Immunoassay kit for detecting SFTSV-NP antigen, antibody and biological material

Abstract

The invention provides an immunokit for detecting SFTSV-NP antigen, an antibody and a biological material, and belongs to the technical field of immunodetection, a pair of new monoclonal antibodies aiming at new bunyavirus NP antigen is obtained through a hybridoma technology, and the monoclonal antibodies comprise a coating antibody and a detection antibody. Western Blot verifies that the antibody has high affinity and high specificity with the new bunyavirus NP protein, so that the antibody can be used for preparing a double-antibody sandwich ELISA detection kit aiming at the new bunyavirus NP antigen to obtain a detection kit with higher detection accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of immune detection, and in particular relates to an immune kit and an antibody for detecting SFTSV-NP antigen. Background Art

[0002] New Bunyavirus, full name Severe Fever with Thrombocytopenia Syndrome Bunyavirus (SFTSV), is a virus first reported and isolated in Henan Province, China. SFTSV is an enveloped negative-sense virus with one RNA chain. Three single-stranded segments make up the SFTSV genome: large segment (L), middle segment (M) and small segment (S). The L segment contains 6368 nucleotides, encoding the RNA-dependent RNA polymerase (RdRp) for viral replication, mediating viral RNA replication and mRNA synthesis; the M segment contains 3378 nucleotides, and the viral envelope glycoproteins (glycoproteins N Gn and C Gc) play a key role in viral assembly and viral particle formation; the S segment consists of 1744 nucleotides and uses two inverted reading frames to encode the nucleocapsid protein (N) and non-structural proteins (NSs).

[0003] Patients infected with the new Bunyavirus will mainly experience systemic poisoning symptoms such as fever, chills, fatigue, and body aches; digestive tract symptoms such as vomiting, hematemesis, and diarrhea. Patients whose condition deteriorates rapidly will soon experience impaired consciousness and respiratory and digestive tract bleeding. In more serious cases, they will die from multiple organ failure such as respiratory failure and disseminated intravascular coagulation. The pathogenic mechanism of SFTSV is still under further exploration. Preliminary studies have shown that SFTSV infection may activate some receptor tyrosine kinases, such as nerve growth factor receptors and vascular endothelial growth factor receptors, and complete the replication and release of the virus through the RTKs-PI3K / Akt-mTOR pathway. Since the clinical symptoms of the new Bunyavirus are not specific and are very similar to the symptoms of many hemorrhagic fever viruses, laboratory testing is necessary.

[0004] So far, there is no safe and effective vaccine for the prevention and treatment of SFTSV virus, so accurate and effective detection and diagnosis methods are particularly important. Existing detection methodologies are mainly divided into two categories: molecular detection and serological detection methods. Molecular detection methods use polymerase chain reaction (PCR) detection as the gold standard and have extremely high sensitivity and specificity. With the introduction of new technologies, many nucleic acid detection technologies with isothermal amplification (RT-LAMP) and CRISPR-Cas systems as the core have emerged in recent years, greatly improving the detection performance. However, molecular detection methods require professional laboratory operators, detection sites and a lot of time, and are not suitable for bedside testing (POCT) and large-scale population screening in epidemic areas. Serological detection methods include enzyme-linked immunosorbent assay (ELISA) and immunochromatography technology, such as the technical solution disclosed in the invention patent with patent number CN201210121521.3. Compared with molecular detection methods, serological detection technology has higher detection efficiency. Based on the high affinity and high specificity of antibody-antigen binding, the monoclonal antibody of SFTSV-NP antigen can specifically bind to NP protein and can be used as SFTSV-NP monoclonal antibody for prevention and treatment. For the serum of patients infected with SFTSV, the antibody serum detection method of enzyme-linked immunosorbent assay (ELISA) that captures NP protein has become the new standard for determining and identifying whether SFTSV is infected. However, the specificity and affinity of the antibodies required for ELISA for SFTSV are insufficient and still need to be improved. Summary of the invention

[0005] Based on the above problems in the prior art, the present invention provides an immune kit for detecting SFTSV-NP antigen, wherein the immune kit is a double antibody sandwich ELISA immune kit, comprising a detection antibody and a coating antibody, wherein the heavy chain of the detection antibody comprises an amino acid sequence as shown in SEQ ID NO.1.

