Preparation and application of anti-digoxin nanobodies

Abstract

The application discloses a digoxin nanobody and a preparation method and application thereof. The amino acid and nucleotide sequences of the digoxin nanobody are shown in SEQ ID NO. 8-9. The antibody has good affinity to a target antigen, and the dissociation constant Kd value is about 73.1 nM, and can be used for digoxin content determination, nucleic acid probe detection and in-vivo digoxin neutralization and detoxification. The antibody has the advantages of small volume, high stability, low cost, low immunogenicity and the like, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the preparation and application of anti-digoxin nanobodies. BACKGROUND

[0002] Cardiac glycosides are a class of steroidal compounds distributed in medicinal plants (such as Digitalis lanata) of the plant families of the Apocynaceae and Scrophulariaceae, and the animal medicine toad venom, which have high medicinal value. Digoxin is a representative drug of cardiac glycosides, which is widely used in the treatment of low-output type congestive heart failure, atrial fibrillation, atrial flutter, paroxysmal supraventricular tachycardia and other cardiovascular diseases in clinic, with definite efficacy and long history (Muk et al. 2020). Domestic and foreign scholars have found that endogenous digoxin exists in the human body, which is closely related to the occurrence and development of cardiovascular diseases, immune system diseases, tumors, etc., and is speculated to be one of the potential biomarkers of diseases (Hamlyn et al. 1982, Manunta et al. 2011, Yang et al. 2023). In addition to physiological and pharmacological activities, digoxin is also a commonly used reagent for non-radioactive nucleic acid probe labeling, which is widely used in nucleic acid molecule hybridization experiments, with many advantages such as no radioactive pollution, high sensitivity, rapid detection, etc. (Hafner et al. 2000).

[0003] Although digoxin is a commonly used drug for clinical cardiovascular diseases, it is generally believed that the treatment window of the drug is relatively narrow, and there is a large individual difference, so that a little carelessness can easily cause acute digoxin poisoning and patient death, so it is necessary to develop high-affinity, low-immunogenicity digoxin neutralizing antibodies to cope with rapid digoxin poisoning in clinic and reduce deaths. Secondly, endogenous digoxin has potential physiological and pathological functions in many diseases, but its content is low, and it is difficult to detect by conventional ELISA methods, so it is necessary to develop high-affinity digoxin antibodies to improve the detection sensitivity of endogenous digoxin and further reveal the mechanism of action of the active substance, and to find potential biomarkers and drug action targets. In addition, given the important application value of digoxin in nucleic acid probe labeling and hybridization detection, it is also necessary to develop digoxin antibodies with stronger affinity and lower cost than traditional antibodies to further improve the detection sensitivity and reduce the detection cost.

[0004] Currently, the antibodies against digoxin are mostly rabbit polyclonal antibodies or mouse hybridoma monoclonal antibodies prepared by traditional methods, which have the disadvantages of large molecular weight, high immunogenicity, weak penetration, long preparation period and high cost. Nanobody is a single-domain heavy chain antibody naturally lacking light chains only existing in camelids (llama, dromedary and guanaco) and some cartilaginous fishes (shark and silver shark), and is the smallest functional antigen-binding fragment. Compared with traditional antibodies, the molecular weight of nanobody is small, and the molecular weight is only about 1 / 10 of that of traditional monoclonal antibodies, about 15 kDa, and can be expressed in a soluble form in microorganisms, greatly shortening the production period and reducing the preparation cost. Nanobody also has low immunogenicity, high heat resistance and acid and alkali resistance, and has great potential application value in the fields of disease detection and treatment (De Meyer et al. 2014). SUMMARY

[0005] The application provides a nanobody of digoxin, which has high affinity with digoxin molecules and can be used for determination of digoxin content, detection of digoxin nucleic acid probe and neutralization and blocking of digoxin in vivo.

