African swine fever virus antibody chemiluminescence immunochromatography detection test strip based on photographing function and preparation method thereof

Abstract

The invention relates to an African swine fever virus antibody chemiluminescence immunochromatography detection test strip based on a photographing function and a preparation method thereof. The test strip is composed of detection test paper and a chemiluminiscence reading device, the detection test paper comprises a supporting bottom plate, a nitrocellulose membrane, a combination pad, a sample pad and an absorption pad are sequentially pasted on the supporting bottom plate, the nitrocellulose membrane is sprayed with a detection line formed by SPA / anti-porcine secondary antibody and a quality control line formed by a P72 monoclonal antibody, the combination pad is provided with an HRP-p72 immune probe, and the sample pad is provided with a chemiluminiscence reading device. The chemiluminiscence reading device comprises a closed cassette and a photographing device, and a pre-opening is formed in the cassette. According to the invention, the chemiluminescence signal quick reading device is matched with the chemiluminescence immunochromatography test paper, so that the on-site detection of the African swine fever virus antibody is realized, the sensitivity is high, the specificity is strong, and the detection result is accurate; the reading device is simple and convenient, additional instruments and equipment and other reagents are not needed, and the cost of expensive instruments and equipment is saved.

Description

Technical Field

[0001] The present invention relates to an immunochromatographic detection method, and in particular to a chemiluminescent immunochromatographic test strip and a detection method for determining results based on a photographing function. Background Art

[0002] ASF, caused by the African swine fever virus (ASFV), is an acute, febrile, highly contagious animal disease with a high mortality rate. Given the lack of a preventive vaccine for ASF, research and development of sensitive, accurate, and simple ASFV antibody detection technologies are urgently needed to prevent the spread of the disease, enabling early detection of ASF-infected pigs, achieving internal decontamination of pig farms, and controlling the outbreak.

[0003] Commonly used African swine fever detection techniques include PCR, enzyme-linked immunosorbent assay (ELISA), and immunochromatographic analysis (IICA). While ELISA offers advantages such as rapidity and high throughput, it is complex to use and requires specialized equipment and specialized technicians, requiring only laboratory-based procedures, making it difficult to implement on-site. Immunochromatographic analysis has been a rapidly developing in vitro diagnostic technique in recent years. Compared to ELISA, it is easier to use, requires no personnel or equipment, and produces results in minutes, making it suitable for on-site testing. Colloidal gold rapid test strips for African swine fever antibodies are already commercially available in China, but market adoption has been suboptimal. Key challenges include low sensitivity and a high rate of false positives, which have become bottlenecks hindering the development of rapid on-site African swine fever antibody detection technology. In recent years, new materials such as quantum dots, fluorescent nanoparticles, and fluorescein have been applied to immunochromatographic analysis, improving sensitivity to some extent. However, these materials are complex to prepare, exhibit poor stability, and rely on a light source, which can affect analytical results. Applying chemiluminescence to immunochromatographic analysis could offer a new approach to improving the sensitivity of immunochromatographic analysis.

