CN118878690A - Preparation and application of an anti-digoxin nanobody - Google Patents

Abstract

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

Description

Preparation and application of an anti-digoxin nanobody

Technical Field

The invention relates to the preparation and application of an anti-digoxin nano antibody.

Background Art

Cardiotonic steroids are steroidal compounds distributed in medicinal plants such as Apocynaceae and Scrophulariaceae (such as Digitalis purpurogena) and animal medicine Toad Venom, and have high medicinal value. Digoxin is a representative drug of cardiotonic steroids. It is widely used in the clinic to treat cardiovascular diseases such as low-output congestive heart failure, atrial fibrillation, atrial flutter, paroxysmal supraventricular tachycardia, etc. It has a definite effect and a long history (Muk et al. 2020). Domestic and foreign scholars have found that endogenous digoxin exists in the human body and is closely related to the occurrence and development of cardiovascular diseases, immune system diseases, tumors, etc. It is speculated to be one of the potential biomarkers of the disease (Hamlyn et al. 1982, Manunta et al. 2011, Yang et al. 2023). In addition to its physiological and pharmacological activities, digoxin is also a commonly used reagent for non-radioactive nucleic acid probe labeling. It is widely used in nucleic acid molecular hybridization experiments and has many advantages such as no radioactive pollution, high sensitivity, and rapid detection (Hafner et al. 2000).

Although digoxin is a commonly used drug for clinical cardiovascular diseases, it is generally believed that the drug has a relatively narrow therapeutic window and large individual differences. If you are not careful, it is easy to cause acute digoxin poisoning and patient death. Therefore, it is necessary to develop digoxin neutralizing antibodies with high affinity and low immunogenicity to deal with rapid digoxin poisoning that occurs clinically 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 with conventional ELISA methods. 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 this active substance, and discover potential biomarkers and drug 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 detection sensitivity and reduce detection costs.

At present, most antibodies against digoxin are 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 cycle and high cost. Nanobodies are single-domain heavy chain antibodies that naturally lack light chains and are only found in camelids (alpacas, Bactrian camels and llamas) and some cartilaginous fish (sharks and chimaeras). They are the smallest functional antigen-binding fragments. Compared with traditional antibodies, nanobodies have a small molecular weight, with a molecular weight of only about 1/10 of that of traditional monoclonal antibodies, about 15 kDa, and can be expressed soluble in microorganisms, greatly shortening the production cycle and reducing preparation costs. Nanobodies also have low immunogenicity, high heat resistance, acid and alkali resistance, and have great potential application value in disease detection and treatment (De Meyer et al. 2014).

Summary of the invention

The invention provides a nano antibody of digoxin, which has a high affinity with digoxin molecules and can be used for digoxin content determination, digoxin nucleic acid probe detection and digoxin neutralization and blocking in vivo.

The digoxin nanobody of the present invention 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.

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

The present invention provides a gene, which encodes the digoxin nanobody of the present invention.

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

The present invention provides an Escherichia coli expression host, which expresses the digoxin nanobody described in the present invention and/or contains the gene or nucleic acid construct described in the present invention.

The present invention provides the use of the digoxin nano-antibody in preparing an ELISA method for detecting digoxin content.

The present invention provides a kit for detecting digoxin. The kit detects digoxin based on a competitive ELISA method, and the kit uses a nano antibody shown in SEQ ID NO.8 as a detection antibody.

The present invention provides an application of using digoxin nano antibody as a secondary antibody to detect a digoxin-labeled nucleic acid probe.

The present invention provides an application of a digoxin nano-antibody capable of neutralizing and antagonizing digoxin toxicity in vivo.

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

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

2. Compared with traditional monoclonal antibodies, this nanobody has many advantages such as stronger affinity and smaller molecular weight, and can be expressed in large quantities in a soluble form through prokaryotic cells at low cost.

