Monoclonal antibody combination for detecting measles virus nucleoprotein and use thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody combination for detecting measles virus nucleoprotein and application thereof. BACKGROUND

[0002] Measles virus (MeV) belongs to the paramyxoviridae family and the measles virus genus, and is a single-stranded negative-strand RNA virus with an envelope. Measles virus has only one serotype and stable antigenicity. The viral genome has six genes, namely N, P, M, F, H and L, which encode nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H) and RNA-dependent RNA polymerase (L). Measles virus is transmitted through the respiratory tract, and the population is generally susceptible. In China, a measles mumps rubella combined attenuated live vaccine has been marketed, and certain immunity can be obtained through vaccination with a measles component vaccine or natural infection, which is an effective means of preventing measles virus infection. After measles virus infection, the human body can produce specific immune response, and the detection of measles virus IgM and IgG antibodies in the serum of the population through immunological methods such as ELISA is the main method for laboratory diagnosis of measles virus at present. In addition, the detection of measles virus pathogenology mainly includes virus isolation, virus antigen and virus nucleic acid detection. Within a few days before or after the onset of measles, measles virus can be detected in throat swabs or blood samples, and the positive rate of virus detection in samples gradually decreases 3-5 days after the onset of rash. Therefore, the combined detection of serology and pathogenology can help improve the accuracy of detection.

[0003] The N protein of measles virus is the main structural protein, which forms a complex with viral RNA, mainly exists in a phosphorylated form, and has a highly stable antigen structure, and is therefore very suitable as an immunological target for virus antigen detection. However, the diagnostic reagents for measles virus infection on the market are mainly antibody detection or nucleic acid detection, and there is no monoclonal antibody and kit for measles virus antigen detection. SUMMARY

[0004] The application provides a monoclonal antibody for detecting measles virus nucleoprotein and application thereof, which has high specificity and good sensitivity, and solves the technical problem of lack of efficient detection tools for measles virus antigens in the prior art.

[0005] In order to achieve the above purpose, the main technical solutions adopted by the application include:

[0006] A monoclonal antibody combination for detecting measles virus nucleoprotein, the monoclonal antibody combination comprising monoclonal antibody 5E2 and monoclonal antibody 5G9;

[0007] The heavy chain variable region of monoclonal antibody 5E2 comprises three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 1-SEQ ID NO. 3, respectively;

[0008] The light chain variable region of monoclonal antibody 5E2 comprises three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 4-SEQ ID NO. 6, respectively;

[0009] The heavy chain variable region of monoclonal antibody 5G9 comprises three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 7-SEQ ID NO. 9, respectively;

[0010] The light chain variable region of monoclonal antibody 5G9 comprises three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 10-SEQ ID NO. 12, respectively.

[0011] In further embodiments, 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.

[0012] In further embodiments, 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.

[0013] In further embodiments, 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.

[0014] In further embodiments, 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.

[0015] In a second aspect, the present application provides use of the above-mentioned monoclonal antibody combination in the preparation of a tool for detecting measles virus nucleoprotein.

[0016] In further embodiments, the tool comprises colloidal gold detection test strips, reagents, kits and antibody chips.

[0017] In a further embodiment, the colloidal gold detection test strip takes monoclonal antibody 5E2 as the capture antibody and monoclonal antibody 5G9 as the labeled antibody.

[0018] In a further embodiment, the colloidal gold detection test strip comprises a nitrocellulose membrane, a colloidal gold pad, a sample pad and an absorbent paper connected to the back plate.

[0019] In a further embodiment, the nitrocellulose membrane is provided with a detection line and a quality control line; the detection line is coated with monoclonal antibody 5E2, and the quality control line comprises goat anti-mouse IgG; the colloidal gold pad is coated with monoclonal antibody 5G9.

