Single-chain antibody of novel coronavirus variant JN.1 spike S protein and application of single-chain antibody

Abstract

The invention discloses a single-chain antibody of a new coronavirus variant JN.1 spike S protein and application of the single-chain antibody. A light chain full-length amino acid sequence of the single-chain antibody is shown as SEQ ID NO1, and a heavy chain full-length amino acid sequence of the single-chain antibody is shown as SEQ ID NO2. The single-chain antibody can be used for diagnosing a new coronavirus variant JN.1 spike S protein and can be used for preparing an ELISA kit, the ELISA kit further comprises a detection antibody, the light chain full-length amino acid sequence of the detection antibody is shown as SEQ ID NO < 3 >, and the heavy chain full-length amino acid sequence of the detection antibody is shown as SEQ ID NO < 4 >.

Description

Technical Field

[0001] The present invention relates to a single-chain antibody against the spike S protein of a novel coronavirus variant JN.1 and its application, belonging to the field of genetic engineering technology. Background Art

[0002] The spike protein (S protein) of the new coronavirus is a key target for the virus to attach to and infect host cells, and plays an important role in the host tropism and virulence of the virus. The host receptor angiotensin-converting enzyme 2 (ACE2) binds to the receptor binding domain (RBD) of the S protein, mediating the virus' entry into host cells. The virus can also spread between different hosts through genetic recombination or mutation of the RBD, resulting in a high mortality rate. If neutralizing antibodies against the virus's S protein can be developed to bind to the S protein, the virus can be prevented from infecting host cells.

[0003] Therefore, this study used the spike S protein of the new coronavirus variant JN.1 to immunize BALB / c mice, extracted total RNA from the spleen of the mice, and then reverse transcribed it into cDNA, PCR amplified VH and VL genes, and amplified the single-chain antibody library gene by overlapping extension PCR. Biotechnology was used to prepare single-chain antibodies against the new coronavirus-specific antigen S protein, and the activity of the recombinant protein was tested by ELISA. This study provides new ideas for targeted biological treatment and diagnosis for the prevention and control of new coronaviruses. Summary of the invention

[0004] The purpose of the present invention is to provide a single-chain antibody against the spike S protein of the new coronavirus variant JN.1 and its application. To prepare monoclonal antibodies against the spike S protein of the new coronavirus variant JN.1, and lay the foundation for further development of targeted biological treatment and diagnosis for the prevention and control of the new coronavirus variant JN.1. And to develop an ELISA kit with monoclonal antibodies for the identification and diagnosis of the new coronavirus.

[0005] The technical solution of the present invention: A single-chain antibody against the spike S protein of the new coronavirus variant JN.1, the full-length amino acid sequence of the light chain of the single-chain antibody is shown in SEQ ID NO1, and the full-length amino acid sequence of the heavy chain of the single-chain antibody is shown in SEQ ID NO2.

[0006] The aforementioned single-chain antibody against the spike S protein of the novel coronavirus variant JN.1 is used in the preparation of antibodies for diagnosing the spike S protein of the novel coronavirus variant JN.1.

[0007] In the aforementioned application, the single-chain antibody can be used to prepare an ELISA kit.

[0008] The aforementioned ELISA kit further comprises a detection antibody, the full-length amino acid sequence of the light chain of the detection antibody is shown in SEQ ID NO3, and the full-length amino acid sequence of the heavy chain of the detection antibody is shown in SEQ ID NO4.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The present invention immunized 5 mice with the spike S protein of the novel coronavirus variant JN.1. After five immunizations, the ELISA binding titers of 4 mice with the immunogen all reached the qualified standard. The SBI240422-2D mice were selected to successfully construct a phage library with a library capacity of 5.39×10 9 cfu. After three rounds of screening, a positive library that binds to the GZZ2 protein and has no cross-talk with the control protein was obtained, and single clones were selected for identification. The positive clones were identified and delivered to downstream expression. Purified single-chain antibodies were obtained after transient expression in 293 cells and purification of the expression supernatant. ELISA detected that all antibodies were bound to the antigen protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : Mouse SBI240422-2D RNA electrophoresis;

[0012] Figure 2 : Heavy and light chain variable region amplification electrophoresis;

[0013] Figure 3 : scFv electrophoresis;

[0014] Figure 4 : scFv ligation vector diagram. DETAILED DESCRIPTION

[0015] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0016] 1 Main instruments and reagents

[0017] 1.1 Cells and experimental animals

[0018] SPF-grade Balb / c mice, 6-8 weeks old, weighing 18-22 g, were purchased from Beijing Biotechnology Co., Ltd., with experimental animal use license number SYXK(Beijing)2020-0011, and were adaptively fed for 7 days. The animal experiment strictly followed the ethical welfare principles and was approved by the Ethics Review Committee of Beijing Yizhuang International Biotechnology Co., Ltd., approval number 2024F01; HEK293 cells were provided by Sino Biological Technology Co., Ltd.