3. This antibody has a small molecular weight, is easy to humanize, and is convenient to couple with a variety of groups, which can greatly expand the actual use function and multi-scenario application value of the antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the SDS-PAGE electrophoresis result of digoxin and BSA conjugate, BSA is bovine serum albumin, DIG-BSA is digoxin-bovine serum albumin conjugate

Figure 2 shows the agarose electrophoresis results of two rounds of PCR. Lanes 1 and 2 on the left: products of the first round of PCR amplification; Lanes 1 and 2 on the right: products of the second round of PCR amplification

FIG3 shows the results of restriction digestion of vectors and fragments, lanes 1 and 2: the results of restriction digestion of PCR products; lane 3: the results of restriction digestion of Pcomb3xss vector.

Figure 4 shows the results of phage ELISA, with positive clones and negative clones selected based on a numerical cutoff of 5 times that of the blank control.

FIG5 shows the IMGT alignment results of candidate clones.

Figure 6 shows the expression and purification of nanobody prokaryotic protein.

FIG7 shows the purification results of the cation exchange resin, where lanes 1-8 correspond to batches of components collected at different retention times on the ion exchange column.

FIG8 is a Western Blot test result, where components 3, 4, and 5 are protein components collected by the cation exchange resin shown in FIG7 .

FIG9 is the isothermal titration calorimetry test results.

FIG. 10 shows the results of micro-thermophoresis detection.

FIG. 11 shows the ELISA standard curve results.

FIG. 12 shows the application results of digoxin nanoantibody in digoxin-labeled nucleic acid detection.

Figure 13 shows the results of stability analysis of Nanobodies.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

Example 1: Conjugation of digoxin-BSA and immunization of alpacas

1. Conjugation of digoxin-BSA

Digoxin was coupled to the BSA carrier using potassium periodate oxidation. The coupled product was detected by SDS-PAGE protein electrophoresis. As shown in Figure 1, the labeled digoxin-BSA electrophoresis had obvious dragging, the main band became lighter, and there was obvious uneven product distribution above 66 kDa, confirming that the coupling was successful.

2. Animal Immunization

The coupled digoxigenin-BSA was used as an immunogen to immunize blank alpacas. The animal immunization scheme is shown in the following table. Blood was collected after each immunization to monitor the immune response.

The serum titer of the immunized alpacas was detected by indirect ELISA. As shown in Table 1, compared with the unimmunized alpacas, the serum titer level of the alpacas antigen increased to 10 ³ times after the first immunization, 10 ⁴ times after the third immunization, and 10 ⁵ times after the fifth immunization. This result shows that the antigen produces a strong immune response in the alpaca.

Table 1 Alpaca serum titer test table

Example 2: Construction of digoxin nanoantibody phage library

Experimental methods

1. cDNA Synthesis and Nested PCR Amplification

After the sixth round of immunization, alpaca venous blood was obtained, lymphocytes were separated by lymphocyte separation solution, total RNA in lymphocytes was extracted by Trizol total RNA extraction kit, and the concentration and purity of RNA were detected by UV spectrophotometer and RNA electrophoresis. RNA was used as a template to obtain a cDNA library by reverse transcription. The amount of RNA used in the reverse transcription 10μL system was 500ng, and the reverse transcription system was prepared as shown in the following table:

Using cDNA as a template, PCR was used to amplify the fragment containing the heavy chain variable region VHH gene. The PCR system was prepared as shown in the following table:

After the system is prepared, it is placed in a PCR instrument for amplification. The reaction procedure is as follows:

Specific amplification primers are as follows:

In order to increase the capacity of the library, the present invention uses the first round of gel recovery products as PCR templates and performs a second round of nested PCR reaction according to the conditions shown in the following table:

After the system is prepared, it is placed in a PCR instrument for amplification. The second round of PCR reaction procedure is as follows:

Specific amplification primers are as follows:

2. Construction of phage library

The present invention adopts pComb3xss phagemid vector as the vector for constructing the phage library. Firstly, the vector and the nested PCR product are simultaneously digested with restriction endonuclease Sfi I.

