A silica-binding protein for directional fixation of antibodies and its preparation method and application

Abstract

The present invention discloses a silica-binding protein for directional fixation of antibodies, and a preparation method and application thereof. The protein consists of a silica-binding tag (Si‑tag) and a protein G domain (2C2) with 2 repeating units, and has the function of stably adhering to the silica surface and efficiently capturing antibodies. This recombinant protein structure can be irreversibly adsorbed on the silica surface through a simple incubation process, and capture the Fc end of monoclonal antibodies (mAbs), thereby achieving directional fixation of antibodies. The present invention provides a methodological basis for high-throughput fixation of antibodies, meets the technical requirements for batch fixation of antibodies of different pathogens / biomarkers on a biosensor platform on a silica surface, and can be used for multiple detection of pathogens / biomarkers in clinical samples.

Description

Technical Field

[0001] The present invention relates to the fields of protein engineering and optical fiber biosensor technology, and in particular to a silica-binding protein-assisted antibody directional immobilization method, and the construction of an optical biosensor system and pathogen detection involving the method. Background Art

[0002] In order to prevent the recurrence of major global public health events, the use of biosensors to monitor epidemics has become a major trend. Immunosensors are a type of biosensor that relies on antibody-target affinity to identify the analyte and can be used to detect disease markers or viruses [1-3]. In existing immunosensor technologies, silica nanomaterials are widely used to manufacture cheap and durable virus optical / electrochemical biosensors because of their lower energy loss than metal materials [4,5]. Methods for immobilizing antibodies on natural silica surfaces are generally divided into several categories, including physical adsorption, bioaffinity immobilization, and covalent binding [6]. Among these methods, streptavidin (SA)-biotin method is one of the popular antibody immobilization methods. However, there are still two major limitations in practical applications: 1) the immobilization direction of biotin-labeled antibodies is random; 2) the use of SA-biotin antibody immobilization method on other biosensor platforms with silica surfaces is very time-consuming.

[0003] Protein A / G affinity antibody immobilization is another common antibody immobilization technology because it can achieve fixed-point immobilization while retaining the biological activity of the antibody, improving the antigen capture ability, which is crucial for achieving consistency and repeatability in biosensing[7]. However, the subsequent antibody binding and sensing performance are greatly affected by the protein A / G immobilization effect, so special bridges are required to reduce the impact of the interface on protein A / G[8]. Currently, most methods for immobilizing protein A / G on silica interfaces are relatively cumbersome, making it difficult to widely apply. Therefore, there is an urgent need to develop efficient, simple, and economical antibody-directed immobilization bridge agents.

[0004] Biolayer interferometry is a technique that uses shifts in the interference spectrum to reflect the binding of substances on the sensor surface. This technique has been used in the rapid detection of neutralizing antibodies in recipients of the novel coronavirus (SARS-CoV-2) vaccine [9,10], SARS-CoV-2 antigens

[11] , and small molecule drugs in clinical samples

[12] . This paper uses biolayer interferometry to verify the reliability of the newly developed bridging protein.

[0005] Definitions of some abbreviations, English and key terms used in this application are as follows:

[0006] cSP: Silica-binding Protein featuring core-functional domains, silica-binding protein with core functional domains;

[0007] Si-tag: Silica-binding tag;

[0008] 2C2: Tandem protein G fragments, with two repeating units of protein G domain;

[0009] mAbs: Monoclonal antibodies, monoclonal antibodies;

[0010] BLI: Biolayer interferometry;

[0011] HRP: Horseradish Peroxidase;

[0012] AMEC: 3-amino-9-ethylcarbazole;

[0013] LOD: Limit of detection, detection limit;

[0014] SA: Streptavidin;

[0015] MPXV: Monkeypox virus;

[0016] SARS-CoV-2: CoronaVirus Disease 2019, new coronavirus;

[0017] VACV:Vacciniavirus, cowpox virus;

[0018] References:

[0019] [1]G.Rabbani,M.E.Khan,A.U.Khan,et al.,Label-free and ultrasensitiveelectrochemical transferrin detection biosensor based on a glassy carbonelectrode and gold nanoparticles,Int.J.Biol.Macromol 256(2024),128312.

[0020] [2]G.Rabbani,M.E.Khan,E.Ahmad,et al.,Serum CRP biomarker detectionbyusing carbon nanotube field-effect transistor(CNT-FET)immunosensor,Bioelectrochemistry 153(2023),108493.

