A Hep-2 cell extract preparation process suitable for preparing fluorescent immunochromatographic test strips for detecting antinuclear antibodies and a method for preparing the test strips

Abstract

The present invention provides a kind of Hep 2 cell extract preparation process and preparation method of test strip suitable for preparing antinuclear antibody fluorescent immunochromatographic test strip. The present invention transfers the Hep 2 cells obtained by culture into PBS buffer, and the obtained Hep 2 cell suspension is centrifuged to obtain Hep 2 cell agglomerates, and the mixture of Hep 2 cell agglomerates and extraction buffer is ultrasonicated in an ice bath, and the mixture after ultrasonication is centrifuged, and the supernatant is collected, and the supernatant is passed through a microfiltration membrane. The obtained filtrate is the Hep 2 cell extract, and the extraction buffer is a HEPES buffer containing isopropanol, trehalose, bovine serum albumin and phenylmethylsulfonyl fluoride. The prepared HEP 2 cell extract is used for preparing antinuclear antibody fluorescent immunochromatographic test strip, which can take into account the reliability of detection sensitivity, specificity and test results.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay analysis, and particularly relates to a process for preparing a Hep-2 cell extract suitable for preparing a fluorescent immunochromatographic test strip for detecting antinuclear antibodies and a method for preparing the test strip. Background Art

[0002] Antinuclear antibodies (ANA) are a general term for autoantibodies that target eukaryotic cell components. They are important biomarkers of autoimmune diseases (AIDs) and the most widely used group of autoantibodies in clinical practice. They are commonly found in patients with systemic (non-organ-specific) AIDs, such as systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, mixed connective tissue disease, and polymyositis / dermatomyositis. ANAs can also be seen in patients with chronic infectious diseases and cancer. Therefore, the detection of ANAs is of great clinical significance for the diagnosis, differential diagnosis, typing, and monitoring of disease activity in AIDs.

[0003] The main methods for detecting antinuclear antibodies include indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA), immunoblotting, and chemiluminescence. Different detection methods have different advantages and disadvantages for clinical application. For example, while indirect immunofluorescence is intuitive, it is highly subjective and requires a high level of expertise from the tester. Interpretation of fluorescence patterns can vary between laboratories. While ELISA and immunoblotting offer high sensitivity, they are complex and time-consuming, and immunoblotting results are primarily semi-quantitative. While chemiluminescence offers the advantages of high sensitivity, specificity, quantification, and automation, it is expensive and requires expensive instrumentation. Currently, there are no fluorescent immunochromatographic test strips for detecting antinuclear antibodies on the market. Fluorescent immunochromatographic technology offers the advantages of ease of use, short detection time, and minimal requirements for both personnel and instrumentation. The development of fluorescent immunochromatographic test strips for detecting antinuclear antibodies could enable rapid detection of antinuclear antibodies, reduce the difficulty and cost of testing, and improve the efficiency of clinical diagnosis and treatment.

[0004] Typically, when developing antinuclear antibody fluorescent immunochromatographic test strips, a stock antigen solution is required. However, no stock antinuclear antibody antigen solution has been developed specifically for use in antinuclear antibody fluorescent immunochromatographic test strips. When using commercially available recombinant antinuclear antibody antigens to prepare antinuclear antibody fluorescent immunochromatographic test strips, the positive miss rate is high. The indirect immunofluorescence assay using Hep-2 cells is an important method for antinuclear antibody detection, suggesting that Hep-2 cell extracts can be used in place of the stock antigen solution. However, different extraction processes lead to differences in the composition of Hep-2 cell extracts, which directly affect the sensitivity of antinuclear antibody detection. Different extraction processes also lead to differences in the stability of Hep-2 cell extracts. The poorer the stability of the Hep-2 cell extracts, the greater the batch-to-batch variability of test strips produced from different batches. These are the key reasons why there are still no reliable antinuclear antibody fluorescent immunochromatographic test strips on the market. Summary of the Invention

[0005] The problem to be solved by the present invention is to provide a preparation process of Hep-2 cell extract suitable for preparing fluorescent immunochromatographic test strips for detecting antinuclear antibodies.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A first aspect of the present invention provides a preparation process for a Hep-2 cell extract. The cultured Hep-2 cells are transferred to a 5-15 mM PBS buffer solution with a pH value of 7.2-7.6. The obtained Hep-2 cell suspension is centrifuged to obtain Hep-2 cell aggregates. A mixture of the Hep-2 cell aggregates and the extraction buffer solution is sonicated in an ice bath. The sonicated mixture is centrifuged to collect the supernatant. The supernatant is passed through a microfiltration membrane to obtain a filtrate, which is the Hep-2 cell extract. The extraction buffer is a 5-15 mM HEPES buffer solution with a pH value of 7.2-7.6, containing 3-8 vol% isopropanol, 3-8 wt% trehalose, 0.3-0.8 wt% bovine serum albumin, and 0.8-1.2 mM phenylmethylsulfonyl fluoride.

