Immunomagnetic bead method kit for enriching staphylococcus aureus enterotoxin B from complex matrix

Abstract

The invention provides an immunomagnetic bead method kit for enriching staphylococcus aureus enterotoxin B from a complex matrix, and belongs to the technical field of food detection. The invention discloses a kit for enriching staphylococcus aureus enterotoxin B (SEB). The kit comprises a carrier coupled with an anti-staphylococcus aureus enterotoxin B antibody and enrichment liquid, the enrichment liquid comprises a 0.05 M phosphate buffer solution containing 0.5%-1.2% of NaCl, 0.08%-0.12% of KCl and 0.08%-0.12% of Tween 20, and the pH value of the enrichment liquid ranges from 7.1 to 7.4. According to the kit, SEB in a to-be-detected sample is enriched under the condition of enrichment liquid through antibody coupling magnetic beads targeting SEB, so that the aims of concentrating and detecting a target substance and purifying the target substance are fulfilled, the detection sensitivity is effectively improved, and matrix interference is reduced.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to an immunomagnetic bead assay kit for enriching Staphylococcus aureus enterotoxin B from complex matrices. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus (SAA) is a Gram-positive coccus widely found in nature and is one of the common foodborne zoonotic pathogens. During food processing and storage, SAA readily contaminates high-protein foods (such as meat and dairy products) and, under suitable conditions, multiplies rapidly and produces toxins, leading to food poisoning incidents. This bacterium can cause skin and soft tissue infections, and its enterotoxins (SEs) pose a significant threat to global food safety. SEs are extremely heat-resistant, difficult to destroy at conventional cooking temperatures, and even trace amounts (ng) can cause severe vomiting, diarrhea, and other poisoning symptoms in humans. Based on antigenic differences, SEs have been identified into 10 serotypes (SEA, SED, SEB, etc.). Among them, SEB type B enterotoxins (SEB) are of particular concern in food safety monitoring due to their superantigenic activity (capable of non-specifically activating a large number of T cells and triggering a cytokine storm), high thermal stability (remaining active even after heating at 100°C for 30 minutes), and resistance to protease digestion (making SEB difficult to completely eliminate during food processing). These characteristics make establishing efficient and accurate SEB testing methods a crucial aspect of food safety control.

[0003] Currently, detection methods for SEB mainly include traditional methods such as microbial culture, immunoassays (e.g., ELISA), and molecular biology techniques (e.g., real-time fluorescence PCR). While these methods are reliable, they suffer from low detection sensitivity. Summary of the Invention

[0004] The purpose of this invention is to provide an immunomagnetic bead assay kit for enriching Staphylococcus aureus enterotoxin B from a complex matrix. By using antibody-conjugated magnetic beads targeting SEB, SEB in the sample to be tested is enriched, thereby concentrating and purifying the target substance, effectively improving detection sensitivity and reducing matrix interference.

[0005] The present invention provides a kit for enriching Staphylococcus aureus enterotoxin B, comprising a carrier coupled with an anti-Staphylococcus aureus enterotoxin B antibody and an enrichment solution; The enrichment solution comprises a 0.05M phosphate buffer containing 0.5%–1.2% NaCl, 0.08%–0.12% KCl, and 0.08%–0.12% Tween 20, with a pH of 7.1–7.4.

[0006] Preferably, the enrichment solution comprises a 0.05M phosphate buffer containing 0.9% NaCl, 0.1% KCl, and 0.1% Tween 20, with a pH of 7.2.

[0007] Preferably, the carrier coupled with the anti-Staphylococcal enterotoxin B antibody includes magnetic beads.

[0008] Preferably, the anti-Staphylococcal enterotoxin B antibody and the carrier are coupled via an amide bond.

[0009] Preferably, it also includes a neutralizing solution and a 0.4~0.6M glycine solution; The neutralization solution comprises 1M Tris buffer with a pH of 8.5.

[0010] This invention provides the application of the kit for enriching Staphylococcus aureus enterotoxin B in detecting Staphylococcus aureus enterotoxin B in samples.

[0011] Preferably, the enrichment temperature for Staphylococcus aureus enterotoxin B is 36~38℃, and the enrichment time is 28~47min.

[0012] Preferably, the elution temperature for enriching Staphylococcus aureus enterotoxin B is 60~70℃.

