A human hepatitis C virus protein and its applications

Abstract

The present invention provides a human hepatitis C virus protein and its applications. The human hepatitis C virus protein provided by the present invention comprises a protein fragment C-a, a protein fragment NS3-a, and a protein fragment NS5-a, and the amino acid sequences of the said protein fragments are defined. The human hepatitis C virus antibody detection kit provided by the present invention has the advantages of simple operation, rapid reaction, high sensitivity, strong specificity, suitability for on-site rapid detection, economy and practicality, etc., and can conduct rapid screening during the epidemic of the virus, and timely detect a large number of people including suspected cases, asymptomatic infected cases in the incubation period, and asymptomatic virus carriers with higher risks quickly, accurately and safely.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a human hepatitis C virus protein and its application, especially to a human hepatitis C virus protein, a test strip for detecting human hepatitis C virus antibodies, a detection kit and its application. Background Art

[0002] Hepatitis C virus (HCV) is an enveloped single-stranded positive-strand RNA virus belonging to the genus Hepatitis C virus of the Flaviviridae family, and is an important human pathogen transmitted through blood. Its chronic infection often leads to chronic hepatitis, persistent liver damage and then serious liver diseases such as liver fibrosis, cirrhosis and hepatocellular carcinoma, bringing heavy health, economic and psychological burdens to society and families.

[0003] According to the data estimate in 2020, there are approximately 70 million HCV-infected people worldwide, of which only 20% are diagnosed and 7% receive treatment. Therefore, expanding the early detection rate of HCV infection and timely conducting early intervention treatment can not only shorten the clinical treatment time more safely and effectively, but also be an important way to control HCV infection and an effective means to reduce the burden of advanced diseases.

[0004] Based on the above background, it is very necessary to develop a new detection device and detection method to improve the sensitivity of HCV detection and assist clinical diagnosis. Summary of the Invention

[0005] The purpose of the present invention is as follows:

[0006] The first purpose of the present invention is to provide a human hepatitis C virus protein;

[0007] The second purpose of the present invention is to provide the application of the above human hepatitis C virus protein in the preparation of a product for detecting human hepatitis C virus antibodies;

[0008] The third purpose of the present invention is to provide a test strip kit to solve at least one of the above problems;

[0009] The fourth purpose of the present invention is to provide the application of a detection kit in detecting human hepatitis C virus.

[0010] In order to achieve the above purpose, the present invention provides a human hepatitis C virus protein, comprising a combination of the following protein fragments;

[0011] Protein fragment C-a, whose amino acid sequence is as shown in SEQ ID NO.1;

[0012] Protein fragment NS3-a, whose amino acid sequence is as shown in SEQ ID NO.2;

[0013] Protein fragment NS5-a, whose amino acid sequence is shown in SEQ ID NO.3;

[0014] Among them, the protein fragments C-a, NS3-a and NS5-a can be combined and linked in any order.

[0015] It should be noted that:

[0016] The amino acid sequence of protein fragment C-a is shown in SEQ ID NO.1, and specifically is as follows: mstnpkpqrktkrntnrrpqdvkfpgggqivggvyllprrgprlgvratrktsersqprgrrqpipkarrpegrawaqpgypwplygnegmgwagwllsprgsrpswgptdprrrsrnlgkvidtltcgfadlmgyiplvgaplggaaralahgvrvledgvnyatgnl.

[0017] The amino acid sequence of protein fragment NS3-a is shown in SEQ ID NO.2, and specifically is as follows:

[0018] egevqvvstatqsflatcvngvcwtvyhgagsktlagpkgpitqmytnvdqdlvgwqappgarsmtpctcgssdlylvtrhadvipvrrrgdgrgsllsprpisylkgssggpllcplghvvgifraavctrgvakavdfvpvesmett.

[0019] The amino acid sequence of protein fragment NS5-a is shown in SEQ ID NO.3, and specifically is as follows:

[0020] sgswlrdvwdwictvlsdfktwlqskllpqlpgvpffscqrgykgvwrgdgimqttcpcgaqitghvkngsmritgpktcsntwhgtfpvnayttgpctpspapnysralwrvaaeeyvevtrvgdfhyvtgmttdnlkcpcqvpapefftevdgvrlhryapacrpllreevvfqvglnqylvgsqlpcepepd.