[0008] In some embodiments, the ultrasound conditions are: ultrasound power 140-160 W, pulse time 4-6 s, interval time 4-10 s, and total duration 4-5 min.

[0009] In some embodiments, the amount of the extraction buffer is (2-8)×10 6 20-80 μL of the extraction buffer is used for each cell. It is further preferred that the amount of the extraction buffer is (4-6)×10 6 Use 40-60 μL of the extraction buffer for each cell.

[0010] In some embodiments, the centrifugation conditions of the Hep-2 cell suspension are: centrifugation temperature 3-5° C., centrifugal force 2000-3000 g, and centrifugation time 25-35 min.

[0011] In some embodiments, the centrifugation conditions of the mixture after ultrasound are: centrifugation temperature 3-5° C., centrifugal force 10,000-20,000 g, and centrifugation time 25-35 min.

[0012] In some embodiments, the pore size of the microfiltration membrane is 0.4-0.6 μm.

[0013] The second aspect of the present invention provides a Hep-2 cell extract prepared by the above preparation process.

[0014] The third aspect of the present invention provides the use of the above-mentioned Hep-2 cell extract in the preparation of fluorescent immunochromatographic test strips for detecting antinuclear antibodies.

[0015] A fourth aspect of the present invention provides a fluorescent immunochromatographic test strip for detecting antinuclear antibodies, comprising a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane and absorbent paper sequentially mounted on the base plate, wherein the nitrocellulose membrane is provided with a detection line and a quality control line, the conjugate pad is coated with fluorescent microsphere-labeled mouse anti-human IgG antibody and fluorescent microsphere-labeled DNP-BSA, and the detection line is formed by the above-mentioned Hep-2 cell extract.

[0016] In some embodiments, the fluorescent microspheres are europium chelated fluorescent microspheres. Further, the particle size of the fluorescent microspheres is 100 nm to 300 nm, more preferably 150 nm to 250 nm, and even more preferably 180 nm to 220 nm.

[0017] In some embodiments, the sample pad is a glass cellulose membrane coated with casein, Tween, trehalose and a heterophilic antibody blocking agent.

[0018] In some embodiments, the conjugate pad is a glass cellulose membrane, and the volume ratio of fluorescent microsphere-labeled mouse anti-human IgG antibody and fluorescent microsphere-labeled DNP-BSA coated thereon is (10-30):1, more preferably (10-20):1, and even more preferably (10-15):1.

[0019] In some embodiments, the quality control line is formed by DNP antibody coated on the nitrocellulose membrane.

[0020] A fifth aspect of the present invention provides an antinuclear antibody detection kit, which comprises the above-mentioned fluorescent immunochromatographic test strip for detecting antinuclear antibodies and a diluent.

[0021] The sixth aspect of the present invention provides an antinuclear antibody quantitative detection system based on fluorescent immunoassay, the antinuclear antibody quantitative detection system includes a fluorescent immunoassay analyzer, the above-mentioned fluorescent immunochromatographic test strip and a diluent, the fluorescent immunochromatographic test strip is provided with an ID card, and the ID card stores calibration data and / or a standard curve.

[0022] In some embodiments, the diluent is a 5-15 mM PB buffer solution containing 40-60 g / L sodium chloride, 0.3-0.8 wt % preservative, and 0.3-0.8 wt % Tween with a pH of 7.2-7.6.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The present invention provides a process for preparing a Hep-2 cell extract suitable for preparing a fluorescent immunochromatographic test strip for detecting antinuclear antibodies. By optimizing and adjusting the extraction buffer formula and the extraction method, the obtained fluorescent immunochromatographic test strip for detecting antinuclear antibodies prepared from the Hep-2 cell extract has high sensitivity, good specificity, and a low positive missed detection rate. The Hep-2 cell extract prepared by the preparation process of the present invention has good stability, and can reduce the batch-to-batch variability of fluorescent immunochromatographic test strips for detecting antinuclear antibodies using different batches of the same batch of Hep-2 cell extracts stored at 4°C within a week. It is unnecessary to prepare the Hep-2 cell extract before each preparation of the fluorescent immunochromatographic test strip for detecting antinuclear antibodies or to repeatedly thaw the Hep-2 cell extract stored at -80°C. This simplifies the operating process, avoids reagent bottle rupture or precipitate formation caused by repeated thawing, improves production efficiency, reduces production costs, and reduces the risk of false negatives caused by fluctuations in storage conditions, thereby facilitating large-scale production and application of fluorescent immunochromatographic test strips for detecting antinuclear antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the structure of the fluorescent immunochromatographic test strip for detecting antinuclear antibodies according to Example 1;

