A nanoantibody for detecting Staphylococcus aureus enterotoxin B and its application

Abstract

The present invention relates to the field of antigen detection technology, and in particular to a nanobody for detecting Staphylococcus aureus enterotoxin B and its application. The nanobody comprises an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2. The applications include: purification, detection, and removal of Staphylococcus aureus enterotoxin B. The present invention screened and obtained two nanobodies for SEB, which have high specificity and high affinity for SEB. Detection of SEB based on these two nanobodies has high specificity and sensitivity, which is of great value in the field of Staphylococcus aureus enterotoxin detection.

Description

Technical Field

[0001] The present invention relates to the technical field of antigen detection, and in particular to a nano antibody for detecting Staphylococcus aureus enterotoxin B and applications thereof. Background Art

[0002] Staphylococcus aureus is one of the most common foodborne pathogens, causing severe staphylococcal food poisoning (SFP). S. aureus enterotoxins (SEs) can cause nausea, severe vomiting, and abdominal cramps, occasionally followed by diarrhea after a brief incubation period. Their molecular weight ranges from 19 to 30 kDa and they are resistant to heat and protease hydrolysis. To date, 29 different SEs and enterotoxin-like substances (SELs) have been reported, with classic SE serotypes including SEA, SEB, SEC, SED, and SEE. Ingestion of 20 to 100 ng of SEB can lead to the release of massive amounts of proinflammatory cytokines, resulting in SFP and even severe toxic shock syndrome (TSS).

[0003] Currently, the main methods for detecting SEs include biological testing, genetic molecular biology testing, mass spectrometry, and immunological testing. The detection method specified in the national standard is enzyme-linked immunosorbent assay (ELISA), which offers advantages such as high throughput, ease of use, and high sensitivity, allowing for direct detection of SEs in samples. Furthermore, compared with other SE detection techniques, ELISA is less susceptible to interference from complex matrices, poses no ethical concerns, and is less susceptible to false positives from contamination. Several commercial SE immunoassay kits are available that can simultaneously detect five classic enterotoxins. The antibodies (primarily IgG) are primarily derived from mouse monoclonal antibodies or rabbit polyclonal antibodies. For example, the enzyme-linked immunosorbent assay for total Staphylococcus aureus enterotoxins, jointly developed by China Agricultural University and Beijing Weideweikang Biotechnology Co., Ltd., uses monoclonal antibodies obtained by immunizing mice with recombinant S. aureus classic enterotoxins.

[0004] Traditional monoclonal antibodies for detecting enterotoxins are obtained by immunizing animals. Screening takes a long time, is costly, and requires skilled operators and specialized cell culture facilities. In particular, the crystallizable fragment (Fc) region of the monoclonal antibody can bind to the surface protein A (SpA) of Staphylococcus aureus, resulting in false positives in ELISA detection of enterotoxins. Nanobodies, however, can avoid the problem of false positives because they lack the Fc region. Currently available kits for Staphylococcus aureus enterotoxins include: Ridascreen from Germany ® set, 3M in the United States @ TECRA ® Staph enterotoxins, Identification Test 3However, these kits all have the problem of overlap between SEB and SEC detection.

[0005] Nanobodies offer numerous advantages: Nanobodies are more likely to bind to concave epitopes, allowing them to bind to antigenic epitopes that traditional antibodies cannot. The extended complementarity determining region 3 (CDR3) and conserved disulfide bonds confer high thermal stability. The hydrophilic amino acids in the framework region 2 (FR2) of nanobodies increase their water solubility and reduce their aggregation potential. They are easy to express and purify in prokaryotes, reducing production costs, overcoming the limitations of in vivo antibody production, and shortening the development cycle. Nanobody VHH fragments lack an Fc region and, in theory, will not bind to and cross-react with the Staphylococcus aureus SpA protein. Therefore, it is necessary to screen for nanobody pairs with high specificity against SEB for the immunological detection of S. aureus enterotoxin B. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a nanobody for detecting Staphylococcus aureus enterotoxin B and its application.

