Shark source single-domain antibody combined with hemoglobin and application of shark source single-domain antibody

Abstract

The invention discloses a shark source single-domain antibody combined with hemoglobin and application of the shark source single-domain antibody. The nucleotide sequence of the single-domain antibody is labeled as SEQ ID NO.1, the amino acid sequence corresponding to the single-domain antibody is recorded as SEQ ID NO.2, the shark-source single-domain antibody is used for detecting hemoglobin concentration, and the detection method comprises the following steps: setting a standard substance group and a sample group, then adding the shark-source single-domain antibody into immune pore plates of the standard substance group and the sample group, and then adding the single-domain antibody into the immune pore plates of the standard substance group and the sample group. And measuring the OD450 values of the standard substance group and the sample group after color development, drawing an OD450 value standard curve according to the OD450 value of the standard substance group, and finally obtaining the hemoglobin concentration of the sample group according to the OD450 value standard curve. The prepared single-domain antibody shows good stability, high specificity and high affinity, and the screened single-domain antibody provides theoretical basis and technical support for subsequent diagnosis and monitoring of related diseases taking hemoglobin as a target spot.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a shark-derived single-domain antibody that binds to hemoglobin and an application thereof. Background Art

[0002] Hemoglobin is a protein present in red blood cells that can combine with oxygen to form oxyhemoglobin and is responsible for transporting oxygen from the lungs to various tissues and organs in the body. Hemoglobin detection can reflect the oxygen carrying capacity of the blood, which is of great significance for evaluating anemia, monitoring the blood status of surgical patients, and guiding blood transfusions. In addition, hemoglobin content is closely related to cardiovascular diseases, colon cancer and other diseases, showing the importance of hemoglobin detection in disease diagnosis, prognosis assessment and treatment monitoring. Commonly used hemoglobin detection methods include optical measurement and immunological methods, and non-invasive hemoglobin monitoring is gradually being introduced. Detection based on optical measurement systems generally requires large and expensive dedicated equipment and professional laboratory equipment and technicians. The results of non-invasive hemoglobin monitoring are affected by many factors, and the accuracy and reliability need to be evaluated in combination with other clinical indicators. The immunological method based on antigen-antibody recognition has higher sensitivity and accuracy. Therefore, it is necessary to develop hemoglobin-targeted antibodies with strong specificity and high sensitivity to improve the accuracy of detection. Traditional antibodies have defects in penetrating living cells, and interference with the function of target proteins also limits their practical application. In 1995, Greenberg et al. discovered that there are new antibodies composed only of heavy chains in cartilaginous fish such as the nurse shark, called immunoglobulin new antigen receptors (IgNAR). The antigen-binding fragment of IgNAR is called the variable domain of immunoglobulin new antigen receptor (VNAR), which is located at the end of a single heavy chain. The molecular weight of VNAR is about 12kDa, which is only one-tenth the size of traditional monoclonal antibodies. It is the smallest antigen-binding unit known so far, also known as a single-domain antibody. Single-domain antibodies have small molecular weight, high solubility, easy recombinant expression, good thermal stability, strong folding ability, good tissue permeability, low aggregation tendency and strong targeted binding ability. In addition, single-domain antibodies can quickly penetrate tissue barriers and maintain their high specificity and affinity, which can effectively overcome the limitations of traditional antibodies, making them an effective supplement to antibody therapy. Therefore, single-domain antibodies have great development potential in the fields of drug development, disease diagnosis and treatment. At present, there are no shark-derived single-domain antibodies that bind to hemoglobin. Summary of the invention

[0003] In order to solve the problems existing in the background technology, the purpose of the present invention is to provide a shark-derived single domain antibody that binds to hemoglobin and its application. The present invention provides a shark-derived heavy chain antibody variable region sequence that can bind to hemoglobin with high affinity, which is crucial for diagnosing diseases, assessing prognosis and monitoring treatment.

[0004] The technical solution adopted by the present invention is as follows:

[0005] 1. 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] 2. Application of a shark-derived single-domain antibody that binds to hemoglobin: Application of the shark-derived single-domain antibody in the preparation of reagents related to detecting hemoglobin concentration.

[0009] The reagents for detecting hemoglobin concentration include anemia diagnostic reagents and colon cancer diagnostic reagents.

