A latex microsphere immunochromatographic test strip for detecting tetrodotoxin and its application

Abstract

This invention provides a latex microsphere immunochromatographic test strip for detecting tetrodotoxin and its application, belonging to the field of rapid detection of marine toxins. The latex microsphere immunochromatographic test strip comprises the following components: a plastic shell, a sample pad, an immunoprobe conjugation pad, a nitrocellulose membrane, and an absorbent pad; the immunoprobe conjugation pad is dotted with latex microsphere immunoprobes labeled with anti-tetrodotoxin monoclonal antibodies; the anti-tetrodotoxin monoclonal antibody is secreted by the anti-tetrodotoxin monoclonal antibody hybridoma cell line 5B9, whose accession number is CGMCC No. 45320. This latex microsphere immunochromatographic test strip has a detection threshold of 1000 ng / mL, a detection limit of 7.8125 ng / mL, high specificity, high sensitivity, good repeatability, stable performance, and low operational requirements, making it highly suitable for rapid detection in food and of great significance for monitoring tetrodotoxin.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection of marine toxin pollution, and relates to a latex microsphere immunochromatographic test strip for detecting tetrodotoxin and its application. Background Technology

[0002] Tetrodotoxin (TTX) is a low-molecular-weight, non-protein marine neurotoxin primarily found in nearly a hundred species of pufferfish belonging to the order Ambimorpha in the subclass Osteichthyes, especially concentrated in the ovaries and liver, and subsequently in the blood, eyes, skin, and other tissues. TTX is an extremely potent neurotoxin, more than 1000 times more toxic than sodium cyanide; 0.5-3 mg can be fatal to an adult. The LD50 in mice after intraperitoneal injection is... 50 The concentration is 80 mg / kg. Because TTX can selectively bind to Na+ on the surface of nerve cell membranes... + At the receptors of the tetrodotoxin channel, it blocks nerve and muscle conduction, leading to nerve paralysis, respiratory failure, and even death. The molecular structure of tetrodotoxin (TTX) is relatively stable and not easily destroyed by ordinary cooking processes. Therefore, in coastal areas, many cases of poisoning due to the consumption of delicious pufferfish occur every year. Although TTX is highly toxic and causes a high mortality rate, its unique properties have attracted much research interest, resulting in further advancements in various fields, particularly in clinical medicine, where its effects have been significant. Currently, commonly used analytical methods for tetrodotoxin determination include mouse biological experiments, fluorescence methods, ultraviolet spectrophotometry, high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC). While these traditional laboratory detection methods have high analytical sensitivity and technological maturity, they typically require complex sample pretreatment, which is time-consuming, labor-intensive, and costly, failing to meet the simple and rapid on-site requirements for detecting tetrodotoxin content in actual samples. Therefore, rapid on-site detection technology for tetrodotoxin has become a research hotspot in related fields in recent years. This invention utilizes activated latex microspheres to conjugate antibodies to prepare an immunoassay probe. Because latex microspheres have a large particle size (200 nm), which is larger than other widely used nanoparticles on the market, their large surface area allows them to bind and conjugate more antibodies. Utilizing this inherent advantage of microspheres, and combined with immunochromatographic test strip technology, a latex microsphere immunochromatographic test strip has been developed. This immunochromatographic test strip is simple to assemble, highly specific, competing only with TTX, and has no cross-reactivity with other marine toxins. It exhibits good stability, high sensitivity, excellent repeatability, and a detection limit lower than the national food safety standard, effectively and accurately detecting tetrodotoxin in food. This has significant practical implications for the safe monitoring of aerobic toxins in food. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a latex microsphere immunochromatographic test strip for detecting tetrodotoxin and its applications. The latex microsphere immunochromatographic test strip prepared by this invention is simple to assemble, highly specific, sensitive, and stable, and can be used for the rapid detection of tetrodotoxin in food.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A hybridoma cell line 5B9 secreting an anti-tetrodotoxin monoclonal antibody, classified and named as anti-tetrodotoxin monoclonal antibody hybridoma cell line 5B9, was deposited on September 28, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 45320.

[0006] A latex microsphere immunochromatographic test strip for detecting tetrodotoxin, comprising the following components: a plastic shell, a sample pad, an immunoprobe conjugation pad, a nitrocellulose membrane, and an absorbent pad; latex microsphere immunoprobes labeled with anti-tetrodotoxin monoclonal antibodies are added to the immunoprobe conjugation pad; the anti-tetrodotoxin monoclonal antibody is secreted by anti-tetrodotoxin monoclonal antibody hybridoma cell line 5B9; the anti-tetrodotoxin monoclonal antibody hybridoma cell line 5B9 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on September 28, 2022, with accession number CGMCC No. 45320.

