Monoclonal antibody combination for detecting measles virus nucleoprotein and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody combination for detecting measles virus nucleoprotein and application of the monoclonal antibody combination. The combination is composed of monoclonal antibodies 5E2 and 5G9 and can efficiently and specifically recognize measles virus nucleoprotein, amino acid sequences of complementary determining regions (CDR) of heavy chain and light chain variable regions of the combination are clear and are shown as SEQ ID NO.1-12 respectively, high specificity and high affinity of the antibodies are guaranteed, the antibody combination can effectively capture and detect target antigens, and the antibody combination can be used for detecting measles virus nucleoprotein. The risk of non-specific binding and cross reaction is obviously reduced. The combination shows excellent sensitivity and specificity in detection platforms such as colloidal gold immunochromatography, and provides a stable and reliable biological recognition tool for rapid and accurate detection of measles viruses.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a combination of monoclonal antibodies for detecting measles virus nucleoprotein and its application. Background Technology

[0002] Measles virus (MeV) belongs to the Paramyxoviridae family and the Measlesvirus genus. It is an enveloped, single-stranded, negative-sense RNA virus. Measles virus has only one serotype and is antigenically stable. The viral genome contains six genes: N, P, M, F, H, and L, which encode the nucleoprotein (N), phosphorylated protein (P), matrix protein (M), fusion protein (F), hemagglutinin (H), and large RNA polymerase (L), respectively. Measles virus is transmitted through the respiratory tract, and the population is generally susceptible. In my country, a combined live attenuated measles-mumps-rubella (MMR) vaccine is available. Immunization with a measles-containing vaccine or natural infection provides some immunity and is an effective means of preventing measles virus infection. After measles virus infection, the human body produces a specific immune response. Immunological methods such as ELISA to detect measles virus IgM and IgG antibodies in serum are currently the main methods for laboratory diagnosis of measles virus. In addition, the detection of the measles virus etiology mainly includes virus isolation, detection of viral antigens, and detection of viral nucleic acids. Measles virus can be detected in throat swabs or blood samples a few days before or up to 3 days after the onset of measles. The positive rate of virus detection in samples gradually decreases 3-5 days after the rash appears. Therefore, combined serological and etiological detection can help improve the accuracy of the test.

[0003] The N protein of measles virus is the main structural protein. It binds to viral RNA to form a complex and exists mainly in phosphorylated form. Its antigenic structure is highly stable, making it very suitable as an immunological target for viral antigen detection. However, most of the diagnostic reagents for measles virus infection on the market are antibody tests or nucleic acid tests, and there are no monoclonal antibodies or kits for detecting measles virus antigens. Summary of the Invention

[0004] This invention provides a highly specific and sensitive monoclonal antibody for detecting measles virus nucleoprotein and its application, solving the technical problem of the lack of efficient detection tools for measles virus antigens in the prior art.

[0005] To achieve the above objectives, the main technical solutions adopted by the present invention include: A monoclonal antibody ensemble for detecting measles virus nucleoprotein, comprising monoclonal antibody 5E2 and monoclonal antibody 5G9; The heavy chain variable region of monoclonal antibody 5E2 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of monoclonal antibody 5E2 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively. The heavy chain variable region of monoclonal antibody 5G9 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively. The light chain variable region of monoclonal antibody 5G9 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0006] In a further embodiment, the amino acid sequence of the heavy chain variable region of monoclonal antibody 5E2 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 5E2 is shown in SEQ ID NO.14.

[0007] In a further embodiment, the amino acid sequence of the heavy chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO.16.

[0008] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5E2 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 5E2 is shown in SEQ ID NO. 18.

[0009] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO. 20.

[0010] Secondly, this application provides the use of the above-mentioned monoclonal antibody combination in the preparation of a tool for detecting measles virus nucleoprotein.

[0011] In a further embodiment, the tools include colloidal gold test strips, reagents, kits, and antibody chips.

[0012] In a further embodiment, the colloidal gold test strip uses monoclonal antibody 5E2 as the capture antibody and monoclonal antibody 5G9 as the labeling antibody.

