A Saxitoxin Monoclonal Antibody, Its Preparation Method and Application

Abstract

The present invention discloses a saxitoxin monoclonal antibody, its preparation method and application, as well as a saxitoxin hapten. Using VLP as a carrier, the structural formula of the saxitoxin hapten is as follows: The present invention successfully prepares a saxitoxin monoclonal antibody through the synthetic saxitoxin artificial hapten. The prepared saxitoxin monoclonal antibody has the advantages of high purity, strong specificity and high titer, can avoid serological cross-reactions between different cells and microbial species or strains, thereby greatly improving the detection specificity and sensitivity of saxitoxin, and has good application prospects; the preparation methods of the saxitoxin hapten and the monoclonal antibody are safe, effective, non-toxic and harmless, and do not require special instrument equipment and harsh and complex reaction conditions.

Description

Technical Field

[0001] The present invention relates to the technical field of saxitoxin detection, and particularly to a saxitoxin monoclonal antibody, a preparation method thereof and an application thereof. Background Art

[0002] Saxitoxins (STX), a derivative of tetrahydropurine, are one of the most toxic marine biotoxins known, with a high lethality rate. The lethal dose for adults is 540 - 1000 μg, and it is one of the main toxins causing paralytic shellfish poisoning (PSP).

[0003] Saxidomus is a benthic shellfish that can live in both fresh water and sea water. Its meat is delicious and nutritious, making it a popular seafood. However, due to the possible presence of highly lethal STX, the detection of saxitoxin content is very important.

[0004] Previously, the commonly used methods for detecting saxitoxin were biological analysis methods and chemical analysis methods. Biological analysis methods are detection methods based on the reactions of cells, tissues, and organs after the action of toxins, including mouse bio - toxicity tests and starfish gastrointestinal operation tests, etc. Due to problems such as complex operation, long time - consuming, high toxicity, and low utilization rate of experimental animals, biological analysis methods have gradually been replaced by chemical analysis methods. Chemical analysis methods mainly include high - performance liquid chromatography coupled with mass spectrometry (HPLC), gas chromatography - mass spectrometry (GC - MS), and liquid chromatography - mass spectrometry (LC - MS), etc. These methods have high accuracy and good repeatability, but have defects such as high cost for a single detection, complex operation, expensive equipment, and long response time.

[0005] In recent years, with the development of molecular biology, the use of immunological methods to detect the content of saxitoxin has gradually replaced biological analysis and chemical analysis methods and become a commonly used detection method. Immunological methods are achieved through the interaction between specific antibody molecules, including ELISA (enzyme - linked immunosorbent assay), immunofluorescence technology, immunoradiometric assay, immunoenzymatic technique, and immunogold colloidal technique, etc. Among them, ELISA technology is currently the most widely used method for detecting saxitoxin. This method uses specific antibodies to recognize saxitoxin, binds the toxin to the antigen, and converts it into an optical signal through the catalytic action of an auxiliary enzyme, thereby achieving the detection of saxitoxin, and has the advantages of simple operation, short time, high sensitivity, and good accuracy.

[0006] However, due to the very small molecular weight of saxitoxin, which is less than 300, it is difficult to prepare monoclonal antibodies. Therefore, almost all the antibodies used in the ELISA detection of saxitoxin are polyclonal antibodies. Polyclonal antibodies have low titers and are prone to cross-reactions, severely limiting the specificity, selectivity, and sensitivity of ELISA detection. Summary of the Invention

[0007] The object of the present invention is to provide a saxitoxin monoclonal antibody, its preparation method and application, so as to further improve the specificity, selectivity, and sensitivity of the ELISA saxitoxin detection kit, and at the same time solve the problem of easy cross-reaction of the ELISA saxitoxin detection kit based on polyclonal antibodies.

[0008] To achieve the above object, the present invention provides a saxitoxin monoclonal antibody, its preparation method and application, a saxitoxin hapten, using VLP as a carrier, and the structural formula of the saxitoxin hapten is:

[0009] A preparation method of the saxitoxin hapten as described above, the steps are as follows:

[0010] (1) Dissolve STX in HCl solution, and dropwise add sodium nitrite solution at low temperature, and monitor the reaction process with starch potassium iodide test paper. Wait until the reaction is complete to obtain solution A;

[0011] (2) Dissolve the VLP carrier in 1×PBS at low temperature, denoted as solution B;

[0012] (3) Dropwise add solution A to solution B under certain conditions. After reacting for a certain time, add 1×PBS to obtain the STX-VLP hapten stock solution;

[0013] (4) Dialyze and purify the STX-VLP hapten stock solution to obtain the pure STX-VLP hapten.

