Shark single-domain antibody binding to hemoglobin and application thereof

Abstract

This invention discloses a shark-derived single-domain antibody that binds to hemoglobin and its application. The nucleotide sequence of the single-domain antibody is labeled SEQ ID NO.1, while the corresponding amino acid sequence is recorded as SEQ ID NO.2. The shark-derived single-domain antibody is used to detect hemoglobin concentration. The detection method includes setting up a standard group and a sample group, then adding the shark-derived single-domain antibody to the immunoassay plates of the standard group and the sample group, and measuring the OD of the standard group and the sample group after color development. 450 Value, based on the OD of the standard group 450 Values, plot OD 450 The standard curve is calculated based on the OD value. 450 The standard curve was used to obtain the hemoglobin concentration of the sample group. The single-domain antibody prepared in this invention exhibits good stability, high specificity, and high affinity. The antibody screened in this invention provides a theoretical basis and technical support for the subsequent diagnosis and monitoring of hemoglobin-targeted diseases.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a shark-derived single-domain antibody that binds to hemoglobin and its applications. Background Technology

[0002] Hemoglobin is a protein found in red blood cells. It binds with oxygen to form oxyhemoglobin, which is responsible for transporting oxygen from the lungs to various tissues and organs throughout the body. Hemoglobin testing reflects the blood's oxygen-carrying capacity and is crucial for assessing anemia, monitoring the blood status of surgical patients, and guiding blood transfusions. Furthermore, hemoglobin levels are closely related to cardiovascular diseases, colon cancer, and other conditions, highlighting the importance of hemoglobin testing in disease diagnosis, prognostic assessment, and treatment monitoring. Common hemoglobin detection methods include optical measurement and immunological methods, with non-invasive hemoglobin monitoring gradually being introduced. Optical measurement systems generally require large and expensive specialized equipment and dedicated laboratory staff. Non-invasive hemoglobin monitoring results are affected by various factors, requiring comprehensive evaluation of accuracy and reliability using other clinical indicators. Immunological methods based on antigen-antibody recognition offer higher sensitivity and accuracy. Therefore, it is necessary to develop highly specific and sensitive hemoglobin-targeting antibodies to improve detection precision. Traditional antibodies have limitations in penetrating living cells, and their interference with target protein function also restricts their practical application. In 1995, Greenberg et al. discovered a novel antibody composed solely of heavy chains in cartilaginous fish such as the wrasse, termed the immunoglobulin new antigen receptor (IgNAR). The antigen-binding fragment of the IgNAR, called the variable domain of the immunoglobulin new antigen receptor (VNAR), is located at the end of a single heavy chain. The VNAR has a molecular weight of approximately 12 kDa, only one-tenth the size of traditional monoclonal antibodies, making it the smallest known antigen-binding unit, also known as a single-domain antibody. Single-domain antibodies are characterized by their small molecular weight, high solubility, ease of recombinant expression, good thermal stability, strong folding ability, good tissue penetration, low polymerization tendency, and strong targeted binding ability. Furthermore, single-domain antibodies can rapidly penetrate tissue barriers while maintaining high specificity and affinity, effectively overcoming the limitations of traditional antibodies, making them an effective complement to antibody therapy. Therefore, single-domain antibodies have enormous development potential in drug development, disease diagnosis, and treatment. Currently, there are no shark-derived single-domain antibodies that bind to hemoglobin. Summary of the Invention

[0003] To address the problems existing in the background art, the present invention aims to provide a shark-derived single-domain antibody that binds to hemoglobin and its applications. The present invention provides a variable region sequence of a shark-derived heavy chain antibody capable of binding to hemoglobin with high affinity, which is crucial for disease diagnosis, prognosis assessment, and treatment monitoring.

[0004] The technical solution adopted in this invention is as follows:

[0005] I. A shark-derived single-domain antibody that binds to hemoglobin:

[0006] The amino acid sequence of the shark-derived single-domain antibody is shown in SEQ ID NO.2.

[0007] The nucleotide sequence of the shark-derived single-domain antibody encoding gene is shown in SEQ ID NO.1.

[0008] II. Application of a shark-derived single-domain antibody that binds to hemoglobin: The application of the shark-derived single-domain antibody in the preparation of reagents for detecting hemoglobin concentration.

