Hep-2 cell extract preparation process suitable for preparing fluorescence immunochromatography test strip for detecting antinuclear antibody and preparation method of test strip

Abstract

The invention provides a preparation process of a Hep-2 cell extract suitable for preparing a fluorescence immunochromatography test strip for detecting antinuclear antibodies and a preparation method of the test strip. The method comprises the following steps: transferring cultured Hep-2 cells into a PBS (Phosphate Buffer Solution), carrying out centrifugal separation on the obtained Hep-2 cell suspension to obtain Hep-2 cell agglomerates, carrying out ultrasonic treatment on a mixture of the Hep-2 cell agglomerates and the extraction buffer solution in an ice bath, centrifuging the mixture subjected to ultrasonic treatment, collecting supernatant, and passing the supernatant through a micro-filtration membrane to obtain filtrate, namely the Hep-2 cell extract. The extraction buffer solution is an HEPES buffer solution containing isopropanol, trehalose, bovine serum albumin and phenylmethylsulfonyl fluoride. The prepared HEP-2 cell extract is used for preparing a fluorescence immunochromatography test strip for detecting antinuclear antibodies, and the detection sensitivity, the specificity and the reliability of a detection result can be considered at the same time.

Description

Technical Field

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

[0002] Antinuclear antibody (ANA) is a general term for autoantibodies targeting eukaryotic cell components. It is an important biological marker for autoimmune diseases (AID) and also a group of autoantibodies most widely used in clinical applications. It is commonly found in patients with systemic (non-organ-specific) AID such as systemic lupus erythematosus, Sjogren's syndrome, systemic sclerosis, mixed connective tissue disease, and polymyositis / dermatomyositis. In addition, ANA can also be seen in patients with chronic infectious diseases and tumors. Therefore, detecting antinuclear antibody has important clinical significance for the diagnosis, differential diagnosis, typing, and disease activity monitoring of AID.

[0003] The main methods for detecting antinuclear antibody are indirect immunofluorescence assay, enzyme-linked immunosorbent assay, immunoblotting method, chemiluminescence method, etc. Different detection methods have different clinical application advantages and disadvantages. For example, although the indirect immunofluorescence assay is intuitive, it is highly subjective and requires a high professional level of the detector. There may be differences in the interpretation of fluorescence patterns by detectors in different laboratories; although the enzyme-linked immunosorbent assay and immunoblotting method have high sensitivity, they are complex in operation and time-consuming in detection, and the detection result of the immunoblotting method is mainly semi-quantitative; although the chemiluminescence method has the advantages of high sensitivity, high specificity, quantification, and automation, the detection cost is high and the detection instrument is expensive. Currently, there is no fluorescence immunoassay chromatography test strip for detecting antinuclear antibody based on fluorescence immunoassay chromatography technology in the market. The fluorescence immunoassay chromatography technology can take into account the advantages of convenient operation, short detection time, and low requirements for detectors and inspection instruments. Developing a fluorescence immunoassay chromatography test strip for detecting antinuclear antibody can achieve rapid detection of antinuclear antibody, reduce the detection difficulty and cost, and promote the improvement of clinical diagnosis and treatment efficiency.

[0004] When developing a fluorescence immunochromatographic test strip for antinuclear antibodies, it is usually necessary to use the antigen stock solution. However, there is currently no antinuclear antibody antigen stock solution developed specifically for the fluorescence immunochromatographic test strip for antinuclear antibodies. After using the commercially available recombinant antinuclear antibody antigen to prepare the fluorescence immunochromatographic test strip for antinuclear antibodies, the positive miss detection rate is relatively high after testing. The indirect immunofluorescence method using Hep-2 cells as the matrix is an important method for antinuclear antibody detection, suggesting that Hep-2 cell extract can be used to replace the antigen stock solution. However, different extraction processes lead to differences in the components of Hep-2 cell extract, directly affecting the detection sensitivity of antinuclear antibodies. Different extraction processes also result in differences in the stability of Hep-2 cell extract. The worse the stability of Hep-2 cell extract, the greater the batch-to-batch difference of test strips in different production batches. These are the key reasons why there is still no fluorescence immunochromatographic test strip for antinuclear antibodies with reliable test results on the current 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 a fluorescence immunochromatographic test strip for detecting antinuclear antibodies.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions: In the first aspect of the present invention, a preparation process of Hep-2 cell extract is provided. The cultured Hep-2 cells are transferred into 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 clumps. The mixture of the Hep-2 cell clumps and the extraction buffer is ultrasonically treated in an ice bath. The ultrasonically treated mixture is centrifuged, and the supernatant is collected. The supernatant is filtered through a microfiltration membrane, and the obtained filtrate 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.