[0014] Preferably, the monitoring of the reaction process with starch potassium iodide test paper in step (1) means stopping the dropwise addition of sodium nitrite solution when the test paper turns blue-gray, and continuing to add sodium nitrite solution when the test paper shows no change.

[0015] Preferably, the concentration of the HCl solution in step (1) is 0.1 M, the concentration of the sodium nitrite solution is 0.01 M, the reaction condition is rotation and mixing reaction at 4°C, the mass-volume ratio of STX to the HCl solution is 10 μmol:1 mL, and before the dropwise addition reaction, both the STX solution and the sodium nitrite solution need to be precooled to 4°C.

[0016] Preferably, the low temperature in step (2) is 4°C, and the concentration of solution B is 2 mg / mL.

[0017] Preferably, the certain conditions in step (3) refer to 4°C, continuous stirring, pH 9-10, the reaction for a certain period of time refers to reacting for 2-4 h, and the volume ratio of the added 1×PBS to the reaction solution volume is 1:1.

[0018] Preferably, the dialysis and purification conditions in step (4) are as follows: the dialysis solution is 1×PBS buffer solution, dialysis is carried out at 4°C for 1-3 d, and the buffer solution is changed 3 times a day, with an interval of not less than 4 h each time.

[0019] A method for preparing a monoclonal antibody against saxitoxin using the above hapten, the steps are as follows:

[0020] S1. Prepare the saxitoxin hapten by the above method;

[0021] S2. Immunize mice with the synthesized hapten as an immunogen;

[0022] S3. Fuse the spleen cells of the immunized mice with mouse myeloma cells;

[0023] S4. Screen to obtain hybridoma cells with positive reaction;

[0024] S5. Inoculate the positive hybridoma cells into mice, collect and purify the ascites antibody to obtain a monoclonal antibody against saxitoxin.

[0025] A monoclonal antibody against saxitoxin prepared by the above method.

[0026] An application of the monoclonal antibody against saxitoxin as described above in the preparation of an ELISA kit for detecting STX.

[0027] Therefore, a monoclonal antibody against saxitoxin provided by the present invention and its preparation method and application have the following technical effects:

[0028] 1. The present invention successfully prepares a monoclonal antibody against saxitoxin through the synthesized artificial saxitoxin hapten. The prepared monoclonal antibody has the advantages of high titer, strong specificity, and high sensitivity, can effectively avoid cross-reaction, and has good application prospects in the inspection of saxitoxin;

[0029] 2. The preparation methods of the saxitoxin hapten and the monoclonal antibody against saxitoxin provided by the present invention are safe, effective, non-toxic and harmless, and do not require special instrument equipment and harsh and complex reaction conditions.

[0030] The technical solutions of the present invention will be further described in detail below through the drawings and examples. Description of the Drawings

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments of the present invention. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 is a schematic diagram of the preparation process of saxitoxin conjugated hapten of the present invention;

[0033] Figure 2 is the effect diagram of the STX-VLP hapten prepared in the first embodiment of the present invention;

[0034] Figure 3 is the absorption wavelength detection result of the STX-VLP hapten prepared in the first embodiment of the present invention;

[0035] Figure 4 is the SDS-PAGE detection result of the monoclonal antibody prepared in the fifth embodiment of the present invention;

[0036] Figure 5 is the standard curve for detecting saxitoxin by competitive ELISA in the sixth embodiment of the present invention. Specific Embodiments

[0037] The following further illustrates the technical solutions of the present invention through drawings and examples.

[0038] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and complete, the following will clearly and completely describe the technical solutions of the present invention through drawings and examples. The following detailed descriptions are all descriptions of examples, aiming to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs.