[0019] 1.2 Main instruments

[0020] ELISA reader, Thermo Corporation (Multiskan Fc); desktop refrigerated centrifuge, Thermo (X4RPro); UV-visible spectrophotometer, Shanghai Youke Instrument (UV756); PCR instrument A300, Hangzhou Longji (A300); 10L imported bioreactor, BBI (B-DCU2L); electric pipette, Brand (26310); inverted microscope, Olympus (CKX41SF); steam generator, Shanghai Huazheng (LDR0034-07); peristaltic pump, LongerPump Company (YZ1515X); nucleic acid protein detector, Shanghai Qingpu Huxi Instrument Factory (HD-21-1); electrophoresis instrument, EPS600 (Tianneng).

[0021] 1.3 Main Reagents

[0022] Complete Freund's adjuvant, Sigma (F5881); Incomplete Freund's adjuvant, BD (263910); Spike S protein of the novel coronavirus variant JN.1 (purchased from Sino Biological, Cat: 40589-V08H59); Horseradish peroxidase-labeled goat anti-mouse IgG Fc, IGL (GGFC-90P); TriPure Isolation Reagent, Roche; reverse transcription kit, restriction endonuclease SfiI, phage vector pComb3x, X-Blue competent cell, Anti-M13 enzyme-labeled secondary antibody, M293TI-1 culture medium (293 cells), SMS-293-SUPI feed solution (293 cells), TF1 transfection reagent, 1× glycine system electrode buffer, Coomassie Brilliant Blue R-250 staining solution, 4× loading buffer (reducing) (4R), 4× loading buffer (non-reducing) (4N), 13% SDS-PAGE gel, 7.5% SDS-PAGE gel, SARS-CoV-2 variant JN.1 spike S protein (Cat: 40589-V08H59), reverse transcription kit, purchased from Sino Biological; 13% protein standard, Thermo (#26610); 7.5% protein standard, Thermo (#26614); horseradish enzyme-labeled rabbit anti-mouse IgG F(ab')2, Jackson Company (315-035-047).

[0023] Example 1 Animal Immunization

[0024] Five female Balb / c mice aged 6-8 weeks (animal numbers SBI240422A, SBI240422B, SBI240422C, SBI240422-2D, SBI240422-2E) were immunized with the spike S protein of the new coronavirus variant JN.1. The single immunization dose was 50 μg. For the first immunization, the immunogen was mixed with an equal volume of complete Freund's adjuvant to make an emulsion, and multiple subcutaneous injections were made in the abdomen. After an interval of 2 weeks, the same dose of immunogen was mixed with an equal volume of incomplete Freund's adjuvant to make an emulsion, and multiple subcutaneous injections were made in the abdomen. Blood was collected one week after the three immunizations, and the serum titer was determined by indirect ELISA. The titer qualification standard was that the blank group (Blank) OD450 (OD450-Blank) was greater than 1.0 when the mouse serum was diluted 1:8000. Among the animals with qualified titers, the animals with high ELISA titers were selected for booster immunization. After 3-4 days, the spleens of the mice were collected and stored in liquid nitrogen for construction of phage library. The animals with unqualified titers were continuously immunized until the titers were qualified, with a maximum of 5 immunizations.

[0025] Example 2 Blood sampling and titer detection

[0026] Serum collection: One week after the last immunization, 50-60 μL of blood was collected from the mouse orbital venous plexus. After standing at 4°C overnight, the blood was centrifuged at 4000 rpm and 4°C for 15 minutes to separate the upper serum for testing.

[0027] ELISA binding titer detection: The indirect ELISA method is used to detect serum titer, and the binding of antiserum to immunogen is detected at the same time. The titer of the spike S protein antigen of the new coronavirus variant JN.1 is qualified by OD450-Blank greater than 1.0 when the mouse serum is diluted 1:8000. Mice with large differences in de-crossed proteins are preferred for library construction. The detection steps are as follows.