[0009] The reagents used to detect hemoglobin concentration include reagents for diagnosing anemia and reagents for diagnosing colon cancer.

[0010] III. A method for detecting hemoglobin concentration using shark-derived single-domain antibodies, comprising the following steps:

[0011] Step S1: First, set up a standard group, a sample group, and a blank group. The standard group is to add hemoglobin standards of different concentrations to the immunoassay plate. The sample group is to add the hemoglobin sample to be tested to the immunoassay plate. The blank group is to add no sample to the immunoassay plate.

[0012] Hemoglobin standards generally refer to hemoglobin assay kits with known concentrations.

[0013] Step S2: Next, the standard group, sample group, and blank group were blocked using bovine serum albumin (BSA). Then, shark-derived single-domain antibody was added to the immunoassay plates of the standard group, sample group, and blank group. After incubation, the immunoassay plates were washed with PBST. Goat anti-human IgG Fc-HRP antibody was added to each well of the immunoassay plate. After incubation, the immunoassay plates were washed again with PBST. Then, TMB was added to each well for color development. After color development, sulfuric acid (H2SO4) was added to terminate the reaction. The absorbance (OD) of the standard group, sample group, and blank group was measured. 450 Values. Next, the absorbance OD values ​​of the standard group and the sample group were calculated separately. 450 Value minus the absorbance OD of the blank group 450 The OD values ​​were obtained for the standard group and the sample group after correction. 450 value;

[0014] Step S3: Based on the concentration of hemoglobin standards in the standard group and the corrected absorbance OD... 450 Values, plot absorbance OD 450 The standard curve of the value is based on the absorbance OD. 450 The standard curve of the values ​​is used to obtain the OD corrected for the sample group. 450 The concentration corresponding to the value is the hemoglobin concentration of the sample group.

[0015] Specifically, the concentration of hemoglobin standards in the standard group is used as the x-axis, and the corresponding corrected absorbance OD is... 450 The absorbance OD was obtained by performing linear regression analysis with the value as the ordinate. 450 The standard curve of the value.

[0016] IV. Preparation and characterization method of shark-derived single-domain antibodies that bind to hemoglobin, including the following steps:

[0017] Step S1: Screen for specific single-domain antibodies;

[0018] Step S2: Recombinant expression of single-domain antibodies to obtain shark-derived single-domain antibodies;

[0019] Step S3: Characterization of shark-derived single-domain antibodies.

[0020] The specific steps of S1 are as follows:

[0021] S11. First, the existing shark-derived single-domain antibody synthesis library was inoculated into liquid culture medium. Then, helper phages were added to the liquid culture medium to promote phage proliferation. After static culture and centrifugation to collect phages, the phages were cultured overnight with shaking to obtain more phages. Then, the phages were enriched by PEG precipitation to obtain the titer of the enriched phages.

[0022] S12. Hemoglobin was dissolved in protein-free GFBE solution to prepare a hemoglobin solution. 0.1 mg / mL of the hemoglobin solution was then coated into immunoassay plates as the hemoglobin group, and the immunoassay plates without hemoglobin solution were used as the negative control group. Phages enriched in S11 were added to the immunoassay plates of the hemoglobin group and the negative control group, respectively, so that the phages bound to the hemoglobin in the hemoglobin group. The immunoassay plates were then washed with PBST (phosphate buffer), and the detached phages from the hemoglobin group and the negative control group were obtained after washing.

[0023] S13. Phages from the hemoglobin group and the negative control group were used to infect TG1 bacteria (Escherichia coli). The TG1-infected phages were then spread on plates and cultured for 12-24 hours to obtain single colonies. The number of single colonies in the hemoglobin group and the negative control group was counted and compared.

[0024] If the number of single colonies in the hemoglobin group is more than 10 times that in the negative control group, it indicates that the number of single colonies in the hemoglobin group meets the requirements and proceeds to S14.

[0025] Otherwise, return to S12 to prepare new phages until the number of single colonies in the hemoglobin group meets the requirements;

[0026] S14. Select a single colony from the hemoglobin group and place it in an immunoplate, add helper phage, and amplify to obtain a monoclonal phage.

