Paralytic saxitoxin and diarrhetic saxitoxin detection test paper and preparation method and application thereof

Abstract

The invention provides paralytic saxitoxin and diarrhetic saxitoxin detection test paper as well as a preparation method and application thereof, and belongs to the technical field of toxin detection. A paralytic shellfish poison antigen STX-BSA and a diarrhetic shellfish poison antigen OA-BSA are prepared and used for immunizing mice to prepare hybridoma cells, then monoclonal antibodies of paralytic shellfish poison and diarrhetic shellfish poison are obtained and sprayed to a combination pad, and the paralytic shellfish poison and diarrhetic shellfish poison is obtained. The paralytic shellfish poison antigen STX-BSA and the diarrhetic shellfish poison antigen OA-BSA are respectively used as detection lines for toxin detection, the detection limit of paralytic shellfish poison is 50 ng / mL, and the detection limit of diarrhetic shellfish poison is 100 ng / mL.

Description

Technical Field

[0001] The invention relates to the technical field of toxin detection, and in particular to a paralytic shellfish toxin and diarrhoeal shellfish toxin detection test paper and a preparation method and application thereof. Background Art

[0002] Paralytic shellfish toxins (PSP) are the most widely distributed and harmful marine biotoxins in the world, mainly produced by unicellular dinoflagellates in the ocean. PSP is a tricyclic compound of tetrahydropurine, which is relatively stable under weak acid and low temperature conditions. Common cooking methods cannot change its structure or reduce its toxicity. After being eaten by humans, shellfish contaminated with PSP will produce symptoms such as paralysis of the limbs and facial muscles, headache and nausea within 30 minutes to 3 hours, and in severe cases, suffocation and death. There are more than 20 types of PSP that have been discovered, among which saxitoxin (STX) accounts for more than 85% of the main components of PSP and is the main toxin. Shellfish can enrich the toxins in red tide algae through filter feeding and convert them into STX in the body. Most countries in the world use STX as the detection indicator for paralytic shellfish poisoning in shellfish products. The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) both stipulate that the limit standard for PSP in shellfish is 80μg STX / 100g.

[0003] Diarrhea shellfish toxin (DSP) is a fat-soluble polycyclic ether bioactive substance produced by some species of toxic red tide algae, Prorocentrum and Prorocentrum. The chemical structure of DSP is characterized by polyether or macrocyclic lactone compounds. According to the carbon skeleton structure of these components, they can be divided into three groups: ① acidic components: soft spongy acid (OA) and its natural derivatives - dinotoxin (DTX-3); ② neutral components: polyether lactone - saxitoxin (PTXI-Vn); ③ other components: sulfonated poisons, yew shellfish toxin (YTX) and its derivatives. OA and DTXs toxins are highly efficient and specific inhibitors of PP1 and PP2a type protein phosphatases, especially PP2a type, which can drastically enhance the phosphorylation of most proteins, causing diarrhea, vomiting, nausea, abdominal pain and headache. The symptoms usually occur 30 minutes to 14 hours after consumption, and the symptoms recover within 48 hours. DSP is not a fatal toxin. It usually only causes mild gastrointestinal diseases, and the symptoms will disappear quickly. It does not have strong acute toxicity, but OA is a strong carcinogen.

[0004] Paralytic shellfish toxins and diarrhetic shellfish toxins are highly toxic to the human body and are a major factor affecting public health. Therefore, it is crucial to strengthen the detection of paralytic shellfish toxins and diarrhetic shellfish toxins in seafood. Summary of the invention

[0005] The purpose of the present invention is to provide a paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper and a preparation method and application thereof, which can detect paralytic shellfish toxin and diarrhetic shellfish toxin.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper, comprising the following steps:

[0008] (1) preparing paralytic shellfish poison antigen STX-BSA and diarrheal shellfish poison antigen OA-BSA;

[0009] (2) immunizing mice with the two antigens prepared in step (1), detecting the serum titers, and selecting mice for booster immunization;

[0010] (3) 3 days after booster immunization, spleen cells of mice were taken and fused with mouse myeloma cells to obtain hybridoma cells, which were screened and injected into mice for further cultivation;

[0011] (4) Ascites was collected from mice with obvious abdominal bulges, and the cells in the ascites were separated by centrifugation and injected into mice for further culture;

[0012] (5) taking ascites of mice injected with ascites cells, detecting antibody titer, screening antibodies, and purifying to obtain monoclonal antibodies against paralytic shellfish poison and monoclonal antibodies against diarrheal shellfish poison;

[0013] (6) coupling the paralytic shellfish poison monoclonal antibody and the diarrheal shellfish poison monoclonal antibody to time-resolved fluorescent microspheres to obtain time-resolved fluorescent microsphere-labeled antibodies;

[0014] (7) Spray the paralytic shellfish toxin time-resolved fluorescent microsphere-labeled antibody and the diarrhetic shellfish toxin time-resolved fluorescent microsphere-labeled antibody evenly on the glass fiber binding pad, and use the STX-BSA prepared in step (1) as the detection line 1, OA-BSA as the detection line 2, and goat anti-mouse IgG as the quality control line to prepare paralytic shellfish toxin and diarrhetic shellfish toxin detection test strips.