[0007] The present invention screened a pair of highly specific anti-SEB nanoantibodies. Using the capture antibody AntiSEB Nb3 and the detection antibody AntiSEB Nb6 as the antibody pair, a double-antibody sandwich assay was constructed to achieve specific detection of SEB. The detection antibody AntiSEB Nb6 is highly specific, recognizing only SEB and not Staphylococcus aureus enterotoxin SEC, thus ensuring highly specific diagnostic results.

[0008] The capture antibody, AntiSEB Nb3, and the detection antibody, AntiSEB Nb6, recognize different epitopes of SEB. AntiSEB Nb6 recognizes an amino acid sequence region that bears no homology between SEB and SEC, resulting in a double-antibody sandwich assay based on this antibody showing no cross-reactivity with SEC. Furthermore, both AntiSEB Nb3 and AntiSEB Nb6 exhibit high affinity for SEB. The extended CDR3 and conserved disulfide bonds impart high thermal stability to these nanobodies, making them easy to store and facilitating the development of subsequent experimental methods.

[0009] In a first aspect, the present invention provides an anti-SEB nanobody (AntiSEB Nb3), wherein the nanobody has at least one of the following CDRs:

[0010] i) heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO. 1;

[0011] ii) heavy chain CDR2 at positions 51 to 58 as shown in SEQ ID NO. 1;

[0012] iii) heavy chain CDR3 at positions 97 to 117 as shown in SEQ ID NO. 1.

[0013] Furthermore, the nanobody has an amino acid sequence as shown in SEQ ID NO.1; preferably, the nanobody is encoded by a nucleotide sequence as shown in SEQ ID NO.3.

[0014] The present invention further provides another anti-SEB nanobody (AntiSEB Nb6), wherein the nanobody has at least one of the following CDRs:

[0015] i) heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO. 2;

[0016] ii) heavy chain CDR2 from positions 51 to 57 as shown in SEQ ID NO. 2;

[0017] iii) heavy chain CDR3 at positions 96 to 113 as shown in SEQ ID NO. 2.

[0018] Furthermore, the nanobody has an amino acid sequence as shown in SEQ ID NO.2; preferably, the nanobody is encoded by a nucleotide sequence as shown in SEQ ID NO.4.

[0019] The amino acid sequence shown in SEQ ID NO.1:

[0020] QVQLVESGGRLVQPGGSLSLSCAASGATFSSYAMAWFRRTPGQEREFVAHITWSGESTHYASSVKGRFTISRDNAKNTLSLHMNNLEPEDTGVYYCASEGNPYYSHYPDISPVRFSSWGQGTLVTVSS.

[0021] The amino acid sequence shown in SEQ ID NO.2:

[0022] QVQLVESGGGLVQTGGSLRLSCAASGDTTLDYYYIGWFRQGPGKEHEGVSCISSTGSTTQYTESVKGRFTISRDNAKKSVYLQMSGLRPEDTAVYYCAAFFVDPRVTTINIQRVCSMSYDSRGQGTQVTVSS.

[0023] The nucleotide sequence shown in SEQ ID NO.3:

[0024] CAGGTGCAGCTGGTAGAGTCTGGGGGAAGATTGGTGCAGCCTGGGGGCTCTCTGAGTCTCTCCTGTGCAGCTTCCGGAGCCACCTTTAGTTCTTATGCCATGGCCTGGTTCCGCCGGACTCCAGGGCAGGAGCGTGAGTTTGTAGCACATATTACATGGAGTGGCGAGAGTACTCACTATGCAAGCTCTGTG AAGGGCCGCTTCACCATCTCCAGAGACAACGCCAAGAACACACTGTCGCTACACATGAACAACCTGGAACCTGAGGACACGGGCGTTTATTGTGCTTCAGAAGGGAACCCCTACTATAGCCACTACCCGGACATAAGTCCCGTGCGCTTCAGTTCCTGGGGCCAGGGGACCCTGGTCACTGTCTCCTCA.

[0025] The nucleotide sequence shown in SEQ ID NO 4:

[0026] CAGGTGCAGCTGGTAGAGTCTGGAGGAGGGTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCGTCTGGAGACACCATCAACAGTTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCAGCTATTAGCTGGAGTGATAATCCTTACTATACAGACTCC GTGAGGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAAGACACAGCCGTTTATTACTGCGCCCGAGGAAAAGCGAAATTTTACATCAGACTAACTGATGACGAGTATACTGACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA.