[0013] In a further embodiment, the colloidal gold test strip includes a nitrocellulose membrane, a colloidal gold pad, a sample pad, and absorbent paper attached to a backing plate.

[0014] In a further embodiment, a detection line and a control line are provided on the nitrocellulose membrane; the detection line is coated with monoclonal antibody 5E2, the control line includes goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 5G9.

[0015] Beneficial effects: This application provides a monoclonal antibody ensemble for detecting measles virus nucleoprotein. The ensemble consists of monoclonal antibodies 5E2 and 5G9, which can efficiently and specifically recognize measles virus nucleoprotein. The complementarity-determining regions (CDRs) of the heavy and light chain variable regions are clearly defined, as shown in SEQ ID NO. 1-12, ensuring high specificity and high affinity of the antibodies. This antibody ensemble can effectively capture and detect the target antigen, significantly reducing the risk of non-specific binding and cross-reaction. This ensemble exhibits excellent sensitivity and specificity in detection platforms such as colloidal gold immunochromatography, providing a stable and reliable biorecognition tool for rapid and accurate detection of measles virus. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Image showing the SDS-PAGE identification results of MeV / NP proteins; Figure 2 The image shows the identification results of the MeV / NP recombinant protein. Figure 3 This is a graph showing the reactivity of monoclonal antibodies on MeV / NP recombinant protein. Figure 4 This is a schematic diagram of colloidal gold assembly; Figure 5 This is the result of specificity analysis of the test strip; Figure 6 This is the result of the sensitivity test of the test strip. Detailed Implementation

[0018] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0019] These embodiments are provided to make the application thorough and complete, and to fully express the scope of the application to those skilled in the art. It should be noted that, unless otherwise specifically stated, the relative arrangement of components and steps, material composition, numerical expressions, and values ​​illustrated in these embodiments should be interpreted as merely exemplary and not as limiting.

[0020] This invention utilizes hybridoma technology to efficiently screen and obtain monoclonal antibodies specific to measles virus nucleoprotein (MeV / NP), and successfully applies them to colloidal gold test strips. The results have been verified to have excellent detection sensitivity and specificity for recombinant MeV / NP proteins, providing a useful reference for the development of diagnostic reagents for measles virus infection.

[0021] The detection method described in this application is not intended for disease diagnosis and treatment.

[0022] The measles virus nucleoprotein in this application includes recombinant measles virus nucleoprotein and natural measles virus nucleoprotein.

[0023] Example 1 1. MeV / NP recombinant protein expression The N gene was amplified from a MeV-positive nucleic acid sample and cloned into the pET32a vector. Its nucleotide sequence is shown in SEQ ID NO.21.

[0024] The amino acid sequence is shown in SEQ ID NO.22: .

[0025] Its cloning primers are: pET32a-MeV-NP-F (as shown in SEQ ID NO.23): GCTGAATCGGATCCATGGCCACACTTTTAAGGAG.

[0026] pET32a-MeV-NP-R (as shown in SEQ ID NO.24): GTGGTGGTGCTCGAGCTAGTCTAGAAGATCTCTGTCATTG.

[0027] Primers were synthesized by Qingke Biotechnology. The PCR amplification program for obtaining the target gene fragment was: 98℃ for 5 min; 98℃ for 20 s, 55℃ for 30 s, 72℃ for 70 s, 30 cycles; 72℃ for 5 min. The target gene fragment was inserted into the pET32a expression vector using molecular cloning technology. Positive clones were screened and sequenced for verification, yielding the recombinant plasmid pET32a-MeV / NP.

[0028] The recombinant plasmid pET32a-MeV / NP was transformed into BL21(DE3) competent cells using standard methods and induced to express the gene. Specifically, the transformed bacteria were plated on LB agar plates (containing 100 μg / mL ampicillin) and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 220 rpm. Then, 1% of the total culture volume was inoculated into LB medium (containing 100 μg / mL ampicillin) and incubated at 37°C with shaking at 220 rpm for approximately 3 hours until OD500 was reached. 600 The concentration was 0.6-0.9, and the final concentration was 0.1 mM IPTG. The cells were collected after induction at 16℃ and 200 rpm for 16 hours.