[0015] Preferably, the preparation method of the paralytic shellfish poison antigen STX-BSA in step (1) is: adding BSA and STX to an acetate buffer, using formaldehyde as a cross-linking agent, and reacting at 37° C. for 72 hours;

[0016] The preparation method of the diarrheal shellfish poison antigen OA-BSA is as follows: adding diarrheal shellfish poison OA to a cross-linking agent, after oscillation reaction, adding BSA-PBS solution, dialyzing at 4° C. overnight to obtain OA-BSA.

[0017] Preferably, the cross-linking agent is prepared by dissolving 50 μg of water-soluble carbodiimide and 30 μg of N-hydroxysuccinimide in 10 μL of pure water.

[0018] Preferably, the conditions for screening mice in step (2) are: the mouse serum titer is greater than 1×10 4 .

[0019] Preferably, the conditions for screening hybridoma cells in step (3) are: the antibody titer of hybridoma cell culture fluid is greater than 1×10 3 .

[0020] Preferably, the conditions for screening the antibodies in the ascites fluid in step (5) are: the titer of the ascites antibody is greater than 1×10 7 .

[0021] Preferably, the pH of the coupling in step (6) is 6.0-7.5; and the mass ratio of the time-resolved fluorescent microspheres to the antibodies during coupling is 1 mg:5-100 μg.

[0022] Preferably, in step (7), the concentrations of the paralytic shellfish poisoning time-resolved fluorescent microsphere-labeled antibody and the diarrheal shellfish poisoning time-resolved fluorescent microsphere-labeled antibody on the conjugate pad are: 0.1 mg / mL to 1 mg / mL, respectively; the spraying volumes are 1 μL to 10 μL, respectively; the concentration of STX-BSA on the detection line 1 is 100 μg / mL to 500 μg / mL, and the spraying volume is 1 μL to 10 μL; the concentration of OA-BSA on the detection line 2 is 100 μg / mL to 500 μg / mL, and the spraying volume is 1 μL to 10 μL; the concentration of sheep anti-mouse IgG on the quality control line C is 100 μg / mL to 500 μg / mL, and the spraying volume is 1 μL to 10 μL.

[0023] The present invention also provides a paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper prepared by the preparation method of the paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper.

[0024] The present invention also provides an application of a paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper in the detection of paralytic shellfish toxin and diarrhetic shellfish toxin.

[0025] Preferably, the paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper carrier has a sample pad, an antibody binding pad, a chromatographic nitrocellulose membrane, and a water absorbent pad fixed thereon in sequence; the chromatographic nitrocellulose membrane is provided with a detection line 1, a detection line 2, and a quality control line, and the quality control line is arranged at one end close to the water absorbent pad.

[0026] The beneficial effects of the paralytic shellfish toxin and diarrhetic shellfish toxin detection test strips of the present invention compared with the prior art are as follows:

[0027] The present invention prepares paralytic shellfish toxin antigen STX-BSA and diarrhetic shellfish toxin antigen OA-BSA, and uses them to immunize mice to prepare hybridoma cells, and then obtains monoclonal antibodies of paralytic shellfish toxins and diarrhetic shellfish toxins, and sprays the monoclonal antibodies of paralytic shellfish toxins and diarrhetic shellfish toxins onto a conjugate pad, and the paralytic shellfish toxin antigen STX-BSA and diarrhetic shellfish toxin antigen OA-BSA are used as detection lines for toxin detection. The paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper of the present invention can detect paralytic shellfish toxins and diarrhetic shellfish toxins in shellfish, and the detection limit of paralytic shellfish toxins is 50ng / mL, and the detection limit of diarrhetic shellfish toxins is 100ng / mL. When the concentration is lower than the detection limit, it is considered as undetected. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0029] Figure 1 is a flow chart of the preparation of the monoclonal antibody in Example 1;

[0030] Figure 2 The molecular structure diagram of the diarrheal shellfish toxin in Example 1;

[0031] Figure 3 The paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper prepared in Example 1, wherein the accompanying drawings are marked with the following numerals: 1 is an antibody binding pad; 2 is an STX detection line; 3 is an OA detection line; 4 is a quality control line; 5 is a sample pad; 6 is a chromatography nitrocellulose membrane; 7 is a carrier plate; and 8 is an absorbent pad. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] The present invention prepares a paralytic shellfish toxin and a diarrhoeal shellfish toxin detection test paper, and the specific preparation method is as follows:

[0038] (1) preparing paralytic shellfish poison antigen STX-BSA and diarrheal shellfish poison antigen OA-BSA;

[0039] (2) immunizing mice with the two antigens prepared in step (1), detecting the serum titers, and selecting mice for booster immunization;

[0040] (3) 3 days after booster immunization, spleen cells of mice were taken and fused with mouse myeloma cells to obtain hybridoma cells, which were screened and injected into mice for further cultivation;

[0041] (4) Ascites was collected from mice with obvious abdominal bulges, and the cells in the ascites were separated by centrifugation and injected into mice for further culture;

[0042] (5) taking ascites of mice injected with ascites cells, detecting antibody titer, screening antibodies, and purifying to obtain monoclonal antibodies against paralytic shellfish poison and monoclonal antibodies against diarrheal shellfish poison;

[0043] (6) coupling the paralytic shellfish poison monoclonal antibody and the diarrheal shellfish poison monoclonal antibody to time-resolved fluorescent microspheres to obtain time-resolved fluorescent microsphere-labeled antibodies;

[0044] (7) Spray the paralytic shellfish toxin time-resolved fluorescent microsphere-labeled antibody and the diarrhetic shellfish toxin time-resolved fluorescent microsphere-labeled antibody evenly on the glass fiber binding pad, and use the STX-BSA prepared in step (1) as the detection line 1, OA-BSA as the detection line 2, and goat anti-mouse IgG as the quality control line to prepare paralytic shellfish toxin and diarrhetic shellfish toxin detection test strips.