[0028] (1) Take an appropriate amount of JN.1 spike S protein antigen for detection, dissolve the immunogen in coating buffer and dilute to 5 μg / mL, then add 100 μL to each well of a 96-well plate using a single-channel pipette, tap the plate to mix the sample, seal it with plastic wrap, and coat it overnight at 4°C;

[0029] (2) Washing: Wash the plate once with washing solution at 200 μL / well and dry the ELISA plate;

[0030] (3) Blocking: Block the ELISA plate with blocking solution at 300 μL / well for 1 h at room temperature;

[0031] (4) Washing: Wash the plate twice with washing solution at 400 μL / well and dry the ELISA plate;

[0032] (5) Sample loading: Add the serially diluted serum sample and sample diluent at 100 μL / well;

[0033] (6) Adding secondary antibody: Add detection antibody at 100 μL / well to a 96-well plate and incubate at room temperature for 2 h;

[0034] (7) Washing: Wash the plate five times with washing solution at 400 μL / well and dry the ELISA plate;

[0035] (8) Color development: Add 200 μL / well of color development solution and incubate at room temperature for 12 min;

[0036] (9) Termination and detection: 50 μL / well of stop solution was added to terminate the reaction, and then the reaction was detected using an ELISA reader at a wavelength of 450 nm.

[0037] Results: Five animals were coated with 5 μg / mL immunogen, and the serum was diluted 8000 times. OD450-Blank>1.0 was the qualified standard for immunity. After three immunizations, the serum titers of mice SBI240422C, SBI240422-2D, and SBI240422-2E all reached the qualified standard, and the phage library could be constructed. SBI240422A and SBI240422B continued to be immunized. After four immunizations, mice SBI240422A and SBI240422B did not reach the qualified standard, so they continued to be immunized. After five immunizations, mouse SBI240422B reached the qualified standard, and SBI240422-2D was selected to construct the phage library. The results of three immunizations of SBI240422-2D are shown in Table 1.

[0038] Table 1 ELISA test results

[0039]

[0040]

[0041] *Blank value: the OD450 value obtained by using sample diluent instead of the sample to be tested; the background value during the reaction detection process.

[0042] Example 3 Preparation, screening and monoclonal identification of phage antibody library

[0043] (1) Preparation of phage antibody library

[0044] The RNA of mouse spleen tissue was extracted with TriPure Isolation Reagent, and reverse transcription was performed with a homemade reverse transcription kit to obtain cDNA. The light chain variable region sequence and heavy chain variable region sequence of mouse antibody were amplified by PCR. The light chain and heavy chain variable region sequences encoding mouse antibody were spliced ​​into nucleotide sequences encoding scFv by overlapping extension splicing PCR method, and then connected to the phage vector pComb3x by homemade restriction endonuclease SfiI digestion, and electroporated into X-Blue competent cells to construct phage display scFv antibody library of immune mice. The antibody library capacity (clone number*dilution multiple*culture volume) and sequence insertion accuracy were calculated.

[0045] The electrophoresis diagram of RNA extraction from mouse SBI240422-2D spleen is shown in Figure 1 The extracted RNA was quantified by UV. The experimental results are shown in Table 2. The results of heavy and light chain variable region amplification electrophoresis are shown in Figure 2 , VH and VL were spliced ​​into scFv, and the electrophoresis of the spliced ​​fragments was shown in Figure 3 , scFv connected to phage vector Figure 4 The constructed phage antibody library has a capacity of 5.39*10 9 cfu.

[0046] Table 2 RNA UV quantification results

[0047] Project ID Organization ID A260 A280 A260 / A280 Concentration (ng / uL) GZZ2 sp-SBI240422-2D 67.603 33.258 2.033 2704

[0048] (2) Phage antibody library screening

[0049] The primary antibody library is infected with helper phages to prepare the original antibody library. The antigen is coated on the ELISA plate, and the original antibody library is added for binding. The non-specific binding is washed with PBST, and then the phage bound to the antigen is eluted with an elution solution. After amplification and precipitation in E. coli, the next round of enrichment screening is carried out. After 2-4 rounds of "adsorption-elution-amplification", the phage library enriched with antigen-positive antibodies is screened. Monoclonal phages are selected from the enriched library for expression, and the binding with the antigen is detected by ELISA method. The high-binding antibody clones that specifically bind to the antigen are screened and sent to the sequencing company for sequencing to obtain the nucleotide sequence.