Prepare 20 μL reaction system as follows:

The vector and the fragment were simultaneously digested at 50°C for 60 minutes and then subjected to agarose electrophoresis to recover the digested vector and the fragment for ligation. The present invention uses T4 ligase for ligation, and 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:

The enzyme ligation system was then connected at 22°C for 30 min, and after the connection was completed, it was transformed into a TG1 competent cell and cultured in a 37°C constant temperature incubator overnight.

Experimental results:

1. Nested PCR Amplification

The present invention adopts nested PCR to amplify VHH gene. As shown in FIG2 , the total RNA extracted above is reverse transcribed to obtain cDNA, and two pairs of specific primers are used to amplify VHH gene fragments in two rounds of PCR. The first round of PCR amplification products are subjected to agarose gel electrophoresis analysis. A DNA band of about 700 bp in size can be seen in lane 1, and the target band is recovered by gel as a template for the next round of PCR. The second round of PCR products are subjected to agarose gel electrophoresis analysis to obtain a DNA band of about 400 bp in size, and the target band product is recovered by gel for subsequent construction of recombinant plasmid. The band sizes of the two rounds of PCR amplification products are consistent with the expected DNA fragment size, and can be used for phage library construction.

2. Library Construction

The present invention adopts Pcomb3xss vector to construct phage library. As shown in the figure, the VHH fragment product after amplification and the phagemid vector pComb3xss are digested, and both are digested by Sfi I enzyme. As shown in Figure 3, clear vector digestion bands are 1700bp and 3200bp in size, which are consistent with the expected vector digestion band size and achieve good digestion effect. The digestion vector and the fragment are connected in proportion to obtain the target phagemid vector.

Example 3: Panning of phage display library and identification of positive clones

Experimental methods:

1. Construction of phage library

The present invention uses M13KO7 helper phage for super infection, scrapes all the library plate clones, dilutes and freezes them with 10mL 2×TY culture medium, takes 100μL of bacterial solution and adds it to a conical flask containing 2×TY liquid culture, and shakes it on a constant temperature shaker until the bacteria grow in the logarithmic phase _OD600 = 0.3-0.5. Add helper phage according to the volume ratio of M13KO7 helper phage: bacterial solution = 1:100, and let it stand in a 37°C constant temperature incubator for 30 minutes. Move the bacterial solution to a 50mL centrifuge tube and centrifuge at 2700rpm for 10 minutes at 4°C. Resuspend the centrifugal precipitate and add it to 250mL 2×TY+100mg/mL ampicillin+50mg/mL kanamycin liquid culture medium. Set the horizontal shaker parameters to 37°C, shake at 225rpm and culture overnight for 16 hours. Collect the culture and centrifuge at 20000g for 30 minutes at 4°C. Add 1/4 volume of PEG8000/NaCl to the supernatant, mix by inversion, and place in an ice bath for no less than 30 minutes. Centrifuge at 4000rpm for 30 minutes at 4°C, and discard the supernatant. Add 1mL PBS solution to the phage precipitate and resuspend it in a 1.5mL Eppendorf tube, centrifuge at 20000g for 1min at 4°C, collect the centrifugal supernatant, take a small amount for the determination of phage titer, and freeze the rest in a -80°C refrigerator. The present invention uses a double-layer agar method to determine the phage titer, dilute the phage with LB culture medium to ^10-1 to ^10-10 pfu, take 1μL of each dilution phage in 100μL of TG1 bacterial solution, mix, stand at 37°C for 20min, add 3mL of top agar, mix and pour on LB solid culture medium plate, culture overnight at 37°C, count plaques and calculate phage titer.

2. Panning of phage library

The above phage-displayed nanoantibody library was affinity panned in the manner of binding, washing, eluting, and amplification, as follows:

1. Antigen coating: Use antigen coating buffer to coat digoxigenin-OVA on a 96-well ELISA plate. The coating concentrations for three rounds are 15 μg/mL, 10 μg/mL and 7 μg/mL, respectively. Coat overnight and wash the plate 5 times with PBST.