[0039] The saxitoxin STX standard product (chromatographically pure ≥ 95%) was purchased from Beijing Epris Technology Development Co., Ltd.; the VLP superantigen protein was purchased from Kactus Biotechnology (Shanghai) Co., Ltd.; the mouse myeloma cell line SP2 / 0 cells were purchased from the Cell Bank of the Chinese Academy of Sciences; female BALB / c mice were purchased from Shanghai Slack Experimental Animal Co., Ltd.; the chemical reagents for preparing PBS and CBS were all purchased from Sigma Company; the BCA kit was purchased from Thermo Fisher Scientific Company, and the enzyme-labeled secondary antibody was purchased from Abcam Company; the UV-2450 continuous spectrophotometer was produced by Shimadzu Corporation of Japan, and the Thermo Multiskan FC microplate reader was produced by Thermo Fisher Scientific Company; the dialysis bag was a 3500D dialysis bag purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0040] Example 1

[0041] Prepare a hapten (STX-VLP) of saxitoxin (STX) as follows:

[0042] (1) Dissolve the purchased STX in dd water according to the instructions. Accurately pipette 10 μmol of STX and dissolve it in 1 mL of 0.1 M HCl, then cool it to 4°C. Then gradually add dropwise the 0.01 M sodium nitrite solution precooled to 4°C (prepared freshly before use), and monitor the reaction with starch potassium iodide test paper. Stop adding when the test paper turns blue-gray. After reacting with rotation at 4°C for 30 min, test with the starch potassium iodide test paper again. If there is no change, continue to add dropwise the sodium nitrite solution while maintaining acidic conditions. Stop adding when the test paper turns blue-gray and continue the reaction for 1 h to obtain Solution A.

[0043] (2) Accurately weigh 1 mg of VLP, add it to 1 mL of 1×PBS at 4°C, and stir to dissolve it completely, denoted as Solution B.

[0044] (3) Under the conditions of 4°C and continuous stirring, gradually add dropwise Solution A to Solution B, and at the same time adjust the pH with 1 M NaOH solution to keep the pH at 9. Stir at 4°C for 3 h, and finally make up the volume of the solution to 3 mL with 1×PBS to obtain the stock solution of the STX-VLP hapten.

[0045] (4) Load the stock solution of the STX-VLP hapten prepared in step (3) into a dialysis bag and dialyze it with 1×PBS buffer at 4°C for 2 d (change the buffer 3 times a day, with an interval of no less than 4 h each time) to obtain the purified STX-VLP hapten. The preparation process is shown in Figure 1 , and the effect diagram of the prepared STX-VLP hapten is shown in Figure 2 .

[0046] (5) Use an ultraviolet spectrophotometer to detect the purified STX-VLP hapten prepared in step (4). Before detection, it is necessary to confirm that the absorption peaks of small molecules and VLP do not overlap. Measure the characteristic absorption wavelength of the carrier protein VLP at a concentration of 1.0 mg / mL; measure the characteristic absorption wavelength of STX at a concentration of 0.1 mg / mL; measure the characteristic absorption wavelength of the STX-VLP antigen at a concentration of 0.1 mg / mL. The results are shown in Figure 3 .

[0047] Example 2

[0048] Prepare an STX-VLP hapten. The method is exactly the same as that in Example 1, except that the pH in step (3) is 10 and the reaction time is 4 h, and the dialysis time in step (4) is 3 d.

[0049] Example 3

[0050] Prepare a STX-VLP hapten. The method is exactly the same as that in Example 1, except that the pH in step (3) is 10 and the reaction time is 2 h, and dialysis is carried out for 1 d in step (4).

[0051] Example 4

[0052] ELISA method for detecting the titer of mouse serum:

[0053] (1) Coating: Dilute the STX-VLP antigen with diluent (50 ng per well), 100 μL per well, and incubate overnight in a refrigerator at 4°C (about 16 - 18 h); discard the liquid in the wells, wash 4 times with PBST, 3 min each time, and pat dry.

[0054] (2) Blocking: Add 5% PBS-BSA blocking solution, 300 μL per well, incubate at 37°C for 2 h, discard the liquid in the wells, wash 4 times with PBST, 3 min each time, and pat dry.

[0055] (3) Sample addition: Dilute the serum serially with antibody diluent, 100 μL per well, incubate at 37°C for 1 h, discard the liquid in the wells, wash 3 times with PBST, 3 min each time, and pat dry.

[0056] (4) Secondary antibody: Dilute HRP-goat anti-mouse IgG according to the dilution factor written in the reagent instruction manual, add 100 μL per well to each well, incubate at 37°C for 1 h, discard the liquid in the wells, wash 3 times with PBST, 3 min each time, and pat dry.