[0021] [3]Y.Zheng,S.Bian,J.Sun,et al.,Label-free LSPR-vertical microcavitybiosensor for on-site SARS-CoV-2detection,Biosensors 12(2022),151.

[0022] [4]M.Mathelié-Guinlet,T.Cohen-Bouhacina,I.Gammoudi,et al.,Silicananoparticles-assisted electrochemical biosensor for the rapid,sensitive andspecific detection ofEscherichia coli,Sens.Actuators B Chem.292(2019)314–320.

[0023] [5]X.Zhang,Y.Zhang,X.Zhang,et al.,Interface design and dielectricresponse behavior ofSiO2 / PB composites with low dielectric constant andultra-low dielectric loss,Sur.Interfaces 22(2021),100807.

[0024] [6]L.S.Puumala,S.M.Grist,J.M.Morales,et al.,Biofunctionalization ofmultiplexed siliconphotonic biosensors,Biosens.Basel.13(2023),53.

[0025] [7]A.Sadiki,S.R.Vaidya,M.Abdollahi,et al.,Site-specific conjugationof native antibody,Antib.Ther.3(2020)271–284.

[0026] [8]X.Song,Z.Fredj,Y.Zheng,et al.,Biosensors for waterborne virusdetection:Challenges and strategies,J.Pharm.Anal.13(2023)1252–1268.

[0027] [9]S.Bian,M.Shang,M.Sawan,Rapid biosensing SARS-CoV-2antibodies invaccinatedhealthy donors,Biosens.Bioelectron.204(2022),114054.

[0028]

[10] S.Bian,M.Shang,Y.Tao,et al.,Dynamic profiling and prediction ofantibody response to SARS-CoV-2booster-inactivated vaccines by microsample-drivenbiosensor andmachine learning,Vaccines 12(2024),352.

[0029]

[11] Y.Tao, S.Bian, P.Wang, et al., Rapid optical biosensing of SARS-CoV-2spike proteins in artificial samples, Sensors 22 (2022), 3768.

[0030]

[12] S.Bian, Y.Tao, Z.Zhu, et al., On-site biolayer interferometry-basedbiosensing ofcarbamazepine in whole blood of epileptic patients, Biosens.Basel.11(2021). Summary of the Invention

[0031] To address the problems of the prior art, the present invention provides a silica-binding protein (cSP) with a core functional domain. This innovative protein, composed of a silica-binding tag (Si-tag) and a protein G domain (2C2) with two repeating units, exhibits stable adhesion to silica surfaces and efficient antibody capture. This recombinant protein structure can be irreversibly adsorbed to silica surfaces through a simple incubation process and capture the Fc termini of monoclonal antibodies (mAbs), thereby achieving directional immobilization of the antibodies.

[0032] The present invention further applies recombinant cSP protein to silica optical fibers and establishes a biosensor based on bio-layer interferometry, which can detect disease markers or viruses (hereinafter referred to as antigens) in clinical samples with high sensitivity. This demonstrates the reliability and robustness of recombinant cSP protein as an antibody bridge.

[0033] In order to achieve the above object, the present invention adopts the following technical solutions:

[0034] (1) Design and preparation of recombinant cSP protein:

[0035] The N-terminal 2C2 segment of the recombinant cSP protein consists of two repeating units of the C2 fragment of Protein G (PDBID: 1FCC) and is used for affinity capture of mAbs.

[0036] The C-terminal Si-tag segment of the recombinant cSP protein consists of amino acids 1-60 and 203-273 of the 50S Escherichia coli ribosomal protein L2 (NCBI RefSeq: WP_000301864.1) [1], which is used to bind to the silica surface.

[0037] The recombinant cSP protein further comprises a flexible connecting peptide linker (EGKSSGSGSESKST) connecting 2C2 and Si-tag.

[0038] The entire cSP coding gene was codon-optimized and inserted into a plasmid vector via the multiple cloning site.

[0039] The plasmid connected to the cSP gene is introduced into the host strain to induce expression and obtain a culture containing the target protein.

[0040] The recombinant cSP protein extract was obtained after purification.

[0041] (2) Preparation of silica fiber probes:

[0042] Since there is no commercial silica optical fiber for bio-layer interferometry technology on the market, the present invention immerses the commercial SA-modified optical fiber in a heated oxidizing solution [2] to remove the SA to obtain an optical fiber probe with an exposed silica surface.