[0050] After three rounds of screening, the binding of the phage library to the antigen (SARS-CoV-2JN.1(Omicron)SpikeS1+S2 trimer Protein) was detected by ELISA, and the test results are shown in Table 3. The ELISA test results showed that after three rounds of screening, a positive library that bound to the antigen protein and had no cross-talk with the control protein was obtained. Monoclonal phages were selected from the enriched positive phage library for expression and ELISA identification.

[0051] Table 3 ELISA test data of bacterial positive library

[0052]

[0053] Example 4 Antibody Construction

[0054] (1) Preparation of heavy and light chain variable region fragments

[0055] The variable region fragments of the light and heavy chains of the scFv antibody were amplified by PCR using self-designed primers. The variable region fragments of the heavy and light chains were added to the membrane binding solution (components of the gel recovery kit), mixed thoroughly and added to the nucleic acid purification column. The solution was discarded after centrifugation at 13000 rpm for 1 min, 700 μL of rinse solution was added, and the solution was discarded after centrifugation at 13000 rpm for 1 min. After spinning for 5 min, the mixture was placed at room temperature for 5 min, and Nuclease-Free Water was added for elution to obtain the purified variable region of the heavy and light chains.

[0056] (2) Cloning construction

[0057] 4 μL of the purified fragment and 1 μL of the vector were added with 5 μL of ligase and mixed thoroughly. After reacting at 50°C for 20 minutes, the fragment was added to 100 μL of competent medium, and then heat-shocked for 90 seconds after ice bath for 30 minutes. The fragment was quickly placed on ice for 3 minutes and then coated on the plate. The monoclonal antibody was amplified and sequenced to obtain the heavy and light chain sequences of the antibody. The heavy and light chain plasmids of the antibody were paired and delivered to HEK293 for transient expression.

[0058] The ELISA data of monoclonal phages selected from the positive phage library for expression and detection are shown in Table 4. The following clones bind well to the antigen protein and have no cross-reaction with the His control protein.

[0059] Table 4 Monoclonal phage expression ELISA detection data

[0060]

[0061]

[0062] The full-length amino acid sequence of the antibody is as follows:

[0063] The full-length amino acid sequence of the light chain of GZZ2-M120

[0064] SEQ ID NO1:

[0065] MGWSCIILFLVATATGVHSDIQMTQSSSFLSVSLGGRVTIACKASDHINSYLAWYQQKPGNAPRLLISGAT

[0066] SLETGIPSRFSGSGSGKDYTLTITSLQTEDFATYYCQQYWDTPPTFGGGTKLEIKRADAAPTVSIFPPSSEQL

[0067] TSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC-

[0068] Full-length amino acid sequence of the heavy chain of GZZ2-M120

[0069] SEQ ID NO2:

[0070] MGWSLILLFLVAVATRVLSEVQLQQSGPELVRPGTSVKVSCQASGYAFTNYLVEWVKQSPGQGLEWIGVIN

[0071] PGSGVFNHNEKFKDKATLTADKSSSTAYMQLSSLTSDDSAVYFCARRSGDGYYPYSLDYWGQGTTVTVSSAKT

[0072] TPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWP

[0073] SETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPE

[0074] VQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKA

[0075] PQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK-

[0076] Full-length amino acid sequence of the light chain of GZZ2-M015

[0077] SEQ ID NO3:

[0078] MGWSCIILFLVATATGVHSDIQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYQQKPDGTVKLLIYHTS

[0079] RLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQANTLPYTFGGGTKLEIKRADAAPTVSIFPPSSEQL

[0080] TSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC-

[0081] Full-length amino acid sequence of the heavy chain of GZZ2-M015

[0082] SEQ ID NO4:

[0083] MGWSLILLFLVAVATRVLSEVQLQQSGPELVRPGASVKLSCKASGYIFITYWMNWVKQRPGQGLEWIGQIF

[0084] PASGSTDYNEMFEGKATLTVDTSSSTAYMQLTSLTSEDSAVYYCARRGGGSAWFPYWGQGTLVTVSAAKTTPP

[0085] SVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSET

[0086] VTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQF

[0087] SWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQV

[0088] YTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGN

[0089] TFTCSVLHEGLHNHHTEKSLSHSPGK-

[0090] Example 5 Cell culture

[0091] HEK293 cells were subcultured in 293 serum-free CD medium. The plasmid DNA to be expressed was mixed with the transfection reagent TF2 and added to the cells. 293 serum-free feed solution was added on the 1st, 3rd, and 5th days after transfection. Shake flask culture conditions: 5% CO 2 , temperature 37°C, shaker speed 175 rpm.