[0029] 2. Purification of MeV / NP recombinant protein Because the expressed recombinant protein carries a histidine tag, it was purified using a protein purification instrument and HisTrap from Suzhou Taidu Biotechnology Co., Ltd. TM Purification was performed using an HP affinity chromatography column. Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0; Buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The fermented bacterial culture was then centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in buffer A and sonicated in ice water for 30 min, with 5-second sonication intervals. The mixture was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a 0.22 μm filter from a GETIC filter and loaded onto the chromatography column. The column was washed with buffer A, followed by gradient elution with buffer B. The elution peak of the target protein was collected and dialyzed overnight at 4°C with buffer A. The purified protein was observed by SDS-PAGE electrophoresis. The electrophoresis results of the purified protein are shown below. Figure 1 Protein concentration was determined using an ultra-micro spectrophotometer and stored at -20°C.

[0030] Figure 1 In the diagram, M represents the protein marker, 1 represents the precipitate after centrifugation following cell disruption, 2 represents the supernatant after centrifugation following cell disruption, 3 represents flow-through, 4 represents elution with 100 mM imidazole, and 5 represents elution with 500 mM imidazole. SDS-PAGE results showed that the elution buffers with 100 mM and 500 mM imidazole exhibited a distinct main band around 70 kDa, consistent with the estimated size of the fusion protein (estimated molecular weight of the fusion protein is 75.9 kDa), and possessed high purity.

[0031] 3. Identification of MeV / NP recombinant proteins Protein collected after elution with 100 mM imidazole was used to coat an ELISA plate. The reaction with MeV / NP positive serum was identified using an indirect ELISA method. MeV / NP positive serum was human serum confirmed positive using a measles virus IgG antibody detection kit. The recombinant protein was first coated in microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6), at a concentration of 1 μg / mL, 50 μL / well, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 3% sucrose + 2% BSA, 150 μL per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST wash buffer (PBS containing 0.05% Tween-20, pH 7.4) and patted dry. MeV / NP positive serum (POS-1#) was diluted with PBS at serial dilutions of 100x, 1000x, 10000x, and 100000x. 50 μL of each solution was added to each well of the antigen-coated microplate. Healthy neonatal serum (NB-1#) was diluted at the same fraction as a negative control. The plates were incubated at 37°C for 30 min. The liquid in the wells was discarded, and the plates were washed four times with PBST. After drying, 50 μL / well of HRP-labeled goat anti-human IgG secondary antibody (Solepro, diluted 5000x with PBS) was added. The plates were incubated at 37°C for 30 min, washed four more times, and dried. 50 μL / well of TMB chromogenic buffer was added, and the plates were incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing MecoWander, 1001SA) was added to stop the reaction. The OD was measured using a microplate reader. 450 nm value. Results are as follows: Figure 2 , Figure 2 NB-1# is serum from a healthy newborn (negative for measles virus IgG antibody), and POS-1# is MeV / NP positive serum. The MeV / NP positive serum still showed a weak positive reaction with the MeV / NP recombinant protein when diluted 10,000 times, indicating that the MeV / NP recombinant protein has good biological activity and can be used for further experiments.

[0032] 4. Mouse immunization Mice were immunized with high-purity MeV / NP recombinant protein, and other recombinant proteins expressed by the pET32a vector were used as screening antigens for monoclonal antibody selection. Specifically, purified MeV / NP recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected at multiple sites into 6-week-old female BALB / c mice at a dose of 30 μg / mouse. At weeks 2 and 4, booster immunizations were administered subcutaneously at multiple sites with the same dose mixed with an equal volume of Freund's incomplete adjuvant. At week 6, mice were immunized by direct injection of insulin (5 μg / mouse) into the spleen. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected for a booster immunization of 20 μg MeV / NP recombinant protein via intraperitoneal pulse, and the spleen was collected 3 days later for hybridoma cell preparation.