[0027] In a second aspect, the present invention provides a nucleic acid encoding the aforementioned Nanobody.

[0028] In a third aspect, the present invention provides a biomaterial, wherein the biomaterial comprises the aforementioned nucleic acid, and the biomaterial is an expression cassette, a vector, or a transgenic cell.

[0029] The transgenic cells described in the present invention do not include transgenic cells that have the ability to independently develop into complete individuals, that is, they do not include plant varieties and animal varieties.

[0030] In a fourth aspect, the present invention provides a kit comprising the aforementioned nanoantibody, or the aforementioned nucleic acid, or the aforementioned biomaterial.

[0031] In a fifth aspect, the present invention provides a nanobody combination comprising the two aforementioned nanobodies.

[0032] Furthermore, in the nanobody combination, AntiSEB Nb3 is a capture antibody and AntiSEB Nb6 is a detection antibody.

[0033] In a sixth aspect, the present invention provides the use of the aforementioned nanoantibody, or the aforementioned nucleic acid, or the aforementioned biomaterial, or the aforementioned nanoantibody combination in the preparation of a diagnostic reagent, a diagnostic kit or a drug.

[0034] Furthermore, the drug is used for purification, detection or removal of Staphylococcus aureus enterotoxin B.

[0035] Furthermore, based on the nanobody combination, Staphylococcus aureus enterotoxin B was detected by a double antibody sandwich method.

[0036] The present invention has the following beneficial effects:

[0037] The present invention screened two antibodies specific for Staphylococcus aureus enterotoxin B, using AntiSEB Nb3 as the capture antibody and AntiSEB Nb6 as the detection antibody. The AntiSEB Nb6 antibody provided by the present invention resolves the crosstalk between SEB and SEC, significantly improving the specificity for detecting Staphylococcus aureus enterotoxin B. Both AntiSEB Nb3 and AntiSEB Nb6 have high affinity for SEB, resulting in high sensitivity in detecting SEB.

[0038] The nanoantibody combination provided by the present invention can be used in detection technologies such as enzyme-linked immunosorbent assay (ELISA), western blot, and colloidal gold method for Staphylococcus aureus enterotoxin B, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0040] Figure 1 This is an SDS-PAGE identification diagram of the purified SEB protein provided in Example 1 of the present invention; wherein M is a marker.

[0041] Figure 2 The elimination screening product provided in Example 1 of the present invention was diluted 10 6 The colonies grown after infecting TG1 were multiplied.

[0042] Figure 3 This is the result of expressing AntiSEB Nb3 and AntiSEB Nb6 using the prokaryotic expression system provided in Example 1 of the present invention.

[0043] Figure 4 This is the SDS-PAGE identification result of the purified Nanobody provided in Example 1 of the present invention.

[0044] Figure 5 This is the binding ability of the purified AntiSEB Nb3 provided in Example 2 of the present invention to the SEB protein.

[0045] Figure 6 This is the binding ability of the purified AntiSEB N6 provided in Example 2 of the present invention to the SEB protein.

[0046] Figure 7 This is the western blot result of AntiSEB Nb3 provided in Example 2 of the present invention against 9 enterotoxins of Staphylococcus aureus.

[0047] Figure 8 This is the western blot result of AntiSEB Nb6 provided in Example 2 of the present invention against 9 enterotoxins of Staphylococcus aureus.

[0048] Figure 9 This is the affinity fitting curve of the affinity between AntiSEB Nb3 and SEB provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0049] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0050] Unless otherwise specified, the experimental methods involved in the following examples are all conventional methods in the art. For example, reference can be made to experimental manuals in the art, or the conditions recommended by the manufacturer's instructions.

