Single-chain antibody of novel coronavirus variant jn.1 spike s protein and application thereof

Abstract

The application discloses a single-chain antibody of a novel coronavirus mutant JN.1 spike S protein and application thereof, a full-length amino acid sequence of a light chain of the single-chain antibody is shown as SEQ ID NO 1, and a full-length amino acid sequence of a heavy chain of the single-chain antibody is shown as SEQ ID NO 2. The single-chain antibody can be used for diagnosing the novel coronavirus mutant JN.1 spike S protein, and can be used for preparing an ELISA kit, and the ELISA kit further comprises a detection antibody, a full-length amino acid sequence of a light chain of the detection antibody is shown as SEQ ID NO 3, and a full-length amino acid sequence of a heavy chain of the detection antibody is shown as SEQ ID NO 4.

Description

TECHNICAL FIELD

[0001] The present application relates to a single-chain antibody of a new coronavirus variant JN.1 spike S protein and its application, and belongs to the technical field of genetic engineering. BACKGROUND

[0002] The spike protein (S protein) of the new coronavirus is a key target for virus adsorption and infection of host cells, and plays an important role in 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, mediates the entry of the virus into the host cell, and the virus can also be transmitted between different hosts through genetic recombination or mutation of the RBD, and cause high mortality. If a neutralizing antibody against the S protein of the virus can be developed to bind the S protein and prevent the virus from infecting host cells.

[0003] Therefore, in the present study, BALB / c mice were immunized with the spike S protein of the new coronavirus variant JN.1, total RNA was extracted from the spleen of the mice, and then reverse transcribed into cDNA, and the VH and VL genes were amplified by PCR, and the single-chain antibody library gene was amplified by overlap extension PCR. The specific antigen S protein of the new coronavirus was prepared by using biotechnology, and the activity of the recombinant protein was detected by ELISA. The present study provides a new idea for the targeted biological treatment and diagnosis of the new coronavirus. SUMMARY

[0004] The present application aims to provide a single-chain antibody of a new coronavirus variant JN.1 spike S protein and its application. The monoclonal antibody against the new coronavirus variant JN.1 spike S protein is prepared, which lays a foundation for further development of targeted biological treatment and diagnosis of the new coronavirus variant JN.1. An ELISA kit with the monoclonal antibody is developed for identification and diagnosis of the new coronavirus.

[0005] The technical solution of the present application is a single-chain antibody of a new coronavirus variant JN.1 spike S protein, wherein the full-length amino acid sequence of the light chain of the single-chain antibody is shown in SEQ ID NO 1, and the full-length amino acid sequence of the heavy chain of the single-chain antibody is shown in SEQ ID NO 2.

[0006] The aforementioned single-chain antibody of a new coronavirus variant JN.1 spike S protein is used for the preparation of an application for diagnosing the new coronavirus variant JN.1 spike S protein.

[0007] The aforementioned application, wherein the single-chain antibody can be used for the preparation of an ELISA kit.

[0008] The foregoing ELISA kit further comprises a detection antibody, a full-length amino acid sequence of a light chain of the detection antibody is shown as SEQ ID NO 3, and a full-length amino acid sequence of a heavy chain of the detection antibody is shown as SEQ ID NO 4.

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

[0010] In the present application, five mice are immunized with the novel coronavirus mutant JN.1 spike S protein, and after five immunizations, the ELISA binding titers of four mice and the immunogen all reach the qualified standard, and the SBI240422-2D mouse is selected to successfully construct a phage library, and the capacity of the library is 5.39x10 9 After 3 rounds of screening, a positive library combined with the GZZ2 protein and without cross with the control protein is obtained, a single clone is selected and identified, and the positive clone is passed to the downstream expression. After 293 cell transient expression and purification of the expression supernatant, a purified single-chain antibody is obtained, and ELISA detection shows that the antibody is combined with the antigen protein. BRIEF DESCRIPTION OF DRAWINGS

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

[0012] Figure 2 : Amplification electrophoresis of heavy and light chain variable regions;

[0013] Figure 3 : scfv electropherogram;

[0014] Figure 4 : scfv connection vector map. DETAILED DESCRIPTION

[0015] The present application will be further described below in combination with examples, but it is not used as a basis for limiting the present application.