[0020] Beneficial effects:

[0021] The present application provides a monoclonal antibody combination for detecting measles virus nucleoprotein, which is composed of monoclonal antibodies 5E2 and 5G9, can efficiently and specifically recognize measles virus nucleoprotein, the complementary determining region (CDR) amino acid sequences of the heavy chain and light chain variable region of which have been determined and are shown as SEQ ID NO. 1-12 respectively, ensuring high specificity and high affinity of the antibody, the antibody combination can effectively capture and detect target antigens, significantly reducing the risk of non-specific binding and cross-reaction. The combination shows excellent sensitivity and specificity in colloidal gold immunochromatography and other detection platforms, providing a stable and reliable biological recognition tool for rapid and accurate detection of measles virus. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiment or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0023] Figure 1 Figure for identification results of MeV / NP protein SDS-PAGE;

[0024] Figure 2 Figure for identification results of MeV / NP recombinant protein;

[0025] Figure 3 Figure for reaction activity of monoclonal antibody on MeV / NP recombinant protein;

[0026] Figure 4 Figure for colloidal gold assembly schematic diagram;

[0027] Figure 5 Figure for specificity analysis results of test strip;

[0028] Figure 6The results of the sensitivity test of the test strip. DETAILED DESCRIPTION

[0029] The embodiments of the present application will be further described in details with reference to the accompanying drawings and examples. The following detailed description of the examples and the accompanying drawings are provided to illustrate the principles of the present application, and should not be used to limit the scope of the present application, which can be implemented in many different forms, not limited to the specific examples disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0030] The present application provides these examples in order to make the present application thorough and complete, and fully express the scope of the present application to those skilled in the art. It should be noted that: unless otherwise specified, the relative arrangement of components and steps, the composition of materials, numerical expressions and values set forth in these examples should be interpreted as merely exemplary, and not as a limitation.

[0031] The present application utilizes the hybridoma technology, and efficiently screens the MeV / NP specific monoclonal antibody, and successfully applies it to the colloidal gold detection test paper, which has been verified to have good detection sensitivity and specificity for MeV / NP recombinant protein, and provides a beneficial reference for the development of measles virus infection diagnostic reagents.

[0032] The detection method of the present application is not used for disease diagnosis and treatment.

[0033] The measles virus nucleoprotein in the present application includes measles virus recombinant nucleoprotein and measles virus natural nucleoprotein.

[0034] Example 1

[0035] 1. MeV / NP recombinant protein expression

[0036] The N gene is amplified from the MeV positive nucleic acid sample and cloned into the pET32a vector, and the nucleotide sequence is shown as SEQ ID NO. 21:

[0037]

[0038] The amino acid sequence is shown as SEQ ID NO. 22:

[0039] MATLLRSLALFKRNKDKPPITSGSGGAIRGIKHIIIVPIPGDSSITTRSRLLDRLVRLIGNPDVSGPKLTGALIGILSLFVESPGQLIQRITDDPDVSIRLLEVVQSDQSQSGLTFASRGTNMEDEADQYFSHDDPISSDQSRFGWFENKEISDIEVQDPEGFNMILGTILAQIWVLLAKAVTAPDTAADSELRRWIKYTQQRRVVGEFRLERKWLDVVRNRIAEDLSLRRFMVALILDIKRTPGNKPRIAEMIWAIDPYFVEAGLPSFILTIKFGIETMYPALGLHEFAGELSTLESLMNLYQQMGETAPYMVILENSIQNKFSAGSYPLLWSYAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSTLASELGITAEDARLVSEIAMHTTEDKISRAVGPRQAQVSFLHGDQSENELPRLGGKEDRRVKQSRGEARESYRETGPSRASDARAAHLPTGTPLDIDTASESSQDPQDSRRSADALLRLQAMAGISEEQGSDTDTPIVYNDRDLLD.

[0040] The cloning primer is:

[0041] pET32a-MeV-NP-F (as shown in SEQ ID NO. 23): GCTGATATCGGATCCATGGCCACACTTTTAAGGAG.

[0042] pET32a-MeV-NP-R (as shown in SEQ ID NO. 24): GTGGTGGTGCTCGAGCTAGTCTAGAAGATCTCTGTCATTG.

[0043] The PCR amplification reaction program for obtaining the target gene fragment is as follows: 98℃ 5min; 98℃ 20s, 55℃ 30s, 72℃ 70s, 30 cycles; 72℃ 5min. The target gene fragment is inserted into the pET32a expression vector by molecular cloning technology, positive clones are screened and verified by sequencing, and the recombinant plasmid pET32a-MeV / NP is obtained.