[0004] The chemiluminescence reaction system is a system in which a chemiluminescent substance absorbs energy during a chemical reaction such as redox, transitioning from the ground state to the excited state. When the excited state molecule returns to the ground state, it releases energy in the form of photons, thereby generating a luminescent signal. The concentration of the luminescent substance and the chemiluminescence intensity are linearly and quantitatively related under certain conditions, so it can be used for quantitative detection. Chemiluminescence does not require any external light source, thus avoiding the interference of other stray light, and has the advantages of low signal-to-noise ratio and high sensitivity. In recent years, chemiluminescence has been applied to immunoassays. Combining the high specificity of antigen-antibody reactions with the high sensitivity of chemiluminescence technology, chemiluminescence immunoassay technology has been formed. This can greatly improve detection sensitivity and has become one of the research hotspots in the field of in vitro detection. It also has good application value in the field of veterinary diagnosis. However, chemiluminescence immunoassay technology relies on professional detection instruments and cannot be used for on-site detection. Therefore, there is an urgent need for a simple and feasible chemiluminescence signal reading method combined with the detection mode of immunochromatographic test strips to successfully apply chemiluminescence immunoassay technology to on-site rapid detection. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide a chemiluminescent immunochromatographic test strip and detection method for result determination based on the camera function of a smartphone. The use of immunochromatographic test strips in combination with a rapid reading device can achieve rapid and highly sensitive on-site detection of African swine fever virus antibodies.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A chemiluminescent immunochromatographic test strip for African swine fever virus antibodies based on a photographic function consists of a test strip and a chemiluminescent reader. The test strip includes a supporting base plate, to which a nitrocellulose membrane, a conjugation pad, a sample pad, and an absorption pad are sequentially adhered. The nitrocellulose membrane is sprayed with a detection line formed by SPA / anti-swine secondary antibody and a quality control line formed by a P72 monoclonal antibody. The conjugation pad is provided with an HRP-p72 immune probe. The chemiluminescent reader includes a closed dark box and a photographic device, and the dark box has a pre-opening.

[0008] The dark box is a test paper packaging box, and the photographing device is a smart phone with a photographing function.

[0009] A method for preparing a chemiluminescent immunochromatographic test strip for African swine fever virus antibodies comprises the following steps:

[0010] (1) Preparation of conjugate pad: HRP conjugation kit was used to conjugate p72 trimer protein to obtain HRP-p72 immunoprobe, which was sprayed onto the pretreated conjugate pad and sealed for storage after drying;

[0011] (2) Sample pad preparation: Soak glass fiber cotton in the sample pad solution, let it stand for 5 minutes, then spread it evenly on a drying plate, dry it, and seal it for storage;

[0012] (3) Preparation of nitrocellulose membrane: Spray the monoclonal antibody solution and SPA onto the nitrocellulose membrane to form the quality control line and the test line respectively, and seal and store after drying;

[0013] (4) Test paper assembly: Glue the nitrocellulose membrane, conjugate pad, sample pad, and absorption pad onto the support base in sequence, with the components partially overlapping, and then cut into test paper strips.

[0014] The preparation method of the pretreated binding pad comprises: spraying a binding pad treatment liquid on the middle of a fiber mat and drying the binding pad to obtain a pretreated binding pad; the binding pad treatment liquid comprises, by mass percentage, 0.8% Na2B4O7·10H2O, 5% BSA, 0.1% PVP-10, 0.1% Triton X-100, and the balance being ultrapure water.

[0015] The nitrocellulose membrane was prepared by spraying a 2.0 mg / mL p72 monoclonal antibody solution onto the nitrocellulose membrane; the flow rate of the monoclonal antibody solution was 1 μL / cm, the concentration of SPA was 0.75 mg / mL, and the membrane was dried at 42° C. for 2 h.

[0016] The detection method of the African swine fever virus antibody chemiluminescent immunochromatographic test strip based on the camera function is as follows: the pig serum sample is diluted 1:100 with normal saline; 100 μL of the diluted sample is added to the sample cup, the test strip is inserted into the sample cup, and the reaction is allowed to stand for 5-10 minutes, and the color developing solution is added to the middle of the nitrocellulose membrane and the color is developed for 4-10 minutes. The test result is photographed using a chemiluminescent reading device, and the result is determined based on the color development of the quality control line and the test line in the photo.

[0017] When only one blue band appears on the quality control line and no blue band appears on the test line, the result is judged as negative; when two blue bands appear on both the quality control line and the test line, the result is judged as positive.

[0018] Beneficial positive effects of the present invention

[0019] 1. Simple and fast. The present invention combines a chemiluminescent signal rapid reading device with chemiluminescent immunochromatographic test paper to achieve on-site detection of African swine fever virus antibodies. During the test, the sample solution is added dropwise below the warning line of the test end of the test strip or inserted into the sample solution to be tested. After standing for 5-10 minutes, the color developing solution is added dropwise. The chemiluminescent reading device is used within 4-10 minutes to achieve rapid and accurate reading of the chemiluminescent test paper.