[0021] Preferably, the combination mode of the protein fragments of the human hepatitis C virus protein includes any one of C-a+NS3-a+NS5-a, C-a+NS5-a+NS3-a, NS3-a+NS5-a+C-a, NS3-a+C-a+NS5-a, NS5-a+NS3-a+C-a or NS5-a +C-a+NS3-a.

[0022] The present invention does not further optimize the combination mode of the protein fragments of the human hepatitis C virus protein, and those skilled in the art can select or adjust according to actual needs.

[0023] The present invention also provides a test strip for detecting human hepatitis C virus antibodies. The test strip for detecting human hepatitis C virus antibodies includes a bottom plate, and a sample pad, a labeling pad, a detection pad and a sample absorption pad which are stacked on the bottom plate in sequence from bottom to top; wherein, the labeling pad and / or the detection pad includes the above-mentioned human hepatitis C virus protein.

[0024] Preferably, the labeling pad contains the above-mentioned human hepatitis C virus protein;

[0025] Preferably, the labeling pad contains chicken IgY antibody, and the chicken IgY antibody is labeled with a labeling agent;

[0026] Preferably, the labeling agent includes any one or a combination of at least two of colloidal gold, colored microspheres, time-resolved fluorescence microspheres or quantum dot microspheres.

[0027] Preferably in any of the above, the average particle size of the colloidal gold particles is 40-100 nm; exemplarily includes 40 nm, 50 nm, 60 nm, 80 nm, 100 nm and the ranges therebetween.

[0028] Preferably in any of the above, the average particle size of the colored microsphere particles is 100-300 nm; exemplarily includes 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and the ranges therebetween.

[0029] Preferably in any of the above, the average particle size of the time-resolved fluorescence microsphere particles is 100-300 nm; exemplarily includes 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and the ranges therebetween.

[0030] Preferably in any of the above, the average particle size of the quantum dot microsphere particles is 100-300 nm; exemplarily includes 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and the ranges therebetween.

[0031] As a preferred embodiment of the present invention, the mass ratio of the human hepatitis C virus protein to colloidal gold is (0.04 - 0.32):1; exemplarily including 0.04:1, 0.10:1, 0.15:1, 0.20:1, 0.25:1, 0.30:1, 0.32:1 and the ranges therebetween.

[0032] As a preferred embodiment of the present invention, the mass ratio of the human hepatitis C virus protein to colored microspheres is (0.1 - 0.4):1; exemplarily including 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the ranges therebetween.

[0033] As a preferred embodiment of the present invention, the mass ratio of the human hepatitis C virus protein to time-resolved fluorescence microspheres is (0.1 - 0.4):1; exemplarily including 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the ranges therebetween.

[0034] As a preferred embodiment of the present invention, the mass ratio of the human hepatitis C virus protein to quantum dot microspheres is (0.1 - 0.4):1; exemplarily including 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the ranges therebetween.

[0035] Preferably, a test line and a quality control line are provided on the test pad; the test line is coated with the human hepatitis C virus protein; the quality control line is coated with goat anti-chicken IgY antibody.

[0036] Preferably in any of the above, the test line is coated with the human hepatitis C virus protein at a concentration of 0.5 - 5 mg / mL. Exemplarily including the human hepatitis C virus protein at concentrations of 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL and the ranges therebetween.

[0037] Preferably in any of the above, the quality control line is coated with goat anti-chicken IgY antibody at a concentration of 0.5 - 5 mg / mL. Exemplarily including goat anti-chicken IgY antibody at concentrations of 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL and the ranges therebetween.

[0038] Preferably, in any of the above, the diluent of the human hepatitis C virus protein and the goat anti-chicken IgY antibody is a 10-50 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose. Exemplarily, the diluent is a PB buffer containing trehalose at 10 mM, 20 mM, 30 mM, 40 mM, 50 mM and ranges therebetween; Exemplarily, each 100 mL of the diluent contains 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 g of trehalose and ranges therebetween.