[0044] The recombinant plasmid pET32a-MeV / NP was transformed into BL21 (DE3) competent cells and induced for expression according to a conventional method. Specifically, the transformed bacteria were spread on LB agar plates (containing 100 μg / mL ampicillin) and incubated at 37°C overnight. A single colony was picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and incubated at 37°C, 220 rpm overnight. The culture was inoculated into LB medium (containing 100 μg / mL ampicillin) at a volume of 1% of the total volume of the medium, and incubated at 37°C, 220 rpm for about 3 hours until the OD 600 was 0.6-0.9. The final concentration of IPTG was 0.1 mM, and the bacteria were induced at 16°C, 200 rpm for 16 hours. The bacteria were then collected.

[0045] 2. Purification of MeV / NP recombinant protein

[0046] The expressed recombinant protein had a histidine tag, and was purified using a protein purification instrument from Suzhou Taide Biological Company, a HisTrap TM HP affinity chromatography column. The A buffer was 50 mM PB, 300 mM NaCl, pH 8.0, and the B buffer was 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The chromatography column was equilibrated with the A buffer, and then the fermented bacteria were centrifuged at 8000 rpm for 10 min. The precipitate was resuspended with the A buffer and ultrasonically broken in ice water for 30 min (5 seconds of ultrasonic treatment every 5 seconds). The supernatant was filtered with a 0.22 micron filter and then loaded onto the chromatography column. The chromatography column was washed with the A buffer, and then eluted with the B buffer. The elution peak of the target protein was collected and dialyzed overnight at 4°C with the A buffer. The purified protein was observed by SDS-PAGE electrophoresis. The electrophoresis results of the purified protein are shown in Figure 1 The protein concentration was determined using an ultramicro spectrophotometer, and the purified protein was stored at -20°C.

[0047] Figure 1 The SDS-PAGE electrophoresis results of the purified protein are shown in FIG. 1. M: protein marker, 1: precipitate after centrifugation of the bacterial cell lysate, 2: supernatant after centrifugation of the bacterial cell lysate, 3: flow-through, 4: 100 mM imidazole elution, and 5: 500 mM imidazole elution. The SDS-PAGE results showed that the eluate of 100 mM and 500 mM imidazole had a clear main band near 70 kDa, which was consistent with the estimated size of the fusion protein (estimated molecular weight of the fusion protein was 75.9 kDa), and had a very high purity.

[0048] 3. Identification of MeV / NP recombinant protein

[0049] The collected protein was coated on an enzyme-labeled plate with 100 mM imidazole, and indirect ELISA method was used to identify its reaction with MeV / NP positive serum. The MeV / NP positive serum was human serum determined as positive by using a measles virus IgG antibody detection kit. The recombinant protein was coated in the microplate (coating buffer: carbonate buffer, sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, constant volume to 1 L of pure water, pH 9.6), and the coating concentration was 1 μg / mL, 50 μL / well, 4°C overnight. The next day, the coating solution was discarded, and 3% sucrose + 2% BSA was used for blocking, 150 μL per well, 37°C for 2 hours, then washed with PBST (0.05% Tween-20 in PBS, pH 7.4) once, and dried. The MeV / NP positive serum (POS-1#) was diluted with PBS at a gradient of 100 times, 1000 times, 10000 times, and 100000 times, and 50 μL was added to the microplate coated with the antigen. At the same time, the healthy newborn serum (NB-1#) was diluted at the same multiples as the negative control, 37°C for 30 min. The liquid in the hole was shaken out, and the plate was washed with PBST 4 times, and dried. Then 50 μL of HRP-labeled goat anti-human IgG secondary antibody (Solebao, diluted 5000 times with PBS) was added to each well, 37°C for 30 min, and the plate was washed 4 times, and dried. Then 50 μL of TMB color developing liquid was added to each well, and the color was developed at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) was added to terminate the reaction, and the OD 450 nm value. The results are shown in Figure 2 , Figure 2 NB-1# is a healthy newborn serum (negative for measles virus IgG antibody detection), and POS-1# is a MeV / NP positive serum. The MeV / NP positive serum still has a weak positive reaction with the MeV / NP recombinant protein at a dilution of 10000 times, indicating that the MeV / NP recombinant protein has good biological activity and can be used for further experiments.

