2”, “1–2 and 4–5”, “1–3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0021] In this document, “and / or” is used to indicate that one or both of the situations described may occur, for example, A and / or B includes (A and B) and (A or B).

[0022] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual such relationship, order or importance between these entities or behaviors.

[0023] In this document, unless otherwise stated, “optional,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0024] In a first aspect, a composition for cell lysis is provided, comprising serum albumin, Triton X-100, ethylenediaminetetraacetic acid (EDTA), and surfactant S-22.

[0025] Serum albumin can act as a stabilizer to enhance the stability of cell lysis products; it can also mimic the physiological environment and regulate the osmotic pressure of the composition. The composition comprises 0.5 to 5 parts of serum albumin by weight, for example, but not limited to, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 parts.

[0026] In an optional embodiment, the serum albumin includes bovine serum albumin (BSA).

[0027] Triton X-100 is a nonionic detergent with the chemical name octylphenoxypolyethoxyethanol. Triton X-100 can disrupt the lipid bilayer structure of cell membranes, releasing cell contents, and dissociate membrane proteins and other protein complexes. The composition comprises 1 to 10 parts by weight of Triton X-100, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts.

[0028] Ethylenediaminetetraacetic acid (EDTA) can form stable complexes with a variety of metal ions and is a commonly used chelating agent. It possesses strong complexing ability and can form stable water-soluble complexes with various metal ions, thereby removing or inhibiting the activity of these metal ions. The composition comprises 0.1 to 1 part EDTA by weight, for example, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 part.

[0029] In an optional embodiment, the ethylenediaminetetraacetic acid salt comprises one or more of a metal salt and an ammonium salt. The metal salt includes, but is not limited to, salts of alkali metals, alkaline earth metals, and transition metals; exemplary metal salts include potassium salts, sodium salts, magnesium salts, calcium salts, or zinc salts. In an optional embodiment, the ethylenediaminetetraacetic acid salt comprises tetrasodium ethylenediaminetetraacetate.

[0030] The molecular formula of surfactant S-22 is C 30 H 62 O 10 S-22 is a nonionic surfactant that promotes protein dissolution. It also possesses wetting and penetrating properties, which are beneficial for the immunochromatographic process. The composition comprises 0.05 to 0.2 parts by weight of surfactant S-22, for example, but not limited to, 0.05, 0.1, 0.15, or 0.2 parts.

[0031] In an optional embodiment, the composition for cell lysis further includes NP-40 (Phenoxypolyethoxyethanol), a nonionic polyoxyethylene surfactant that can dissolve lipids on the cell membrane, thereby disrupting the cell membrane structure and releasing proteins and other molecules from inside the cell.

[0032] In an optional embodiment, the composition comprises 0.5 to 2 parts of NP-40 by weight, for example, but not limited to, 0.5, 0.6, 0.8, 0.9, 1.0, 1.2, 1.5, 1.8 or 2 parts.

[0033] In optional embodiments, the composition may further include a buffering component and / or a preservative.

[0034] "Buffer component" refers to a component in an aqueous solution or composition that, when an acid or base is added to the solution or composition, enables the aqueous solution or composition to resist and / or inhibit changes in pH. The buffer component may be selected from buffer salts or nonionic buffer components. In an optional embodiment, the buffer component comprises tris(hydroxymethyl)aminomethane (Tris), preferably comprising 0.1 to 1 part of tris(hydroxymethyl)aminomethane, for example, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 part.

[0035] In an optional embodiment, the preservative includes sodium azide, preferably 0.05 to 0.3 parts of sodium azide, for example, but not limited to 0.05, 0.1, 0.15, 0.2, 0.25 or 0.3 parts.

[0036] In an optional embodiment, the composition further includes NaCl, preferably 4 to 5 parts of NaCl, for example, but not limited to 4, 4.5 or 5 parts.

[0037] In an optional embodiment, the composition comprises, by weight parts: 4-5 parts NaCl, 0.5-5 parts bovine serum albumin, 1-10 parts Triton X-100, 0.1-1 parts tetrasodium EDTA, 0.05-0.3 parts sodium azide, 0.1-1 parts tris(hydroxymethyl)aminomethane, 0.05-0.2 parts surfactant S-22, and 0.5-2 parts NP-40.