[0026] In the figure: 1. Base plate; 2. Sample pad; 3. Conjugate pad; 4. Nitrocellulose membrane; 5. Absorbent paper; 6. Quality control line; 7. Antinuclear antibody test line. DETAILED DESCRIPTION

[0027] In order to adapt to the fluorescent immunochromatography technology, the inventors of this application have conducted extensive research on the preparation process of HEP-2 cell extracts, including the optimization and adjustment of the extraction buffer formula and the extraction method. This has resulted in a fluorescent immunochromatographic test strip for detecting antinuclear antibodies prepared from the obtained HEP-2 cell extracts with high sensitivity, good specificity, and a low positive missed detection rate. In addition, the obtained HEP-2 cell extracts have good stability, which can reduce the batch-to-batch differences between different batches of fluorescent immunochromatographic test strips for detecting antinuclear antibodies using the same batch of HEP-2 cell extracts stored at 4°C within a week. Based on this, the present application also provides a fluorescent immunochromatographic test strip for detecting antinuclear antibodies based on Hep-2 cell extracts that is fast and has reliable test results.

[0028] The fluorescent immunochromatographic test strip for detecting antinuclear antibodies of the present invention is based on an indirect immunofluorescence method. If a sample to be tested contains antinuclear antibodies, the antinuclear antibodies in the sample to be tested will bind to mouse anti-human IgG labeled with fluorescent microspheres in a sample pad and chromatograph along a nitrocellulose membrane through capillary action. When reaching a detection area, the antinuclear antibodies are captured by a HEP-2 cell extract fixed on a corresponding detection line. The amount of fluorescent microsphere labels bound to the detection line is proportional to the amount of antinuclear antibodies in the sample. The content of antinuclear antibodies in the sample to be tested is calculated using a fluorescent immunoassay based on the fluorescence intensity and a standard curve.

[0029] The present invention is further described below with reference to the following examples. However, the present invention is not limited to the following examples. The technical features involved in the various embodiments of the present invention may be combined with each other as long as they do not conflict with each other. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific applications. The implementation conditions not specified are conventional conditions in the industry.

[0030] The experimental methods in the following examples and comparative examples, unless otherwise specified, are conventional methods, and the experimental materials used, unless otherwise specified, are purchased from conventional biochemical reagent manufacturers.

[0031] It should be noted that in the following examples and comparative examples, when phenylmethylsulfonyl fluoride is involved, the components other than phenylmethylsulfonyl fluoride can be pre-mixed and used, and phenylmethylsulfonyl fluoride is added before resuspending the cell pellet.

[0032] Example 1: This example provides a method for preparing a Hep-2 cell extract, as follows:

[0033] Hep-2 cells (ATCC source) were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C, 5% CO2, and humidity ≥95%. 6After 10 flasks that meet the cell growth requirements, remove the original culture medium and replace it with an equal volume of 10 mM PBS buffer (pH 7.4, sterile). Use a sterile cell scraper to gently scrape the surface of the culture flask from one corner of the culture flask to make the Hep-2 cells fall off the flask wall to form a Hep-2 cell suspension. Combine the Hep-2 cell suspensions in the 10 culture flasks.

[0034] The harvested Hep-2 cell suspension was centrifuged at 3000 rpm at 4°C. The supernatant was removed, and the cell pellet was collected and resuspended in 500 μL of pre-chilled 4°C extraction buffer (10 mM HEPES buffer, pH 7.4, sterile) containing 5 vol% isopropanol, 5 wt% trehalose, 0.5 wt% bovine serum albumin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The resuspension was placed on ice for 15 min and then sonicated in an ice bath at 150 W, 5 s pulse duration, 5 s interval duration, for a total of 5 min. After sonication, the cell suspension was centrifuged at 15,000 g for 30 min at 4°C. The supernatant was collected and filtered through a 0.45 μm filter. The filtrate, the HEP-2 cell extract, was stored at 4°C until ready for use.

[0035] Comparative Example 1: This comparative example provides a method for preparing a Hep-2 cell extract, which is as follows:

[0036] Hep-2 cells were cultured in DMEM medium containing 10 wt% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C, 5% CO2, and humidity ≥95%. 6 After that, take 10 culture flasks that meet the cell growth requirements, aspirate the original culture medium and replace it with an equal volume of 10mM PBS buffer (pH 7.4) as the original culture medium, use a sterile cell scraper to start from one corner of the culture flask and gently scrape along the surface of the culture flask to make the Hep-2 cells fall off the flask wall to form a Hep-2 cell suspension, and combine the Hep-2 cell suspensions in the 10 culture flasks.