[0043] (3) Preparation of cSP-mAb fiber optic probe:

[0044] The recombinant cSP protein and primary antibody (1 st mAb) were sequentially immobilized on the surface of the silica fiber probe to obtain the cSP-mAb fiber probe.

[0045] (4) Prepare the sample to be tested:

[0046] Different concentrations of the antigen protein to be tested are added to the sample dilution buffer, serum sample, and saliva sample respectively to obtain several samples to be tested with different antigen concentrations.

[0047] (5) Antigen capture and detection:

[0048] The cSP-mAb optical fiber probe is brought into contact with the sample to be tested, and the antigen protein in the sample to be tested specifically binds to the 1 on the optical fiber probe. st mAb binding.

[0049] (6) Signal amplification:

[0050] The cSP-mAb optical fiber probe is sequentially contacted with the HRP-labeled secondary antibody (2 nd In the solution of mAb-HRP) and AMEC biochromogenic reagent (signal enhancer), the HRP on the secondary antibody reacts with the AMEC biochromogenic reagent to produce precipitation, thereby amplifying the binding signal.

[0051] (7) Obtaining the standard curve and detection limit:

[0052] The test data of the gradient antigen solution obtained above were used to draw a standard curve using GraphPad Prism software (version 9.02) from GraphPad Software, USA, in the "non-linear regression and single-site specific binding" mode.

[0053] The limit of detection (LOD) is defined as the antigen concentration equal to the mean of the blank control plus three times the standard deviation. This value is calculated using the following formula:

[0054] y=x*A / (x+B)

[0055] Where x is the target concentration, y is the binding shift (spectral shift), and A and B represent fitting parameters. Enter "blank shift + 3*STD" as "y" into the formula to obtain the LOD; blank shift is the data from the blank group, and STD is the standard deviation of nonspecific binding (n>3).

[0056] In the steps of obtaining the samples to be tested, the serum sample is diluted 100-fold using a high-salt sample dilution buffer, and the saliva sample is diluted 20-fold using a high-salt sample dilution buffer; wherein the high-salt sample dilution buffer is a 10 mM pH 7.4 phosphate buffer containing 274 mM NaCl, 0.02% by mass of Tween-20, and 0.1% by mass of bovine serum albumin (BSA).

[0057] In the biosensor detection method based on recombinant cSP protein provided by the present invention, the concentration of the antigen protein is 0 ng / mL to 100 ng / mL.

[0058] In the biosensor detection method based on recombinant cSP protein provided by the present invention, the AMEC biochromogenic agent is 3-amino-9-ethylcarbazole.

[0059] 2 of the present invention nd mAb-HRP is st mAb paired 2 nd mAb conjugated to HRP.

[0060] In the biosensor detection method based on recombinant cSP protein provided by the present invention, the AMEC signal enhancer is diluted 10-fold before contacting with the cSP-mAb fiber optic probe: the 10-fold dilution is a 10-fold dilution relative to the guidance scheme given by the ImmPACT AMEC Red commercial kit: 9 μL Reagent 1 and 8 μL Reagent 2 are added to 5 mL of ImmPACT AMEC Red diluent.

[0061] References:

[0062] [1]S.Kim,KIJoo,BHJo,et al.,Stability-controllable self-immobilization of carbonic anhydrase fused with a silica-binding tag ontodiatom biosilica for enzymatic CO2 capture and utilization,ACSAppl.Mater.Interfaces 12(2020)27055–27063.

[0063] [2] C. Liu, DL Steer, H. Song, et al., Superior binding of proteins onasilica surface: Physical insight into the synergetic contribution of polyhistidine and asilica-bindingpeptide, J. Phys. Chem. Lett. 13 (2022) 1609–1616.

[0064] Beneficial effects:

[0065] The antibody-directed immobilization technology proposed in this paper, based on recombinant cSP protein, provides a methodological basis for high-throughput antibody immobilization. It meets the technical requirements for batch immobilization of antibodies targeting different pathogens / biomarkers on a silica-surface biosensor platform and can be used for multiplex detection of pathogens / biomarkers in clinical samples.