[0006] Preferably, the light chain of the detection antibody comprises the amino acid sequence shown in SEQ ID NO.2.

[0007] Preferably, the heavy chain of the coated antibody comprises the amino acid sequence shown in SEQ ID NO.3.

[0008] Preferably, the light chain of the coated antibody comprises the amino acid sequence shown in SEQ ID NO.4.

[0009] Preferably, the detection antibody and the coating antibody are both IgG monoclonal antibodies.

[0010] The present invention also provides an ELISA immune detection antibody for detecting SFTSV-NP antigen, wherein the heavy chain of the detection antibody comprises the amino acid sequence shown in the sequence SEQ ID NO.1; preferably, the light chain of the detection antibody comprises the amino acid sequence shown in the sequence SEQ ID NO.2.

[0011] The present invention also provides an ELISA immune coated antibody for detecting SFTSV-NP antigen, wherein the heavy chain of the coated antibody comprises the amino acid sequence shown in the sequence SEQ ID NO.3; and the light chain of the coated antibody comprises the amino acid sequence shown in the sequence SEQ ID NO.4.

[0012] The present invention also provides a biological material for detecting SFTSV-NP antigen, wherein the biological material contains or carries a nucleotide sequence expressing an antibody as shown in SEQ ID NO.1-4, and the biological material is a nucleotide sequence, a plasmid vector, a viral vector or a cell. The present invention has the beneficial effects of obtaining a pair of monoclonal antibodies targeting the new Bunyavirus NP antigen through hybridoma technology, including a coating antibody and a detection antibody, and verifying the high affinity and high specificity of the antibodies to the new Bunyavirus NP protein through Western Blot. Therefore, the antibodies can be used for the preparation of a double antibody sandwich ELISA detection kit targeting the new Bunyavirus NP antigen to obtain a detection kit with higher detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram of the detection principle of the ELISA detection kit of the present invention.

[0014] Figure 2 This is a diagram showing the Western Blot identification results of two monoclonal antibodies.

[0015] Figure 3 The standard curves for the ELISA assays of the two antibodies.

[0016] Figure 4 The cut-off values ​​of the two antibodies are shown in Figure 2.

[0017] Figure 5 These are the specificity test results for the two antibodies.

[0018] Figure 6 The sensitivity test results of the two antibodies are shown in Figure 2. DETAILED DESCRIPTION

[0019] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Example 1: An immunological kit for detecting SFTSV-NP antigen.

[0021] An immune kit for detecting SFTSV-NP antigen, the immune kit is a double antibody sandwich ELISA immune kit, comprising a detection antibody and a coating antibody, the detection antibody and the coating antibody are both IgG monoclonal antibodies, the heavy chain of the detection antibody comprises the amino acid sequence shown in the sequence SEQ ID NO.1, and the light chain comprises the amino acid sequence shown in the sequence SEQ ID NO.2; the heavy chain of the coating antibody comprises the amino acid sequence shown in the sequence SEQ ID NO.3, and the light chain comprises the amino acid sequence shown in the sequence SEQ ID NO.4.

[0022] The principle of the kit is as follows Figure 1 As shown, the method of use is as follows: the coated antibody is coated on the ELISA plate and then blocked, the sample to be tested is added, the detection antibody is added after incubation, the color developing solution is added after incubation, and the test result of the sample to be tested is obtained by analyzing the result of the ELISA instrument.

[0023] Example 2: An ELISA immunoassay antibody for detecting SFTSV-NP antigen.

[0024] An ELISA immune detection antibody for detecting SFTSV-NP antigen, wherein the heavy chain of the detection antibody comprises the amino acid sequence shown in the sequence SEQ ID NO.1, and the light chain comprises the amino acid sequence shown in the sequence SEQ ID NO.2.