[0007] In some embodiments, the conditions of the ultrasonic treatment are: ultrasonic power 140-160 W, pulse time 4-6 s, interval time 4-10 s, and total duration 4-5 min.

[0008] In some embodiments, the dosage of the extraction buffer is 20-80 μL of the extraction buffer for (2-8)×10 6 cells. Further preferably, the dosage of the extraction buffer is 40-60 μL of the extraction buffer for (4-6)×10 6 cells.

[0009] In some embodiments, the centrifugation conditions for the Hep-2 cell suspension are as follows: centrifugation temperature is 3 - 5°C, centrifugal force is 2000 - 3000g, and centrifugation time is 25 - 35 min.

[0010] In some embodiments, the centrifugation conditions for the mixture after ultrasonic treatment are as follows: centrifugation temperature is 3 - 5°C, centrifugal force is 10000 - 20000g, and centrifugation time is 25 - 35 min.

[0011] In some embodiments, the pore size of the microfiltration membrane is 0.4 - 0.6 μm.

[0012] The second aspect of the present invention provides a Hep-2 cell extract prepared by the above preparation process.

[0013] The third aspect of the present invention provides the application of the above Hep-2 cell extract in the preparation of a fluorescence immunochromatographic test strip for detecting antinuclear antibodies.

[0014] The fourth aspect of the present invention provides a fluorescence immunochromatographic test strip for detecting antinuclear antibodies, which includes a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and a blotting paper sequentially mounted on the base plate. The nitrocellulose membrane is provided with a test line and a control line. The conjugate pad is coated with a mouse anti-human IgG antibody labeled with fluorescent microspheres and DNP-BSA labeled with fluorescent microspheres. The test line is formed by the above Hep-2 cell extract.

[0015] In some embodiments, the fluorescent microspheres are europium chelate fluorescent microspheres. Further, the particle size of the fluorescent microspheres is 100 nm - 300 nm, more preferably 150 nm - 250 nm, and even more preferably 180 nm - 220 nm.

[0016] In some embodiments, the sample pad is made of a glass cellulose membrane, which is coated with casein, Tween, trehalose, and an anti-heterophilic antibody blocker.

[0017] In some embodiments, the conjugate pad is made of a glass cellulose membrane, and the volume ratio of the mouse anti-human IgG antibody labeled with fluorescent microspheres and DNP-BSA labeled with fluorescent microspheres coated thereon is (10 - 30):1, more preferably (10 - 20):1, and even more preferably (10 - 15):1.

[0018] In some embodiments, the control line is formed by a DNP antibody coated on the nitrocellulose membrane.

[0019] The fifth aspect of the present invention provides an antinuclear antibody detection kit, which includes the above fluorescence immunochromatographic test strip for detecting antinuclear antibodies and a diluent.

[0020] The sixth aspect of the present invention provides a quantitative detection system for antinuclear antibodies based on fluorescence immunoassay. The antinuclear antibody quantitative detection system includes a fluorescence immunoassay analyzer, the above-mentioned fluorescence immunochromatographic test strip, and a diluent. An ID card is provided on the fluorescence immunochromatographic test strip, and calibration data and / or a standard curve are stored in the ID card.

[0021] In some embodiments, the diluent is a 5-15 mM PB buffer solution with a pH value of 7.2-7.6 containing 40-60 g / L sodium chloride, 0.3-0.8 wt% preservative, and 0.3-0.8 wt% Tween.

[0022] The present invention has the following advantages compared with the prior art: The present invention provides a preparation process for Hep-2 cell extract suitable for preparing a fluorescence immunochromatographic test strip for detecting antinuclear antibodies. Through the optimization and adjustment of the extraction buffer formula and extraction method, the fluorescence immunochromatographic test strip for detecting antinuclear antibodies prepared from the obtained Hep-2 cell extract has high sensitivity, good specificity, and low positive miss detection rate. The Hep-2 cell extract prepared by the preparation process of the present invention has good stability, which can reduce the batch-to-batch difference of the fluorescence immunochromatographic test strips for detecting antinuclear antibodies prepared from different batches of Hep-2 cell extracts stored at 4°C for the same week. There is no need to freshly prepare Hep-2 cell extract or repeatedly thaw the Hep-2 cell extract stored at -80°C before each preparation of the fluorescence immunochromatographic test strip for detecting antinuclear antibodies, thereby simplifying the operation process, avoiding reagent bottle bursting or precipitate formation caused by repeated thawing, being beneficial to improving production efficiency, reducing production costs, and reducing the risk of false negatives caused by fluctuations in storage conditions, and being beneficial to the large-scale production and application of the fluorescence immunochromatographic test strip for detecting antinuclear antibodies. Description of the Drawings