[0045] Example 1

[0046] Example 1 of the present invention prepares a paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper, and the specific preparation method is as follows:

[0047] (1) Preparation of Antigen

[0048] A. Preparation of paralytic shellfish poisoning antigen STX-BSA

[0049] Since STX has a molecular weight of 299 and a small molecular weight, it is only antigenic but not immunogenic, and cannot be used for mouse immunization and ELISA plate coating. It needs to be coupled with the corresponding protein to prepare the corresponding immune antigen and coating antigen. The lysine residue of the protein can form a primary amine with the guanidine group of STX through formaldehyde, so that STX is coupled with the protein to obtain the paralytic shellfish poison antigen. The process is as follows: BSA and STX are added to the acetate buffer to obtain a concentration of 2mg / mL BSA and 1mg / mLSTX, 1mg / mLSTX 300ul is added to 600uL of 2mg / mL BSA, 10 drops of 70% formaldehyde are added, and the reaction is carried out at 37°C for 72 hours to obtain the paralytic shellfish poison antigen.

[0050] Hapten-N + H2+HCOH+carrier protein-NH2→hapten-N + H2-HCOH-NH2-carrier protein.

[0051] B. Preparation of OA-BSA antigen

[0052] Because diarrhetic shellfish toxins are small in molecular weight (see Figure 2 ), only antigenicity but no immunogenicity, cannot be used for mouse immunization and ELISA plate coating, and needs to be coupled with corresponding proteins to prepare corresponding immune antigens and coating antigens.

[0053] 500 μg MC-LR(OA) was dissolved in 0.5 mL DMSO to prepare a 1 mg / mL solution;

[0054] Prepare the crosslinker solution: water-soluble carbodiimide-NHS solution, the concentration of which is 50 μg EDC (water-soluble carbodiimide) and 30 μg NHS (N-hydroxysuccinimide) in 10 μL pure water.

[0055] Coupling method: Weigh 100 μg OA, add 10 μL of the above crosslinker solution, shake for 15 minutes, then add 300 μL of 1 mg / mL BSA (KLH)-PBS solution to each tube, let stand at 4°C overnight, and dialyze against pH 7.2 0.01 mol / L PBS in dialysis bags the next day, changing the solution 4 times. Obtain the diarrheal shellfish poison antigen OA-BSA (KLH), and calculate the antibody concentration according to the amount of antibody added and the volume.

[0056] (2) Preparation of monoclonal antibodies to paralytic shellfish toxins (see the procedure for Figure 1 ):

[0057] A. Immunization of BALB / c mice

[0058] Animals: 6 female SFP grade BALB / c mice weighing 12-15 g.

[0059] Antigen emulsification: 6 paralytic shellfish toxin antigens 1.5mg diluted with PPS or saline to 1.2-1.5mL, then add an equal volume of Freund's adjuvant, and emulsify repeatedly with a 16-gauge needle until it does not dissolve. Each injection is about 0.4mL, and each is subcutaneously injected at 6 points, in the armpit, groin and back of the mouse. (Use Freund's complete adjuvant for the first vaccination, and use Freund's incomplete adjuvant for all subsequent vaccinations).

[0060] B. ELISA screening of positive immune mice

[0061] 50 μL of whole blood was collected from the mouse eyeball and centrifuged, 3 μL of supernatant was taken and diluted to 4.5 mL with PBS.

[0062] Coating immunogen: Take the plate rack and install the plate, and mark the name of the coating material on the right side of the plate. Take a beaker, add 10mL of coating solution, add 2.5μL of the corresponding antigen (final concentration is 2-5μg / mL), and mix thoroughly. Take a gun tank, pour the corresponding coating solution, and add antigen with a gun, 100μL / well. Incubate at 37℃ for 2 hours (or 4℃ overnight).

[0063] Shake off the coating solution in the strips. Dry on newspapers. Add washing solution (PBST) with a pipette, 200 μL / well, shake on a shaker for 3 minutes, shake off the washing solution, and dry on newspapers. Repeat the washing 3 times.

[0064] Closure:

[0065] Prepare 4% blocking solution: add 60mL PBS to the blocking solution bottle, add 2.4mL newborn calf serum, and mix thoroughly. Use a spray gun to add blocking solution to the strips, 200μL / well. Incubate at 37℃ for 2 hours (or 4℃ overnight). Wash 3 times with washing solution and dry. Put it in a self-sealing bag with the corresponding mark and store at -20℃.

[0066] C. ELISA to detect antibody titer

[0067] Take out three strips of each antigen and place them on the strip rack as shown in Table 1. Add 100 μL / well PBS to the wells marked with PBS in the table.

[0068] Table 1 Amount of mouse serum dilution added to the strips

[0069]

[0070] Add the corresponding 1:1500 mouse serum dilution to the corresponding wells in the 4 rows of "1, 2, 7, 8", 100μL / well. Adjust the range of the gun to 100μL, press 6 gun tips, blow and beat more than 6 times in the second row to mix thoroughly; then draw 100μL to the third row and blow and beat more than 6 times; continue to dilute in this way until the fifth row, mix more than 6 times and remove 100μL. The operation of wells 8-11 is the same as above. Put the plate rack into a self-sealing bag and incubate it in a 37℃ constant temperature incubator for 45min. Wash 3 times with washing solution after incubation and pat dry. Prepare 10mL enzyme-labeled secondary antibody (1:10000): take a small beaker, measure 10mL PBS, take 1μL secondary antibody (goat anti-mouse IgG) and add it to the beaker, blow and beat, mix thoroughly, add secondary antibody to the gun, 100μL / well.

