Nanobodies, antibody compositions and applications thereof for detecting Staphylococcus aureus enterotoxin H

Abstract

This invention belongs to the field of immunological testing and discloses nanobodies, antibody compositions, and their applications for detecting Staphylococcus aureus enterotoxin H. The invention successfully screened for Staphylococcus aureus enterotoxin H-specific antibodies and used a checkerboard titration method to identify capture antibodies, AntiSEH-Nb4, and detection antibodies, AntiSEH-Nb3. The resulting antibodies are effective for detecting food poisoning caused by non-classical enterotoxin H, providing a highly specific means of detecting Staphylococcus aureus enterotoxin H. This not only improves the accuracy and reliability of detection but also facilitates the rapid diagnosis and prevention of outbreaks of related foodborne diseases.

Description

Technical Field

[0001] The present invention belongs to the field of immunological detection, and in particular relates to a nano antibody for detecting Staphylococcus aureus enterotoxin H, an antibody composition 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. They range in molecular weight from 19 to 30 kDa and are resistant to thermophilic and proteolytic hydrolysis. To date, 29 different SEs and enterotoxin-like serotypes (SELs) have been described, including the classic SE serotypes SEA, SEB, SEC, SED, and SEE. However, in recent years, food poisoning incidents caused by nonclassical enterotoxins have increased globally, garnering widespread attention. Studies have shown that S. aureus strains isolated from certain food poisoning incidents often lack the genes for classic SEs. However, in-depth genetic analysis has revealed that these strains commonly carry genes for "nonclassical SEs." This finding suggests that nonclassical SEs may play an equally important pathogenic role in SFP outbreaks as classic SEs. Furthermore, the occurrence of food poisoning may be the result of the combined action of one or more non-classical SEs, which further highlights the importance of non-classical SEs in food safety and public health. Therefore, strengthening the detection and research of non-classical SEs is of great significance for the prevention and control of food poisoning caused by Staphylococcus aureus.

[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] Currently available commercially available test kits for Staphylococcus aureus enterotoxin include: Ridascreen from Germany ® set, 3M in the United States @ TECRA® Staph enterotoxins, Identification Test 3 , France's VIDAS ® SET3 However, most of these kits can only detect classical enterotoxins, not non-classical enterotoxins. Among them, only the French VIDAS® SET3 kit can detect SEH, but the enzyme-linked fluorescence immunoassay (ELFA) method it uses has the following limitations: First, ELFA requires specialized fluorescence detection equipment, which may increase experimental costs and operational complexity; second, the fluorescence signal may be quenched by factors such as light and oxidation, affecting the accuracy of the test results; finally, compared with the enzyme-linked immunosorbent assay (ELISA), the fluorescence reagent is less stable and requires more stringent environmental and storage conditions. There is an urgent need to develop domestically produced detection kits with independent intellectual property rights.

[0005] Traditional monoclonal antibodies for enterotoxin detection are obtained by immunizing animals. Screening is time-consuming, costly, and requires skilled operators and specialized cell culture facilities. In particular, the crystallizable fragment (Fc) region of monoclonal antibodies can bind to Staphylococcus aureus surface protein A (SpA), leading to false positives in ELISA detection of enterotoxin. Nanobodies offer several advantages: Nanobodies prefer 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, are unlikely to bind to and cross-react with the Staphylococcus aureus SpA protein. Therefore, it is necessary to screen nanoantibody pairs with good specificity against Staphylococcus aureus enterotoxin H (SEH) for immunological detection of SEH. Summary of the Invention

[0006] The purpose of the present invention is to provide a nanobody, an antibody composition and applications thereof for detecting Staphylococcus aureus enterotoxin H.

[0007] To achieve the purpose of the present invention, in a first aspect, the present invention provides a nanobody for detecting Staphylococcus aureus enterotoxin H (nanobody Nb3, AntiSEH-Nb3), wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the variable regions of the nanobody are SRAFSQNA (SEQ ID NO: 5), SMRTGKT (SEQ ID NO: 6), and CETWGVGGQG (SEQ ID NO: 7), respectively.

[0008] Furthermore, the amino acid sequence of the Nanobody Nb3 is shown in SEQ ID NO: 2.

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding said Nanobody.

[0010] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding Nanobody Nb3 is shown in SEQ ID NO:4.