[0033] 5. Screening of hybridoma cells All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway to the bottom of the well, clones that reacted positively with the MeV / NP recombinant protein were selected by indirect ELISA. Since the immunogen was a prokaryotic expression source of the pET32a vector containing a His tag, background components needed to be screened to select specific cell lines targeting the MeV / NP protein. Positive cells were cloned to a monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0034] Indirect ELISA method for screening positive clones: MeV / NP recombinant protein and other recombinant proteins of the pET32a vector (pET32a-HPV18 / E7, His tag, preparation process as described in our patent CN120352624B) were coated in microplates (coating buffer: carbonate buffer: sodium carbonate 1.59g, sodium bicarbonate 2.93g, diluted to 1L of pure water, pH 9.6), coating concentration 1μg / mL, incubated overnight at 4℃; the next day, the coating buffer was discarded, and the plates were blocked with 3% sucrose + 2% BSA, 150 μL per well, incubated at 37℃ for 2 hours, then washed once with PBST wash buffer (PBS containing 0.05% Tween-20, pH 7.4), and patted dry. 50 μL of cell culture supernatant was added, and the reaction was carried out at 37℃ for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, pat dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, pat dry, and add 50 μL / well of TMB chromogenic buffer for incubation at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) to stop the reaction. Measure the OD using a microplate reader. 450nm value. Positive cell lines that reacted with the MeV / NP recombinant protein but not with the control recombinant protein were selected for subsequent experiments. The screening process is shown in Table 1.

[0035] Table 1: Screening results of monoclonal antibodies

[0036] After the selected hybridoma cell lines were expanded and cultured, 0.2 ml (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were used to collect ascites fluid approximately 10 days later, when the mice’s abdomens were noticeably swollen.

[0037] 6. Purification of monoclonal antibodies Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, add 4 ml of acetate-sodium acetate buffer (0.06 M, pH 4.5), mix well, and slowly add 10 μl of n-octanoic acid while stirring. After the addition is complete, continue stirring for 30 minutes. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the supernatant. Filter the supernatant through defatted cotton, and add saturated ammonium sulfate at a final volume ratio of 50% (V / V) while stirring. After the addition is complete, continue stirring for 30 minutes, and let it precipitate overnight at 2–8°C. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the precipitate. After the precipitate was completely dissolved in binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), it was filtered through a 0.22 μm filter. The filtered sample was then pumped slowly through a peristaltic pump into a Protein L purification column equilibrated with binding buffer. The column was connected to a protein purification instrument, and the sample was washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled off. Elution was then performed with elution buffer (0.1 M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1 M Tris-HCl at pH 9 and placed in a dialysis bag (MW: 8000-14000). Dialysis was performed at 2-8 °C in 20 mM PBS pH 7.4 solution for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody.

[0038] The identification method is the same as the indirect ELISA detection described above. The purified antibody was diluted to 1 μg / ml, and the binding reaction between the antibody and the antigen was identified. The results are shown in [Figure number missing]. Figure 3 The binding ability of purified monoclonal antibodies to antigens was detected by indirect ELISA. Most clonal antibodies showed strong reactivity to MeV / NP recombinant proteins, while no significant reaction was observed to irrelevant antigens such as HPV18 / E7 recombinant proteins, indicating that the obtained monoclonal antibodies have good specificity and high affinity. Figure 3 The specific data is shown in Table 2.

[0039] Table 2: Results of antigen-binding activity assay for purified monoclonal antibodies

[0040] 7. Preparation of test strips coated with different MeV / NP monoclonal antibodies: The selected MeV / NP monoclonal antibodies were scribed onto nitrocellulose membranes of different sizes (20mm × 300mm). Diluted monoclonal antibodies (diluted to 1.5 mg / mL with PBS at pH 7.4) were sprayed horizontally in a linear pattern using a scribing instrument, with a spray volume of 0.8 μL / cm per line, forming the detection line (T line). Goat anti-mouse IgG antibodies, diluted to 1 mg / mL with 0.01M PBS at pH 7.4, were sprayed horizontally in a linear pattern at 6mm intervals, coating the nitrocellulose membrane at a volume of 0.8 μL / cm, forming the control line (C line).