[0066] Compared with the existing technology, the present invention utilizes silica-binding proteins that can directionally fix antibodies and fiber optic bio-layer interferometry technology, combined with effective signal amplification, to establish a fast (less than 15 minutes from antibody fixation to result output), high-sensitivity (taking the exemplary MPXV detection as an example, the LODs are: LOD in buffer = 0.62 ng / mL, LOD in serum = 0.77 ng / mL, LOD in saliva = 0.80 ng / mL), simple, and high-throughput (using an eight-channel bio-layer interferometry analyzer for multiple sample and multiple antigen detection) fully automatic optical biosensor detection method, which can detect antigen proteins in human serum and saliva. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Figure 1 shows the design and purification of the recombinant cSP protein proposed in this invention. 1-A is a schematic diagram of the 3D structure of the recombinant cSP protein; 1-B is a schematic diagram of the gene structure of the pET26b(+) plasmid into which the cSP fusion gene is inserted; 1-C is a diagram showing the expression, purification, and characterization of the recombinant cSP protein (SDS-PAGE). MW: protein molecular weight standard; CL: whole cell lysate; P1-P2: purified cSP protein.

[0068] Figure 2 Figure 2 shows the construction and characterization of the cSP-mAb fiber used in the recombinant cSP protein-based MPXV biosensor detection proposed in this invention. Figure 2-A depicts the relationship between oxidation treatment time and the resulting silica fiber; Figure 2-B shows representative results of silica fiber sequentially binding recombinant cSP protein and mAb; and Figure 2-C shows the capture ability of recombinant cSP protein-modified silica fiber for mAbs from different species.

[0069] Figure 3 Flowchart of the MPXV biosensor detection based on recombinant cSP protein proposed in the present invention.

[0070] Figure 4 Figure 4-A compares the standard curves generated by the SA-mAb fiber and the cSP-mAb fiber for MPXV detection in buffer and spiked clinical samples. Figure 4-B shows the cross-reactivity assay. Figure 4-C describes the performance of the recombinant cSP protein-based biosensor technology for detecting MPXV in spiked saliva and serum.

[0071] Figure 5This paper analyzes the development prospects of cSP-based optical biosensing methods for the detection of various viruses. Figure 5-A describes an example of high-throughput detection of recombinant cSP protein-based optical biosensing methods on a bio-layer interferometer. Figure 5-B demonstrates high-throughput immobilization of mAbs on a silica biosensor platform using recombinant cSP protein. DETAILED DESCRIPTION

[0072] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific examples, which can help to better understand the present invention. The contents described in the embodiments are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0073] 1. Design and preparation of recombinant cSP protein:

[0074] The N-terminal 2C2 segment of the recombinant cSP protein is composed of two repeating units of the C2 fragment of Protein G (PDBID: 1FCC) and is used for affinity capture of mAbs. The C-terminal Si-tag segment of the recombinant cSP protein is composed of amino acids 1-60 and 203-273 of the 50S Escherichia coli ribosomal protein L2 (NCBI RefSeq: WP_000301864.1) and is used for binding to the silica surface. The recombinant cSP protein also includes a flexible linker peptide linker (EGKSSGSGSESKST) connecting 2C2 and Si-tag (such as Figure 1 -A).

[0075] After codon optimization, the entire cSP coding gene was inserted into the plasmid vector pET26b(+) via the NdeⅠ / XhoⅠ sites (e.g. Figure 1 -B). The cSP-pET26b(+) plasmid connected with the cSP gene was introduced into the competent Escherichia coli BL21(DE3).

[0076] A single colony containing the cSP-pET26b(+) plasmid was picked and cultured in 4 mL of LB medium containing 10 μg / mL kanamycin. The culture was continued at 37°C and 200 rpm until the OD 600 =0.6, and then 1 mL of culture was transformed into 100 mL of fresh LB medium containing kanamycin and cultured at 37 °C and 200 rpm until OD 600 =0.6, followed by the addition of 0.05 mM IPTG and induction at 15°C, 180 rpm for 16 h. The cells were then centrifuged at 4°C, 4000 rpm for 20 min to enrich the cells, and washed twice with PBS buffer.

[0077] Obtain cell culture containing target protein and purify cSP protein according to the instructions of commercial His-tag protein purification kit, and perform 15% SDS-PAGE analysis and identification on the purified product (as shown in FIG Figure 1 -C).

[0078] The obtained cSP protein was stored in the elution buffer provided by the kit and stored at -80°C until use.