2. Blocking: Block with 5% BSA and wash the plate 5 times with PBST.

3. Adjust the phage titer to 10 ^-11 pfu/mL, add 100 μL of phage, incubate for 2 hours, and wash the plate 10 times.

4. Elution: Add 100 μL of glycine for elution. After 15 minutes, add Tris-HCl for neutralization to end the elution.

5. Infection: Eluate is infected with XL1-Blue Escherichia coli

6. Detection Titer

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

8. Proceed to the next round of screening, a total of three rounds.

3. Identification of positive clones

The screening of positive clones was performed using phage ELISA. Specifically, 60 clones were selected from the bacterial plate in the third round of screening, and superinfected in a 96-well deep-well plate with M13K07 auxiliary phage for 14 hours. 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 minutes; the plate was washed 6 times with PBST, and HRP-labeled anti-M13 monoclonal antibody was added, 100 μL/well, and incubated at 37°C for 45 minutes; the plate was washed 6 times with PBST, and TMB colorimetric solution was added, 100 μL/well, and incubated at 37°C for 10 minutes; 2 mol/L dilute sulfuric acid was added, 50 μL/well, the reaction was terminated, and the OD ₄₅₀ value was immediately read with an ELISA reader. The wells that were 5 times higher than the negative control were sequenced to obtain the final nanoantibody sequence.

Experimental results:

1. Enrichment and panning of phage libraries

After the original library is constructed, the present invention will perform 3 rounds of bio-panning on the phage library. In the first round of panning, the coating antigen digoxin-OVA concentration is 15 μg/mL; in the second round of panning, the coating antigen concentration is 10 μg/mL; in the third round of panning, the coating antigen concentration is 7 μg/mL. The results are shown in Table 2. Compared with the first round, the number of positive clones in the third round of panning is enriched by about 1000 times, the recovery rate is significantly improved, and the input-elution ratio of phages is close to stability in the second and third rounds of panning, further indicating that positive clones are significantly enriched.

Table 2 Phage enrichment parameters

2. Phage ELISA and screening of positive clones

The present invention screens positive clones by an indirect ELISA method. Monoclones are selected from the enriched library for multiple rounds of screening, and the periplasmic expression products of the monoclones are detected by indirect ELISA. As shown in FIG4 , an index of 5 times or more of the blank control OD ₄₅₀ value is selected as a dividing line, and multiple positive clones are finally selected for DNA sequencing. The best clone is selected for subsequent experiments. It encodes a nano antibody against digoxin.

Analysis of its amino acid sequence showed that it had a typical stable structure of a nanobody, and the amino acid sequences of its framework region FR and complementary determining region CDR were shown in FIG5 , respectively.

Example 4: Expression and purification of nanobodies

Experimental methods

1. Construction of prokaryotic expression vector

The present invention uses pET-28a as an expression vector, selects NheI and XhoI as restriction sites, uses the above positive clone as a template, adds restriction sites NheI-BamHI at both ends to perform PCR, performs agarose electrophoresis on the PCR product, recovers the fragments on the gel, and determines the concentration.

The PCR amplification system is shown in the following table:

The reaction system is as follows:

The specific amplification primer sequences are as follows:

The pET-28a plasmid and the above gel recovery product were digested with Nhe I and Xho I, and then digested for 30 min at 37°C by agarose electrophoresis. The digested fragments were recovered by gel and the concentration was determined. The digestion system is shown in the following table:

The vector and fragment after enzyme digestion were connected at a molar ratio of 1:4. The connection reaction system is shown in the following table.

After ligation at 22°C for 30 min, the cells were transformed into BL21(DE3) competent cells, and positive clones were obtained for sequencing the next day.

