Construction methods of Protein A phage display antibody libraries, Protein A antibodies and their applications

Abstract

The application provides a construction method of a Protein A immunized phage display antibody library, and the steps comprise: (1) immunizing a cock with a Protein A protein, extracting total RNA of lymphocytes, and reversely transcribing the total RNA into cDNA; (2) amplifying heavy chain variable region and light chain variable region fragments and splicing into scFv genes by using a PCR technology; (3) constructing a pCANTAB5E-2sfi1-scFv recombinant plasmid, electrically transforming the recombinant plasmid into TG1 competent cells, and constructing a phage single-chain antibody library; and (4) specifically enriching the phage single-chain antibody library, and screening positive clones from the phage single-chain antibody library. The application screens a chicken-derived single-chain antibody with high affinity by using a phage display antibody library technology, converts the single-chain antibody into an IgY form, provides a development method for antibody raw materials of a Protein A immunized detection kit, and screens 13 monoclonal antibodies against Protein A by using a phage display technology. Two of the 13 monoclonal antibodies are screened out and used for double-antibody sandwich ELISA pairing (IgY-80 and IgY-81), and the ELISA has high sensitivity and strong specificity.

Description

Technical Field

[0001] This invention patent relates to a method for constructing a Protein A immune phage display antibody library, a Protein A monoclonal antibody and its application, and a Protein A detection kit containing the antibody, belonging to the field of bioengineering technology. Background Technology

[0002] Staphylococal Protein A (SPA), also known as Protein A, is a cell wall protein of Staphylococcus aureus with a molecular weight of 42 kDa. It specifically binds to the Fc region of human and mammalian antibodies (mainly IgG) and is commonly used in large-scale antibody production to purify IgG. Protein A is classified into natural Protein A, natural recombinant Protein A, and non-natural recombinant Protein A. These Protein A proteins are typically coupled with agarose, resin, etc., to prepare affinity chromatography columns. In antibody drug production, Protein A is inevitably eluted along with the target antibody protein during downstream affinity chromatography, resulting in Protein A residue in the final purification step. This impurity can severely affect product quality, efficacy, and safety.

[0003] WHO, ICH (International Council for Harmonisation of Pharmaceutical Registration Standards for Human Use), Chinese Pharmacopoeia, USP <130> All of these publications set limits on the residual amount of Protein A. The 2020 edition of the *Pharmacopoeia of the People's Republic of China*, Volume III, under Section 3.2.4 "Process-Related Impurities" of the "General Treatise on Recombinant Monoclonal Antibody Products for Human Use," states that "appropriate methods should be used to detect host proteins, host cell and vector DNA, Protein A, and other process-related impurities in the test sample." The *Nimotuzumab Injection*, under Section 3.1.3.3 "Protein A Residual A," states that "the residual amount of Protein A should not exceed 0.001% of the total protein content, determined by enzyme-linked immunosorbent assay (General Rule 3429)." The 2005 *General Principles for the Review of Analytical Method Validation Technology for Quality Control of Biological Products* issued by the Center for Drug Approval recommends that ELISA be used to detect the residual amount of Protein A in fusion proteins and monoclonal antibody products.

[0004] Because Protein A is an essential part of quality control in biopharmaceutical manufacturing, and residue detection is required in process research and final product quality control, it is necessary to develop a Protein A detection kit based on the ELISA method. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a Protein A immune phage display antibody library.

[0006] A method for constructing a Protein A phage display antibody library, characterized by the following steps: (1) Immunize roosters with Protein A, extract total RNA from lymphocytes, and reverse transcribe it into cDNA; (2) The heavy chain variable region and light chain variable region fragments were amplified by PCR and spliced ​​into the scFv gene; (3) Construct the pCANTAB5E-2SFi1-scFv recombinant plasmid, electroporate it into TG1 competent cells, and construct a phage single-chain antibody library; (4) Specific enrichment of phage single-chain antibody library, from which positive clones are screened; (5) The positive single-chain antibody was converted into IgY antibody and expressed in a eukaryotic expression system; (6) Purification of IgY antibody (7) IgY antibody is used as a raw material in ELISA detection kits IgY-VL-F TCGGGTGGCGGTGGATCCGGCGGTGGCGGTTCGGCCGTGACGTTGGACGAGGCCTGACTCAGCCGTCGTCGGTGTC IgY-sfi1-VL-R GGCCAGATCGGCCTAGGACGGTCAGGGTYG IgY-sfi1-VH-F GGCCCAGCCGGCCGCCGTGACGTTGGACGAG IgY-VH-R CACCGCCACCCGAGCCACCGCCACCGGAGGAGACGATGACTTCGGTCC In step (2), the upstream primer IgY-sfi1-VH-F and the downstream primer IgY-VH-R of the heavy chain VH are mixed for PCR, and the upstream primer IgY-VL-F and the downstream primer IgY-sfi1-VL-R of the light chain VL are mixed for PCR. The obtained PCR products of the light chain VL and the heavy chain VH are recovered and mixed in equal amounts as templates. The corresponding upstream primer of the heavy chain and the downstream primer of the light chain in the primer table are used for PCR.

