Nanometer antibody for detecting staphylococcus aureus enterotoxin H, antibody composition and application thereof

Abstract

The invention belongs to the field of immunological detection, and discloses a nano antibody for detecting staphylococcus aureus enterotoxin H, an antibody composition and application thereof. According to the present invention, the specific antibody of the staphylococcus aureus enterotoxin H is successfully screened, and the chessboard titration method is adopted to determine the capture antibody AntiSEH-Nb4 and the detection antibody AntiSEH-Nb3; the obtained antibody can effectively detect food poisoning caused by non-classical intestines, and a high-specificity detection means is provided for staphylococcus aureus enterotoxin H. The accuracy and reliability of detection are improved, and rapid diagnosis and prevention of outbreak of related food-borne diseases are facilitated.

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 and can cause severe staphylococcal food poisoning (SFP). Staphylococcus aureus enterotoxins (SEs) can cause nausea, severe vomiting and abdominal cramps, and occasionally diarrhea after a short incubation period. Its molecular weight is between 19-30kD and it is resistant to high temperature and protease hydrolysis. So far, a total of 29 different SEs and enterotoxin-like toxins (SELs) have been reported, and the classic SEs serotypes include SEA, SEB, SEC, SED and SEE. However, in recent years, food poisoning incidents caused by non-classical enterotoxins have gradually increased worldwide, attracting widespread attention. Studies have shown that Staphylococcus aureus strains isolated from certain food poisoning incidents often do not carry the classic SEs gene, but through in-depth genetic analysis, it was found that these strains generally carry the "non-classical SEs" gene. This finding suggests that non-classical SEs may play an equally important pathogenic role as classic SEs in SFP outbreaks. In addition, 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] At present, the main methods for detecting SEs include biological detection, genetic molecular biology detection, mass spectrometry detection and immunological detection. The detection method specified in the national standard is enzyme-linked immunosorbent assay (ELISA), which has the advantages of high throughput, easy operation, high sensitivity, etc., and can directly detect SEs in samples. In addition, compared with other technologies for detecting SEs, the ELISA method is not easily interfered by complex matrices, does not involve ethical issues, and is not easily contaminated to produce false positives. Several existing commercial SEs immunological detection kits can detect 5 classic enterotoxins at the same time, and the antibodies (mainly IgG) are mainly derived from mouse monoclonal antibodies or rabbit polyclonal antibodies. For example, the enzyme-linked immunosorbent assay kit for the total amount of Staphylococcus aureus enterotoxin jointly developed by China Agricultural University and Beijing Weideweikang Biotechnology Co., Ltd. uses monoclonal antibodies obtained by immunizing mice with recombinant Staphylococcus aureus classic enterotoxins.

[0004] Currently available commercial test kits for Staphylococcus aureus enterotoxin include: Ridascreen from Germany ® set, 3M of the United States @ TECRA® Staph enterotoxins, Identification Test 3 , VIDAS of France ® SET3 etc. However, most of these kits can only detect classical enterotoxins, but 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 stability of the fluorescent reagent is poor and requires more stringent environmental and storage conditions. At present, there is an urgent need to develop domestic detection kits with independent intellectual property rights in my country.

[0005] Traditional monoclonal antibodies for detecting enterotoxins are obtained by immunizing animals, which takes a long time to screen, is costly, and requires skilled operators and specialized cell culture facilities. In particular, the crystallizable fragment (Fc) region of monoclonal antibodies can bind to the surface protein A (SpA) of Staphylococcus aureus, resulting in false positives in the ELISA method for detecting enterotoxins. Nanobodies have many advantages: Nanobodies are more inclined to bind to concave epitopes and can bind to antigenic epitopes that traditional antibodies cannot bind to; the extension of the complementarity determining region 3 (CDR3) and the conservative disulfide bonds make nanobodies have higher thermal stability; the hydrophilic amino acids in the framework region 2 (FR2) of the nanobody increase the water solubility of the nanobody and reduce the aggregation ability; it is easy to express and purify in prokaryotes, reduce production costs, break through the limitations of in vivo antibody preparation, and shorten the development cycle. The nanobody VHH fragment does not contain the Fc region, and theoretically will not bind to the SpA protein of Staphylococcus aureus and produce cross-reactions. 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 application thereof for detecting Staphylococcus aureus enterotoxin H.

[0007] In order to achieve the purpose of the present invention, in the 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 biological materials containing the nucleic acid molecule, wherein the biological materials include but are not limited to expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or host cells.

[0012] In a fourth aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate is obtained by conjugating the nanobody with a marker, and the marker 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; 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.