[0027] S15. Coat the immunoplate containing the monoclonal phage with hemoglobin solution, then add horseradish peroxidase-labeled phage-specific antibody to the immunoplate. Next, add TMB (3,3',5,5'-tetramethylbenzidine) chromogenic solution to perform a colorimetric reaction on the monoclonal phage. Measure the absorbance OD. 450 Monoclonal phages with a count greater than 1 were sequenced, and repetitive sequences were removed using sequence alignment software. The resulting nucleotide sequences after sequencing and repetitive sequence removal were used as the nucleotide sequences encoding the shark-derived single-domain antibody. Among them, the nucleotide sequences obtained from S1 were hemoglobin-specific.

[0028] Specifically, S2 is:

[0029] The coding gene of the shark-derived single-domain antibody was constructed into the eukaryotic expression vector pTT5-TEV-Fc, and then expressed using HEK293F (human embryonic kidney cells). The shark-derived single-domain antibody (i.e., a single-domain antibody-Fc fusion protein specifically targeting hemoglobin) was purified by agarose gel rProtein A affinity chromatography column.

[0030] The specific steps of step S3 are as follows:

[0031] S31. Hemoglobin antigen was coated onto an immunoassay plate at a concentration of 1 μg / mL. Protein-free solution served as the negative control. The plate was incubated overnight at 4°C. The next day, the liquid was removed, and the plate was washed with phosphate-buffered saline (PBS), then blocked with MPBS (phosphate buffer with 5% milk). Subsequently, the expressed antibody was serially diluted to multiple concentrations and added to the wells for incubation. After incubation, the wells were washed with PBST (phosphate buffer with 0.1% Tween-20), and then incubated with goat anti-human IgG Fc-HRP antibody. After washing again, TMB was used for color development, and the incubation was terminated with sulfuric acid (H2SO4). The OD was measured. 450 The value was calculated, and the half-maximal effective concentration (EC50) of the antibody was obtained by fitting the values. 50 value.

[0032] S32. The sensor is pre-wetted in 200 μL PBST for 10 min. After each round of antibody-antigen binding / dissociation, the sensor is regenerated with 10 mM glycine (pH 2.0) to restore it.

[0033] S33. Hemoglobin antigen was coated onto an immunoassay plate at a concentration of 1 μg / mL and incubated overnight at 4°C. The coating liquid was removed, and the plate was washed with PBS and blocked with MPBS. Untreated antibody served as the negative control, while antibodies treated under different conditions served as the experimental group. Untreated and treated antibodies were added to the wells and incubated. After incubation, the wells were washed with PBST, and then incubated with goat anti-human anti-IgG Fc-HRP antibody, followed by washing. TMB was used for color development, and the incubation was stopped with H2SO4. OD was measured. 450 The antibody binding activity of the experimental group and the positive control group was compared to assess the physicochemical stability of the antibodies.

[0034] S34. Different mammalian hemoglobin antigens were coated onto an immunoassay plate at a concentration of 1 μg / mL. GFBE protein-free solution was used as a negative control. Coating was performed overnight at 4°C. The next day, the liquid was removed, and the plate was washed with PBS and blocked with MPBS. Subsequently, the expressed antibody was added to the wells at serially diluted concentrations and incubated. After incubation, the wells were washed with PBST, incubated with anti-IgG Fc-HRP antibody, and then washed again. TMB was used for color development, and the reaction was stopped with H2SO4. OD was measured. 450 Value. The EC50 of the antibody was obtained by fitting. 50 The cross-reactivity of antibodies was assessed by comparing their binding ability to hemoglobin from different mammals.

[0035] This invention utilizes a pre-constructed shark-derived single-domain antibody synthesis library. Through biopanning of hemoglobin, a high-affinity single-domain antibody was finally isolated and named 13F-1A. Its nucleotide and amino acid sequences are as follows:

[0036] Nucleotide sequence:

[0037] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCTACAAAGAAGGAGAGCTTATCAAATGGCGGACGAT ACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATTCGCGGCAGTTCTCCTTCTGGCTGGGCAGGGCCGCGTTCGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0038] amino acid sequence:

[0039] TQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYSRQFSFWLGRAAFEGGGTILTVKP

[0040] The single-domain antibody includes two complementarity determining regions (CDR1 and CDR3), namely the first complementarity determining region CDR1 and the second complementarity determining region CDR3, and the sequences of the two complementarity determining regions are as follows:

[0041] CDR1: SSYALGST

[0042] CDR3: SRQFSFWLGRAAF

[0043] After fusing the single-domain antibody 13F-1A with the human IgG1 Fc fragment, it was cloned into the pTT5 vector and expressed secretoryly in mammalian 293F cells. Three days after expression, the 13F-1A-Fc fusion protein in the culture supernatant was purified using an rProtein A affinity chromatography column, and the antibody yield was found to be above 65 mg / L.