[0051] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources, for example:

[0052] Example 1

[0053] The present invention provides SEB nanobodies, which are obtained by screening through the following process:

[0054] 1. SEB protein expression and purification

[0055] The present invention extracts DNA from strain DC53060 stored in the Chinese Center for Disease Control and Prevention, and amplifies enterotoxin by PCR. seb The PCR amplification conditions were as follows: 95°C for 5 min, 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, and 72°C for 5 min, for a total of 35 cycles. seb The PCR product size is 720 bp. BamH I and Hind The PCR product and expression vector pET28a(+) were digested with enzyme III and ligated. The recombinant plasmid was transformed into Escherichia coli BL21(DE)3. After successful ligation, the recombinant plasmid was induced to express with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). The expressed SEB protein was purified using a HisTrap HP column and identified by SDS-PAGE (e.g. Figure 1 (As shown), SEB protein was successfully expressed, yielding a highly pure soluble protein with a molecular weight of 32 kD. The SEB protein concentration was determined using a BCA protein assay, yielding 4 mg of protein, which served as the antigen for antibody screening.

[0056] 2. Nanobody library selection

[0057] Recombinant SEB protein antibodies were screened from the natural nanoantibody phage display library constructed earlier. First, the ELISA plate was coated with recombinant SEB protein (4 μg / well in the first round, 3 μg / well in the second round, 3 μg / well in the third round, 2 μg / well in the fourth round, and 1 μg / well in the fifth round). 8 replicate wells were set for antigen and 4 replicate wells for blank control (coating solution). The plate was incubated at 4°C overnight. The coating solution was discarded and the plate was washed 6 times with PBST. 200-300 μl of blocking solution PBS (containing 2% skim milk powder) was added to each well and blocked at 37°C for 1 hour. After washing, the phage display library was diluted to 10 with the blocking solution. 12 -10 13pfu, add 100μl phage library to each well and place at 37℃ for 1h. Wash 9 times with PBST (0.1% (v / v) in the first round, 0.1% (v / v) in the second round, 0.2% (v / v) in the third round, 0.3% (v / v) in the fourth round, and 0.3% (v / v) in the fifth round) and wash once with PBS. Add 150μl 0.1M glycine (pH=2.2) to each well and elute for 20min at room temperature. Add 1MTris (pH=8.0) solution for rapid neutralization. The eluted phages were infected with Escherichia coli TG1 cells in the logarithmic growth phase and shaken slowly at 37℃ at 180rpm for 1h. Add about 10 12 After 40 min of incubation at 37°C with 50 μg / ml of M13KO7 helper phage, add ampicillin and kanamycin at a final concentration of 25 μg / ml, incubate overnight at 30°C at 220 rpm, centrifuge at 12,000 rpm for 20 min, add 1 / 4 volume of PEG / NaCl solution to the supernatant, and precipitate on ice for more than 4 h. 20 μl of phage suspension was taken to calculate the titer, and clones that bind to SEB were screened through five rounds of panning ( Figure 2 ), the product titer was measured after the fifth round of panning, and the calculated fifth round output was 3.59×10 9 pfu.

[0058] 3. Phage-ELISA

[0059] Phage-ELISA was used to analyze the response of different clones to SEB. Single colonies grown on the above plates were randomly selected and cultured in 96-well deep-well plates (containing LB medium) at 37°C until OD 600 Reach 0.6. Add about 10 12 Incubate with pfu of M13KO7 overnight at 30°C. Coat an ELISA plate with recombinant SEB protein (0.5 μg / well). Add 100 μl of the phage display library from the deep-well plate to the ELISA plate and incubate at 37°C for 1 hour. After washing, add a 1:10,000 dilution of HRP-conjugated mouse anti-M13 phage antibody and incubate at 37°C for 1 hour. After washing, add 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution to each well and measure absorbance at 450 nm. Select clones with a significant difference in absorbance between the positive and control wells for sequencing.

[0060] 4. Construction of prokaryotic expression vector to express nanobodies

[0061] First, the enriched nanobody fragments were amplified by PCR using the following conditions: 95°C for 5 minutes, 95°C for 30 seconds, 53°C for 30 seconds, 72°C for 45 seconds, and 72°C for 5 minutes, for a total of 35 cycles. The AntiSEB Nb3 PCR product was 384 bp in size, with the sequence shown in SEQ ID NO. 3; the AntiSEB Nb6 PCR product was 372 bp in size, with the sequence shown in SEQ ID NO. 4.

[0062] Plasmid pET-28a(+) and VHH amplified fragment were BamH I and Hind The ligation product was digested with enzyme III at 37°C for 2 h. The ligation product was transformed into E. coli BL21(DE)3 and plated on LB plates containing 50 μg / ml kanamycin and cultured at 37°C for 12 h. Single clones were screened for protein expression and expression verification.