[0013] Preferably, the test sample includes at least one of the following: water, food, and biological fluids.

[0014] The present invention provides a kit for detecting Staphylococcus aureus, including the kit for enriching Staphylococcus aureus enterotoxin B.

[0015] This invention provides a kit for enriching Staphylococcus aureus enterotoxin B, comprising a carrier conjugated with an anti-Staphylococcus aureus enterotoxin antibody and an enrichment solution. The enrichment solution comprises 0.05M phosphate buffer containing 0.5%–1.2% NaCl, 0.08%–0.12% KCl, and 0.08%–0.12% Tween 20, with a pH of 7.1–7.4. This invention conjugates the anti-Staphylococcus aureus enterotoxin B antibody with the carrier, and under specific enrichment solution conditions, uses the anti-Staphylococcus aureus enterotoxin B antibody to capture Staphylococcus aureus enterotoxin B in the detection sample. Then, the carrier's properties are utilized to separate the target substance from the detection sample, achieving the purpose of concentrating and purifying the target substance, thereby improving detection sensitivity and reducing matrix interference. In this embodiment, the enrichment solution formulation is optimized to obtain an enrichment solution formulation with the greatest difference in binding and elution, ensuring sufficient enrichment of Staphylococcus aureus enterotoxin B in the detection sample, thereby greatly improving the detection sensitivity of Staphylococcus aureus enterotoxin B.

[0016] This invention provides a kit for detecting Staphylococcus aureus, including a kit for enriching Staphylococcus aureus enterotoxin B. Based on this kit for enriching Staphylococcus aureus enterotoxin B in the detection sample, Staphylococcus aureus enterotoxin B is enriched and impurities are removed. Using a reagent for detecting Staphylococcus aureus enterotoxin B, the detection sensitivity is significantly improved compared to the unenriched group. In this embodiment, water, milk, and urine are used as examples. The kit for enriching Staphylococcus aureus enterotoxin B in the detection sample is used to pre-enrich three positive samples constructed by the labeling method before routine detection. Verification results show that the direct detection sensitivity for water samples is below 10 pg / ml. After pretreatment, the sensitivity reaches 1 pg / ml. Direct detection of milk samples is invalid, but after pretreatment, the detection sensitivity reaches 2 pg / ml. Direct detection of urine samples shows false positives, but after pretreatment, the detection sensitivity reaches 5 pg / ml. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of chromatography combined with immunomagnetism; Figure 2 The effects of enrichment time and temperature on the enrichment effect are shown; where 1 is enrichment at 23℃ for 15 min, 2 is enrichment at 23℃ for 30 min, 3 is enrichment at 23℃ for 45 min, 4 is enrichment at 37℃ for 15 min, 5 is enrichment at 37℃ for 30 min, 6 is enrichment at 37℃ for 45 min, 7 is enrichment at 45℃ for 15 min, 8 is enrichment at 45℃ for 30 min, and 9 is enrichment at 45℃ for 45 min. Figure 3 The results are from a simulation experiment on a complex matrix sample. Detailed Implementation

[0018] The present invention provides a kit for enriching Staphylococcus aureus enterotoxin B, comprising a carrier coupled with an anti-Staphylococcus aureus enterotoxin B antibody and an enrichment solution; The enrichment solution comprises a 0.05M phosphate buffer containing 0.5%–1.2% NaCl, 0.08%–0.12% KCl, and 0.08%–0.12% Tween 20, with a pH of 7.1–7.4.

[0019] In this invention, the enrichment solution preferably comprises a 0.05M phosphate buffer containing 0.9% NaCl, 0.1% KCl, and 0.1% Tween 20, with a pH of 7.2.

[0020] In this embodiment of the invention, the pH value of the enrichment solution (7.2-8.0) has little effect on the immune response of SEB and antibodies, but has a significant impact on the elution effect. SEB elution is more complete at pH 7.2. The reason for this is that the enrichment solution at pH 8.0 ultimately interferes with the pH of glycine, resulting in a reduced elution effect. Increasing sodium ions, potassium ions, and Tween 20 in the enrichment solution increases the B1 value, while the B2 value is not significantly different from the control. This may be because the glycine elution effect has reached saturation, and the enrichment gain effect is not fully manifested by elution.

