Staphylococcus aureus specific nanobody and sandwich elisa detection method and application thereof

Abstract

The application discloses Staphylococcus aureus specific nanobodies and a sandwich ELISA detection method and application thereof, and belongs to the technical field of foodborne pathogenic bacteria detection. The Staphylococcus aureus specific nanobodies provided by the application include at least one of Nb55, Nb90, Nb156 and 3Nb156. The application constructs a sandwich ELISA based on HRP-labeled monovalent or trivalent nanobodies to detect Staphylococcus aureus, which effectively avoids unnecessary interaction between the crystallizable fragment and the immunoglobulin binding protein, and is successfully used for detection in actual samples, has the advantages of time saving, low cost, sensitivity and the like, and has important reference value for wide promotion of nanobodies in the field of foodborne pathogenic bacteria detection.

Description

Technical Field

[0001] This application belongs to the field of foodborne pathogen detection technology, and particularly relates to Staphylococcus aureus-specific nanobodies and their sandwich ELISA detection methods and applications. Background Technology

[0002] Foodborne illnesses frequently break out globally, with foodborne pathogens being a major contributing factor. These pathogens can directly or indirectly contaminate food, leading to serious consequences. Staphylococcus aureus (S. aureus), a common and highly dangerous foodborne pathogen, is widely distributed in the natural environment. It has been reported to be one of the leading infectious factors causing high mortality rates in 135 countries. According to statistics from the U.S. Centers for Disease Control and Prevention, from 1998 to 2020, the United States recorded 12,139 cases of illness caused by Staphylococcus aureus. Staphylococcus aureus is an aerobic or facultative anaerobic, non-spore-forming Gram-positive coccus that primarily colonizes the skin and mucous membranes of humans and animals. This bacterium exhibits strong variability, greatly enhancing its invasiveness and causing a wide variety of infectious symptoms. Although it typically colonizes harmlessly in healthy individuals, Staphylococcus aureus possesses significant pathogenic potential, capable of causing a range of diseases from mild skin infections to severe systemic infections, such as sepsis, endocarditis, meningitis, pneumonia, and toxic shock syndrome. Its pathogenicity is primarily attributed to various virulence factors, including hemolysins, leukocidins, enterotoxins, plasma coagulases, and deoxyribonucleases. Furthermore, Staphylococcus aureus is undemanding in its growth requirements and highly adaptable, readily contaminating various food products. Therefore, rapid and sensitive diagnosis of Staphylococcus aureus is crucial for reducing infection transmission, ensuring food safety at its source, and safeguarding public health.

[0003] To date, various technologies based on different mechanisms have been developed for the detection of Staphylococcus aureus in food. Microbial culture is a traditional method for pathogen detection; although accurate and inexpensive, it is time-consuming, labor-intensive, and inefficient, making it unsuitable for large-scale sample testing. Molecular biology methods, such as polymerase chain reaction (PCR), detect pathogens by amplifying specific nucleic acid fragments, offering advantages in accuracy, specificity, and sensitivity. However, the widespread application of this method is limited by cumbersome sample preparation, susceptibility to false positives due to impurities in the sample, and the need for expensive equipment and highly trained operators. Immunoassays based on pathogen-specific antibody recognition effectively compensate for the shortcomings of traditional microbial culture and molecular biology methods due to their ease of operation, rapid detection, and high sensitivity and efficiency. Traditional antibodies, including polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs), are commonly used as biorecognition elements in immunoassays. However, they have inherent limitations, such as batch-to-batch variability, long preparation cycles, and high production costs. More importantly, the immunoglobulin-binding proteins on the surface of Staphylococcus aureus inevitably interact with the crystallizable (Fc) fragments of traditional antibodies, which can lead to false positives and hinder the application of immunoassay for Staphylococcus aureus. Therefore, researchers are constantly working to find novel, high-performance immunoassay reagents to replace traditional antibodies.

[0004] In 1993, heavy chain antibodies (HCAbs) lacking the first constant region of the light and heavy chains were discovered in camels. Cloning the variable domain of HCAb yields a single-domain antibody called a VHH (Variable Domain of Heavy Chain) or nanobody (Nb), which retains complete antigen-binding properties. Although nanobodies are the smallest genetically engineered fragments capable of binding antigens, only about 15 kDa, they exhibit powerful biological properties. They possess high stability, maintaining activity even under extreme conditions such as high temperature and acidity / alkali, which is unmatched by traditional antibodies. Furthermore, the long and flexible CDR3 of nanobodies allows them to precisely recognize and tightly bind to antigens, even targeting antigenic epitopes that are difficult for traditional antibodies to reach, resulting in excellent specificity and affinity. As a new member of the antibody family, nanobodies stand out due to their unique structure and superior performance. Crucially, nanobodies lack crystallizable fragments, which can effectively avoid false positives in Staphylococcus aureus. Combined with the powerful properties of nanobodies themselves, this opens up new avenues for designing immunoassay methods for Staphylococcus aureus.