[0038] In an optional embodiment, the composition comprises, by weight parts: 4 parts NaCl, 1 part bovine serum albumin, 5 parts Triton X-100, 0.5 parts tetrasodium EDTA, 0.2 parts sodium azide, 0.6 parts tris(hydroxymethyl)aminomethane, 0.1 parts surfactant S-22, and 1 part NP-40.

[0039] In an optional embodiment, the composition further includes a solvent, including but not limited to water, buffer solution, or non-buffer solution.

[0040] In an optional embodiment, the composition further includes a solvent, wherein the composition is a cell lysis buffer; the concentrations of each component in the composition are: NaCl 4-5% wt, for example, but not limited to 4, 4.5, or 5% wt; bovine serum albumin 0.5-5% wt, for example, but not limited to 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% wt; Triton X-100 1–10% wt, for example, but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% wt; 0.1–1% wt of tetrasodium ethylenediaminetetraacetate, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% wt; 0.05–0.3% wt of sodium azide, for example, but not limited to 0.05, 0.1, 0.15, 0.2, 0.25, or 0.3% wt; 0.1–1% wt of tris(hydroxymethyl)aminomethane, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% wt; surfactant S-22 The solvent is 0.05 to 0.2% wt, for example, but not limited to 0.05, 0.1, 0.15, or 0.2% wt; and NP-40 is 0.5 to 2% wt, for example, but not limited to 0.05, 0.1, 0.2, 0.3, 0.5, 0.6, 0.8, 0.9, 1.0, 1.2, 1.5, 1.8, or 2% wt. In this embodiment, the solvent may optionally be water.

[0041] In an optional embodiment, the composition further includes a solvent, and the composition is a cell lysis buffer; the concentrations of each component in the composition are: NaCl 4%wt, serum albumin 1%wt, Triton X-100 5%wt, tetrasodium EDTA 0.5%wt, sodium azide 0.2%wt, tris(hydroxymethyl)aminomethane 0.6%wt, surfactant S-22 0.1%wt, and NP-40 1%wt. In this embodiment, the solvent may optionally be water.

[0042] In a second aspect, the use of the composition for cell lysis described in the first aspect in the lysis of epithelial cells for non-diagnostic and therapeutic purposes, or in the preparation of kits for detecting epithelial cell lysis products, is also provided.

[0043] In an optional embodiment, the detection of epithelial cell lysis products includes the detection of internal reference proteins.

[0044] In optional embodiments, the internal reference protein includes, but is not limited to, one or more of glyconuclease P, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-microglobulin, β-tubulin, α-tubulin, 18sRNA, β-actin, and TATA-binding protein.

[0045] Thirdly, a detection kit is also provided, which includes the composition for cell lysis described in the first aspect, enabling the detection of cell lysis products.

[0046] In an optional embodiment, the detection kit further includes reagents for detecting cell lysis products.

[0047] In optional embodiments, the test kit further includes test reagents for any of the following detection methods: immunofluorescence staining, flow cytometry, immunoblotting, immunohistochemistry, ELISA, immunochromatography, immunoprecipitation, co-precipitation, or immunomagnetic bead detection.

[0048] In an optional embodiment, the detection kit further includes reagents for detecting an internal reference protein.

[0049] In an optional embodiment, the internal reference protein includes one or more of glyconuclease P, glyceraldehyde-3-phosphate dehydrogenase, β-microglobulin, β-tubulin, α-tubulin, 18sRNA, β-actin actin, and TATA-binding protein.

[0050] In an optional embodiment, the test kit includes immunochromatographic strips.

[0051] In an optional embodiment, the control line of the immunochromatographic test strip uses an internal reference protein as the detection target molecule.

[0052] In optional embodiments, the markers on the immunochromatographic test strip include, but are not limited to, one or more of fluorescent particles, colored particles, latex particles, quantum dots, chemiluminescent reagents, fluorescent molecules, colloidal gold, colloidal silver, colloidal selenium, horseradish peroxidase, and alkaline phosphatase, preferably including fluorescent particles, latex particles, or colloidal gold. The composition for cell lysis provided in the first aspect effectively eliminates the background of immunochromatographic test strips using fluorescent particles and latex particles, improves the chromatographic membrane surface, improves readings, and makes results clearer and easier to obtain.