[0037] The collected Hep-2 cell suspension was centrifuged at 3000 rpm at 4°C. The supernatant was removed and the cell pellets were collected. The cell pellets were resuspended in 500 μL of pre-chilled 4°C 10 mM PBS buffer (pH 7.4, sterile). The resulting resuspension was placed on ice for 15 minutes. NP-40 solution (10%, sterile, Shanghai Yuanye Biotechnology Co., Ltd.) was added to the resuspension at a volume of 5 vol%. The mixture was vortexed for 10 seconds and the resulting homogenate was centrifuged at 3000 rpm at 4°C for 10 minutes to separate the supernatant and cell pellets.

[0038] The resulting cell pellet was resuspended in 500 μL of pre-chilled 4°C secondary extraction solution (10 mM sterile PBS buffer (pH 7.4) containing 1 wt% ethylphenyl polyethylene glycol (NP-40), 0.5 wt% sodium deoxycholate, and 0.5 wt% Tween-20). The resulting resuspension was placed on ice for 30 minutes, then vortexed for 10 minutes. The resulting homogenate was centrifuged at 14,000 rpm for 30 minutes at 4°C, and the supernatant was separated.

[0039] The supernatants obtained from the two separations were combined, and 5 wt % trehalose and 0.5 wt % bovine serum albumin were added to obtain a Hep-2 cell extract, which was placed in a refrigerator at 4° C. for later use.

[0040] Comparative Example 2: This comparative example provides a method for preparing a Hep-2 cell extract. The preparation method is basically the same as that in Example 1, except that the extraction buffer used in preparing the Hep-2 cell extract is different. The extraction buffer used in this comparative example is 25 mM Tris buffer (pH 7.6, sterile) containing 150 mM NaCl, 1 wt% NP40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 1 mM phenylmethylsulfonyl fluoride (PMSF).

[0041] Comparative Example 3: This comparative example provides a method for preparing a Hep-2 cell extract. The preparation method is basically the same as that in Example 1, except that the extraction buffer used in preparing the Hep-2 cell extract is different. The extraction buffer used in this comparative example is 10 mM PBS buffer (pH 7.4, sterile) containing 5 vol% methanol, 5 wt% trehalose, 0.5 wt% bovine serum albumin and 1 mM phenylmethylsulfonyl fluoride.

[0042] Comparative Example 4: This comparative example provides a method for preparing a Hep-2 cell extract, which is basically the same as that in Example 1, except that the extraction buffer used in preparing the Hep-2 cell extract is different. The extraction buffer used in this comparative example is 50 mM Tris buffer (pH 7.5, sterile) containing 5 vol% glycerol, 5 wt% trehalose, 0.5 wt% casein and 1 mM phenylmethylsulfonyl fluoride.

[0043] Application Example 1: This application example provides a fluorescent immunochromatographic test strip for detecting antinuclear antibodies.

[0044] like Figure 1As shown, the fluorescent immunochromatographic test strip for detecting antinuclear antibodies in this embodiment includes a base plate 1, a sample pad 2, a conjugate pad 3, a nitrocellulose membrane 4, and absorbent paper 5, which are sequentially attached to the base plate 1. The nitrocellulose membrane 4 (Sartorius, Germany) is provided with a quality control line 6 and an antinuclear antibody detection line 7. The base plate 1 is made of PVC, and the sample pad 2 and conjugate pad 3 are both made of glass cellulose membrane (Ahlstrom, Cat. No. 1UN14ER100025NT). The fluorescent immunochromatographic test strip for detecting antinuclear antibodies in this embodiment is provided with sample wells (not shown). The structural configuration of the fluorescent immunochromatographic test strip, including the arrangement of the sample wells, is conventional in the art and will not be described in detail here.

[0045] The preparation method of the fluorescent immunochromatographic test strip for detecting antinuclear antibodies in this application example is as follows:

[0046] 1. Preparation of sample pad 2

[0047] The glass fiber membrane was evenly moistened with the sample pad blocking solution, then placed in an environment with a humidity of less than 30% and baked at 37°C for 15 hours before cutting. The sample pad blocking solution consisted of 50 mM Tris buffer (pH 8.5) containing 0.3 wt% casein, 1 wt% Tween-20, 5 wt% trehalose, and 0.1 mg / mL heterophilic antibody blocking agent (Baode Kangti (Beijing) Biotechnology Co., Ltd., Cat. No. 3KC028).