[0021] In this invention, the carrier coupled with the anti-Staphylococcus aureus enterotoxin B antibody preferably comprises magnetic beads. The magnetic beads comprise superparamagnetic nanoparticles (such as Fe3O4). The anti-Staphylococcus aureus enterotoxin B antibody and the carrier are preferably coupled via an amide bond. The carrier preferably comprises carboxylated immunomagnetic beads. The anti-Staphylococcus aureus enterotoxin B antibody comprises at least one of a monoclonal antibody against Staphylococcus aureus enterotoxin B, a polyclonal antibody against Staphylococcus aureus enterotoxin B, or a nanobody against Staphylococcus aureus enterotoxin B. In this embodiment of the invention, the method for enriching and detecting Staphylococcus aureus enterotoxin B in a sample based on the kit is illustrated using an anti-Staphylococcus aureus enterotoxin B monoclonal antibody as an example.

[0022] In this invention, it is preferable to further include a neutralizing solution and a 0.4-0.6M glycine solution, which may be a 0.5M glycine solution. The neutralizing solution preferably includes a 1M Tris buffer solution with a pH of 8.5.

[0023] This invention provides the application of the kit for enriching Staphylococcus aureus enterotoxin B in the detection of Staphylococcus aureus enterotoxin B in samples.

[0024] In this invention, the method for enriching and detecting Staphylococcus aureus enterotoxin B in a sample preferably includes the following steps: washing a carrier coupled with an anti-Staphylococcus aureus enterotoxin B antibody and mixing it with the sample; performing a capture reaction to separate the carrier that has captured Staphylococcus aureus enterotoxin B; incubating and eluting with a glycine solution and a neutralizing solution; removing the carrier to obtain a separation solution rich in Staphylococcus aureus enterotoxin B, which is then used for detection by a detection reagent.

[0025] In this invention, the enrichment temperature for Staphylococcus aureus enterotoxin B is preferably 36-38°C, and can be 37°C. The enrichment time is preferably 28-47 min, and can be 30-45 min, or 35-40 min. The elution temperature for enriching Staphylococcus aureus enterotoxin B is preferably 60-70°C, and can be 60-65°C. The elution time is preferably 8-15 min, and can be 10-12 min.

[0026] In this invention, the test sample preferably includes at least one of the following: water, food, and biological fluids. The food includes dairy products. The dairy products include milk powder, milk, yogurt, etc. The biological fluids preferably include blood, urine, tissue fluid, cerebrospinal fluid, cerebrospinal fluid, cerebral tissue fluid, etc.

[0027] The present invention provides a kit for detecting Staphylococcus aureus, including the kit for enriching Staphylococcus aureus enterotoxin B.

[0028] In this invention, the kit preferably further includes a detection reagent for Staphylococcus aureus enterotoxin B. This invention does not impose any particular limitation on the type of detection reagent for Staphylococcus aureus enterotoxin B; any detection reagent for Staphylococcus aureus enterotoxin B well-known in the art can be used, such as a time-resolved fluorescence immunochromatographic assay card.

[0029] The following detailed description, in conjunction with embodiments, of an immunomagnetic bead assay kit for enriching Staphylococcus aureus enterotoxin B from a complex matrix provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 1. Experimental Materials The main equipment and materials required for the experiment are as follows: time-resolved fluorescence immunochromatographic assay kit (Chengdu Weirui Biotechnology Co., Ltd.), fluorescence detector (Chengdu Weirui Biotechnology Co., Ltd.), magnetic rack (Shanghai Tuohe Electromechanical Technology Co., Ltd.), vortex oscillator (Dalong Xingchuang Experimental Instrument Co., Ltd.), constant temperature water bath (Shanghai Qiuzuo Technology Co., Ltd.), rotary incubator (Wuxi Woxin Instrument Co., Ltd.), carboxyl magnetic beads (Guangzhou Yixin Biotechnology), and SEB antibody (Wuxi Ditengmin Biotechnology Co., Ltd.).