[0007] In step (4), the specific enrichment of the phage single-chain antibody library refers to taking the antigen with PBS, mixing it well, coating it with a 96-well plate, using 5% BSA as the blocking solution, adding the phage single-chain antibody library, and incubating at room temperature. Elution: Add 100 mM HCl, incubate at room temperature for 5 min, then collect by forcefully blowing with a pipette tip. Add 1 M Tris-HCl to the collection tube to neutralize the pH, and then add 1% BSA solution to dilute. Elution product amplification: The elution product was added to TG1 bacterial culture and cultured. M13KO7 helper phage was added and cultured for a longer period. The bacterial culture was centrifuged, and the precipitate was resuspended in 2×YT medium and cultured overnight with shaking. The next day, the bacterial culture was centrifuged, and the supernatant was transferred to a new centrifuge tube. PEG-NaCl was added, and the mixture was allowed to stand on ice to precipitate. After centrifugation, the supernatant was removed, and the precipitate was resuspended in 1% BSA solution. The precipitate was transferred to another EP tube, centrifuged, and the precipitate was removed. The supernatant was the amplified and enriched product. Repeat the above steps for four rounds of screening, increasing the rigor of each round to enrich phages with affinity.

[0008] IgY-pVH-F: GCACTTGTCACGAATTCGGGCCGTGACGTTGGACGAGTCC IgY-pVH-R: GGAGGAGACGATGACTTCGGT IgY-pVL-F: GCACTTGTCACGAATTCACAGGCAGCGCTGACTCAGCCG IgY-pVL-R: TAGGACGGTCAGGGTTGTCCCGG In step (5), converting the single-chain antibody into IgY specifically means cloning the light and heavy chain antibody genes of the single-chain antibody into the pfuse expression vector (which has the heavy chain and light chain constant region genes of IgY pre-inserted) to construct a complete IgY heavy chain and light chain expression vector.

[0009] The present invention also discloses an IgY monoclonal antibody against Protein A, named IgY-80 and IgY-81 respectively.

[0010] Protein A antibody IgY-80 has the following amino acid sequence: The heavy chain variable region sequence is as follows: AVTLDESGGGLQTPGGTLSLLVCKASGFTFSDRGMHWVRQAPGKGLEWVAGTYTSGSITYYAPAVNGRATISRDNGQSTLRLQLNNLRAEDSGTYYCARDFGSGWNAGSIDAWGHGTEVIVSS (SEQ ID No. 1), The amino acid sequence of the heavy chain variable region CDR1 is: DRGMH. The amino acid sequence of the heavy chain variable region CDR2 is: GTYTSGSITYYAPAVNG. The amino acid sequence of the heavy chain variable region CDR3 is: DFGPSGWNAGSIDA.

[0011] The light chain variable region sequence is as follows: QAALTQPSSVSANLGGTVKITCSGGGSRYGYMYGWIQQTSPGSAPVTVIYDNTNRPSNIPSRFSGSLSGSTNTLTITGVQAEDEAVYFCGSYEGTTLTFGAGTTLTVL (SEQ ID No. 2), The amino acid sequence of the CDR1 variable region of the light chain is: SGGGSRYGYMYG. The amino acid sequence of the light chain variable region CDR2 is: DNTNRPS. The amino acid sequence of the CDR3 variable region of the light chain is: GSYEGTTLT.

[0012] The amino acid sequence of Protein A antibody IgY-81 is as follows: The heavy chain variable region sequence is as follows: AVTLDESGGGLQTPGGALSLVCKASGFTFSSNGMGWVRQAPGKGLEYVASISNDGSLTDYASPVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKSPGGGWLADPIDAWGRGTEVIVSS (SEQ ID No. 3), The amino acid sequence of the heavy chain variable region CDR1 is: SNGMG. The amino acid sequence of the heavy chain variable region CDR2 is: SISNDGSLTDYASPVKG. The amino acid sequence of the heavy chain variable region CDR3 is: SPGGGWLADPIDA; The light chain variable region sequence is as follows: QAALTQPASVSANPGETVQITCSGGGSSRYYGWYQQKSPGSAPVTLIYDNINRPSGIPSRFSGSTSGSTGTLSITGVRAEDEAVYYCGSFDSSGTGIFGAGTTLTVL (SEQ ID No. 4), The amino acid sequence of the CDR1 variable region of the light chain is: SGGGSSRYYG. The amino acid sequence of the light chain variable region CDR2 is: DNINRPS. The amino acid sequence of the CDR3 variable region of the light chain is: GSFDSSGTGI.

[0013] This invention also discloses the application of the above-mentioned Protein A antibody IgY-80 and Protein A antibody IgY-81 in the detection of Protein A in samples by ELISA.