[0011] In a third aspect, the present invention provides a biological material containing the nucleic acid molecule, wherein the biological material includes but is not limited to an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a host cell.

[0012] In a fourth aspect, the present invention provides an antibody conjugate, which is obtained by conjugating the nanobody to a label, and the label is selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radioactive label.

[0013] In a fifth aspect, the present invention provides an antibody composition for detecting Staphylococcus aureus enterotoxin H, the antibody composition comprising Nanobody Nb3 and Nanobody Nb4 for detecting Staphylococcus aureus enterotoxin H;

[0014] The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the variable region of the nanobody Nb4 are EFPFNYYT (SEQ ID NO: 8), IIKSDGST (SEQ ID NO: 9), and AVRYGSSCPGSDRADA (SEQ ID NO: 10), respectively.

[0015] Furthermore, the amino acid sequence of the nanobody Nb4 is shown in SEQ ID NO: 1.

[0016] In a sixth aspect, the present invention provides the use of nanoantibodies Nb3 or nucleic acid molecules encoding the nanoantibodies or biological materials containing the nucleic acid molecules or the antibody conjugates or the antibody compositions in the preparation of reagents for the qualitative or quantitative detection of Staphylococcus aureus enterotoxin H (including non-disease diagnosis and treatment purposes).

[0017] In a seventh aspect, the present invention provides a Staphylococcus aureus enterotoxin H double antibody sandwich enzyme-linked immunosorbent assay kit, which uses nanobody Nb3 as the detection antibody and nanobody Nb4 as the capture antibody, i.e., capture antibody AntiSEH-Nb4 and detection antibody AntiSEH-Nb3.

[0018] Furthermore, the kit further comprises at least one of a substrate color developing solution, a blocking solution and a stop solution.

[0019] In one embodiment of the present invention, the labeling of the Nanobody Nb3 can be carried out by a variety of markers, including but not limited to: biotin tag (Avi-tag), horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-galactosidase, lysozyme, malate dehydrogenase, etc., preferably using a biotin tag (Avi-tag).

[0020] Furthermore, the kit further comprises at least one of a substrate color developing solution, a blocking solution and a stop solution.

[0021] Commonly used substrates include, but are not limited to, o-phenylenediamine (OPD), tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, TMB) and ABTS [2,2'-azino-di-(3-ethylbenziazobinesulfonate-6)], etc., with TMB being preferred.

[0022] The blocking solution may be a 5% BSA (bovine serum albumin) solution or the like.

[0023] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0024] This study successfully screened for antibodies specific for Staphylococcus aureus enterotoxin H (ESH), and identified capture antibody AntiSEH-Nb4 and detection antibody AntiSEH-Nb3 using a checkerboard titration method. The resulting antibodies are effective for detecting food poisoning caused by nonclassical enterotoxin H, providing a highly specific method for detecting S. aureus enterotoxin H. This not only improves the accuracy and reliability of detection but also facilitates the rapid diagnosis and prevention of related foodborne disease outbreaks.

[0025] The antibody of the present invention can be used in detection techniques such as enzyme-linked immunosorbent assay (ELISA), western blot, and colloidal gold method for Staphylococcus aureus enterotoxin H. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The purified SEH protein in the preferred embodiment of the present invention is shown in FIG. Wherein, M is a protein molecular weight marker; and 1 is a recombinant protein after SEH purification.

[0027] Figure 2 The titer of the product after the fifth round of panning in the preferred embodiment of the present invention is measured. 6 The colonies grown after infecting TG1 were multiplied.

[0028] Figure 3 The results of expressing AntiSEH-Nb4 using a eukaryotic expression system in a preferred embodiment of the present invention are shown in Figure 1. M: protein molecular weight marker. R: AntiSEH-Nb4.

[0029] Figure 4 The results of expressing AntiSEH Avi-Nb3 using a eukaryotic expression system in a preferred embodiment of the present invention are shown in Figure 1. M: protein molecular weight marker. 1: AntiSEH Avi-Nb3.

[0030] Figure 5 Western blot analysis of purified AntiSEH-Nb4, recombinant SEH, and eight Staphylococcus aureus enterotoxins from a preferred embodiment of the present invention. M: 180 kDa protein marker; 1: SEA; 2: SEB; 3: SEC; 4: SED; 5: SEE; 6: SEG; 7: SEK; 8: SEH; 9: SEQ.