[0020] 2. The results are displayed visually, intuitively, and accurately. During testing, a blue band appears on the control line (C) but not on the test line (T), indicating a negative result. A blue band appears on both the control line (C) and the test line (T), indicating a positive result. The color depth of the test line is proportional to the antibody titer within a certain range. The results are visually, intuitively, accurately, and simply, making false positives and false negatives less likely to be misjudged.

[0021] 3. High sensitivity and strong specificity. The titer of the ASFV antibody chemiluminescence test paper for detecting ASFV positive serum can reach 1:1280×10 4 , which is 8 times that of the ASFV antibody colloidal gold detection test paper; at the same time, the ASFV antibody chemiluminescence detection test paper of the present invention has good specificity and the detection results are accurate.

[0022] 4. The rapid test strips of the present invention are used in combination with a chemiluminescent reading device. The reading device is simple and convenient. It can be a combination of a smartphone with a camera function and a test strip packaging box (with a pre-opened hole on the top). No additional instruments or other reagents are required, which enables on-site rapid detection of chemiluminescent test strips, improves detection sensitivity, and saves expensive instruments and equipment costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram of a chemiluminescent signal reading device for the ASFV antibody chemiluminescent test strip of the present invention;

[0024] Figure 2 This is the sensitivity test result of the ASFV antibody chemiluminescence test paper of the present invention;

[0025] Figure 3 The sensitivity test results of the ASFV antibody colloidal gold test strips used as the control;

[0026] Figure 4 This is the specific detection result of the ASFV antibody chemiluminescence detection test paper of the present invention. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are further described in detail below with reference to the examples.

[0028] 1. Main reagents and consumables: p72 trimer protein was homemade by the Institute of Animal Disease Prevention and Control, Henan Academy of Agricultural Sciences (Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences), ECL colorimetric solution was purchased from Xinsaimei Biotechnology Co., Ltd., and HRP-coupled protein kit was purchased from Abcom.

[0029] 2. Main instruments and equipment: Honor GT80 mobile phone was purchased from Shenzhen Zhixin New Information Technology Co., Ltd.; 93-3 timed constant temperature bidirectional magnetic stirrer was purchased from Shanghai Yarong Biochemical Instrument Factory; electric blast drying oven was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd.; SIGMA4-16K centrifuge was purchased from Sigma, Germany; heating magnetic stirrer was purchased from IKA, Germany; BioDot-XYZ3060 three-dimensional spray system and BioDot-CM 4000 chopper were purchased from Bio-Dot, USA.

[0030] Example 1 Chemiluminescent immunochromatographic test strips for result determination based on smartphone camera function

[0031] 1. Establishment of chemiluminescent immunochromatographic test strip detection mode

[0032] The chemiluminescent immunochromatographic test paper was prepared based on the principle of indirect immunochromatographic test paper. SPA / anti-swine secondary antibody was sprayed on the NC membrane to form a detection line, P72 monoclonal antibody was used as the quality control line, and HRP-p72 was used as the immune probe to establish the test paper.

[0033] 2. Test strip preparation method

[0034] (1) Preparation of conjugate pad: Use a spraying device to spray the conjugate pad treatment solution at a rate of 8 μL / cm2 onto the center of an 8 mm wide fiber pad. Bake at 42°C for 50 min to obtain a pretreated blank conjugate pad, which is then sealed and stored.

[0035] The binding pad treatment solution is composed of the following components by mass percentage: Na2B4O7·10H2O 0.8%, BSA 5%, PVP-10 0.1%, Triton X-100 0.1%, and the balance is ultrapure water.