[0031] 2 Experimental Methods 2.1 Preparation of carboxylated immunomagnetic bead-conjugated antibodies 1) Take 1 mg of thoroughly shaken carboxylated magnetic beads and add them to a 2 mL centrifuge tube containing 1 mL MES to wash the magnetic beads; after separation with a magnetic rack, resuspend the magnetic beads in 900 μL MES; 2) Activation: Disperse the magnetic beads by sonication, add freshly prepared activation reagents NHS (100 mg / mL) and EDC (100 mg / mL) solutions respectively, mix well by vortexing, fix in a rotary incubator and incubate at room temperature for 25 min; magnetically separate and discard the supernatant, add 1 mL MES to wash the magnetic beads, and then add 500 μL MES to reconstitute the magnetic beads; 3) Conjugation: Take 0.5 mg (250 μL) of activated carboxylated immunomagnetic beads into two 2 mL centrifuge tubes, and add 250 μL of pre-diluted SEB monoclonal antibody (the name of the monoclonal antibody is "SEB monoclonal antibody", purchased from Wuxi Ditengmin Biotechnology Co., Ltd.). Add PBS buffer to make up to 500 μL, mix thoroughly with a vortex mixer, fix on an inverted incubator, and conjugate at 37℃ for 2 h. After magnetic separation, discard the supernatant, and wash thoroughly with 500 μL PBS buffer 3 times to remove unbound antibody. 4) Blocking: Add 500 μL of magnetic bead blocking solution to a centrifuge tube containing carboxylated immunomagnetic beads, vortex to mix thoroughly, fix on an inverted incubator, and invert at 37°C for 30 min; magnetically separate and discard the supernatant, add 500 μL of PBS buffer to wash thoroughly 3 times, and finally add 500 μL of PBS buffer to resuspend the magnetic beads and store at 4°C for later use.

[0032] 2.2 Enrichment of Staphylococcus aureus type B enterotoxin 1) Cleaning the magnetic beads: Take 20 μg of carboxylated immunomagnetic beads conjugated with SEB monoclonal antibody into a 5 mL centrifuge tube, add 2 mL of sample, vortex to mix thoroughly, let stand on a magnetic rack to collect the magnetic beads, discard the supernatant, repeat the above operation twice, and collect the cleaned carboxylated immunomagnetic beads. 2) SEB extraction and elution: Add the sample to the sample processing solution, vortex to mix, and centrifuge at 4000 r / min for 10 min. Take 5 mL of the supernatant and add it to a centrifuge tube containing carboxylated immunomagnetic beads, vortex to mix, and incubate in a centrifuge at 42℃ for 1 h. Then, place the SEB-magnetic bead mixture on a magnetic rack for 5 min, discard the supernatant, and add 100 μL of neutralization solution (i.e., 1 M Tris pH 8.5 neutralization solution), and incubate at 70℃ with shaking for 15 min. 3) SEB collection and detection: Collect magnetic beads using a magnetic rack at 70℃, add the supernatant to the sample well of the SEB time-resolved fluorescence detection reagent card, perform chromatography reaction for 15 min, and use a portable high-sensitivity fluorescence analyzer to detect the fluorescence signal and interpret the results.

[0033] 2.3 Optimization of conditions for the enrichment of Staphylococcus aureus type B enterotoxin In the SEB antibody magnetic bead enrichment conditions, key factors were optimized, including different enrichment solutions (i.e., enrichment reaction solutions, which provide a liquid environment for the enrichment reaction; the composition and ratio of the enrichment solution are detailed in section 1.2.3.1), the amount of enrichment magnetic beads used, the enrichment temperature, and the enrichment time, in order to enrich the most SEB in the shortest time.

[0034] 2.3.1 Effect of enrichment solution on enrichment effect Different components in the enrichment solution can affect the immune response. To explore the most suitable immune response environment, the ionic strength, pH, and surfactant were optimized and compared, and a suitable enrichment solution was initially selected. The experimental design selected four conditions that affect the immune response: NaCl, KCl, TWEEN-20, and pH. Glycine was used for elution, followed by dilution with fluorescent detection reagent card diluent. Finally, 80 μl of the sample was loaded onto a high-sensitivity fluorescent detection card for detection. During the experiment, the high-sensitivity fluorescent detection card was used to detect the SEB solution before enrichment (T0), the enrichment solution (T0-1), the SEB solution after enrichment (T1), and the SEB solution after elution (T2), with a blank elution negative control (T2-1). The optimal ratio B1 was calculated using formula I: B1 = T0 / T1 (T0 > 2). T0-1); B2=T2 / T2-1. The experimental group with the largest B1 and B2 is the optimal experimental group, and the experimental results are shown in Table 1.