[0014] Furthermore, the amino acid sequence of the nanobody Nb4 is shown in SEQ ID NO:1.

[0015] In a sixth aspect, the present invention provides the use of nanobody Nb3 or a nucleic acid molecule encoding the nanobody or a biological material containing the nucleic acid molecule or the antibody conjugate or the antibody composition in the preparation of a reagent for qualitative or quantitative detection of Staphylococcus aureus enterotoxin H (including non-disease diagnosis and treatment purposes).

[0016] In the seventh aspect, the present invention provides a Staphylococcus aureus enterotoxin H double antibody sandwich enzyme-linked immunosorbent assay kit, using nano antibody Nb3 as the detection antibody and nano antibody Nb4 as the capture antibody, that is, capture antibody AntiSEH-Nb4 and detection antibody AntiSEH-Nb3.

[0017] Furthermore, the kit also includes at least one of a substrate color developing solution, a blocking solution and a stop solution.

[0018] In a specific 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 label (Avi-tag), horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-galactosidase, lysozyme, malate dehydrogenase, etc., preferably using a biotin label (Avi-tag).

[0019] Furthermore, the kit also includes at least one of a substrate color developing solution, a blocking solution and a stop solution.

[0020] 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., preferably TMB.

[0021] The blocking solution may be a 5% BSA (bovine serum albumin) solution or the like.

[0022] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention successfully screened out Staphylococcus aureus enterotoxin H specific antibodies, and used the chessboard titration method to determine the capture antibody AntiSEH-Nb4 and the detection antibody AntiSEH-Nb3. The obtained antibodies can effectively detect food poisoning caused by non-classical enterotoxin, and provide a highly specific detection method for Staphylococcus aureus enterotoxin H. Not only does it improve the accuracy and reliability of the detection, it also helps to quickly diagnose and prevent the outbreak of related foodborne diseases.

[0023] The antibody of the present invention can be used in detection techniques such as enzyme-linked immunosorbent assay (ELISA), western blot, colloidal gold method, etc. for Staphylococcus aureus enterotoxin H. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The purified SEH protein in the preferred embodiment of the present invention, wherein M is a protein molecular weight marker; 1 is a recombinant protein after SEH purification.

[0025] 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.

[0026] Figure 3 The results of expressing AntiSEH-Nb4 using a eukaryotic expression system in a preferred embodiment of the present invention are shown in FIG.

[0027] 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 FIG. 1 , where M is a protein molecular weight marker. 1: AntiSEH Avi-Nb3.

[0028] Figure 5 The western blot results of purified AntiSEH-Nb4, recombinant protein SEH and 8 enterotoxins of Staphylococcus aureus in the preferred embodiment of the present invention are shown in Figure 1. Among them, M: 180 KDa protein marker; 1: SEA; 2: SEB; 3: SEC; 4: SED; 5: SEE; 6: SEG; 7: SEK; 8: SEH; 9: SEQ.

[0029] Figure 6 The western blot results of purified AntiSEH-Nb3, recombinant protein SEH and 8 enterotoxins of Staphylococcus aureus in the preferred embodiment of the present invention are shown in Figure 1. Among them, M: 180 KDa protein marker; 1: SEA; 2: SEB; 3: SEC; 4: SED; 5: SEE; 6: SEG; 7: SEK; 8: SEH; 9: SEQ.

[0030] Figure 7 The binding ability of purified AntiSEH-Nb4 with recombinant SEH protein and natural SEH protein in the preferred embodiment of the present invention is shown in Figure 1. Wherein, M: 180 KDa protein marker; 1: SEH recombinant protein; 2: DC53015; 3: DC53034; 4: DC53138; 5: DC53140.

[0031] Figure 8The binding ability of purified AntiSEH-Nb3 with recombinant SEH protein and natural SEH protein in the preferred embodiment of the present invention is shown in Figure 1. Wherein, M: 180 KDa protein marker; 1: SEH recombinant protein; 2: DC53015; 3: DC53034; 4: DC53138; 5: DC53140.

[0032] Fig. 9 Thermal stability results of purified AntiSEH-Nb4 and purified AntiSEH-Nb3 in preferred embodiments of the present invention. DETAILED DESCRIPTION

[0033] The present invention uses the natural nanoantibody library constructed in the early stage to screen out a pair of anti-SEH nanoantibody pairs with good specificity. The present invention uses the capture antibody AntiSEH-Nb4 (SEQ ID NO: 1) and the detection antibody AntiSEH Avi-Nb3 (SEQ ID NO: 2) as an antibody pair, and constructs a double antibody sandwich method to achieve specific detection of SEH. The capture antibody and the detection antibody can specifically recognize SEH and do not react with other enterotoxins, thereby ensuring the specificity of the diagnostic results.