[0010] 3. A method for detecting hemoglobin concentration using a shark-derived single domain antibody comprises the following steps:

[0011] Step S1, first, setting a standard group, a sample group and a blank group, wherein the standard group adds different concentrations of hemoglobin standards to the immunoplate, the sample group adds the hemoglobin sample to be tested to the immunoplate, and the blank group does not add any sample to the immunoplate;

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

[0013] Step S2: Then, the standard group, sample group and blank group were blocked with bovine serum albumin (BSA), and then shark-derived single domain antibodies were added to the immunoplates of the standard group, sample group and blank group. After incubation, the immunoplates were washed with PBST, and goat anti-human IgG Fc-HRP antibodies were added to each well of the immunoplates. After incubation, the immunoplates were washed with PBST again, and then TMB was added to each well for color development. After color development, sulfuric acid (H2SO4) was added to terminate the reaction, and the absorbance OD of the standard group, sample group and blank group was measured respectively. 450 Then, the absorbance OD of the standard group and the sample group were 450 The absorbance OD of the blank group was subtracted from the value 450 The corrected absorbance OD of the standard group and sample group was obtained. 450 value;

[0014] Step S3: according to the concentration of the hemoglobin standard in the standard group and the corrected absorbance OD 450 Value, plot absorbance OD 450 The standard curve of the absorbance OD 450 The standard curve of the values ​​was used to obtain the OD value after correction of the sample group. 450 The concentration corresponding to the value is the hemoglobin concentration of the sample group.

[0015] Specifically, the concentration of the hemoglobin standard in the standard group is used as the horizontal axis, and the corresponding corrected absorbance OD 450 The absorbance OD was obtained by linear regression analysis. 450 The standard curve of the values.

[0016] 4. A method for preparing and characterizing a shark-derived single domain antibody that binds to hemoglobin, comprising the following steps:

[0017] Step S1: Screening 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 step S1 is specifically as follows:

[0021] S11. First, the existing shark-derived single-domain antibody synthetic library is inoculated into a liquid culture medium, and then a helper phage is added to the liquid culture medium to promote the proliferation of the phage. After static culture and centrifugal collection of the phage, the phage is shaken and cultured overnight to obtain more phages, and then the phages are enriched by PEG precipitation method to obtain the titer of the enriched phages;

[0022] S12, dissolving hemoglobin in a protein-free solution GFBE to prepare a hemoglobin solution, and then coating the 0.1 mg / mL hemoglobin solution in an immunoplate as a hemoglobin group, and using an immunoplate not coated with the hemoglobin solution as a negative control group, respectively adding S11-enriched phages to the immunoplates of the hemoglobin group and the negative control group, so that the phages bind to the hemoglobin in the hemoglobin group, and then washing the immunoplate with PBST (phosphate buffer), and after washing, obtaining the detached phages in the hemoglobin group and the negative control group respectively;

[0023] S13. The phages of the hemoglobin group and the negative control group were used to infect TG1 bacteria (Escherichia coli), and then the phages infected with TG1 bacteria were spread on a plate. After culturing for 12-24 hours, single colonies were obtained. The number of single colonies of the hemoglobin group and the negative control group was counted and compared:

[0024] If the number of single colonies in the hemoglobin group is greater than 10 times that of the negative control group, it means that the number of single colonies in the hemoglobin group meets the requirement, and the process goes to S14;

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

[0026] S14, selecting a single colony in the hemoglobin group and placing it in an immune well plate, adding a helper phage, and amplifying to obtain a monoclonal phage;

[0027] S15. Use hemoglobin solution to coat the immunoplate where the monoclonal phage is located, then add horseradish peroxidase-labeled phage-specific antibodies to the immunoplate, and then add TMB (3,3',5,5'-tetramethylbenzidine) colorimetric solution to develop the monoclonal phage. Measure the absorbance OD 450 The monoclonal phage with a gene expression of S1>1 is sequenced, and the repeated sequences are removed using sequence alignment software. The nucleotide sequence obtained after sequencing and removing the repeated sequences is used as the nucleotide sequence of the coding gene of the shark-derived single domain antibody. Among them, the nucleotide sequence obtained by S1 has hemoglobin specificity.

[0028] The S2 is specifically:

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

[0030] The step S3 is specifically as follows:

[0031] S31. Hemoglobin antigen was coated on the immunoplate at a concentration of 1 μg / mL. The negative control group was a protein-free solution. The plate was coated overnight at 4°C. The next day, the liquid was removed, the plate was washed with phosphate buffer PBS, and the plate was blocked with MPBS (phosphate buffer plus 5% milk). Subsequently, the expressed antibody was diluted to multiple concentrations and added to the wells for incubation. After incubation, the wells were washed with PBST (phosphate buffer plus 0.1% Tween-20), incubated with goat anti-human IgG Fc-HRP antibody, washed again, developed with TMB, terminated with sulfuric acid H2SO4, and the OD was measured. 450 The half effective concentration (EC) of the antibody was obtained by fitting. 50 value.