[0005] Enzyme-linked immunosorbent assay (ELISA) is one of the most widely used immunoassay techniques. For the detection of foodborne pathogens, a double-antibody sandwich ELISA is commonly used. Currently, double nanobody sandwich ELISA is widely used to detect pathogens such as Salmonella enteritidis (S. enteritidis), Vibrio parahaemolyticus (V. parahaemolyticus), and Listeria monocytogenes (L. monocytogenes). However, these methods have certain limitations, such as long detection time and low sensitivity. Summary of the Invention

[0006] To overcome the aforementioned deficiencies in existing technologies, this application provides Staphylococcus aureus-specific nanobodies and their sandwich ELISA detection method and application. This application obtains specific nanobodies (Nb55, Nb90, and Nb156) as capture antibodies through screening, expression, and purification. Horseradish peroxidase (HRP) is used to label the corresponding nanobodies as detection antibodies. By pairing them together and systematically optimizing reaction conditions, a sandwich ELISA without the need for additional HRP-labeled secondary antibodies is established. Furthermore, to explore the potential of multivalent nanobodies as detection antibodies in improving ELISA detection performance, a trivalent nanobodies with a flexible linker (GGGGS)3- are designed. A sandwich ELISA based on HRP-labeled trivalent nanobodies is constructed to detect Staphylococcus aureus. This method effectively avoids unnecessary interactions between crystallizable fragments and immunoglobulin-binding proteins and has been successfully applied to the detection of actual samples. It has advantages such as time-saving, low cost, and high sensitivity, and has important reference value for the widespread application of nanobodies in the detection of foodborne pathogens.

[0007] To achieve the above-mentioned objectives, this application provides the following technical solution:

[0008] On the one hand, this application provides Staphylococcus aureus-specific nanobodies, wherein the specific nanobodies include at least one of Nb55, Nb90, Nb156, and 3Nb156;

[0009] The complete amino acid sequence of Nb55 is shown in SEQ ID No. 1;

[0010] The complete amino acid sequence of Nb90 is shown in SEQ ID No. 2;

[0011] The complete amino acid sequence of Nb156 is shown in SEQ ID No. 3;

[0012] The complete amino acid sequence of 3Nb156 is shown in SEQ ID No. 4.

[0013] Optionally, the method for constructing 3Nb156 includes: using a homologous recombination method to tandem Nb156 with a flexible linker -(GGGGS)3- to amplify trivalent Nb156.

[0014] In this application, multivalent modification of nanobodies can effectively enhance antibody affinity. By increasing their size, multivalent nanobodies generate more antigen-binding sites, thereby enhancing their binding affinity and conferring better performance on immunoassays.

[0015] Secondly, this application provides the application of the above-mentioned Staphylococcus aureus-specific nanobody in the identification of Staphylococcus aureus surface antigen.

[0016] Thirdly, this application provides the application of the above-mentioned Staphylococcus aureus-specific nanobody in the preparation of reagents and / or kits for detecting Staphylococcus aureus infection in food.

[0017] Fourthly, this application provides a method for detecting Staphylococcus aureus using a sandwich ELISA based on the aforementioned Staphylococcus aureus-specific nanobody, comprising the following steps:

[0018] (1) HRP labeling of the above Staphylococcus aureus-specific nanobody yields at least one of HRP-Nb55, HRP-Nb90, HRP-Nb156, and HRP-3Nb156;

[0019] (2) Using at least one of Nb55, Nb90, and Nb156 as the capture antibody, Staphylococcus aureus as the antigen, and at least one of HRP-Nb55, HRP-Nb90, HRP-Nb156, and HRP-3Nb156 as the detection antibody, the process is as follows: coating with capture antibody, blocking, incubating with antigen, incubating with detection antibody, color development, and terminating color development.

[0020] In this application, step (1) involves in vitro HRP labeling of monovalent and trivalent nanobodies. Compared to the preparation of in vivo nanobodies and HRP fusion proteins, this method offers advantages such as flexible operation, shorter experimental cycle, and higher labeling efficiency. Furthermore, using HRP-labeled nanobodies can help eliminate the need for additional HRP-labeled secondary antibodies, which will effectively shorten the detection time and reduce reagent usage.

[0021] Optionally, in step (2), Nb55 is used as the capture antibody, and HRP-Nb156 or HRP-3Nb156 is used as the detection antibody.

[0022] Optionally, in step (2), when Nb55 is used as the capture antibody and HRP-Nb156 is used as the detection antibody, the concentration of Nb55 is 0.5-10 μg / mL, the dilution factor of HRP-Nb156 is 1:500-16000, the reagent used in the blocking step is skim milk powder, the concentration of skim milk powder is 1-5%, the blocking time is 1-2 h, the incubation time of the antigen is 30-120 min, and the incubation time of the detection antibody is 30-120 min.

[0023] Optionally, in step (2), when Nb55 is used as the capture antibody and HRP-Nb156 is used as the detection antibody, the concentration of Nb55 is independently selected from any value or a range between 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, and 10 μg / mL; the dilution factor of HRP-Nb156 is independently selected from any value or a range between 1:500, 1:1000, 1:2000, 1:4000, 1:8000, and 1:16000; the reagent used in the blocking step is skim milk powder. The concentration of the skim milk powder is independently selected from any value of 1%, 3%, 5%, or any range between two of them; the blocking time is independently selected from any value of 1h, 1.2h, 1.5h, 1.8h, 2h, or any range between two of them; the antigen incubation time is independently selected from any value of 30min, 45min, 60min, 90min, 120min, or any range between two of them; and the antibody incubation time is independently selected from any value of 30min, 45min, 60min, 90min, 120min, or any range between two of them.

[0024] Optionally, in step (2), when Nb55 is used as the capture antibody and HRP-Nb156 is used as the detection antibody, the concentration of Nb55 is 5 μg / mL, the dilution factor of HRP-Nb156 is 1:4000, the reagent used in the blocking step is skim milk powder with a concentration of 5%, the blocking time is 1 h, the incubation time of the antigen is 60 min, and the incubation time of the detection antibody is 60 min.