[0031] Figure 6 Western blot analysis of purified AntiSEH-Nb3, recombinant SEH, and eight Staphylococcus aureus enterotoxins from a preferred embodiment of the present invention. M: 180 kDa protein marker; 1: SEA; 2: SEB; 3: SEC; 4: SED; 5: SEE; 6: SEG; 7: SEK; 8: SEH; 9: SEQ.

[0032] Figure 7 The binding capacity of purified AntiSEH-Nb4 to recombinant SEH protein and native SEH protein in a preferred embodiment of the present invention is shown in Figure 5. Wherein, M: 180 KDa protein marker; 1: SEH recombinant protein; 2: DC53015; 3: DC53034; 4: DC53138; 5: DC53140.

[0033] Figure 8The binding capacity of purified AntiSEH-Nb3 to recombinant SEH protein and native SEH protein in a preferred embodiment of the present invention is shown in Figure 5. Wherein, M: 180 KDa protein marker; 1: SEH recombinant protein; 2: DC53015; 3: DC53034; 4: DC53138; 5: DC53140.

[0034] Figure 9 Thermal stability results of purified AntiSEH-Nb4 and purified AntiSEH-Nb3 in preferred embodiments of the present invention. DETAILED DESCRIPTION

[0035] The present invention utilizes a previously constructed natural nanoantibody library to screen for a highly specific anti-SEH nanoantibody pair. This antibody pair, consisting of the capture antibody AntiSEH-Nb4 (SEQ ID NO: 1) and the detection antibody AntiSEH Avi-Nb3 (SEQ ID NO: 2), is constructed using a double-antibody sandwich assay to achieve specific detection of SEH. The capture and detection antibodies specifically recognize SEH and do not react with other enterotoxins, ensuring the specificity of the diagnostic results.

[0036] The capture antibody AntiSEH-Nb4 and the detection antibody AntiSEH Avi-Nb3 recognize different epitopes of SEH; the present invention previously created a natural nanoantibody library with a sufficiently large library capacity, so the screened antibodies can obtain higher affinity; the extension of the nanoantibody CDR3 and the conserved disulfide bond give the nanoantibody a higher thermal stability, so the nanoantibody screened by the present invention is not only easy to preserve, but also conducive to the establishment of subsequent experimental methods; the invention effectively makes up for the shortcomings of domestic test kits in SEH diagnosis, and provides an important supplement to my country's diagnostic technology in this field.

[0037] The present invention adopts the following technical solutions:

[0038] In a first aspect, the present invention provides an anti-SEH nanobody (AntiSEH-Nb4), wherein the nanobody has at least one of the following CDRs:

[0039] i) heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO: 1;

[0040] ii) heavy chain CDR2 from positions 51 to 58 of SEQ ID NO: 1;

[0041] iii) heavy chain CDR3 at positions 97 to 112 as shown in SEQ ID NO: 1.

[0042] 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.

[0043] The present invention also provides another anti-SEH nanobody (AntiSEH-Nb3), wherein the nanobody has at least one of the following CDRs:

[0044] i) heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO: 2;

[0045] ii) heavy chain CDR2 at positions 51 to 57 as shown in SEQ ID NO: 2;

[0046] iii) heavy chain CDR3 at positions 95 to 104 as shown in SEQ ID NO: 2.

[0047] 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.

[0048] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0049] Example 1 Screening of anti-SEH nanobody pairs

[0050] 1. SEH protein expression and purification

[0051] DNA was extracted from Staphylococcus aureus DC53321 stored in the Chinese Center for Disease Control and Prevention, and enterotoxin was amplified by PCR. seh Gene (GenBank: MN752207.1). PCR amplification conditions were: 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. seh The PCR amplification product of the gene was 657 bp in size. H I-HF and Xho The PCR product and expression vector pET30a (+) were digested with I enzyme and ligated. The recombinant plasmid was transformed into Escherichia coli BL21 (DE) 3. After successful ligation, the recombinant plasmid was induced to express with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). The expressed SEH protein was purified using a HisTrap HP column and identified by SDS-PAGE ( Figure 1), SEH protein was successfully expressed, yielding a highly pure soluble protein with a molecular weight of 35 kD. The SEH protein concentration was determined using a BCA protein assay, yielding 5 mg of protein, which served as the antigen for antibody screening.