[0023] Figure 1 It is a schematic structural diagram of the fluorescence immunochromatographic test strip for detecting antinuclear antibodies in Application Example 1; In the figure: 1, bottom plate; 2, sample pad; 3, conjugate pad; 4, nitrocellulose membrane; 5, absorbent paper; 6, control line; 7, antinuclear antibody detection line. Detailed Embodiments

[0024] In order to adapt to the fluorescence immunoassay chromatography technology, the inventors of the present application have conducted a large number of studies on the preparation process of Hep-2 cell extracts, including the optimization and adjustment of the extraction buffer formulation and extraction method, so that the fluorescence immunoassay chromatography test strips for detecting antinuclear antibodies prepared from the obtained Hep-2 cell extracts have high sensitivity, good specificity, and low positive miss detection rate. In addition, the obtained Hep-2 cell extracts have good stability, which can reduce the batch-to-batch differences of the fluorescence immunoassay chromatography test strips for detecting antinuclear antibodies using different batches of the same batch of Hep-2 cell extracts stored at 4 °C within one week. Based on this, the present application also provides a fluorescence immunoassay chromatography test strip for detecting antinuclear antibodies based on Hep-2 cell extracts, which is fast and has reliable detection results.

[0025] The fluorescence immunoassay chromatography test strip for detecting antinuclear antibodies of the present invention is based on the indirect immunofluorescence method. If the test sample contains antinuclear antibodies, the antinuclear antibodies in the test sample will bind to the fluorescein microsphere-labeled mouse anti-human IgG in the sample pad and chromatograph along the nitrocellulose membrane by capillary action. When reaching the detection area, they will be captured by the Hep-2 cell extracts immobilized on the corresponding detection line. The amount of fluorescein microsphere-labeled bound on the detection line is proportional to the amount of antinuclear antibodies in the sample. The content of antinuclear antibodies in the test sample is calculated by a fluorescence immunoassay analyzer according to the fluorescence intensity and the standard curve.

[0026] The present invention will be further described below in conjunction with the embodiments. However, the present invention is not limited to the following embodiments. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are the conventional conditions in the industry.

[0027] In the following examples and comparative examples, the experimental methods are all conventional methods unless otherwise specified, and the experimental materials used are all purchased from conventional biochemical reagent manufacturers unless otherwise specified.

[0028] It should be noted that in the following examples and comparative examples, for those involving phenylmethylsulfonyl fluoride, other components except phenylmethylsulfonyl fluoride can be premixed and reserved, and phenylmethylsulfonyl fluoride is added before resuspending the cell clumps.

[0029] Example 1: This example provides a method for preparing Hep-2 cell extracts, which is specifically as follows: Under the culture conditions of 37 °C, 5% CO2, and humidity ≥ 95%, Hep-2 cells (from ATCC source) were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. When the cells in the cell culture flask reached about 5×10 6After that, take 10 culture flasks that meet the cell growth requirements, aspirate the original culture medium respectively and replace it with 10 mM PBS buffer (pH 7.4, sterile) of the same volume as the original culture medium. Starting from a corner of the culture flask with a sterile cell scraper, gently scrape along the surface of the culture flask to make the Hep-2 cells detach from the flask wall, forming a Hep-2 cell suspension. Combine the Hep-2 cell suspensions in the 10 culture flasks.

[0030] Centrifuge the collected Hep-2 cell suspension at 4 °C and 3000 rpm, discard the supernatant, collect the cell pellet, transfer the cell pellet to 500 μL of pre-cooled 4 °C extraction buffer for resuspension. The extraction buffer is 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). Place the obtained resuspension on ice for 15 min, then perform ultrasonic treatment in an ice bath. The ultrasonic power is 150 W, the pulse time is 5 s, the interval time is 5 s, and the total duration is 5 min. After the ultrasonic treatment, centrifuge at 4 °C and 15000 g for 30 min, collect the supernatant, filter it using a 0.45 μm filter membrane, and the obtained filtrate is the Hep-2 cell extract, which is placed in a 4 °C refrigerator for standby.

[0031] Comparative Example 1: This comparative example provides a method for preparing a Hep-2 cell extract, which is as follows: Under the culture conditions of 37 °C, 5% CO2, and humidity ≥ 95%, culture Hep-2 cells using DMEM medium containing 10 wt% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin. When the cells in the cell culture flask reach about 5×10 6 After that, take 10 culture flasks that meet the cell growth requirements, aspirate the original culture medium respectively and replace it with 10 mM PBS buffer (pH 7.4) of the same volume as the original culture medium. Starting from a corner of the culture flask with a sterile cell scraper, gently scrape along the surface of the culture flask to make the Hep-2 cells detach from the flask wall, forming a Hep-2 cell suspension. Combine the Hep-2 cell suspensions in the 10 culture flasks.