[0053] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0054] Example 1

[0055] Prepare the following lysis buffer according to Table 1:

[0056] Table 1

[0057] Ingredients / Concentration (%wt) lysis buffer 1 lysis buffer 2 pyrolysis solution 3 NaCl 4 4 4 Bovine serum albumin 1 1 1 Triton X-100 5 5 5 Tetrasodium ethylenediaminetetraacetate 0.5 0.5 0.5 Sodium azide 0.2 0.2 0.2 Tris(hydroxymethyl)aminomethane - 0.6 0.6 Surfactant S-22 - - 0.1

[0058] Fifty negative nasal swab samples were collected. Each swab was placed in a lysis tube and soaked in 400 μL of physiological saline for at least 5 minutes. The lysis tube was squeezed 20 times with the thumb and forefinger to extract the swab and obtain the nasal swab eluent. The 50 nasal swab eluents were mixed to form a homogenized sample solution as the negative matrix for loading the sample. The lysis buffer in Table 1 was used for the detection of influenza A virus latex immunochromatographic test strips. The homogenized negative sample solution was mixed with the lysis buffer in Table 1 at a volume ratio of 1:1. After mixing, the mixture was allowed to stand for more than 15 minutes before loading the sample. The loading volume was 100 μL. Three or more parallel samples were used for testing. The results were taken as the colorimetric value or average value of more than 95% of the parallel samples.

[0059] Interpretation of latex immunochromatographic assay results: The colorimetric values ​​of the test line and control line are referenced from a colorimetric card disclosed in the invention patent with publication number CN111909257A (application number CN202010736865.X, invention title "A method for synthesizing calcipopolypeptide antigen and its application"). Higher numbers indicate deeper color development. The colorimetric value range is 1-10, with a reading of 3.5 or higher considered a visible line. The varying shades of the C and T lines in the subsequent table are represented using this reading method. Therefore, the criterion for determining a visible line is whether the colorimetric value of the C and T lines reaches a reading of 3.5 on the colorimetric card, where "-" indicates a negative result.

[0060] The experimental results are shown in the table below:

[0061] Table 2

[0062] pyrolysis solution C-line output value lysis buffer 1 7 lysis buffer 2 7+ pyrolysis solution 3 8- PBS control group - saline control group -

[0063] The results of Example 1 show that for the influenza A virus latex immunochromatographic test strip, lysis buffer 3 enhanced the C-line signal value, indicating that the sample solution contained more GAPDH, and also indicating that the sample lysis buffer 3 of Example 1 can more effectively lyse the cells in human nasal swabs and release the contents.

[0064] Example 2

[0065] Prepare the following lysis buffer according to Table 3:

[0066] Table 3

[0067] Ingredients / Concentration (%wt) pyrolysis solution 3 pyrolysis solution 4 NaCl 4 4 Bovine serum albumin 1 1 Triton X-100 5 5 Tetrasodium ethylenediaminetetraacetate 0.5 0.5 Sodium azide 0.2 0.2 Tris(hydroxymethyl)aminomethane 0.6 0.6 Surfactant S-22 0.1 0.1 NP-40 N / A 1

[0068] The lysis buffers in Table 3 were used to test the samples from Example 1 using the novel coronavirus fluorescence immunochromatographic test and the novel coronavirus latex immunochromatographic test strip. The sample processing method was the same as in Example 1. Three or more parallel samples were used for testing, and the results were taken as the colorimetric values ​​or average values ​​of more than 95% of the parallel samples.

[0069] For the interpretation of the results of the fluorescence immunochromatographic assay reagent, the reading value comes from the dry immunofluorescence analyzer of Wuxi Tianzong Yijun Biotechnology, model: TFL1600. The value indicates the intensity of the fluorescence signal, and the larger the value, the stronger the fluorescence signal.

[0070] The method for interpreting the results of the latex immunochromatographic assay is the same as in Example 1.