[0052] 2. Nanobody library selection

[0053] Recombinant SEH protein antibodies were screened from the natural nanoantibody phage display library constructed in the early stage. First, the ELISA plate was coated with recombinant SEH 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 13 pfu, 100 μl of phage library was added to each well and placed at 37°C for 1 hour. 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. 150 μl of 0.1M glycine (pH 2.2) was added to each well and eluted at room temperature for 20 minutes. 1M Tris (pH 8.0) solution was added for rapid neutralization. The eluted phage was infecting Escherichia coli TG1 cells in the logarithmic growth phase and shaken slowly at 180 rpm at 37°C for 1 hour. About 10 12 After 40 min of standing at 37°C, 50 μg / ml of ampicillin and 25 μg / ml of kanamycin were added to the cells. The cells were cultured overnight at 30°C at 220 rpm and centrifuged at 12,000 rpm for 20 min. 1 / 4 volume of PEG / NaCl solution was added to the supernatant and the cells were precipitated on ice for more than 4 h. 20 μl of the phage suspension was taken to calculate the titer. Clones that bound to SEH were screened through five rounds of panning ( Figure 2 ), the product titer was measured after the fifth round of panning, and the calculated output of the fifth round was 2.32×10 9 pfu.

[0054] 3. Phage-ELISA

[0055] Phage-ELISA was used to analyze the response of different clones to SEH. 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 the OD 600 Reach 0.6. Add about 10 12Incubate with pfu of M13KO7 helper phage at 30°C overnight. Coat an ELISA plate with recombinant SEH protein (0.5 μg / well). Transfer 100 μl of the phage display library from the deep-well plate to the 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.

[0056] 4. Construction of eukaryotic expression vector to express nanobodies

[0057] First, a His tag was added to the N-terminus of the enriched nanoantibodies, and then PCR amplification was performed under the following conditions: 95°C for 5 min, 95°C for 30 s, 53°C for 30 s, 72°C for 45 s, and 72°C for 5 min, for a total of 35 cycles. The size of the AntiSEH-Nb4 PCR product was 369 bp (SEQ ID NO: 3), and the size of the AntiSEH-Nb3 PCR product was 345 bp (SEQ ID NO: 4). The amino acid sequences of the nanoantibodies AntiSEH-Nb4 and AntiSEH-Nb3 are shown in SEQ ID NO: 1 and 2, respectively. The plasmid PCDNA3.4 and VHH amplified fragments were Bam H I-HF and Xho I double enzyme digestion, reaction conditions: 37℃ for 2h. The ligation product was transformed into E. coli JM108, plated on LB plates containing 50μg / ml ampicillin, and cultured at 37℃ for 12h. Single clones were screened for protein expression and expression verification.

[0058] Cell culture, transfection and expression: CHO-K1 cells were cultured in a CO2 incubator at 37°C, 120 rpm, and 8% CO2. 145M cells were taken and centrifuged to remove the supernatant (20 ml for expression). About 0.5 ml of electroporation solution was added to the cells, mixed, and then a plasmid at a concentration of 500 ng / ul was added. After the above cell plasmid suspension was thoroughly mixed, 1 ml was taken and added to a 1 ml electroporation tube, and the electroporation tube was placed in an electroporator for electroporation. After the electroporation was completed, the cells in the electroporation tube were divided into a shake flask containing 20 ml of culture medium prepared in advance and incubated statically for 40 minutes. After the incubation was completed, the shake flask was placed in 37°C, 270 rpm, and 8% CO2 for incubation. After 24 hours, feed / sodium butyrate / double antibody were added and cultured for 3-7 days. The expressed anti-SEH nanobody protein was purified using a HisTrap HP column and identified by SDS-PAGE ( Figure 3), obtaining a highly pure soluble protein. The anti-SEH nanobody protein was successfully expressed. AntiSEH-Nb4 has a molecular weight of 16 kD, and the amino acid sequence of the nanobody is shown in SEQ ID NO: 1. The concentration of the purified protein was determined using a BCA protein assay. 3 mg of AntiSEH-Nb4 protein was obtained for subsequent nanobody evaluation.