[0039] Preferably, the human hepatitis C virus protein on the conjugate pad is the same as the human hepatitis C virus protein on the test pad.

[0040] Preferably, in any of the above, a test line and a control line are provided on the test pad; the test line is coated with the human hepatitis C virus protein; the control line is coated with goat anti-chicken IgY; the conjugate pad contains the same human hepatitis C virus protein, and the human hepatitis C virus protein on the conjugate pad is labeled with a label; the conjugate pad further contains a chicken IgY antibody, and the chicken IgY antibody on the conjugate pad is labeled with a label.

[0041] The present invention also provides a human hepatitis C virus antibody detection test strip and a housing including any of the above, and the human hepatitis C virus antibody detection test strip is disposed inside the housing.

[0042] Preferably, the housing includes a detachable upper cover and a lower cover.

[0043] Preferably, in any of the above, the upper cover is provided with an observation window and a sample addition hole.

[0044] Preferably, in any of the above, the lower cover is provided with a test card mounting strip area.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The human hepatitis C virus antibody detection kit provided by the present invention has the advantages of simple operation, rapid reaction, high sensitivity, strong specificity, suitability for on-site rapid detection, economy and practicality, etc., and is of great significance for the early diagnosis of HCV infection, prediction of disease progression, judgment of prognosis, and screening and evaluation of the efficacy of anti-HCV drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is the upper cover of the human hepatitis C virus antibody detection kit in Example 1 of the present invention;

[0048] Figure 2This is the lower cover of the human hepatitis C virus antibody detection kit in Embodiment 1 of the present invention;

[0049] Figure 3 This is the structural diagram of the human hepatitis C virus antibody detection test strip in Embodiment 1 of the present invention;

[0050] Figure 4 This is the detection result display diagram in Embodiment 2 of the present invention;

[0051] Icon: 1 - Observation window; 2 - Sample addition hole; 3 - Test card installation strip area; 4 - Bottom plate; 5 - Detection pad; 6 - Sample absorption pad; 7 - Marking pad; 8 - Sample pad; 9 - Quality control line; 10 - Detection line. Detailed implementation manners

[0052] The following will describe the implementation schemes of the present invention in detail in combination with the implementation manners and embodiments. However, those skilled in the art will understand that the following implementation manners and embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. Those not specifying specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicating the manufacturer are all conventional products that can be obtained through commercial purchase.

[0053] The embodiment of the present invention provides a human hepatitis C virus protein, which includes protein fragment C-a, protein fragment NS3-a, and protein fragment NS5-a;

[0054] The amino acid sequence of protein fragment C-a is shown in SEQ ID NO.1;

[0055] The amino acid sequence of protein fragment NS3-a is shown in SEQ ID NO.2;

[0056] The amino acid sequence of protein fragment NS5-a is shown in SEQ ID NO.3;

[0057] Among them, protein fragment C-a, protein fragment NS3-a, and protein fragment NS5-a can be combined and connected in any order.

[0058] In some embodiments, the combination mode of the protein fragments of the human hepatitis C virus protein is any one of the following combinations:

[0059] C-a + NS3-a + NS5-a;

[0060] C-a + NS5-a + NS3-a;

[0061] NS3-a + NS5-a + C-a;

[0062] NS3-a + C-a + NS5-a;

[0063] NS5-a + NS3-a + C-a;

[0064] NS5-a + C-a + NS3-a.

[0065] An embodiment of the present invention provides a human hepatitis C virus antibody detection test strip, which includes a bottom plate, and a sample pad, a labeling pad, a detection pad, and a sample absorption pad that are sequentially stacked on the bottom plate from top to bottom.

[0066] In some embodiments, the labeling pad contains the above-mentioned human hepatitis C virus protein, and the human hepatitis C virus protein is labeled with a label, and the label can detect and identify the position or concentration where the label is located.