[0071] The experimental results are shown in the table below:

[0072] Table 4

[0073]

[0074] Based on the results of Example 2 and Figure 1 As can be seen, when using latex immunochromatographic test strips, the membrane surface of group 3 with lysis buffer was reddish, while the membrane surface of group 4 with lysis buffer was clean, and the C line was clearer. This indicates that the addition of NP-40 to the buffer in the COVID-19 latex immunochromatographic test strips reduced the matrix effect. When using fluorescent immunochromatographic test strips, fluorescent particles remained on the membrane surface of group 3 with lysis buffer, while the membrane surface of group 4 with lysis buffer was clean, the readings were clearer, and the readings were improved. Figure 2 The conclusion that can be drawn is that the addition of NP-40 to the buffer reduces the matrix effect, significantly eliminates the background, improves the detection signal value, improves the membrane surface, and reduces the matrix effect to a certain extent.

[0075] Example 3

[0076] Prepare the following lysis buffer according to Table 5:

[0077] Table 5

[0078]

[0079]

[0080] The lysis buffer in Table 5 was used to test the sample from Example 1 using the influenza A virus latex immunochromatographic test strip. The sample was tested according to the method in Example 1, and the results are shown in Table 6.

[0081] Table 6

[0082] pyrolysis solution C-line output value 5% pyrolysis solution 9 6 pyrolysis solution 8+ pyrolysis solution 7 8+ PBS control group -

[0083] The results of Example 3 showed that lysis buffers 5 to 7 could effectively lyse human epithelial cells and fully release the GAPDH therein. Among them, lysis buffer 5 had the highest reading, indicating that lysis buffer 5 had the best lysis effect.

[0084] Example 4

[0085] Referring to Example 3 of Chinese Patent Application CN118624896A (Application No. CN202410857418.8, entitled "Immunochromatographic test strip that ensures sampling validity and its application in the preparation of human sample detection products"), the eluent of negative oral exudate was used as the detection matrix. The collected negative oral exudate homogenized eluent was used as a negative sample for testing using HIV colloidal gold immunochromatographic test strip and lysis buffer 5, and the appearance of the C-line was observed. All experimental groups used three or more parallel samples for testing, and the experimental results are shown in Table 7 below.

[0086] Table 7

[0087] pyrolysis solution C-line output value PBS 7 physiological saline 7 5% pyrolysis solution 8

[0088] The results of Example 4 show that when lysis buffer 5 was used as a diluent for sample loading, the C-line was stronger than that of physiological saline and PBS as diluents. Therefore, this indicates that the HSA target was higher in the lysis buffer 5 loading system than in PBS and physiological saline, thus proving that the lysis buffer 5 formulation can effectively lyse oral epithelial cells in negative samples and release the detection target.

[0089] Example 5

[0090] Hepatitis B surface antigen colloidal gold immunochromatographic test strips were used, with whole blood from hepatitis B surface antigen-negative individuals as the negative sample. The C-line detection target was hemoglobin in whole blood erythrocytes. The homogenized eluate of collected hepatitis B surface antigen-negative individuals was also used as a negative sample for testing, and the C-line elution was observed. All experimental groups used three or more parallel samples for testing. The experimental results are shown in Table 8.

[0091] Table 8

[0092] pyrolysis solution C-line output value PBS 8 physiological saline 8 5% pyrolysis solution 8

[0093] The results of Example 5 demonstrate that lysis buffer 5 can be used as a diluent for sample loading, and the C-line readings showed no significant difference compared to those using physiological saline and PBS as diluents. Therefore, it can be concluded that the hemoglobin target content in human whole blood erythrocytes loaded with lysis buffer 5 in this loading system is no different from that in human whole blood treated with PBS and physiological saline, thus proving that the lysis buffer 5 formulation is more suitable for lysing oral epithelial cells in negative samples to release the detection target.

[0094] Example 6

[0095] The effects of different concentrations of sodium azide in the lysis buffer on the sample lysis efficiency were compared. The lysis buffer was prepared according to Table 9, and the influenza A virus latex immunochromatographic test strip was used for detection. The detection method was the same as in Example 1, and the detection results are shown in Table 9.