[0041] 8. Colloidal gold pairing of MeV / NP monoclonal antibodies Preparation of antibody-colloidal gold labeled complex: Antibody labeling: Colloidal gold solution was prepared using the trisodium citrate reduction method. The specific procedure was as follows: 100 mL of 0.01% chloroauric acid solution was heated to boiling, and then 1 mL of 1% trisodium citrate solution was quickly added until the solution turned wine-red. Boiling was continued for 5 minutes, and the colloidal gold particles were allowed to stabilize before cooling to room temperature. To optimize the conjugation efficiency between the antibody and colloidal gold, 1 mL of colloidal gold solution was placed in a centrifuge tube, and 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL of 0.2M potassium carbonate solution were added respectively to adjust the pH environment of the colloidal gold solution. The optimal conjugation effect was found to be 5 μL. After mixing, 5 μg of the MeV / NP monoclonal antibody to be labeled was added, and the mixture was quickly mixed and incubated at room temperature for 10 min. Then, 10 μL of 10% (w / v) bovine serum albumin (BSA) was added to block non-specific binding sites, and the mixture was incubated at room temperature for another 10 min. Add 10 μL of 10% (w / v) polyethylene glycol 20000 (PEG20000) to enhance labeling stability. After mixing, centrifuge at 12000 rpm for 10 min and discard the supernatant. Resuspend the lower precipitate in 1 / 10 volume of reconstitution solution (0.01 M phosphate buffer + 1% BSA + 2% sucrose, pH 7.4) to obtain the antibody-colloidal gold labeled complex. Store at 4°C protected from light for later use.

[0042] 9. Screening of paired monoclonal antibodies Nitrocellulose membranes streaked with different monoclonal antibodies against MeV / NP were individually paired with different colloidal gold-labeled monoclonal antibodies. MeV / NP protein was diluted to 20 ng / mL for detection, while HPV18 / E7 recombinant protein was diluted to 20 ng / mL as a negative antigen for detection. Combinations showing the strongest color development for MeV / NP protein and not reacting with the control protein were screened. The screening process is shown in Tables 3 and 4. Therefore, the optimal pairing for detecting MeV / NP recombinant protein was determined to be 5E2 streaking and 5G9 gold labeling.

[0043] Table 3: Results of screening paired monoclonal antibodies using MeV / NP recombinant protein 1

[0044] Table 4: Results of screening paired monoclonal antibodies using MeV / NP recombinant protein 2

[0045] - indicates a negative result, meaning no color develops; + / ++ / +++ indicates a positive result, meaning a color reaction occurs. The more + signs there are, the deeper the color, and the stronger the positive reaction.

[0046] Tables 3 and 4 show the screening results using MeV / NP recombinant protein diluted to a concentration of 20 ng / ml as a positive antigen. The detection results for HPV18 / E7 recombinant protein and blank dilution were negative and are not shown. The results show that the combination of 5E2 monoclonal antibody streaking and 5G9 monoclonal antibody labeling resulted in the deepest staining of MeV / NP recombinant protein, making it the optimal pairing. That is, the combination of monoclonal antibody anti-5E2 as the capture antibody and monoclonal antibody 5G9 as the labeling antibody can specifically recognize MeV / NP recombinant protein.

[0047] 10. Preparation and Assembly of Colloidal Gold Test Strips Preparation of gold-labeled pads: A 6mm x 300mm glass fiber membrane RB65 was treated with Tris-HCl containing 0.5% BSA and 0.5% NP40 at pH 8. The prepared colloidal gold-labeled antibody was then uniformly dropped onto the glass fiber at a rate of 1200ul / strip. After air drying, it was dried at 37℃ for 2 hours for later use. See Figure 4 , Figure 4This is a schematic diagram of the colloidal gold assembly. A 60mm x 300mm PVC backing plate is used as a support. Sample pads, gold-labeled pads (colloidal gold pads), nitrocellulose membranes, and absorbent paper are attached to the backing plate. The nitrocellulose membrane is coated with two lines and dried at 37℃ for 12 hours before use. The nitrocellulose membrane is coated with a detection line (monoclonal antibody 5E2) and a control line (goat anti-mouse IgG). The colloidal gold pad is coated with monoclonal antibody 5G9. The assembled plate is cut into 4mm strips using a strip cutter and wrapped with colloidal gold plastic casings. The sample pad is exposed at the sample application well of the plastic casing, while the control and detection lines are exposed at the result observation wells. The colloidal gold test strip is now assembled.