[0007] The preparation method of the above-mentioned latex microsphere immunochromatographic test strip for detecting tetrodotoxin includes the following steps:

[0008] (1) Preparation of latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody: Take 15 μL of polystyrene latex microsphere suspension, add 1 mL of 0.05 M MES buffer (pH 6.0), and centrifuge at 4 °C and 12000 rpm for 10 min; discard the supernatant, resuspend the precipitate in 1 mL of 0.05 M MES buffer (pH 6.0), add 1.5 mg EDC, and shake at 25 °C and 160 rpm for 15 min, then centrifuge at 12000 rpm and 4 °C for 10 min; discard the supernatant, resuspend the precipitate in 0.5 mL of 50 g / L glycine buffer (pH 6.5), add 10 μL of 1.25 mg / mL anti-tetrodotoxin monoclonal antibody, and incubate at 160 rpm for 2 h at room temperature; after incubation, centrifuge twice at 4 °C and 12000 rpm each time. min; discard the supernatant, add 1 mL of 50 g / L glycine buffer (pH 6.5) to the precipitate, and shake at 160 rpm for 30 min at room temperature to quench the microspheres; centrifuge twice at 12000 rpm at 4℃, 10 min each time; discard the supernatant, and blow the precipitate with 20 μL of 50 g / L glycine buffer (pH 6.5) to obtain the latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody, and store at 4℃ for later use;

[0009] (2) Pretreatment of immunoprobe binding pad and sample pad: Cut the untreated immunoprobe binding pad and sample pad into strips 1.3 cm wide with scissors, place them in a large petri dish, submerge them in the pre-prepared blocking solution for 2 hours, then transfer them to a 37°C constant temperature incubator for 2 hours. After removing them from the incubator, filter out the blocking solution and continue to dry them in the incubator at 37°C. After drying, store them at 4°C. For subsequent use, cut them to a length of 1.3 cm and a width of 4 mm. The composition of the blocking solution is: 5wt% BSA + 1vol% Tween-20.

[0010] (3) Streaking on nitrocellulose membrane: Goat anti-mouse secondary antibody was used to streak C lines on nitrocellulose membrane. The final concentration of goat anti-mouse secondary antibody on C lines was 0.5 mg / mL. After cutting, the content of goat anti-mouse secondary antibody on each 4 mm wide strip was 32 ng. Complete antigen OVA-TTX was used to streak T lines on nitrocellulose membrane. The final concentration of complete antigen OVA-TTX on T lines was 32.80 mg / mL. After cutting, the content of complete antigen OVA-TTX on each 4 mm wide strip was 20.992 ng. The distance between C and T lines on the same nitrocellulose membrane was 0.5 cm.

[0011] (4) Preparation of immune probe binding pad: 4 μL of latex microsphere immune probe labeled with anti-tetrodotoxin monoclonal antibody prepared in step (1) was dropped onto the pretreated immune probe binding pad;

[0012] (5) Assembly of immunochromatographic test strips: The nitrocellulose membrane after scribing in step (3), the immunoprobe binding pad prepared in step (4), the sample pad pretreated in step (2), and the absorbent pad are assembled on the base plate in sequence; the immunoprobe binding pad and the sample pad are overlapped and pasted, with a 2 mm gap between the same end, and the absorbent pad overlaps the nitrocellulose membrane by 2 mm at both ends. The plastic outer shell is covered, dried and sealed, and stored at 4℃.

[0013] The method for preparing the anti-tetrodotoxin monoclonal antibody in step (1) is as follows: inject the anti-tetrodotoxin monoclonal antibody hybridoma cell line 5B9 in the logarithmic growth phase into the peritoneal cavity of paraffin-sensitized Balb / c mice. When the mouse abdomen is distended and shows signs of tension, the ascites is extracted and purified to obtain the anti-tetrodotoxin monoclonal antibody.

[0014] The application of the hybridoma cell line 5B9 mentioned above in the preparation of products for detecting tetrodotoxin.

[0015] The above-mentioned latex microsphere immunochromatographic test strip is used in the detection of tetrodotoxin.

[0016] The detection principle of the latex microsphere immunochromatographic test strip for detecting tetrodotoxin described in this invention is as follows: When the test strip is first used for detection, the prepared latex microsphere immunoprobe is first dropped onto the immunoprobe binding pad, and after drying, it is then followed according to the schematic diagram (…). Figure 1 The sample is assembled using the following method, and a drop is placed on the sample pad. The liquid flows towards the absorbent paper via chromatography. When it passes the T-line, any latex microsphere immunoprobes that have not yet bound to the antigen in the sample bind completely to the antigen at the T-line and remain there, resulting in color development at the T-line. The liquid continues to flow forward. When it passes the C-line, both latex microsphere immunoprobes that have bound to the antigen in the sample and those that have not bound bind to the antigen bind to the C-line until saturation. The bound gold-labeled probes remain at the C-line, resulting in color development at the C-line. The entire colorimetric reaction takes approximately 10 minutes.