[0016] 1 Main instruments and reagents

[0017] 1.1 Cells and experimental animals

[0018] SPF grade Balb / c mice, 6-8 weeks old, weighing 18-22g, purchased from Sibeifeng (Beijing) Biotechnology Co., Ltd., experimental animal use license number SYXK(Jing)2020-0011, adaptively fed for 7 days, strictly in accordance with the animal experiment ethics and welfare principles, approved by the Beijing Yizhuang International Biomedicine Technology Co., Ltd. Ethical Review Committee, approval number 2024F01; HEK293 cells were provided by Yiqiao God Science and Technology Co., Ltd.

[0019] 1.2 Main instruments

[0020] Multiskan Fc, Thermo; X4R Pro, Thermo; UV756, Shanghai Youke Instruments; A300, Hangzhou Langji; B-DCU2L, BBI; 26310, Brand; CKX41SF, Olympus; LDR0034-07, Shanghai Huazheng; YZ1515X, Longer Pump; HD-21-1, Shanghai Qingpu Huxi Instruments; EPS600, Tian Neng.

[0021] 1.3 Main reagents

[0022] Complete Freund's Adjuvant, Sigma (F5881); Incomplete Freund's Adjuvant, BD (263910); SARS-CoV-2 Spike S Protein (JN.1 variant), purchased from Yiqiao Biotech (Cat: 40589-V08H59); Goat Anti-Mouse IgG Fc, HRP-labeled, IGL (GGFC-90P); TriPure Isolation Reagent, Roche; Reverse Transcription Kit, Restriction Enzyme Sfil, Phage Vector pComb3x, X-Blue Competent, Anti-M13 HRP-labeled Secondary Antibody, M293TI-1 Medium (293 cells), SMS-293-SUPI Feed Solution (293 cells), TF1 Transfection Reagent, 1x Glycine System Electrode Buffer, Coomassie Brilliant Blue R-250 Staining Solution, 4x Loading Buffer (Reducing) (4R), 4x Loading Buffer (Non-reducing) (4N), 13% Concentration SDS-PAGE Gel, 7.5% Concentration SDS-PAGE Gel, SARS-CoV-2 Spike S Protein (JN.1 variant) (Cat: 40589-V08H59), Reverse Transcription Kit, purchased from Yiqiao Shenzhou; 13% Protein Standard, Thermo (#26610); 7.5% Protein Standard, Thermo (#26614); Rabbit Anti-Mouse IgG F(ab')2, HRP-labeled, Jackson (315-035-047).

[0023] Example 1 Animal immunization

[0024] New coronavirus variant JN.1 spike S protein immunizes 5 female Balb / c mice aged 6-8 weeks (animal numbers SBI240422A, SBI240422B, SBI240422C, SBI240422-2D, SBI240422-2E), single immunization dose 50 μg, the first immunization emulsifier is prepared by mixing equal volume of immunogen and complete Freund's adjuvant, and the immunization is performed by subcutaneous injection in multiple points of the abdomen. After 2 weeks, the same dose of immunogen is mixed with equal volume of incomplete Freund's adjuvant to prepare emulsifier, and the immunization is performed by subcutaneous injection in multiple points of the abdomen. One week after 3 immunizations, blood is taken, and the serum titer is determined by indirect ELISA. The qualified standard for titer is that the OD450-Blank of mouse serum diluted by 1:8000 is greater than 1.0. The animals with high ELISA titer are preferentially selected for booster immunization, and the mouse spleen is taken after 3-4 days for nitrogen preservation to construct phage library. The animals with unqualified titer continue to be immunized until the titer is qualified, and the immunization is performed for a maximum of 5 times.

[0025] Blood collection and titer detection in Example 2

[0026] Serum collection: one week after the last immunization, 50-60 μL of blood is taken from the orbital venous plexus of the mouse. After standing at 4°C overnight, the upper serum is separated by centrifugation at 4000 rpm and 4°C for 15 min.