[0048] 2. Preparation of conjugate pad 3

[0049] Fluorescent microspheres (Europium-chelated fluorescent microspheres, particle size 200 nm, Hangzhou Boyue Biotechnology Co., Ltd., Cat. No. EU0200C1) were added to 1 mL of 50 mM MES buffer (pH 6.0) to a solids content of 0.1%. After ultrasonic mixing, the supernatant was removed by centrifugation. 0.5 mL of 50 mM MES buffer (pH 6.0) was added, ultrasonic mixing was performed, and then 50 μL of 10 mg / mL NHS solution and 25 μL of 10 mg / mL EDC solution were added. Activation was performed for 30 min, and the supernatant was removed by centrifugation. 1 mL of 50 mM MES buffer (pH 6.0) was added, ultrasonic mixing was performed, and the supernatant was removed by centrifugation. 0.5 mL of 50 mM MES buffer (pH 6.0) was added again, ultrasonic mixing was performed, and mouse anti-human IgG antibody was added to a final concentration of 100 μg / mL. The mixture was incubated on a shaker at 25°C, 250 rpm, for 2 h. The supernatant was removed by centrifugation, and 0.5 mL of blocking buffer was added. The cells were shaken at 25°C, 250 rpm, and incubated for 1 h. The supernatant was removed by centrifugation, and 1 mL of blocking buffer was added again for washing. The supernatant was removed by centrifugation to obtain fluorescent microsphere-labeled mouse anti-human IgG antibody (Beijing Borsi Biotechnology Co., Ltd., Catalog No. B1423). The blocking buffer consisted of 50 mM HEPES buffer (pH 8.5) containing 1 wt% bovine serum albumin. The fluorescent microsphere-labeled mouse anti-human IgG antibody was stored in a storage buffer (50 mM Tris buffer (pH 8.5) containing 150 mM NaCl, 10 wt% trehalose, 0.5 wt% Tween-20, and 0.5 wt% casein), mixed by ultrasonication, and used until ready for use.

[0050] Fluorescent microspheres (Europium-chelated fluorescent microspheres, particle size 200 nm, Hangzhou Boyue Biotechnology Co., Ltd., Catalog No. EU0200C1) were added to 1 mL of 50 mM MES buffer (pH 6.0) to a solids content of 0.1%. The supernatant was removed by centrifugation. 0.5 mL of 50 mM MES buffer (pH 6.0) was added and ultrasonically mixed. Then, 50 μL of 10 mg / mL NHS solution and 25 μL of 10 mg / mL EDC solution were added. Activate the solution for 30 minutes and centrifuge to remove the supernatant. 1 mL of 50 mM MES buffer (pH 6.0) was added and ultrasonically mixed. The supernatant was removed by centrifugation. 0.5 mL of 50 mM MES buffer (pH 6.0) was added again, and after ultrasonic mixing, DNP-BSA (Nanjing Fuxiao Biotechnology Co., Ltd., Catalog No. T112505R) was added to a final concentration of 100 μg / mL. The cells were incubated on a shaker at 25°C, 250 rpm, for 2 h. The supernatant was removed by centrifugation, and 0.5 mL of blocking buffer was added. The cells were incubated on a shaker at 25°C, 250 rpm, for 1 h. The supernatant was removed by centrifugation, and 1 mL of blocking buffer was added again for washing. The supernatant was removed by centrifugation to obtain fluorescent microsphere-labeled DNP-BSA. The blocking buffer was 50 mM HEPES buffer (pH 8.5) containing 1 wt% bovine serum albumin. The fluorescent microsphere-labeled DNP-BSA was stored in a preservation solution containing 150 mM NaCl, 10 wt% trehalose, 0.5 wt% Tween-20, and 0.5 wt% casein in 50 mM Tris buffer (pH 8.5), and ultrasonically mixed for later use.

[0051] Prepare the gold spray solution: 60% by volume of fluorescent microsphere-labeled mouse anti-human IgG antibody, 3% by volume of fluorescent microsphere-labeled DNP-BSA, and 37% by volume of the preservation solution. Use a gold sprayer to evenly apply the gold spray solution to the glass fiber membrane at a rate of 2 μL / cm. Bake at 37°C for 15 hours and place in a desiccating oven until ready for use. Cut the glass fiber membrane to the desired size. Use a gold sprayer to evenly apply the gold spray solution to the glass fiber membrane at a rate of 2 μL / cm. Bake at 37°C for 15 hours and place in a desiccating oven until ready for use.

[0052] 3. Preparation of Nitrocellulose Membrane 4

[0053] DNP antibody was diluted with 10 mM PB buffer (pH 7.4) containing 5 wt% trehalose to a final concentration of 2 mg / mL. A nitrocellulose membrane was streaked with the DNP antibody coating solution at a rate of 1 μL / cm using a streaker. The HEP-2 cell extract prepared in Example 1 was streaked with the streaker at a rate of 1 μL / cm. The nitrocellulose membrane was dried in a 37°C oven for 1-2 hours, then transferred to a drying oven and dried at 60°C to form control line 6 (coated with DNP antibody) and antinuclear antibody detection line 7 (coated with HEP-2 cell extract). The membrane was then placed in a drying oven until ready for use.