[0096] Table 9

[0097]

[0098] Based on the above experimental results, it can be seen that the test strip has the highest sensitivity when the concentration of sodium azide is 0.2%wt. When the concentration of sodium azide is between 0.05%wt and 0.3%wt, the overall sensitivity of the test strip is affected by less than half a color degree, and its main function is to prevent corrosion of the solution. The preferred concentration of sodium azide is 0.2%wt.

[0099] Example 7

[0100] The effects of different concentrations of surfactant S-22 in the lysis buffer on the lysis effect of the samples were compared. The lysis buffer was prepared according to Table 10, and the influenza A virus latex immunochromatographic test strip was used for detection. The detection method was the same as in Example 1, and the detection results are shown in Table 10.

[0101] Table 10

[0102]

[0103]

[0104] S-22 acts as a wetting and penetrating agent, which is beneficial to the immunochromatographic process. As shown in the table above, surfactant concentrations in the range of 0.05%wt-0.1%wt improve the detection sensitivity of the test strip. However, the test strip exhibits the highest sensitivity at a concentration of 0.1%wt.

[0105] Example 8

[0106] The effects of different concentrations of tris(hydroxymethyl)aminomethane in the lysis buffer on the lysis efficiency of the samples were compared. The lysis buffer was prepared according to Table 11, and the influenza A virus latex immunochromatographic test strip was used for detection. The detection method was the same as in Example 1, and the detection results are shown in Table 11.

[0107] Table 11

[0108]

[0109] Tris(hydroxymethyl)aminomethane primarily functions as a stabilizer for the pH of solutions. As shown in the table above, the amount of tris(hydroxymethyl)aminomethane added significantly affects the sensitivity of the test strips, with an optimal Tris content of 0.6% wt yielding the best results.

[0110] Example 9

[0111] The effects of different concentrations of NP-40 in the lysis buffer on the sample lysis efficiency were compared. The lysis buffer was prepared according to Table 12, and the influenza A virus latex immunochromatographic test strip was used for detection. The detection method was the same as in Example 1, and the detection results are shown in Table 12.

[0112] Table 12

[0113]

[0114] The table above shows that when the amount of NP-40 added is between 0.5% wt and 1% wt, the sensitivity to reagents is improved. The improvement is more significant when the amount added is 1% wt.

[0115] The immunochromatographic test strips used in the above embodiments are as follows:

[0116] Influenza A virus latex immunochromatographic test strip:

[0117] The quality control line (C-line) system described in Chinese patent application CN116223796A (application number CN202310156113.X, invention title "Virus Combined Detection Immunochromatographic Test Strip, Detection Pen and Detection Product") is adopted. The structure of the influenza A virus latex immunochromatographic test strip is as follows:

[0118] The latex immunochromatographic assay includes a sample pad, a conjugation pad, a detection pad, and an absorbent pad, which are sequentially arranged on a fixed base plate along the chromatography direction of the sample to be tested.

[0119] The test pad is coated with a first-line influenza A virus antibody (Hangzhou Xuke Biotechnology, catalog number R1403) and a first-line human internal reference protein antibody (Hangzhou Huaan Biotechnology Co., Ltd. GAPDH antibody, catalog number ET1601-4). Both the first-line influenza A virus antibody and the first-line human internal reference protein antibody are labeled with fluorescent particles. Along the chromatographic direction of the sample to be tested, the test pad includes a test line T and a sampling control line C. The test line T is coated with a second-line influenza A virus antibody (Hangzhou Xuke Biotechnology, catalog number R1401), and the sampling control line C is coated with a second-line human internal reference protein antibody (Hangzhou Huaan Biotechnology Co., Ltd. GAPDH antibody, catalog number ET1601-4). The first-line and second-line influenza A virus antibodies recognize the influenza A virus N protein (nucleocapsid protein). The first-line and second-line human internal reference protein antibodies recognize glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0120] The concentration of the second influenza A virus antibody in the coating solution of the test pad was 0.7 mg / mL, and the concentration of the second human internal reference protein antibody was 1 mg / mL. The coating solution of the test pad was a solution containing 0.9 wt% NaCl, 0.13 wt% Na₂HPO₄, and 3 wt% sucrose. The spray volume of the test pad coating solution on the test line T and the sampling control line C was 1 μL / cm.