[0079] 2. Preparation of Bare Silicon Fiber Probes and Functional Testing of Recombinant cSP Protein

[0080] (1) Preparation of bare silicon fiber probe:

[0081] Commercial SA optical fibers are available from Sartorius Stedim Biotech GmbH. The commercial SA optical fibers were immersed in an oxidizing solution and heated at 80°C. The oxidizing solution consisted of deionized water, 30% H₂O₂ solution, and 25%-28% ammonia water, mixed in a ratio of 3:1:1. The present invention optimized the heating time, with a time gradient of 30, 60, 90, and 120 minutes.

[0082] The treated optical fiber was immersed in the solution containing 5 μg / mL recombinant cSP protein, 10 μg / mL antigen-specific monoclonal antibody (1 st The PCR amplification step was repeated for 80 seconds in a solution containing 1 μg / mL of mAb and 1 μg / mL of antigen. All three solutions were prepared using a sample diluent consisting of 0.02% Tween-20 and 0.1% bovine serum albumin (BSA) in 10 mM phosphate buffer, pH 7.4. A 60-second wash with the sample diluent was added between each step.

[0083] The results are shown in Figure 2 -A. After 90min and 120min of oxidation treatment, 1 st The spectral shifts for mAb capture and antigen binding showed similarities. Therefore, we next used a 90-min oxidation treatment time for the silica fiber and performed cSP modification.

[0084] (2) Functional testing of recombinant cSP protein

[0085] The present invention verifies that recombinant cSP protein is an essential element for the fixation of antibodies on silica surfaces. The implementation conditions include immersing in sample diluent for pre-cleaning for 60 seconds, immersing in cSP solution (5 μg / mL) for 80 seconds, immersing in sample diluent for washing for 60 seconds, and immersing in 1 st mAb solution (10 μg / mL) for 80 seconds, then immersed in sample diluent for 60 seconds. stmAb solutions were prepared from sample diluent. Figure 2 -B shows that the spectral shift signal representing mAb binding can only be detected on the bare silica fiber incubated with recombinant cSP protein.

[0086] This study also validated the ability of recombinant cSP protein-modified silica fibers to bind mAbs from different species. Two mAbs each from human, goat, donkey, rabbit, and mouse were used. A cSP solution (5 μg / mL) and a 10 μg / mL solution of each mAb were prepared using sample diluent, following the same testing procedures as above.

[0087] Figure 2 -C shows that the bare silica fiber probe modified with recombinant cSP protein has strong binding affinity with different types of mAbs, and the binding strength trend is similar to that of Protein G.

[0088] 3. Example 1 (MPXV detection)

[0089] To demonstrate the ease of biosensing using recombinant cSP protein, we established an optical biosensing method for MPXV. This assay can specifically detect the MPXV A29L antigen in various media, including sample dilutions, saliva, and serum. The protocol is shown in Table 1.

[0090] Table 1

[0091]

[0092] (1) Establishment of MPXV detection method in sample dilution

[0093] As shown in Table 1, the total time from sample to result for the cSP-based optical biosensing technology is 12 min: MPXV specificity 1 st mAb was attached to the surface of bare silica fiber probe via recombinant cSP protein, which took only 340s; detection of MPXVA29L protein took 140s; and finally, the MPXV-specific 2 nd The signal amplification step achieved by mAb-HRP and AMEC signal enhancer can be completed within 220s. nd The concentration of mAb-HRP solution and AMEC enhancer were adjusted to control background interference and maximize positive signal (the spectral shift caused by background interference was controlled within about 1 nm, and the spectral shift caused by 100 ng / mL MPXVA29L protein was controlled within 40 nm). Figure 3 The whole process of detecting MPXVA29L protein in sample dilution was summarized.

[0094] (a) Preparation of test samples and test solutions:

[0095] cSP solution (5 μg / mL), targeting the A29L antigen of MPXV st mAb solution (10 μg / mL) and 2 nd The mAb-HRP solution (1.25 μg / mL) was prepared in sample diluent. The MPXV A29L antigen to be tested was diluted to a gradient concentration (0–100 ng / mL) in sample diluent. The signal enhancer AMEC was diluted 10-fold before contacting the cSP-mAb fiber optic probe. This 10-fold dilution corresponds to the 10-fold dilution protocol provided in the ImmPACT AMEC Red commercial kit: 9 μL of Reagent 1 and 8 μL of Reagent 2 were added to 5 mL of ImmPACT AMEC Red diluent. All washes during the assay were performed in sample diluent.