[0006] The digoxin nanobody of the application contains CDR1 shown by SEQ ID NO. 1: GRSISGFA, CDR2 shown by SEQ ID NO. 2: IMWSGRDT, CDR3 shown by SEQ ID NO. 3: AAATRLPLNSASSYNI, FR1 shown by SEQ ID NO. 4: EVQLVDSGGGLVQP, FR2 shown by SEQ ID NO. 5: MGWFRQGPGKEREFVSS, FR3 shown by SEQ ID NO. 6: YYADSVKGRFTISRDPAKNTVYLQMNSLKPEDTAVYYC and FR4 shown by SEQ ID NO. 7: WGQGTQVTVSS.

[0007] The amino acid sequence of the digoxin nanobody is SEQ ID NO. 8:

[0008]

[0009] The application provides a gene encoding the digoxin nanobody of the application.

[0010] The nucleotide sequence of the gene is SEQ ID NO. 9:

[0011]

[0012] The application provides an E. coli expression host which expresses the digoxin nanobody of the application, and / or comprises the gene or nucleic acid construct of the application.

[0013] The application provides the use of the digoxin nanobody in the preparation of an ELISA method for detecting the content of digoxin.

[0014] The application provides a kit for detecting digoxin, which detects digoxin based on a competitive ELISA method, and uses the nanobody shown in SEQ ID NO. 8 as a detection antibody.

[0015] The application provides the use of the digoxin nanobody as a secondary antibody for detecting a nucleic acid probe labeled with digoxin.

[0016] The application provides the use of the digoxin nanobody which can neutralize and antagonize the toxicity of digoxin in vivo.

[0017] Compared with the prior art, the application has the following advantages:

[0018] 1. The digoxin nanobody provided by the application has strong affinity (dissociation equilibrium constant Kd≈73nM) with digoxin, and can efficiently bind to a digoxin antigen.

[0019] 2. Compared with a traditional monoclonal antibody, the nanobody has stronger affinity, smaller molecular weight and many other advantages, and can be expressed in a large amount of soluble form by a prokaryotic cell, and has low cost.

[0020] 3. The antibody has small molecular weight, is easy to be humanized, is convenient to be coupled with various groups, and can greatly expand the actual use function and multi-scene application value of the antibody. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is an SDS-PAGE electrophoresis result of a digoxin-BSA conjugate, BSA is bovine serum albumin, and DIG-BSA is a digoxin-bovine serum albumin conjugate.

[0022] Figure 2 It is an agarose electrophoresis result of two rounds of PCR, lane 1 and lane 2 in the left figure are first-round PCR amplification products, and lane 1 and lane 2 in the right figure are second-round PCR amplification products.

[0023] Figure 3 It is a vector and fragment enzyme digestion result, lane 1 and lane 2 are PCR product enzyme digestion results, and lane 3 is a Pcomb3xss vector enzyme digestion result.

[0024] Figure 4 It is a phage ELISA result, and the positive clones and negative clones are selected according to a value of more than 5 times the blank control as a demarcation line.

[0025] Figure 5 The alignment results for the candidate clone IMGT.

[0026] Figure 6 To express and purify prokaryotic proteins for nanobodies.

[0027] Figure 7 The results of purification by cation exchange resin are shown. Lanes 1-8 correspond to batches of components collected at different retention times on the ion exchange column.

[0028] Figure 8 The results are from Western blotting analysis; components 3, 4, and 5 are... Figure Seven The protein components collected by the cation exchange resin shown are illustrated.

[0029] Figure 9 The results are from the isothermal titration calorimetric method.

[0030] Figure 10 This is the result of micro-thermophoretic dynamic detection.

[0031] Figure 11 This is the result of the ELISA standard curve.

[0032] Figure 12 The results show the application of digoxigenin nanobody in the detection of digoxigenin-labeled nucleic acid.

[0033] Figure 13 The results show the stability analysis of the nanobody. Detailed Implementation

[0034] The present invention will be further described below with reference to the embodiments, but the description of the embodiments does not limit the scope of protection of the present invention in any way.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0036] Unless otherwise specified, all substances or instruments used in the following examples can be obtained from conventional commercial sources.

[0037] Example 1: Digoxin-BSA conjugation and alpaca immunization

[0038] 1. Coupling between digoxin and BSA

[0039] Digoxin was conjugated to the BSA vector using the potassium periodate oxidation method. The conjugation products were detected by SDS-PAGE protein electrophoresis. Figure 1As shown, the labeled digoxin-BSA electrophoresis has obvious smear, the main band is light, and there are obvious uneven product distribution above 66 kDa, confirming successful coupling.