[0025] Example 3: An ELISA immune-coated antibody for detecting SFTSV-NP antigen.

[0026] An ELISA immune coated antibody for detecting SFTSV-NP antigen, wherein the heavy chain of the coated antibody comprises the amino acid sequence shown in the sequence SEQ ID NO.3, and the light chain comprises the amino acid sequence shown in the sequence SEQ ID NO.4.

[0027] Example 4: A biological material for detecting SFTSV-NP antigen.

[0028] A biological material for detecting SFTSV-NP antigen, wherein the biological material contains or carries a nucleotide sequence expressing an antibody as shown in SEQ ID NO.1-4, and the biological material is a nucleotide sequence, a plasmid vector, a viral vector or a cell.

[0029] The following is a detailed description of the preparation process of the antibodies described in Examples 2 and 3 and the related tests and evaluations of the antibodies. The results are as follows: Figure 2-6 shown.

[0030] 1. Preparation of new bunyavirus NP protein.

[0031] According to the gene sequence information of the new bunyavirus NP protein published by NCBI (> NC_043452.1: c1674-937 Severe fever with thrombocytopenia syndrome virus strain HNXH segment S, complete sequence), the gene sequence was re-optimized, the new gene was obtained by the whole gene synthesis method, and inserted into the expression vector by double enzyme digestion. Two restriction endonucleases, Xho I and Nde I, were used for double enzyme digestion. The digestion product was inserted into the expression vector pET-30a (+), and the ligation product was transformed into Escherichia coli DH5α competent cells. A single colony was picked from a kanamycin-containing resistant plate and inoculated into liquid culture medium for culture. After the bacterial solution was verified by PCR, it was expanded, the plasmid was extracted by alkaline lysis method and digested, and the size was correct. After sequencing, the DH5SV01- strain and recombinant plasmid were saved. The recombinant plasmid was transferred into the expression strain BL21 cells for propagation, and isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 200ug / ml to induce expression at 25°C and 200rpm. The bacteria were collected, the cells were ultrasonically broken and lysed, the protein was collected, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to verify the correct expression of the protein. After verification, a large amount of expression was performed. After the induction was completed, the bacteria were centrifuged and collected, and PBS was added for ultrasonic lysis. After lysis, TritonX-100 was added to a final concentration of 1%, placed on ice and stirred for 1h, and then centrifuged at high speed. The supernatant was taken and the expressed recombinant protein was purified using an AKTA purifier and a cytiva HisTrap purification column according to the instructions for experimental consumables. The obtained recombinant protein was concentrated with a Millipor ultrafiltration tube to obtain the final new bunyavirus NP recombinant protein. The recombinant protein will be used to prepare monoclonal antibodies against the new bunyavirus NP protein.

[0032] 2. Inactivation of new Bunyavirus.

[0033] The inoculation and inactivation experiments of SFTSV virus were carried out in a biosafety level 3 laboratory (P3 laboratory). The Vero cells stored in the laboratory were revived, and the revived T25 cell culture flasks were placed in a cell culture incubator at 37°C and 5% CO2. An appropriate amount of 10% DMEM medium was added to culture and proliferate to 80% of the T25 cell culture flask, and then subcultured. When the cells were cultured to form a dense monolayer, the SFTSV strain was inoculated with MOI=0.01, and the cell lesions were observed under a microscope every day. After complete lesions occurred in 5-7 days, the virus was harvested and the virus titer was determined. β-propiolactone was added to the virus collection solution at 1:4000, and it was inactivated at 4°C for 48h, then placed in a 37°C water bath for 2h, and the inactivated virus culture was stored at -20°C.

[0034] 3. Preparation of monoclonal antibodies against the NP protein of the new bunyavirus.