2. Prokaryotic expression of nanobodies

The correctly sequenced colonies were selected to induce protein expression at 4°C with 100 mM IPTG for 16 h. The bacterial solution was ultrasonically disrupted and the supernatant was collected and purified through a nickel column. The impurities were washed away with a washing solution, and the nanoantibodies were eluted with an eluent. PBS was added to the obtained nanoantibody solution for ultrafiltration (3600 rpm/min, 10 min, repeated twice), and the liquid in the small tube of the ultrafiltration tube was collected.

The target protein was further purified using AKTA protein purifier ion exchange chromatography, with a buffer system of 0.02MTris-HCl, 1 M NaCl, pH 8.0. The system flow rate was set to 0.85mL/min according to the instrument pressure, and the sample loop was used to load the sample after pre-equilibration of the chromatographic column, and the effluent fractions at different times were collected using an automatic fraction collector. SDS-PAGE electrophoresis analysis was performed to detect different component proteins, and the protein fractions were frozen and stored in a -80℃ refrigerator.

Experimental results:

1. Expression and purification of nanobodies

The present invention constructs a prokaryotic expression recombinant plasmid, and induces it with 100 mM IPTG for 16 h at 4° C. The protein lysate and the purified protein eluate are subjected to SDS-PAGE electrophoresis analysis. The results are shown in FIG6 .

2. Further purification of nanobodies

The present invention uses AKTA protein purification instrument and ion exchange chromatography to further purify the target protein. As shown in Figure 7, SDS-PAGE electrophoresis analyzed protein samples with different retention times, and obtained target protein samples with purity > 95% in the 3rd, 4th and 5th tube proteins. At the same time, WB analysis results show that there is an obvious nano antibody band near 14kDa as shown in Figure 8.

Example 5: Determination of digoxin nanobody binding capacity

Experimental methods:

1. Isothermal titration calorimetry to detect the affinity of nanobodies

Dilute the digoxin nanoantibody to a concentration of 20 μM (dissolved in PBS) and place it in the sample pool. Place PBS solution in the reference pool. Dissolve digoxin powder in PBS to a concentration of 200 μM and place it in the titration syringe. After placement, perform titration and sample addition according to the requirements of the operating manual to determine the Kd value.

2. MST detection of nanoantibody binding

The present invention detects the affinity of antibody protein and antigen by micro-thermophoresis (MST) technology, and the method requires at least one of the ligand molecule or receptor protein to be fluorescently labeled. The present invention selects Monolith RED-NHS second-generation protein labeling kit to label the target antibody.

(I) Protein labeling

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

(II) Determination of labeling efficiency

The labeling efficiency was calculated according to the following formula:

The labeling efficiency (DOL) is calculated as follows:

First, dilute the digoxin solution in a gradient, prepare a PBS solution containing 0.05% Tween-20, dilute digoxin to 1 μM with the solution, and pipette 10 μL into a 0.2 mL PCR tube, and mark the PCR tube as PCR tube No. 1 and number it sequentially until No. 16. Add 10 μL of PBS solution containing 0.05% Tween-20 to PCR tubes No. 2 to 16, and gradiently dilute PCR tube No. 1 to PCR tube No. 16, so that the digoxin ligand concentration of the latter tube is exactly half of that of the previous tube. Dilute the labeled nanoantibody to be tested to a concentration of 10 nM, add 10 μL of the labeled nanoantibody solution to each PCR tube, and mix thoroughly with a pipette. Make the concentration of the antibody protein to be tested in each PCR tube 5 nM. Add an equal volume of labeled antibody protein to the PCR tube, suck it into the capillary, and detect the affinity of the antibody and the ligand by MST. Use a special capillary to absorb the liquid in the above PCR tube, and be careful not to absorb bubbles. Set parameters on the machine to detect the affinity of nanoantibodies to digoxin.

Experimental results:

1. Isothermal titration calorimetry to detect the affinity of nanobodies

The present invention measures the affinity of the nanobody and digoxin by isothermal titration calorimetry, as shown in Figure 9, at 298.15 K, the reaction ΔH = -4.28 × 10 ⁶ cal/mol, ΔS = -1.12 × 10 ⁴ cal/mol/deg, ΔG = -9.26 Kcal/mol. The calculated Kd value is 160 nM.