[0035] Table 1. Effect of enrichment solution on enrichment effect

[0036] Note: Enrichment solution 1 is 0.05M phosphate buffer (pH 7.2); Enrichment solution 2 is Enrichment solution 1 containing 0.9% NaCl; Enrichment solution 3 is Enrichment solution 1 containing 0.9% NaCl and 0.1% KCl; Enrichment solution 4 is Enrichment solution 1 containing 0.9% NaCl, 0.1% KCl, and 0.1% Tween 20. Enrichment solution 5 is 0.05M phosphate buffer (pH 8.0).

[0037] Compared to the five enrichment groups, enrichment group 1 showed smaller differences in B1 and larger differences in B2. The smaller difference in B1 indicates that the pH of the enrichment solution (7.2-8.0) has little impact on the immune response of SEB and antibodies. The larger difference in B2 indicates a difference in elution efficiency. This is likely because the enrichment solution at pH 8.0 ultimately interferes with the pH of glycine, leading to a decrease in elution efficiency and consequently, a lower B2. Increasing the sodium, potassium, and Tween 20 ions in the enrichment solution significantly increased B1, but the difference in B2 was not significant. This is likely because the elution efficiency of glycine had already reached saturation, and the enrichment gain was not fully realized. Therefore, enrichment solution 4 was ultimately selected as the optimal enrichment solution.

[0038] 2.3.2 Effects of enrichment time and temperature on enrichment effect A 40 pg / ml SEB solution was prepared using enrichment buffer and divided evenly into 9 portions. Enrichment temperatures were set at room temperature (23℃), 37℃, and 45℃, with enrichment periods of 15 min, 30 min, and 45 min at each temperature. Before enrichment, the fluorescence signal value (S0) of the SEB solution at this concentration was detected using an SEB fluorescence card. After enrichment in each experimental group, the values ​​of the enriched SEB solution were detected again using the card, resulting in values ​​of S1, S2, ..., S9. Following the same elution method (elution with a 70℃ high-temperature neutralization solution), the fluorescence signal of the SEB eluent was detected again using a card, resulting in values ​​of q1, q2, ..., q9. The experimental results were recorded as follows: Figure 2 And Table 2.

[0039] Table 2 Optimization Experiment Results

[0040] Among the nine enrichment conditions, the highest fluorescence detection values ​​(q) were observed after enrichment at 37℃ for 30 min and 37℃ for 45 min. However, the s value was low when enriched at 45℃, but the q value did not increase after elution. The reason for this may be that prolonged enrichment at high temperature affects the antigenicity of SEB, leading to an overall decrease in the detection signal.

[0041] 2.4 Optimization of SEB elution conditions 2.4.1 Effect of different elution methods on SEB elution Two SEB elution methods were selected: Method 1, elution under acidic conditions, and Method 2, elution under high-temperature conditions. The elution effects of the two methods were compared, and the optimal elution conditions were optimized. In the experimental group, an SEB solution with a final concentration of 40 pg / ml was prepared using the optimal enrichment buffer. One 1 ml aliquot of SEB solution was used for enrichment and acidic elution experiments, and another 1 ml aliquot of SEB solution was used for the same enrichment and high-temperature elution. Two negative control groups were set up according to the two enrichment and elution methods. The ratio of the detection signal in the positive group to the negative group was used as the key screening factor N. The larger the ratio, the better the elution conditions.

[0042] Acidic elution: Prepare a 0.5M glycine solution and a 1M Tris pH 8.5 neutralization solution. After enriching the antigen with magnetic beads, add 50 μL of glycine solution and react for 20 min. After the magnetic beads are adsorbed by the magnetic rack, collect the supernatant, add 20 μL of neutralization solution, mix and react for a period of time, then add 40 μL of neutralization solution to dilute and mix well before detection. Calculate N1.

[0043] High-temperature elution: Prepare a 60℃ constant temperature water bath. After enriching the antigen with magnetic beads, add 100μL of neutralization solution and place in the constant temperature water bath. React for 10 min. Adsorb the magnetic beads with a magnetic rack at 70℃, collect the supernatant, and after restoring to room temperature, add 80μL of supernatant to the fluorescent detection card for detection. Calculate N2. The experimental results of different elution methods are shown in Table 3.