[0071] Put it in a ziplock bag and incubate it in a 37℃ incubator for 30min. Shake off the secondary antibody, dry it, wash it 6 times with detergent, and pat it dry as much as possible for the last time. Prepare OPD (1.5-2mg / mL): Measure 5mL OPD buffer and pour it into a brown bottle, weigh 7.5-10mg OPD, crush it and add it to the brown bottle, stir it away from light, and fully dissolve it. Finally, add 2μL H2O2 and mix well. Select 1 blank well and add 50μL OPD solution. Observe whether it develops color after 3min. If it does, it proves that the plate is not clean. Rewash it and wash it several times. Test again until no color develops within 3min before proceeding to the next step. Add OPD solution with a gun, 50μL / well. Press "vertical row" to add, not "horizontal row". Color development at room temperature in the dark or in a 37℃ incubator (about 15min), observe the color development at any time, and stop the color development as appropriate.

[0072] Turn on the microplate reader, select the OD490 test item (note: the control well should not be set to 0), and read the OD value of the strip. Note: The OD value of the negative control well must be controlled below 0.05, and above 1 is considered a positive valid well.

[0073] Copy the data to the serum titer test template table. Analyze and save the data.

[0074] If the positive serum titer is greater than 1W, continue to boost for fusion, and boost immunization 3 days before fusion.

[0075] D. Fusion of immune mouse spleen cells and mouse myeloma cells SP2 / 0

[0076] Preparation steps of feeder cells: Mixed cells of peritoneal macrophages and spleen cells of BALB / c mice are used as feeder cells, and the steps are as follows:

[0077] 1) Place sterilized centrifuge tubes, aluminum boxes containing tweezers and scissors, newspaper boxes, test tube racks, homogenizers, etc. on the operating table, sterilize them under UV for more than half an hour, turn off the UV, and blow the table for more than 5 minutes.

[0078] 2) Prepare the workbench: light the alcohol lamp, peel off the newspaper outside the scissors and tweezers box, take two 50mL sterile centrifuge tubes, pour out the water, put the lid on the left, and put the tube on the right test tube rack; open the sterile homogenizer, burn the head and tail of the cross first, then burn the head of the homogenizer, place the cross in the centrifuge tube, and put the homogenizer in the tube rack for use; take out 1640, burn two graduated tubes, and pad the foam board with newspaper.

[0079] 3) Take a blank BALB / c mouse, bleed its eyeball, kill it by dislocating the cervical vertebrae, and soak it in 75% alcohol for 5 minutes.

[0080] 4) In the clean bench, clamp the mouse's tail to the foam board with hemostatic forceps, turn the mouse's abdomen upward, and use a needle to fix the four soles of the mouse to the foam board padded with sterilized newspaper. Use hemostatic forceps (burned by the flame of an alcohol lamp) to clamp a set of small scissors and tweezers from the aluminum box, hold the tweezers in the left hand and the scissors in the right hand, extract, cut, and tear the abdominal skin to completely expose the chest and abdomen.

[0081] 5) Change to a set of small scissors and tweezers. Use the small tweezers to pinch the peritoneum in the middle of the mouse's abdomen and lift it upwards. Use the small scissors to cut a small incision of about 3 mm behind the lifted part. Place the small tweezers on the peritoneum and the small scissors on the edge of the small aluminum box lid for later use.

[0082] 6) Use a 5mL pipette to draw up 2.5mL of RPMI-1640 basal culture medium, lift the peritoneum with small forceps, insert the tip of the pipette through the small cut, and inject the culture medium into the mouse's abdominal cavity. After blowing and aspirating several times, aspirate the culture medium and transfer it to a 50mL centrifuge tube. Repeat this process once.

[0083] 7) Cross the hind limbs of the mouse and fix them (with the left hind limb on top to facilitate exposure of the spleen). Use small tweezers to pinch the peritoneum away from the spleen and lift it upwards. Use small scissors to make a small cut behind the lifted part. Insert the tip of the scissors through the small cut, open the scissors and stretch the small cut to both sides. Then insert small tweezers through the stretched small cut, clamp and hang the peritoneum and tear it forward until the spleen is completely exposed.

[0084] 8) Change to a set of small scissors and forceps, use small tweezers to grasp the spleen and gently lift it up, use small scissors to cut the connective tissue to separate the spleen, then use small scissors to cut some small holes on the edge of the spleen and transfer the spleen to the glass homogenizer.

[0085] 9) Add 8 mL of RPMI-1640 to a glass homogenizer and homogenize the spleen (tilt at a certain angle to prevent bacteria from falling vertically into the homogenizer) until no red tissue blocks are visible, mix well, let stand for 1 min, wait for the white tissue blocks to sink to the bottom of the tube, aspirate the upper layer of cell suspension into a 50 mL centrifuge tube containing macrophages, add 10 mL of RPMI-1640 to the glass homogenizer, mix well, let stand for 1 min, aspirate the upper layer of cell suspension, and transfer to a 50 mL centrifuge tube.