[0040] 2. Animal immunization

[0041] The coupled digoxin-BSA was used as an immunogen to immunize the blank llama. The animal immunization scheme is shown in the following table. After each immunization, blood was collected to monitor the immune response.

[0042]

[0043]

[0044] The immune llama serum titer was detected by indirect ELISA method, as shown in Table 1, compared with the unimmunized llama, the llama antigen serum titer level increased to 10 3 times after the first immunization, the serum titer level increased to 10 4 times after the third immunization, and the serum titer level reached 10 5 times after the fifth immunization. This result shows that the antigen produces a strong immune response in the llama.

[0045] Table 1 Llama serum titer detection table

[0046]

[0047] Example Two: Construction of a digoxin nanobody phage library

[0048] Experimental method

[0049] 1. Synthesis of cDNA and nested PCR amplification

[0050] The llama vein blood after the sixth round of immunization was taken, and the lymphocytes were separated by lymphocyte separation medium to obtain total RNA in the lymphocytes by Trizol total RNA extraction kit, and the concentration and purity of RNA were detected by ultraviolet spectrophotometer and RNA electrophoresis. The RNA was used as a template to obtain a cDNA library by reverse transcription. The amount of RNA used in the reverse transcription of 10 μL system was 500 ng, and the reverse transcription system was prepared according to the following table:

[0051]

[0052] The cDNA was used as a template to amplify the fragment containing the heavy chain variable region VHH gene by PCR, and the PCR system was prepared according to the following table:

[0053]

[0054] The system is prepared, placed in PCR instrument for amplification, and the reaction procedure is shown as follows:

[0055]

[0056] The specific amplification primer is as follows:

[0057]

[0058] In order to increase the capacity of the library, the first round of gel recovery product is used as a PCR template, and the second round of nested PCR reaction is carried out according to the conditions shown in the following table:

[0059]

[0060] The system is prepared, placed in PCR instrument for amplification, and the second round of PCR reaction procedure is shown as follows:

[0061]

[0062] The specific amplification primer is as follows:

[0063]

[0064] 2. Construction of phage library

[0065] The pComb3xss phagemid vector is used as a phage library construction vector, and the vector and the nested PCR product are simultaneously digested by restriction endonuclease Sfi I.

[0066] The 20 μL reaction system is prepared as follows:

[0067]

[0068]

[0069] The vector and the fragment are simultaneously digested at 50℃ for 60 min, and then agarose electrophoresis is carried out, and the digested vector and the fragment are recovered for a ligation reaction. The T4 ligase is used for ligation, 20 ng of the digested vector and 8 ng of the digested insert (molar ratio of 1:4) are taken for ligation, and the ligation system is prepared according to the following table:

[0070]

[0071] Then the ligation system is connected at 22℃ for 30 min, and after the ligation is completed, it is transformed into TG1 competent cells and cultured in a 37℃ constant temperature incubator overnight.

[0072] Experimental results:

[0073] 1. Nested PCR amplification

[0074] This invention uses nested PCR to amplify the VHH gene, such as... Figure 2 The total RNA extracted above was reverse transcribed to obtain cDNA, and the VHH gene fragment was amplified by two rounds of PCR using two pairs of specific primers. The first round of PCR products were analyzed by agarose gel electrophoresis. A DNA band of approximately 700 bp was visible in lane 1. The target band was recovered from the gel and used as a template for the next round of PCR. The second round of PCR products were analyzed by agarose gel electrophoresis, yielding a DNA band of approximately 400 bp. The target band was recovered from the gel for subsequent construction of recombinant plasmids. The band sizes of both rounds of PCR amplification products matched the expected DNA fragment size and were suitable for phage library construction.

[0075] 2. Building the Library

[0076] This invention uses the Pcomb3xss vector to construct a phage library. As shown in the figure, the amplified VHH fragment product is digested with the phage vector pComb3xss, and both are digested with Sfi I enzyme. Figure 3 The clearly visible vector restriction enzyme bands were 1700bp and 3200bp in size, respectively, which met the expected size and achieved good restriction enzyme digestion results. The digested vector and fragment were ligated in the correct proportions to obtain the target phage particle vector.