[0067] The human hepatitis C virus antibody detection test strip provided by the present invention has the advantages of simple operation, rapid reaction, high sensitivity, strong specificity, suitability for on-site rapid detection, economy and practicality, etc., and is of great significance for the early diagnosis of HCV infection, prediction of disease progression, judgment of prognosis, and screening and evaluation of the efficacy of anti-HCV drugs.

[0068] In some embodiments, the label includes but is not limited to at least one of colloidal gold, colored microspheres, time-resolved fluorescence microspheres, or quantum dot microspheres;

[0069] In some embodiments, the particle size of the colloidal gold can be, for example, but not limited to, 40 nm, 60 nm, 80 nm, or 100 nm;

[0070] In some embodiments, the particle sizes of the colored microspheres, time-resolved fluorescence microspheres, and quantum dot microspheres can be, for example, but not limited to, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm;

[0071] In some embodiments, the mass ratio of the human hepatitis C virus protein conjugated with colloidal gold can be, for example, but not limited to, 0.04:1, 0.10:1, 0.15:1, 0.20:1, 0.25:1, 0.30:1, 0.32:1; the mass ratio of the human hepatitis C virus protein to the colored microspheres, time-resolved fluorescence microspheres, or quantum dot microspheres can be, for example, but not limited to, 0.1:1, 0.2:1, 0.3:1, 0.4:1.

[0072] In some embodiments, by adjusting the particle size and dosage of the label, the sensitivity of the test strip is made higher.

[0073] In some embodiments, a test line (T line) and a quality control line (C line) are provided on the detection pad;

[0074] The test line is coated with the aforementioned human hepatitis C virus protein, which can be, for example, but not limited to, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL;

[0075] The quality control line is coated with, for example, but not limited to, goat anti-chicken IgY antibody at 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL;

[0076] In some embodiments, the diluent of the human hepatitis C virus protein and the goat anti-chicken IgY antibody is a PB buffer containing trehalose, which can be, for example, but not limited to, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, and each 100 mL of the diluent contains, for example, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 g of trehalose.

[0077] In some preferred embodiments, the human hepatitis C virus protein on the labeling pad is the same as the human hepatitis C virus protein on the test pad.

[0078] The test strip provided by the present invention is based on the principle of antigen-antibody reaction. After labeling the human hepatitis C virus monoclonal protein fragment with colloidal gold, colored microspheres, time-resolved fluorescence microspheres or quantum dots, the labeling agent is solidified on the glass cellulose membrane. The same human hepatitis C virus is coated on the test pad (such as NC membrane). Based on the principle of antigen-antibody reaction, it can be detected by the naked eye or a supporting instrument within the detection time. If there is human hepatitis C virus antibody in the sample, a double-protein fragment sandwich structure is formed, forming a visible band with the naked eye or a light intensity signal in the instrument. If there is no human hepatitis C virus antibody in the sample, no band appears on the NC membrane or there is no light intensity signal in the instrument. The positive and negative are judged according to the presence or absence of the signal, or the antibody content is predicted according to the light intensity signal intensity.

[0079] Using the test strip provided by the present invention for detection, the detection result can be obtained within 10 - 30 minutes for the whole process, which is fast and efficient, helping medical staff to obtain the detection result in time, making a comprehensive judgment according to the result and disposing in time, avoiding panic and reducing the spread of the epidemic.

[0080] The embodiment of the present invention provides a kit for detecting human hepatitis C virus antibody, which includes a test strip for detecting human hepatitis C virus antibody and a housing, and the test strip for detecting human hepatitis C virus antibody is arranged inside the housing.

[0081] The human hepatitis C virus antibody detection kit provided by the present invention contains a human hepatitis C virus antibody detection test strip, and thus has all the beneficial effects of the human hepatitis C virus antibody detection test strip.

[0082] In some embodiments, the housing includes a detachable upper cover and a lower cover;

[0083] The upper cover is provided with an observation window and a sample addition hole, and the lower cover is provided with a test card mounting strip area.

