An immunological kit, antibodies and biological materials for detecting SFTSV-NP antigen

Abstract

The application provides an immunological kit, antibodies and biological materials for detecting SFTSV-NP antigen, belongs to the technical field of immunological detection, and a pair of new monoclonal antibodies for the NP antigen of the new Bunyavirus is obtained through a hybridoma technology, including a coating antibody and a detection antibody; the high affinity and high specificity of the new Bunyavirus NP protein are verified through Western Blot, so that the preparation of a double-antibody sandwich ELISA detection kit for the NP antigen of the new Bunyavirus can be realized, and a detection kit with higher detection accuracy can be obtained.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, and particularly relates to an immunoassay kit and antibody for detecting SFTSV-NP antigen. Background Technology

[0002] 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 a single RNA strand. The SFTSV genome consists of three single-stranded segments: a large segment (L), a medium segment (M), and a small segment (S). The L segment, containing 6368 nucleotides, encodes RNA-dependent RNA polymerase (RdRp) for viral replication, mediating viral RNA replication and mRNA synthesis. The M segment, containing 3378 nucleotides, contains viral envelope glycoproteins (glycoproteins NGn and CGc), which play a crucial role in viral assembly and viral particle formation. The S segment, consisting of 1744 nucleotides, uses two reverse reading frames to encode nucleocapsid proteins (N) and non-structural proteins (NSs).

[0003] Patients infected with the novel Bunyavirus (SFTSV) primarily experience systemic symptoms of poisoning, such as fever, chills, fatigue, and body aches; as well as gastrointestinal symptoms like vomiting, hematemesis, and diarrhea. Patients with rapidly deteriorating conditions may quickly develop altered consciousness, respiratory and gastrointestinal bleeding, and in more severe cases, death due to respiratory failure, disseminated intravascular coagulation (DIC), and multiple organ failure. The pathogenic mechanism of SFTSV is still under investigation. Preliminary studies suggest that SFTSV infection may activate certain receptor tyrosine kinases, such as nerve growth factor receptor and vascular endothelial growth factor receptor, and complete viral replication and release through the RTKs-PI3K / Akt-mTOR pathway. Because the clinical symptoms of the novel Bunyavirus are not specific and are very similar to those of many hemorrhagic fever viruses, laboratory testing is essential.

[0004] Currently, there are no safe and effective vaccines for the prevention and treatment of SFTSV virus, making accurate and effective detection and diagnostic methods particularly important. Existing detection methodologies are mainly divided into two categories: molecular detection and serological detection methods. Molecular detection methods, with polymerase chain reaction (PCR) as the gold standard, have extremely high sensitivity and specificity. With the introduction of new technologies, many nucleic acid detection technologies based on isothermal amplification (RT-LAMP) and CRISPR-Cas systems have emerged in recent years, greatly improving detection performance. However, molecular detection methods require professional laboratory personnel, testing facilities, and significant time, making them unsuitable for point-of-care testing (POCT) and large-scale population screening in epidemic areas. Serological detection methods include enzyme-linked immunosorbent assay (ELISA) and immunochromatographic techniques, such as the technical solution disclosed in invention patent CN201210121521.3. Compared to molecular detection methods, serological detection technologies offer higher detection efficiency. Based on the high affinity and specificity of antibody-antigen binding, monoclonal antibodies against SFTSV-NP antigen can specifically bind to the NP protein and can be used for both prevention and treatment of SFTSV-NP. For serum samples from patients infected with SFTSV, enzyme-linked immunosorbent assay (ELISA) for capturing the NP protein has become the new standard for detecting and identifying SFTSV infection. However, the specificity and affinity of the antibodies required for current SFTSV ELISA methods are insufficient and need further improvement. Summary of the Invention

[0005] In view of the above-mentioned problems in the existing technology, the present invention provides an immunoassay kit for detecting SFTSV-NP antigen. The immunoassay kit is a double antibody sandwich ELISA immunoassay kit, comprising a detection antibody and a coating antibody. The heavy chain of the detection antibody contains a nucleotide sequence as shown in SEQ ID NO.1.

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

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

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

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

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

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

[0012] The present invention also provides a biological material for detecting SFTSV-NP antigen, the biological material comprising or carrying a nucleotide sequence expressing an antibody as shown in SEQ ID NO. 1-4, wherein the biological material is a nucleotide sequence, a plasmid vector, a viral vector, or a cell.