[0027] ELISA binding titer detection: indirect ELISA method is used for serum titer detection, and the binding of antiserum and immunogen is also detected. The qualified standard for titer is that the OD450-Blank of mouse serum diluted by 1:8000 is greater than 1.0. The mice with large difference value from cross protein are preferentially selected for library construction. The detection steps are as follows.

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

[0029] (2) Wash the plate: wash the plate once with 200 μL / well of washing solution, and dry the enzyme-labeled plate;

[0030] (3) Blocking: block the enzyme-labeled plate with 300 μL / well of blocking solution at room temperature for 1 h;

[0031] (4) Wash the plate: wash the plate twice with 400 μL / well of washing solution, and dry the enzyme-labeled plate;

[0032] (5) Sample addition: add 100 μL / well of gradient-diluted serum sample and sample diluent;

[0033] (6) Add secondary antibody: add detection antibody, 100 μL / well into 96-well plate, co-incubate at room temperature for 2 h;

[0034] (7) Wash plate: then wash plate with washing solution, 400 μL / well, 5 times, and dry enzyme-labeled plate;

[0035] (8) Color development: add color development solution, 200 μL / well, and place at room temperature for 12 min;

[0036] (9) Termination and detection: add termination solution, 50 μL / well, to terminate reaction, and then detect with enzyme-labeled instrument, and detection wavelength is 450 nm.

[0037] Results: 5 animals were coated with 5 μg / mL immunogen, and serum was diluted 8000 times. OD450-Blank>1.0 was the qualified standard for immunization. After three times of immunization, 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 times of immunization, SBI240422A and SBI240422B did not reach the qualified standard, and continued to be immunized. After five times of immunization, SBI240422B reached the qualified standard. SBI240422-2D was selected to construct the phage library, and the results of three times of immunization of SBI240422-2D are shown in Table 1.

[0038] Table 1 ELISA detection results

[0039]

[0040]

[0041] Blank value: the OD450 value obtained by using sample diluent instead of sample to be detected, which is the background value in 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 spleen tissue of the mouse was subjected to RNA extraction with TriPure Isolation Reagent reagent, and cDNA was obtained by reverse transcription with a self-made reverse transcription kit. The light chain variable region sequence and the heavy chain variable region sequence of the mouse antibody were amplified by PCR, the light chain and the heavy chain variable region sequence coding the mouse antibody were spliced into a nucleotide sequence coding scFv by overlap extension splicing PCR, and the nucleotide sequence was subjected to enzyme digestion with self-made restriction endonuclease Sfil and then connected to phage vector pComb3x. The phage display scFv antibody library of the immunized mouse was constructed by electrotransformation of X-Blue competent cells. The library capacity (clone number * dilution factor * culture volume) and the sequence insertion accuracy were calculated.

[0045] The RNA extraction electropherogram of the spleen of the mouse SBI240422-2D is shown in Figure 1 The RNA extraction electropherogram of the spleen of the mouse SBI240422-2D is shown in Figure 2 The VHand VLwere spliced into scFv, and the electropherogram of the spliced fragment is shown in Figure 3 The scFv was connected to the phage vector, and the connection result is shown in Figure 4 The library capacity of the constructed phage antibody library was 5.39*10 9 cfu.

[0046] Table 2: UV quantitative results of RNA

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

[0048] (2) Screening of phage antibody library

[0049] After the primary antibody library was infected with helper phage, the original antibody library was prepared. The antigen was coated on an ELISA plate, the original antibody library was added for binding, and PBST was used for washing to remove non-specific binding. Then, the phage bound to the antigen was eluted with an elution solution, amplified in E. coli, and precipitated for the next round of enrichment screening. After 2-4 rounds of “adsorption-elution-amplification”, the phage library enriched with antigen-positive antibodies was obtained. Single clone phage was picked from the enriched library for expression, and the binding with the antigen was detected by ELISA. High-binding force antibody clones specifically binding to the antigen were screened, and the nucleotide sequence was obtained by sequencing by a sequencing company.

[0050] After 3 rounds of screening, the binding of the phage library with the antigen (SARS-CoV-2 JN.1 (Omicron) Spike S1+S2 trimer Protein) was detected by ELISA, and the detection results are shown in Table 3. The ELISA detection results showed that the positive library obtained after 3 rounds of screening was bound to the antigen protein and did not cross with the control protein. Single clone phage was picked from the enriched positive phage library for expression and ELISA identification.