[0035] 6-8 week old healthy Balb / c female mice were selected and injected according to the pre-specified immunization scheme as the immunogen to immunize BALB / c mice. The spleen lymphocytes of the successfully immunized mice were extracted and fused with mouse myeloma cells SP2 / 0 by cell fusion technology. After two rounds of subclone screening, a hybridoma cell line that stably secreted monoclonal antibodies against the new Bunyavirus NP antigen was obtained, thereby obtaining monoclonal antibodies against the new Bunyavirus NP antigen.

[0036] The HIS-SFTSV-NP antigen purified by prokaryotic expression was used to perform phased immunization on experimental mice.

[0037] The specific steps of animal immunization experiments include: 1. Balb / c mice with the same mean body weight and age were randomly divided into two groups: the group with aluminum adjuvant (aluminum hydroxide adjuvant) and the group without aluminum adjuvant.

[0038] 2. Before the experiment, the pre-immune serum of each mouse was collected (pre-immune serum was collected on the fifth day, blood was collected through the eyeball, and an appropriate amount of blood was collected to ensure the normal state of the mice), and the collected serum was stored at -80°C.

[0039] 3. Preparation of the aluminum adjuvant (aluminum hydroxide adjuvant) group: Before immunization, each antigen was diluted to the corresponding dose (75 μg / mouse) in 75 μL PBS, and mixed with alum adjuvant (1 mg / mouse), according to the volume of antigen: adjuvant = 3:1 (i.e., 25 μl adjuvant was added to 75 μl immunogen diluent); the adjuvant was shaken before use, and the injection adjuvant (25 μl) was slowly added to the immunogen solution; after the adjuvant and the immunogen diluent were fully mixed, the two were fully mixed for 30 minutes. The adjuvant effectively adsorbed the antigen; the subsequent operation was carried out according to the immunization animal experiment.

[0040] 4. Group without aluminum adjuvant: The antigen was diluted in 100 μL PBS to the corresponding dose in the above table (75 μg / mouse), and 100 μL of immunogen was added. The subsequent operation was carried out according to the immune animal experiment.

[0041] 5. Subcutaneous injection at intervals of 2 weeks: The experimental design is a 3-time immunization method, but blood is collected from the eyeball 7 days after each immunization, and part of the mouse supernatant is obtained by centrifugation. The serum titer is first tested. 7 days after the last immunization, blood is collected from the heart to obtain the maximum blood volume, and the supernatant is obtained by centrifugation and stored at -80°C; 6. Detect serum titer.

[0042] The indirect ELISA was used to detect the titer of the immunized mouse serum using the new Bunyavirus NP antigen as the coating antigen. Indirect ELISA method: 50 μl of the coating antigen diluted with the coating solution at 1 μg / ml was added to each well of the ELISA plate. After coating overnight at 4°C, the plate was washed 3 times with the washing solution (PBST) (the same below). 200 μl of the blocking solution (5% skim milk powder) was added to each well, and placed in a 37°C incubator for 2 h. After washing, 50 μl of the diluted serum was added to each well, and then reacted in a 37°C incubator for 30 min. After washing, 50 μl of the goat anti-mouse IgG-HRP solution was added, and reacted in a 37°C incubator for 30 min. After washing, 100 μl of the substrate solution was added, and the color was developed in a 37°C incubator in the dark for 10 min. Finally, 2 mol / L H2SO4, 50 μL was added to terminate the reaction, and the A450 value was read on an ELISA reader. When the orbital blood titers of mice immunized three times are all >62500 after three immunizations, and the titer of three mice can reach more than 50% at 1:12500, fusion can be arranged.

[0043] Immune spleen cells were fused with myeloma cell line SP2 / 0 cells, and the fused cells were selected by HAT selection medium (HAT selection medium contains hypoxanthine, aminopterin and thymidine), and the fused cells were subjected to ELISA positive screening and subcloning; the screened positive monoclonal antibodies were purified from ascites using Protein A / G antibody purification columns to obtain multiple monoclonal antibodies. The ELISA titer of the purified antibodies was >1: 128, 000, and the purity was >90%.