[0077] Example 3: Screening of phage display libraries and identification of positive clones

[0078] Experimental methods:

[0079] 1. Construction of phage libraries

[0080] This invention uses M13KO7 helper phage for superinfection. All clones from the library plate are scraped off, diluted with 10 mL of 2×TY medium, and frozen. 100 μL of the bacterial culture is added to an Erlenmeyer flask containing 2×TY liquid culture medium and incubated on a shaker until the bacteria reach the logarithmic growth phase (OD). 600= 0.3-0.5. According to the volume ratio of M13KO7 helper phage: bacterial liquid = 1:100, the helper phage was added, and the 37°C constant temperature incubator was incubated for 30 min. The bacterial liquid was moved to a 50 mL centrifuge tube and centrifuged at 2700 rpm for 10 min at 4°C. The centrifugal precipitate was resuspended and added to 250 mL of 2xTY + 100 mg / mL ampicillin + 50 mg / mL kanamycin liquid medium. The horizontal shaker parameters were set to 37°C and 225 rpm shaking culture overnight for 16 h. The culture was collected and centrifuged at 20000 g for 30 min at 4°C. The supernatant was added to 1 / 4 volume of PEG8000 / NaCl, mixed well by inverting, and placed in an ice bath for not less than 30 min. Centrifugation was performed at 4000 rpm for 30 min at 4°C, and the supernatant was discarded. The phage precipitate was resuspended in 1 mL of PBS solution and placed in a 1.5 mL Eppendorf tube, centrifuged at 20000 g for 1 min at 4°C, and the centrifugal supernatant was collected. A small amount was used for determination of the phage titer, and the rest was stored in a -80°C refrigerator. The phage titer was determined by the double-layer agar method. The phage was diluted to 10 -1 pfu / mL, 100 μL of the phage was added, and the incubation was performed for 2 h, and the plate was washed 10 times. -10 pfu / mL, 100 μL of the phage was added, and the incubation was performed for 2 h, and the plate was washed 10 times.

[0081] 2. Phage library panning

[0082] According to the combination, washing, elution, and amplification, the phage display nanobody library was subjected to affinity panning, and the specific process was as follows:

[0083] 1. Antigen coating: The digoxin-OVA was coated on a 96-well enzyme-labeled plate using an antigen coating buffer, and the coating concentration was 15 μg / mL, 10 μg / mL, and 7 μg / mL in turn, and the coating was performed overnight, and the plate was washed 5 times with PBST.

[0084] 2. Blocking: 5% BSA blocking, and the plate was washed 5 times with PBST.

[0085] 3. The phage titer was adjusted to 10 -11 pfu / mL, 100 μL of the phage was added, and the incubation was performed for 2 h, and the plate was washed 10 times.

[0086] 4. Elution: 100 μL of glycine was added for elution, 15 min later, Tris-HCl was added for neutralization, and the elution was completed.

[0087] 5. Infection: The eluate was used to infect XL1-Blue E. coli

[0088] 6. Titer detection

[0089] 7. Amplification: Spread the infected bacteria on a plate, then collect all colonies, and superinfect with M13KO7 helper phage.

[0090] 8. Proceed to the next round of screening, for a total of three rounds.

[0091] 3. Identification of positive clones

[0092] Positive clones were screened using a phage ELISA. Specifically, 60 clones were selected from the third-round screening plates and transferred to 96-well deep-well plates. M13K07 helper phage was added for superinfection for 14 h. After centrifugation, 100 μL of the supernatant was transferred to a 96-well ELISA plate pre-coated with digoxigenin-OVA and incubated at 37°C for 45 min. The plates were washed 6 times with PBST, and 100 μL of HRP-labeled anti-M13 monoclonal antibody was added to each well, and the plates were incubated at 37°C for 45 min. After washing 6 times with PBST, 100 μL of TMB chromogenic solution was added to each well, and the plates were incubated at 37°C for 10 min. Finally, 50 μL of 2 mol / L dilute sulfuric acid was added to terminate the reaction, and the OD values ​​were immediately read using an ELISA reader. 450 Value. Wells with a value 5 times higher than the negative control were sequenced to obtain the final nanobody sequence.