[0050] 4. Mouse immunization

[0051] The mice were immunized with MeV / NP recombinant protein with high purity, and other recombinant proteins expressed by pET32a vector were used as counter-screening antigens for monoclonal antibody screening. Specifically, the purified MeV / NP recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected into 6-week-old female BALB / c mice at a dose of 30 μg per mouse. At 2 and 4 weeks, the mice were boosted by subcutaneous injection of the same dose of the protein mixed with an equal volume of Freund's incomplete adjuvant. At 6 weeks, the mice were immunized by directly injecting the spleen with 5 μg of the protein. Seven days after the last immunization, the serum of the mice was collected for antibody titer detection. The mice with high titers were selected and boosted by intraperitoneal injection of 20 μg of MeV / NP recombinant protein. Three days later, the spleen of the mice was collected for hybridoma cell preparation.

[0052] 5. Screening of hybridoma cells

[0053] After the fusion of all the spleen cells of the immunized mice with SP2 / 0 myeloma cells in the logarithmic growth phase, the cells were cultured in HAT medium. When the fused cells grew to 1 / 2 of the bottom of the well, positive clones that reacted with MeV / NP recombinant protein were screened by indirect ELISA. Since the immunogen was derived from prokaryotic expression of pET32a vector and contained His tag, counter-screening was required to screen specific cell lines targeting MeV / NP protein. The positive cells were cloned into a single clone by limiting dilution, and the cell lines were expanded and cryopreserved.

[0054] Screening of positive clones by indirect ELISA:

[0055] MeV / NP recombinant protein and other recombinant proteins of pET32a vector (pET32a-HPV18 / E7, His tag, preparation process see the company's patent CN120352624B) were coated in a microplate (coating buffer: carbonate buffer: sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, constant volume to 1 L of pure water, pH 9.6), coating concentration was 1 μg / mL, 4°C overnight; the next day, the coating solution was discarded, and 3% sucrose + 2% BSA was used for blocking, 150 μL per well, 37°C incubation for 2 hours, then washed the plate once with PBST (0.05% Tween-20 in PBS, pH 7.4), and dried. Add 50 μL of cell culture supernatant, 37°C for 30 min. Shake off the liquid in the well, wash the plate with PBST 4 times, dry, then add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solebo, diluted 5000 times with PBS), 37°C for 30 min, wash the plate again 4 times, dry, then add 50 μL / well of TMB color developing liquid, color develop at room temperature for 10 min, finally add 50 μL of TMB stop solution (Beijing Meikewande, 1001SA), stop the reaction, and use an enzyme-labeled instrument to measure OD 450 nm values. Select positive cell strains that react with MeV / NP recombinant protein and do not react with control recombinant protein for subsequent experiments. The screening process is shown in Table 1.

[0056] Table 1: Screening results of monoclonal antibodies

[0057]

[0058] After the screened hybridoma cell strains were expanded and cultured, 0.2 ml (containing 2.5×10 6 cells) was injected into the abdominal cavity of female BALB / c mice, and about 10 days later, when the mouse abdomen was significantly enlarged, a sterile syringe needle was used to collect ascites.

[0059] 6. Purification of monoclonal antibodies

[0060] The ascites is centrifuged at 12000 r / min for 10 minutes, 1 ml of supernatant is taken, 4 ml of acetic acid-sodium acetate buffer (0.06M, pH 4.5) is added, mixed uniformly, then 10 μl of n-octanoic acid is slowly added while stirring, after adding, continue stirring for 30 minutes, centrifuge at 12000 r / min for 30 minutes at 2-8°C, take the supernatant. The supernatant is filtered with defatted cotton, saturated ammonium sulfate is added at a ratio of 50% (V / V) of the final volume, stirring while adding, after adding, continue stirring for 30 minutes, precipitate overnight at 2-8°C, centrifuge at 12000 r / min for 30 minutes at 2-8°C, take the precipitate. After the precipitate is completely dissolved with binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), filter through a 0.22 um filter, pump the filtered sample into a Protein L purification column equilibrated with binding buffer at low speed through a peristaltic pump, connect the protein purification instrument, wash with 5-10 column volumes of binding buffer until the ultraviolet absorption peak is washed flat, then elute with elution buffer (0.1M glycine, pH 2.7), collect the elution peak, adjust the collected sample to neutral with 1M Tris-HCl, pH 9, load into a dialysis bag (MW: 8000-14000), dialyze in 20 mM PBS pH 7.4 solution at 2-8°C for 16 hours. Transfer the liquid in the dialysis bag to a centrifuge tube, centrifuge at 12000 r / min for 5 minutes, the supernatant is the purified monoclonal antibody.