[0121] Novel Coronavirus Fluorescent Immunochromatographic Test Strip:

[0122] The quality control line (C-line) system described in Chinese patent application CN116223796A (application number CN202310156113.X, invention title "Virus Combined Detection Immunochromatographic Test Strip, Detection Pen and Detection Product") is adopted. The structure of the fluorescent immunochromatographic test strip is as follows:

[0123] The fluorescence immunochromatographic assay includes a sample pad, a conjugation pad, a detection pad, and an absorbent pad, which are sequentially arranged on a fixed base plate along the chromatography direction of the sample to be tested.

[0124] The test pad is coated with a first novel coronavirus antibody (Hangzhou Xuke Biotechnology Co., Ltd., catalog number RM0032) and a first human internal reference protein antibody (Hangzhou Huaan Biotechnology Co., Ltd. GAPDH antibody, catalog number: ET1601-4)). Both the first novel coronavirus antibody and the first human internal reference protein antibody are labeled with fluorescent particles. The test pad includes a test line T and a sampling control line C along the chromatographic direction of the sample. The test line T is coated with a second novel coronavirus antibody (Hangzhou Xuke Biotechnology Co., Ltd., catalog number RM0033), and the sampling control line C is coated with a second human internal reference protein antibody (Hangzhou Huaan Biotechnology Co., Ltd. GAPDH antibody, catalog number: ET1601-4)). The first and second novel coronavirus antibodies recognize the novel coronavirus N protein (nucleocapsid protein). The first and second human internal reference protein antibodies recognize glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0125] The concentration of the second novel coronavirus antibody in the coating solution of the test pad was 0.5 mg / mL, and the concentration of the second human internal reference protein antibody was 1 mg / mL. The coating solution of the test pad was a solution containing 0.9 wt% NaCl, 0.13 wt% Na₂HPO₄, and 3 wt% sucrose. The spray volume of the coating solution on the test line T and the sampling control line C was 1 μL / cm.

[0126] Novel Coronavirus Latex Immunochromatographic Test Strips:

[0127] The quality control line (C-line) system described in Chinese patent application CN116223796A (application number CN202310156113.X, invention title "Virus Combined Detection Immunochromatographic Test Strip, Test Pen and Detection Product") is adopted. The structure of the immunochromatographic test strip is as follows:

[0128] The latex immunochromatographic assay includes a sample pad, a conjugation pad, a detection pad, and an absorbent pad, which are sequentially arranged on a fixed base plate along the chromatography direction of the sample to be tested.

[0129] The test pad is coated with a first novel coronavirus antibody (Hangzhou Xuke Biotechnology Co., Ltd., catalog number RM0032) and a first human internal reference protein antibody (Hangzhou Huaan Biotechnology Co., Ltd. GAPDH antibody, catalog number: ET1601-4)). Both the first novel coronavirus antibody and the first human internal reference protein antibody are labeled with fluorescent particles. The test pad includes a test line T and a sampling control line C along the chromatographic direction of the sample. The test line T is coated with a second novel coronavirus antibody (Hangzhou Xuke Biotechnology Co., Ltd., catalog number RM0033), and the sampling control line C is coated with a second human internal reference protein antibody (Hangzhou Huaan Biotechnology Co., Ltd. GAPDH antibody, catalog number: ET1601-4)). The first and second novel coronavirus antibodies recognize the novel coronavirus N protein (nucleocapsid protein). The first and second human internal reference protein antibodies recognize glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0130] The concentration of the second novel coronavirus antibody in the coating solution of the test pad was 1.0 mg / mL, and the concentration of the second human internal reference protein antibody was 1 mg / mL. The coating solution of the test pad was a solution containing 0.9 wt% NaCl, 0.13 wt% Na₂HPO₄, and 3 wt% sucrose. The spray volume of the coating solution on the test line T and the sampling control line C was 1 μL / cm.

[0131] HIV colloidal gold immunochromatographic test strip:

[0132] This colloidal gold immunochromatographic test strip is an HIV colloidal gold immunochromatographic test strip, using the immunochromatographic test strip described in Experimental Group 3 of Example 6 in Chinese Patent Application CN118091132A (application number CN202410258366.2, invention title "CN202410258366.2"). The C line and T line are coated with human serum albumin-specific antibodies, which were purchased from Hangzhou Xuke Biotechnology Co., Ltd., with catalog numbers RM0083 and RM0084, respectively. Other conditions are the same as the test strip provided in CN118091132A.