[0051] Table 3 Bacterial body positive library ELISA detection data

[0052]

[0053] Construction of antibodies of Example 4

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

[0055] The light and heavy chain variable region fragments of the scFv antibody were amplified by PCR using self-designed primers, and the heavy and light chain variable region fragments were added to the membrane binding solution (gel recovery kit components), mixed thoroughly, and then added to a nucleic acid purification column. After centrifugation at 13000 rpm for 1 min, the liquid was discarded, 700 μL of rinse solution was added, and centrifugation was performed at 13000 rpm for 1 min. After discarding the liquid, the column was emptied for 5 min, and then left at room temperature for 5 min. Nuclease-Free Water was added for elution, and the purified heavy and light chain variable regions were obtained.

[0056] (2) Cloning and construction

[0057] After purification, 4 μL of the fragment and 1 μL of the vector were mixed thoroughly after adding 5 μL of ligase, and then incubated at 50°C for 20 min. The mixture was then added to 100 μL of competent cells, which were incubated on ice for 30 min, and then heat shocked at 90s. The mixture was quickly placed on ice for 3 min, and then plated and incubated at 37°C overnight. The obtained monoclonal was amplified and sequenced, and the heavy and light chain sequences of the antibody were obtained. The heavy and light chain plasmids of the antibody were paired and delivered to HEK293 for transient expression.

[0058] The ELISA data of the monoclonal phage selected from the positive phage library after expression and detection are shown in Table 4. The following clones have good binding with the antigen protein and no cross with the His control protein.

[0059] Table 4 ELISA detection data of monoclonal phage expression

[0060]

[0061]

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

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

[0064] SEQ ID NO 1:

[0065] MGWSCIILFLVATATGVHSDIQMTQSSSFLSVSLGGRVTIACKASDHINSYLAWYQQKPGNAPRLLISGAT

[0066] SLETGIPSRFSGSGSGKDYTLTITSLQTEDFATYYCQQYWDTPPTFGGGTKLEIKRADAAPTVSIFPPSSEQL

[0067] TSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC-

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

[0069] SEQ ID NO 2:

[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 NO 3:

[0078] MGWSLILLFLVAVATRVLSEVQLQQSGPELVRPGASVKLSCKASGYIFITYWMNWVKQRPGQGLEWIGQIF

[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 with 293 serum-free CD medium, and the plasmid DNA to be expressed was mixed with transfection reagent TF2 and added to the cells. The 293 serum-free feeding solution was added on the 1st, 3rd and 5th day after transfection. The shake flask culture conditions were 5% CO2, temperature 37°C, and shaking speed 175 rpm.

[0092] Example 6 Monoclonal antibody purification

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

[0094] 2. Antibody purification: The centrifuged cell culture supernatant was purified using a protein A affinity purification method, and the mouse monoclonal antibody was harvested. The operation steps are as follows:

[0095] (1) Connection system: Select the appropriate size of the protein A column according to the expression amount, and connect it with the purification system;

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

[0097] (3) Equilibrium chromatography column: Equilibrate 3 CV of AC Binding (50 mM Tris, 100 mM NaCI, pH 8.0) to the UV baseline stable;

[0098] (4) Sample loading: Adjust the appropriate flow rate for sample loading, generally 1 / 3 column volume flow rate;

[0099] (5) Elution: Elute with 5-10 CV of elution liquid until the UV baseline is stable, and the flow rate is the same as that of sample loading;

[0100] (6) Elution: Elute with AC Elution elution liquid, and collect according to the UV peak;

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

[0102] (8) Equilibrium: Equilibrate 3 CV of AC Binding (50 mM Tris, 100 mM NaCI, pH 8.0) to neutral;

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

[0104] (10) Rinse with base: rinse the column with equilibration buffer until the pH at the outlet is neutral;

[0105] (11) Storage: store the column in 25% ethanol equilibration buffer 2 CV.