[0063] Two monoclonal clones of nanobodies were picked and induced to express with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG), as shown in Figure 3 The secretion of AntiSEB Nb3 and AntiSEB Nb6 is shown in Figure 2. The expressed nanobody protein supernatant was purified using a HisTrapHP column and identified by SDS-PAGE to obtain highly pure soluble protein. Figure 4 As shown, AntiSEB Nb3 has a molecular mass of 19 kD and a sequence as shown in SEQ ID NO. 1; AntiSEB Nb6 has a molecular mass of 19 kD and a sequence as shown in SEQ ID NO. 2. The concentration of the purified protein was determined using a BCA protein assay. 4 mg of AntiSEB Nb3 and 3 mg of AntiSEB Nb6 protein were obtained, respectively, for subsequent nanobody evaluation.

[0064] Example 2

[0065] In this example, the Nanobody provided in Example 1 was functionally evaluated, and the process was as follows:

[0066] 1. Evaluation of the binding ability of nanobodies to SEB protein

[0067] Western blot experiments were used to evaluate the binding ability of the purified nanobody to SEB protein. The SEB obtained after SDS-PAGE in step 1 above was transferred to a PVDF membrane and blocked with skim milk powder for 1 hour at room temperature. After washing, it was incubated with the purified nanobody at room temperature for 1 hour and detected with a 1:5000 dilution of HRP-labeled rabbit anti-camelid antibody. The color was developed with DAB substrate. AntiSEB Nb3 (1μg / ml) (as Figure 5 as indicated) and AntiSEB Nb6 (1 μg / ml) (as indicated) Figure 6 shown).

[0068] 2. Cross-reaction between nanobodies and other enterotoxins

[0069] The SEB obtained after SDS-PAGE in step 1 of Example 1 and the SEA, SEC, SED, SEE, SEG, SEH, SEK, and SEQ proteins stored in the laboratory were transferred to a PVDF membrane and blocked with skim milk powder for 1 hour at room temperature. After washing, they were incubated with nanobodies with binding activity respectively at room temperature for 1 hour, and detected with a 1:5000 dilution of HRP-labeled rabbit anti-camelid antibody. The color was developed with DAB substrate, and AntiSEB Nb3 (1 μg / ml) (as Figure 7 as indicated) and AntiSEB Nb6 (1 μg / ml) (as indicated) Figure 8 The AntiSEB Nb3 of the present invention can effectively identify the recombinant protein SEB and can also identify the recombinant protein SEC, and its band is located at 32kD, indicating that the AntiSEB Nb3 antibody can simultaneously detect the recombinant SEB protein and the recombinant SEC protein (as shown). Figure 7 The AntiSEB Nb6 of the present invention can effectively recognize the recombinant protein SEB, and its band is located at 32kD. It has no cross-reaction with other recombinant enterotoxins SEA, SEC, SED, SEE, SEG, SEH, SEK, and SEQ proteins, indicating that the AntiSEB Nb6 antibody has good specificity for the recombinant SEB protein (as shown). Figure 8 shown).

[0070] 3. Checkerboard titration method to determine the best antibody pair

[0071] Nine nanobodies with binding activity were labeled with HRP. The titers of the detection antibodies were determined indirectly, with the titers of AntiSEB HRP-Nb1 being 1:64,000; AntiSEB HRP-Nb2 being 1:1,000; AntiSEB HRP-Nb3 being 1:16,000; AntiSEB HRP-Nb4 being 1:1,000; AntiSEB HRP-Nb5 being 1:32,000; AntiSEB HRP-Nb6 being 1:64,000; AntiSEB HRP-Nb7 being 1:1,000; and AntiSEB HRP-Nb8 being 1:1,000. ELISA plates were coated with unlabeled nanobodies as capture antibodies (2 μg / well) and incubated overnight at 4°C. After washing, the plates were blocked with 5% skim milk powder at 37°C for 1 hour. After washing, 2 μg of antigen was added to each well and blocked at 37°C for 1 hour. After washing, HRP-labeled antibodies were added as detection antibodies and blocked at 37°C for 1 hour. After washing, 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added to each well, and the absorbance at 450 nm was measured. Antibody pairs with significant differences in absorbance between positive and control wells were selected as candidate antibody pairs for constructing an enzyme-linked immunosorbent assay (ELISA) double antibody sandwich assay. The optimal antibody pair screened by this method used AntiSEBNb3 as the capture antibody and AntiSEB HRP-Nb6 as the detection antibody.