[0032] Centrifuge the collected Hep-2 cell suspension at 4 °C and 3000 rpm, discard the supernatant, collect the cell pellet, transfer the cell pellet to 500 μL of pre-cooled 4 °C 10 mM PBS buffer (pH 7.4, sterile) for resuspension. Place the obtained resuspension on ice for 15 min. Add NP-40 solution (10%, sterile, Shanghai Yuanye Bio-Technology Co., Ltd.) to the resuspension, and the addition amount is 5 vol%. Vortex for 10 s, and centrifuge the obtained homogenate at 4 °C and 3000 rpm for 10 min to separate the supernatant and the cell pellet.

[0033] Transfer the obtained cell mass to 500 μL of pre-cooled secondary extraction solution at 4 °C for resuspension. The secondary extraction solution is 10 mM PBS buffer (pH 7.4, sterile) containing 1 wt% ethyl phenyl polyethylene glycol (NP-40), 0.5 wt% sodium deoxycholate, and 0.5 wt% Tween-20. Place the obtained resuspension on ice for 30 min, then vortex for 10 min. Centrifuge the obtained homogenate at 14,000 rpm for 30 min at 4 °C to separate the supernatant.

[0034] Combine the supernatants separated twice, add 5 wt% trehalose and 0.5 wt% bovine serum albumin to obtain Hep-2 cell extract, and place it in a refrigerator at 4 °C for standby.

[0035] Comparative Example 2: This comparative example provides a method for preparing Hep-2 cell extract. The preparation method is basically the same as that in Example 1, except that the extraction buffer used for preparing 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).

[0036] Comparative Example 3: This comparative example provides a method for preparing Hep-2 cell extract. The preparation method is basically the same as that in Example 1, except that the extraction buffer used for preparing 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.

[0037] Comparative Example 4: This comparative example provides a method for preparing Hep-2 cell extract. The preparation method is basically the same as that in Example 1, except that the extraction buffer used for preparing 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.

[0038] Application Example 1: This application example provides a fluorescence immunoassay chromatographic test strip for detecting antinuclear antibody.

[0039] As Figure 1As shown in the figure, the fluorescence immunochromatographic test strip for detecting antinuclear antibodies in the embodiment of the present application includes a bottom plate 1, and a sample pad 2, a conjugate pad 3, a nitrocellulose membrane 4, and an absorbent paper 5 that are sequentially lapped on the bottom plate 1. A quality control line 6 and an antinuclear antibody detection line 7 are provided on the nitrocellulose membrane 4 (Sartorius AG, Germany). The material of the bottom plate 1 is PVC, and the materials of the sample pad 2 and the conjugate pad 3 are both glass fiber membranes (Ahlstrom, product number 1UN14ER100025NT). A sample adding hole (not shown in the figure) is provided on the fluorescence immunochromatographic test strip for detecting antinuclear antibodies in this embodiment. The structural setting of the fluorescence immunochromatographic test strip including the setting of the sample adding hole is a conventional technical means in the art and will not be elaborated here.

[0040] The preparation method of the fluorescence immunochromatographic test strip for detecting antinuclear antibodies in the embodiment of the present application is as follows: 1. Preparation of the sample pad 2 The glass fiber membrane is evenly moistened with the sample pad blocking solution, and then placed in an environment with a humidity of less than 30%, and baked at 37 °C for 15 h, and then cut for standby. The sample pad blocking solution is a 50 mM Tris buffer solution (pH 8.5) containing 0.3 wt% casein, 1 wt% Tween-20, 5 wt% trehalose, and 0.1 mg / mL heterophilic antibody blocker (Baodekangti (Beijing) Biotechnology Co., Ltd., product number 3KC028).

[0041] 2. Preparation of the conjugate pad 3 Add fluorescent microspheres (europium chelate fluorescent microspheres, particle size 200 nm, Hangzhou Boyue Biotechnology Co., Ltd., product number EU0200C1) to 1 mL of 50 mM MES buffer (pH 6.0) to a solid content of 0.1%. After ultrasonic mixing, centrifuge to remove the supernatant. Add 0.5 mL of 50 mM MES buffer (pH 6.0), ultrasonic mix, then add 50 μL of 10 mg / mL NHS solution and 25 μL of 10 mg / mL EDC solution, and activate for 30 min. Centrifuge to remove the supernatant. Add 1 mL of 50 mM MES buffer (pH 6.0), ultrasonic mix, and centrifuge to remove the supernatant. Add 0.5 mL of 50 mM MES buffer (pH 6.0) again, ultrasonic mix, and add mouse anti-human IgG antibody at a final concentration of 100 μg / mL. Place on a shaker at 25 °C and 250 r / min for 2 h. Centrifuge to remove the supernatant, add 0.5 mL of blocking solution, place on a shaker at 25 °C and 250 r / min for 1 h, centrifuge to remove the supernatant, add 1 mL of blocking solution again for washing, centrifuge to remove the supernatant, to obtain mouse anti-human IgG antibody labeled with fluorescent microspheres (Beijing Borsi Biotechnology Co., Ltd., product number B1423). The blocking solution is 50 mM HEPES buffer (pH 8.5) containing 1 wt% bovine serum albumin. Store the mouse anti-human IgG antibody labeled with fluorescent microspheres in the preservation solution, ultrasonic mix, and set aside. The preservation solution is 50 mM Tris buffer (pH 8.5) containing 150 mM NaCl, 10 wt% trehalose, 0.5 wt% Tween-20, and 0.5 wt% casein.