[0093] Experimental results:

[0094] 1. Enrichment and panning of bacteriophage libraries

[0095] After the original library was constructed, the phage library was subjected to three rounds of biological panning. In the first round, the concentration of the coating antigen digoxigenin-OVA was 15 μg / mL; in the second round, the concentration was 10 μg / mL; and in the third round, the concentration was 7 μg / mL. The results are shown in Table 2. Compared with the first round, the third round of panning enriched the number of positive clones by approximately 1000 times, and the recovery rate was significantly improved. Furthermore, the ratio of phage input to elution was nearly stable in the second and third rounds of panning, further indicating that the positive clones were significantly enriched.

[0096] Table 2. Phage enrichment parameters

[0097]

[0098] 2. Phage ELISA and screening of positive clones

[0099] This invention uses an indirect ELISA method to screen positive clones. Single clones are selected from an enriched library through multiple rounds of screening, and the peripheral blood expression products of the single clones are detected by indirect ELISA. For example... Figure 4 As shown, a blank control OD was selected. 450The index of 5 times or more is the boundary line, and finally multiple positive clones are selected for DNA sequencing. The best clone is selected for subsequent experiments. It encodes a nanobody against digoxin.

[0100] The amino acid sequence is analyzed, and it has a typical stable structure of nanobody, and the amino acid sequences of the framework region FR region and the complementarity determining region CDR region are as shown in Figure 5

[0101] Example Four: Expression and purification of nanobody

[0102] Experimental method

[0103] 1. Construction of prokaryotic expression vector

[0104] The present application uses pET-28a as an expression vector, and the enzyme cutting site is selected as NheI and XhoI. The above positive clone is used as a template for PCR, and the enzyme cutting site NheI-BamHI is added at both ends. The PCR product is subjected to agarose electrophoresis, the fragment is recovered from the gel, and the concentration is measured.

[0105] The PCR amplification system is shown in the following table:

[0106]

[0107] The reaction system is as follows:

[0108]

[0109]

[0110] The specific amplification primer sequence is as follows:

[0111]

[0112] The pET-28a plasmid and the above gel recovery product are cut with Nhe I and Xho I, and subjected to agarose electrophoresis detection at 37℃ for 30min. The enzyme cutting fragment is recovered from the gel, and the concentration is measured. The enzyme cutting system is shown in the following table:

[0113]

[0114] The enzyme-cutting vector and fragment are connected at a molar ratio of 1:4, and the connection reaction system is shown in the following table,

[0115]

[0116] After 30min of connection at 22℃, it is transformed into BL21(DE3) competent cells, and the next day the positive clone is sequenced.

[0117] 2. Prokaryotic expression of nanobody​

[0118] The colony with correct sequencing was induced for protein expression, 100 mM IPTG was used to induce for 16 h at 4°C, the supernatant was collected after the bacteria were broken by ultrasonic, and the supernatant was purified by a nickel column, the impurities were washed away by a washing solution, the nanobody was eluted by adding an elution solution, and PBS was added to the obtained nanobody solution for ultrafiltration (3600 rpm / min, 10 min, repeated twice), and the liquid in the ultrafiltration tube was collected.

[0119] The AKTA protein purification instrument was used for ion exchange chromatography to further purify the target protein, the buffer system was 0.02 M Tris-HCl, 1 M NaCl, pH 8.0, the system flow rate was 0.85 mL / min according to the pressure setting of the instrument, the sample loop was used for loading after pre-equilibrating the chromatographic column, and the components at different times were collected by using an automatic component collector. The protein components were detected by SDS-PAGE electrophoresis analysis, and the protein components were stored in a refrigerator at -80°C.

[0120] Experimental results:

[0121] 1. Expression and purification of nanobody

[0122] The prokaryotic expression recombinant plasmid was constructed, 100 mM IPTG was used to induce for 16 h at 4°C, and SDS-PAGE electrophoresis analysis was performed on the protein lysate and the elution solution after purification, and the results are shown in Figure 6 .