[0059] 5. Biotinylated expression of detection antibodies

[0060] A His tag was added to the N-terminus of AntiSEH-Nb3, and a short peptide tag consisting of 15 amino acids (Gly-Leu-Asn-Asp-Ile-Phe-Glu-Ala-Gln-Lys-Ile-Glu-Trp-His-Glu) was added to the C-terminus to perform site-specific biotinylation on AntiSEH-Nb3. The remaining expression steps were the same as step 4. The expressed anti-SEH nanobody protein was purified using a HisTrap HP column and identified by SDS-PAGE ( Figure 4 ), the molecular mass of AntiSEH-Nb3 is 19kD, and the amino acid sequence of the nanobody is shown in SEQ ID NO: 2. After the protein was labeled with biotin using an in vitro kit, biotinylated AntiSEH-Nb3 (AntiSEH Avi-Nb3) was obtained for evaluation of subsequent methods.

[0061] The anti-SEH nanobody (AntiSEH-Nb4) provided by the present invention has at least one of the following CDRs:

[0062] i) heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO: 1;

[0063] ii) heavy chain CDR2 from positions 51 to 58 of SEQ ID NO: 1;

[0064] iii) heavy chain CDR3 from positions 96 to 115 as shown in SEQ ID NO: 1.

[0065] 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.

[0066] The present invention also provides another anti-SEH nanobody (AntiSEH-Nb3), wherein the nanobody has at least one of the following CDRs:

[0067] i) heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO: 2;

[0068] ii) heavy chain CDR2 at positions 51 to 57 as shown in SEQ ID NO: 2;

[0069] iii) heavy chain CDR3 at positions 95 to 104 as shown in SEQ ID NO: 2.

[0070] 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.

[0071] 6. Nanobody Evaluation

[0072] Western blot experiments were used to evaluate the ability of the purified nanoantibody to bind to SEH protein and cross-react with 8 other enterotoxins. The SEH obtained after SDS-PAGE in step 1 above and the laboratory-stored SEA, SEB, SEC, SED, SEE, SEG, SEK, and SEQ recombinant proteins were transferred to a PVDF membrane and blocked with skim milk powder for 1 hour at room temperature. After washing, the membrane was incubated with the purified nanoantibody and incubated at room temperature for 1 hour. The membrane was detected with a 1:5000 dilution of HRP-labeled rabbit anti-camelid antibody and developed with DAB substrate. AntiSEH-Nb4 (1 μg / ml) ( Figure 5 ) and AntiSEH-Nb3 (1 μg / ml) ( Figure 6 The AntiSEH-Nb4 of the present invention can only effectively recognize the recombinant protein SEH, and its band is located at 35kD, indicating that the AntiSEH-Nb4 antibody can specifically detect the recombinant SEH protein ( Figure 5 The AntiSEH-Nb3 of the present invention recognizes the conformational epitope of the recombinant protein SEH and has no cross-reaction with other recombinant enterotoxins SEA, SEB, SEC, SED, SEE, SEG, SEK, and SEQ proteins, indicating that the AntiSEH-Nb3 antibody has good specificity for the recombinant SEH protein ( Figure 6 ).

[0073] Western blot experiments were used to evaluate the ability of the purified nanoantibody to bind to the natural SEH protein. The SEH recombinant protein was subjected to SDS-PAGE electrophoresis with the enterotoxin supernatant of the natural SEH strains (Staphylococcus aureus) DC53015, DC53034, DC53138, and DC53140, and then transferred to a PVDF membrane. The membrane was blocked with skim milk powder for 1 hour at room temperature. After washing, the membrane was incubated with the purified nanoantibody and incubated for 1 hour at room temperature. The membrane was detected with a 1:5000 dilution of HRP-labeled rabbit anti-camelid antibody and developed with DAB substrate. AntiSEH-Nb4 (1μg / ml) ( Figure 7) and AntiSEH-Nb3 (1 μg / ml) ( Figure 8 ).

[0074] 7. Checkerboard titration method to determine the best antibody pair

[0075] Five active nanobodies were biotinylated for site-specific labeling. The titers of the detection antibodies were determined indirectly, with a titer of 1:5000 for all five. Unlabeled nanobodies were coated onto ELISA plates (2 μg / well) as capture antibodies and incubated overnight at 4°C. After washing, the plates were blocked with 5% bovine serum albumin for 1 hour at 37°C. After washing, 2 μg of antigen was added to each well and blocked at 37°C for 1 hour. After washing, biotinylated site-specific labeled antibodies were added as detection antibodies and blocked at 37°C for 1 hour. After washing, HRP-streptavidin was added at a 1:20,000 dilution and washed, blocking at 37°C for 1 hour. 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 best antibody pair screened by the present invention is AntiSEH-Nb4 as the capture antibody and AntiSEH Avi-Nb3 as the detection antibody.