[0086] 10) After mixing the mixed cells, centrifuge horizontally at 1200r / min for 5 minutes, pour off the supernatant, add 5mL 1640 and mix well, add horizontally to wash the cells (wash away the fibroblasts inside). If the cells are not clean enough, change a graduated tube and wash again.

[0087] 11) Centrifuge horizontally at 1200 rpm for 5 min, resuspend the feeder cells in 20 mL of RPMI-1640 complete culture medium containing 1% HAT, cover the bottle with a stopper, and place in a CO2 constant temperature incubator for later use.

[0088] Preparation of myeloma cells:

[0089] SP2 / 0 myeloma cells were prepared by homogenizing SP2 / 0 myeloma cells grown in BALB / c mice and then used for fusion. The preparation steps are as follows:

[0090] 1) Prepare the workbench: same as feeder cell preparation.

[0091] 2) BALB / c mice with subcutaneous myeloma on their backs were killed by cervical dislocation and soaked in 75% alcohol for 5 minutes.

[0092] 3) Add about 17 mL of lymphocyte separation solution into a sterile 50 mL centrifuge tube.

[0093] 4) Fix the mouse with its back facing up, and refer to the preparation method of feeder cells to separate bean-sized myeloma. First, add 6 mL of 1640 to the homogenizer, grind slowly, and pump up and down repeatedly for about 4 times. Slowly take out the cross and discard it in the waste liquid tank.

[0094] 5) Take about 4 mL of 1640 in a homogenizer, gently blow it with a graduated tube, let it stand for about 1 minute, use a graduated tube to aspirate the supernatant into a clean centrifuge tube, and slowly drip it horizontally (to wash the cells); use another clean graduated tube to add about 5 mL of 1640 to the homogenizer, mix well, let it stand for 1 minute, and aspirate it into the centrifuge tube as above.

[0095] 6) Take 15mL of myeloma cell suspension and gently add it along the wall to the upper layer of lymphocyte separation solution. Note: Make sure the two liquids are in a layered state and do not mix the liquids (the dropping method is the same as cell separation).

[0096] 7) After horizontal centrifugation at 1800 r / min for 15 minutes, take out the centrifuge tube. You can see a layer of white cells at the junction of the myeloma cell suspension and the lymphocyte separation fluid. These are the myeloma cells. Transfer all the upper, middle and lower layers to a clean centrifuge tube and centrifuge at 1200 r / min for 5 minutes.

[0097] 8) Resuspend the myeloma cells in 30 mL of RPMI-1640 basal culture medium and count them using a hemocytometer. Myeloma cell concentration = (number of cells in the four corners + the center square) × 5 × 10 4 Pieces / mL.

[0098] Preparation of immune splenocytes:

[0099] The spleen cells of the immunized mice were separated and homogenized to prepare immune spleen cells. The method was the same as that of preparing spleen cells in feeder cell preparation, except that the immunized spleen was wrapped by a large amount of connective tissue and was more difficult to separate than the normal spleen. If it could not be separated completely, it could be separated in small pieces. Cell fusion was performed using 50% PEG with a molecular weight of 1450 as a fusion agent. Cell fusion was performed according to conventional methods. The operation steps were as follows:

[0100] 1) Take 1×10 7 SP2 / 0 myeloma cells and immune spleen cells (generally, the number of spleen cells in a mouse is 10 8 The cells were mixed (i.e., tumor cells: spleen cells = 1:10), resuspended in 15 mL RPMI-1640 basal culture medium, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded.

[0101] 2) Take out the sterilized absorbent paper from the clean bench, and place the 50mL centrifuge tube containing the mixed cells of myeloma cells and immune spleen cells upside down on the absorbent paper to control the water droplets (very important).

[0102] 3) Take out 50% PEG incubated to 37°C from the CO2 incubator, draw 1mL with a 1mL pipette, hold the 50mL centrifuge tube containing the mixed cells in the left hand, place it in a pre-prepared 37°C water bath, slowly add PEG to the mixed cells, gently stir while adding, add within 90s, add slowly, stir slowly while adding, let all the cells at the bottom be suspended, do not blow), then let it stand in the water bath for 60s, insert the centrifuge tube into the centrifuge tube rack, remove the water bath, take out 50mL RPMI-1640 basal culture medium incubated to 37°C from the CO2 incubator, draw 10mL with a pipette and slowly add it along the wall to the fused cells, stir gently while adding (do not blow) to disperse the cell clumps, add 1mL first, then add 2mL, then add 3mL, and finally add the remaining 4mL. After adding the first 10mL, continue to add the remaining 40mL along the wall of the tube. After adding, tighten the cap and slowly invert it several times to mix the cells.

[0103] 4) Centrifuge at 1200 rpm for 5 min, discard the supernatant, and resuspend the feeder cells in 20 mL of RPMI-1640 containing 1% HAT. Use complete culture medium (incubation) to resuspend the fused cells and mix well (be gentle, use a pipette to gently stir the cells, do not blow).

[0104] 5) Add the resuspended cell solution to the prepared 80 mL semi-solid culture medium, shake the glass bottle to mix the cells (do not pipette), and use a 20 mL syringe to evenly distribute them to 9 6-well plates, 1.5 mL / well, and place them in a CO2 incubator for culture (place a saturated copper sulfate solution at the bottom of the incubator).

[0105] 6) Small colonies can be observed 7 days after fusion. When the colonies grow into small white spots (about 14 days), the cells can be picked and transferred to a 96-well culture plate and cultured with HAT medium.