[0044] The 13F-1A antibody binds to hemoglobin with high affinity. ELISA assays showed that the affinity of the disclosed 13F-1A-Fc antibody for hemoglobin was 0.42 nM. Biolayer interferometry (BLI) experiments indicated that the equilibrium dissociation constant (K0) between hemoglobin and the 13F-1A-Fc antibody... D The value is 42.3 nM.

[0045] The effects of different pH values, temperatures, urea concentrations, and human plasma-like medium (HPLM) on the stability of the single-domain antibody were determined using enzyme-linked immunosorbent assay (ELISA). The results showed that the 13F-1A-Fc antibody has good physicochemical stability. Cross-reactivity of 13F-1A-Fc with other mammalian hemoglobins was assessed using non-competitive ELISA; no significant cross-reactivity was found with porcine, bovine, ovine, or rabbit hemoglobins.

[0046] Compared with the prior art, the present invention has the following advantages:

[0047] 1. Since the single-domain antibody of the present invention is derived from shark heavy chain antibody, it has the characteristics of high stability, high expression level and high affinity. The shark-derived single-domain antibody can stably detect the concentration of hemoglobin.

[0048] 2. The shark-derived single-domain antibody of the present invention has a small molecular weight, high solubility, easy recombination and expression, good thermal stability, strong folding ability, good tissue permeability, low polymerization tendency and strong targeting binding ability.

[0049] 3. The single-domain antibodies screened by this invention exhibit good stability, high specificity, and high affinity, providing a theoretical basis and technical support for the diagnosis and monitoring of related diseases targeting hemoglobin. Attached Figure Description

[0050] Figure 1 This is a graph showing the results of a monoclonal phage ELISA test.

[0051] Figure 2 This is an image of the SDS-PAGE gel electrophoresis results of the 13F-1A-Fc antibody;

[0052] Figure 3 This is an enzyme-linked immunosorbent assay (ELISA) to measure the binding affinity between 13F-1A-Fc antibody and hemoglobin;

[0053] Figure 4 It uses antibody affinity assay (BLI) to detect the affinity map between single-domain antibodies and hemoglobin;

[0054] Figure 5 The results show the physicochemical stability of the 13F-1A-Fc antibody under different conditions, as determined by ELISA.

[0055] Figure 6 This is a graph showing the cross-reactivity analysis of the 13F-1A-Fc antibody with other mammalian hemoglobins. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0057] Example 1: Screening and characterization of shark-derived single-domain antibodies that bind to hemoglobin

[0058] I. Screening of single-domain antibodies targeting hemoglobin

[0059] Step S1: The shark-derived single-domain antibody synthesis library was inoculated into 2×TY liquid medium containing 100 μg / mL ampicillin (Amp) and 2% glucose (G) (volume 100 mL) to allow the initial OD to be adjusted. 600 Approximately 0.1. Incubate with shaking at 37℃ and 200 rpm until OD... 600 Approximately 0.5–0.6 (OD) 600 The absorbance value refers to the absorbance of the solution at a wavelength of 600 nm. Then add 10 μL of 10 12 PFU-containing KM13 helper phages were incubated at 37°C for 45 min. After incubation, the bacterial culture was centrifuged for 10 min (37°C, 3500g), and the supernatant was discarded. The precipitated bacterial culture was resuspended in 200 mL of 2TY / Amp / kanamycin sulfate (Kana) / 0.1% G medium and cultured at 25°C and 200 rpm with shaking for 16 h. After incubation, the entire bacterial culture was centrifuged at 4°C and 3500g for 30 min. The entire supernatant was collected and added to 20% PEG / NaCl (polyethylene glycol / sodium chloride) at a ratio of 4:1. After mixing, the culture was incubated on ice for 1 h and centrifuged for 30 min (4°C, 3500g). The supernatant was discarded, and the precipitate was resuspended in autoclaved PBS (phosphate buffered saline solution). The precipitate was vortexed and transferred to a centrifuge tube, and centrifuged for 10 min (4°C, 12000 rpm). Transfer the supernatant to a new centrifuge tube and measure the OD. 260 value.