[0025] Optionally, in step (2), when Nb55 is used as the capture antibody and HRP-3Nb156 is used as the detection antibody, the concentration of Nb55 is 0.5-10 μg / mL, the dilution factor of HRP-3Nb156 is 1:500-16000, the reagent used in the blocking step is skim milk powder, the concentration of skim milk powder is 1-5%, the blocking time is 1-2 h, the incubation time of the antigen is 30-120 min, and the incubation time of the detection antibody is 30-120 min.

[0026] Optionally, in step (2), when Nb55 is used as the capture antibody and HRP-3Nb156 is used as the detection antibody, the concentration of Nb55 is independently selected from any value or a range between 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, and 10 μg / mL, and the dilution factor of HRP-3Nb156 is independently selected from any value or a range between 1:500, 1:1000, 1:2000, 1:4000, 1:8000, and 1:16000, and the reagent used in the blocking step is skim milk powder. The concentration of the skim milk powder is independently selected from any value of 1%, 3%, 5%, or any range between two of them; the blocking time is independently selected from any value of 1h, 1.2h, 1.5h, 1.8h, 2h, or any range between two of them; the antigen incubation time is independently selected from any value of 30min, 45min, 60min, 90min, 120min, or any range between two of them; and the antibody incubation time is independently selected from any value of 30min, 45min, 60min, 90min, 120min, or any range between two of them.

[0027] Optionally, in step (2), when Nb55 is used as the capture antibody and HRP-3Nb156 is used as the detection antibody, the concentration of Nb55 is 5 μg / mL, the dilution factor of HRP-3Nb156 is 1:8000, the reagent used in the blocking step is skim milk powder with a concentration of 3%, the blocking time is 1 h, the incubation time of the antigen is 60 min, and the incubation time of the detection antibody is 45 min.

[0028] Fifthly, this application provides the application of the above-mentioned Staphylococcus aureus sandwich ELISA detection method in the preparation of foodborne pathogen detection kits.

[0029] Compared with the prior art, this application has the following advantages:

[0030] This application establishes a sandwich ELISA for the detection of Staphylococcus aureus using Nb55 as the capture antibody and paired HRP-Nb156 as the detection antibody, without the need for an additional HRP-labeled secondary antibody. The MNb-ELISA established using HRP-Nb156 can detect levels as low as 9.11 × 10⁻⁶. 4Staphylococcus aureus at CFU / mL. This newly developed sandwich ELISA features low cost and rapid detection. To further improve the sensitivity of the detection method, this application constructed 3Nb156 with a flexible linker -(GGGGS)3- using a tandem linker strategy and labeled it with HRP. A TNb-ELISA was constructed based on HRP-3Nb156, and under optimal reaction conditions, the limit of detection (LOD) was 2.84 × 10⁻⁶. 4 The CFU / mL concentration was increased by 3.2 times compared to MNb-ELISA. This method exhibits good selectivity for Staphylococcus aureus and shows no cross-reactivity with other foodborne pathogens. Furthermore, the established TNb-ELISA demonstrates good reproducibility and high reliability, and has been successfully applied to dairy and meat samples, with recoveries ranging from 89.49% to 110.37% and a coefficient of variation (CV) within 9.18%. In summary, this application provides an excellent strategy for improving the sensitivity of detection methods. The established sandwich ELISA shortens detection time and reduces experimental costs, providing solid technical support for the application of nanobodies in the immunoassay of foodborne pathogens. It lays a good foundation for subsequent related research and practical detection work, promoting the development of foodborne pathogen immunoassay technology towards a more efficient and economical direction. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The SDS-PAGE and structural model of 3Nb156 in this application (Note: A represents SDS-PAGE; B represents structural model);

[0033] Figure 2 The specificity and freeze-thaw stability of HRP-labeled monovalent and trivalent nanobodies in this application are shown (Note: A represents the specificity result; B represents the freeze-thaw stability result);

[0034] Figure 3 This refers to the pairing results of the capture antibody and the detection antibody in the sandwich ELISA of this application;

[0035] Figure 4The effect of different concentrations of capture antibody and detection antibody on the results in the sandwich ELISA of this application (Note: A represents MNb-ELISA; B represents TNb-ELISA);

[0036] Figure 5 The results show the effect of different blocking conditions on the sandwich ELISA in this application (Note: A represents MNb-ELISA; B represents TNb-ELISA);

[0037] Figure 6 The results show the effect of different antigen incubation times on the sandwich ELISA in this application (Note: A represents MNb-ELISA; B represents TNb-ELISA);

[0038] Figure 7 The results show the effect of different detection antibody incubation times on the sandwich ELISA in this application (Note: A represents MNb-ELISA; B represents TNb-ELISA);

[0039] Figure 8 The standard curve for the sandwich ELISA detection of Staphylococcus aureus in this application (Note: A represents MNb-ELISA; B represents TNb-ELISA);

[0040] Figure 9 This serves as a validation result for the specificity of the TNb-ELISA in this application;

[0041] Figure 10 This is the stability validation result for the TNb-ELISA in this application (Note: A indicates intra-batch assay; B indicates inter-batch assay).

[0042] Figure 11 The results are for the repeatability validation of the TNb-ELISA in this application (Note: A indicates intraday analysis; B indicates interday analysis). Detailed Implementation

[0043] The present application is further illustrated below with reference to specific embodiments. The following descriptions are merely a few embodiments of the present application and are not intended to limit the present application in any way. Although the present application discloses preferred embodiments as follows, they are not intended to limit the present application. Any modifications or variations made by those skilled in the art without departing from the scope of the technical solution of the present application using the disclosed technical content are equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

[0044] Unless otherwise specified, the raw materials used in the embodiments of this application are all purchased commercially and used directly without any special treatment.