[0014] The present invention also discloses a Protein A detection kit containing the aforementioned Protein A antibody IgY-80 and / or Protein A antibody IgY-81.

[0015] The method for constructing a Protein A phage display antibody library of the present invention can screen for chicken single-chain antibodies with high affinity and can construct a library with a capacity of 9.0 × 10⁻⁶. 9 The recombinant phage titer was 1.0 × 10⁻⁶. 13 A phage single-chain antibody library with pfu / ml and a recombination rate of 92.3%. The single-chain antibodies of this invention can be obtained through expression in *E. coli* BL21. The IgY antibody of this invention can be obtained through the 293F expression system. Attached Figure Description

[0016] Figure 1 This is an electrophoresis diagram of the extracted total RNA.

[0017] Figure 2 This is a gel electrophoresis image of the PCR products of the light chain VL and heavy chain VH genes.

[0018] Figure 3 These are the results of the IgY-80 affinity test.

[0019] Figure 4 These are the results of the IgY-81 affinity test.

[0020] Figure 5 This is a gel electrophoresis image of the PCR product of the scFv gene.

[0021] Figure 6 These are electrophoresis images of the pCANTAB5E-2sfi1 plasmid before and after double enzyme digestion.

[0022] Figure 7 It is a flat plate diagram of the electroconversion of the connected products.

[0023] Figure 8 This is a colony PCR identification image.

[0024] Figure 9 This is a purification diagram of the recombinant protein IgY.

[0025] Figure 10 This is the standard curve of the detection sensitivity of the Protein A detection kit for Protein A. Detailed Implementation

[0026] 1. Biomaterials Rooster, Brilliant Breeding Farm 2. Experimental reagents and consumables Protein A Shanghai Sangon Biotech C610039 Freund's complete adjuvant Sigma F5881 Freund's incomplete adjuvant Sigma F5506 PEG8000 Sigma P5413 Ultrapure RNA extraction kit (DNase I) Kangwei Century CW0597S PCR amplification primers Shanghai Sangon Biotech SanPrep Column DNA Gel Recovery Kit Omega D2500 Plasmid miniprep kit Omega D6943 PCR product purification kit Shanghai Sangon Biotech B110093 20× PBS Shanghai Sangon Biotech B548117 Tween 20 Shanghai Sangon Biotech A100777 Poly(Vinyl Alcohol) Shanghai Sangon Biotech A502784-0250 Sfi1 restriction endonuclease JapanTakara 1244A M13KO7 helper phage UK NEB N0315S Escherichia coli (E. coli) TG1 Electro competent cells Shanghai Weidi DE1055 E. coli DH5α competent cells Shanghai Weidi DL1001 pCANTAB5E phage vector Shanghai Kelei Biotechnology KL-ZL3112 HB2151 Escherichia coli host bacteria Beijing Serrui PB0221 PrimeSTAR® GXL DNA Polymerase JapanTakara R050A Anhydrous ethanol Sinopharm 10009218 chloroform Sinopharm 10006818 Glycerol Sinopharm 10010618 Carbenicillin sodium Solarborg 4800-94-6 Kanamycin sulfate YEASEN 60206ES10 DEPC-treated Water YEASEN 10601ES76 50x TAE buffer YEASEN 60116ES76 Agarose YEASEN 10208ES60 5000bp DNA marker YEASEN 10504ES60 YeaRed Nucleic Acid Gel Stain YEASEN 10202ES76 Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix Component YEASEN 11119ES60 T4 ligase YEASEN 10300ES80 Bovine serum albumin YEASEN 36101ES25 Sodium chloride YEASEN 60372ES76 TMB colorimetric solution YEASEN 36602ES60 Example 1: Construction of a phage display antibody library 1.1 Extraction of total RNA from animal immune cells and spleen cells

[0027] Approximately 2 kg of roosters were immunized subcutaneously with Protein A every two weeks. For the initial immunization, Protein A was emulsified with Freund's complete adjuvant; for booster immunizations, it was emulsified with incomplete adjuvant. The initial immunization dose was 0.5 mg of protein, followed by 0.25 mg for each subsequent immunization. Two months after immunization, three days before spleen harvesting, a 0.25 mg protein was administered subcutaneously as a pulse immunization. Three days after the pulse immunization, 30-50 mg of spleen tissue was harvested. Total RNA was extracted using a DNase I ultrapure RNA extraction kit. The sample was thoroughly homogenized in liquid nitrogen with 1 ml of TRIzon Reagent added, or 1 ml of TRIzon Reagent was added to the spleen tissue and homogenized. After adding TRIzon Reagent to the sample, the sample was repeatedly pipetted several times to ensure complete lysis. The sample was incubated at room temperature for 5 min to allow complete separation of the protein and nucleic acid complexes. Chloroform was added at a ratio of 200 μl of chloroform per 1 ml of TRIzon Reagent, the tube was capped, and the tube was vigorously shaken for 15 s. The sample was then incubated at room temperature for 2 min. Centrifuge at 12,000 rpm (~13,400×g) at 4℃ for 10 min. At this point, the sample will be divided into three layers: a red organic phase, a middle layer, and a colorless aqueous phase on top. The RNA is mainly in the upper aqueous phase. Transfer the upper aqueous phase to a new RNase-Free centrifuge tube (provided by the customer).