[0106] Example 7 Antibody concentration and purity detection

[0107] Start the micro-spectrophotometer, and use phosphate buffer to point sample. When the absorbance value at 280 nm wavelength is between ±0.015, it indicates that the instrument baseline is stable. Then, sequentially point the purified antibody sample, record the OD280 and OD320 absorbance values, and detect the value of (OD280-OD320) divided by the IgG extinction coefficient (1.414). The obtained value is the concentration (unit: mg / mL) of the detection sample.

[0108] Use sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 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 electrophoresis gel according to their different molecular weights, thereby verifying the purity of the target antibody. Take 5 μg of the detection sample and add 5 μL of 4R / 4N, heat in a 100°C water bath, and then centrifuge at 10000 rpm. Add enough 1x 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 marker (Thermo Scientific, #26610) to the sample well. Connect the power supply, first electrophorese at a constant voltage of 100 V until the bromophenol blue dye enters the separation gel from the concentrated gel, then adjust the current to 140 V and continue electrophoresis to the bottom of the gel plate, and turn off the power. Soak the gel in the staining solution, heat in the microwave oven for 60 s, and place on a gently shaking platform at room temperature for staining. Replace the decolorizing solution to cover the gel, heat in the microwave oven for 60 s, and place on a gently shaking platform at room temperature for decolorization. Repeat the decolorization operation until a blue band and clean background are obtained.

[0109] Example 8 ELISA binding detection

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

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

[0112] (3) Wash the plate: 300 μL / well of washing solution, wash the plate twice, and dry at the end;

[0113] (4) Antibody dilution: GZZ2 antibody (GZZ2-M015, GZZ2-M120) was diluted to 1 pg / mL, 100 pL was added to the corresponding well plate, mixed well, and reacted at room temperature for 2 h;

[0114] (5) Washing plate: 300 pL / well washing solution, washing plate 3 times, and the last one was patted dry;

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

[0116] (7) Washing plate: 300 pL / well washing solution, washing plate 3 times, and the last one was patted dry;

[0117] (8) Color development: mix A and B liquids in a ratio of 1:1, add 200 pL per well, and incubate at room temperature for 20 min in the dark;

[0118] (9) Termination: add 50 pL of termination solution per well, and immediately measure the OD450 value at 450 nm wavelength.

[0119] The purified antibody ELISA detection results are shown in Table 5.

[0120] Table 5 Purified antibody ELISA detection results

[0121]

[0122] Example 9 Purified antibody labeling

[0123] 1 Activated HRP

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

[0125] 2) According to the volume of the HRP solution, weigh the appropriate amount of sodium periodate (Shenguo Biotechnology Lot: AA27BA0032) in a ratio of 1:1-2;

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

[0127] 4) Replace the solution with an ultrafiltration centrifuge tube to store the activated HRP in 0.01 M PBST solution at pH 7.4, replace 2-3 times (original buffer residue <1%);

[0128] 5) Adjust the HRP to the appropriate concentration, aliquot, and store for future use.

[0129] 2 Coupling of antibody with HRP

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

[0131] 2) After diluting the antibody: HRP at a ratio of 1:20000, shake table reaction at room temperature for 1 h in the dark, and reserve;

[0132] 3) Add 3% sodium borohydride solution (freshly prepared), 4°C, avoid light, react for 1 h;

[0133] 4) Add 10% saturated ammonium sulfate, 4°C, avoid light, stand still;

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

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

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

[0137] 8) Select the appropriate dilution factor according to 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 Results of HRP labeling of purified antibodies

[0140]

[0141]

[0142] Example 10 Pairwise detection

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

[0144] 2) Blocking: Shake off the liquid in the plate, pat dry, 2% BSA, 300 μL / well, seal after incubation at room temperature for 1 h;

[0145] 3) Wash the plate: 300 μL / well of washing solution (0.01M pH 7.4 PBST solution), wash the plate twice, and pat dry at the end;

[0146] 4) antigen dilution: antigen 40589-V08H59 was diluted (diluent is 0.1% BSA-PBST pH 7.4) to 1 ng / mL and 10 ng / mL, blank control is sample diluent (diluent is 0.1% BSA-PBST pH 7.4), 100 μL each was added to the corresponding well plate, mixed well, and reacted at room temperature for 2 h;