[0106] 7) Use 1640 complete culture medium containing 1% HAT, 3 drops / well. Around the 10th day, when the colonies grow to occupy 2 / 3 of the bottom of the well and the culture medium turns yellow, antibody detection can be performed.

[0107] Positive clone cells with an antibody titer of more than 1000 in the cell culture fluid were screened for subculture and preservation.

[0108] E. Preparation of monoclonal antibody ascites by intraperitoneal injection of cells

[0109] 1) SP2 / 0 hybridoma cells frozen in liquid nitrogen tanks were revived and subcultured in tissue culture bottles. When the cells grew to the logarithmic growth phase, the supernatant was removed by centrifugation, and the precipitated cells were washed once by centrifugation with physiological saline. After appropriate dilution, they were injected into BALB / c mice that had been intraperitoneally injected with dexamethasone 7 to 10 days ago. Each mouse was injected with 1×106 hybridoma cells. 2) 7 to 14 days after intraperitoneal injection of tissue culture monoclonal antibody hybridoma cells, select BALB / c mice with obvious abdominal bulges, pull their necks to kill them, and fix them in a supine position on a mouse rack in a clean bench. Soak the mice in 75% alcohol for 1 minute. Use sterile ophthalmic scissors to carefully cut open the abdominal skin and muscles to completely expose the peritoneum. Use a 5mL disposable syringe filled with an appropriate amount of heparin to quickly aspirate the ascites into a cell centrifuge tube and shake it immediately to prevent agglutination.

[0110] 3) Centrifuge the collected ascites at 1200 r / min for 7 minutes, and carefully remove the supernatant, which is the pure mouse ascites containing monoclonal antibodies. Wash the precipitated cells once with sterile saline, and centrifuge at 1200 r / min for 7 minutes each time to obtain a pure cell precipitate.

[0111] 4) Add sterile physiological saline to the purified cell precipitate to the original volume of ascites. Gently shake and carefully add the cell suspension along the tube wall into a test tube containing an equal amount of cell separation solution, taking care not to break up the liquid surface of the cell separation solution. Centrifuge at 1500r / min for 10min in a horizontal centrifuge. A thin but very obvious milky white cell layer can be obtained between the cell suspension and the separation solution, extending above the cell layer. Carefully aspirate this layer of cells into a cell centrifuge tube.

[0112] 5) Wash the cells in the cell centrifuge tube once with sterile saline, centrifuge at 1500r / min for 7min each time. Dilute the obtained cell pellet with an appropriate amount of sterile saline. After counting under an inverted microscope, add an appropriate amount of double antibody and sterile saline to make the cell count around 5×10 6 Each BALB / c mouse was intraperitoneally injected with about 1 mL of ascites-separated cells.

[0113] 6) 7 to 14 days after the injection of ascites cells, the ascites of the bulging abdomen was collected and the above hybridoma cell isolation and injection into the peritoneal cavity of the mouse were repeated. The antibody titer of the ascites was determined by enzyme-linked immunosorbent assay (ELISA) and the titer was 1×10 7 The above ascites was used as antibody.

[0114] F. Antibody Purification

[0115] 1) Prepare saturated ammonium sulfate solution (SAS): slowly add 767g (NH4)2SO4 to 1L distilled water while stirring. Adjust the pH of the solution to 7.0 with ammonia or sulfuric acid. This is a 100% saturated ammonium sulfate solution (4.1 mol / L, 25°C).

[0116] 2) Precipitation: Centrifuge the ascites at 20,000 r / min for 30 min to remove cell debris, retain the supernatant and measure the volume; slowly add SAS to the sample solution while stirring to a concentration of 0.5:1 (v / v); place the solution on a magnetic stirrer and stir for 6 hours or overnight (4°C); centrifuge at 3,000 r / min for 30 min (4°C), retain the supernatant; add SAS to the supernatant to a concentration of 0.5:1 (v / v), and centrifuge again to obtain a precipitate.

[0117] 3) Dialysis: Dissolve the precipitate in a small amount of PBS. After the precipitate is dissolved, place it in a dialysis bag and dialyze it with PBS for 24 to 48 hours (4°C). Change the dialysis buffer every 3-6 hours to completely remove the ammonium sulfate; centrifuge the dialysate, determine the protein content in the supernatant, and screen antibodies with a protein content of 2 mg / mL-20 mg / mL to obtain monoclonal antibodies.

[0118] (3) Preparation of monoclonal antibodies against diarrhetic shellfish toxins

[0119] Prepare the monoclonal antibody of diarrhetic shellfish toxin by referring to the preparation method of monoclonal antibody of paralytic shellfish toxin in steps (2) and (3)

[0120] (4) Preparation of test strips

[0121] A. Time-resolved fluorescent microsphere-labeled antibodies

[0122] 1) The time-resolved fluorescent microspheres were coupled with the paralytic shellfish toxin monoclonal antibody and the diarrhetic shellfish toxin monoclonal antibody respectively by the EDC-NHS two-step method: 1 mg of the fluorescent microspheres was diluted with 1 mL of phosphate buffer (10 mM, pH 7.0); 10 ul of freshly prepared EDC (10 mg / mL) and NHS (10 mg / mL) were added, and the mixture was stirred at room temperature for 30 min; the mixture was centrifuged at 10000 rpm for 20 min, the supernatant was discarded, and the precipitate was dissolved by shaking with 1 mL of boric acid buffer (20 mM, pH 8.0), and 20 μg of the antibody was added to the mixture, mixed and stirred for 3 h, and then 0.1 mL of 0.3 mol / L glycine was added for blocking for 2 h, and the mixture was centrifuged at 10000 r / min for 30 min to collect the precipitate; the precipitate was re-dissolved with 1 mL of reconstitution solution (PB buffer containing 2% sucrose, 5% BSA, 2% Tween-20 and 1% ProClin300), and stored at 4°C for later use.