[0036] The p72 trimer protein was coupled with a commercial HRP coupling kit from abcom according to the instructions. After coupling, the HRP-p72 immunoprobe was obtained, which was then sprayed onto the pretreated conjugate pad, dried at 42°C for 50 minutes, and sealed for storage.

[0037] (2) Sample pad preparation: Soak glass fiber cotton with a size of 1.8 cm × 30 cm in the sample pad solution, let it stand for 5 minutes, then spread it evenly on a drying plate, dry it at 42°C for 4 hours, and store it in a sealed container.

[0038] (3) Preparation of nitrocellulose (NC) membrane:

[0039] The control line (C) and test line (T) were sprayed using a BioDot-XYZ3060 3D spraying system. Specific parameters were as follows: a flow rate of 1 μL / cm was set, and a 2.0 mg / mL p72 monoclonal antibody solution was sprayed onto the upper end of the NC membrane to form the control line C; a 0.75 mg / mL SPA solution was sprayed onto the lower end of the NC membrane to form the test line T. The membrane was dried at 42°C for 2 hours and then sealed for storage.

[0040] (4) Assembly of the test strip: Attach the NC membrane, conjugate pad, sample pad, and absorbent pad to the support base in sequence, with each component overlapping by 2.0 mm. Use a cutting machine to cut the test strip into 3.0 mm × 60 mm strips, add desiccant, and seal for storage.

[0041] 3. Test paper detection principle of the present invention

[0042] The chemiluminescent reaction system is a system in which the chemiluminescent substance absorbs energy during chemical reactions such as oxidation-reduction, and transitions from the ground state to the excited state. When the excited state molecules return to the ground state, they release energy in the form of photons, thereby generating a luminescent signal. The color developer luminol emits blue light, which is visible to the naked eye in a dark environment. With the help of a mobile phone camera and a dark box (a test paper packaging box with a pre-opening at the top), take a picture of the test paper, and then visually determine the result through the photo. The chemiluminescent reading device of the present invention consists of a mobile phone and a dark box. Its structure and operation method can be found in Figure 1 .

[0043] 4. Test paper detection method and result determination

[0044] Detection method: Dilute the porcine serum sample 1:100 with normal saline; take 100 μL of the diluted sample and add it to the sample cup. Insert the test paper into the sample cup and let it react for 10 minutes. Then, add 70 μL of luminol colorimetric solution in the middle of the NC membrane of the test paper and let it develop for 4-10 minutes. Use a chemiluminescence reader to take a picture of the test paper and judge the result by the color development of the C and T lines in the photo.

[0045] Result determination: When only one blue band appears on the quality control line (C) and no blue band appears on the test line (T), the result is judged as negative; when blue bands appear on both the quality control line (C) and the test line (T), that is, two blue bands appear, the result is judged as positive, and the color depth of the test line is proportional to the antibody titer within a certain range.

[0046] Example 2: Sensitivity and specificity detection of test paper

[0047] (1) Sensitivity of ASFV antibody chemiluminescence test strips

[0048] The sensitivity of the test strips was evaluated by measuring the antibody titer of ASFV-positive serum. ASFV-positive serum samples were diluted in saline in multiples, then tested with chemiluminescent test strips. The results were photographed using a chemiluminescent reader, and the highest dilution factor at which the T line developed was used as the sensitivity of the test strips.

[0049] See the results Figure 2 , showing that the detection sensitivity of the test paper can reach 1:1280×10 4 The control was ASFV antibody colloidal gold test paper. The test line, quality control line, detection antigen (p72 trimer) and other raw materials of the colloidal gold test paper were the same as those of the chemiluminescence test paper. The test results are shown in Figure 3 , the sensitivity is 1:160×10 4 It can be seen that the sensitivity of the ASFV antibody test paper based on chemiluminescence is 8 times higher than that of the colloidal gold test paper.