[0057] (5) Color development: Add 100 μL of freshly prepared TMB color developer to each well and incubate in the dark for 30 min.

[0058] (6) Termination: Incubate for 1 h to terminate the reaction.

[0059] (7) Detection: Detect with an enzyme-labeled instrument at a wavelength of 450 nm.

[0060] Example 5

[0061] Prepare a monoclonal antibody against saxitoxin, including the following steps:

[0062] S1. Immunize mice. Take 3 8-week-old female BALB / c mice and immunize them with the purified immunizing antigen obtained in Example 1.

[0063] The immune antigen was mixed with an equal amount of Freund's complete adjuvant, and primary immunization was carried out by combined multi-point intraperitoneal and subcutaneous injection, with 50 μg of the immune antigen injected into each mouse. Two weeks after the primary immunization, the immune antigen was mixed with an equal amount of incomplete Freund's adjuvant for booster immunization, and the immunization dose and route were the same as those of the primary immunization. Booster immunization was carried out once every 2 weeks, and after a total of 3 booster immunizations, 50 μg of the antigen was directly injected intraperitoneally for booster immunization. On the 7th day after each immunization, the antibody production in the mice was detected by the ELISA detection method described in Example 4.

[0064] S2. Culture SP2 / 0 myeloma cells. Take out one vial of SP2 / 0 myeloma cells stored in liquid nitrogen, immediately transfer it to a 37°C constant temperature water bath, and gently shake the cryopreservation tube from time to time. Take it out when the cells melt to a semi-ice crystal state. Operate in a sterile environment, transfer the SP2 / 0 cells in the cryopreservation tube into a 50 mL sterile centrifuge tube, slowly add 10 mL of pre-warmed 1640 complete medium dropwise to the centrifuge tube, centrifuge at 1000 r / min for 5 min, and discard the supernatant. Gently disperse the cell mass, resuspend the cells with 5 mL of medium and transfer them to a T75 cell culture flask. Additionally, add 5 mL of medium, shake the cell flask in a "cross" direction, place it in a 5% CO2 cell culture incubator, and culture it at 37°C. Observe the cell state under a microscope, and when the density is about 80%, passage the SP2 / 0 cells.

[0065] S3. Prepare immune mouse spleen cells. On the 3rd day after booster immunization by intraperitoneal injection of 50 μg of the antigen, take the sera of 3 immune mice respectively, and perform serum ELISA detection by the method described in Example 4. Select the immune mouse with the highest serum ELISA titer. In a biosafety cabinet, use sterilized scissors and forceps to cut open the outer skin of the mouse, replace with a new set of sterilized scissors and forceps to cut open the abdominal cavity of the mouse, and then carefully take out the spleen with another set of sterilized scissors and forceps, and cut off the excess fat. Prepare a sterile 15 mL centrifuge tube, add 10 mL of DMEM medium, put the spleen into this centrifuge tube, and carefully discard the excess medium after the spleen is wetted. Then aspirate 10 mL of DMEM medium into a sterile petri dish, grind the spleen with a frosted glass slide to make a single cell suspension, filter it through a 200-mesh nylon mesh into a sterile centrifuge tube, add 30 mL of DMEM to a 50 mL sterile centrifuge tube, rinse the nylon mesh with a pipette, centrifuge the centrifuge tube containing the spleen cell suspension at 1500 rpm for 5 min, discard the supernatant, gently disperse the cell mass by hand, then resuspend it with 30 mL of DMEM medium and centrifuge again, and then discard the supernatant, gently disperse the cell mass by hand, and resuspend it with 10 mL of DMEM medium.

[0066] S4. Cell fusion. Centrifuge the well-grown SP2 / 0 cells in step S2 at 1000 rpm for 5 min and collect them in a 50 mL centrifuge tube. Gently disperse the SP2 / 0 cell clumps, resuspend them in 30 mL of DMEM medium, centrifuge again, then resuspend in 10 mL of DMEM medium. Then mix the spleen cell suspension with the SP2 / 0 cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, and gently disperse the cell clumps. Place it in a 37 °C water bath environment, pipette 1 mL of PEG1500 fusogen into the centrifuge tube, add 1 mL within 1 min. At this time, the cell state is red homogeneous quicksand-like, and when rotating the tube wall, it feels like frosted glass.