[0045] Unless otherwise specified, the analytical methods in the embodiments all adopt conventional instrument or equipment settings and conventional analytical methods.

[0046] Example 1

[0047] I. Experimental Materials and Equipment

[0048] 1. Test strains and vectors

[0049] Escherichia coli (E. coli) DH5α was purchased from Guangdong Provincial Microbial Culture Collection Center, GDMCC NO.: 1.2606. The vector pSTEP2-10×His was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. Staphylococcus aureus CICC 21600, Escherichia coli CICC 21530, Salmonella enteritidis CICC 21513, Listeria monocytogenes CICC 21633, Shigella flexneri (S. flexneri) CICC 21534, Cronobacter sakazakii (C. sakazakii) CICC 21645, and Yersinia enterocolitica (Y. enterocolitica) CICC 21669 were purchased from China Industrial Microbial Culture Collection Center.

[0050] 2. Test reagents

[0051] The main reagents are shown in Table 1.

[0052] Table 1 Main Reagents

[0053]

[0054]

[0055] 3. Test instruments and equipment

[0056] The main instruments and equipment are listed in Table 2.

[0057] Table 2 Main Instruments and Equipment

[0058]

[0059]

[0060] II. Experimental Methods and Results

[0061] 1. Construction of trivalent nanobodies

[0062] Homologous recombination technology was used to tandem Nb156 with the flexible linker -(GGGGS)3-, amplifying trivalent Nb156 (3Nb156), which was then ligated into the pSTEP2-10×His vector via EcoRI and BamHI restriction endonuclease sites. The resulting plasmid was then transformed into *E. coli* DH5α competent cells, and single clones were randomly selected to confirm sequence accuracy. PCR verification and sequencing further confirmed the inserted DNA. The recombinant plasmid was transfected into HEK293 cells for transient expression, purified using nickel affinity chromatography, and desalted using a gravity column. SDS-PAGE was used to analyze protein purity and size. The concentration of 3Nb156 was determined using Nanodrop and stored at -20°C for later use.

[0063] Results: 3Nb156 was prepared using a mammalian cell expression system, with an expression level of 15 mg / L. SDS-PAGE analysis showed that 3Nb156 exhibited a characteristic band of 45 kDa. Figure 1 In section A), the molecular weight is approximately three times that of monovalent Nb156, indicating that trivalent Nb156 was successfully prepared. The structural model is as follows: Figure 1 As shown in B in the table. The physicochemical properties and secondary structure prediction results of 3Nb156 are shown in Table 3. Its overall average hydrophilicity is negative (-0.509), and its aliphatic index and instability index are 56.88 and 35.65, respectively, indicating that 3Nb156 is a stable hydrophilic protein. The α-helix (36.0%) and β-sheet (44.8%) are more abundant in 3Nb156 than the turn (16.3%), and the β-sheet is the main secondary structure of 3Nb156.

[0064] Table 33 Physicochemical properties and secondary structure of Nb1 56

[0065]

[0066] 2. HRP labeling, specificity, and freeze-thaw stability analysis of nanobodies

[0067] HRP tags:

[0068] Monovalent nanobodies (Nb55, Nb90, and Nb156) and trivalent nanobodies (3Nb156) were HRP-labeled according to the kit procedure and stored at -20°C for subsequent experiments. (The complete amino acid sequence of Nb55 (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) is shown in SEQ ID No. 1: QVRLVESGGGSVQAGGSLRLSCEAAGYSYSDYTMGWFRQAPGKEREGVALVDRDGTATYIDSVKGRFTISRDNAKNTLFLQMNGLKPEDTAMYYCAALDMTVAEALRVPPPPLRGRGTQVTVS S;

[0069] The complete amino acid sequence of Nb90 (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) is shown in SEQ ID No. 2: QVQLVESGGGSVQAGGSLRLSCAASAYTINNYYMGWFRKAPGKMHEGVAAIDSVGVTSYADSVKGQFTISKDKAKNTLYLQMNSLKPDDSAMYYCAADYRSSGFTVDADRVLNLAFRGQGTQVTVS S;

[0070] The complete amino acid sequence of Nb156 (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) is shown in SEQ ID No. 3: EVQLVESGGGSVQPGGSLRLSCAASGFTFKDDEMSWVRQVPGKGLEWVASISNGGTFADYAGSVKGRFTISRSNDKNTLYLEMNSLKTEDTAVYYCARDRRGENRGQGTQVTVS S;

[0071] The complete amino acid sequence of 3Nb156 (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) is shown in SEQ ID. Shown in No.4: EVQLVESGGGSVQPGGSLRLSCAASGFTFKDDEMSWVRQVPGKGLEWVASISNGGTFADYAGSVKGRFTISRSNDKNTLYLEMNSLKTEDTAVYYCARDRRGENRGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCAASGFTFKDDEMSWVRQVPGKGLEWVASISNGG TFADYAGSVKGRFTISRSNDKNTLYLEMNSLKTEDTAVYYCARDRRGENRGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCAASGFTFKDDEMSWVRQVPGKGLEWVASISNGGTFADYAGSVKGRFTISRSNDKNTLYLEMNSLKTEDTAVYYCARDRRGENRGQGTQVTVSS).