[0061] The identification method is the same as the above-mentioned indirect ELISA detection, the purified antibody is diluted to 1 ug / ml, the binding reaction of the antibody and the antigen is identified, and the results are shown in Figure 3 The binding ability of the purified monoclonal antibody and the antigen is detected by indirect ELISA, most of the cloned antibodies show strong reactivity on MeV / NP recombinant protein, and there is no significant reaction on irrelevant antigen HPV18 / E7 recombinant protein, indicating that the obtained monoclonal antibody has good specificity and high affinity. Figure 3 The specific data are shown in Table 2.

[0062] Table 2: Detection results of the binding activity of purified monoclonal antibody and antigen

[0063]

[0064] 7. Preparation of test strips coated with MeV / NP different monoclonal antibodies:

[0065] The selected MeV / NP monoclonal antibodies were respectively drawn on the nitrocellulose membrane with different specifications of 20 mm x 300 mm, using a membrane drawing instrument to spray the diluted monoclonal antibodies (diluted to 1.5 mg / mL with PBS at pH 7.4) in a horizontal line, with a spraying amount of 0.8 μL / cm, to form the test line (T line). After 6 mm interval, the goat anti-mouse IgG antibody was sprayed in a horizontal line with the same spraying amount, diluted to 1 mg / mL with 0.01 M PBS at pH 7.4, coated on the nitrocellulose membrane at an amount of 0.8 uL / cm, to form the control line (C line).

[0066] 8. Colloidal gold pairing of MeV / NP monoclonal antibodies

[0067] Preparation of antibody-colloidal gold labeling complex:

[0068] Antibody labeling: The colloidal gold solution was prepared by the trisodium citrate reduction method. Specifically, 100 mL of 0.01% chloroauric acid solution was heated and boiled, then 1 mL of 1% trisodium citrate solution was quickly added until the solution color was wine red. Boiling was continued for 5 minutes, and after the colloidal gold particles were stable, the solution was cooled to room temperature for standby. To optimize the coupling efficiency of the antibody and colloidal gold, 1 mL of colloidal gold solution was taken in a centrifuge tube, and 1 uL, 2 uL, 3 uL, 4 uL, 5 uL, and 6 uL of 0.2 M potassium carbonate solution were added to adjust the pH environment of the colloidal gold solution in a gradient. Finally, 5 uL was selected when the coupling effect was optimal. After mixing, 5 ug of the MeV / NP monoclonal antibody to be labeled was added, and after rapid mixing, it was incubated at room temperature for 10 min. Then, 10 uL of 10% (w / v) bovine serum albumin (BSA) was added to block the non-specific binding sites, and the incubation was continued at room temperature for 10 min. Then, 10 uL of 10% (w / v) polyethylene glycol 20000 (PEG20000) was added to enhance the labeling stability, and after mixing, it was centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The lower precipitate was resuspended with 1 / 10 volume of resuspension solution (0.01 M phosphate buffer + 1% BSA + 2% sucrose, pH 7.4), to obtain the antibody-colloidal gold labeling complex, which was stored at 4°C in the dark for standby.

[0069] 9. Screening of paired monoclonal antibodies

[0070] The nitrocellulose membrane marked with MeV / NP different monoclonal antibodies was matched with different colloidal gold labeled monoclonal antibodies one by one. The MeV / NP protein was diluted to 20 ng / mL for detection, and the HPV18 / E7 recombinant protein was diluted to 20 ng / mL as a negative antigen for detection, and the combination of deep color development for MeV / NP protein and no reaction with the control protein was screened. The screening process is shown in Table 3 and Table 4. Therefore, the combination of 5E2 membrane and 5G9 gold label is the best combination for detecting MeV / NP recombinant protein.