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

[0033] S33, hemoglobin antigen was coated on the immunoplate at a concentration of 1 μg / mL, coated overnight at 4°C, the coating liquid was removed, washed with PBS, blocked with MPBS, the untreated antibody was used as the negative control, and the antibodies treated with different conditions were used as the experimental group. The untreated and treated antibodies were added to the wells for incubation, washed with PBST after incubation, incubated with goat anti-human anti-IgG Fc-HRP antibody, and then washed. TMB color development, H2SO4 termination, OD measurement 450 The antibody binding activity of the experimental group and the positive control group was compared to evaluate the physicochemical stability of the antibody.

[0034] S34. Different mammalian hemoglobin antigens were coated onto the immunoplate at a concentration of 1 μg / mL, and GFBE protein-free solution was used as a negative control. The plates were coated overnight at 4°C. The next day, the liquid was removed, washed with PBS, and blocked with MPBS. Subsequently, the expressed antibodies were diluted to multiple concentrations and added to the wells for incubation. 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, H2SO4 was used for termination, and OD was measured. 450 The EC value of the antibody was obtained by fitting. 50 The cross-reactivity of antibodies was evaluated by comparing their binding abilities to different mammalian hemoglobins.

[0035] The present invention utilizes the constructed shark-derived single domain antibody synthetic library, and finally isolates a high-affinity single domain antibody by bio-panning of hemoglobin, and names it 13F-1A. The nucleotide sequence and amino acid sequence are as follows:

[0036] Nucleotide sequence:

[0037] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCTACAAAGAAGGAGAGCTTATCAAATGGCGGACGAT ACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATTCGCGGCAGTTCTCCTTCTGGCTGGGCAGGGCCGCGTTCGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0038] Amino Acid Sequence:

[0039] TQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYSRQFSFWLGRAAFEGGGTILTVKP

[0040] The single domain antibody comprises two complementary antigen determining regions (CDR1 and CDR3), namely, a first complementary antigen determining region CDR1 and a second complementary antigen determining region CDR3. The sequences of the two complementary antigen determining regions are as follows:

[0041] CDR1:SSYALGST

[0042] CDR3:SRQFSFWLGRAAF

[0043] The single-domain antibody 13F-1A was fused with the human IgG1 Fc segment and cloned into the pTT5 vector. The mammalian cell 293F was used for secretory expression. After 3 days of expression, the 13F-1A-Fc fusion protein in the culture supernatant was purified using an rProtein A affinity chromatography column. The antibody production was found to be above 65 mg / L.

[0044] The 13F-1A antibody can bind to hemoglobin with high affinity. The ELISA test showed that the affinity of the 13F-1A-Fc antibody disclosed in the present invention for binding to hemoglobin was 0.42 nM. The biolayer interferometry (BLI) test showed that the equilibrium dissociation constant (K) between hemoglobin and the 13F-1A-Fc antibody was 0.42 nM. D ) is 42.3nM.

[0045] The effects of different pH, temperature, urea concentration, and human plasma-like medium (HPLM) on the stability of single-domain antibodies were determined by ELISA, and the results showed that the 13F-1A-Fc antibody had good physical and chemical stability. The cross-reactivity of 13F-1A-Fc with hemoglobin from other mammals was evaluated by non-competitive ELISA, and it had no significant cross-reactivity with porcine, bovine, sheep, and rabbit hemoglobin.

[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 antibodies, 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 recombinant expression, good thermal stability, strong folding ability, good tissue permeability, low aggregation tendency and strong targeted binding ability.