[0072] HRP-Nb specificity:

[0073] Staphylococcus aureus and six common foodborne pathogens (including Escherichia coli, Salmonella enteritidis, Listeria monocytogenes, Shigella flexneri, Cronobacter sakazakii, and Yersinia enterocolitica) were used as antigens, and the specificity of HRP-Nb was verified by direct ELISA. First, the foodborne pathogen suspensions were diluted to 1×10⁻⁶ with 1×PBS. 8 CFU / mL, 100 μL / well for coating, incubated overnight at 4℃; washed 3 times with 0.1% PBST, added 300 μL of 5% skim milk powder to each well, blocked at 37℃ for 1 h; after washing 3 times with 0.1% PBST, added 100 μL / well of the four HRP-Nb compounds to the microplate, incubated at 37℃ for 1 h; washed 6 times with 0.1% PBST, added 100 μL of TMB chromogenic solution to each well, chromogenic at 37℃ for 15 min; then immediately added 50 μL of stop solution to each well to terminate the reaction, and measured the absorbance at 450 nm. The selectivity coefficient was calculated according to formula (1):

[0074] Selectivity coefficient (%) = [k (干扰剂) / k (S.aureus) ]×100 (1)

[0075] In the formula: k (干扰剂) The absorbance value for non-Staphylococcus aureus as antigens; k (S.aureus)The absorbance value is that of Staphylococcus aureus as an antigen; the selectivity coefficient of Staphylococcus aureus as an antigen is 100%.

[0076] HRP-Nb freeze-thaw stability:

[0077] Dilute the Staphylococcus aureus culture with 1×PBS to a final concentration of 1×10⁻⁶. 8 CFU / mL, add 100 μL to each well, coat overnight at 4℃; wash 3 times with 0.1% PBST, add 300 μL of 5% skim milk powder to each well, block at 37℃ for 1 h; wash 3 times with 0.1% PBST, add 100 μL of HRP-Nb with different freeze-thaw cycles to each well, incubate at 37℃ for 1 h; wash 6 times with 0.1% PBST, add 100 μL of TMB chromogenic solution to each well, develop color at 37℃ in the dark for 15 min; immediately add 50 μL of stop solution to each well to terminate the reaction, and measure the absorbance at 450 nm. Calculate the residual antibody activity according to formula (2):

[0078] Residual antibody activity (%) = (A1 / A2) × 100 (2)

[0079] In the formula: A1 is the absorbance value of HRP-Nb after freeze-thaw treatment; A2 is the absorbance value of HRP-Nb without freeze-thaw treatment; the activity of HRP-Nb without freeze-thaw treatment is 100%.

[0080] Results: The development of immunoassay methods requires ensuring the good detection performance of the necessary biological reagents to guarantee the accuracy and reproducibility of results. Therefore, the specificity and freeze-thaw stability of HRP-labeled monovalent and trivalent nanobodies were determined. Figure 2 As shown in A, HRP-Nb55, HRP-Nb90, HRP-Nb156, and HRP-3Nb156 specifically recognize Staphylococcus aureus, but show no cross-reactivity with the other six common foodborne pathogens. Regarding freeze-thaw stability, the four HRP-Nb groups retained over 80% of their binding activity after 10 freeze-thaw cycles. Figure 2 (B in the text). These experimental results fully demonstrate that HRP-labeled nanobodies possess precise targeting and recognition characteristics and high stability, providing a solid and reliable foundation for the successful establishment of subsequent sandwich ELISA.

[0081] III. Establishment of Sandwich ELISA and the Effect of Different Reaction Conditions

[0082] Nanobodies were used as capture antibodies, Staphylococcus aureus as antigen, and HRP-Nb was added as detection antibody. The specific method is as follows:

[0083] Coating capture antibody: Diluted nanobodies, 100 μL / well, coated overnight at 4°C.

[0084] Blocking: Wash the plate 3 times with 0.1% PBST, add 5% skim milk powder, 300 μL / well, and incubate at 37°C for 1 h.

[0085] Antigen incubation: Wash the plate 3 times with 0.1% PBST, then add 100 μL / well of 1×10⁻⁶ PBST. 8 CFU / mL bacterial culture was incubated at 37°C for 1 hour, with a negative control set up simultaneously.

[0086] Antibody incubation: Wash the plate 5 times with 0.1% PBST, add 100 μL / well of diluted HRP-Nb, and incubate at 37°C for 1 h.

[0087] Color development: Wash the plate 6 times with 0.1% PBST, add 100 μL of TMB color development solution per well, and develop for 15 min.

[0088] Termination of color development: Add 50 μL of stop solution per well to terminate the color development and measure the absorbance value at 450 nm.

[0089] 1. Pairing of capture antibodies and detection antibodies

[0090] To apply nanobodies to the detection of macromolecular analytes with multiple antibody binding sites, such as pathogenic bacteria, screening nanobodies that bind to different epitopes is essential. This application uses three nanobodies (Nb55, Nb90, and Nb156) as capture antibodies and corresponding HRP-Nb (HRP-Nb55, HRP-Nb90, and HRP-Nb156) as detection antibodies in a checkerboard assay. Different capture antibodies were paired with different detection antibodies. The influence of different antibody pairings was studied based on the highest absorbance ratio (P / N) between positive wells and negative control wells to select the optimal antibody combination and construct a sandwich ELISA (MNb-ELISA) based on HRP-labeled monovalent nanobodies. Furthermore, based on the best-paired HRP-Nb, a sandwich ELISA (TNb-ELISA) based on HRP-labeled trivalent nanobodies was constructed.