[0071] Table 3: Results of screening paired monoclonal antibodies with MeV / NP recombinant protein 1

[0072]

[0073] Table 4: Results of screening paired monoclonal antibodies with MeV / NP recombinant protein 2

[0074]

[0075] - represents negative, i.e. no color development; + / ++ / +++ represents positive, i.e. color development reaction, the more the number of +, the darker the color development, i.e. the stronger the positive reaction.

[0076] Table 3 and Table 4 are the screening results using MeV / NP recombinant protein diluted to 20 ng / ml as a positive antigen. The detection results of HPV18 / E7 recombinant protein and blank diluent are negative, and no display is performed. The results show that the 5E2 monoclonal antibody membrane combined with the 5G9 monoclonal antibody label has the deepest color development for the MeV / NP recombinant protein, and is used as the best combination. That is, the combination of monoclonal antibody 5E2 as a capture antibody and monoclonal antibody 5G9 as a labeled antibody can specifically recognize MeV / NP recombinant protein.

[0077] 10. Preparation and assembly of colloidal gold detection test paper

[0078] Preparation of gold label pad: 6mm X 300mm glass fiber membrane RB65 is used, which is treated with Tris-HCl containing 0.5% BSA and 0.5% NP40, pH 8. The prepared colloidal gold labeled antibody is uniformly added to the glass fiber at 1200ul / strip, naturally dried, and then placed in a 37°C oven for 2h for standby;

[0079] 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.

[0080] 11. Test strip specificity test

[0081] Recombinant protein samples: MeV / NP recombinant protein and HPV18 / E7 recombinant protein were diluted to 100 ng / mL with sample dilution buffer for detection.

[0082] 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.

[0083] The sample dilution solution was an aqueous solution of 0.05M Tris + 0.1% NP40 + 1.5% NaCl + 0.1% SDS, pH 8.0.

[0084] 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.

[0085] 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.

[0086] 12. Sensitivity test of test strips

[0087] 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 show that the colloidal gold test paper still has weak color development at a recombinant protein concentration of 1 ng / mL, while the blank diluent, i.e., the sample diluent (0.05M Tris+0.1% NP40+1.5% NaCl+0.1% SDS, pH 8.0) (0 ng / mL) has no color development, indicating that the minimum detection limit of the test paper card for MeV / NP recombinant protein is 1 ng / mL.

[0088] In summary, the present application obtains a pair of monoclonal antibodies that specifically recognize MeV / NP protein by hybridoma technology. The combined antibody can efficiently recognize MeV / NP recombinant protein and has good specificity and sensitivity. The present application applies the monoclonal antibody pair to an immunodetection platform and constructs a rapid detection test strip or test card based on colloidal gold immunochromatography technology. The test paper has high sensitivity for MeV / NP recombinant protein and no cross-reaction with other proteins.

[0089] 13. Gene sequence of the monoclonal antibody

[0090] Total RNA of the hybridoma cells was extracted using an RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers. Mouse antibody variable region universal primers were designed, and VH and VL genes were amplified by 2 rounds of PCR. In the 3rd round of PCR, an Age1 and Bsiw1 enzyme cutting site was introduced into the primers, and the PCR product was purified by cutting gel and then connected to the pUC19 vector. The TOP10 strain was transformed and cultured at 37℃ for 14h, and then a single colony was picked for sequencing to obtain the gene sequence of the light and heavy chains of the monoclonal antibody.

[0091] Gold-labeled monoclonal antibody 5G9:

[0092] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5G9 is shown in SEQ ID NO. 20:

[0093] GACATCCTGATGACCCAATCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAGGCCTCCATCTCTTGCAGATCTAGTCAGAGAATTGTCCATAGTCATGGAAACACCTATTTAGCATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAGGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAGCTGAAACGTACGGTG.

[0094] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5G9 is shown in SEQ ID NO. 15:

[0095] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGGINPSNGGTNYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAADGYSSSSYMEYWGQGTQVTVSS.