[0067] S5. Terminate fusion. Pipette 9 mL of DMEM medium to terminate fusion, which is divided into three stages. The first stage is to add 1 mL within the first 1 min, the second stage is to add 1 mL within 1 min, and the third stage is to add the remaining 7 mL of medium within 3 min. Then let it stand and stabilize in a 37 °C water bath for 5 min, and then centrifuge at 800 rpm for 5 min.

[0068] S6. Selective culture of fused cells. Resuspend the fused cells with HAT culture medium and add them to a pre-prepared 96-well culture plate containing feeder cells at 100 μL / well. Place it in a cell culture incubator with 5% CO2 and culture at 37 °C, setting NS-1 and spleen cells as controls. Replace 1 / 2 of the culture medium in the wells with freshly prepared HAT culture medium every 3 days. When all the control cells die, replace it with HT culture medium and continue culturing. Screen for positive clones by indirect ELISA 18 days after fusion, and the method is the same as that described in Example 4. Collect the supernatants of the wells with growing fused cells for detection, using VLP as the negative control and PBS as the blank control.

[0069] S7. Clone hybridoma cells by limiting dilution method. Adjust the density of hybridoma cells to 3 - 10 cells / mL and add them to a 96-well culture plate containing feeder cells, with 0.1 mL added to each well. When the cell colonies at the bottom of the wells are 1 - 2 mm in size, aspirate the supernatant of the wells where hybridoma cells grow. Detect the antibodies in the supernatant according to the antibody screening method. Transfer the cells in the wells with positive antibodies to a 24-well culture plate for expansion culture for 2 - 4 days. Repeat the above steps, and repeat the cloning of the expanded positive cells 2 - 3 times until the positive well rate of hybridoma cells reaches 100%, which is considered a monoclonal, that is, the successfully established cell line. Expand the selected positive monoclonal for culture and cryopreserve it according to the specification of 10 6 cells / tube.

[0070] S8. Preparation of ascites. The positive hybridoma cells obtained in step S7 are collected and inoculated into the peritoneal cavity of female BALB / c mice, 0.5 mL is injected into each mouse. Observe the growth status of the mice carefully. Ascites can be collected 10 days later when the abdomen of the mice becomes swollen. Collect the ascites of the mice into a centrifuge tube, centrifuge at 8000 r / min for 20 min, and aspirate the middle ascites layer to detect whether the ascites is successfully prepared by the ELISA detection method described in Example 4.

[0071] S9. Purification of the successfully prepared ascites. Purification is carried out by Protein G affinity chromatography. After the Protein G affinity chromatography column is equilibrated with 10 mmol / L PBS, the ascites is taken and passed through the column, and then the column is washed with 10 mmol / L PBS until its OD value is about 0, and eluted with 0.1 mol / L glycine hydrochloride solution. Collect the eluate, measure the OD values of each collection tube, and detect the antibody concentration using a BCA protein quantification kit.

[0072] Example 6

[0073] Establish a competitive ELISA detection method for STX

[0074] (1) Design the number of coated plates according to experimental needs and make marks on the strips.

[0075] (2) Dilute the saxitoxin-VLP conjugate with PBS coating solution to 1 μg / mL. After mixing, add it to the strips, 100 μL per well, and incubate overnight in a 4°C refrigerator.

[0076] (3) After coating, discard the coating solution, wash the plate 3 times, add 200 μL of blocking solution to each well, and incubate in a 37°C incubator for 1 h. Take out the enzyme-labeled plate, discard the internal liquid, and wash the plate 1 time.

[0077] (4) Take 1 μg / mL of STX as the starting concentration, and continuously dilute it with 1×PBS to obtain solutions of 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39 ng / mL, 100 μL per well. Then add 100 μL of the STX monoclonal antibody prepared in Example 5 to each well, and set up a blank control and a negative control. Then place the enzyme-labeled plate in a 37°C incubator for 1 h. Among them, the blank control is 200 μL of PBS, and the negative control is 100 μL of PBS + 100 μL of antibody.

[0078] (5) Take out the enzyme-labeled plate, discard the internal liquid, wash the plate 3 times, add 100 μL of diluted goat anti-mouse enzyme-labeled secondary antibody to each well, dilute it at a ratio of 1:10000 according to the instructions, and incubate in a 37°C incubator for 1 h.