[0076] Example 2 Anti-SEH-Nb3 antibody affinity test for SEH

[0077] Surface Plasmon Resonance (SPR) was used to detect the affinity of the antibody. The instrument model for this experiment was Biacore X100. Different concentrations of ligand solution were input, and then the ligand solutions of different concentrations were placed in the sample rack according to the software prompts to start the experiment. The optimal ligand concentration was selected based on the experimental results for the next step of the experiment. The name and concentration of the analyte were input, and then the analyte solutions of different concentrations were placed in the sample rack according to the software prompts to start the experiment. The optimal analyte concentration was selected based on the experimental results for the next step of the experiment. The capture reagent, ligand, analyte, and regeneration reagent solutions were then placed in the sample rack according to the software prompts to start the experiment. The real-time kinetic data of the binding and dissociation of the antigen and antibody at each concentration were recorded, and the curve of the response signal changing with time was recorded. Enter the Kinetics / Affinity analysis interface, click Kinetics analysis, select the 1:1 interaction model, and calculate the binding constant of the antibody and antigen ( k a ) is 1.008E+5, the dissociation constant ( k d ) is 4.217E-4. According to the formula K D = k d / k a Calculate the affinity constant ( K D ) is 4.185E-9, indicating that AntiSEH-Nb3 has a high affinity for SEH.

[0078] Example 3 Specificity and sensitivity investigation

[0079] Based on the above results, the present invention determined a double antibody sandwich method using AntiSEH-Nb4 as the capture antibody and AntiSEH Avi-Nb3 as the detection antibody.

[0080] The specific experimental methods are as follows:

[0081] (1) AntiSEH-Nb4 was coated on a 96-well ELISA plate at 1.25 μg / mL (100 μL / well) using pH 9.6 carbonate buffer and incubated overnight at 4°C.

[0082] (2) After washing with 0.05% PBST (containing 0.05% Tween-20), the membrane was blocked with a blocking solution containing 5% BSA (200 μL / well) at room temperature for 1 h to block nonspecific binding sites;

[0083] (3) After washing three times with 0.05% PBST, the SEH standard was serially diluted 2-fold with an initial concentration of 10 μg / mL. Three replicate wells were set for each gradient, and 100 μL of dilution was added to each well. The cells were incubated at room temperature with shaking at 700 rpm for 1 h to promote antigen-antibody binding.

[0084] (4) After five intensive washes, 78.125 μg / mL AntiSEH Avi-Nb3 detection antibody (100 μL / well) was added and incubated under the same conditions;

[0085] (5) After six rigorous washes, diluted (1:1000) SA-PolyHRP80 (100 μL / well, shaken at 700 rpm for 30 min) was added to achieve signal amplification;

[0086] (6) Wash the plate six times with 0.05% PBST, pat dry, add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB), and incubate at 37°C in the dark with shaking for 10-15 minutes. Immediately add 50 μL of sulfuric acid (2M H2SO4) to terminate the color reaction, and read the absorbance (OD) value at a wavelength of 450 nm within 30 minutes. 1. Specificity experiments have verified that this method does not cross-react with common foodborne strains such as Salmonella, Bacillus, Listeria monocytogenes, Escherichia coli O157:H7, and Shigella flexneri type 2a during the detection process, and has high specificity.

[0087] 2. After sensitivity experiment verification, the detection limit (LOD) of this method for Staphylococcus aureus enterotoxin H (SEH) can reach 0.42 ng / mL.

[0088] The present invention tested 43 strains containing only SEH. seh All SEH expressed by the strains containing the gene were detected. seh One strain that expressed the gene but did not express the enterotoxin tested negative, showing 100% sensitivity and specificity.

[0089] Example 4

[0090] The thermal stability of the two nanobodies, AntiSEH-Nb4 and AntiSEH-Nb3, was evaluated using an indirect ELISA. The antigen-binding activity of the two antibodies was measured after incubation at 63°C, 75°C, and 95°C for 10 minutes, respectively. Antibodies stored at room temperature served as controls. The thermal stability of the nanobodies was assessed by comparing the activity of the nanobodies in the different temperature-treated groups with that of the control group.