[0054] 4. Assembly

[0055] The sample pad 2, conjugate pad 3, nitrocellulose membrane 4 and absorbent paper 5 are sequentially overlapped at specific positions on the bottom plate 1. After assembly, they are cut into a fixed width of 3.9 mm to obtain a fluorescent immunochromatographic test strip for detecting antinuclear antibodies.

[0056] Comparative Application Example 1: This comparative application example provides a fluorescent immunochromatographic test strip for detecting antinuclear antibodies. Its structure and preparation method are similar to those of Example 1, except that the HEP-2 cell extract prepared in Comparative Example 1 is used instead of the HEP-2 cell extract prepared in Example 1.

[0057] Comparative Application Example 2: This comparative application example provides a fluorescent immunochromatographic test strip for detecting antinuclear antibodies. Its structure and preparation method are similar to those of Example 1, except that the HEP-2 cell extract prepared in Comparative Example 2 is used instead of the HEP-2 cell extract prepared in Example 1.

[0058] Comparative Application Example 3: This comparative application example provides a fluorescent immunochromatographic test strip for detecting antinuclear antibodies. Its structure and preparation method are similar to those of Example 1, except that the HEP-2 cell extract prepared in Comparative Example 3 is used instead of the HEP-2 cell extract prepared in Example 1.

[0059] Comparative Application Example 4: This comparative application example provides a fluorescent immunochromatographic test strip for detecting antinuclear antibodies. Its structure and preparation method are similar to those of Example 1, except that the HEP-2 cell extract prepared in Comparative Example 4 is used instead of the HEP-2 cell extract prepared in Example 1.

[0060] Comparative Application Example 5: This comparative application example provides a fluorescent immunochromatographic test strip for detecting antinuclear antibodies. Its structure and preparation method are similar to those of Application Example 1, except that the HEP-2 cell extract prepared in Example 1 is replaced with a coating solution containing recombinant antinuclear antibody antigen (Qingdao Henderson Biotechnology Co., Ltd., Catalog No.: HDS-D2001). The coating material on antinuclear antibody detection line 7 is replaced with recombinant antinuclear antibody antigen, as described in Example 1 for the DNP antibody coating method.

[0061] Performance Testing

[0062] 1. Sensitivity test

[0063] Antinuclear antibody fluorescent immunochromatographic test strips for detecting antinuclear antibodies (ANA calibrators) of varying concentrations were used, respectively, for application example 1 and comparative examples 1-5. The concentrations of the ANA calibrators were 0 U / mL, 10 U / mL, 25 U / mL, 50 U / mL, 100 U / mL, and 200 U / mL, respectively. The detection method was as follows: Each concentration gradient was diluted 20-fold with a diluent consisting of 10 mM PB buffer (pH 7.4) containing 45.0 g / L sodium chloride, 0.5 wt% ProClin 300, and 0.5 wt% Tween-20. 80 μL of the diluted calibrator was added to the sample well. After a 15-minute timer, the fluorescence intensity of the ANA test line 7 (denoted as the T value) and the fluorescence intensity of the control line 6 (denoted as the C value) on the test strip were measured using a fluorescent immunoassay analyzer. Each calibrator concentration was tested three times, and the average value was taken. The signal intensities were compared, and the average T / C ratio was calculated. See Table 1 for details. The antinuclear antibody standard curve was fitted as follows: y = (AD) / [1 + (x / C^B)] + D, where y is the T / C value and x is the antinuclear antibody concentration, as shown in Table 2. The fluorescence immunoassay analyzer used for the test was the DL300 dry-type fluorescence immunoassay analyzer produced by Dialab (Zhangjiagang) Biotechnology Co., Ltd.

[0064]

[0065] Table 1 shows that the fluorescence intensity of the antinuclear antibody fluorescent immunochromatographic test strip of Example 1 is the highest when detecting antinuclear antibody calibrators of different concentrations, indicating that the antinuclear antibody fluorescent immunochromatographic test strip of Example 1 has the highest sensitivity.

[0066]

[0067] Table 2 shows that the standard curves of the antinuclear antibody fluorescent immunochromatographic test strips of Example 1 and Comparative Example 3 have the highest fitting degree and better linear range. Combined with Table 1, the sensitivity and linear range of the antinuclear antibody fluorescent immunochromatographic test strip of Example 1 are both the best.

[0068] 2. Repeatability test

[0069] The DL300 dry-type fluorescent immunoassay analyzer has a processor that can read the contents of the ID card and process the sample test results. To facilitate subsequent testing, the antinuclear antibody standard curves in Table 2 were burned onto the ID card, and the ID card was inserted into the corresponding antinuclear antibody test strip. The complete test steps are as follows:

[0070] (1) Insert the antinuclear antibody fluorescent immunochromatographic test strip into the dry fluorescent immunoassay analyzer DL300, which reads the standard curve in the ID card, and then pull out the antinuclear antibody fluorescent immunochromatographic test strip.