[0084] In the present invention, the shapes of the observation window and the sample addition hole are not specifically limited, and those skilled in the art can adjust them according to actual needs. Exemplarily, the observation window can be square, located above the test line and the quality control line of the test strip for observing the test results. The sample addition hole can be, for example, a circular hole with a diameter of 0.5 - 1 cm, located above the sample pad.

[0085] In some embodiments, sample addition for the kit test:

[0086] Unless otherwise specified in the following examples, the diluent for human hepatitis C virus protein and goat anti - chicken IgY antibody is 20 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.5 g of trehalose.

[0087] Example 1

[0088] This example provides a human hepatitis C virus antibody detection kit, including a housing and a test strip located inside the housing. Among them, the housing includes a detachable upper cover and a lower cover. As Figure 1 shown, the upper cover is provided with an observation window 1 and a sample addition hole 2; as Figure 2 shown, the lower cover is provided with a test card mounting strip area 3; as Figure 3 shown, the human hepatitis C virus antibody detection test strip includes a bottom plate 4 and a sample pad 8, a labeled pad 7, a test pad 5, and a sample absorption pad 6 stacked in sequence on the bottom plate; a test line 10 and a quality control line 9 are provided on the test pad.

[0089] Example 2

[0090] This example provides a colloidal gold - labeled protein fragment for preparing a human hepatitis C virus antibody detection kit, and the kit structure is as shown in Example 1:

[0091] 1 Main materials

[0092] 1.1 Protein fragments: human hepatitis C virus protein (protein fragment combination is C-a+NS3-a+NS5-a), used for labeling and detection line coating respectively; chicken IgY antibody: purchased from Nanjing Jingda Biotechnology Co., Ltd., used for labeling; goat anti-chicken IgY antibody: purchased from Nanjing Jingda Biotechnology Co., Ltd., used for nitrocellulose membrane quality control line coating;

[0093] 1.2 Nitrocellulose membrane: NC membrane is a product of Sartorius;

[0094] 1.3 Other consumables: PVC boards and other consumables are products of Beacon Labs; commonly used reagents are all analytical grade reagents and can be purchased through platforms such as Aladdin.

[0095] 2 Methods

[0096] 2.1 Preparation of colloidal gold labeling pad:

[0097] The steps for preparing the colloidal gold labeling pad are as follows:

[0098] (1) Take 1 mL of colloidal gold solution with a particle size of 40 nm and adjust the pH to 8.5 with 0.2 M K2CO3;

[0099] (2) Add 25 μg of human hepatitis C virus protein and 5 μg of chicken IgY antibody. The mass ratio of total protein to colloidal gold particles is 0.12:1. Adjust the rotary shaker to a certain speed. Rotate and label at room temperature for 1.5 h. Then add 20 μL of blocking solution.

[0100] (3) Centrifuge at 12,000 rpm for 15 min and discard the supernatant;

[0101] (4) Add 100 μL of colloidal gold solution;

[0102] (5) The above concentrate was diluted in a ratio of 1:7, sprayed with gold, and placed in a drying oven at 37°C for 2 h for later use.

[0103] 2.2 NC membrane coating:

[0104] Human hepatitis C virus protein was diluted to 1.5 mg / mL and goat anti-chicken IgY antibody was diluted to 2 mg / mL using 0.02 M PB containing 0.5% trehalose. Then, the detection line T and the quality control line C were drawn on the nitrocellulose membrane using a film sprayer. After coating, the NC membrane was dried in an oven at 37°C for 24 h for later use.

[0105] 2.3 Assembly of the kit:

[0106] Place the coated nitrocellulose membrane in the middle of the plastic support plate and paste it in the drying chamber. Lap and paste the labeled colloidal gold pad on one side of the T line of the nitrocellulose membrane (lap 1 / 3 of the colloidal gold pad), and lap and paste the sample pad on the other side of the colloidal gold pad (lap 1 / 5 of the colloidal gold pad); lap and paste the sample absorption pad on one side of the C line of the nitrocellulose membrane (lap 1 / 10 of the sample absorption pad); then use a cutting machine to cut the plastic plate with the pasted membrane into test strips of a certain width, and then put them into the test card to form a kit for detecting human hepatitis C virus antibodies.