[0048] 11. Test strip specificity test Recombinant protein samples: MeV / NP recombinant protein and HPV18 / E7 recombinant protein were diluted to 100 ng / mL with sample dilution buffer for detection.

[0049] Natural antigen samples: Measles virus culture (measles virus L4 strain inoculated into chicken embryo cells, cultured until lesions appear, harvested and inactivated), and chicken embryo cell lysate (supernatant after repeated freeze-thaw lysis and centrifugation, with protein concentration measured at A280nm), were diluted to 100ng / mL with sample diluent for detection.

[0050] The sample dilution solution was an aqueous solution of 0.05M Tris + 0.1% NP40 + 1.5% NaCl + 0.1% SDS, pH 8.0. Take 80 μL of the diluted sample and add it to the sample well of the test strip. Determine the result within 20 minutes. If both the T line and C line show clear red bands, the result is positive; if only the C line shows color, the result is negative; if the C line does not show color, the result is invalid.

[0051] Depend on Figure 5 It can be seen that the test strip can effectively detect MeV / NP recombinant protein and natural antigen (measles virus culture), while showing no cross-reactivity with HPV18 / E7 recombinant protein and chicken embryo cells, indicating that the test strip has good specificity.

[0052] 12. Sensitivity test of test strips The MeV / NP recombinant protein was diluted at concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.1 ng / mL before detection. Figure 6The results showed that the colloidal gold test strip still showed weak color development at a recombinant protein concentration of 1 ng / mL, while the blank dilution, i.e. the sample dilution (0.05M Tris + 0.1% NP40 + 1.5% NaCl + 0.1% SDS, pH 8.0) (0 ng / mL), did not show color development, indicating that the test strip card's limit of detection for MeV / NP recombinant protein is 1 ng / mL.

[0053] In summary, this invention utilizes hybridoma technology to screen and obtain monoclonal antibody pairs that specifically recognize MeV / NP proteins. These combined antibodies can efficiently recognize MeV / NP recombinant proteins, exhibiting good specificity and sensitivity. This invention applies the monoclonal antibody pairs to an immunoassay platform, constructing rapid test strips or test cards based on colloidal gold immunochromatography technology. These test strips demonstrate high sensitivity to MeV / NP recombinant proteins and show no cross-reactivity with other proteins.

[0054] 13. Gene sequence of monoclonal antibodies Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibody.

[0055] Gold-labeled monoclonal antibody 5G9: The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 5G9 is shown in SEQ ID NO.20: GACATCCTGATGACCCAATCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAGGCCTCCATCTCTTGCAGATCTAGTCAGAGAATTGTCCATAGTCATGGAAACACCTATTTAGCATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAA CCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAGGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAGCTGAAACGTACGGTG.

[0056] The amino acid sequence of the variable region of the light chain of monoclonal antibody 5G9 is shown in SEQ ID NO.16: DILMTQSPLSLPVSLGDQASISCRSSQRIVHSHGNTYLAWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLELKRTV.

[0057] Light chain CDR area annotation: The amino acid sequence of the complementarity-determining region (CDR-L1) of the light chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO. 10: CDR-L1: RSSQRIVHSHGNTYLA; The amino acid sequence of the complementarity-determining region (CDR-L2) of the light chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO. 11: CDR-L2: KVSNRFS; The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO. 12: CDR-L3: FQGSHVPWT.

[0058] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO.19: GAGGTGAAGCTGGAGGAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAGGATCTCCTGCAAGGCTTCTGGTTATAACCTTCACAGACTATTCAATGCAGTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAGAGTGGATGGGCTGGATAAACACTGAGACAGGTGAGCCAGCATA TACAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGGTAGGAGTAACTTCTATTACTACGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACTGTCTCTGCA.

[0059] Heavy chain variable region amino acid sequence: The amino acid sequence of the heavy chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO.15: EVKLEESGPELKKPGETVRISCKASGYTFTDYSMQWVKQAPGKGLEWMGWINTETGEPAYTDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCGRSNFYYYAMDYWGQGTSVTVSA.