[0050] (2) Specificity of ASFV antibody chemiluminescence test strips

[0051] ASFV antibody chemiluminescence test strips were used to detect ASFV, PRRSV, PPV, PCV2, PRV, CSFV positive serum and ASFV negative serum, and photos were taken to identify the specificity of the test strips. Figure 4 The test paper only reacts with ASFV positive serum, and has no reaction with PRRSV, PPV, PCV2, PRV, CSFV positive serum and ASFV negative serum. Therefore, the ASFV antibody chemiluminescent detection test paper established by the present invention has good specificity.

Claims

1. A chemiluminescent immunochromatographic test strip for African swine fever virus antibodies based on a camera function, characterized in that: The test paper consists of two parts: a test strip and a chemiluminescence reading device. The test strip includes a supporting base plate, on which a nitrocellulose membrane, a conjugation pad, a sample pad, and an absorption pad are sequentially adhered. The nitrocellulose membrane is sprayed with a detection line formed by SPA / anti-pig secondary antibody and a quality control line formed by P72 monoclonal antibody; the conjugation pad is provided with an HRP-p72 immune probe; the chemiluminescence reading device includes a closed dark box and a photographing device, and the dark box has a pre-opening.

2. The method for preparing the African swine fever virus antibody chemiluminescence immunochromatographic test strip based on the camera function according to claim 1, characterized in that: The dark box is a test paper packaging box, and the photographing device is a smart phone with a photographing function.

3. A method for preparing the African swine fever virus antibody chemiluminescent immunochromatographic test strip according to claim 1, characterized in that: The following steps are involved: (1) Preparation of conjugate pad: HRP conjugation kit was used to conjugate p72 trimer protein to obtain HRP-p72 immunoprobe, which was sprayed onto the pretreated conjugate pad and sealed for storage after drying; (2) Sample pad preparation: Soak glass fiber cotton in the sample pad solution, let it stand for 5 minutes, then spread it evenly on a drying plate, dry it, and seal it for storage; (3) Preparation of nitrocellulose membrane: Spray the monoclonal antibody solution and SPA onto the nitrocellulose membrane to form the quality control line and the test line respectively, and seal and store after drying; (4) Test paper assembly: Glue the nitrocellulose membrane, conjugate pad, sample pad, and absorption pad onto the support base in sequence, with the components partially overlapping, and then cut into test paper strips.

4. The preparation method according to claim 3, characterized in that The preparation method of the pretreated binding pad comprises: spraying a binding pad treatment liquid on the middle of a fiber pad and drying the binding pad to obtain a pretreated binding pad; the binding pad treatment liquid comprises, by mass percentage, 0.8% Na2B4O7·10H2O, 5% BSA, 0.1% PVP-10, 0.1% Triton X-100, and the balance being ultrapure water.

5. The preparation method according to claim 3, characterized in that The nitrocellulose membrane preparation method is as follows: A 2.0 mg / mL p72 monoclonal antibody solution was sprayed onto the nitrocellulose membrane; the flow rate of the monoclonal antibody solution was 1 μL / cm, the concentration of SPA was 0.75 mg / mL, and the membrane was dried at 42° C. for 2 h.

6. A method for detecting African swine fever virus antibody chemiluminescence immunochromatographic test strips based on a camera function according to claim 1, characterized in that: The porcine serum sample was diluted 1:100 with physiological saline; 100 μL of the diluted sample was added to the sample cup, the test paper was inserted into the sample cup, and the reaction was allowed to stand for 5-10 minutes. The color developing solution was added to the middle of the nitrocellulose membrane and the color was developed for 4-10 minutes. The test results were photographed using a chemiluminescence reader, and the results were determined based on the color of the quality control line and the test line in the photo.

7. The detection method of the African swine fever virus antibody chemiluminescence immunochromatographic test strip based on the camera function according to claim 6, characterized in that: The result determination method is: when only one blue band appears on the quality control line and no blue band appears on the test line, the result is determined to be negative; when two blue bands appear on both the quality control line and the test line, the result is determined to be positive.

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