[0092] Example 6 Monoclonal Antibody Purification

[0093] 1. Sample preparation: The collected cell supernatant was centrifuged using a desktop centrifuge at 4000 g for 30 min, and the cell supernatant was collected; the cell supernatant was filtered using a 0.45 μM filter membrane.

[0094] 2. Antibody purification: Purify the cell culture supernatant after centrifugation using protein A affinity purification method to harvest mouse monoclonal antibodies. The steps are as follows:

[0095] (1) Connection system: Select a protein A column of appropriate specifications according to the expression level and connect it to the purification system;

[0096] (2) Water balance: Wash with ultrapure water for 3 CV and replace the 25% ethanol storage solution;

[0097] (3) Equilibration of the column: AC Binding (50 mM Tris, 100 mM NaCI, pH 8.0) equilibration solution for 3 CV until the UV baseline is stable;

[0098] (4) Sample loading: Adjust the flow rate to a suitable level, usually 1 / 3 column volume;

[0099] (5) Elution: Elution with eluent for 5-10 CV until UV baseline is stable, the flow rate is the same as the sample loading flow rate;

[0100] (6) Elution: elution with AC Elution buffer and collection based on UV peaks;

[0101] (7) Neutralization: Add 2M Tris, pH 8.0 to neutralize the eluted antibody;

[0102] (8) Equilibration: AC Binding (50 mM Tris, 100 mM NaCI, pH 8.0) equilibrated to neutrality for 3 CV;

[0103] (9) CIP cleaning: CIP (NaOH) cleaning for more than 5CV;

[0104] (10) Alkali flushing: flush alkali with equilibrium liquid until the pH at the outlet is neutral;

[0105] (11) Storage: Equilibrate the column with 25% ethanol for 2CV.

[0106] Example 7 Antibody concentration and purity detection

[0107] Start the micro-spectrophotometer and spot the sample with phosphate buffer. When the absorbance value is between ±0.015 at a wavelength of 280 nm, the instrument baseline is stable. Spot the purified antibody samples in turn, record the OD280 and OD320 absorbance values, and divide the value of (OD280-OD320) by the IgG extinction coefficient (1.414). The resulting value is the concentration of the test sample (in mg / mL).

[0108] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to verify the purity of the purified antibody according to the proportion of heavy and light chain bands. This method separates the proteins in the test sample in the electrophoresis gel according to their different molecular weights, thereby verifying the purity of the target antibody. Take 5μg of the test sample and add 5μL 4R / 4N, heat it in a 100℃ water bath, and then centrifuge it at 10000rpm. Add enough 1×SDS electrode buffer (SDS: sodium dodecyl sulfate) to the electrophoresis tank, and use a micropipette to add the treated sample solution and protein molecular weight standard (Thermo Scientific, #26610) to the sample well. Connect the power supply, first electrophoresed at a constant voltage of 100V until the bromophenol blue dye enters the separation gel from the stacking gel, then adjust the current to 140V and continue electrophoresis to the bottom of the gel plate, and turn off the power supply. Soak the gel in the staining solution, heat it in a microwave for 60s, and stain it at room temperature on a gently shaking platform. Replace the destaining solution to cover the gel, heat it in a microwave for 60 seconds, destain it at room temperature on a gently shaking platform, and repeat the destaining operation until a blue band and a clean background are obtained.

[0109] Example 8 ELISA binding detection

[0110] (1) Coating: Coating antigen 40589-V08H59 0.1 μg / mL, 1 μg / mL, 100 μL / well, overnight at 4°C;

[0111] (2) Blocking: shake off the liquid in the plate and pat dry, add 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h;

[0112] (3) Washing: 300 μL / well washing solution, wash the plate twice, and pat dry for the last time;

[0113] (4) Antibody dilution: dilute the GZZ2 antibody (GZZ2-M015, GZZ2-M120) to 1 μg / mL, add 100 μL of each to the corresponding well plate, mix well, and react at room temperature for 2 h;

[0114] (5) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry for the last time;

[0115] (6) Add secondary antibody: dilute Rabbit Anti-Mouse IgG F(ab)2 / HRP secondary antibody to the working concentration, 100 μL / well, mix well, and incubate at room temperature for 1 h;

[0116] (7) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry for the last time;

[0117] (8) Color development: Mix solution A and solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 20 min;

[0118] (9) Stopping: Add 50 μL of stop solution to each well and immediately measure the OD450 value at a wavelength of 450 nm.