[0049] 3. The single-domain antibodies screened by the present invention exhibit good stability, high specificity and high affinity, providing a theoretical basis and technical support for the subsequent diagnosis and monitoring of related diseases targeting hemoglobin. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a graph of monoclonal phage ELISA results;

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

[0052] Figure 3 This is a graph showing the binding affinity of 13F-1A-Fc antibody to hemoglobin measured by ELISA;

[0053] Figure 4 The affinity diagram between single domain antibodies and hemoglobin is detected by BLI using antibody affinity determination;

[0054] Figure 5 The results of ELISA testing the physical and chemical stability of 13F-1A-Fc antibody under different conditions;

[0055] Figure 6 This is a cross-reactivity analysis chart of the 13F-1A-Fc antibody with hemoglobin from other mammals. DETAILED DESCRIPTION

[0056] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0057] Example 1. Screening and characterization of shark-derived single-domain antibodies binding to hemoglobin

[0058] 1. Screening of single-domain antibodies targeting hemoglobin

[0059] Step S1: Inoculate the shark-derived single-domain antibody synthetic library into 2×TY liquid culture medium containing 100 μg / mL ampicillin (Amp) and 2% glucose (G) (volume: 100 mL) to make the initial OD 600 The culture was shaken at 37°C and 200 rpm until the OD 600 About 0.5~0.6(OD 600 The value refers to the absorbance value of the solution at 600nm wavelength). Then add 10μL 10 12 pfu of KM13 helper phage, and let it stand at 37°C for 45 minutes. After the standing, centrifuge the bacterial solution for 10 minutes (37°C, 3500g), and remove the supernatant. Resuspend the precipitated bacteria in 200mL of 2TY / Amp / Kanamycin sulfate (Kana) / 0.1%G medium, and shake and culture at 25°C and 200rpm for 16 hours. After the end, centrifuge the entire bacterial solution at 4°C and 3500g for 30 minutes. Take all the supernatant, add 20% PEG / NaCl (polyethylene glycol / sodium chloride) in a ratio of 4:1, mix well, let it stand in an ice bath for 1 hour, and centrifuge for 30 minutes (4°C, 3500g). Discard the supernatant, resuspend the precipitate with PBS (phosphate buffered saline) sterilized by high temperature and high pressure, vortex the precipitate and place it in a centrifuge tube, and centrifuge it for 10 minutes (4°C, 12000rpm). Transfer the supernatant to a new centrifuge tube and measure the OD 260 value.

[0060] Step S2, panning. Coat the hemoglobin antigen at a concentration of 0.1 mg / mL in the immunowell plate, and set up a negative control of protein-free solution GFBE. After standing overnight at 4°C, discard the coating solution and wash three times with 280 μL of PBS. Then add 280 μL of MPBS (phosphate buffer solution) and block the reaction at room temperature for 2 hours. Then wash twice with 280 μL of PBST (phosphate buffer), add 1×10 11 pfu The phage solution prepared above was incubated at 80 rpm for 1 h, the waste liquid was discarded, and 280 μL of PBST was used for 20 washes. 100 μL of trypsin (0.5 mg / mL) was added to the hemoglobin group Hb and negative control wells, and the phages were eluted at 80 rpm for 1 h. Then 100 μL of the eluted phage was taken to infect 900 μL OD 600 About 0.5% TG1 bacterial solution was incubated in a 37°C water bath for 45 min. 50 μL and 5 μL of the infected bacterial solution were spread on LB / Amp plates respectively and cultured overnight.

[0061] Step S3, phage ELISA screening of anti-hemoglobin single domain antibody monoclonal:

[0062] Step S31, preparation of monoclonal phage: 95 monoclones were picked from the overnight culture plate and inoculated into 100 μL 2×TY / Amp / 2% G respectively. Another well was used as a negative control and cultured in a 96-well culture plate with shaking for 6-8 hours (37°C, 250 rpm). Subsequently, 2.5 μL of bacterial solution was taken from each well and re-inoculated into 100 μL 2×TY / Amp / 2% G / KM13 culture medium (2.5 μL KM13 was added to 12.5 mL 2×TY / Amp / 2% G), and cultured again with shaking for 1.5 hours (37°C, 250 rpm), and then allowed to stand at 37°C for 45 minutes. An equal amount of 30% glycerol was added to the remaining 97 μL bacterial solution wells, and the mixed solution was stored in a -80°C refrigerator. After standing, the bacterial solution was thoroughly mixed and 50 mL was discarded, centrifuged at 3200g for 20 minutes, and the supernatant was completely removed. The pellet was resuspended in 200 μL 2TY / Amp / Kana / 0.1% G medium and cultured for 20 h (25°C, 250 rpm). After the culture, the plate was centrifuged for 40 min (4°C, 3200 g), and the monoclonal phage supernatant was transferred to a new 96-well plate and stored at 4°C.