[0017] The beneficial effects of this invention are:

[0018] (1) This invention is the first to propose a latex microsphere immunochromatographic test strip technology for detecting tetrodotoxin by covalently coupling anti-tetrodotoxin monoclonal antibodies with latex microspheres;

[0019] (2) The latex microsphere immunochromatographic test strip provided by the present invention can detect tetrodotoxin in food. The limit of detection is 7.8125 ng / mL, the detection threshold (complete disappearance of T line) is 1000 ng / mL, and the detection time is only 10 min. It meets the requirements of national food safety standards and can be used for actual on-site testing. Attached Figure Description

[0020] Figure 1 Schematic diagram of the structure of a latex microsphere immunolayer test strip.

[0021] Figure 2 Determination of the optimal pH for latex microsphere activation buffer.

[0022] Figure 3 Determination of the optimal EDC amount for activated latex microsphere immunoprobes.

[0023] Figure 4 Determination of the optimal glycine buffer pH for latex microsphere immunoprobes.

[0024] Figure 5 Determination of the optimal amount of antibody conjugated to latex microsphere immunoprobes.

[0025] Figure 6 Selection of the optimal dilution for line C of latex microsphere immunolayer test strips.

[0026] Figure 7 Selection of the optimal dilution for the T-line of latex microsphere immunochromatographic test strips.

[0027] Figure 8 Determination of the optimal probe amount for latex microsphere immunolayer test strips.

[0028] Figure 9 Specificity determination of latex microsphere immunochromatographic test strips.

[0029] Figure 10 Sensitivity determination of latex microsphere immunochromatographic test strips.

[0030] Figure 11 : Actual sample testing of latex microsphere immunolayer test strips. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments. The illustrative embodiments and descriptions herein are used to explain the present invention, but are not intended to limit the present invention.

[0032] Example 1: Preparation of anti-tetrodotoxin monoclonal antibody

[0033] The anti-tetrodotoxin monoclonal antibody involved in this invention is secreted by the anti-tetrodotoxin monoclonal antibody hybridoma cell line 5B9; the anti-tetrodotoxin monoclonal antibody hybridoma cell line 5B9 has been deposited at the China General Microbiological Culture Collection Center, the deposit address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is September 28, 2022, and the deposit number is CGMCC No. 45320.

[0034] 1. Preparation of ascites

[0035] (1) Take a 1 mL sterile syringe and inject 500 μL of paraffin into the peritoneal cavity of a Balb / c mouse about 12 weeks old to sensitize it. Lubricate the peritoneal cavity of the mouse thoroughly. When injecting, insert the syringe vertically into the back of the peritoneal cavity of the mouse. Disinfect the injection site with alcohol before and after injection to avoid bleeding.

[0036] (2) One week after sensitization, take the 5B9 anti-tetrodotoxin monoclonal antibody hybridoma cell line that has just grown to the logarithmic phase and has filled about 80% of the culture dish. Discard the supernatant, use a pipette to take 1 mL of fresh 1640 medium to wash the cells from the bottom of the culture dish, mix them evenly, use a 1 mL sterile syringe to draw the cell suspension from one well of a 6-well plate, and then vertically inject it into the peritoneal cavity of the sensitized mouse. Note that the needle must be inserted into the peritoneal cavity accurately and without obstruction. Do not inject into the peritoneal tissue to avoid tumor formation and waste of the mouse.

[0037] (3) About a week after injecting cells into the peritoneum of mice, under normal circumstances, the abdominal distension of mice can be clearly observed, and the mice will be lethargic and dislike exercise. At this time, it is necessary to start preparing to collect ascites.

[0038] (4) Prepare blood collection needles, 1.5 mL EP tubes, cotton and other items. Hold the mouse with one hand and wipe the blood collection site with an alcohol swab. With the other hand, carefully insert the blood collection needle into one side of the abdominal cavity. Place the blood collection needle against the EP tube and collect the ascites. You can see the ascites flowing into the tube along the blood collection needle. The color is reddish. This may be due to excessive ascites production and hemolysis, so the color is reddish. However, it does not affect the properties of the antibodies in the ascites. When collecting blood, the mouse should be fixed to prevent the mouse from struggling and causing loss of ascites. Each mouse can only collect about 1.5 mL of ascites at a time. After the tail blood is collected, put the mouse back into the cage. Ascites can continue to be produced. Ascites can be collected again the next day until the mouse dies.

[0039] (5) After the mouse ascites fluid was taken out and balanced, it was centrifuged at 12,000 rpm for 20 min in a 4℃ centrifuge. After taking it out, it can be observed that the liquid in the centrifuge tube is divided into three layers. The top layer is a layer of fat. Carefully take out the middle layer of ascites fluid, put it into a new 1.5 mL EP tube, mark it, and store it in a -20℃ refrigerator.