[0091] The specific experimental method is as follows (repeated 3 times):

[0092] (1) Recombinant protein H was coated on a 96-well ELISA plate at 10 μg / mL using pH 9.6 carbonate buffer and incubated overnight at 4°C.

[0093] (2) After washing with 0.05% PBST (phosphate buffered saline-Tween 20), the membranes were blocked with a blocking solution containing 5% BSA (200 μL / well) at 37°C for 1 h.

[0094] (3) After washing the ELISA plate three times with 0.05% PBST buffer, add the antibody sample (1 μg / mL) that has been heat-treated at 63°C, 75°C, and 95°C for 10 minutes and stored at room temperature to the ELISA plate wells and incubate at 37°C for 1 hour.

[0095] (4) After washing five times with 0.05% PBST, add 1:10,000 anti-VHH secondary antibody (Rabbit Anti-Camelid VHH Antibody (HRP)) and incubate at 37°C for 1 h;

[0096] (5) Wash the plate six times with 0.05% PBST, pat dry, add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB), and incubate at 37°C in the dark for 10-15 minutes with shaking;

[0097] (6) Immediately add 50 μL of sulfuric acid (2 M H2SO4) to terminate the color development reaction and read the absorbance (OD) value at a wavelength of 450 nm within 30 minutes.

[0098] To evaluate the thermal stability of AntiSEH-Nb4 and AntiSEH-Nb3, this study incubated the two antibodies at different temperatures (63°C, 75°C, and 95°C) for 10 minutes. The activity changes were then detected by direct ELISA and compared with samples stored at room temperature. The experimental results showed that the antigen binding activity of AntiSEH-Nb4 and AntiSEH-Nb3 did not change significantly under 63°C and 75°C conditions; it is worth noting that even under high temperature treatment at 95°C, AntiSEH-Nb4 can still maintain more than 80% of its activity. These data fully demonstrate that AntiSEH-Nb4 and AntiSEH-Nb3 have excellent temperature stability, among which AntiSEH-Nb4 shows stronger thermal tolerance ( Figure 9 ).

[0099] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A nanobody for detecting Staphylococcus aureus enterotoxin H, characterized in that The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the variable region of the nanobody are SRAFSQNA, SMRTGKT, and CETWGVGGQG, respectively.

2. The Nanobody according to claim 1, characterized in that The amino acid sequence of the nanobody is shown in SEQ ID NO:

2.

3. A nucleic acid molecule encoding the Nanobody according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleotide sequence is shown in SEQ ID NO:

4.

5. The biological material containing the nucleic acid molecule according to claim 4, characterized in that The biological material is an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a host cell.

6. An antibody conjugate, characterized in that The antibody conjugate is obtained by conjugating the nanobody according to claim 1 or 2 to a label, and the label is selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radioactive label.

7. An antibody composition for detecting Staphylococcus aureus enterotoxin H, characterized in that: The antibody composition comprises the Nanobody according to claim 1 or 2 and the Nanobody Nb4 for detecting Staphylococcus aureus enterotoxin H; The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the variable region of the nanobody Nb4 are EFPFNYYT, IIKSDGST, and AVRYGSSCPGSDRADA, respectively.

8. The antibody composition according to claim 7, characterized in that The amino acid sequence of the nanobody Nb4 is shown in SEQ ID NO:

1.

9. Use of the nanobody according to claim 1 or 2, or the nucleic acid molecule according to claim 4, or the biomaterial according to claim 5, or the antibody conjugate according to claim 6, or the antibody composition according to claim 7 or 8 in the preparation of a reagent for qualitative or quantitative detection of Staphylococcus aureus enterotoxin H; The applications are for non-disease diagnosis and treatment purposes.

10. Staphylococcus aureus enterotoxin H double antibody sandwich enzyme-linked immunosorbent assay kit, characterized in that: The nanobody according to claim 1 or 2 is used as the detection antibody, and the nanobody Nb4 according to claim 7 or 8 is used as the capture antibody.

11. The double antibody sandwich enzyme-linked immunosorbent assay kit according to claim 10, characterized in that: The kit further comprises at least one of a substrate color developing solution, a blocking solution and a stopping solution.

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