[0063] Step S32, monoclonal phage ELISA detection: dilute hemoglobin to 1 μg / mL with GFBE, take 100 μL to coat the 96-well immunoplate, and set up a protein-free solution GFBE as a negative control, and coat overnight at 4°C. Wash 3 times with PBS, add 300 μL MPBS to each well, and block at room temperature for 2 hours. Add 100 μL of the phage MPBS mixture prepared above to each well and incubate at room temperature for 1 hour. Wash the plate 4 times with PBST. Use MPBS to moderately dilute the HRP-anti M13 antibody (diluted in MPBS at a ratio of 1:8000), add 100 μL to each well of the above immunoplate, and incubate at room temperature for 1 hour. Wash the plate 4 times with PBST. Add 100 μL TMB colorimetric substrate to each well, wrap it with aluminum foil to avoid light, and react at room temperature for 5 minutes. Add 50 μL of 1M H2SO4 to each well to terminate the reaction and measure OD 450 Value, the result is Figure 1 shown.

[0064] Step S33: All OD 450 The positive clones with values ​​greater than 1 were sent to the company for sequencing, and the sequencing results were analyzed and compared. Finally, a single-domain antibody sequence was screened out and named as the above shark-derived single-domain antibody 13F-1A.

[0065] 2. Expression and purification of single domain antibodies and their Fc fusion proteins

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

[0067] 3. Characterization of Single Domain Antibodies

[0068] Step S1: On the immunoplate, the hemoglobin antigen was coated at a concentration of 1 μg / mL, and the protein-free solution GFBE was used as a negative control, and both were coated overnight at 4°C. The next day, the protein coating solution and the liquid in the protein-free control wells were removed, and the wells were washed with PBS twice and blocked with MPBS for 2 hours. The purified 13F-1A-Fc antibody was added at an initial concentration of 10 3 nM as the starting point, and serial dilution to 10 -4 nM, a total of 22 gradient concentrations were set. All dilution steps were performed in MPBS. After blocking, each concentration gradient antibody protein solution was added to the pre-coated wells (100 μL per well) and incubated at 80 rpm for 1 hour. Only an equal amount of MPBS was added to the control wells. To ensure data reliability, three parallel wells were set for each concentration. After incubation, the wells were washed 4 times with PBST. 100 μL of anti-IgG Fc-HRP antibody (diluted in MPBS at a ratio of 1:10000) was added to each well, incubated at 80 rpm for 1 hour, and washed again with PBST 3 times. After TMB color development and termination with 1moL / L sulfuric acid H2SO4 solution, OD was measured. 450 The result is Figure 3 As shown in Figure 2, the affinity of 13F-1A-Fc antibody to hemoglobin is good, and the half effective concentration EC 50 The value is 0.42nM.

[0069] Step S2: Characterizing the affinity between single domain antibodies and hemoglobin using biofilm interferometry (BLI)

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

[0071] Step S3: Characterization of the physicochemical stability of the single domain antibody using ELISA

[0072] This experiment uses ELISA to detect the binding of antibodies to antigens to quantify the stability of antibodies. Untreated antibodies are used as controls to compare the stability of antibodies under different treatment conditions.

[0073] Step S31: In order to evaluate the tolerance of the expressed 13F-1A-Fc at different pH values, the antibody was diluted in a PBS buffer adjusted to a pH value of 3.0 to 13.0 using hydrochloric acid HCl or sodium hydroxide NaOH, and incubated at room temperature for 1 hour. Subsequently, the acid-base tolerance of the antibody sample was evaluated by measuring the binding activity at each pH value.

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

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

[0076] Step S34: In addition, to simulate the plasma environment, the experiment used human plasma-like medium (HPLM) and PBS as a control, and the 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 evaluate its stability in the plasma environment.

[0077] Figure 5 A- Figure 5 D are the stability results in different temperatures, pH values, urea concentrations and human plasma-like medium (HPLM). Figure 5 It can be seen that the binding activity of 13F-1A-Fc drops to 30% at 70°C and almost completely loses its activity at 90°C, and its thermal stability is poor under high temperature environment. Under extremely acidic conditions (pH 2), the binding activity of 13F-1A-Fc is almost lost. In the range of pH 4 to 10, its antibody binding activity shows a certain fluctuation relative to neutral conditions (pH 7). When the pH is 12, its binding activity is significantly reduced to 60-70%. The binding activity of 13F-1A-Fc did not change significantly in 2-8 mol / L urea. In addition, the binding activity of 13F-1A-Fc is less affected by HPLM culture medium.