[0091] The results are as follows Figure 3 As shown, the ELISA achieved the highest P / N ratio when the capture antibody was Nb55 and the detection antibody was HRP-Nb156, indicating the optimal antibody pairing. These antibodies demonstrated high affinity for different epitopes of Staphylococcus aureus, forming a stable sandwich structure. Therefore, Nb55 was selected as the capture antibody, and HRP-Nb156 and HRP-3Nb156 were chosen as the detection antibodies for the subsequent establishment of MNb-ELISA and TNb-ELISA detection methods.

[0092] 2. Effects of different capture antibody and detection antibody concentrations

[0093] To construct a highly sensitive detection system, selecting appropriate concentrations of capture and detection antibodies is essential. The main method involves diluting the capture and detection antibodies by a certain factor, performing a checkerboard assay, and measuring the absorbance at 450 nm. Negative and positive results are affected by antibody concentration; higher concentrations increase negative values, while lower concentrations result in lower positive values. Therefore, to obtain high detection sensitivity, the optimal concentrations of capture and detection antibodies corresponding to the highest P / N ratio are selected as the optimal scheme for constructing the detection system. In this application, after selecting the combination of capture and detection antibodies, the nanobody was diluted to 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, and 10 μg / mL, and HRP-Nb was diluted at ratios of 1:500, 1:1000, 1:2000, 1:4000, 1:8000, and 1:16000 to study the effect of different capture and detection antibody concentrations. The optimal working concentration of the capture antibody and detection antibody combination was selected based on the highest P / N value. The results are as follows: Figure 4 As shown.

[0094] according to Figure 4 As shown in Figure A, the optimal concentration of the capture antibody Nb55 in MNb-ELISA is 5 μg / mL, and the optimal dilution of the detection antibody HRP-Nb156 is 1:4000. The optimal concentration of the capture antibody Nb55 in TNb-ELISA is 5 μg / mL, and the optimal dilution of the detection antibody HRP-3Nb156 is 1:8000. Figure 4 (B in the text). Compared to MNb-ELISA, TNb-ELISA reduces the concentration of detection antibodies used, thus lowering the cost of the experiment.

[0095] 3. The impact of different enclosure conditions

[0096] Six different blocking agents, namely skim milk powder at concentrations of 1%, 3%, and 5% and BSA at concentrations of 1%, 3%, and 5%, were used and blocked at 37°C for 1 hour. The above process was repeated, and the blocking was carried out at 37°C for 2 hours. The effect of different blocking conditions was studied, and the optimal blocking condition was selected based on the highest P / N value.

[0097] The results showed that the negative value of BSA blocking was high, resulting in significantly higher P / N values ​​for both methods using skim milk powder blocking compared to BSA, indicating that skim milk powder was more effective at blocking than BSA. In MNb-ELISA, the highest P / N value was observed with 5% skim milk powder blocking for 1 hour. Figure 5 In the A), while TNb-ELISA showed the highest P / N value for 3% skim milk powder blocked for 1 hour (in A), Figure 5(B in the text). This indicates that the binding of antigen and antibody can be affected by different blocking conditions, which in turn can lead to changes in the sensitivity of the method. Therefore, 5% and 3% skim milk powder were subsequently selected as the optimal blocking conditions for MNb-ELISA and TNb-ELISA, respectively, at 37°C for 1 hour.

[0098] 4. Effect of incubation time on different antigens

[0099] The same concentration of antigen was added to the ELISA plate and incubated at 37°C for 30 min, 45 min, 60 min, 90 min and 120 min respectively to study the effect of different antigen incubation times. The optimal antigen incubation time was determined by the P / N ratio.

[0100] The results are as follows Figure 6 As shown, when the MNb-ELISA antigen incubation time was 60 min, the P / N value had basically stabilized. Further incubation did not significantly change the P / N value, and it even showed a decreasing trend. Figure 6 (A) Similarly, the P / N value of TNb-ELISA antigen incubation reached its maximum at 60 min, and then decreased (A). Figure 6 (B in the text). Therefore, the optimal incubation time for the antigen in both MNb-ELISA and TNb-ELISA is 60 min.

[0101] 5. The effect of different antibody incubation times

[0102] This application uses Nb directly coupled to HPR as the detection antibody, and the reaction time between the detection antibody and the antigen is crucial to the sensitivity of the method. This application sets the incubation time of the detection antibody to 30 min, 45 min, 60 min, 90 min, and 120 min, and studies the effect of different detection antibody incubation times. The optimal incubation time of the detection antibody is determined by the P / N ratio.

[0103] Results of antibody detection at different incubation times in MNb-ELISA are as follows: Figure 7 As shown in Figure A, the P / N value gradually increases with increasing incubation time, reaching its peak at 60 min and then decreasing. This is because the binding time between the detection antibody and the target antigen is insufficient, and the reaction is not complete. Excessive binding time may lead to dissociation of the antigen-antibody complex and changes in antibody activity. Therefore, the optimal incubation time for the detection antibody in MNb-ELISA is determined to be 60 min. For TNb-ELISA, this may be because the trivalent antibody, used as the detection antibody, increases the affinity for the antigen, thus shortening the incubation time. The detection antibody reaches its maximum P / N value at 45 min of incubation. Figure 7(as shown in section B). Therefore, 60 min and 45 min were selected as the optimal incubation times for the MNb-ELISA and TNb-ELISA, respectively. Compared with MNb-ELISA, TNb-ELISA further shortened the detection time.

[0104] IV. Validation of Sandwich ELISA

[0105] 1. Standard Curve

[0106] Staphylococcus aureus bacterial suspension was serially diluted 3-fold with 1×PBS, 1×10⁻⁶. 8 The initial concentration was CFU / mL. Based on the established optimal reaction conditions for MNb-ELISA and TNb-ELISA, different concentrations of Staphylococcus aureus were detected. Standard curves for both detection methods were constructed with bacterial concentration as the X-axis and absorbance as the Y-axis. The lowest level of deviation (LOD) was calculated by adding three times the standard deviation (SD) to the blank value.