[0096] (b) Preparation of cSP-mAb fiber-optic probe:

[0097] As shown in Table 1, the prepared silica fiber probe was immersed in the sample dilution buffer, shaken and washed for 60 seconds, then immersed in the cSP solution for 80 seconds, and then shaken and washed for 60 seconds. In order to specifically recognize and bind to the antigen, the MPXV-specific 1 targeting MPXVA29L antigen was added to the silica fiber probe. st mAb, the implementation procedure is still immersed in MPXV specific 1 st The cells were placed in mAb solution for 80 s, followed by shaking for 60 s as a pre-wash before antigen testing.

[0098] (c) Antigen capture and detection:

[0099] As shown in Table 1, the cSP-MPXV specific 1 st The mAb-loaded silica fiber probe was immersed in the antigen solution for 80 s and then washed with shaking for 60 s.

[0100] (d) Signal amplification:

[0101] As shown in Table 1, in the signaling method step, the silica fiber probe bound to the antigen is immersed in the MPXV specific 2 nd The cells were then incubated in mAb-HRP solution for 80 seconds, followed by a 60-second wash step, and finally, the signal was enhanced with AMEC signal enhancer for 80 seconds.

[0102] (e) Obtaining the standard curve and detection limit:

[0103] The test data of the gradient antigen solution obtained above were used to draw a standard curve using GraphPad Prism software (version 9.02) from GraphPad Software, USA, in the "non-linear regression and single-site specific binding" mode.

[0104] The limit of detection (LOD) is defined as the antigen concentration equal to the mean of the blank control plus three times the standard deviation. This value is calculated using the following formula:

[0105] y=x*A / (x+B)

[0106] Where x is the target concentration, y is the binding shift (spectral shift), and A and B represent fitting parameters. Enter "blank shift + 3*STD" as "y" into the formula to obtain the LOD; blank shift is the data from the blank group, and STD is the standard deviation of nonspecific binding (n>3).

[0107] (f) Comparison of detection performance between cSP-based optical biosensors and commercial SA chips:

[0108] The cSP-based optical biosensor test and result calculation were the same as steps (a) to (e). The calculated LOD of the cSP-based optical biosensor for MPXVA29L protein was 0.62 ng / mL.

[0109] When using commercial SA chips for comparison with cSP-based optical biosensing technology, step (b) needs to be adjusted. st mAb was immobilized on the surface of SA chip. st The mAb biotinylation operation procedure can refer to the commercial biotinylation kit available on the market. st The subsequent steps (c)-(e) can be performed only after the mAb layer spectral shift is adjusted to be close to the data in Table 1. The calculated LOD of the commercial SA chip for MPXVA29L protein is 0.52 ng / mL.

[0110] like Figure 4 As shown in Figure 1-A, the signal trend and LOD of the cSP-based optical biosensor are close to those of the commercial SA chip. This convergence in performance demonstrates the reliability of the cSP-based optical biosensor technology.

[0111] (g) Cross-reactivity test:

[0112] The specificity of cSP-based optical biosensor for MPXV was evaluated using three popular viral proteins. The cross-reactions of the present invention selected the vaccinia virus (VACV) A27L protein, SARS-CoV-2 XBB.1.5RBD protein, and influenza A H3N2 nucleoprotein, which are homologous to the MPXVA29L protein. The above-mentioned MPXVA29L protein solution and the above-mentioned three test protein solutions were prepared using sample buffer at a concentration of 5 ng / mL. The test steps are the same as steps (a)-(d). Figure 4 As shown in Figure 2B, the cSP-based fiber optic biosensor does not cross-react with viral proteins other than MPXV.

[0113] (2) Detection of MPXV in spiked clinical samples (serum and saliva)

[0114] When using cSP-based optical biosensing technology to detect spiked serum and saliva samples, the same steps as for sample buffer testing were followed, as shown in Table 1. However, the present invention used a high-salt sample dilution buffer to dilute the samples to mitigate potential nonspecific interference from serum and saliva components with the cSP-based optical biosensor. The high-salt sample dilution buffer consisted of 10 mM phosphate buffer, pH 7.4, containing 274 mM NaCl, 0.02% Tween-20, and 0.1% bovine serum albumin (BSA). Serum samples were diluted 100-fold using the high-salt sample dilution buffer, while saliva samples were diluted 20-fold.