[0042] Add fluorescent microspheres (Europium chelated fluorescent microspheres, particle size 200nm, Hangzhou Boyue Biotechnology Co., Ltd., catalog number EU0200C1) to 1mL 50mM MES buffer (pH 6.0) until the solid content is 0.1%, and centrifuge to remove the supernatant. Add 0.5mL 50mM MES buffer (pH 6.0), mix by ultrasound, add 50μL 10mg / mL NHS solution and 25μL 10mg / mL EDC solution, activate for 30min, and centrifuge to remove the supernatant. Add 1mL 50mM MES buffer (pH 6.0), mix by ultrasound, and centrifuge to remove the supernatant. 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 number T112505R) with a final concentration of 100 μg / mL was added, and the mixture was incubated for 2 h at 25°C and 250 r / min on a shaker. The supernatant was removed by centrifugation, 0.5 mL of blocking solution was added, and the mixture was incubated for 1 h at 25°C and 250 r / min on a shaker. The supernatant was removed by centrifugation, and 1 mL of blocking solution was added again for washing. The supernatant was removed by centrifugation to obtain DNP-BSA labeled with fluorescent microspheres. The blocking solution was 50 mM HEPES buffer (pH 8.5) containing 1 wt% bovine serum albumin. The fluorescent microsphere-labeled DNP-BSA was stored in a storage solution, ultrasonically mixed, and set aside. The storage solution was 50 mM Tris buffer (pH 8.5) containing 150 mM NaCl, 10 wt % trehalose, 0.5 wt % Tween-20, and 0.5 wt % casein.

[0043] Prepare the gold spray solution: the volume of mouse anti-human IgG antibody labeled with fluorescent microspheres accounts for 60%, the volume of DNP-BSA labeled with fluorescent microspheres accounts for 3%, and the volume of the preservation solution accounts for 37%. Use a gold sprayer to evenly spray the gold spray solution on the glass cellulose membrane at 2μL / cm, bake it at 37℃ for 15h, and place it in a drying oven for use. Cut the glass cellulose membrane to the required specifications. Use a gold sprayer to evenly spray the gold spray solution on the glass cellulose membrane at 2μL / cm, bake it at 37℃ for 15h, and place it in a drying oven for use.

[0044] 3. Preparation of Nitrocellulose Membrane 4 Dilute the DNP antibody with 10 mM PB buffer (pH 7.4) containing 5 wt% trehalose to obtain a DNP antibody coating solution with a final concentration of 2 mg / mL, and use a membrane scribing instrument to scribe the DNP antibody coating solution on the nitrocellulose membrane at 1 μL / cm. Use a membrane scribing instrument to scribe the HEP-2 cell extract prepared in Example 1 on the nitrocellulose membrane at 1 μL / cm. Place the nitrocellulose membrane in an oven at 37 °C and dry it for 1 h to 2 h, then transfer it to a drying oven and dry it at 60 °C to form a quality control line 6 (coated with DNP antibody) and an antinuclear antibody detection line 7 (coated with HEP-2 cell extract), and place it in the drying oven for later use.

[0045] 4. Assembly Lap the sample pad 2, the conjugate pad 3, the nitrocellulose membrane 4, and the absorbent paper 5 in sequence at specific positions on the bottom plate 1. After assembly, cut it with a fixed width of 3.9 mm to obtain a fluorescence immunoassay strip for detecting antinuclear antibodies.

[0046] Application Comparative Example 1: This application comparative example provides a fluorescence immunoassay strip for detecting antinuclear antibodies. Its structure is the same as that of Application Example 1, and its preparation method is basically the same as that of Application Example 1, except that the HEP-2 cell extract prepared in Comparative Example 1 is used to replace the HEP-2 cell extract prepared in Example 1.

[0047] Application Comparative Example 2: This application comparative example provides a fluorescence immunoassay strip for detecting antinuclear antibodies. Its structure is the same as that of Application Example 1, and its preparation method is basically the same as that of Application Example 1, except that the HEP-2 cell extract prepared in Comparative Example 2 is used to replace the HEP-2 cell extract prepared in Example 1.