[0107] The results are as follows Figure 8 As shown, all values ​​exhibit an "S" shape. A four-parameter logistic equation was used for fitting, where x represents the concentration of Staphylococcus aureus culture and y represents the absorbance value at 450 nm. Based on the LOD being the blank value plus three times the standard deviation, the lowest LOD for Staphylococcus aureus determined by TNb-ELISA was 2.84 × 10⁻⁶. 4 CFU / mL Figure 8 B in the text is MNb-ELISA (LOD: 9.11 × 10⁻⁶). 4 3.2 times (CFU / mL) Figure 8 (A) This suggests that high-affinity and large-size trivalent nanobodies may help reduce the LOD of ELISA, making the signal response during detection faster and more sensitive. Therefore, further methodological validation will only be performed on TNb-ELISA.

[0108] 2. Specificity

[0109] To evaluate the specificity of the established TNb-ELISA, Staphylococcus aureus and six common foodborne pathogens (including Escherichia coli, Salmonella enteritidis, Listeria monocytogenes, Shigella flexneri, Cronobacter sakazakii, and Yersinia enterocolitica) were used as antigens, and the bacterial concentration was set at 1×10⁻⁶. 8 The concentration of CFU / mL was used for sandwich ELISA detection, and the selectivity coefficient was calculated using the same formula (1) to assess whether the method specifically detects Staphylococcus aureus.

[0110] The results are as follows Figure 9As shown, the established TNb-ELISA exhibits good specificity against Staphylococcus aureus. The selectivity coefficients for the other six common foodborne pathogens are all less than 2.77%, with no significant cross-reactivity. This is mainly attributed to the high affinity and high specificity of Nb55 and Nb156.

[0111] 3. Stability

[0112] Validating the stability of the detection method is crucial for the accuracy of experimental results. Therefore, six intra-batch assays were performed on three concentrations of Staphylococcus aureus and a negative control, and six inter-batch assays were also performed.

[0113] Intra-batch stability: Take plates from the same batch and dilute the bacterial suspension to 1×10⁻⁶. 8 CFU / mL, 3×10 7 CFU / mL and 1×10 7 Three concentration gradients of CFU / mL were used, with six replicates for each concentration. The absorbance at 450 nm was measured, and a negative control was set up.

[0114] Inter-batch stability: Take plates from different batches and dilute the bacterial suspension to 1×10⁻⁶. 8 CFU / mL, 3×10 7 CFU / mL and 1×10 7 Three concentration gradients of CFU / mL were used, with six replicates for each concentration. The absorbance at 450 nm was measured, and a negative control was set up.

[0115] Intra-batch test results as follows Figure 10 As shown in A, the CVs were 2.14%, 2.08%, 3.14%, and 3.57%, respectively. Inter-batch determination results are as follows... Figure 10 As shown in B, the CV values ​​are all less than 4.19%. The experimental results demonstrate that this method exhibits excellent stability for the detection of Staphylococcus aureus.

[0116] 4. Repeatability

[0117] Intraday repeatability: Dilute the bacterial culture to 1×10⁻⁶ 8 CFU / mL, 3×10 7 CFU / mL and 1×10 7 Three concentration gradients of CFU / mL were performed, with five replicates within one day. The absorbance at 450 nm was compared at the same concentration, and a negative control was also set up.

[0118] Daytime repeatability: Dilute the bacterial culture to 1×10⁻⁶ 8 CFU / mL, 3×10 7 CFU / mL and 1×10 7Three concentration gradients of CFU / mL were performed, with five replicates over 20 days. The absorbance at 450 nm was compared at the same concentration, and a negative control was also included.

[0119] The results are as follows Figure 11 As shown, the CV for intraday detection of Staphylococcus aureus was 2.41–3.87%. Figure 11 (A) In daytime detection, the CV for Staphylococcus aureus was less than 6.14% (in section A). Figure 11 (B in the text). Experimental results show that this method has good reproducibility and can be effectively used for the detection of Staphylococcus aureus.

[0120] 5. Actual sample testing

[0121] To evaluate the practicality and reliability of TNb-ELISA in food testing, milk and chicken samples contaminated with different concentrations of Staphylococcus aureus were tested. Milk and chicken were purchased from local markets and their sterility was confirmed by plate counting. Subsequently, 1 mL (or 1 g) of sample was mixed with 9 mL of 1×PBS in a sterile centrifuge tube and vortexed for 5 min to ensure complete dissolution. The pretreated samples were centrifuged at 10,000 rpm for 10 min at 4°C to remove the precipitate and the top fat layer. Staphylococcus aureus was diluted to 1×10⁻⁶ with the supernatant from the food matrix. 8 CFU / mL, 1×10 7 CFU / mL and 1×10 6 The target bacteria were detected using a sandwich ELISA at CFU / mL, and the recovery rate and CV were calculated.

[0122] Table 4. Determination of Staphylococcus aureus in actual samples

[0123]

[0124]

[0125] As shown in Table 4, the recoveries of these samples ranged from 89.49% to 110.37%, with CVs all less than 9.18%. These experimental results demonstrate that this method exhibits excellent detection performance in real samples and is feasible for the detection of Staphylococcus aureus in food, showing broad application prospects.