[0119] The results of ELISA test of purified antibodies are shown in Table 5.

[0120] Table 5 Purified antibody ELISA test results

[0121]

[0122] Example 9 Purification and labeling of antibodies

[0123] 1. Activation of HRP

[0124] 1) Prepare HRP solution with 5 mg HRP and 0.20 mL of freshly prepared 0.1 M sodium acetate buffer;

[0125] 2) Weigh sodium periodate (Sangon Biotechnology Lot: AA27BA0032) according to the volume of HRP solution in a ratio of 1:1-2;

[0126] 3) Dissolve sodium periodate in HRP solution and react for 1 hour at room temperature in the dark;

[0127] 4) Use an ultrafiltration centrifuge tube to replace the solution with 0.01M PBST solution with pH 7.4 to activate HRP, and replace the solution 2 to 3 times (the original buffer solution remains <1%);

[0128] 5) Adjust HRP to the appropriate concentration, divide into smaller pieces and set aside.

[0129] 2. Conjugation of Antibodies with HRP

[0130] 1) Ultrafilter the coupled antibody into 0.01M pH 7.4 PBST solution (ultrafilter 3 times) and adjust to the appropriate concentration (0.8-1.3 mg / ml);

[0131] 2) Detection antibody: HRP was diluted at a ratio of 1:20000, and then reacted in a shaker at room temperature in the dark for 1 hour for later use;

[0132] 3) Add 3% sodium borohydride solution (prepared immediately before use) and react at 4°C in the dark for 1 hour;

[0133] 4) Add 10% saturated ammonium sulfate and place at 4°C in the dark;

[0134] 5) Centrifuge, discard the supernatant and keep the precipitate;

[0135] 6) Wash twice with saturated ammonium sulfate solution (add PBS first, mix well, then add saturated ammonium sulfate solution), centrifuge and remove supernatant;

[0136] 7) Dissolve the precipitate with PBS and centrifuge to remove the precipitate (sterile filtration (0.22 μm filter membrane) in large quantities);

[0137] 8) Select the appropriate dilution factor based on experience, measure OD280 and OD403, and calculate the concentration (OD280-0.3*OD403)*0.62.

[0138] The results of HRP labeling of purified antibodies are shown in Table 6.

[0139] Table 6 Purified antibody HRP labeling results

[0140]

[0141]

[0142] Example 10 Pairing Detection

[0143] 1) Coating: GZZ2 antibody (GZZ2-M120) 2 μg / mL, 100 μL / well, overnight at 4°C;

[0144] 2) Blocking: shake off the liquid in the plate and pat dry, add 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 hour;

[0145] 3) Washing: 300 μL / well washing solution (0.01M pH 7.4 PBST solution), wash the plate twice, and pat dry for the last time;

[0146] 4) Antigen dilution: Antigen 40589-V08H59 was diluted (the diluent was 0.1% BSA-PBST pH7.4) to 1 ng / mL and 10 ng / mL, and the blank control was the sample diluent (the diluent was 0.1% BSA-PBST pH7.4). 100 μL of each was added to the corresponding well plate, mixed evenly, and reacted at room temperature for 2 h;

[0147] 5) Washing: 300 μL / well washing solution (0.01M pH 7.4 PBST solution), wash the plate 3 times, and pat dry for the last time;

[0148] 6) Add secondary antibody: dilute the GZZ2 labeled antibody (GZZ2-M015-HRP) to the working concentration (0.5 μg / mL), 100 μL / well, mix well, and incubate at room temperature for 1 hour;

[0149] 7) Washing: 300 μL / well washing solution (0.01M pH 7.4 PBST solution), wash the plate 3 times, and pat dry for the last time;

[0150] 8) Color development: Mix color development solution A (DH2) and color development solution B (TMB) at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 20 min;

[0151] 9) Stop: Add 50 μL of stop solution (2 M sulfuric acid) to each well and immediately measure the OD450 value at a wavelength of 450 nm.

[0152] The results of antibody pairing test are shown in Table 7.