[0040] 2. Ascites purification

[0041] Ascites fluid was diluted 1:10 with equilibration buffer (Na2HPO4 3.5814 g / L, NaCl 4.383 g / L; pH=7.0). The diluted ascites fluid was filtered through a 0.45 μm filter and then passed through a Protein G affinity chromatography column. The procedure was performed according to the instructions for commercial Protein G affinity chromatography. The purified antibody was dialyzed in a dialysis bag with PBS (NaCl 8.0 g / L, KCl 0.2 g / L, Na2HPO4 1.44 g / L, KH2PO4 0.24 g / L; pH=7.4) for 3 days, followed by dialyzed with ultrapure water for 1 day. Finally, the antibody was concentrated using PEG20000 to obtain the anti-tetrodotoxin monoclonal antibody. The anti-tetrodotoxin monoclonal antibody was stored at -20°C for later use. The purity of the antibody was verified by 13wt% SDS-PAGE gel electrophoresis. The purified antibody showed bands at 25 kDa and 50 kDa, corresponding to the light and heavy chains of the IgG antibody, respectively, and there were almost no impurities, indicating good purification. After complete dialyzing with ultrapure water, the antibody was freeze-dried, and the lyophilized powder was collected to obtain the anti-tetrodotoxin monoclonal antibody. The anti-tetrodotoxin monoclonal antibody was stored at -20°C for later use.

[0042] Example 2: Preparation of latex microsphere immunoprobes

[0043] 1. Determination of the optimal pH value for the MES buffer system for activating latex microspheres

[0044] First, take 15 μL of polystyrene latex microsphere suspension (purchased from Bang Laboratories USA, CAS: 9003-53-6, particle size approximately 200 nm under electron microscopy) into six 1.5 mL EP tubes, and then add 1 mL of 0.05 mol / L MES buffer (0.05 M) at pH values ​​of 5, 5.5, 6.0, 6.5, 7.0, and 7.5 respectively, and label the corresponding pH values. Wash the latex microspheres by blowing them up, and centrifuge at 4℃ and 12000 rpm for 10 min, and discard the supernatant. Next, resuspend the precipitate in 1 mL of MES buffer (0.05 M) at the appropriate pH value, then add 1 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) to the resuspended solution. Incubate at 25°C and 160 rpm for 15 min with shaking, ensuring thorough mixing to prevent clumping (clumping indicates experimental failure). Then, centrifuge at 12000 rpm at 4°C for 10 min, discard the supernatant, add 0.5 mL of glycine buffer (50 g / L) at pH 6.5 to the precipitate, and finally add 15 μL of 1 mg / mL anti-tetrodotoxin monoclonal antibody. Incubate at 160 rpm for 2 h at room temperature. Finally, centrifuge twice at 12000 rpm for 10 min each time at 4°C, collect the precipitate, add 0.5 mL of glycine buffer (50 g / L) at pH 6.5, and incubate at 160 rpm for 30 minutes at room temperature. The latex microspheres were quenched at 12000 rpm for 10 min at 4 °C. The supernatant was discarded, and the precipitate was resuspended in 20 μL of glycine buffer (50 g / L) at pH 6.5 to obtain the latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody. The immunoprobe was stored at 4 °C for later use.

[0045] The latex microsphere immunochromatographic test strip comprises a plastic shell, a sample pad, an immunoprobe binding pad, a nitrocellulose membrane, and an absorbent pad. The plastic shell contains the immunochromatographic test strip, which is composed of the nitrocellulose membrane, immunoprobe binding pad, sample pad, and absorbent pad, sequentially assembled on a base plate. The immunoprobe binding pad and sample pad are overlapped and staggered, with a 2 mm gap between the same ends. The absorbent pad overlaps the nitrocellulose membrane end-to-end by 2 mm. The plastic shell is then closed, dried, and sealed, and stored at 4°C. The immunoprobe is located on the probe binding pad. Figure 1 (Illustrative diagram).

[0046] The test strip results show that the pH of the MES (2-morpholinoethanesulfonic acid) buffer has a significant impact on the activation of carboxyl groups on the latex microspheres. At pH 5.5, the test strip shows almost no band or a very faint band, while at pH 6.0, the band is most prominent, indicating that the optimal pH for latex microsphere activation is 6.0. Figure 2 ).

[0047] 2. Determination of EDC (Electrode Discharge) of the Optimal Activated Latex Microsphere Immunoprobe