[0126] This application establishes a sandwich ELISA for the detection of Staphylococcus aureus using Nb55 as the capture antibody and paired HRP-Nb156 as the detection antibody, without the need for an additional HRP-labeled secondary antibody. The MNb-ELISA established using HRP-Nb156 can detect levels as low as 9.11 × 10⁻⁶. 4CFU / mL of Staphylococcus aureus. This newly developed sandwich ELISA features low cost and rapid detection. To further improve the sensitivity of the detection method, a tandem linker 3Nb156 with a flexible linker -(GGGGS)3- was constructed and HRP-labeled. A TNb-ELISA was constructed based on HRP-3Nb156, achieving a LOD of 2.84 × 10⁻⁶ under optimal reaction conditions. 4 The CFU / mL concentration was increased by 3.2 times compared to MNb-ELISA. This method exhibits good selectivity for Staphylococcus aureus and shows no cross-reactivity with other foodborne pathogens. Furthermore, the established TNb-ELISA demonstrates good reproducibility and high reliability, and has been successfully applied to dairy and meat samples, with recoveries ranging from 89.49% to 110.37% and CVs within 9.18%. In summary, this application provides an excellent strategy for improving the sensitivity of detection methods. The established sandwich ELISA shortens detection time and reduces experimental costs, providing solid technical support for the application of nanobodies in the immunoassay of foodborne pathogens. It lays a good foundation for subsequent related research and practical detection work, promoting the development of foodborne pathogen immunoassay technology towards a more efficient and economical direction.

[0127] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. Staphylococcus aureus-specific nanobody, characterized in that, The specific nanobody includes at least one of Nb55, Nb90, Nb156, and 3Nb156; The complete amino acid sequence of Nb55 is shown in SEQ ID No. 1; The complete amino acid sequence of Nb90 is shown in SEQ ID No. 2; The complete amino acid sequence of Nb156 is shown in SEQ ID No. 3; The complete amino acid sequence of 3Nb156 is shown in SEQ ID No.

4.

2. The Staphylococcus aureus-specific nanobody according to claim 1, characterized in that, The method for constructing 3Nb156 includes: using homologous recombination to tandem Nb156 with the flexible linker -(GGGGS)3- to amplify trivalent Nb156.

3. The application of the Staphylococcus aureus-specific nanobody as described in claim 1 or 2 in the identification of Staphylococcus aureus surface antigen.

4. The use of the Staphylococcus aureus-specific nanobody according to claim 1 or 2 in the preparation of reagents and / or kits for detecting Staphylococcus aureus infection in food.

5. A method for detecting Staphylococcus aureus using a sandwich ELISA based on Staphylococcus aureus-specific nanobody as described in claim 1 or 2, characterized in that, Includes the following steps: (1) HRP labeling of the Staphylococcus aureus-specific nanobody according to claim 1 or 2 to obtain at least one of HRP-Nb55, HRP-Nb90, HRP-Nb156, and HRP-3Nb156; (2) Using at least one of Nb55, Nb90, and Nb156 as the capture antibody, Staphylococcus aureus as the antigen, and at least one of HRP-Nb55, HRP-Nb90, HRP-Nb156, and HRP-3Nb156 as the detection antibody, the process is as follows: coating with capture antibody, blocking, incubating with antigen, incubating with detection antibody, color development, and terminating color development.

6. The method for detecting Staphylococcus aureus using a sandwich ELISA according to claim 5, characterized in that, In step (2), Nb55 is used as the capture antibody, and HRP-Nb156 or HRP-3Nb156 is used as the detection antibody.

7. The method for detecting Staphylococcus aureus using a sandwich ELISA according to claim 6, characterized in that, In step (2), when Nb55 is used as the capture antibody and HRP-Nb156 is used as the detection antibody, the concentration of Nb55 is 0.5-10 μg / mL, the dilution factor of HRP-Nb156 is 1:500-16000, the reagent used in the blocking step is skim milk powder, the concentration of skim milk powder is 1-5%, the blocking time is 1-2 h, the incubation time of the antigen is 30-120 min, and the incubation time of the detection antibody is 30-120 min. Preferably, in step (2), when Nb55 is used as the capture antibody and HRP-Nb156 is used as the detection antibody, the concentration of Nb55 is 5 μg / mL, the dilution factor of HRP-Nb156 is 1:4000, the reagent used in the blocking step is skim milk powder with a concentration of 5%, the blocking time is 1 h, the incubation time of the antigen is 60 min, and the incubation time of the detection antibody is 60 min.

8. The method for detecting Staphylococcus aureus using a sandwich ELISA according to claim 6, characterized in that, In step (2), when Nb55 is used as the capture antibody and HRP-3Nb156 is used as the detection antibody, the concentration of Nb55 is 0.5-10 μg / mL, the dilution factor of HRP-3Nb156 is 1:500-16000, the reagent used in the blocking step is skim milk powder, the concentration of skim milk powder is 1-5%, the blocking time is 1-2 h, the incubation time of the antigen is 30-120 min, and the incubation time of the detection antibody is 30-120 min; Preferably, in step (2), when Nb55 is used as the capture antibody and HRP-3Nb156 is used as the detection antibody, the concentration of Nb55 is 5 μg / mL, the dilution factor of HRP-3Nb156 is 1:8000, the reagent used in the blocking step is skim milk powder with a concentration of 3%, the blocking time is 1 h, the incubation time of the antigen is 60 min, and the incubation time of the detection antibody is 45 min.

9. The application of the Staphylococcus aureus sandwich ELISA detection method according to any one of claims 5 to 8 in the preparation of a foodborne pathogen detection kit.

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