2. Determination of Nanobody Affinity by Microthermophoresis

The present invention uses Monolith RED-NHS second-generation protein labeling kit to label the antibody protein with nucleic acid dye. Microthermophoresis is used to determine the affinity between the candidate antibody protein and digoxin. As shown in Figure 10, the affinity determination is achieved in a relatively short time by measuring the fluorescence change produced by the reaction of the protein with the small molecule. The results show that the protein fluorescence is evenly distributed, indicating that the fluorescent protein labeling effect is good, and the Kd of the nano antibody and digoxin is measured to be 73.1nM.

Example 6: Detection of digoxin by competitive binding ELISA

Experimental methods:

1. Exploration of the optimal antibody concentration

Add 100 μL of digoxin-OVA at a concentration of 200 ng/mL to the adsorbed 96-well ELISA plate and coat overnight; discard the liquid in the well, wash 5 times with PBST, add 100 μL of OVA to block at room temperature for 1 hour; discard the liquid in the well, wash 5 times with PBST, add a series of concentrations of biotin-labeled digoxin nanoantibodies and incubate at room temperature for 2 hours; discard the liquid in the well, wash 5 times with PBST, add 100 μL of streptomycin-HRP, incubate at room temperature for 1 hour; discard the liquid in the well, wash 5 times with PBST, add 100 μL of TMB color development liquid, incubate at 37°C for 10 minutes, and then add 50 μL of color stop solution. The absorbance was detected at 450 nm by an enzyme reader; the optimal antibody concentration was selected for subsequent detection experiments.

2. Competitive ELISA Binding Assay

Drawing of standard curve:

Add 100 μL of digoxin-OVA at a concentration of 20 ng/mL to a high-absorption 96-well ELISA plate and coat overnight; discard the liquid in the wells, wash 5 times with PBST, add 100 μL of OVA and block at room temperature for 1 hour; discard the liquid in the wells, wash 5 times with PBST, and for the standard group, add 100 μL of digoxin at concentrations of 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, and 0.6 ng/mL. For the blank control group (B0), add 100 μL of PBS. For the test group, add 100 μL of the serum sample to be tested. Add 1 μg/mL of biotin-labeled digoxin nanoantibody to each sample well and incubate at room temperature for 2 hours; discard the liquid in the well, wash 5 times with PBST, add 100 μL of streptomycin-HRP, and incubate at room temperature for 1 hour; discard the liquid in the well, wash 5 times with PBST, add 100 μL of TMB color development liquid, incubate at 37°C for 10 minutes, and then add 50 μL of color development stop solution. The absorbance is detected at 450nm by an enzyme reader; the average value (B) of the absorbance value of each concentration standard solution and sample obtained is divided by the absorbance value (B0) of the first blank (0 standard) and then multiplied by 100%, that is, the percentage absorbance value; draw a standard curve with the logarithm of digoxin concentration as the X-axis and the percentage absorbance value as the Y-axis.

Sample recovery experiment:

Prepare 0.1 ml of digoxin solution with concentrations of 2 ng/mL, 20 ng/mL, and 200 ng/mL, and detect the absorbance of the above-mentioned samples. According to the average absorbance of the sample wells measured, the logarithm of the digoxin concentration can be obtained from the standard curve, and the antilogarithm is the digoxin concentration in the sample. Calculate the recovery rate (Recovery) and coefficient of variation (RSD) of digoxin samples with different concentrations.

Experimental results:

The antigen was immobilized, and the working concentration of the antibody was determined to be around 1 μg/mL, and the optimal antibody concentration was selected for subsequent detection experiments. A competitive ELISA standard curve diagram was established based on the amino acid sequence of the nanoantibody VHH-A9 shown in SEQ ID NO: 8. The average OD ₄₅₀ of the blank standard was recorded as B0, and the average OD ₄₅₀ of the digoxin standard of different concentrations was recorded as B. A linear fit was performed with the logarithmic value of the standard concentration as the horizontal axis and the B/B0 ratio as the vertical axis. The standard curve drawn is shown in Figure 11.