[0013] The beneficial effects of this invention are as follows: A pair of monoclonal antibodies against the new Bunyavirus NP antigen were obtained through hybridoma technology, including a coating antibody and a detection antibody. Western blotting verified their high affinity and high specificity for the new Bunyavirus NP protein. Therefore, they can be used to prepare a double-antibody sandwich ELISA detection kit against the new Bunyavirus NP antigen to obtain a detection kit with higher detection accuracy. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the detection principle of the ELISA detection kit of the present invention.

[0015] Figure 2 The image shows the Western blotting results of two monoclonal antibodies.

[0016] Figure 3 This is a standard curve for ELISA detection of two antibodies.

[0017] Figure 4 The cut-off values ​​for the two antibodies are shown.

[0018] Figure 5 These are the results of a specificity test for the two antibodies.

[0019] Figure 6 The results show the sensitivity test results for the two antibodies. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0021] Example 1: An immunoassay kit for detecting SFTSV-NP antigen.

[0022] An immunoassay kit for detecting SFTSV-NP antigen, the immunoassay kit being a double-antibody sandwich ELISA immunoassay kit, comprising a detection antibody and a coating antibody, both of which are IgG monoclonal antibodies, wherein the heavy chain of the detection antibody contains a nucleotide sequence as shown in SEQ ID NO.1, and the light chain contains a nucleotide sequence as shown in SEQ ID NO.2; the heavy chain of the coating antibody contains a nucleotide sequence as shown in SEQ ID NO.3, and the light chain contains a nucleotide sequence as shown in SEQ ID NO.4.

[0023] The principle of the reagent kit is as follows Figure 1 As shown, the usage method is as follows: after coating the ELISA plate with the antibody, block it, add the sample to be tested, incubate it, add the detection antibody, incubate it again, add the colorimetric solution, and obtain the detection result of the sample by analyzing the results of the ELISA reader.

[0024] Example 2: An ELISA immunoassay antibody for detecting SFTSV-NP antigen.

[0025] An ELISA immunoassay antibody for detecting SFTSV-NP antigen, wherein the heavy chain of the antibody comprises a nucleotide sequence as shown in SEQ ID NO.1, and the light chain comprises a nucleotide sequence as shown in SEQ ID NO.2.

[0026] Example 3: An ELISA-coated antibody for detecting SFTSV-NP antigen.

[0027] An ELISA-coated antibody for detecting SFTSV-NP antigen, wherein the heavy chain of the coated antibody comprises a nucleotide sequence as shown in SEQ ID NO.3, and the light chain comprises a nucleotide sequence as shown in SEQ ID NO.4.

[0028] Example 4: A biomaterial for detecting SFTSV-NP antigen.

[0029] A biological material for detecting SFTSV-NP antigen, the biological material comprising or carrying a nucleotide sequence expressing an antibody as shown in SEQ ID NO. 1-4, wherein the biological material is a nucleotide sequence, a plasmid vector, a viral vector, or a cell.

[0030] The preparation process of the antibodies described in Examples 2 and 3, as well as the related tests and evaluations of the antibodies, are explained in detail below. The results are as follows: Figure 2-6 As shown.

[0031] I. Preparation of the new Bunyavirus NP protein.

[0032] Based on the published NCBI sequence information of the novel Bunyavirus NP protein gene (>NC_043452.1:c1674-937Severe fever with thrombocytopenia syndrome virus strain HNXH segment S, complete sequence), the gene sequence was re-optimized, and a new gene was obtained using a whole-gene synthesis method. This new gene was then inserted into an expression vector using a double enzyme digestion method. Xho I and Nde I restriction endonucleases were used for double digestion. The digestion products were inserted into the expression vector pET-30a(+), and the ligation product was transformed into Escherichia coli DH5α competent cells. Single colonies were picked from kanamycin-resistant plates and inoculated into liquid culture medium. After PCR verification of the bacterial culture, amplification was performed. The plasmid was extracted using an alkaline lysis method and verified by enzyme digestion. After confirming the correct size, sequencing was performed. Once the sequencing results were correct, the DH5SV01- strain and recombinant plasmid were preserved. The recombinant plasmid was transformed into expression strain BL21 cells for amplification. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 200 μg / ml, and expression was induced at 25°C and 200 rpm. Cells were collected, lysed by sonication, and the protein was collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to verify correct protein expression. After verification, large-scale expression was performed. After induction, the cells were centrifuged, lysed by sonication with PBS, and Triton X-100 was added to a final concentration of 1%. The mixture was incubated on ice for 1 hour with stirring, followed by high-speed centrifugation. The supernatant was collected and purified using an AKTA purification system and a Cytiva HisTrap purification column according to the instructions of the experimental consumables. The obtained recombinant protein was concentrated using a Millipor ultrafiltration tube to obtain the final neo-Bunyavirus NP recombinant protein. This recombinant protein will be used to prepare a monoclonal antibody against the neo-Bunyavirus NP protein.