[0048] Application Comparative Example 3: This application comparative example provides a fluorescence immunoassay strip for detecting antinuclear antibodies. Its structure is the same as that of Application Example 1, and its preparation method is basically the same as that of Application Example 1, except that the HEP-2 cell extract prepared in Comparative Example 3 is used to replace the HEP-2 cell extract prepared in Example 1.

[0049] Application Comparative Example 4: This application comparative example provides a fluorescence immunoassay strip for detecting antinuclear antibodies. Its structure is the same as that of Application Example 1, and its preparation method is basically the same as that of Application Example 1, except that the HEP-2 cell extract prepared in Comparative Example 4 is used to replace the HEP-2 cell extract prepared in Example 1.

[0050] Application Comparative Example 5: This application comparative example provides a fluorescence immunochromatographic test strip for detecting antinuclear antibodies. Its structure is the same as that of Application Example 1, and its preparation method is basically the same as that of Application Example 1, except that the coating solution containing recombinant antinuclear antibody antigen (Qingdao Henderson Biotechnology Co., Ltd., product number: HDS-D2001) is used to replace the HEP-2 cell extract prepared in Example 1, and the coating on the antinuclear antibody detection line 7 is replaced with recombinant antinuclear antibody antigen according to the coating method of the DNP antibody in Example 1.

[0051] Performance Test 1. Sensitivity Test The fluorescence immunochromatographic test strips for detecting antinuclear antibodies of Application Example 1 and Application Comparative Examples 1-5 were respectively used to detect antinuclear antibody calibrators (referred to as ANA calibrators) with different concentrations. The concentrations of the antinuclear antibody 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 times with a diluent, and the diluent was a 10 mM PB buffer solution (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 application hole. After 15 minutes, the fluorescence intensity of the antinuclear antibody detection line 7 (denoted as T value) and the fluorescence intensity of the quality control line 6 (denoted as C value) on the test strip were detected with a fluorescence immunoassay analyzer. Each concentration calibrator was tested 3 times, and the average value was taken. The signal intensities were compared and the average value of T / C was calculated. See Table 1 for details. By fitting the antinuclear antibody standard curve: y = (A - D) / [1 + (x / C^B)] + D, where y is the T / C value and x is the concentration value of the antinuclear antibody. See Table 2 for details. The fluorescence immunoassay analyzer used for detection was the dry fluorescence immunoassay analyzer DL300 produced by DiaLabo (Zhangjiagang) Biotechnology Co., Ltd.

[0052]

[0053] Table 1 shows that when the fluorescence immunochromatographic test strip for detecting antinuclear antibodies of Application Example 1 detected antinuclear antibody calibrators with different concentrations, the fluorescence intensity was the highest, indicating that the fluorescence immunochromatographic test strip for detecting antinuclear antibodies of Application Example 1 had the highest sensitivity.

[0054]

[0055] Table 2 shows that the fitting degrees of the standard curves of the fluorescence immunochromatographic test strips for detecting antinuclear antibodies of Application Example 1 and Application Comparative Example 3 were the highest, and the linear range was better. Combining Table 1, the sensitivity and linear range of the fluorescence immunochromatographic test strip for detecting antinuclear antibodies of Application Example 1 were both the best.

[0056] 2. Repeatability Test The dry fluorescence immunoassay analyzer DL300 has a processor that can read the content of the ID card and process the sample test results. For the convenience of subsequent tests, the anti-nuclear antibody standard curves in Table 2 are respectively burned into the ID cards, and the ID cards are inserted into the corresponding test strips for anti-nuclear antibody detection. The complete operation steps of the test are as follows: (1) Insert the fluorescence immunoassay chromatography test strip for anti-nuclear antibody detection into the dry fluorescence immunoassay analyzer DL300. The dry fluorescence immunoassay analyzer DL300 reads the standard curve in the ID card, and then pulls out the fluorescence immunoassay chromatography test strip for anti-nuclear antibody detection.

[0057] (2) After diluting the sample to be tested 20 times with the diluent, take 80 μL and add it to the sample addition hole of the fluorescence immunoassay chromatography test strip for anti-nuclear antibody detection, and let it stand for 15 min. The diluent is a 10 mM PB buffer solution (pH 7.4) containing 45.0 g / L sodium chloride, 0.5 wt% ProClin 300, and 0.5 wt% Tween-20.