[0060] Step S2, Panning. Hemoglobin antigen was coated onto immunoplates at a concentration of 0.1 mg / mL, with a protein-free GFBE negative control included. After incubation overnight at 4°C, the coating solution was discarded, and the plates were washed three times with 280 μL of PBS. Then, 280 μL of MPBS (phosphate buffered saline) was added, and the reaction was blocked at room temperature for 2 hours. Subsequently, the plates were washed twice with 280 μL of PBST (phosphate buffered saline), and 1 × 10⁻⁶ mg / mL of PBS was added to the hemoglobin wells and the negative control wells. 11 The phage solution prepared above was incubated at 80 rpm for 1 h, the waste liquid was discarded, and the solution was washed 20 times with 280 μL of PBST. 100 μL of trypsin (0.5 mg / mL) was added to the hemoglobin group (Hb) and negative control wells, and the solution was shaken at 80 rpm for 1 h to elute the phage. Then, 100 μL of the eluted phage was used to infect 900 μL of OD200 solution. 600 Incubate approximately 0.5 μL of TG1 bacterial suspension in a 37°C water bath for 45 min. Spread 50 μL and 5 μL of the infected bacterial suspension onto LB / Amp plates and incubate overnight.

[0061] Step S3: Phage ELISA screening for single-domain antibody monoclonal antibodies against hemoglobin:

[0062] Step S31: Preparation of monoclonal phages: Pick 95 monoclonal phages from the overnight culture plate and inoculate them into 100 μL of 2×TY / Amp / 2% G, with one well serving as a negative control. Incubate in a 96-well plate with shaking for 6–8 h (37℃, 250 rpm). Then, aspirate 2.5 μL of bacterial culture from each well and re-inoculate into 100 μL of 2×TY / Amp / 2% G / KM13 medium (add 2.5 μL of KM13 to 12.5 mL of 2×TY / Amp / 2% G), and incubate again with shaking for 1.5 h (37℃, 250 rpm), followed by standing at 37℃ for 45 min. Add an equal volume of 30% glycerol to each well containing the remaining 97 μL of bacterial culture, and store the mixture at -80℃. After standing, thoroughly mix the bacterial culture and discard 50 mL. Centrifuge at 3200 g for 20 min to completely remove the supernatant. The precipitate was resuspended in 200 μL of 2TY / Amp / Kana / 0.1% G medium and incubated for 20 h (25℃, 250 rpm). After incubation, the phage was centrifuged for 40 min (4℃, 3200 g), and the monoclonal phage supernatant was transferred to a new 96-well plate and stored at 4℃.

[0063] Step S32, Monoclonal Phage ELISA Detection: Dilute hemoglobin with GFBE to 1 μg / mL, and coat each well of a 96-well immunoplate with 100 μL of the diluted solution. Use a protein-free GFBE solution as a negative control. Coat overnight at 4°C. Wash three times with PBS, add 300 μL of MPBS to each well, and block at room temperature for 2 h. Add 100 μL of the prepared phage-MPBS mixture to each well and incubate at room temperature for 1 h. Wash four times with PBST. Appropriately dilute HRP-anti-M13 antibody (1:8000 dilution in MPBS) with MPBS, add 100 μL to each well of the immunoplate, and incubate at room temperature for 1 h. Wash four times with PBST. Add 100 μL of TMB substrate to each well, wrap in aluminum foil to protect from light, and react at room temperature for 5 min. Stop the reaction by adding 50 μL of 1M H2SO4 to each well and measure the OD. 450 Value, result as Figure 1 As shown.

[0064] Step S33, All ODs 450 Positive clones with a value greater than 1 were sent to the company for sequencing. The sequencing results were analyzed and compared, and finally a single-domain antibody sequence was obtained, which was named shark-derived single-domain antibody 13F-1A as shown above.