[0034] 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 make the nanoantibody have 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.

[0035] The present invention adopts the following technical solution: 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: i) the 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 112 as shown in SEQ ID NO:1.

[0036] 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.

[0037] The present invention also provides another anti-SEH nanobody (AntiSEH-Nb3), wherein the nanobody has at least one of the following CDRs: i) the heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO: 2; ii) heavy chain CDR2 at positions 51 to 57 as shown in SEQ ID NO: 2; iii) heavy chain CDR3 at positions 95 to 104 as shown in SEQ ID NO:2.

[0038] 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.

[0039] 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.

[0040] Example 1 Screening of anti-SEH nanoantibody pairs 1. SEH protein expression and purification DNA was extracted from Staphylococcus aureus DC53321 stored in the Chinese Center for Disease Control and Prevention, and PCR was used to amplify enterotoxin seh Gene (GenBank: MN752207.1). PCR amplification conditions were: 95°C 5 min, 95°C 30 s, 55°C 30 s, 72°C 45 s, 72°C 5 min, for a total of 35 cycles. seh The size of the PCR amplification product of the gene was 657 bp. H I-HF and XOt I digested the PCR product and the expression vector pET30a (+), and after ligation, 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 with HisTrap HP column and identified by SDS-PAGE ( Figure 1 ), SEH protein was successfully expressed, and a high-purity soluble protein with a molecular weight of 35kD was obtained. The concentration of SEH protein was determined using the BCA protein kit, and a total of 5mg of protein was obtained as an antigen for screening antibodies.

[0041] 2. Nanobody library selection Recombinant SEH protein antibodies were screened from the natural nanoantibody phage display library constructed earlier. 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 for antigen and 4 replicate wells for blank control (coating solution) were set up 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% skimmed milk powder) was added to each well, and the plate was 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, 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 (pH2.2) to each well and elute for 20min at room temperature, then add 1M Tris (pH8.0) solution for rapid neutralization. The eluted phage infects Escherichia coli TG1 cells in the logarithmic growth phase, and shake slowly at 37℃ 180rpm for 1h. Add about 10 12 pfu of M13KO7 helper phage was placed at 37°C for 40 min, and then ampicillin and kanamycin were added at a final concentration of 50ug / ml and 25ug / ml, and cultured overnight at 220rpm and 30°C. The supernatant was centrifuged at 12000rpm for 20min, and 1 / 4 volume of PEG / NaCl solution was added to the supernatant. The supernatant was precipitated on ice for more than 4h. 20μl of phage suspension was taken to calculate the titer, and clones that bind to SEH were screened through five rounds of panning ( Figure 2 ), the product titer was measured after the fifth round of panning, and the fifth round output was calculated to be 2.32×10 9 pfu.

[0042] 3. Phage-ELISA 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 OD 600 Reach 0.6. Add about 10 12pfu of M13KO7 helper phage, incubate at 30°C overnight. Coat the ELISA plate with recombinant SEH (0.5μg / well) protein, take 100μl of phage display library from the deep-well plate and add it to the ELISA plate, incubate at 37°C for 1h. After washing the plate, add 1:10000 diluted HRP-labeled mouse anti-M13 phage antibody and incubate at 37°C for 1h. After washing the plate, add 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution to each well and measure the absorbance at 450 nm. Select clones with significant differences in absorbance between the positive well and the control well for sequencing.

[0043] 4. Construction of eukaryotic expression vector to express nanobodies 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 5min, 95°C 30s, 53°C 30s, 72°C 45s, 72°C 5min, for a total of 35 cycles. The size of the AntiSEH-Nb4 PCR product was 369bp (SEQ ID NO: 3), and the size of the AntiSEH-Nb3 PCR product was 345bp (SEQ ID NO: 4). The amino acid sequences of the nanoantibodies AntiSEH-Nb4 and AntiSEH-Nb3 are shown in SEQ ID NOs: 1 and 2, respectively. The plasmid PCDNA3.4 and VHH amplified fragments were subjected to Bam H I-HF and XOt I double enzyme digestion, reaction conditions: 37℃ for 2h. The ligation product was transformed into E. coli JM108, and then cultured on LB plates containing 50μg / ml ampicillin at 37℃ for 12h, and single clones were screened for protein expression and expression verification.