[0058] (3) Insert the fluorescence immunoassay chromatography test strip for anti-nuclear antibody detection into the dry fluorescence immunoassay analyzer DL300 again. The dry fluorescence immunoassay analyzer DL300 reads the fluorescence intensity (T value) of the detection line 7 for Kanghe antibody and the fluorescence intensity (C value) of the quality control line 6. The processor of the dry fluorescence immunoassay analyzer DL300 automatically calculates the T / C value and converts it into the corresponding anti-nuclear antibody concentration for output according to the standard curve information in the ID card.

[0059] The fluorescence immunoassay chromatography test strips (including ID cards) for anti-nuclear antibody detection and the dry fluorescence immunoassay analyzer DL300 in Application Example 1 and Application Comparative Examples 1-5 were respectively used to detect anti-nuclear antibody calibrators with concentrations of 20 U / mL and 100 U / mL. Each test strip was repeatedly detected 10 times for each concentration of anti-nuclear antibody calibrator, and the average value (Mean, U / mL) and coefficient of variation (CV, %) were calculated. The results are summarized in Table 3.

[0060]

[0061] Table 3 shows that the test results of the fluorescence immunoassay chromatography test strip for anti-nuclear antibody detection in Application Example 1 are more accurate and have the smallest coefficient of variation. Compared with other application comparative examples, Application Example 1 can more accurately and reliably quantitatively analyze the content of anti-nuclear antibody in the sample.

[0062] 3. Stability Test The HEP-2 cell extracts used in Application Example 1 and Application Comparative Examples 1 to 4 above were all prepared on the day of use (within 24 hours of storage at 4°C). In actual production, it is impossible to ensure that each batch of test strips uses the HEP-2 cell extract prepared on the same day. Therefore, samples were taken after storing the HEP-2 cell extracts in Application Example 1 and Comparative Examples 1 to 4 at 4°C for 4 days, 7 days, 10 days, and 14 days respectively, and fluorescence immunochromatographic test strips for detecting antinuclear antibodies were prepared according to the corresponding application examples, and were used to detect antinuclear antibody calibrators with concentrations of 20 U / mL and 100 U / mL. Each test was set with 10 parallels. The corresponding antinuclear antibody concentration measurement values were calculated according to the standard curve in Table 2, and the average value (Mean, U / mL) was calculated. The deviation values of the test results of the test strips prepared from the HEP-2 cell extracts stored for different times compared with the test strips prepared from the HEP-2 cell extracts on the day of preparation (see the average values in Table 3) were calculated. The results are shown in Table 4.

[0063]

[0064] The results in Table 4 show that except for the relatively large deviation results of Comparative Example 2 on the 10th day and the 14th day, the deviations of Application Example 1 and Comparative Examples 1, 3, and 4 were less than 10% after storing at 4°C for 4 days, 7 days, 10 days, and 14 days, indicating that Application Example 1 and Comparative Examples 1, 3, and 4 have good stability. Among them, the deviations of Application Example 1 after storing at 4°C for 4 days and 7 days were both less than 1%, which can reduce the batch-to-batch differences of the fluorescence immunochromatographic test strips for detecting antinuclear antibodies in different batches using the same batch of HEP-2 cell extracts stored at 4°C within one week, and there is no need to freshly prepare the HEP-2 cell extract or repeatedly thaw the HEP-2 cell extract stored at -80°C before each preparation of the fluorescence immunochromatographic test strip for detecting antinuclear antibodies.

[0065] 4. Negative and positive coincidence rate test: The fluorescence immunochromatographic test strips (including ID cards) for detecting antinuclear antibodies in Application Example 1 and Application Comparative Examples 1 to 5 and the dry fluorescence immunoassay analyzer DL300 were used to detect clinical serum samples, and the results were compared with those detected by using the antinuclear antibody IgG detection kit (indirect immunofluorescence method) of EUROIMMUN Medical Diagnostics (China) Co., Ltd., which is recognized in the industry. The detection method of the antinuclear antibody IgG detection kit (indirect immunofluorescence method) of EUROIMMUN Medical Diagnostics (China) Co., Ltd. refers to its instruction manual.

[0066] The numbers of positive and negative samples detected by each anti-nuclear antibody fluorescence immunochromatographic test strip (including ID card) and the anti-nuclear antibody IgG detection kit (indirect immunofluorescence method) of EUROIMMUN Medizinische Labordiagnostika AG (China) are shown in Table 5. The positive and negative coincidence rates of the detection results of each anti-nuclear antibody fluorescence immunochromatographic test strip (including ID card) and the anti-nuclear antibody IgG detection kit (indirect immunofluorescence method) of EUROIMMUN Medizinische Labordiagnostika AG (China) are shown in Table 6.