[0028] Add an equal volume of 70% ethanol (prepared with RNase-free water), approximately 600-700 μl, to the obtained aqueous solution and mix thoroughly by inverting. Add the entire solution obtained in the previous step to the adsorption column (Spin Columns RM) already loaded into the collection tube. If the solution cannot be added all at once, it can be added in multiple batches. Centrifuge at 12,000 rpm for 20 s, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Add 350 μl of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 20 s, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Prepare the DNase I mixture: Take 52 μl of RNase-Free Water, add 8 μl of 10× Reaction Buffer and 20 μl of DNase I (1 U / μl), mix well, and prepare a reaction solution with a final volume of 80 μl. Add 80 μl of DNase I mixture directly to the adsorption column and incubate at 20-30℃ for 15 min. Add 500 μl of Buffer RW2 (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 20 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Centrifuge at 12,000 rpm for 2 min and discard the waste liquid in the collection tube. Let the adsorption column air dry completely at room temperature for 3 min. Place the adsorption column in a new RNase-free centrifuge tube, add 50 μl of RNase-Free Water to the middle of the adsorption column, incubate at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min, collect the RNA solution, and run 1 μl of the sample for electrophoresis to detect the extraction effect. The results are as follows. Figure 1 The remaining RNA samples were stored at -80°C for later use.

[0029] 1.2 Cloning and splicing of the scFv gene

[0030] This invention utilizes a reverse transcription kit from Yisheng Biotechnology Co., Ltd., using RNA extracted from rooster spleen cells as a template. Following the reverse transcription reaction system shown in Table 2-1, the kit includes RNA template, RNase-free water, and 4×Hifair. ®III. SuperMix, 25 ℃ for 5 min, incubated at 55 ℃ for reverse transcription for 15 min, heated at 85 ℃ for 5 min to obtain cDNA. Primers in Table 2-2 were synthesized by Shanghai Sangon Biotech. Both the light chain variable region gene and the heavy chain variable region gene used chicken cDNA as templates. PCR was performed using a mixture of upstream and downstream primers for the light chain, and a mixture of upstream and downstream primers for the heavy chain. The PCR reaction components are shown in Table 2-3, and the PCR reaction was performed according to the reaction conditions in Table 2-4. The obtained PCR products of the light chain VL and heavy chain VH were loaded onto a 1.2% agarose gel, and the amplification results were observed by electrophoresis. The results are shown below. Figure 2 The product was recovered using the OMEGA gel recovery kit following the kit's experimental procedures, and stored at -20°C.

[0031] The recovered heavy and light chain genes from the previous step were mixed in equimolar amounts and used as a template. PCR was performed using the corresponding upstream primer for the heavy chain VH gene and the downstream primer for the light chain, and the resulting fragments were spliced ​​into scFv. The PCR reaction system is shown in Table 2-5, and the reaction conditions were the same as those for VH gene amplification in Table 2-4. After the reaction, the samples were loaded and detected by agarose gel electrophoresis. The results are shown below. Figure 5 The product was recovered using the OMEGA gel recovery kit following the kit's experimental procedures, and stored at -20°C.

[0032] RNase-free ddH2O 14 μl 4×Hifair® Ⅲ SuperMix 5 μl Total RNA (1128 ng / μl) 1 μl IgY-VL-F TCGGGTGGCGGTGGATCCGGCGGTGGCGGTTCGGCCGTGACGTTGGACGAGGCCTGACTCAGCCGTCGTCGGTGTC IgY-sfi1-VL-R GGCCAGATCGGCCTAGGACGGTCAGGGTYG IgY-sfi1-VH-F GGCCCAGCCGGCCGCCGTGACGTTGGACGAG IgY-VH-R CACCGCCACCCGAGCCACCGCCACCGGAGGAGACGATGACTTCGGTCC Y represents C / T Prime STAR GXL Premix (2x) 25 μl Primer F (10 μM) 1 μl Primer R (10 μM) 1 μl Template (cDNA) 1 μl Sterilized water 22 μl Total 50 μl VL 98℃ 10s 55℃ 15s 68℃ 42s 4℃ 30 VH 98℃ 10s 60℃ 15s 68℃ 42s 4℃ 30 Prime STAR GXL Premix (2×) 25 μl Primer F (10 μM) 1 μl Primer R (10 μM) 1 μl Template (VH+VL) 2 μl Sterilized water 21 μl Total 50 μl 1.3 Construction of pCANTAB5E-2sfi1-scFv recombinant plasmid