[0071] (2) Dilute the sample 20-fold with diluent, then add 80 μL to the sample well of the antinuclear antibody fluorescent immunochromatographic test strip and let it stand for 15 minutes. The diluent is 10 mM PB buffer (pH 7.4) containing 45.0 g / L sodium chloride, 0.5 wt% ProClin 300, and 0.5 wt% Tween-20.

[0072] (3) Insert the antinuclear antibody fluorescent immunochromatographic test strip into the dry fluorescent immunoassay analyzer DL300 again. The dry fluorescent immunoassay analyzer DL300 reads the fluorescence intensity (T value) of the Kanghe antibody detection line 7 and the fluorescence intensity (C value) of the quality control line 6. The processor of the dry fluorescent immunoassay analyzer DL300 automatically calculates the T / C value through the standard curve information in the ID card and converts it into the corresponding antinuclear antibody concentration output.

[0073] Antinuclear antibody fluorescent immunochromatographic test strips (including ID cards) for detecting antinuclear antibodies of Application Example 1 and Application Comparative Examples 1 to 5 and dry fluorescent immunoassay analyzer DL300 were used to detect antinuclear antibody calibrators with concentrations of 20 U / mL and 100 U / mL, respectively. Each test strip was tested 10 times for each concentration of antinuclear antibody calibrator, and the mean value (Mean, U / mL) and coefficient of variation (CV, %) were calculated. The results are statistically shown in Table 3.

[0074]

[0075] Table 3 shows that the detection results of the antinuclear antibody fluorescent immunochromatographic test strip of Application Example 1 are more accurate and have the smallest coefficient of variation. Compared with other application comparison examples, Application Example 1 can more accurately and reliably quantitatively analyze the antinuclear antibody content in the sample.

[0076] 3. Stability test

[0077] The HEP-2 cell extracts used in Application Example 1 and Comparative Examples 1-4 were all prepared on the same day (stored at 4°C for less than 24 hours). In actual production, it is not possible to guarantee that each batch of test strips will use HEP-2 cell extracts prepared on the same day. Therefore, samples were taken from the HEP-2 cell extracts in Example 1 and Comparative Examples 1-4 after storage at 4°C for 4 days, 7 days, 10 days, and 14 days, respectively, to prepare fluorescent immunochromatographic test strips for detecting antinuclear antibodies according to the corresponding application examples. These were used to detect antinuclear antibody calibrators with concentrations of 20 U / mL and 100 U / mL, respectively. Ten replicates were set up for each test. The corresponding antinuclear antibody concentrations were calculated using the standard curve in Table 2, and the average values ​​(mean, U / mL) were calculated. The deviations of the test strips prepared from HEP-2 cell extracts stored at different times relative to the test strips prepared from HEP-2 cell extracts prepared on the same day (see the average values ​​in Table 3) were calculated. The results are shown in Table 4.

[0078]

[0079] The results in Table 4 show that, except for Comparative Example 2, which had a larger deviation on days 10 and 14, the deviations of Example 1 and Comparative Examples 1, 3, and 4 were less than 10% after storage at 4°C for 4 days, 7 days, 10 days, and 14 days, indicating that Example 1 and Comparative Examples 1, 3, and 4 had better stability. Specifically, the deviations of Example 1 after storage at 4°C for 4 days and 7 days were both less than 1%. This can reduce the inter-batch variability of antinuclear antibody fluorescent immunochromatographic test strips tested using the same batch of HEP-2 cell extracts stored at 4°C within a week, eliminating the need to prepare HEP-2 cell extracts freshly before each preparation of antinuclear antibody fluorescent immunochromatographic test strips or to repeatedly thaw HEP-2 cell extracts stored at -80°C.

[0080] 4. Positive and negative compliance test:

[0081] Clinical serum samples were tested using the antinuclear antibody fluorescent immunochromatographic test strips (including ID cards) of Application Example 1 and Comparative Examples 1-5 and a DL300 dry-type fluorescent immunoassay analyzer, and the results were compared with those obtained using the industry-recognized antinuclear antibody IgG detection kit (indirect immunofluorescence assay) from Omen Medical Diagnostics (China) Co., Ltd. The detection method of the antinuclear antibody IgG detection kit (indirect immunofluorescence assay) from Omen Medical Diagnostics (China) Co., Ltd. is described in its instructions.