[0096] The CDR regions of the heavy chain variable region of monoclonal antibody 5G9 are labeled as follows:

[0097] 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, respectively:

[0098] CDR-H1 : GFTFSSYWMS;

[0099] 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, respectively:

[0100] CDR-H2: INPSNGGTNYADSVKG;

[0101] 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, respectively:

[0102] CDR-H3: ADGYSSSSYMEYWGQGTQVTVSS.

[0103] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5G9 is shown as SEQ ID NO. 19:

[0104] GAGGTGAAGCTGGAGGAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAGGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATGCAGTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAGAGTGGATGGGCTGGATAAACACTGAGACAGGTGAGCCAGCATATACAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGGTAGGAGTAACTTCTATTACTACGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACTGTCTCTGCA.

[0105] Heavy chain variable region amino acid sequence:

[0106] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5G9 is shown as SEQ ID NO. 15:

[0107] EVKLEESGPELKKPGETVRISCKASGYTFTDYSMQWVKQAPGKGLEWMGWINTETGEPAYTDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCGRSNFYYYAMDYWGQGTSVTVSA.

[0108] Heavy chain CDR region annotation:

[0109] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5G9 is shown as SEQ ID NO. 15:

[0110] CDR-H1: DYSMQ;

[0111] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5G9 is shown as SEQ ID NO. 15:

[0112] CDR-H2: WINTETGEPAYTDDFKG;

[0113] 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:

[0114] CDR-H3: SNFYYYAMDY.

[0115] Scratch-resistant monoclonal antibody 5E2:

[0116] The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 5E2 is shown in SEQ ID NO.18:

[0117] GACATCAAGATGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAGGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAAATTACATACACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCCAACCTGGCTTC TGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGTAGTTATGCACCGACGTTCGGTGGAGGCACCAAGCTGGAAATAAAACGTACGGTG.

[0118] The amino acid sequence of the variable region of the light chain of monoclonal antibody 5E2 is shown in SEQ ID NO.14:

[0119] DIKMTQSPAIMSASPGERVTITCSASSSVNYIHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYAPTFGGGTKLEIKRTV.

[0120] Light chain CDR area annotation:

[0121] The amino acid sequence of the light chain variable region CDR-L1 of monoclonal antibody 5E2 is shown in SEQ ID NO.4:

[0122] CDR-L1: SASSSVNYIH;

[0123] The amino acid sequence of the light chain variable region CDR-L2 of monoclonal antibody 5E2 is shown in SEQ ID NO. 5:

[0124] CDR-L2: STSNLAS;

[0125] The amino acid sequence of the light chain variable region CDR-L3 of monoclonal antibody 5E2 is shown in SEQ ID NO. 6, respectively:

[0126] CDR-L3: QQRSSYAPT.

[0127] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5E2 is shown in SEQ ID NO. 17:

[0128] GAGGTGCAGCTGGTGGAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGTACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTAGTACTAACTCCAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCCACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGAAGGAGGGGAAACTGGGTGGGCCTCTTTGACTACTGGGGCCAAGGCACCACTCTCACCGTCTCCTCA.

[0129] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5E2 is shown in SEQ ID NO. 13

[0130] EVQLVESGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPVHGLEWIGEILPGSGSTNSNEKFKGKATFTADTSSNTAHMQLSSLTSEDSAVYYCARRRGNWVGLFDYWGQGTTLTVSS.

[0131] Heavy chain CDR region annotation:

[0132] The amino acid sequence of the heavy chain variable region complementarity determining region CDR-H1 of monoclonal antibody 5E2 is shown in SEQ ID NO. 1, respectively:

[0133] CDR-H1: SYWIE;

[0134] The amino acid sequence of the heavy chain variable region complementarity determining region CDR-H2 of monoclonal antibody 5E2 is shown in SEQ ID NO. 2, respectively:

[0135] CDR-H2: EILPGSGSTNSNEKFKG; CDR-H3: RRGNWVGLFDY.

[0136] The amino acid sequences of the heavy chain variable region complementarity determining regions CDR-H3 of monoclonal antibody 5E2 are set forth in SEQ ID NO. 2, respectively:

[0137] 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 is coated with goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 5G9.

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