[0033] pCANTAB5E-2sfi1 was transformed into DH5α and amplified to prepare plasmids. Following the restriction enzyme digestion system of Takara Pharmaceuticals (Japan), the pCANTAB5E-2sfi1 plasmid and scFv fragment were digested with Sfi1, as shown in Table 2-6 below. The mixture was thoroughly mixed and placed in a 50℃ water bath for 2 h. After digestion, the pCANTAB5E-2sfi1 plasmid was observed by 1.2% agarose gel electrophoresis. The results are shown below. Figure 6 The product was recovered using an OMEGA gel extraction kit. The enzyme-digested scFv gene fragment and pCANTAB5E-2sfi1 plasmid were ligated using YEASEN T4 ligase according to the reaction system in Table 2-7, and ligated at 20°C for 2 hours.

[0034] 10×M buffer 16μl Sfi1 enzyme 8 μl pCANTAB5E-2sfi1 / scFv 8 μg Sterilized water Add to 160 μl 10× T4 DNA Ligase buffer 80 μl T4 DNA Ligase 80μl pCANTAB5E-2sfi1(S) 4 μg ScFv(S) 2 μg Sterilized water Add to 800 μl 1.4 Determination of TG1, library capacity, and recombination rate of the ligation product conversion competent cells

[0035] After thawing TG1 competent cells on ice, 1 μl of pCANTAB5E-2sfi1-scFv ligation product was added and gently mixed. The mixture was then poured along the tube wall into an ice-cold electroporation cuvette. After incubation for 5-10 min, the cuvette was placed in an electroporator. The voltage was set to 2.5 kV, and the electroporation time was maintained at a constant voltage for approximately 5.0 ms. Immediately after electroporation, 1 ml of preheated SOC liquid medium was added for resuspending. The cells were then activated and cultured at 37°C with shaking for 1 h. In this experiment, 20 tubes were transformed in parallel, with 1 μl of cells per tube, for a total of 20 transformations, yielding 20 ml of transformed competent cells.

[0036] After transformation and resuscitation, 100 μl of the bacterial culture was mixed and diluted 10-fold serially. Another 100 μl of each diluted culture was then plated onto a 2×YT-C plate (containing 50 mg / ml Carb). The plate was incubated overnight at 37°C with the plate inverted. The library volume was calculated, and the transformation results of the ligation product were as follows: Figure 7 The following day, 50 single colonies from well-dispersed dilution plates were picked and incubated overnight at 37°C with shaking at 200 rpm / min in 2×YT-C (containing 50 mg / ml Carb) liquid medium with good dispersion. 1 μl of bacterial culture from each of the 50 expanded single colonies was used as a template for PCR according to the scFv gene splicing PCR system. The PCR products were analyzed by electrophoresis and gel imaging, and the recombination rate of the antibody library was calculated. The PCR identification results are as follows: Figure 8 .

[0037] Calculation formula: Storage capacity = Number of single colonies × Dilution factor × Total volume (Formula 1-1) Recombination rate (%) = positive clones / number of single clones picked (Formula 1-2).

[0038] 1.5 Construction of phage scFv antibody library

[0039] 1.5.1 Assisting in the preparation of bacteriophage strains The overnight cultured TG1 bacterial suspension was inoculated into 5 ml of SOB medium at a ratio of 1:100 and cultured at 37°C with shaking at 200 rpm until the OD600 reached 0.4-0.6. Then, 5 μl of M13KO7 helper phage was added, and the suspension was cultured at 37°C with shaking at 200 rpm for 1 h. Next, 5 μl of kanamycin (50 mg / ml) was added, and the suspension was cultured at 37°C with shaking at 200 rpm for 1 h. The cultured bacterial suspension was then transferred to 100 ml of 2×YT liquid medium, and kanamycin (50 mg / ml) was added to a final concentration of 50 μg / ml. The suspension was then cultured overnight at 37°C with shaking at 200 rpm. The following day, the overnight culture was aliquoted into 50 ml centrifuge tubes and centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was transferred to a sterile 500 ml Erlenmeyer flask, and PEG / NaCl (1 / 5 volume of the initial culture, 20 ml in this operation) was added. After mixing, the mixture was aliquoted into new 50 ml centrifuge tubes and precipitated on ice for 2 h. After centrifugation at 12,000 rpm for 10 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in 2 ml (1 / 50 volume) of 1×PBS. The precipitate was transferred to a sterile centrifuge tube and centrifuged again at 12,000 rpm for 5 min at 4°C. The supernatant was carefully collected, filtered through a 0.22 μM filter for sterilization, and 50% glycerol was added to the final concentration. The mixture was then aliquoted into sterile centrifuge tubes and stored at -20°C.