[0147] 5) plate washing: 300 μL / well washing solution (0.01M pH 7.4 PBST solution), plate washing 3 times, the last time was patted dry;

[0148] 6) addition of secondary antibody: dilute project GZZ2 labeled antibody (GZZ2-M015-HRP) to the use concentration (0.5 μg / mL), 100 μL / well, mix well, incubate at room temperature for 1 h;

[0149] 7) plate washing: 300 μL / well washing solution (0.01M pH 7.4 PBST solution), plate washing 3 times, the last time was patted dry;

[0150] 8) color development: mix color A liquid (DH2) and color B liquid (TMB) liquid at 1:1, add 200 μL per well, incubate at room temperature for 20 min in the dark;

[0151] 9) termination: add 50 μL termination liquid (2M sulfuric acid) per well, immediately measure OD450 value at 450 nm wavelength.

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

[0153] Table 7 Antibody pairing detection results

[0154]

[0155] Example 11 Establishment of ELISA reagent detection

[0156] 1) coating: coat GZZ2-M120 antibody 2 μg / mL, 100 μL / well, 4°C coating overnight;

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

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

[0159] 4) drying: shake off the liquid in the plate and pat dry, dry, bag, seal;

[0160] 5) plate washing: 300 μL / well washing solution (0.01M pH 7.4 PBST solution), plate washing 2 times, the last time was patted dry;

[0161] 6) Standard Graded Dilution: Take 5 μL of stock 40589-V08H59 protein into 495 μL of 1x sample diluent labeled tube A, take 100 μL of tube A into 900 μL of 1x sample diluent labeled tube B, take 2.27 μL of stock into 997.73 μL of 1x sample diluent to make 1000 μL of 6000 pg / mL highest concentration standard (OD value at the top of the curve around 2.5 is the standard for judgment). Use 500 μL of 1x sample diluent as diluent, make six two-fold serial dilutions of the 6000 pg / mL highest concentration standard in six separate test tubes: after mixing the 6000 pg / mL highest concentration standard, take 500 μL of pipette into the next test tube, and so on. Use 1x sample dilution buffer as zero standard (0 pg / mL). Mix well, react at room temperature for 2 h (the diluent used in this step is 0.1% BSA-PBST pH 7.4);

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

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

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

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

[0166] 11) Termination: add 100 μL of termination solution (2M sulfuric acid) per well, immediately measure the OD value at 450 nm wavelength. The results of performance verification of ELISA reagent detection are shown in Table 8

[0167] Table 8 Results of performance verification of ELISA reagent detection

[0168]

[0169]

[0170] Conclusion:

[0171] Immunize 5 mice with the protein (Cat: 40589-V08H59), after five immunizations, the ELISA binding titers of 4 mice with the immunogen all meet the qualified standard, select SBI240422-2D mouse to construct phage library. The phage library is successfully constructed, and the library capacity is 5.39x109cfu. After three rounds of screening, a positive library that binds to GZZ2 protein and does not cross with the control protein is obtained. Single clones are picked and identified, and the positive clones are passed to the downstream expression. After 293 cell transient expression and expression supernatant purification, two strains of purified antibodies are obtained. ELISA detection of antibody and antigen binding showed that the two antibodies were bound to the antigen protein. The two antibodies were labeled with HRP and then paired for detection, and were coated into a kit. The detection sensitivity was 6.8 pg / mL.

Claims

1. A single-chain antibody of the S protein of the SARS-CoV-2 variant JN.1, characterized in that: The full-length amino acid sequence of the light chain of the single-chain antibody is shown as SEQ ID NO. 1, and the full-length amino acid sequence of the heavy chain of the single-chain antibody is shown as SEQ ID NO.

2.

2. The single-chain antibody of the S protein of the JN.1 spike protein of the novel coronavirus mutant strain according to claim 1 is used for preparing an ELISA kit for detecting the S protein of the JN.1 spike protein of the novel coronavirus mutant strain.

3. Use according to claim 2, characterized in that: The ELISA kit further comprises a detection antibody, and the full-length amino acid sequence of the light chain of the detection antibody is shown as SEQ ID NO. 3, and the full-length amino acid sequence of the heavy chain of the detection antibody is shown as SEQ ID NO. 4.

Images