[0033] II. Inactivation of the new Bunyavirus.

[0034] Both the inoculation and inactivation experiments of SFTSV virus were conducted in a biosafety level 3 (P3) laboratory. Vero cells preserved in the laboratory were resuscitated, and the resuscitated T25 cell culture flasks were placed in a 37°C, 5% CO2 cell culture incubator. An appropriate amount of 10% DMEM medium was added, and the cells were cultured until they reached 80% capacity in the T25 cell culture flasks, followed by subculturing. Once a dense monolayer of cells formed, the SFTSV strain was inoculated at an MOI of 0.01, and cytopathic effects were observed daily under a microscope. After 5-7 days, when complete cytopathic effects occurred, the virus was harvested, and the viral titer was determined. The virus collection solution was mixed with β-propiolactone at a ratio of 1:4000 and inactivated at 4°C for 48 hours, followed by a 2-hour water bath at 37°C. The inactivated virus culture was then stored at -20°C.

[0035] III. Preparation of monoclonal antibodies against the NP protein of the new Bunyavirus.

[0036] Healthy female Balb / c mice aged 6-8 weeks were selected and immunized according to a pre-specified immunization protocol as immunogens. Splenic lymphocytes were extracted from the successfully immunized mice and fused with mouse myeloma cells SP2 / 0 using cell fusion technology. After two rounds of subclonal selection, a hybridoma cell line stably secreting monoclonal antibodies against the novel Bunyavirus NP antigen was obtained, thus yielding the monoclonal antibody against the novel Bunyavirus NP antigen.

[0037] The purified HIS-SFTSV-NP antigen was expressed in prokaryotes and used to immunize experimental mice in stages.

[0038] The specific steps of animal immunization experiments include:

[0039] 1. Balb / c mice with the same weight and age were randomly divided into two groups: an aluminum adjuvant group (aluminum hydroxide adjuvant) and a non-aluminum adjuvant group.

[0040] 2. Before the experiment, pre-immune serum was collected from each mouse (pre-immune serum was collected on the fifth day by blood collection through the eyeball, and an appropriate amount of blood was collected to ensure the normal condition of the mice). The collected serum was stored at -80℃.

[0041] 3. Preparation of aluminum adjuvant (aluminum hydroxide adjuvant) group: Before immunization, each antigen was diluted in 75 μL PBS to the corresponding dose (75 μg / mouse) and mixed with alum adjuvant (1 mg / mouse) at a volume ratio of antigen:adjuvant = 3:1 (i.e., 25 μl adjuvant was added to 75 μl of immunogen dilution). The adjuvant was shaken well before use, and the 25 μl adjuvant was slowly added dropwise to the immunogen solution. After thorough mixing of the adjuvant and immunogen dilution, the mixture was allowed to sit for 30 minutes to allow for effective adsorption of the antigen. Subsequent procedures were performed according to the animal immunization experiment procedures.

[0042] 4. Group without aluminum adjuvant: The antigen was diluted in 100 μL PBS to the corresponding dose in the table above (75 μg / mouse), and 100 μL of immunogen was added. Subsequent procedures were carried out according to the animal immunization experiment procedures.

[0043] 5. Subcutaneous injection at 2-week intervals: The experiment was designed as a 3-immunization method, but blood was collected from the eyeballs 7 days after each immunization injection. Part of the mouse supernatant was obtained by centrifugation and the serum titer was first tested. 7 days after the last immunization, the maximum blood volume was collected from the heart, and the supernatant was obtained by centrifugation and stored at -80℃.

[0044] 6. Detect serum titer.

[0045] Indirect ELISA was performed to detect the serum titer of immunized mice using the novel Bunyavirus NP antigen as the coating antigen. Indirect ELISA method: 50 μl of coating antigen diluted with coating buffer (1 μg / ml) was added to each well of the ELISA plate. After coating overnight at 4°C, the plate was washed three times with washing buffer (PBST). 200 μl of blocking buffer (5% skim milk powder) was added to each well, and the plate was incubated at 37°C for 2 h. After washing, 50 μl of diluted serum was added to each well, and the plate was incubated at 37°C for 30 min. After washing, 50 μl of goat anti-mouse IgG-HRP solution was added, and the plate was incubated at 37°C for 30 min. After washing, 100 μl of substrate solution was added, and the plate was incubated at 37°C in the dark for 10 min. Finally, 50 μL of 2 mol / L H2SO4 was added to stop the reaction, and the A450 value was read using an ELISA reader. After three immunizations, the orbital blood titer of mice was >62500. Three mice could only reach more than 50% titer at a ratio of 1:12500, so fusion could be arranged.