[0040] 1.5.2 Assisted phage titration The prepared phage solution was serially diluted 10-fold using 1% BSA (in 1×PBS) as the diluent, and the diluted value was taken. -4 10 -6 10 -8 10 -10 10 -12 10 μl of bacterial suspension was added to TG1 with an OD600 of 0.4-0.6. The diluted solution and TG1 were used as negative controls. The mixture was incubated at 37°C for 30 min, followed by shaking for 30 min. After incubation, the bacterial suspensions of the five dilution gradients and the two negative controls were plated onto a plate containing kanamycin (50 mg / ml) and incubated at 37°C. The next day, the number of clones on the plate was calculated, and the phage titer (pfu / ml) was calculated using the following formula. Generally, a titer of 10-1 is required. 12 Only then can subsequent experiments be conducted. Calculation formula: Phage titer = Number of single clones × Dilution factor × 10 (Formula 1-3).

[0041] 1.5.3 Construction of the primary phage antibody library Take 20 ml of the remaining culture from the transformation and preservation and add it to 380 ml of 2×YT-C (25 μg / ml Carb) medium. Incubate at 37°C and 200 rpm for 1 h using a shaker. Add Carb to a final concentration of 50 μg / ml and continue incubating for another h. Add 10 μl of helper phage M13KO7 to the above culture at a multiplicity of infection (MOI) of 20:1. Gently shake at 37°C and 60 rpm for 30 min to facilitate phage infection, then incubate with shaking for another 30 min. Transfer the culture to a sterile centrifuge tube in a sterile laminar flow hood and centrifuge at 5000 rpm for 15 min. Carefully discard the supernatant. Resuspend the bacterial pellet in 400 ml of 2×YT-CK (50 mg / ml Carb and 50 mg / ml Kan) medium under aseptic conditions and incubate with shaking overnight. The next day, all the overnight bacterial culture was transferred into sterile 50 ml centrifuge tubes and centrifuged at 12,000 rpm at 4°C for 20 min.

[0042] Transfer the supernatant to a new sterile Erlenmeyer flask, add PEG-NaCl (80 ml) to 1 / 5 volume of the initial bacterial culture, and incubate on ice for 1 h to precipitate. Centrifuge at 12000 rpm, 4°C for 20 min, remove the supernatant, and invert the centrifuge tube for 2-5 min to remove as much excess liquid as possible. Resuspend the precipitate in 1% BSA (in 1×PBS) at 1 / 50 volume of the initial bacterial culture, transfer to a 1.5 ml sterile tube, centrifuge at 12000 rpm for 5 min, remove the precipitate, and transfer the supernatant to a new 1.5 ml sterile tube. The supernatant is the primary antibody library; add glycerol to 50% and store at -20°C. Determine the titer of the recombinant phage using the same method as in 1.5.2.

[0043] Implementation Case 2: Phage Antibody Library Screening

[0044] 2.1 Screening of specific antibodies using ELISA plate screening method

[0045] Add 10 μg / ml Protein A to two wells of an ELISA plate, 100 μL per well, and incubate overnight at 4°C. Remove the coating buffer from the 96-well plate. Add 200 μL of 5% BSA to each well. A blank control is also included; add 200 μL of 5% BSA directly to the blank wells as blocking buffer. Incubate on a shaker at room temperature for 2 hours. Remove the BSA blocking buffer by inverting the plate. Wash 10 times with 200 μL PBST. Add 100 μL of phage antibody library to both the antigen and BSA control wells. Incubate on a shaker at room temperature for 2 hours. Discard the antibody library solution. Add 200 μL of PBST to each well and wash 10 times. Pat dry. Add 100 μL of 100 mM HCl to each well. Incubate at room temperature for 5 minutes. Elute the product and neutralize with 40 μL of 1M Tris-HCl. Mix well. Add the eluted product to a container containing 1 mL of HCl. In TG1 (OD600 = 0.4-0.6) bacterial culture medium, incubate at 37℃ and 220 rpm for 1 h. Then, take 20 μL of each solution for appropriate dilution and perform titer experiments on LB agar plates containing Carb resistance. Incubate overnight at 37℃. Finally, add 1 μL of helper phage to each protein tube (ensuring a final concentration of 10). 10 (Use helper phages at pfu / ml), incubate at 37℃, 220rpm for 1 hour; transfer the culture medium from approximately 1 ml of the above EP tube into 50 ml of 2×YT-CK (50 mg / ml Carb and 50 mg / ml Kan) medium, incubate overnight at 37℃, 220rpm, and collect phages the next day according to the library construction method. Repeat the above operation four times. The first round uses the original antibody library solution; the antibody libraries added in the second and third rounds are collected from the overnight culture from the previous day. Obtain the results of the titer experiment in each round and compare whether there is enrichment in each round of screening; verify the success of screening based on the titer results, and then select single clones for PCR colony identification.