[0123] 2. Further purification of nanobody

[0124] The AKTA protein purification instrument was used for ion exchange chromatography to further purify the target protein, as shown in Figure 7 , SDS-PAGE electrophoresis analysis was performed on the protein samples at different retention times, and the target protein samples with a purity of >95% were obtained in the 3rd, 4th and 5th tubes. At the same time, the WB analysis results are shown in Figure 8 , there is an obvious nanobody band near 14 kDa.

[0125] Example Five: Determination of the binding force of digoxin nanobody

[0126] Experimental method:

[0127] 1. Isothermal titration calorimetry for detecting the affinity of nanobody

[0128] The digoxin nanobody was diluted to a concentration of 20 μM (dissolved in PBS), and was placed in a sample cell. PBS solution was placed in a reference cell. Digoxin powder was dissolved in PBS to a concentration of 200 μM, and was placed in a titration syringe. After placement, titration was performed according to the operation manual, and the Kd value was determined.

[0129] 2. MST detects nanobody binding force

[0130] The present application detects the affinity of antibody protein and antigen by micro-thermal swing (MST) technology, which requires at least one fluorescent label of ligand molecules or receptor protein. The present application selects Monolith RED-NHS second-generation protein labeling kit to label the target antibody.

[0131] (I) Protein labeling

[0132] In a 1.5 mL Eppendorf tube, add 7 μL of RED-NHS second-generation dye and 7 μL of NHS labeling buffer, and mix well by blowing and sucking to obtain a dye solution with a final concentration of 300 μM. Take another new 1.5 mL Eppendorf tube, and add 90 μL of protein sample (concentration 10 μM). Take 10 μL of the above dye solution and add it to the protein sample, and mix well by blowing and sucking to obtain 100 μL of dye protein solution, and incubate at room temperature in the dark for 30 min.

[0133] (II) Determination of labeling efficiency

[0134] The labeling efficiency is calculated according to the following formula:

[0135]

[0136] The labeling efficiency (DOL) is calculated according to the following formula:

[0137]

[0138] First, dilute the digoxin solution by gradient, prepare a PBS solution containing 0.05% Tween-20, dilute the digoxin to 1 μM with the solution, and take 10 μL into a 0.2 mL PCR tube, and mark the PCR tube as No. 1 PCR tube, and sequentially number up to No. 16. Add 10 μL of PBS solution containing 0.05% Tween-20 to No. 2 to No. 16 PCR tubes, and dilute No. 1 PCR tube to No. 16 PCR tube by gradient, so that the digoxin ligand concentration of the latter tube is exactly half of the former tube. Dilute the labeled nanobody to be tested to a concentration of 10 nM, and add 10 μL of the labeled nanobody solution to each PCR tube, and mix well by blowing with a pipette. The concentration of the antibody protein to be tested in each PCR tube is 5 nM. Add an equal volume of labeled antibody protein to the PCR tube, and suck it into a capillary tube, and detect the affinity of the antibody and the ligand by MST. Use a special capillary tube to suck the liquid in the above-mentioned PCR tube, and pay attention not to suck air bubbles. Set the parameters on the machine, and detect the affinity of the nanobody and digoxin.

[0139] Experimental results:

[0140] 1. Isothermal titration calorimetry for detecting the affinity of nanobody

[0141] The present application determines the affinity of nanobody and digoxin by isothermal titration calorimetry, as shown in the following formula: Figure 9 The reaction ΔH = -4.28 x 10 6 cal / mol, ΔS = -1.12 x 10 4 cal / mol / deg, and ΔG = -9.26 Kcal / mol are determined at 298.15 K temperature. The Kd value is calculated to be 160 nM.