[0065] II. Expression and purification of single-domain antibodies and their Fc fusion proteins

[0066] Primers were designed to fuse the N-terminus of the nanobody gene sequence with the IFNα protein signal peptide to guide secretory expression, and the C-terminus of the nanobody gene sequence was fused with human IgG1 Fc. A TEV restriction site was introduced between the two sequences, and the mixture was then cloned into the mammalian expression vector pTT5. The constructed recombinant vector was transiently transfected into HEK293F mammalian cells using PEI. After 3 days of culture, the supernatant was collected, and the 13F-1A-Fc fusion protein in the supernatant was purified using rProtein A affinity chromatography and analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 2 As shown, high-purity 13F-1A-Fc antibody was obtained after purification.

[0067] III. Characterizing Single-Domain Antibodies

[0068] Step S1: On an immunoassay plate, hemoglobin antigen was coated at a concentration of 1 μg / mL, with protein-free GFBE solution as a negative control. Coating was performed overnight at 4°C. The next day, the protein coating solution and the liquid in the protein-free control wells were removed. After washing twice with PBS, the plate was blocked with MPBS for 2 hours. The purified 13F-1A-Fc antibody was then injected at an initial concentration of 10 μg / mL. 3 Starting with nM, through continuous dilution to 10 -4 A total of 22 concentration gradients were set up, with nM. All dilution steps were performed in MPBS. After blocking, antibody protein solutions of each concentration gradient were added to pre-coated wells (100 μL per well) and incubated at 80 rpm for 1 h. Control wells contained only an equal volume of MPBS. To ensure data reliability, three parallel wells were set up for each concentration. After incubation, the wells were washed four times with PBST. 100 μL of anti-IgG Fc-HRP antibody (diluted 1:10000 in MPBS) was added to each well, incubated at 80 rpm for 1 h, and washed three more times with PBST. OD was measured after TMB colorimetric development and termination with 1 mol / L sulfuric acid (H2SO4) solution. 450 Value. Result as follows Figure 3 As shown, the 13F-1A-Fc antibody has good affinity for hemoglobin, with a half-maximal effective concentration (EC50) of 1 / 2. 50 The value is 0.42nM.

[0069] Step S2: Characterize the affinity between single-domain antibodies and hemoglobin using biomembrane interference (BLI) technology.

[0070] Given the highly specific affinity between Protein A and the Fc domain, a Protein A sensor was used in the experiment. Antibody immobilization was achieved through its specific binding to the Fc domain of the single-domain antibody Fc fusion protein. First, to ensure effective sensor operation, the sensor was pre-wetted with 200 μL of phosphate-buffered saline (PBST) for 10 min. After each experimental cycle of antibody-antigen binding and dissociation, the sensor was regenerated using 10 mM glycine solution (pH 2.0) to restore its initial state and ensure reusability. Subsequently, to investigate the interaction between the antibody and antigen at different concentrations, the antigen solution was serially diluted to four concentration gradients, and corresponding binding experiments were performed. Figure 4 It can be seen that the equilibrium dissociation constant K between 13F-1A-Fc and hemoglobin is... D The value is 42.3 nM. For example... Figure 4 As shown, the interaction between 13F-1A and hemoglobin was measured using a Protein A sensor. The solid line represents the real-time kinetic curve. BLItz Pro™ software was used to perform fitting analysis based on a 1:1 binding model to obtain the binding constant (ka), dissociation constant (kd), and equilibrium dissociation constant (K). D The kinetic curves of different antibody concentration gradients correspond from top to bottom to the concentrations marked on the right from top to bottom.

[0071] Step S3: Characterize the physicochemical stability of single-domain antibodies using ELISA.

[0072] This experiment used ELISA to detect the binding of antibodies to antigens in order to quantify antibody stability. Untreated antibodies served as a control, used to compare the stability of antibodies under different treatment conditions.

[0073] Step S31: To assess the tolerance of the expressed 13F-1A-Fc at different pH values, the antibody was diluted in PBS buffer with pH adjusted to 3.0 to 13.0 using HCl or NaOH and incubated at room temperature for 1 hour. Subsequently, its acid-base tolerance was assessed by measuring the binding activity of antibody samples at each pH value.

[0074] Step S2: To further investigate the stability of 13F-1A-Fc at different temperatures, the antibody was diluted in PBS and incubated for 5 minutes at different temperatures, including 4℃, 25℃, 37℃, 50℃, 70℃, and 90℃. After the antibody returned to room temperature and bound to the coated antigen, its binding activity was measured.