[0046] Antigen coating concentration (μg / ml) 10 10 10 10 Number of washes 10 10 10 10 Table 3-2 Results of 4 Rounds of Screening

[0047] 2.2 Gator Prime assay for IgY antibody affinity The purified IgY antibody was used to detect its affinity using Gator Prime. The specific procedure was as follows: Protein A antigen was labeled with biotin, dialyzed overnight, and its concentration was measured. Biotin-Protein A was first immobilized with an SA probe, and then bound to the prepared lysis buffer. The affinity test results for the two positive monoclonal antibodies used in the ELISA kit are shown below. Figure 3 and Figure 4As shown.

[0048] Implementation Case 3: Cis-expression, purification, and identification of IgY antibodies

[0049] The above 13 positive phage clones were sent for sequencing. After obtaining the scFv gene sequence, the CDR1, CDR2, and CDR3 regions were analyzed using Immunoglobulin BLAST and Nucleotide nucleotide BLAST analysis software in NCBI BLAST (Numbering Scheme: Kabat). Based on the scFv sequencing results, an IgY expression vector was constructed. Cis-transformation expression and purification were performed as follows: 3.1 Cell culture and preparation before transfection

[0050] 1) Place HEK-293 cells in a 5% CO2 constant-temperature shaker at 37°C and 120 rpm for incubation. Before passage, perform cell counting and observe cell viability, selecting cells with a density of 3-6 × 10⁶ cells / year. 6 Cells / ml were used for passage culture.

[0051] 2) Cell density and viability need to be determined before transient transfection. To ensure effective transfection, it is recommended to use cells in the exponential growth phase (density approximately 3-6 × 10⁻⁶). 6 Transfection with cells (cells / ml) and cells with a viability greater than 98%; 3) Cells do not need to be centrifuged if the cell density is 4 × 10⁻⁶. 6 If the cell density is >4×10⁶ cells / ml, direct transfection is possible; if the cell density is >4×10⁶ cells / ml, transfection is not possible. 6 If the cell density is 4 × 10⁻⁶ / ml, it needs to be diluted with fresh CD05 culture medium to achieve a cell density of 4 × 10⁻⁶ / ml. 6 cell / ml 4) Place the shake flask in a 5% CO2 constant temperature shaker and incubate at 37℃ and 120rpm for 10 minutes before starting transfection.

[0052] 3.2 Transient transfection

[0053] 1) Prepare two 15ml sterile centrifuge tubes. Add 5ml of CD05 medium and 250μg of sterile plasmid DNA to one tube and gently pipette to mix. Take the other centrifuge tube, add 5ml of CD05 medium and 750μl of PEI transfection reagent, and gently pipette to mix. 2) Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid and mix by pipetting. 3) Plasmid-vector complex was prepared by standing at room temperature for 10 minutes; 4) Remove the cells from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and return the cells to the CO2 constant temperature shaker for oscillation culture.

[0054] 3.3 Product Expression and Detection

[0055] 1) Transient transfection with CD05 profeed medium can increase the expression level of the product; add it once 24 hours after transfection. 2) Measure the product expression level on day 5 post-transfection; 3) The expression level of recombinant IgY protein reaches its peak around day 5 after transfection, and the supernatant is collected.

[0056] 3.4 Anti-Protein A IgY Purification

[0057] 1) After the instantaneous fermentation broth, centrifuge 4000g and collect the supernatant, then filter through a 0.45um filter membrane; 2) Install the chromatography column and add 1 ml of Protein A packing material; 3) Ultrapure water rinsing packing material 10CV, PBS equilibration packing material 10CV; 4) After filtration, load the supernatant onto the sample, controlling the flow rate at 1 ml / min; 5) Equilibrate with PBS at 10 CV; 6) Elute 15CV with 0.1M Gly, then neutralize the eluent with 1 / 10 1M Tris (pH 8.5).

[0058] Nanodrop OD280 measured protein concentration. The amounts of IgY-6, IgY-14, IgY-27, IgY-33, IgY-34, IgY-53, IgY-66, IgY-71, IgY-80, IgY-81, IgY-91, IgY-94, and IgY-95 were 0.72mg and 0.061 respectively. mg, 0.13 mg, 0.92 mg, 0.47 mg, 0.055 mg. The purification results of IgY are as follows: Figure 9 The IgY antibody was diluted to 1 μg / ml and then serially diluted twofold. Protein A was used to coat the plates, followed by BSA blocking. The diluted IgY samples were then tested. HRP-labeled anti-IgY antibody was used as the secondary antibody. BSA coating and blocking served as controls. The ELISA results are shown in the table below. All 13 IgY antibodies bound to Protein A but not to the BSA control.

[0059] Table 1-10 ELISA Results

[0060] Implementation Case 4: The two IgY antibodies finally selected were paired to form an immunoassay kit. The paired experiment procedure based on the ELISA experimental principle is as follows: 4.1 Coating: The coated antibody IgY-81 was stored in PBS containing 50% glycerol at -20°C. An appropriate amount of antibody was diluted with PBS to 1 μg / mL, and 100 μL was coated per well. The mixture was incubated overnight (16 h) at 4°C and shaken at 100 rpm. After overnight coating, the wells were washed three times with PBST (200 μL per well).