[0107] 2.4 Detection:

[0108] Step 1: Take out the kit and the sample to be tested, and equilibrate to room temperature;

[0109] Step 2: Open the sealed aluminum foil bag, take out the kit and place it flat on the table;

[0110] Step 3: At Figure 1 Add 2 drops of the sample (about 80 - 100 μL) to the sample adding hole;

[0111] Step 4: Start timing with a timer, and read the result after 10 min; note that if the sample does not laterally chromatograph or percolate within 1 min after adding the sample, it may be due to the sample being too viscous, and the sample needs to be pretreated with physiological saline.

[0112] 3 Results

[0113] Under the action of lateral chromatography, when there is human hepatitis C virus antibody in the sample, the test line will show color, and at the same time the quality control line will show color ( Figure 4 a in); when there is no human hepatitis C virus antibody in the sample, the test line does not show color, and the quality control line shows color ( Figure 4 b in); after loading the sample, if the quality control line does not show color, regardless of whether the test line shows color or not, the result is judged invalid ( Figure 4 c in, Figure 4 d in).

[0114] 4 Use the kit provided in Example 2 to detect the blood samples of HCV patients, and the results are consistent with those shown in 3. It shows the effectiveness of the human hepatitis C virus protein provided by the present invention in the detection of HCV antibodies.

[0115] Example 3

[0116] This embodiment provides a kit for detecting human hepatitis C virus antibodies by preparing protein fragments labeled with time-resolved fluorescent microspheres; using the monoclonal protein of human hepatitis C virus provided in this embodiment to prepare a kit for detecting human hepatitis C virus antibodies, the kit includes a test card and a test strip, the test card is divided into an upper cover and a lower bottom, the test strip embeds time-resolved fluorescent microspheres labeled with human hepatitis C virus protein in the fluorescence pad, and the test line is coated with human hepatitis C virus protein, and the double protein sandwich method is used to quantitatively detect human hepatitis C virus antibodies in the sample.

[0117] 1 Preparation operation process of the kit:

[0118] The surface of the time-resolved fluorescent microspheres is labeled with the human hepatitis C virus protein (the protein fragment combination is C-a + NS3-a + NS5-a) prepared by the method of the present invention. The specific embodiments are as follows:

[0119] Labeling of time-resolved fluorescent microglobulin fragments: 1 mL of 1% carboxyl time-resolved fluorescent microspheres, add 9 mL of MES buffer, then add 25 μL of EDC solution (10 mg / mL) and 25 μL of NHS solution (10 mg / mL), shake and react at room temperature for 30 min, and centrifuge to collect the precipitate. Add HEPES complex solution, ultrasonically disperse evenly and then add 1 mL of 1 mg / mL human hepatitis C virus protein, shake and react at room temperature for 120 min, and centrifuge to collect the precipitate; then add 1 mL of blocking solution, shake and react at room temperature for 120 min. Centrifuge to collect the microsphere precipitate and redissolve it with the complex solution.

[0120] Preparation of the fluorescence pad: The labeled time-resolved fluorescent microspheres are diluted with the microsphere complex solution, and the fluorescence pad is sprayed with a gold-spraying and membrane-drawing instrument, spraying 3 μL / cm, spraying spacing 6 mm, and after spraying, put it in a low humidity environment at 37 °C (<30%) and dry for 2 h.

[0121] Coating the CT line on the NC membrane: The concentration of the human hepatitis C virus protein used for the T line is 1.5 mg / mL, and the concentration of the goat anti-chicken IgY antibody used for the C line is 1 mg / mL, and it is drawn at 1 μL / cm. After completion, put it in a low humidity environment at 37 °C (<30%) and dry for 24 h.

[0122] Treatment of the sample pad: The sample pad treatment solution is composed of buffer salts, sustained-release agents, co-solvents, blocking agents, etc. The specific formula is 20 mM Tris buffer, and each 100 mL of Tris buffer contains 1 g of BSA, 0.5 g of Tween 20, and 2 g of sucrose. According to every 20 cm 2 Use 1 mL of the sample treatment solution for treatment. After uniform treatment, put it in a low humidity environment at 37 °C (<30%) and dry for 2 h.