[0048] First, take 15 μL of latex microsphere suspension into five 1.5 mL EP tubes, add 1 mL of MES buffer (0.05 M) at the optimal pH (pH 6.0) determined in step 1 above, wash the latex microspheres, centrifuge at 4 °C and 12000 rpm for 10 min, and discard the supernatant. Next, the precipitate was resuspended in 1 mL of 0.05 M MES buffer (pH 6.0). Then, 0.5, 1.0, 1.5, 2.0, and 2.5 mg of EDC were added to the resuspended solution, labeled by the mass of EDC added. The mixture was then incubated at 25°C and 160 rpm for 15 min with shaking, ensuring thorough mixing to prevent clumping (clumping indicates experimental failure). The mixture was then centrifuged at 12000 rpm for 10 min at 4°C, the supernatant was discarded, and 0.5 mL of 50 g / L glycine buffer (pH 6.5) was added to resuspend the precipitate. Finally, 15 μL of 1.25 mg / mL anti-tetrodotoxin monoclonal antibody was added, and the mixture was incubated at 160 rpm for 2 h at room temperature. Finally, the mixture was centrifuged at 12000 rpm for 10 min at 4°C, the supernatant was discarded, and 0.5 mL of 50 g / L glycine buffer (pH 6.5) was added. The mixture was then incubated at 160 rpm for 30 h at room temperature. The latex microspheres were quenched at 12000 rpm for 10 min at 4℃. The supernatant was discarded, and the precipitate was resuspended in 20 μL of glycine buffer (50 g / L) at pH 6.5 to obtain the latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody. The immunoprobe was stored at 4℃ for later use. According to the test strip results, when 15 μL of latex microsphere stock solution was added and 1 mL of MES buffer (0.05 M) at pH 6.0 was added, the amount of EDC required for carboxyl activation was 1.5 mg. Under the same conditions, the C and T lines showed the most obvious color development. Figure 3 ).

[0049] 3. Determination of the optimal pH of glycine buffer for latex microsphere immunoprobes

[0050] First, take 15 μL of latex microsphere suspension into three 1.5 mL EP tubes, add 1 mL of MES buffer (0.05 M) at pH 6.0 to each tube, wash the latex microspheres by blowing, centrifuge at 12000 rpm for 10 min at 4℃, and discard the supernatant. Next, the precipitate was resuspended in 1 mL of 0.05 M MES buffer at pH 6.0. Then, 1.5 mg of EDC was added to the resuspended solution. Next, three 50 g / L glycine buffer solutions with different pH values ​​(6.0, 6.5, and 7.0) were prepared (50 g of glycine was weighed and diluted to 1 L with double-distilled water as the stock solution; then, three 40 mL volumetric flasks were dispensed, and the glycine solutions in each flask were adjusted to pH 6.0, 6.5, and 7.0 with 1 M NaOH, and labeled accordingly). The precipitate was resuspended in the three flasks, and 15 μL of 1.0 mg / mL anti-tetrodotoxin monoclonal antibody was added. The mixture was incubated at 160 rpm for 2 h at room temperature. Finally, the mixture was centrifuged twice at 12000 rpm at 4℃, 10 μL each time. After 30 minutes, the precipitate was collected and 0.5 mL of glycine buffer (50 g / L) corresponding to the pH was added. The mixture was shaken at 160 rpm for 30 minutes at room temperature to quench the latex microspheres. The microspheres were then centrifuged twice at 12000 rpm for 10 minutes each time at 4 ℃. The supernatant was discarded, and the precipitate was resuspended in 20 μL of glycine buffer (50 g / L) corresponding to the pH. The resulting latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody was stored at 4 ℃ for later use. According to the test strip results, the C and T lines of the test strip only showed color when the pH of the glycine buffer was 6.5, and the color was obvious. At pH values ​​of 6.0 and 7.0, the C and T lines of the test strip did not show color or showed very faint color. Figure 4 ).

[0051] 4. Determination of the optimal antibody amount for latex microsphere immunoprobes

[0052] First, take 15 μL of latex microsphere suspension into four 1.5 mL EP tubes, add 1 mL of MES buffer (0.05 M) at pH 6.0 to each tube, wash the latex microspheres by blowing, centrifuge at 12000 rpm for 10 min at 4℃, and discard the supernatant. Next, the precipitate was resuspended in 1 mL of MES buffer (0.05 M) at pH 6.0. Then, 1.5 mg of EDC was weighed and added to the resuspended solution. The resuspended solution was placed in a constant temperature shaker at 25℃ and 160 rpm for 15 min, ensuring thorough mixing to prevent clumping (clumping indicates experimental failure). The solution was centrifuged at 12000 rpm and 4℃ for 10 min, and the supernatant was discarded. The precipitate was resuspended in 0.5 mL of 50 g / L glycine solution at pH 6.5. Then, 5, 10, 15, and 20 μL of four different volumes of anti-tetrodotoxin monoclonal antibody (1.25 mg / mL) were added, labeled according to the volume of antibody added. The latex microspheres were incubated at 160 rpm for 2 h at room temperature; then centrifuged twice at 12000 rpm for 10 min each time at 4℃, the supernatant was discarded, and the precipitate was collected. 0.5 mL of glycine (pH 6.5, 50 g / L) was added, and the mixture was shaken at 160 rpm for 30 min at room temperature to quench the latex microspheres; then centrifuged twice again at 12000 rpm for 10 min each time at 4℃; the supernatant was discarded, and the precipitate was resuspended in 20 μL of glycine buffer (pH 6.5, 50 g / L), finally obtaining the latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody. This immunoprobe was stored at 4℃ for later use. The final results showed that ( Figure 5 When the volume of latex microsphere stock solution added is 15 μL, the optimal amount of antibody added is 10 μL. At this point, the C and T lines are close to saturation, consistent with the C and T line colors of the strips with 15 and 20 μL of anti-TTX monoclonal antibody added. Compared to the immunochromatographic test strips with 5 μL of monoclonal antibody added, the C and T lines are significantly darker, indicating that the amount of conjugated antibody is insufficient.