[0072] 4. AntiSEB Nb3 antibody and SEB affinity determination experiment:

[0073] Antibody affinity was determined using biolayer interferometry (BLI). The instrument used in this experiment was an OCTET RH16, using an AR2G sensor and a buffer system of PBST + 0.1% BSA. Anti-SEB Nb3 was immobilized on the sensor surface at a concentration of 10 μg / mL. SEB antigen was then sequentially diluted to 300 nM, 150 nM, 75 nM, 37.5 nM, and 18.8 nM and loaded onto the sensor. Real-time kinetic data for antigen-antibody binding and dissociation were recorded at each concentration, with the response signal plotted as a time-dependent curve. The sensor surface was rinsed with a specific regeneration buffer to release bound SEB antigen, allowing for multiple binding / dissociation cycles. The experimental data were exported and analyzed, and a kinetic model was fitted. Using the analysis software Octet BLI Discovery 13.0, the association rate constant (k on ) was calculated to be 1.27E+05, and the dissociation rate constant (k off ) was calculated to be 3.03E-04. The affinity constant (KD) calculated according to the formula KD=koff / kon was 2.38E-09, indicating that AntiSEB Nb3 has a high affinity for SEB.

[0074] The results are shown in the following table and Figure 9 As shown:

[0075] Table 1 Affinity test results of AntiSEB Nb3 and SEB

[0076]

[0077] 5. Specificity and sensitivity

[0078] Based on the above results, the present invention determined a double antibody sandwich method using AntiSEB Nb3 as the capture antibody and AntiSEB HRP-Nb6 as the detection antibody.

[0079] 1. Specificity experiments have shown that this method does not cross-react with common foodborne strains such as Vibrio parahaemolyticus, Salmonella, Bacillus, Escherichia coli O157:H7, Listeria monocytogenes and Shigella flexneri type 2a during the detection process, and has high specificity.

[0080] 2. After sensitivity experiment verification, the detection limit (LOD) of this method for Staphylococcus aureus enterotoxin B (SEB) can reach 5.57 ng / mL.

[0081] 3. The present invention tested 24 strains containing only SEB and 24 strains containing only SEC. All strains containing SEB were detected, and all strains containing SEC were negative, showing 100% sensitivity and specificity.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An anti-SEB nanobody, characterized in that The Nanobody comprises the following CDRs: i) heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO. 2; ii) heavy chain CDR2 from positions 51 to 57 as shown in SEQ ID NO. 2; iii) heavy chain CDR3 at positions 96 to 113 as shown in SEQ ID NO.

2.

2. The Nanobody according to claim 1, characterized in that The nanobody has the amino acid sequence as described in SEQ ID NO.

2.

3. A nucleic acid, characterized in that The nucleic acid is used to encode the Nanobody according to claim 1 or 2.

4. A biomaterial, characterized in that The biological material comprises the nucleic acid according to claim 3, and the biological material is an expression cassette, a vector or a transgenic cell.

5. A kit, characterized in that Comprising the nanobody of claim 1 or 2, or the nucleic acid of claim 3, or the biomaterial of claim 4.

6. A nanobody combination, characterized in that Comprising the Nanobody of claim 1 or 2, and a second Nanobody; The second Nanobody comprises the following CDRs: i) heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO. 1; ii) heavy chain CDR2 at positions 51 to 58 as shown in SEQ ID NO. 1; iii) heavy chain CDR3 at positions 97 to 117 as shown in SEQ ID NO.

1.

7. The Nanobody combination according to claim 6, characterized in that The second nanobody comprises the amino acid sequence shown in SEQ ID NO.

1.

8. Use of the Nanobody according to claim 1 or 2, or the nucleic acid according to claim 3, or the biomaterial according to claim 4, or the Nanobody combination according to claim 6 in the preparation of a diagnostic reagent or diagnostic kit for SEB.

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