[0115] Due to the difference in sample dilution, the MPXV specificity 1 st The fiber pre-cleaning procedure after mAb binding has been adjusted. High-salt sample dilution buffer is now used for pre-cleaning, and the pre-cleaning time is extended to 200 seconds. Other testing and calculation procedures remain unchanged.

[0116] like Figure 4 As shown in Figure 5-C, the concentration of MPXVA29L protein detected in diluted serum and saliva ranged from 0 to 100 ng / mL, with LODs of 0.77 ng / mL and 0.80 ng / mL, respectively.

[0117] 4. Example 2

[0118] Because the recombinant cSP protein designed in the present invention has universal mAb binding ability, mAbs targeting various viruses can be easily and firmly immobilized on the silica surface. Therefore, the recombinant cSP protein can be used for multiple virus detection.

[0119] Figure 5This paper introduces the methods of immobilizing mAbs using recombinant cSP protein in different detection modes and possible detection implementation schemes. Figure 5 For example, when using a multi-channel bio-layer interferometer, the multiple wells of a 96-well plate can be aligned with the instrument's multiple channels, allowing silica fiber probes to bind to mAbs specific to different viruses. Combined with the detection scheme described in Example 1, it is possible to test 32 samples across 8 channels, or eight viruses in four samples, with each test run completed in less than 15 minutes.

[0120] Figure 5 -B introduces another detection mode based on recombinant cSP protein. cSP protein and virus-specific mAbs can be added to specific areas of the biosensor array as needed to achieve the immobilization of multiple virus-specific mAbs in a single channel.

[0121] The amino acid sequence of the recombinant cSP protein and the DNA sequence of its encoding gene are as follows: DNA sequence (SEQ ID NO: 2)

[0122] atgacaacttacaaacttgttattaatggtaaaacattgaaaggcgaaacaactactgaagctgttgatgctgctactgcagaa

[0123] aaagtcttcaaacaatacgctaacgacaacggtgttgacggtgaatggacttacgacgatgcgactaagacctttacagtta

[0124] ctgaaacaacttacaaacttgttattaatggtaaaacattgaaaggcgaaacaactactgaagctgttgatgctgctactgcag

[0125] aaaaagtcttcaaacaatacgctaacgacaacggtgttgacggtgaatggacttacgacgatgcgactaagacctttacagt

[0126] tactgaagagggtaaatctagcggaagtgggtcagaatcgaaaagcaccatggcggttgtgaagtgcaaaccgacgagc

[0127] ccgggtcgtcgccacgtggtgaaagttgtcaacccggagctgcacaagggcaagccgttcgcccctttgctggagaaaaa

[0128] ctctaagtccggtggccgtaataacaacggccgcattaccacccgtcacatcggcggcggtcataaacaaggtggcggc

[0129] ggcagccgtgttctgggtaaagctggtgcggcgcgttggcgtggtgtgcgtccgaccgttcgcgggaccgcaatgaatcc

[0130] ggtcgatcatccgcatggtggcggcgaaggtcgcaactttggtaagcacccagtaactccgtggggtgttcagaccaaag

[0131] gtaagaagacccgtagcaataaacgtacggacaagttcatcgtgcgccgtagatccaagctcgagcaccaccaccacca

[0132] ccactga DNA sequence (SEQ ID NO: 3, the coding gene after codon optimization)

[0133] atgactacatataagctagtaataaatggaaaaacgttgaagggtgagactacgactgaggcggtcgacgcggcaaccgc

[0134] tgaaaaagtttttaaacaatatgcgaatgataacggtgtggacggcgagtggacgtacgacgacgcgaccaagacctttac

[0135] cgttaccgaaaccacgtacaaactggttatcaacggcaaaaccctgaaaggtgaaaccacgactgaggcggtggacgct

[0136] gccaccgcagaaaaggtgttcaagcagtatgcgaacgataatggcgttgatggtgaatggacctacgatgatgccaccaa

[0137] gaccttcaccgtgaccgaggagggtaaatctagcggaagtgggtcagaatcgaaaagcaccatggcggttgtgaagtgc