[0142] 2. Microscale thermophoresis for determining the affinity of nanobody

[0143] The present application labels the antibody protein with nucleic acid dye by using Monolith RED-NHS second-generation protein labeling kit. The affinity between the candidate antibody protein and digoxin is determined by microscale thermophoresis. As shown in the following formula, the affinity is determined in a short time by measuring the fluorescence change generated by the reaction of the protein and the small molecule, and the results show that the fluorescence distribution of the protein is uniform, indicating that the fluorescence protein labeling effect is good, and the Kd of the nanobody and digoxin is measured to be 73.1 nM. Figure 10

[0144] Example Six: Competition binding method ELISA for detecting digoxin

[0145] Experimental method:

[0146] 1. Exploration of the optimal antibody concentration

[0147] 100 μL of digoxin-OVA with a concentration of 200 ng / mL was added to the adsorption 96-well ELISA plate, and coated overnight; the liquid in the wells was discarded, and washed with PBST for 5 times, 100 μL of OVA was added for room temperature blocking for 1 h; the liquid in the wells was discarded, and washed with PBST for 5 times, a series of concentrations of biotin-labeled digoxin nanobody was added for room temperature incubation for 2 h; the liquid in the wells was discarded, and washed with PBST for 5 times, 100 μL of streptomycin-HRP was added for room temperature incubation for 1 h; the liquid in the wells was discarded, and washed with PBST for 5 times, 100 μL of TMB color developing liquid was added, and incubated at 37°C for 10 min, and then 50 μL of color developing termination liquid was added. The absorbance was detected by the enzyme label instrument at 450 nm; the optimal antibody concentration was selected for the subsequent detection experiment.

[0148] 2. Competition ELISA binding experiment

[0149] Preparation of standard curve:

[0150] ​Doxin-OVA 100 μL with a concentration of 20 ng / mL was added to a high adsorption 96-well ELISA plate for overnight coating; the liquid in the wells was discarded, and the wells were washed with PBST for 5 times, 100 μL of OVA was added for 1 h of room temperature blocking; the liquid in the wells was discarded, and the wells were washed with PBST for 5 times, for the standard group, 100 μL of doxin with a concentration of 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.6 ng / mL was added. For the blank control group (B0), 100 μL of PBS was added. For the detection group, 100 μL of the serum sample to be detected was added. 1 μg / mL of biotin-labeled doxin nanobody was added to each sample well for 2 h of room temperature incubation; the liquid in the wells was discarded, and the wells were washed with PBST for 5 times, 100 μL of streptomycin-HRP was added for 1 h of room temperature incubation; the liquid in the wells was discarded, and the wells were washed with PBST for 5 times, 100 μL of TMB color developing liquid was added, and after 10 min of 37°C incubation, 50 μL of color developing termination liquid was added. The absorbance was detected by a microplate reader at 450 nm; the average value of the absorbance of each concentration standard solution and sample (B) was divided by the absorbance value of the first blank (0 standard) (B0), and then multiplied by 100%, that is, the percentage absorbance value; the logarithmic value of the doxin concentration was taken as the X axis, and the percentage absorbance value was taken as the Y axis to draw a standard curve graph.

[0151] Sample addition recovery rate experiment:

[0152] A 0.1 ml doxin solution with a concentration of 2 ng / mL, 20 ng / mL, and 200 ng / mL was prepared, and the absorbance value of the above-mentioned sample to be detected was detected. According to the average absorbance value of the sample well measured, the logarithmic value of the doxin concentration can be obtained from the standard curve, and the inverse logarithm is the doxin concentration in the sample. The recovery rate (Recovery) and the coefficient of variation (RSD) of doxin samples with different concentrations were calculated.

[0153] Experimental results:

[0154] The antigen was immobilized, and the working concentration of the antibody was determined to be about 1 μg / mL. The optimal antibody concentration was selected for subsequent detection experiments. The standard curve of the competitive ELISA established based on the nanobody VHH-A9 with the amino acid sequence shown in SEQ ID NO: 8. The OD 450 The average value is B0, and the OD 450 The average value is B. The logarithmic value of the standard concentration was taken as the abscissa, and the B / B0 ratio was taken as the ordinate. Linear fitting was performed, and the standard curve was drawn as shown in Figure 11 .

[0155] The results of the recovery rate and coefficient of variation calculated by adding 2 ng / mL, 20 ng / mL, and 200 ng / mL of digoxin, respectively, are shown in Table 3.