[0044] Table 3 Comparison of the two elution methods

[0045] After the antibody binds to the antigen, it dissociates, and then the antigen is detected and analyzed using an immunoassay. Two factors need to be considered: dissociation efficiency and post-dissociation antigen activity. In this study, both acidic elution and high-temperature elution were performed under low-requirement conditions. Based on the principle that a higher N value is more beneficial for the detection of the fluorescent detection card, high-temperature elution was ultimately selected as the optimal elution method for SEB.

[0046] 2.4.2 Effect of different elution temperatures on elution efficiency The enrichment solution was prepared with an SEB concentration of 40 pg / ml. After enrichment under the optimized conditions described above, the solution was eluted in water baths at 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ before detection. The effect of different elution temperatures on the detection results was observed, and the experimental results are shown in Table 4.

[0047] Table 4 Effect of elution temperature on elution

[0048] The highest detection value of the SEB fluorescent test card is achieved at an elution temperature of 60℃. Too low a temperature results in insufficient elution and lower detection sensitivity, while too high a temperature will destroy the antigen's immunogenicity, also leading to a decrease in the detection value.

[0049] 3. Comparison of enrichment chromatography and direct dilution loading results SEB was detected in serum, milk, and water, with comparisons made between two sample addition methods: direct addition and addition after enrichment and purification using magnetic beads. The test results of the two sets of simulated negative and positive samples are shown below. Figure 3As shown in the figure. During detection, the enrichment solution was used for a 3-fold dilution. One group of samples was directly added 80 μl to the SEB fluorescent card for detection, while the other group was detected using the SEB fluorescent card after enrichment and elution. Before immunomagnetic bead enrichment, the milk sample showed no detectability when detected using the SEB fluorescent card. The sensitivity of water and serum samples using the SEB fluorescent card was low, with positive / negative discrimination ratios of 2 and 4, respectively. After immunomagnetic bead enrichment, the milk sample transformed from an ineffective sample to a high-discrimination sample (discrimination ratio above 30), while the positive / negative discrimination ratios for water and serum samples were 20 and 8, respectively, showing a significant improvement in discrimination.

[0050] Example 2 Validation of the enrichment effect of Staphylococcus aureus type B enterotoxin This validation protocol provides a reference for the sample pretreatment techniques used by customs port laboratories in detecting various biotoxins using time-resolved fluorescence immunochromatography, and offers suggestions on key technical steps in the pretreatment process. When preparing samples for validation, reference standards of known concentrations (or contents) can be used for serial dilution and testing. For intercepted substances discovered during port passenger and mail inspections, real samples can be used for validation.

[0051] 1. Experimental Materials 1.1 Instrument Preparation (1) Prepare a centrifuge (compatible with 10mL centrifuge tubes) and set its parameters as follows: temperature 4℃, speed 4000rpm, centrifugation time 10min. Before centrifuging the sample, the centrifuge should be started in advance to ensure that the working temperature reaches a low temperature of 4℃ during use.

[0052] (2) Design and print an appropriate number of labels. The specifications and quantity of the labels should be adjusted according to the needs of subsequent sample processing. Affix the printed labels to the enrichment tubes (10mL centrifuge tubes).

[0053] (3) Prepare a magnetic rack, vortex shaker, and 37°C rotary incubator that are compatible with the enrichment tube specifications.

[0054] (4) Prepare the time-resolved fluorescence detector and its matching test reagents.

[0055] (5) Constant temperature water bath, set to 60℃.

[0056] 1.2 Preparation of Reagents and Consumables (1) Prepare toxin enrichment magnetic beads (immunocarboxyl magnetic beads coupled with corresponding capture antibodies, as described in Example 1) and enrichment reagent (enrichment solution 4 described in Example 1).

[0057] (2) Prepare reference samples of Staphylococcus aureus type B enterotoxin and other toxins with known concentrations (or contents). (Note: The concentration (or content) can be determined using detection techniques such as ELISA. The detection operation standards can be referred to the customs health and quarantine industry standards.)

[0058] (3) Substrate: water, milk, urine.

[0059] 2. Experimental Methods 2.1 Experimental Method Description This embodiment establishes a toxin detection method based on enrichment with carboxylated immunomagnetic beads (IMB) combined with lanthanide fluorescent immunochromatography (TRFIA). By optimizing the magnetic bead-antibody coupling process, efficient enrichment of multiple toxins in complex samples is achieved. This method utilizes the antigen-antibody specific reaction and the magnetic responsiveness of magnetic beads to rapidly separate and concentrate target toxins, and combines this with the fluorescence properties of lanthanides for detection, offering the following advantages: (1) Applicable to samples with low concentration and complex matrix (water, milk, urine) for port testing (2) Reduces the use of toxic reagents, and is simple and efficient to meet the needs of rapid screening for trace toxins. (3) Completion time limit: Sample pretreatment should be completed within 12 hours.