[0044] Two monoclonal antibodies were obtained, namely, a coating antibody (named SV07-28) and a detection antibody (named SV09-35). The SV09-35 heavy chain is shown in SEQ ID NO. 1, with a length of 352 bp, and the light chain is shown in SEQ ID NO. 2, with a length of 322 bp; the SV07-28 heavy chain is shown in SEQ ID NO. 3, with a length of 346 bp, and the SV07-28 light chain is shown in SEQ ID NO. 4, with a length of 322 bp.

[0045] 4. Double Antibody Sandwich ELISA Detection of New Bunyavirus NP Antigen 1. Standard curve drawing The established method was used: the absorbance value and the new bunyavirus NP antigen protein concentration fitting curve were used as regression analysis equation, and the standard curve was as follows Figure 3 As shown, repeated detection found that within the linear range, the absorbance values ​​showed a good linear relationship, R2=0.9746; 2.1 times the negative average value was taken as the judgment result.

[0046] 2. Confirm the cut-off value The average value of the test results was calculated by testing the OD450nm of 24 new bunyavirus-negative sera ( ) is 0.056, the standard deviation (SD) is 0.008, and the +3SD value is calculated to be 0.056+3×0.008=0.08. Therefore, when OD450nm≥0.08, the test result of the sample is determined to be positive, and when OD450nm<0.08, the test result of the sample is determined to be negative. The results are as follows Figure 4 shown.

[0047] 3. Specificity test Under appropriate conditions, positive sera of dengue virus, Zika virus and chikungunya virus were tested, and positive sera of new bunyavirus were used as positive control to verify the specificity of the ELISA test kit. The results are as follows Figure 5 shown.

[0048] 4. Sensitivity test The new bunyavirus positive serum was diluted 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200 respectively, and negative and positive controls were set up at the same time. The ELISA detection kit was used for determination, and the OD450nm value was read to verify its immunological reaction sensitivity. The results are as follows Figure 6 shown.

[0049] The above-mentioned embodiment only expresses one implementation mode of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. An immune kit for detecting SFTSV-NP antigen, the immune kit is a double antibody sandwich ELISA immune kit, comprising a detection antibody and a coating antibody, characterized in that: The heavy chain of the detection antibody comprises the amino acid sequence shown in SEQ ID NO.

1.

2. An immune kit for detecting SFTSV-NP antigen according to claim 1, characterized in that, The light chain of the detection antibody comprises the amino acid sequence shown in SEQ ID NO.

2.

3. An immune kit for detecting SFTSV-NP antigen according to claim 2, characterized in that, The heavy chain of the coated antibody comprises the amino acid sequence shown in SEQ ID NO.

3.

4. An immune kit for detecting SFTSV-NP antigen according to claim 3, characterized in that, The light chain of the coated antibody comprises the amino acid sequence shown in SEQ ID NO.

4.

5. An immune kit and antibody for detecting SFTSV-NP antigen according to claim 4, characterized in that, The detection antibody and the coating antibody are both IgG monoclonal antibodies.

6. An ELISA immunoassay antibody for detecting SFTSV-NP antigen, characterized in that: The heavy chain of the detection antibody comprises the amino acid sequence shown in SEQ ID NO.

1.

7. An ELISA immunoassay antibody for detecting SFTSV-NP antigen according to claim 6, characterized in that: The light chain of the detection antibody comprises the amino acid sequence shown in SEQ ID NO.

2.

8. An ELISA immune coated antibody for detecting SFTSV-NP antigen, characterized in that: The heavy chain of the coated antibody comprises the amino acid sequence shown in SEQ ID NO.

3.

9. An ELISA immune coated antibody for detecting SFTSV-NP antigen according to claim 8, characterized in that: The light chain of the coated antibody comprises the amino acid sequence shown in SEQ ID NO.

4.

10. A biological material for detecting SFTSV-NP antigen, characterized in that: The biological material contains or carries a nucleotide sequence expressing an antibody as shown in SEQ ID NO. 1-4, and the biological material is a nucleotide sequence, a plasmid vector, a viral vector or a cell.

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