[0046] Immunosplenic cells were fused with myeloma cell line SP2 / 0. Fusion cells were screened using HAT selective medium (containing hypoxanthine, aminopterin, and thymine), and ELISA was performed to screen for positive results and subcloning. Ascites fluid was collected from the selected positive monoclonal cells, and antibodies were purified using a Protein A / G antibody purification column to obtain multiple monoclonal antibodies. The purified antibodies had an ELISA titer >1:128,000 and a purity >90%.

[0047] Two monoclonal antibodies were obtained: a coating antibody (named SV07-28) and a detection antibody (named SV09-35). The heavy chain of SV09-35 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 heavy chain of SV07-28 is shown in SEQ ID NO. 3, with a length of 346 bp, and the light chain of SV07-28 is shown in SEQ ID NO. 4, with a length of 322 bp.

[0048] IV. Double-antibody sandwich ELISA detection of novel Bunyavirus NP antigen

[0049] 1. Plotting Standard Curves

[0050] The established method involved regression analysis of the absorbance value versus the concentration of the new Bunyavirus NP antigen protein, with the standard curve shown below. Figure 3 As shown, repeated testing revealed a good linear relationship in absorbance values ​​within the linear range, with R² = 0.9746; the result was determined by taking 2.1 times the average negative value.

[0051] 2. Cut-off value confirmation

[0052] The average value of the test results was calculated by detecting the OD450nm of 24 negative serum samples for the novel Bunyavirus. The OD450nm value is 0.056, and the standard deviation (SD) is 0.008. The +3SD value is calculated to be 0.056 + 3 × 0.008 = 0.08. Therefore, when OD450nm ≥ 0.08, the sample is considered positive; when OD450nm < 0.08, the sample is considered negative. The results are as follows: Figure 4 As shown.

[0053] 3. Specificity test

[0054] Under suitable conditions, positive sera for dengue virus, Zika virus, and chikungunya virus were detected, with positive sera for novel Bunyavirus as a positive control, to verify the specificity of this ELISA kit. Results are as follows: Figure 5 As shown.

[0055] 4. Sensitivity Test

[0056] New Bunyavirus-positive sera were diluted 1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200, with negative and positive controls. The ELISA kit was used for assay, and the OD450nm value was read to verify the immunological sensitivity. Results are as follows: Figure 6 As shown.

[0057] The above-described embodiments are merely one implementation of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An immunological kit for detecting SFTSV-NP antigen, which is a double antibody sandwich ELISA immunological kit comprising a detection antibody and a coating antibody, characterized in that, The heavy chain of the detection antibody comprises a nucleotide sequence as shown in sequence SEQ ID NO. 1, and the light chain of the detection antibody comprises a nucleotide sequence as shown in sequence SEQ ID NO.

2.

2. The immunoassay kit for detecting SFTSV-NP antigen according to claim 1, characterized by, The heavy chain of the coating antibody comprises a nucleotide sequence as shown in sequence SEQ ID NO. 3, and the light chain of the coating antibody comprises a nucleotide sequence as shown in sequence SEQ ID NO.

4.

3. The immunoassay kit and antibodies for detecting SFTSV-NP antigen according to claim 2, characterized by, Both the detection antibody and the coating antibody are IgG monoclonal antibodies.

4. An ELISA immuno-detection antibody for detecting SFTSV-NP antigen, characterized in that, The heavy chain of the detection antibody comprises a nucleotide sequence as shown in sequence SEQ ID NO. 1, and the light chain of the detection antibody comprises a nucleotide sequence as shown in sequence SEQ ID NO.

2.

5. An ELISA-immunocovering antibody for detecting SFTSV-NP antigen, characterized in that, The heavy chain of the coating antibody comprises a nucleotide sequence as shown in sequence SEQ ID NO. 3, and the light chain of the coating antibody comprises a nucleotide sequence as shown in sequence SEQ ID NO.

4.

6. A biological material for detecting SFTSV-NP antigen, characterized by, The biological material comprises or carries nucleotide sequences expressing the antibodies as shown in sequences 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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