[0053] Example 3 Performance verification of latex microsphere immunochromatographic test strips

[0054] Preparation of latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody: Take 15 μL of polystyrene latex microsphere suspension, add 1 mL of 0.05 M MES buffer (pH 6.0), and centrifuge at 12000 rpm for 10 min at 4 °C. Discard the supernatant, resuspend the precipitate in 1 mL of 0.05 M MES buffer (pH 6.0), add 1.5 mg EDC, and react with shaking at 160 rpm for 15 min at 25 °C. Centrifuge at 4 °C for 10 min at 12000 rpm. Discard the supernatant, resuspend the precipitate in 0.5 mL of 50 g / L glycine buffer (pH 6.5), add 10 μL of 1.25 mg / mL anti-tetrodotoxin monoclonal antibody, and incubate at 160 rpm for 2 h at room temperature. After incubation, centrifuge twice at 12000 rpm for 10 min each time at 4 °C. Discard the supernatant, add 1... The microspheres were quenched by shaking at 160 rpm for 30 min at room temperature with 50 g / L glycine buffer at pH 6.5. The microspheres were then centrifuged twice at 12000 rpm for 10 min each time at 4 °C. The supernatant was discarded, and the precipitate was lifted with 20 μL of 50 g / L glycine buffer at pH 6.5. This yielded the latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody, which was stored at 4 °C for later use.

[0055] 1. Pretreatment of probe binding pad and sample pad

[0056] Cut the untreated probe binding pad and sample pad into strips 1.3 cm wide. Place them in a large petri dish and submerge them in the pre-prepared blocking solution (5 wt% BSA + 1 vol% Tween-20) for 2 hours. Then transfer them to a 37°C incubator for 2 hours of incubation. After removing them from the incubator, filter out the blocking solution and continue drying at 37°C until constant weight. Store at 4°C for later use. For subsequent use, cut the strips to a length of 1.3 cm and a width of 4 mm.

[0057] 2. Selection of the optimal dilution for line C of latex microsphere immunochromatographic test strips

[0058] 2 mg / mL of goat anti-mouse secondary antibody (purchased from Beijing Bio-Sen Biotechnology Co., Ltd., product number: bs-0293G) was diluted with 0.01 M PBS at pH 7.4 according to the final concentration of C line, resulting in four concentrations of 1, 0.67, 0.5, 0.4, and 0.33 mg / mL. 10 μL of each concentration was taken and streaked on the NC membrane. When streaking with a gold sputtering instrument, the streaking speed was 0.16 mL / cm, and each NC membrane was streaked 3 times. After the NC membrane with the lines drawn was dried in a 37°C incubator for 20 min, it was cut into strips 4 mm wide. Next, 3 μL of latex microsphere immunoassay probe labeled with anti-tetrodotoxin monoclonal antibody was added to the probe binding pads of four assembled latex microsphere immunoassay strips. 100 μL of 0.01 M PBS (pH 7.4) was slowly added to the sample pads, and the mixture was incubated at room temperature for 10 min. The color intensity of the C line on the test strips was observed. Finally, based on observation, 0.5 mg / mL (i.e., 0.2 ng of goat anti-mouse secondary antibody on each 4 mm wide strip) was selected as the optimal C line concentration. Figure 6 ).

[0059] 3. Selection of the optimal dilution for the T-line of latex microsphere immunochromatographic test strips

[0060] The complete antigen OVA-TTX (prepared under the optimal reaction conditions as described in the literature [Cong Lei. Preparation of tetrodotoxin-specific monoclonal antibody [D]. Shanghai Ocean University, 2011]) was diluted with 0.01 M PBS (pH 7.4) according to the final concentration at the T line, resulting in four concentrations: 54.67, 46.86, 41.00, 36.44, and 32.80 μg / mL. 10 μL of each concentration was streaked onto an NC membrane. When streaking with a gold sputtering apparatus, the streaking speed was 0.16 μL / cm, and each NC membrane was streaked 3 times. After the NC membrane with the lines drawn was dried in a 37℃ incubator for 20 min, it was cut into strips with a width of 4 mm. Next, 3 μL of latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody was added to the probe binding pads of the four assembled immunochromatographic test strips. 100 μL of 0.01M PBS (pH 7.4) was slowly added to the sample pads, and the mixture was allowed to react at room temperature for 10 min. The color intensity of the T line on the test strips was observed. Finally, based on observation, 32.80 μg / mL was selected as the optimal T line concentration. Figure 7 ).