[0153] Table 7 Antibody pairing test results

[0154]

[0155] Example 11 Establishment of ELISA reagent detection

[0156] 1) Coating: Coat with GZZ2-M120 antibody 2 μg / mL, 100 μL / well, overnight at 4°C;

[0157] 2) Blocking: shake off the liquid in the plate and pat dry, add 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 hour;

[0158] 3) Protective agent: shake off the liquid in the plate and pat dry, add 300 μL / well of 1% gelatin, seal and incubate at room temperature for 1 hour;

[0159] 4) Drying: shake off the liquid in the board, pat it dry, dry it, bag it, and seal it;

[0160] 5) Washing: 300 μL / well washing solution (0.01M pH 7.4 PBST solution), wash the plate twice, and pat dry for the last time;

[0161] 6) Serial dilution of standard: 5 μL of stock solution 40589-V08H59 protein was added to 495 μL 1× sample diluent and marked as tube A, 100 μL of tube A was added to 900 μL 1× sample diluent and marked as tube B, and 2.27 μL of stock solution was added to 997.73 μL 1× sample diluent to prepare 1000 μL 6000 pg / mL highest concentration standard (the OD value at the highest point of the curve is around 2.5 as the criterion for judgment). Using 500 μL 1× sample diluent as diluent, six two-fold serial dilutions of the 6000 pg / mL highest concentration standard were made in 6 separate test tubes: after mixing the 6000 pg / mL highest concentration standard, 500 μL was pipetted into the next test tube, and so on. 1× sample dilution buffer was used as zero standard (0 pg / mL). Mix well and react at room temperature for 2 h (the diluent used in this step is 0.1% BSA-PBST pH 7.4);

[0162] 7) Washing: 300 μL / well washing solution (0.01M pH 7.4 PBST solution), wash the plate 3 times, and pat dry for the last time;

[0163] 8) Add secondary antibody: add GZZ2-M015 / HRP labeled antibody to 0.5ug / ml (1:800), 100μL / well, mix well, and incubate at room temperature for 1h;

[0164] 9) Washing: 300 μL / well washing solution (0.01M pH 7.4 PBST solution), wash the plate 3 times, and pat dry for the last time;

[0165] 10) Color development: Mix color development solution A (DH2) and color development solution B (TMB) at a ratio of 1:1, add 100 μL to each well, and incubate at room temperature in the dark for 20 min;

[0166] 11) Stop: Add 100 μL of stop solution (2M sulfuric acid) to each well and immediately measure the OD value at a wavelength of 450 nm. The results of ELISA reagent detection performance verification are shown in Table 8

[0167] Table 8 ELISA reagent detection performance verification results

[0168]

[0169]

[0170] in conclusion:

[0171] Protein (Cat: 40589-V08H59) was used to immunize 5 mice. After five immunizations, the ELISA binding titer of 4 mice to the immunogen reached the qualified standard, and SBI240422-2D mice were selected to construct the phage library. The phage library was successfully constructed with a library capacity of 5.39x109cfu. After three rounds of screening, a positive library that bound to the GZZ2 protein and had no crossover with the control protein was obtained. Single clones were selected for identification, and the positive clones were identified for downstream expression. After transient expression in 293 cells and purification of the expression supernatant, 2 purified antibodies were obtained. The binding of antibodies to antigens was detected by ELISA. The results showed that both antibodies bound to the antigen protein. The two antibodies were labeled with HRP and then paired for detection, and coated to form a kit. The sensitivity was 6.8pg / mL.

Claims

1. A single-chain antibody against the spike S protein of the novel coronavirus variant JN.1, characterized in that: The full-length amino acid sequence of the light chain of the single-chain antibody is shown in SEQ ID NO1, and the full-length amino acid sequence of the heavy chain of the single-chain antibody is shown in SEQ ID NO2.

2. The use of the single-chain antibody against the spike S protein of the novel coronavirus variant JN.1 according to claim 1 in the preparation of antibodies for diagnosing the spike S protein of the novel coronavirus variant JN.

1.

3. The use according to claim 2, characterized in that: The single-chain antibody can be used for ELISA monitoring.

4. The use according to claim 3, characterized in that: The ELISA test also includes a detection antibody, the full-length amino acid sequence of the light chain of the detection antibody is shown in SEQ ID NO3, and the full-length amino acid sequence of the heavy chain of the detection antibody is shown in SEQ ID NO4.

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