[0060] 2.2 Operating Procedures (1) Sample processing solution preparation: Negative samples are water, milk, and urine. The preparation of positive samples is shown in Table 5.

[0061] Table 5. Preparation of SEB Positive Samples

[0062] (2) Extraction: Dilute the positive and negative samples in half with enrichment buffer, vortex to mix, and centrifuge at 4000 r / min for 10 min. Take 5 mL of supernatant, add 4 μl of carboxylated immunomagnetic beads, vortex to mix, and incubate in a rotary incubator at 37℃ for 30 min. Then, place the toxin-magnetic bead mixture on a magnetic rack for 5 min, discard the supernatant, add 100 μL of neutralization solution, shake in a constant temperature water bath at 60℃ for 10 min, and then place on a magnetic rack for magnetic separation for 5 min. Collect all supernatant for later use.

[0063] (3) Toxin collection and detection: After the supernatant has returned to room temperature, add 80 μl of supernatant to the sample well of the toxin time-resolved fluorescence detection reagent card, perform chromatographic reaction for 15 min, use a toxin detector to detect the fluorescence signal and interpret the result, compare the result with the result of the untreated sample and record it.

[0064] 2.3 Verification Results "Direct detection" refers to adding 80 μl of the enrichment solution directly to the sample well of the test card after diluting the sample by 2 times, and then performing chromatography for 15 min before detection. "Enrichment detection" refers to enriching the sample with magnetic beads and then eluting it before detection. The detection results are shown in Table 6.

[0065] Table 6. Validation Results of Pretreatment for Staphylococcus aureus Enterotoxin Type B

[0066] The validation results showed that the direct detection sensitivity of water samples was below 10 pg / ml. After pretreatment, the sensitivity reached 1 pg / ml. Direct detection of milk samples was invalid, but after pretreatment, the detection sensitivity reached 2 pg / ml. Direct detection of urine samples resulted in false positives, but after pretreatment, the detection sensitivity reached 5 pg / ml.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for enriching Staphylococcus aureus enterotoxin B, characterized in that, This includes a vector and enrichment solution conjugated with an anti-Staphylococcal enterotoxin B antibody; The enrichment solution comprises a 0.05M phosphate buffer containing 0.5%–1.2% NaCl, 0.08%–0.12% KCl, and 0.08%–0.12% Tween 20, with a pH of 7.1–7.

4.

2. The kit for enriching Staphylococcus aureus enterotoxin B according to claim 1, characterized in that, The enrichment solution comprises a 0.05M phosphate buffer containing 0.9% NaCl, 0.1% KCl, and 0.1% Tween 20, with a pH of 7.

2.

3. The kit for enriching Staphylococcus aureus enterotoxin B according to claim 1, characterized in that, The carrier coupled with anti-Staphylococcal enterotoxin B antibody includes magnetic beads.

4. The kit for enriching Staphylococcus aureus enterotoxin B according to claim 1, characterized in that, The anti-Staphylococcal enterotoxin B antibody and the vector are coupled via amide bonds.

5. The kit for enriching Staphylococcus aureus enterotoxin B according to claim 1, characterized in that, It also includes neutralizing solution; The neutralization solution is a 1M Tris buffer solution with a pH of 8.

5.

6. The use of the kit for enriching Staphylococcus aureus enterotoxin B according to any one of claims 1 to 5 in detecting the enrichment of Staphylococcus aureus enterotoxin B in samples.

7. The application according to claim 6, characterized in that, The enrichment temperature for Staphylococcus aureus enterotoxin B was 36-38℃, and the enrichment time was 28-47 min.

8. The application according to claim 6, characterized in that, The elution temperature for enriching Staphylococcus aureus enterotoxin B is 60~70℃.

9. The application according to any one of claims 6 to 8, characterized in that, The test samples include at least one of the following: water, food, and biological fluids.

10. A kit for detecting Staphylococcus aureus, characterized in that, The kit includes the method for enriching Staphylococcus aureus enterotoxin B as described in any one of claims 1 to 5.

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