[0078] Step S4, characterizing the cross-reactivity between the single domain antibody and hemoglobin from different mammalian sources using non-competitive ELISA;

[0079] On the immunoplate, hemoglobin from different mammalian sources was coated at a concentration of 1 μg / mL, and a protein-free solution GFBE was used as a negative control, all at 4°C overnight. The next day, the protein coating solution and the liquid in the protein-free control wells were removed, and the wells were washed with PBS twice and blocked with MPBS for 2 hours. The 13F-1A-Fc antibody protein was added at an initial concentration of 10 3 nM as the starting point, and serial dilution to 10 -4nM, a total of 22 gradient concentrations were set. All dilution steps were performed in MPBS. After blocking, each concentration gradient antibody protein solution was added to the pre-coated wells (100 μL per well) and incubated at 80 rpm for 1 hour. Only an equal amount of MPBS was added to the control wells. To ensure data reliability, three parallel wells were set for each concentration. After incubation, the wells were washed 4 times with PBST. 100 μL of anti-IgG Fc-HRP antibody (diluted in MPBS at a ratio of 1:10000) was added to each well, incubated at 80 rpm for 1 hour, and washed again with PBST 3 times. After TMB color development and termination with 1moL / L H2SO4 solution, OD was measured 450 The result is Figure 6 As shown, the 13F-1A-Fc antibody has a good affinity for human hemoglobin and has no significant cross-reaction with pig, bovine, sheep, and rabbit hemoglobin.

[0080] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is 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 in the scope of protection 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] Organism 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] Organism 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. A shark-derived single domain antibody binding to hemoglobin according to claim 1, characterized in that: The nucleotide sequence of the shark-derived single-domain antibody encoding gene is shown in SEQ ID NO.

1.

3. The use of a shark-derived single domain antibody that binds to hemoglobin according to any one of claims 1 to 2, characterized in that: The shark-derived single-domain antibody is used in the preparation of reagents related to detecting hemoglobin concentration.

4. The use of a shark-derived single domain antibody binding to hemoglobin according to claim 3, characterized in that: The reagent for detecting hemoglobin concentration is an anemia diagnostic reagent.

5. The use of a shark-derived single domain antibody binding to hemoglobin according to claim 3, characterized in that: The reagent for detecting hemoglobin concentration is a colon cancer diagnostic reagent.

6. A method for detecting hemoglobin concentration using a shark-derived single domain antibody as described in any one of claims 1-2, characterized in that: The following steps are involved: Step S1, first, setting a standard group, a sample group and a blank group, wherein the standard group adds different concentrations of hemoglobin standards to the immunoplate, the sample group adds the hemoglobin sample to be tested to the immunoplate, and the blank group does not add any sample to the immunoplate; Step S2: adding shark-derived single domain antibodies to the standard group, sample group and blank group, and measuring the absorbance OD of the standard group and sample group after adding shark-derived single domain antibodies 450 value; Step S3: according to the absorbance OD of the standard group and the sample group 450 The hemoglobin concentration of the sample group to be tested is obtained.

7. The method for detecting the hemoglobin concentration of a shark-derived single domain antibody according to claim 6, characterized in that: The step S2 is specifically as follows: the standard group, the sample group and the blank group are blocked with bovine serum albumin BSA, and then the shark-derived single domain antibody is added to the immunoplate of the standard group, the sample group and the blank group, and the immunoplate is washed with PBST after incubation, and goat anti-human IgG Fc-HRP antibody is added to each well of the immunoplate, and the immunoplate is washed with PBST again after incubation, and then TMB is added to each well for color development, and sulfuric acid H2SO4 is added to terminate the reaction after color development, and the absorbance OD of the standard group, the sample group and the blank group is measured respectively. 450 Then, the absorbance OD of the standard group and the sample group were 450 The absorbance OD of the blank group was subtracted from the value 450 The corrected absorbance OD of the standard group and sample group was obtained. 450 value.

8. The method for detecting the hemoglobin concentration of a shark-derived single domain antibody according to claim 6, characterized in that: The step S3 is specifically as follows: according to the concentration of the hemoglobin standard in the standard group and the corrected absorbance OD 450 Value, plot absorbance OD 450 The standard curve of the absorbance OD 450 The standard curve of the values ​​was used to obtain the OD value after correction of the sample group. 450 The concentration corresponding to the value is the hemoglobin concentration of the sample group.

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