[0079] (6) Take out the enzyme-linked immunosorbent assay (ELISA) plate, discard the internal liquid, wash the plate 4 times. First, add 100 μL of TMB chromogenic solution to each well, and incubate at 37 °C for 30 min.

[0080] (7) After color reading is completed, add 100 μL of 1 M HCl solution to each well to terminate the reaction. Immediately read the absorbance at a wavelength of 450 nm using an ELISA reader. The detection results of each well are shown in Table 1, and the prepared standard curve is shown in Figure 5 .

[0081] Table 1 Results of competitive ELISA

[0082]

[0083] Result analysis

[0084] I. Analysis of the hapten prepared

[0085] Since the conjugate contains both the absorbance group of STX and the absorbance group of the carrier protein, the maximum absorption wavelength of STX-VLP is between the maximum absorption wavelengths of STX and VLP. As Figure 3 can be seen, the STX standard (0.1 mg / mL) shows peaks at 234 nm and 265 nm, and its absorbance values are 1.17 and 0.68 respectively; VLP (1 mg / mL) shows peaks at 278 nm and 346 nm, and its absorbance values are 0.91 and 0.11 respectively; STX-VLP shows a peak at 266 nm, with an absorbance value of 0.15 and a concentration of 68.4 μg / mL. The maximum absorption wavelength of the hapten prepared in Example 1 is between STX and VLP, meeting the expectation. The antigen is successfully prepared and can be used for subsequent immune reactions.

[0086] II. Identification of monoclonal antibody against STX

[0087] (1) Titer identification

[0088] Using the STX-VLP conjugate prepared in Example 1 as the coating antigen, dilute it to 1 μg / mL with CBS buffer and add 100 μL to each well of a 96-well ELISA plate. Incubate overnight at 4°C. Discard the residual liquid, wash three times, and pat dry. Add 300 μL of blocking solution to each well of the 96-well ELISA plate and incubate at 37°C for 3 h. The blocked 96-well ELISA plate can be stored at 4°C for several weeks for later use. When measuring, discard the blocking solution, wash the plate three times, and pat dry. Add antibodies diluted at 1:4K, 1:8K, 1:16K, 1:32K, 1:64K, 1:128K, and 1:256K, 100 μL per well, and incubate at 37°C for 1 h. Take out the ELISA plate from the incubator, wash the plate and pat dry, add appropriately diluted enzyme-labeled goat anti-mouse secondary antibody, 100 μL per well, and incubate at 37°C for 45 min. Wash the plate and pat dry, add 100 μL of TMB substrate solution to each well, incubate at 37°C for 15 min, add 50 μL of stop solution to each well to terminate the reaction, measure the OD value of each well at a wavelength of 450 nm, and determine the titer of the prepared monoclonal antibody. The antibody titer is defined as the ratio of the OD450 of the sample detection well to the OD450 of the negative control well (P / N) ≥ 2.1. The results are shown in Table 2.

[0089] Table 2 Identification Results of Monoclonal Antibody Titers

[0090] Dilution factor OD value 4K 2.374 8K 1.961 16K 1.561 32K 0.851 64K 0.564 128K 0.369 256K 0.192 512K 0.091 Negative control 0.048 Positive control 3.731

[0091] As can be seen from Table 2, both the prepared monoclonal antibody against saxitoxin and the saxitoxin hapten are successful, and the antibody titer is 256K.

[0092] (2) SDS-PAGE Electrophoresis Identification

[0093] Purify the IgG antibody in mouse ascites with a Protein A FF affinity chromatography column. A protein peak can be eluted at pH 4.2, which is the antibody protein. Two protein bands were obtained by SDS-PAGE identification. One band is darker in color and the other is lighter, with sizes at 50 kD and 25 kD respectively, which is consistent with the actual sizes of the IgG heavy chain and light chain. The results are shown in Figure 4 .

[0094] III. Evaluate the STX Competitive ELISA Detection Method Established in Example VI

[0095] As can be seen from Table 1 and Figure 5 it can be seen that the competitive ELISA detection method shows good linear fitting in the range of 1.5 ng / mL to 25 ng / mL. After calculation, the fitting curve is determined to be y = -0.252ln(x) + 1.0096, indicating that this monoclonal antibody can specifically recognize saxitoxin.