[0133] Hepatitis B surface antigen colloidal gold immunochromatographic test strip:

[0134] The chromatographic test strip used was the one described in Example 6 of Chinese Patent Application CN118091132A (application number CN202410857418.8, entitled "Immunochromatographic test strip that can ensure sampling validity and its application in the preparation of human sample detection products").

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for cell lysis, characterized in that, The composition comprises, by weight parts: 4-5 parts NaCl, 0.5-5 parts bovine serum albumin, 1-10 parts Triton X-100, 0.1-1 parts tetrasodium ethylenediaminetetraacetate, 0.05-0.3 parts sodium azide, 0.1-1 parts tris(hydroxymethyl)aminomethane, 0.05-0.2 parts surfactant S-22, and 0.5-2 parts NP-40; The cells in question are epithelial cells.

2. The composition for cell lysis according to claim 1, characterized in that, The composition comprises, by weight parts: 4 parts NaCl, 1 part bovine serum albumin, 5 parts Triton X-100, 0.5 parts tetrasodium EDTA, 0.2 parts sodium azide, 0.6 parts tris(hydroxymethyl)aminomethane, 0.1 parts surfactant S-22, and 1 part NP-40.

3. The composition for cell lysis according to claim 2, characterized in that, The composition further includes a solvent; The concentrations of each component in the solvent of the composition are as follows: NaCl 4~5%wt, bovine serum albumin 0.5~5%wt, Triton X-100 1~10%wt, tetrasodium ethylenediaminetetraacetate 0.1~1%wt, sodium azide 0.05~0.3%wt, tris(hydroxymethyl)aminomethane 0.1~1%wt, surfactant S-22 0.05~0.2%wt, and NP-40 0.5~2%wt.

4. The composition for cell lysis according to claim 3, characterized in that, The concentrations of each component in the solvent in the composition are as follows: NaCl 4%wt, serum albumin 1%wt, Triton X-100 5%wt, tetrasodium ethylenediaminetetraacetate 0.5%wt, sodium azide 0.2%wt, tris(hydroxymethyl)aminomethane 0.6%wt, surfactant S-22 0.1%wt, and NP-40 1%wt.

5. The use of the composition for cell lysis according to any one of claims 1 to 4 in the lysis of epithelial cells for non-diagnostic and therapeutic purposes, or in the preparation of a kit for detecting epithelial cell lysis products.

6. A test kit, characterized in that, The composition comprising any one of claims 1 to 4 for cell lysis.

7. The detection kit according to claim 6, characterized in that, It also includes reagents for detecting cell lysis products.

8. The detection kit according to claim 7, characterized in that, The test kit also includes test reagents for any of the following detection methods: immunochromatographic assay, flow cytometry assay, immunoblotting assay, immunohistochemistry assay, ELISA assay, immunoprecipitation assay, or immunomagnetic bead assay. The immunohistochemical detection includes immunofluorescence staining detection; The immunoprecipitation assay includes immunocoprecipitation assay.

9. The detection kit according to claim 8, characterized in that, The test kit includes immunochromatographic strips.

10. The detection kit according to claim 9, characterized in that, The markers in the immunochromatographic test strip include one or more of the following: fluorescent particles, colored particles, latex particles, chemiluminescent reagents, fluorescent molecules, colloidal gold, colloidal silver, colloidal selenium, horseradish peroxidase, and alkaline phosphatase. The fluorescent particles include quantum dots.

11. The detection kit according to claim 9, characterized in that, The control line of the immunochromatographic test strip uses internal reference protein as the detection target molecule.

12. The detection kit according to claim 8, characterized in that, The test kit also includes reagents for detecting internal reference proteins.

13. The detection kit according to claim 12, characterized in that, The internal reference protein includes one or more of the following: glyconuclease P, glyceraldehyde-3-phosphate dehydrogenase, β-microglobulin, β-tubulin, α-tubulin, 18sRNA, β-actin actin, and TATA-binding protein.

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