2 ng/mL, 20 ng/mL, and 200 ng/mL of digoxin were added, and the calculated recovery rates and coefficients of variation are shown in Table 3.

Table 3 Digoxin detection and recovery rate

Example 7: Nanobody Detection of Digoxigenin-Labeled Nucleic Acid Probe

Experimental methods:

Nanobody detection of digoxigenin-labeled nucleic acid probes:

Dissolve the digoxigenin-labeled nucleic acid probe to 128 μM as the starting concentration of the experiment. Digoxigenin-labeled nucleic acid probes were diluted in sequence so that the concentration of each subsequent tube was 1/2 of the previous tube. Then, 1 μL of the gradient diluted digoxigenin-labeled nucleic acid probe solution was transferred with a multichannel pipette and carefully dropped onto the NC membrane. After UV crosslinking for 30 minutes using a UV crosslinker, the biotin-labeled nanobody was incubated overnight. Wash three times with PBST, add streptavidin-HRP, incubate at room temperature for 1 hour, and use ECL luminescence detection after washing three times with PBST.

Determination of Nanobody Stability:

The nanobody and commercially available digoxigenin monoclonal antibody were placed in an incubator at 37°C for 24 h or 72 h, respectively, and then incubated with the digoxigenin-labeled nucleic acid probe on the NC membrane to analyze and detect the minimum detection concentration of the nanobody and commercially available monoclonal antibody.

Experimental results:

As shown in the results of Figure 12, the digoxin nanobody VHH-A9 can specifically recognize the digoxin nucleic acid probe, and the detection signal intensity gradually weakens with the decrease of the concentration of the digoxin-labeled nucleic acid probe, and the lowest detection concentration is about ^2-1 pM.

As shown in Figure 13, after being placed at 37°C for 24 hours, the ability of the control digoxin monoclonal antibody (Dig-mAb) to detect digoxin-labeled nucleic acid probes decreased, and the minimum detection concentration dropped to 24 pM. After being placed for 72 hours, the binding ability was basically lost, and the minimum detection concentration dropped to 2 ⁷ pM. In contrast, the binding ability of the digoxin nanobody was basically unchanged after being placed at 37°C for 24 hours, and the minimum detection concentration was about 2 ² pM. Even after being placed at 37°C for 72 hours, the minimum detection concentration of 2 ⁴ pM was still maintained. This result shows that the stability of the digoxin nanobody is significantly better than the commercially available digoxin monoclonal antibody.

Example 8: Nanobodies rescue digoxin poisoning

Experimental methods:

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

Experimental results:

As shown in Table 5, intraperitoneal injection of digoxin (3.6 mg/kg) caused the death of about half of the mice. The nanoantibody (10 mg/kg) injected into 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). The injection of the nanoantibody alone had no significant effect on the survival of the mice. The results show that the nanoantibody of the present invention can reduce the toxicity caused by digoxin, increase the survival rate of mice, and rescue digoxin poisoning.

Table 4 Animal mortality rate

^** P＜0.01; statistically significant.

Claims (5)

1. The digoxin nanometer antibody is characterized in that the amino acid sequence is shown as SEQ ID NO. 8.

2. A coding gene of the digoxin nano-antibody as set forth in claim 1, whose nucleotide sequence is shown in SEQ ID No. 9.

3. A method for preparing the digoxin nanobody as set forth in claim 1 by genetic engineering means.

4. Use of the digoxin nanobody as set forth in claim 1 in the establishment of a digoxin assay kit or diagnostic kit.

5. Use of the digoxin nanobody as set forth in claim 1 for alleviating digoxin poisoning in vivo and for detecting a nucleic acid probe.