[0123] 2) Optimization of coupling pH

[0124] 0.01 mol / L PBS buffer with pH values ​​of 6.0, 6.5, 7.0, and 7.5 were used to activate the fluorescent microspheres, and then the antibodies were labeled in boric acid buffer (20 mM, pH 8.0). Test strips were prepared using the labeled antibodies, and the fluorescence intensity of the negative dilution test of the reagent strips prepared by labeling the antibodies in microsphere activation buffers with different pH values ​​was detected. The test was repeated 5 times for each pH value, and the average value was taken. The optimal labeling pH value was determined according to the signal intensity.

[0125] The corresponding fluorescence intensities of the four pH gradient labeled antibodies prepared under the same conditions for the test strips were 61579, 51445, 36789, and 19845. The fluorescent microspheres were activated at pH 6.0.

[0126] 3) Optimization of saturation labeling amount

[0127] Keeping the amount of fluorescent microspheres unchanged, 5, 10, 20, 50, and 100 μg of antibodies were added for coupling, and the optimal saturation labeling amount was determined based on the fluorescence intensity results of the blank dilution test. The fluorescence intensities of the test paper fluorescent paper obtained by the five antibody labeling amounts were 6238, 12387, 23489, 60239, and 63482, respectively. The optimal labeling amount was 50 μg.

[0128] B. Optimization of coated antigens on NC membrane

[0129] The amount of fluorescent microspheres in each test reagent card was kept unchanged, and the test line on the NC membrane was coated with 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL and 1.0 mg / mL of STX-BSA antigen, respectively, with the best coating antigen dosage being 0.1 mg / mL.

[0130] C. Assembly of test strips

[0131] The composition of the fluorescent immunochromatographic test strip is as follows: STX and OA monoclonal antibodies labeled with fluorescent microspheres are evenly sprayed on the glass fiber antibody binding pad in a 1:1 ratio, with a total of 10 μL sprayed, and the concentration is 0.1 mg / mL, and then dried at 37 degrees for 2 hours; STX-BSA antigen, OA-BSA antigen, and goat anti-mouse IgG are diluted to 0.1 mg / mL and used as coating solutions for detection line 1, detection line 2, and quality control line (C line), respectively, and sprayed on the corresponding positions on the nitrocellulose membrane in turn, with a spraying amount of 5 μL, and then dried at 37 degrees for 2 hours.

[0132] Test strip structure Figure 3 As shown, 1 is the antibody binding pad; 2 is the STX detection line; 3 is the OA detection line; 4 is the quality control line; 5 is the sample pad; 6 is the chromatography nitrocellulose membrane; 7 is the carrier plate; and 8 is the water absorbent pad.

[0133] Example 2

[0134] Example 2 of the present invention tested the performance of the test strip prepared in Example 1, and the specific method was as follows:

[0135] (1) Preparation of standard curve:

[0136] Standard solutions of paralytic shellfish toxin (STX) and diarrhetic shellfish toxin (OA) were prepared respectively, with STX standard concentrations (0, 2.5, 5, 10, 20, 40, 80, 200 ng / mL) and OA standard concentrations (0, 13, 26, 66, 133, 266 ng / mL). Three parallels were measured for each mass concentration, and the results were read using a dry fluorescence analyzer. The logarithm of the T / C ratio of solutions with different concentrations was the abscissa, and the logarithm of other spiked mass concentrations was the ordinate. The quantitative standard curves of STX and OA fluorescent immunochromatographic test strips were drawn, and the results are shown in Tables 2 and 3.

[0137] Table 2 STX standard curve

[0138]

[0139]

[0140] Let p = OD / OD0, q = 1-p;

[0141] y=ln(p / q)

[0142] x = lg concentration

[0143] Where: OD is the reaction value, OD0 is the mean of the reaction values ​​with a concentration of 0.

[0144] The standard equation is: y = a + b * x, where a = 3.371177; b = -2.026168; r 2 =0.995495.

[0145] Table 3OA standard curve

[0146] ng / mL Concentration ng / g T / G(OD) 0 0 1.751312176 13 78 1.457172145 26 156 1.148726001 66 396 0.764565031 133 798 0.502136855 266 1596 0.330454194

[0147] Let p = OD / OD0, q = 1-p;

[0148] y=ln(p / q)

[0149] x = lg concentration

[0150] Where: OD is the reaction value, OD0 is the mean of the reaction values ​​with a concentration of 0.

[0151] The standard equation is: y = a + b * x, where a = 4.027566; b = -2.306061; r2 =0.990845.

[0152] (2) Sample processing

[0153] A. Use a knife to pry open the adductor muscle to open the shell, take out the shell meat, then wash it with clean water and crush the tissue with a pounder;

[0154] B. Weigh 1.0 g of the mashed tissue sample, put it into a 10 mL centrifuge tube, add 1 mL of methanol, shake well and mix for 2 min; centrifuge at 4000 r / min for 5 min;

[0155] C. Take 100uL of supernatant and add it to a 1.5mL centrifuge tube. Add 200uL of sample diluent and mix well to obtain the test solution.