[0123] Test strip assembly: An NC membrane, a sample absorption pad, a fluorescence pad, and a sample pad are sequentially pasted onto a PVC board. The sample absorption pad and the fluorescence pad each press the NC membrane by 1 - 2 mm, and the sample pad presses the fluorescence pad by 1 - 2 mm. After assembly, the test strip is cut into a width of 4 ± 0.4 mm and loaded into a cartridge. The cartridge and a desiccant are placed into an aluminum foil bag and then sealed. After labeling and boxing, the finished test card can be obtained.

[0124] 2 Detection process of the kit

[0125] Place the test card on a clean and flat tabletop, aspirate 80 - 100 μL of the treated sample and add it dropwise to the sample addition end of the test card, and set up a PBS control group at the same time.

[0126] Import the standard curve of the test strip. After 10 min, use a fluorescence immunoassay analyzer to scan the detection area to obtain a fluorescence signal, and the corresponding titer of the human hepatitis C virus antibody will be displayed after detection.

[0127] 3 Detection results

[0128] Select 3 negative samples and 3 low - value positive samples each, dilute them 2, 10, 50, 250, and 1250 times respectively with 0.01 M PBS (pH 7.2), and then use the above - mentioned finished test card for detection. The results show that there are still significant differences between the test results of the low - value positive samples diluted 250 times and the negative samples. The test results are shown in Table 1:

[0129] Table 1

[0130] 。

[0131] Example 4

[0132] This example provides a kit for detecting human hepatitis C virus antibodies by preparing a protein fragment labeled with colored microspheres. Using the human hepatitis C virus monoclonal protein provided in this example, a test strip for detecting human hepatitis C virus antibodies is prepared. The test strip embeds the human hepatitis C virus protein labeled with colored microspheres on the labeling pad, and the detection line is coated with the human hepatitis C virus protein. The double - protein sandwich method is used to qualitatively detect the human hepatitis C virus antibodies in the sample.

[0133] 1 Preparation of the test strip:

[0134] 1.1 Protein fragment labeled with colored microspheres

[0135] (1) Adjust the pH of 100 - nm - sized colored microspheres to 8.0 with 0.1 mol / L K2CO3;

[0136] (2) Human hepatitis C virus protein-labeled microspheres: Take 1 mL of the above-mentioned solution with adjusted pH, add 30 μg of human hepatitis C virus protein (the protein fragment combination is C-a + NS3-a + NS5-a), and the mass ratio of the total protein to the colored microspheres is 0.3:1. After reacting at room temperature for 1 h, centrifuge and discard the supernatant;

[0137] Chicken IgY protein fragment-labeled microspheres: Add 5 μg of chicken IgY to 1 mL of the above-mentioned solution with adjusted pH. The mass ratio of the protein fragment to the colored microspheres is 0.05:1. After reacting at room temperature for 1 h, centrifuge and discard the supernatant;

[0138] (3) Add 1 mL of 20% BSA and incubate for 2 h, then centrifuge and discard the supernatant;

[0139] (4) After redissolving the above-mentioned microspheres with 100 μL of the reconstitution solution at pH 8.0, mix the two kinds of microspheres at a ratio of 5:1, and dilute the mixture with the reconstitution solution at a ratio of 15%.

[0140] 1.2 Preparation of the conjugate pad

[0141] Dilute the labeled colored microsphere protein fragment complex, and use a gold-spraying and membrane-stripping instrument to spray the conjugate pad at a rate of 7.5 μL / cm with a spraying spacing of 6 mm. After spraying, place it in an oven at 37°C with low humidity (<30%) and dry for 2 h.

[0142] 1.3 Preparation of the sample pad

[0143] The sample pad treatment solution consists of buffer salts, sustained-release agents, co-solvents, blocking agents, etc.; the specific formula is 20 mM Tris, 1% BSA, 0.5% Tween 20, 2% sucrose; for every 20 cm 2 Use 1 mL of the sample treatment solution for treatment. After uniform treatment, place it in an oven at 37°C with low humidity (<30%) and dry for 2 h.