[0061] 4. Determination of the optimal probe amount for latex microsphere immunolayer test strips

[0062] Based on the optimized dilution ratios for the C-line (0.5 mg / mL) and T-line (32.80 μg / mL) mentioned above, the NC membrane was dried in a 37°C incubator for 20 min, then cut into strips 4 mm wide. Next, 1, 2, 3, 4, and 5 μL of latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody were added to the probe binding pads of the four assembled immunochromatographic test strips, respectively. 100 μL of 0.01 M PBS (pH 7.4) was slowly added to the sample pads, and the mixture was allowed to react at room temperature for 10 min. The color intensity of the C and T lines was observed. Finally, based on observation, 4 μL was selected as the optimal latex microsphere probe loading amount. Figure 8 ).

[0063] 5. Specificity determination of latex microsphere immunochromatographic test strips

[0064] According to the optimized dilution ratios of the C-line (0.5 mg / mL) and T-line (32.80 μg / mL) mentioned above, streaks were simultaneously applied to the NC membrane. The streaked NC membrane was then dried in a 37°C incubator for 15 min before being cut into strips. Next, 4 μL of latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody was added to the probe binding pad, and the test strip was assembled. μ-KIIIA-CTX (conotoxin), aB-VxXXIVA-CTX (conotoxin), APETx_2 (anemone toxin), BSD1 (anemone toxin), SN311 (sea snake toxin), OA (okada leucosin), and TTX (tetrodotoxin) were diluted to a final concentration of 2000 ng / mL using 0.01 M PBS (pH 7.4). 100 μL of each was added to the sample pad of the test strip, and the strips were incubated at room temperature for 10 min. The disappearance of the T-line was observed to determine the specificity of the test strip. The results showed that the test strip only competitively reacted with tetrodotoxin (TTX), with the T line disappearing, while there was no obvious cross-reactivity with other marine toxins, indicating that the test strip had good specificity. Figure 9 ).

[0065] 6. Sensitivity determination of latex microsphere immunochromatographic test strips

[0066] According to the optimized C-line (0.5 mg / mL) and T-line (32.80 μg / mL) dilution ratios, streaks were simultaneously applied to the NC membrane. The streaked NC membrane was then dried in a 37°C incubator for 15 min before being cut into strips. Next, 4 μL of latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody was added to the probe binding pad, and the test strip was assembled. Tetrodotoxin (TTX) was diluted with 0.01 M PBS (pH 7.4) to final concentrations of 2000 ng / mL, 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, 7.8125 ng / mL, 3.90625 ng / mL, and 0 ng / mL. Subsequently, 100 μL of each sample was added to the sample pad of the test strip and allowed to react at room temperature for 10 min. The disappearance of the T line was observed to determine the detection range of the test strip. Results showed that when the added TTX was 1000 ng / mL, the T line disappeared significantly, indicating that the epitope of the anti-tetrodotoxin monoclonal antibody was completely bound to the hapten TTX in the sample. With each valence dilution, the T line gradually darkened. The detection limit of this immunochromatographic test strip was 7.8125 ng / mL. Figure 10 ).

[0067] Example 4: Actual Sample Testing

[0068] According to the optimized dilution ratios of the C line (0.5 mg / mL) and T line (32.80 μg / mL) mentioned above, streaks were simultaneously applied to the NC membrane. After streaking, the NC membrane was dried in a 37℃ incubator for 15 min before cutting into strips. 4 μL of latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody was added to the probe binding pad, and the test strip was assembled. Common fish species such as yellow croaker (Sample 1), grass carp (Sample 2), perch (Sample 3), and pufferfish (Sample 4) were purchased as test subjects. 10 g of fish tissue was taken, chopped with scissors, placed in a mortar, and ground with methanol-acetic acid solution (1% (v / v) acetic acid + 99% (v / v) methanol). The ground material and liquid were collected, and the volume was adjusted to 50 mL with methanol-acetic acid solution. After mixing, the mixture was incubated at room temperature for 30 min, then centrifuged at 8000 r / min for 10 min, and the supernatant was collected. In addition, 1 mL of 2000 ng / mL tetrodotoxin (TTX) was used as a control. Then, 100 μL of filtrate from yellow croaker, grass carp, perch, and pufferfish was added to the sample pad of the test strip, and the mixture was allowed to react at room temperature for 10 min. The disappearance of the T line on the test strip was observed. The results showed that compared with the control group (sampled in 0.01 M pH 7.4 PBS buffer), the T line color of the four samples (yellow croaker, grass carp, perch, and pufferfish) did not change significantly and was not detected, indicating that the samples did not contain tetrodotoxin. Meanwhile, in the sample spiked with 2000 ng of TTX, the T line on the test strip disappeared significantly compared with the control group. Figure 11 This indicates that the test strip has excellent stability and can be used for on-site testing of actual samples. It has practical significance for detecting tetrodotoxin in food.