[0044] Cell culture, transfection and expression: CHO-K1 cells were cultured in a CO2 incubator at 37°C, 120rpm, 8% CO2; 145M cells were taken, centrifuged and the supernatant was removed (20ml for expression); about 0.5ml of electroporation solution was added to the cells, mixed and then a plasmid with a concentration of 500ng / ul was added; the above cell plasmid suspension was fully mixed and 1ml was added to a 1ml 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 20ml culture medium prepared in advance, and incubated statically for 40min; after the incubation was completed, the shake flask was placed in 37°C, 270rpm, 8% CO2 for culture, and feed / sodium butyrate / double antibody was added after 24h, and the culture was continued for 3-7d; the expressed anti-SEH nanoantibody protein was purified using a HisTrap HP column and identified by SDS-PAGE ( Figure 3), a high-purity soluble protein was obtained, and the anti-SEH nanobody protein was successfully expressed. The molecular weight of AntiSEH-Nb4 was 16kD, and the amino acid sequence of the nanobody AntiSEH-Nb4 was shown in SEQ ID NO: 1. The concentration of the purified protein was determined using a BCA protein kit. 3 mg of AntiSEH-Nb4 protein was obtained for subsequent nanobody evaluation.

[0045] 5. Biotinylated expression of detection antibodies 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 weight of AntiSEH-Nb3 is 19kD, and the amino acid sequence of the nanobody is shown in SEQ ID NO: 2. After the protein is labeled with biotin by an in vitro kit, biotinylated AntiSEH-Nb3 (AntiSEH Avi-Nb3) is obtained for evaluation of subsequent methods.

[0046] The anti-SEH nanobody (AntiSEH-Nb4) provided by the present invention has at least one of the following CDRs: i) the 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 96 to 115 as shown in SEQ ID NO:1.

[0047] 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.

[0048] The present invention also provides another anti-SEH nanobody (AntiSEH-Nb3), wherein the nanobody has at least one of the following CDRs: i) the heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO: 2; ii) heavy chain CDR2 at positions 51 to 57 as shown in SEQ ID NO: 2; iii) heavy chain CDR3 at positions 95 to 104 as shown in SEQ ID NO:2.

[0049] 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.

[0050] 6. Evaluation of Nanobodies 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 SEA, SEB, SEC, SED, SEE, SEG, SEK, and SEQ recombinant 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, incubate with purified nanoantibodies, incubate at room temperature for 1 hour, detect with HRP-labeled rabbit anti-camelid antibody diluted 1:5000, and develop with DAB substrate. AntiSEH-Nb4 (1μg / ml) ( Figure 5 ) and AntiSEH-Nb3(1μg / 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 ). 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 ).

[0051] Western blot experiments were used to evaluate the ability of purified nanoantibodies to bind to natural SEH proteins. The recombinant SEH protein was subjected to SDS-PAGE electrophoresis with the enterotoxin supernatants of SEH natural strains (Staphylococcus aureus) DC53015, DC53034, DC53138, and DC53140, and then transferred to a PVDF membrane and blocked with skim milk powder for 1 hour at room temperature. After washing, the membrane was incubated with purified nanoantibodies at room temperature for 1 hour, 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(1μg / ml)( Figure 8 ).

[0052] 7. Determine the best antibody pair by chessboard titration Five kinds of nanoantibodies with binding activity were biotinylated and site-specifically labeled. The titer of the detection antibody was determined by the indirect method, and the titer of the five detection antibodies was 1:5000. The unlabeled nanoantibody was coated on the ELISA plate (2μg / well) as the capture antibody, incubated at 4℃ overnight, and then blocked with 5% bovine serum albumin at 37℃ for 1h after washing; after washing, 2μg of antigen was added to each well and blocked at 37℃ for 1h; after washing, the biotinylated site-specific labeled antibody was added as the detection antibody and blocked at 37℃ for 1h; after washing, 1:20000 HRP-streptavidin was added and the plate was washed and blocked at 37℃ for 1h; 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added to each well, and the absorbance at 450 nm was measured. The antibody pairs with significant differences in absorbance between the positive wells and the control wells were selected as candidate antibody pairs for constructing the enzyme-linked immunosorbent double antibody sandwich method. The best antibody pair screened by the present invention is AntiSEH-Nb4 as the capture antibody and AntiSEH Avi-Nb3 as the detection antibody.

[0053] Example 2 AntiSEH-Nb3 antibody and SEH affinity determination experiment 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 different concentrations of ligand solution were placed in the sample rack according to the software prompts to start the experiment. The most suitable ligand concentration was selected for the next step of the experiment based on the experimental results. The name and concentration of the analyte were input, and then different concentrations of analyte solution were placed in the sample rack according to the software prompts to start the experiment. The most suitable analyte concentration was selected for the next step of the experiment based on the experimental results. The capture reagent, ligand, analyte, and regeneration reagent solution were then placed in the sample rack according to the software prompts to start the experiment. The real-time kinetic data of antigen-antibody binding and dissociation at each concentration were recorded, and the response signal curve over 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.