[0061] 4.2 Blocking: Block with 1% BSA, 200 μL / well, react at 37°C for 2 h, wash three times with PBST (200 μL per well) after blocking; Standard addition and incubation: Add 100 μL of Protein A standard (prepared with PBS to concentrations of 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 pg / mL) to each well, with two replicates for each concentration gradient. Incubate at 37°C for 1 h. After incubation, wash 5 times with PBST (200 μL per well).

[0062] 4.3 Biotin-conjugated antibody incubation: Add 100 μL of pre-prepared working concentration (12.5 ng / mL) of IgY-80 (prepared with PBS) to the microplate and incubate at 37°C for 1 h. After incubation, wash 5 times with PBST (200 μL per well).

[0063] 4.4 HRP-conjugated Streptavidin Incubation: Add 100 μL of pre-prepared HRP-conjugated Streptavidin (prepared with PBS, diluted 10,000 times) to the microplate, and incubate at 37°C for 40 min. After incubation, wash 7 times with PBST (200 μL per well).

[0064] 4.5 Color development: 10 min before color development, bring the substrate solution TMB to room temperature, 100 μL / well, and incubate at 37°C in the dark for 15 min.

[0065] 4.6 Termination: Add 50 μL of termination solution (0.5 mol / L sulfuric acid) to each well.

[0066] 4.7 Reading: Read the dual-wavelength absorption values ​​at 450 / 630 nm within 10 minutes.

[0067] After pairing, IgY-80 and IgY-81 were selected as the raw materials for the ELISA kit. The final standard curve is shown below. Figure 10 As shown in the figure. The results show that this antibody pair has extremely high sensitivity for the detection of Protein A protein.

Claims

1. A Protein A antibody IgY-80, characterized in that... Its amino acid sequence is as follows: The heavy chain variable region sequence is as follows: AVTLDESGGGLQTPGGTLSLVCKASGFTFSDRGMHWVRQAPGKGLEWVAGTYTSGSITYYAPAVNGRATISRDNGQSTLRLQLNNLRAEDSGTYYCARDFGSGWNAGSIDAWGHGTEVIVSS, The amino acid sequence of the heavy chain variable region CDR1 is: DRGMH. The amino acid sequence of the heavy chain variable region CDR2 is: GTYTSGSITYYAPAVNG. The amino acid sequence of the heavy chain variable region CDR3 is: DFGPSGWNAGSIDA; The light chain variable region sequence is as follows: QAALTQPSSVSANLGGTVKITCSGGGSRYGYMYGWIQQTSPGSAPVTVIYDNTNRPSNIPSRFSGSLSGSTNTLTITGVQAEDEAVYFCGSYEGTTLTFGAGTTLTVL, The amino acid sequence of the CDR1 variable region of the light chain is: SGGGSRYGYMYG. The amino acid sequence of the light chain variable region CDR2 is: DNTNRPS. The amino acid sequence of the CDR3 variable region of the light chain is: GSYEGTTLT.

2. The application of the Protein A antibody IgY-80 according to claim 1 in detecting Protein A in a sample by ELISA, wherein the application is for non-disease diagnostic purposes.

3. A Protein A antibody IgY-81, characterized in that... Its amino acid sequence is as follows: The heavy chain variable region sequence is as follows: AVTLDESGGGLQTPGGALSLVCKASGFTFSSNGMGWVRQAPGKGLEYVASISNDGSLTDYA SPVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKSPGGGWLADPIDAWGRGTEVIVSS, The amino acid sequence of the heavy chain variable region CDR1 is: SNGMG. The amino acid sequence of the heavy chain variable region CDR2 is: SISNDGSLTDYASPVKG. The amino acid sequence of the heavy chain variable region CDR3 is: SPGGGWLADPIDA; The light chain variable region sequence is as follows: QAALTQPASVSANPGETVQITCSGGGSSRYYGWYQQKSPGSAPVTLIYDNINRPSGIPSRFSG STSGSTGTLSITGVRAEDEAVYYCGSFDSSGTGIFGAGTTLTVL, The amino acid sequence of the CDR1 variable region of the light chain is: SGGGSSRYYG. The amino acid sequence of the light chain variable region CDR2 is: DNINRPS. The amino acid sequence of the CDR3 variable region of the light chain is: GSFDSSGTGI.

4. The application of the Protein A antibody IgY-81 according to claim 3 in the detection of Protein A in a sample by ELISA, wherein the application is for non-disease diagnostic purposes.

5. A Protein A detection kit, characterized in that... Contains Protein A antibody IgY-80 as described in claim 1 and / or Protein A antibody IgY-81 as described in claim 3.

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