[0144] 1.4 Coating of the C / T lines

[0145] The concentration of the protein used for the T line is 1.5 mg / mL of human hepatitis C virus protein (the concentration of the protein used for the C line is 2 mg / mL of goat anti-chicken IgY protein fragment), and it is drawn at 1 μL / cm. After completion, place it in an oven at 37°C with low humidity (<30%) and dry for 24 h.

[0146] 1.5 Assembly of the test strip

[0147] On the PVC board, paste the sample pad, NC membrane, conjugate pad, and sample pad in sequence. The sample pad and the conjugate pad each press the NC membrane by 1 - 2 mm, and the sample pad presses the conjugate pad by 1 - 2 mm; after assembly, cut the test strip into a width of 4 ± 0.4 mm and install it in the cartridge; put the cartridge and the desiccant into an aluminum foil bag and seal it; label and pack it into a box to obtain the finished kit.

[0148] 2 Detection

[0149] Place the test card on a clean and flat tabletop, aspirate 80 - 100 μL of the treated sample and add it dropwise to the sample application end of the test card, and set up a PBS control group; observe the window of the test card after 10 minutes. If there are 2 lines in the window, it is positive, and if there is only one C line, it is negative.

[0150] 3 Test Results

[0151] Select 3 negative samples and 3 low - value positive samples each, dilute them 2, 10, 50, 250, and 1250 times respectively with 0.01 M PBS (pH 7.2), and then test them with the above - mentioned finished test kit. The results show that the test result of the low - value positive sample is still positive after being diluted 250 times. The test results are shown in Table 2:

[0152] Table 2

[0153] 。

[0154] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A human hepatitis C virus protein, characterized in that The combination connection mode of the human hepatitis C virus protein is C-a+NS3-a+NS5-a, wherein: Protein fragment Ca, whose amino acid sequence is shown in SEQ ID NO.1; Protein fragment NS3-a, the amino acid sequence of which is shown in SEQ ID NO.2; The amino acid sequence of protein fragment NS5-a is shown in SEQ ID NO.

3.

2. Use of the human hepatitis C virus protein according to claim 1 in the preparation of a human hepatitis C virus antibody detection product.

3. A test strip for detecting antibodies against human hepatitis C virus, characterized in that: The human hepatitis C virus antibody detection test strip comprises a bottom plate and a sample pad, a marking pad, a detection pad and a sample suction pad which are sequentially stacked on the bottom plate from bottom to top; Wherein, the label pad and / or the detection pad comprises the human hepatitis C virus protein according to claim 1.

4. The human hepatitis C virus antibody detection test strip according to claim 3, characterized in that: The label pad contains chicken IgY antibody, and the chicken IgY antibody is labeled with a marker; The marker includes any one of colloidal gold, color microspheres, time-resolved fluorescent microspheres or quantum dot microspheres, or a combination of at least two of them.

5. The human hepatitis C virus antibody detection test strip according to claim 3, characterized in that: The detection pad is provided with a detection line and a quality control line; The detection pad includes the human hepatitis C virus protein of claim 1, and the detection line is coated with the human hepatitis C virus protein at a concentration of 0.5 to 5 mg / mL; The quality control line is coated with goat anti-chicken IgY antibody with a concentration of 0.5-5 mg / mL.

6. The human hepatitis C virus antibody detection test strip according to claim 3, characterized in that: The label pad and the detection pad both include the human hepatitis C virus protein according to claim 1.

7. A detection kit, characterized in that: The detection kit comprises a shell and a detection test paper arranged inside the shell, and the detection test paper is the human hepatitis C virus antibody detection test strip according to any one of claims 3-6.

8. The detection kit according to claim 7, characterized in that The housing comprises an upper cover and a lower cover which are detachably connected; The upper cover is provided with an observation window and a sample adding hole, and the lower cover is provided with a detection card strip installation area.

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