[0075] Step S33: To evaluate the stability of 13F-1A-Fc at different urea concentrations, different amounts of urea were added to PBS to form a concentration gradient of 0–8 mol / L. The antibody was dissolved in the urea and incubated overnight at room temperature. Its binding activity was measured, with GFBE solution used as a protein-free control.

[0076] Step S34: In addition, to simulate a plasma environment, the experiment used human plasma-like medium (HPLM) and PBS as controls. Antibody 13F-1A-Fc was incubated at 37°C for 0, 2, and 6 hours. Subsequently, the binding activity of the antibody in the two media was compared to assess its stability in a plasma environment.

[0077] Figure 5 A- Figure 5 The figures D represent the stability results at different temperatures, pH values, urea concentrations, and human plasma-like media (HPLM). Figure 5 It was found that the binding activity of 13F-1A-Fc decreased to 30% at 70℃ and almost completely lost its activity at 90℃, indicating poor thermal stability under high-temperature conditions. Under extremely acidic conditions (pH 2), the binding activity of 13F-1A-Fc was almost completely lost. Within the pH range of 4 to 10, its antibody binding activity showed some fluctuations relative to neutral conditions (pH 7). At pH 12, its binding activity significantly decreased to 60–70%. The binding activity of 13F-1A-Fc did not change significantly in 2–8 mol / L urea. Furthermore, the binding activity of 13F-1A-Fc was less affected by HPLM medium.

[0078] Step S4: Characterize the cross-reactivity between single-domain antibodies and hemoglobins from different mammalian sources using a non-competitive ELISA.

[0079] In immunochromatograms, hemoglobins from different mammalian sources were coated at a concentration of 1 μg / mL, with protein-free GFBE solution used as a negative control. Coating was performed overnight at 4°C. The next day, the protein coating solution and the liquid in the protein-free control wells were removed. After washing twice with PBS, the plates were blocked with MPBS for 2 hours. The 13F-1A-Fc antibody protein was then added at an initial concentration of 10 μg / mL. 3 Starting with nM, through continuous dilution to 10 -4A total of 22 concentration gradients were set up, with nM. All dilution steps were performed in MPBS. After blocking, antibody protein solutions of each concentration gradient were added to pre-coated wells (100 μL per well) and incubated at 80 rpm for 1 h. Control wells contained only an equal volume of MPBS. To ensure data reliability, three parallel wells were set up for each concentration. After incubation, the wells were washed four times with PBST. 100 μL of anti-IgG Fc-HRP antibody (diluted 1:10000 in MPBS) was added to each well, incubated at 80 rpm for 1 h, and washed three more times with PBST. OD was measured after TMB colorimetric development and termination with 1 mol / L H2SO4 solution. 450 Value. Result as follows Figure 6 As shown, the 13F-1A-Fc antibody has a good affinity for human hemoglobin and no significant cross-reactivity with porcine, bovine, ovine, and rabbit hemoglobin.

[0080] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0081] SEQ ID NO.1:

[0082] Name: Nucleotide sequence of shark-derived single-domain antibody

[0083] DNA type: other DNA

[0084] Biological source: Chiloscyllium plagiosum

[0085] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCTACAAAGAAGGAGAGCTTATCAAATGGCGGACGAT ACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATTCGCGGCAGTTCTCCTTCTGGCTGGGCAGGGCCGCGTTCGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0086] SEQ ID NO.2:

[0087] Name: Amino acid sequence of shark-derived single-domain antibody

[0088] Sequence type: AA

[0089] Biological source: Chiloscyllium plagiosum

[0090] TQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKKES LSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYSRQFSFWLGRAAFE GGGTILTVKP.

Claims

1. A shark-derived single-domain antibody that binds to hemoglobin, characterized in that: The amino acid sequence of the shark-derived single-domain antibody is shown in SEQ ID NO.

2.

2. The application of the shark-derived single-domain antibody that binds to hemoglobin as described in claim 1, characterized in that: Application of the shark-derived single-domain antibody in the preparation of reagents for detecting hemoglobin concentration.

3. The application of the shark-derived single-domain antibody that binds to hemoglobin according to claim 2, characterized in that: The reagents used to detect hemoglobin concentration are diagnostic reagents for anemia.

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