[0096] IV. Evaluate the Accuracy of the STX Competitive ELISA Detection Method Established in Example VI

[0097] Dilute the STX standard product with 1×PBS to a solution with a concentration of 50 ng / mL, denoted as the spiked sample; the negative sample is 1×PBS. Detect the OD values of the 50 ng / mL negative sample, 50 ng / mL standard product, and spiked sample on an enzyme-labeled instrument at a wavelength of 450 nm, and calculate the recovery rate according to the formula: P = measured value of the spiked sample / spiked amount × 100%. The results are shown in Table 3.

[0098] Table 3 Recovery Rate Detection

[0099]

[0100] As can be seen from Table 3, the recovery rate is between 85 - 115%, meeting the requirements specified in the Chinese Pharmacopoeia.

[0101] Therefore, through the synthetic saxitoxin artificial hapten, the present invention successfully prepared a monoclonal antibody against saxitoxin. The prepared monoclonal antibody against saxitoxin has the advantages of high purity, strong specificity, and high titer, can avoid serological cross-reactions between different cells and microbial species or strains, thus greatly improving the detection specificity and sensitivity of saxitoxin, and has good application prospects; the preparation methods of the saxitoxin hapten and monoclonal antibody are safe, effective, non-toxic and harmless, and do not require special instrument equipment and harsh and complex reaction conditions.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A saxitoxin hapten, characterized in that, Using VLP as a carrier, the saxitoxin hapten is STX-VLP, and its structural formula is: .

2. A preparation method of the saxitoxin hapten as described in claim 1, characterized in that, The steps are as follows: (1) Dissolve STX in HCl solution, and dropwise add sodium nitrite solution at 4°C, and monitor the reaction process with starch potassium iodide test paper. After the reaction is complete, obtain solution A; (2) Dissolve the VLP vector in 1×PBS at 4°C, denoted as solution B; (3) Dropwise add solution A to solution B under certain conditions. After reacting for a certain time, add 1×PBS to obtain the STX-VLP hapten stock solution; (4) Dialyze and purify the STX-VLP hapten stock solution to obtain the pure STX-VLP hapten.

3. The preparation method of a saxitoxin hapten according to claim 2, characterized in that: The monitoring of the reaction process with starch potassium iodide test paper in step (1) means stopping adding sodium nitrite solution when the test paper turns blue-gray, and continuing to add sodium nitrite solution when the test paper shows no change.

4. The preparation method of a saxitoxin hapten according to claim 2, characterized in that: The concentration of the HCl solution in step (1) is 0.1 M, the concentration of the sodium nitrite solution is 0.01 M, the reaction condition is rotary mixing reaction at 4°C, the molar volume ratio of STX to the HCl solution is 10 μmol:1 mL. Before the dropwise addition reaction, both the STX solution and the sodium nitrite solution need to be pre-cooled to 4°C.

5. The preparation method of a saxitoxin hapten according to claim 2, characterized in that: The concentration of solution B in step (2) is 2 mg / mL.

6. The preparation method of a saxitoxin hapten according to claim 2, wherein: The certain conditions in step (3) refer to 4°C, continuous stirring, pH 9-10. The reaction for a certain time refers to reacting for 2-4 h. The volume ratio of the added 1×PBS to the reaction solution volume is 1:

1.

7. The preparation method of a saxitoxin hapten according to claim 2, characterized in that, The dialysis and purification conditions in step (4) are: the dialysis solution is 1×PBS buffer solution, dialysis at 4°C for 1-3 d, and change the buffer solution 3 times a day, with an interval of no less than 4 h each time.

8. A method for preparing a monoclonal antibody against saxitoxin using the hapten described in claim 1, characterized in that, The steps are as follows: S1. Prepare the saxitoxin hapten by the method described in any one of claims 2-7; S2. Immunize mice with the synthesized hapten as an immunogen; S3. Fuse the spleen cells of the immunized mice with mouse myeloma cells; S4. Screen to obtain positive hybridoma cells; S5. Inoculate the positive hybridoma cells into mice, collect and purify the ascites antibody to obtain the monoclonal antibody against saxitoxin.

9. A monoclonal antibody against saxitoxin prepared by the method described in claim 8.

10. Use of a monoclonal antibody against saxitoxin as described in claim 9 in the preparation of an ELISA kit for detecting STX.

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