[0060] Heavy chain CDR region annotation: The amino acid sequence of the complementarity-determining region (CDR-H1) of the heavy chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO. 7: CDR-H1: DYSMQ; The amino acid sequence of the complementarity-determining region (CDR-H2) of the heavy chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO. 8: CDR-H2: WINTETGEPAYTDDFKG; The amino acid sequence of the complementarity-determining region (CDR-H3) of the heavy chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO. 9: CDR-H3: SNFYYYAMDY.

[0061] Scratch-resistant monoclonal antibody 5E2: The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 5E2 is shown in SEQ ID NO.18: GACATCAAGATGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAGGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAAATTACATACACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCCAACCTGGCTTC TGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGTAGTTATGCACCGACGTTCGGTGGAGGCACCAAGCTGGAAATAAAACGTACGGTG.

[0062] The amino acid sequence of the variable region of the light chain of monoclonal antibody 5E2 is shown in SEQ ID NO.14: DIKMTQSPAIMSASPGERVTITCSASSSVNYIHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYAPTFGGGTKLEIKRTV.

[0063] Light chain CDR area annotation: The amino acid sequence of the light chain variable region CDR-L1 of monoclonal antibody 5E2 is shown in SEQ ID NO.4: CDR-L1: SASSSVNYIH; The amino acid sequence of the light chain variable region CDR-L2 of monoclonal antibody 5E2 is shown in SEQ ID NO. 5: CDR-L2: STSNLAS; The amino acid sequence of the light chain variable region CDR-L3 of monoclonal antibody 5E2 is shown in SEQ ID NO. 6: CDR-L3: QQRSSYAPT.

[0064] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5E2 is shown in SEQ ID NO.17: GAGGTGCAGCTGGTGGAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGTACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTAGTACTAACTCC AATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCCACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGAAGGAGGGGAAACTGGGTGGCCTCTTTGACTACTGGGGCCAAGGCACCACTCTCACCGTCTCCTCA.

[0065] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5E2 is shown in SEQ ID NO. 13. EVQLVESGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPVHGLEWIGEILPGSGSTNSNEKFKGKATFTADTSSNTAHMQLSSLTSEDSAVYYCARRRGNWVGLFDYWGQGTTLTVSS.

[0066] Heavy chain CDR region annotation: The amino acid sequence of the complementarity-determining region (CDR-H1) of the heavy chain variable region of monoclonal antibody 5E2 is shown in SEQ ID NO. 1: CDR-H1: SYWIE; The amino acid sequence of the complementarity-determining region (CDR-H2) of the heavy chain variable region of monoclonal antibody 5E2 is shown in SEQ ID NO. 2: CDR-H2: EILPGSGSTNSNEKFKG; The amino acid sequence of the complementarity-determining region (CDR-H3) of the heavy chain variable region of monoclonal antibody 5E2 is shown in SEQ ID NO. 2: CDR-H3: RRGNWVGLFDY.

Claims

1. A monoclonal antibody combination for detecting measles virus nucleoprotein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 5E2 and monoclonal antibody 5G9. The heavy chain variable region of the monoclonal antibody 5E2 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 5E2 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively. The heavy chain variable region of the monoclonal antibody 5G9 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively. The light chain variable region of the monoclonal antibody 5G9 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

2. The monoclonal antibody combination for detecting measles virus nucleoprotein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting measles virus nucleoprotein according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5G9 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 5G9 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting measles virus nucleoprotein according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting measles virus nucleoprotein according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5G9 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5G9 is shown in SEQ ID NO.

20.

6. The use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for detecting measles virus nucleoprotein.

7. The application according to claim 6, characterized in that, The tools include colloidal gold test strips, reagents, kits, and antibody chips.

8. The application according to claim 7, characterized in that, The colloidal gold test strip uses monoclonal antibody 5E2 as the capture antibody and monoclonal antibody 5G9 as the labeling antibody.

9. The application according to claim 8, characterized in that, The colloidal gold test strip includes a nitrocellulose membrane, a colloidal gold pad, a sample pad, and absorbent paper attached to a backing plate.

10. The application according to claim 9, characterized in that, The nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 5E2, the control line includes goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 5G9.

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