[0156] (3) Sample measurement procedure

[0157] Take 80uL of the test solution and add it to the reagent card, incubate it at room temperature in the range of 20-26 degrees for 8 minutes, and then insert it into the fluorescent immunoassay analyzer to read the results. The results are shown in Tables 4 and 5.

[0158] Table 4 Test strip STX test data

[0159]

[0160] Table 5 Test strip OA test data

[0161]

[0162]

[0163] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a test strip for detecting paralytic shellfish toxins and diarrhoeal shellfish toxins, characterized in that: The steps include: (1) Preparation of paralytic shellfish poison antigen STX-BSA and diarrhoeal shellfish poison antigen OA-BSA; (2) Immunizing mice with the two antigens prepared in step (1), detecting their serum titers, and selecting mice for booster immunization; (3) Three days after booster immunization, spleen cells from mice were taken and fused with mouse myeloma cells to obtain hybridoma cells. The hybridoma cells were screened and injected into mice for further culture. (4) Collect ascites from mice with obvious abdominal bulges, separate the cells from the ascites by centrifugation, and then inject them into mice for further culture; (5) taking the ascites of the mice injected with the ascites cells, testing the antibody titer, screening the antibodies, and purifying them to obtain monoclonal antibodies against paralytic shellfish poisoning and monoclonal antibodies against diarrheal shellfish poisoning; (6) coupling the paralytic shellfish poison monoclonal antibody and the diarrheal shellfish poison monoclonal antibody to time-resolved fluorescent microspheres to obtain time-resolved fluorescent microsphere-labeled antibodies; (7) Spray the paralytic shellfish toxin time-resolved fluorescent microsphere-labeled antibody and the diarrhetic shellfish toxin time-resolved fluorescent microsphere-labeled antibody evenly on the glass fiber binding pad, and use the STX-BSA prepared in step (1) as the detection line 1, OA-BSA as the detection line 2, and goat anti-mouse IgG as the quality control line to prepare paralytic shellfish toxin and diarrhetic shellfish toxin detection test strips.

2. The method for preparing the paralytic shellfish toxin and diarrhetic shellfish toxin test strip according to claim 1, characterized in that: The preparation method of the paralytic shellfish poison antigen STX-BSA in step (1) is as follows: adding BSA and STX to an acetate buffer, using formaldehyde as a cross-linking agent, and reacting at 37° C. for 72 hours; The preparation method of the diarrheal shellfish poison antigen OA-BSA is as follows: adding diarrheal shellfish poison OA to a cross-linking agent, after oscillation reaction, adding BSA-PBS solution, dialyzing at 4°C overnight to obtain OA-BSA.

3. The method for preparing the paralytic shellfish toxin and diarrhetic shellfish toxin test strip according to claim 2, characterized in that: The cross-linking agent was prepared by dissolving 50 μg of water-soluble carbodiimide and 30 μg of N-hydroxysuccinimide in 10 μL of pure water.

4. The method for preparing the paralytic shellfish toxin and diarrhetic shellfish toxin test strip according to claim 1, characterized in that: The conditions for screening mice in step (2) are: the mouse serum titer is greater than 1×10 4 The conditions for screening hybridoma cells in step (3) are: the antibody titer of the hybridoma cell culture fluid is greater than 1×10 3 .

5. The method for preparing the paralytic shellfish toxin and diarrhetic shellfish toxin test strip according to claim 1, characterized in that: The conditions for screening the antibodies in the ascites in step (5) are: the titer of the ascites antibody is greater than 1×10 7 .

6. The method for preparing the paralytic shellfish toxin and diarrhetic shellfish toxin test strip according to claim 1, characterized in that: In step (6), the pH of the coupling is 6.0-7.5; during the coupling, the mass ratio of the time-resolved fluorescent microspheres to the antibody is 1 mg:5-100 μg.

7. The method for preparing the paralytic shellfish toxin and diarrhetic shellfish toxin test strip according to claim 1, characterized in that: In step (7), the concentrations of the paralytic shellfish poisoning time-resolved fluorescent microsphere-labeled antibody and the diarrheal shellfish poisoning time-resolved fluorescent microsphere-labeled antibody on the binding pad are 0.1 mg / mL to 1 mg / mL, respectively; the spraying volumes are 1 μL to 10 μL, respectively; the concentration of STX-BSA on the detection line 1 is 100 μg / mL to 500 μg / mL, and the spraying volume is 1 μL to 10 μL; the concentration of OA-BSA on the detection line 2 is 100 μg / mL to 500 μg / mL, and the spraying volume is 1 μL to 10 μL; the concentration of goat anti-mouse IgG on the quality control line C is 100 μg / mL to 500 μg / mL, and the spraying volume is 1 μL to 10 μL.

8. A paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper prepared according to the preparation method of the paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper according to any one of claims 1 to 7.

9. The paralytic shellfish toxin and diarrhetic shellfish toxin test strip according to claim 8, characterized in that: The paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper includes a carrier plate, on which a sample pad, an antibody binding pad, a chromatographic nitrocellulose membrane, and a water absorbent pad are fixed in sequence; a detection line 1, a detection line 2, and a quality control line are arranged on the chromatographic nitrocellulose membrane, and the quality control line is arranged at one end close to the water absorbent pad.

10. Use of the paralytic shellfish toxin and diarrhetic shellfish toxin detection test paper according to any one of claims 8 to 9 in the detection of paralytic shellfish toxins and diarrhetic shellfish toxins.

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