[0082] The number of positive and negative samples detected by each antinuclear antibody fluorescent immunochromatographic test strip (including ID card) and the antinuclear antibody IgG detection kit (indirect immunofluorescence method) of Omen Medical Diagnostics (China) Co., Ltd. is shown in Table 5, and the positive and negative coincidence rates of the test results of each antinuclear antibody fluorescent immunochromatographic test strip (including ID card) and the antinuclear antibody IgG detection kit (indirect immunofluorescence method) of Omen Medical Diagnostics (China) Co., Ltd. are shown in Table 6.

[0083]

[0084]

[0085] The results in Tables 5 and 6 show that the negative coincidence rates for Application Example 1 and Comparative Examples 1, 3, and 4 were all 100%, significantly higher than those for the other two comparison groups. Application Example 1 had the highest positive coincidence rate and the highest total coincidence rate, while the positive coincidence rates and total coincidence rates for the other comparison groups were relatively poor. The positive missed detection rate for Comparative Example 5 was very high.

[0086] Based on the above results, the Hep-2 cell extract prepared in Example 1 is more suitable for forming a detection line for the fluorescent immunochromatographic test strip for detecting antinuclear antibodies, so that the fluorescent immunochromatographic test strip for detecting antinuclear antibodies in Example 1 can take into account the performance of sensitivity, specificity, stability, etc. Application Example 1 provides a fluorescent immunochromatographic test strip for detecting antinuclear antibodies based on Hep-2 cell extracts, which is fast and has reliable detection results.

[0087] The above detailed description of the present invention is intended to enable those skilled in the art to understand the contents of the present invention and implement them, but it does not limit the scope of protection of the present invention. The present invention is not limited to the above embodiments. Any equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A process for preparing a Hep-2 cell extract, characterized in that: The cultured Hep-2 cells are transferred to a 5-15 mM PBS buffer solution with a pH of 7.2-7.

6. The obtained Hep-2 cell suspension is centrifuged to obtain Hep-2 cell pellets. The mixture of the Hep-2 cell pellets and the extraction buffer solution is sonicated in an ice bath. The sonicated mixture is centrifuged to collect the supernatant. The supernatant is passed through a microfiltration membrane to obtain the filtrate as the Hep-2 cell extract. The extraction buffer is a 5-15 mM HEPES buffer containing 3-8 vol% isopropanol, 3-8 wt% trehalose, 0.3-0.8 wt% bovine serum albumin and 0.8-1.2 mM phenylmethylsulfonyl fluoride with a pH value of 7.2-7.

6.

2. The preparation process of the Hep-2 cell extract according to claim 1, characterized in that: The ultrasonic conditions are as follows: ultrasonic power 140-160 W, pulse time 4-6 s, interval time 4-10 s, and total duration 4-5 min.

3. The preparation process of the Hep-2 cell extract according to claim 1 or 2, characterized in that: The amount of the extraction buffer is 2~8×10 6 Use 20-80 μL of the extraction buffer for each cell; And / or, the centrifugation conditions of the Hep-2 cell suspension are: centrifugation temperature 3-5°C, centrifugal force 2000-3000g, and centrifugation time 25-35min; and / or, the centrifugal conditions of the mixture after ultrasound are: centrifugal temperature 3-5° C., centrifugal force 10,000-20,000 g, and centrifugal time 25-35 min; And / or, the pore size of the microfiltration membrane is 0.4-0.6 μm.

4. A method for preparing a fluorescent immunochromatographic test strip for detecting antinuclear antibodies, the fluorescent immunochromatographic test strip comprising a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper sequentially mounted on the base plate, wherein the nitrocellulose membrane is provided with a test line and a quality control line, wherein: The conjugate pad is coated with fluorescent microsphere-labeled mouse anti-human IgG antibody and fluorescent microsphere-labeled DNP-BSA, the detection line is formed by Hep-2 cell extract, and the preparation method comprises preparing the Hep-2 cell extract using the preparation process described in any one of claims 1 to 3.

5. The method for preparing the fluorescent immunochromatographic test strip for detecting antinuclear antibodies according to claim 4, wherein: The fluorescent microspheres are europium chelated fluorescent microspheres; And / or, the particle size of the fluorescent microspheres is 100 nm to 300 nm; And / or, the sample pad is a glass cellulose membrane coated with casein, Tween, trehalose and heterophilic antibody blocking agent; And / or, the conjugate pad is a glass cellulose membrane, and the volume ratio of the fluorescent microsphere-labeled mouse anti-human IgG antibody and the fluorescent microsphere-labeled DNP-BSA coated thereon is (10-30):1; And / or, the quality control line is formed by DNP antibody coated on the nitrocellulose membrane.

6. The method for preparing the fluorescent immunochromatographic test strip for detecting antinuclear antibodies according to claim 4, wherein: The antinuclear antibody fluorescent immunochromatographic test strip is provided with an ID card, and the ID card stores calibration data and / or a standard curve.

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