[0067]

[0068]

[0069] The results in Table 5 and Table 6 show that the negative coincidence rates of Application Example 1, Application Comparative Example 1, Application Comparative Example 3, and Application Comparative Example 4 are all 100%, which are significantly higher than those of the other two groups of comparative examples. Among them, Application Example 1 has the highest positive coincidence rate and the highest total coincidence rate, and the positive coincidence rates and total coincidence rates of the remaining application comparative examples are relatively poor. The positive miss detection rate of Application Comparative Example 5 is very high.

[0070] Based on the above results, the Hep-2 cell extract prepared in Example 1 is more suitable for detecting the formation of the test line of the anti-nuclear antibody fluorescence immunochromatographic test strip, so that the anti-nuclear antibody fluorescence immunochromatographic test strip of Application Example 1 can take into account performance such as sensitivity, specificity, and stability. Application Example 1 provides a rapid and reliable anti-nuclear antibody fluorescence immunochromatographic test strip based on Hep-2 cell extract.

[0071] The above has made a detailed description of the present invention, aiming to enable those skilled in the art to understand the content of the present invention and implement it. However, it cannot be used to limit the protection scope of the present invention. Moreover, the present invention is not limited to the above embodiments. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A preparation process of Hep-2 cell extract, characterized in that, Transfer the cultured Hep-2 cells into a 5-15 mM PBS buffer with a pH value of 7.2 - 7.

6. The obtained Hep-2 cell suspension is centrifuged to obtain Hep-2 cell aggregates. The mixture of the Hep-2 cell aggregates and the extraction buffer is sonicated in an ice bath. After sonication, the mixture is centrifuged, and the supernatant is collected. The supernatant is filtered through a microfiltration membrane, and the resulting filtrate is the Hep-2 cell extract. The extraction buffer is a 5-15 mM HEPES buffer 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.

2. The preparation process of the Hep-2 cell extract according to claim 1, characterized in that, The conditions for sonication are: sonication 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 dosage of the extraction buffer is 20 - 80 μL of the extraction buffer for every (2 - 8) × 10 6 cells; And / or, the centrifugation conditions for the Hep-2 cell suspension are: centrifugation temperature 3-5 °C, centrifugal force 2000-3000 g, and centrifugation time 25-35 min; And / or, the centrifugation conditions for the mixture after sonication are: centrifugation temperature 3-5 °C, centrifugal force 10000-20000 g, and centrifugation time 25-35 min; And / or, the pore size of the microfiltration membrane is 0.4-0.6 μm.

4. A Hep-2 cell extract prepared by the preparation process according to any one of claims 1 to 3.

5. Use of the Hep-2 cell extract according to claim 4 in the preparation of a fluorescence immunoassay chromatographic test strip for detecting antinuclear antibodies.

6. A fluorescence immunochromatographic test strip for detecting antinuclear antibodies, comprising a bottom plate and a sample pad, a conjugate pad, a nitrocellulose membrane and a blotting paper sequentially mounted on the bottom plate, wherein a test line and a quality control line are provided on the nitrocellulose membrane, and it is characterized in that, The conjugate pad is coated with a mouse anti-human IgG antibody labeled with fluorescent microspheres and DNP-BSA labeled with fluorescent microspheres, and the test line is formed by the Hep-2 cell extract according to claim 4.

7. The fluorescence immunochromatographic test strip for detecting antinuclear antibody according to claim 6, wherein, The fluorescent microspheres are europium chelate fluorescent microspheres; And / or, the particle size of the fluorescent microspheres is 100 nm - 300 nm; And / or, the sample pad uses a glass cellulose membrane, which is coated with casein, Tween, trehalose, and heterophilic antibody blocker; And / or, the conjugate pad uses a glass cellulose membrane, and the volume ratio of the mouse anti-human IgG antibody labeled with fluorescent microspheres and DNP-BSA labeled with fluorescent microspheres coated thereon is (10-30):1; And / or, the control line is formed by DNP antibody coated on the nitrocellulose membrane.

8. An anti-nuclear antibody detection kit, characterized in that, The antinuclear antibody detection kit includes the fluorescence immunoassay chromatographic test strip for detecting antinuclear antibodies according to claim 6 or 7 and a diluent.

9. A quantitative detection system for antinuclear antibodies based on fluorescence immunoassay, characterized in that, The antinuclear antibody quantitative detection system includes a fluorescence immunoassay analyzer, the fluorescence immunoassay chromatographic test strip according to claim 6 or 7, and a diluent. An ID card is provided on the fluorescence immunoassay chromatographic test strip, and calibration data and / or a standard curve are stored in the ID card.

10. The anti-nuclear antibody detection kit according to claim 8 or the anti-nuclear antibody quantitative detection system according to claim 9, characterized in that, The diluent is a 5-15 mM PB buffer with a pH value of 7.2 - 7.6 containing 40-60 g / L sodium chloride, 0.3-0.8 wt% preservative, and 0.3-0.8 wt% Tween.

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