[0138] aaaccgacgagcccgggtcgtcgccacgtggtgaaagttgtcaacccggagctgcacaagggcaagccgttcgcccctt

[0139] tgctggagaaaaactctaagtccggtggccgtaataacaacggccgcattaccacccgtcacatcggcggcggtcataaa

[0140] caaggtggcggcggcagccgtgttctgggtaaagctggtgcggcgcgttggcgtggtgtgcgtccgaccgttcgcggga

[0141] ccgcaatgaatccggtcgatcatccgcatggtggcggcgaaggtcgcaactttggtaagcacccagtaactccgtggggt

[0142] gttcagaccaaaggtaagaagacccgtagcaataaacgtacggacaagttcatcgtgcgccgtagatccaagctcgagca

[0143] ccaccaccaccaccactga

[0144] Amino acid sequence (SEQ ID NO: 1)

[0145] MTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATK

[0146] TFTVTETTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYD

[0147] DATKTFTVTEEGKSSGSGSESKSTMAVVKCKPTSPGRRHVVKVVNPELHKGK

[0148] PFAPLLEKNSKSGGRNNNGRITTRHIGGGHKQGGGGSRVLGKAGAARWRGV

[0149] RPTVRGTAMNPVDHPHGGGEGRNFGKHPVTPWGVQTKGKKTRSNKRTDKFIVRRRSKLEHHHHHH*。

Claims

1. A silica-bound protein for directional immobilization of antibodies, characterized in that: The protein is a recombinant silica binding protein, i.e., a recombinant cSP protein, and its amino acid sequence is SEQ ID NO:

1. Its N-terminus is composed of 2 repeating units of the C2 fragment of Protein G, which is used for affinity capture of mAbs and is named as the 2C2 segment. The C-terminus of the recombinant cSP protein is composed of the 1st to 60th and 203rd to 273rd amino acids of the 50S Escherichia coli ribosomal protein L2, which is used for binding to the silica surface and is named as the Si-tag segment. The protein also includes a flexible connecting peptide linker EGKSSGSGSESKST connecting the 2C2 segment and the Si-tag segment. The amino acid sequence of the C2 fragment is numbered as 1FCC in the PDB database, and the amino acid sequence of the 50S Escherichia coli ribosomal protein L2 is numbered as WP_000301864.1 in the NCBI database.

2. The silica-bound protein for directional antibody immobilization according to claim 1, characterized in that: The protein preparation method is as follows: the entire cSP coding gene SEQ ID NO: 2 is codon optimized and then inserted into a plasmid vector via a multiple cloning site; The plasmid connected to the cSP gene is introduced into the host strain to induce expression, thereby obtaining a culture containing the target protein; The recombinant cSP protein extract was obtained after purification.

3. The use of the silica-bound protein for directional antibody fixation as claimed in claim 1, characterized in that: The protein was used to immobilize monoclonal antibodies on a silica surface.

4. The use according to claim 3, characterized in that: The method for using the protein is: immersing the silica in a solution containing the protein and the monoclonal antibody to be fixed, or immersing the silica in a solution containing the protein and a solution containing the monoclonal antibody to be fixed in sequence.

5. The use according to claim 4, characterized in that The solution containing the protein and the monoclonal antibody to be fixed, the solution containing the protein, and the solution containing the monoclonal antibody to be fixed are all prepared from a sample diluent, and the sample diluent is a 10mM pH 7.4 phosphate buffer containing 0.02% Tween-20 by mass and 0.1% bovine serum albumin (BSA) by mass.

6. The use of the silica-bound protein for directional antibody fixation according to claim 1, characterized in that: The protein is used for biosensing detection for the purpose of non-disease diagnosis and treatment.

7. The use according to claim 6, characterized in that: The method of biosensor detection for the purpose of non-disease diagnosis and treatment is: after the protein is used to fix a monoclonal antibody on a silica surface, it is added to a sample to be tested as a probe to detect whether the sample to be tested contains an antigen that specifically binds to the monoclonal antibody.

8. The use according to claim 7, characterized in that: The sample to be tested is a serum sample or a saliva sample diluted with a high-salt sample dilution buffer.

9. The use according to claim 8, characterized in that The high-salt sample dilution buffer is a 10 mM pH 7.4 phosphate buffer containing 274 mM NaCl, 0.02% by mass of Tween-20, and 0.1% by mass of bovine serum albumin (BSA).

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