[0054] Example 3 Specificity and sensitivity investigation Based on the above results, the present invention determines the double antibody sandwich method using AntiSEH-Nb4 as the capture antibody and AntiSEH Avi-Nb3 as the detection antibody.

[0055] The specific experimental methods are as follows: (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; (2) After washing with 0.05% PBST (containing 0.05% Tween-20), the cells were blocked with a blocking solution containing 5% BSA (200 μL / well) at room temperature for 1 h to block nonspecific binding sites; (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. 100 μL of dilution was added to each well and incubated at room temperature with shaking at 700 rpm for 1 h to promote antigen-antibody binding. (4) After five intensive washes, add 78.125 μg / mL AntiSEH Avi-Nb3 detection antibody (100 μL / well) and incubate under the same conditions; (5) After six stringent washes, diluted (1:1000) SA-PolyHRP80 (100 μL / well, 700 rpm shaking for 30 min) was added to achieve signal amplification; (6) Wash the plate six times with 0.05% PBST, pat dry, add 100uL of 3,3',5,5'-tetramethylbenzidine (TMB), and incubate at 37℃ in the dark for 10-15min; immediately add 50uL of sulfuric acid (2M H2SO4) to terminate the color reaction, and read the absorbance (OD) value at a wavelength of 450nm within 30min. 1. After specificity experimental verification, 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.

[0056] 2. After sensitivity experiment verification, the detection limit (LOD) of this method for detecting Staphylococcus aureus enterotoxin H (SEH) can reach 0.42 ng / mL.

[0057] The present invention tested 43 strains containing only SEH. seh All SEH expressed by strains containing the gene were detected. sehOne strain that did not express the enterotoxin gene was negative, showing 100% sensitivity and specificity.

[0058] Example 4 The indirect ELISA method was used to evaluate the thermal stability of the two nanobodies, AntiSEH-Nb4 and AntiSEH-Nb3. The two antibodies were incubated at 63°C, 75°C, and 95°C for 10 minutes, and their antigen binding activity was measured. The antibodies stored at room temperature were used as controls. The thermal stability of the nanobodies was evaluated by comparing the activity differences between the groups treated at different temperatures and the control group.

[0059] The specific experimental method is as follows (repeated 3 times): (1) Use pH 9.6 carbonate buffer to coat the recombinant protein H at 10 ug / mL on a 96-well ELISA plate and incubate overnight at 4°C; (2) After washing with 0.05% PBST (phosphate buffered saline-Tween 20), the cells were blocked with a blocking solution containing 5% BSA (200 μL / well) at 37°C for 1 h. (3) After washing the ELISA plate three times with 0.05% PBST buffer, add the antibody samples (1 μg / mL) that have been heat-treated at 63°C, 75°C, and 95°C for 10 minutes and stored at room temperature to the wells of the ELISA plate and incubate at 37°C for 1 hour. (4) After washing five times with 0.05% PBST, add 1:10000 anti-VHH secondary antibody (Rabbit Anti-CamelidVHH Antibody (HRP)) and incubate at 37°C for 1 h; (5) Wash the plate six times with 0.05% PBST, pat dry, add 100uL of 3,3',5,5'-tetramethylbenzidine (TMB), and incubate at 37°C in the dark for 10-15min with shaking; (6) Immediately add 50uL of sulfuric acid (2M H2SO4) to terminate the color reaction and read the absorbance (OD) value at a wavelength of 450nm within 30min.

[0060] In order 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, then detected their activity changes by direct ELISA, and compared them 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 exhibits stronger thermal tolerance ( Fig. 9 ).

[0061] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

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. A 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 virus vector, an engineering 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 with a label, and the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

7. An antibody composition for detecting Staphylococcus aureus enterotoxin H, characterized in that: The antibody composition comprises the Nanobody of 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; Furthermore, the amino acid sequence of the nanobody Nb4 is shown in SEQ ID NO:

1.

8. Use of the nanobody according to claim 1 or 2, the nucleic acid molecule according to claim 4, the biomaterial according to claim 5, the antibody conjugate according to claim 6, or the antibody composition according to claim 7 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.

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

10. The double antibody sandwich ELISA kit according to claim 9, characterized in that: The kit further comprises at least one of a substrate color developing solution, a blocking solution and a stop solution.

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