[0069] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A latex microsphere immunochromatographic test strip for detecting tetrodotoxin, characterized in that: The latex microsphere immunochromatographic test strip comprises the following components: a plastic shell, a sample pad, an immunoprobe conjugation pad, a nitrocellulose membrane, and an absorbent pad; the immunoprobe conjugation pad is topped with latex microsphere immunoprobes labeled with anti-tetrodotoxin monoclonal antibodies; the anti-tetrodotoxin monoclonal antibody is secreted by the anti-tetrodotoxin monoclonal antibody hybridoma cell line 5B9; the anti-tetrodotoxin monoclonal antibody hybridoma cell line 5B9 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on September 28, 2022, with accession number CGMCC No. 45320; The preparation method of the latex microsphere immunochromatographic test strip for detecting tetrodotoxin includes the following steps: (1) Preparation of latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody: Take 15 μL of polystyrene latex microsphere suspension, add 1 mL of 0.05 M MES buffer (pH 6.0), and centrifuge at 4 °C and 12000 rpm for 10 min; discard the supernatant, resuspend the precipitate in 1 mL of 0.05 M MES buffer (pH 6.0), add 1.5 mg EDC, and shake in a constant temperature shaker at 25 °C and 160 rpm for 15 min, then centrifuge at 12000 rpm and 4 °C for 10 min; discard the supernatant, resuspend the precipitate in 0.5 mL of 50 g / L glycine buffer (pH 6.5), add 10 μL of 1.25 mg / mL anti-tetrodotoxin monoclonal antibody, and incubate at room temperature in a shaker at 160 rpm for 2 h; after incubation, centrifuge at 4 °C and 12000 rpm for 2 h. Centrifuge twice, 10 min each time; discard the supernatant, add 1 mL of 50 g / L glycine buffer (pH 6.5) to the precipitate, and shake at 160 rpm for 30 min at room temperature to quench the microspheres; then centrifuge twice at 12000 rpm at 4 °C, 10 min each time; discard the supernatant, and blow the precipitate with 20 μL of 50 g / L glycine buffer (pH 6.5) to obtain the latex microsphere immunoprobe labeled with anti-tetrodotoxin monoclonal antibody, and store at 4 °C for later use; (2) Pretreatment of immunoprobe binding pad and sample pad: Cut the untreated immunoprobe binding pad and sample pad into strips 1.3 cm wide, place them in a large petri dish, submerge them in the pre-prepared blocking solution for 2 hours, then transfer them to a 37°C constant temperature incubator for 2 hours. After removing them from the incubator, filter out the blocking solution and continue to dry them in the incubator at 37°C. After drying, store them at 4°C. For subsequent use, cut them to a length of 1.3 cm and a width of 4 mm. The composition of the blocking solution is: 5 wt% BSA + 1 vol% Tween-20. (3) Streaking on nitrocellulose membrane: Goat anti-mouse secondary antibody was used to streak C lines on nitrocellulose membrane. The final concentration of goat anti-mouse secondary antibody on C lines was 0.5 mg / mL. After cutting, the content of goat anti-mouse secondary antibody on each 4 mm wide strip was 32 ng. Complete antigen OVA-TTX was used to streak T lines on nitrocellulose membrane. The final concentration of complete antigen OVA-TTX on T lines was 32.80 mg / mL. After cutting, the content of complete antigen OVA-TTX on each 4 mm wide strip was 20.992 ng. The distance between C and T lines on the same nitrocellulose membrane was 0.5 cm. (4) Preparation of immune probe binding pad: 4 μL of latex microsphere immune probe labeled with anti-tetrodotoxin monoclonal antibody prepared in step (1) was dropped onto the pretreated immune probe binding pad; (5) Assembly of immunochromatographic test strips: The nitrocellulose membrane after scribing in step (3), the immunoprobe binding pad prepared in step (4), the sample pad pretreated in step (2), and the absorbent pad are assembled on the base plate in sequence; the immunoprobe binding pad and the sample pad are overlapped and pasted, with a 2mm gap between the same end, and the absorbent pad overlaps the nitrocellulose membrane by 2mm at both ends. The plastic outer shell is covered, dried and sealed, and stored at 4℃.

2. The method for preparing a latex microsphere immunochromatographic test strip for detecting tetrodotoxin according to claim 1, characterized in that: The method for preparing the anti-tetrodotoxin monoclonal antibody in step (1) is as follows: inject the anti-tetrodotoxin monoclonal antibody hybridoma cell line 5B9 in the logarithmic growth phase into the peritoneal cavity of paraffin-sensitized Balb / c mice. When the mouse abdomen is distended and shows signs of tension, ascites fluid is extracted and purified to obtain the anti-tetrodotoxin monoclonal antibody.

3. The application of the latex microsphere immunochromatographic test strip according to claim 1 in the detection of tetrodotoxin.

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