[0156] Table 3: Table of detection of digoxin and recovery rate

[0157]

[0158] Example Seven: Nanobody detection of digoxin-labeled nucleic acid probe

[0159] Experimental method:

[0160] Nanobody detection of digoxin-labeled nucleic acid probe:

[0161] The digoxin-labeled nucleic acid probe was dissolved to a concentration of 128 μΜ as the starting concentration for the experiment. The digoxin-labeled nucleic acid probe was then diluted in a gradient, with each subsequent tube having a concentration of 1 / 2 of the previous tube. Subsequently, 1 μL of the gradient-diluted digoxin-labeled nucleic acid probe solution was pipetted using a multichannel pipette and carefully dropped onto the NC membrane. After ultraviolet crosslinking for 30 min using an ultraviolet crosslinker, the biotin-labeled nanobody was incubated overnight. After washing three times with PBST, streptavidin-HRP was added and incubated at room temperature for 1 h. After washing three times with PBST, ECL luminescence detection was performed.

[0162] Determination of nanobody stability:

[0163] The nanobody and a commercially available digoxin monoclonal antibody were placed in an incubator at 37°C for 24 h or 72 h, respectively, and then co-incubated with the digoxin-labeled nucleic acid probe on the NC membrane. The minimum detection concentration of the nanobody and the commercially available monoclonal antibody was analyzed and detected.

[0164] Experimental results:

[0165] As shown in the results in Figure 12 , the digoxin nanobody VHH-A9 could specifically recognize the digoxin nucleic acid probe, and the detection signal intensity gradually decreased as the concentration of the digoxin-labeled nucleic acid probe decreased. The minimum detection concentration was about 2 -1 pM.

[0166] As shown in the results in Figure 13 , after being placed at 37°C for 24 h, the ability of the control digoxin monoclonal antibody (Dig-mAb) to detect the digoxin-labeled nucleic acid probe decreased, and the minimum detection concentration decreased to 24 pM. After being placed at 37°C for 72 h, the binding ability of the control digoxin monoclonal antibody was basically lost, and the minimum detection concentration decreased to 2 7 pM. In contrast, the binding ability of the digoxin nanobody did not change substantially after being placed at 37°C for 24 h, and the minimum detection concentration was about 2 2 pM. Even after being placed at 37°C for 72 h, the minimum detection concentration remained at 2 4pM minimum detectable concentration. This result shows that the stability of the digoxin nanobody is significantly better than that of the commercial digoxin mAb.

[0167] Example Eight: Nanobody rescues digoxin poisoning

[0168] Experimental method:

[0169] Male C57BL / 6 mice with a body weight of 18-22 g were randomly divided into three groups, nanobody group: 10 mg / kg of nanobody VHH-A9 was injected through the tail vein; digoxin group: 3.6 mg / kg of digoxin was injected intraperitoneally to form a digoxin poisoning model; digoxin-nanobody group: 3.6 mg / kg of digoxin was injected intraperitoneally, and 10 mg / kg of nanobody VHH-A9 was injected through the tail vein at the same time. The survival rate of the test animals in each group was observed and counted.

[0170] Experimental results:

[0171] As shown in Table 5, intraperitoneal injection of digoxin (3.6 mg / kg) caused about half of the mice to die, and the nanobody (10 mg / kg) injected through the tail vein at the same time as the intraperitoneal injection of digoxin significantly reduced the mortality rate of the test mice (P<0.01). Injection of the nanobody alone had no significant effect on the survival of the mice. The results show that the nanobody of the present application can reduce the toxicity caused by digoxin, increase the survival rate of the mice, and rescue digoxin poisoning.

[0172] Table 4 Animal mortality table

[0173]

[0174]

[0175] ** P<0.01; statistically significant.

Claims

1. A digoxin nanobody, characterized in that, The amino acid sequence is shown as SEQ ID NO.

8.

2. A gene encoding the nanobody of digoxin according to claim 1, and the nucleotide sequence is shown as SEQ ID NO.

9.

3. A method for preparing the nanobody of digoxin according to claim 1 by genetic engineering.

4. The use of the nanobody of digoxin according to claim 1 in the preparation of a kit for detecting the content of digoxin or a diagnostic kit.

5. The use of the nanobody of digoxin according to claim 1 in the detection of a nucleic acid probe labeled with digoxin.

Images