CN116675696A - Saxitoxin monoclonal antibody, and preparation method and application thereof - Google Patents

Abstract

The application discloses a saxitoxin monoclonal antibody, a preparation method and application thereof, and a saxitoxin hapten which takes VLP as a carrier, wherein the saxitoxin hapten has the structural formula:the method successfully prepares the monoclonal antibody of the saxitoxin through the synthesized artificial hapten of the saxitoxin, and the prepared saxitoxin monoclonal antibody has the advantages of high purity, strong specificity and high titer, and can avoid different cellsAnd the serological cross reaction between microorganism species or strains, thereby greatly improving the detection specificity and sensitivity of the saxitoxin and having good application prospect; the preparation method of the saxitoxin hapten and the monoclonal antibody is safe, effective, nontoxic and harmless, and does not need special instruments and equipment and harsh and complex reaction conditions.

Description

Saxitoxin monoclonal antibody, and preparation method and application thereof

Technical Field

The application relates to the technical field of saxitoxin detection, in particular to a saxitoxin monoclonal antibody, a preparation method and application thereof.

Background

Saxitoxin (STX), a derivative of tetrahydropurine, is one of the most toxic marine toxins known to have a high mortality rate, and has a lethal dose of 540-1000 μg for adults, which is one of the main toxins of shellfish neuroparalysis poisoning (Paralytic Shellfish Poisoning, PSP).

The saxifrage is benthic shellfish which can live in fresh water and sea water, has delicious meat and rich nutrition, and is a seafood food popular with the masses. However, due to the high mortality STX that may be contained therein, it is important to detect saxitoxin content.

Previously, the commonly used saxitoxin detection method was a biological and chemical analysis method. The bioassay method is a detection method based on the reaction of cells, tissues and organs after toxin action, and comprises a mouse biotoxicity test, a starfish gastrointestinal tract operation test and the like. The biological analysis method has the problems of complex operation, long time consumption, high toxicity, low utilization rate of experimental animals and the like, and is gradually replaced by the chemical analysis method. The chemical analysis method mainly comprises a high performance liquid chromatography-mass spectrometry (HPLC), a gas chromatography-mass spectrometry (GC-MS), a liquid chromatography-mass spectrometry (LC-MS) and the like, and has the defects of high accuracy, good repeatability, high cost of one-time detection, complex operation, expensive equipment, long response time and the like.

In recent years, with the development of molecular biology, the immunological method for detecting the content of saxitoxin gradually replaces biological analysis and chemical analysis methods, and is a common detection method. Immunological methods are realized by utilizing the interaction between specific antibody molecules, and include ELISA (enzyme-linked immunosorbent assay) and immunofluorescence techniques, immunoradiometric techniques, immunoenzymatic techniques, immunocolloidal gold techniques and the like. The ELISA technology is the most widely used saxitoxin detection method at present, and the method recognizes saxitoxin through a specific antibody, combines the toxin with an antigen, and converts the toxin into an optical signal by utilizing the catalysis of auxiliary enzyme, so that the saxitoxin detection is realized, and the method has the advantages of simplicity in operation, short time, high sensitivity, good accuracy and the like.

However, because the molecular weight of saxitoxin is small and is less than 300, the difficulty in preparing monoclonal antibodies is high, so that the antibodies used for ELISA detection of saxitoxin are almost all polyclonal antibodies, the polyclonal antibodies have low titer, cross reaction is easy to occur, and the specificity, the specificity and the sensitivity of ELISA detection are severely limited.

Disclosure of Invention

The application aims to provide a saxitoxin monoclonal antibody, a preparation method and application thereof, so as to further improve the specificity, the specificity and the sensitivity of an ELISA saxitoxin detection kit, and solve the problem that the ELISA saxitoxin detection kit based on polyclonal antibodies is easy to cross react.

In order to achieve the above purpose, the application provides a saxitoxin monoclonal antibody, a preparation method and application thereof, and a saxitoxin hapten which takes VLP as a carrier, wherein the saxitoxin hapten has the structural formula:

the preparation method of the saxitoxin hapten comprises the following steps:

(1) Dissolving STX in HCl solution, dropwise adding sodium nitrite solution at low temperature, monitoring the reaction process by using starch potassium iodide test paper, and obtaining solution A after the reaction is complete;

(2) VLP carrier was dissolved in 1 XPBS under low temperature conditions and designated as solution B;

(3) Dropwise adding the solution A into the solution B under a certain condition, reacting for a certain time, and adding 1 XPBS to obtain STX-VLP hapten stock solution;

(4) And dialyzing and purifying the STX-VLP hapten stock solution to obtain a pure STX-VLP hapten product.

Preferably, the step (1) of monitoring the reaction process by using the starch potassium iodide test paper means stopping dripping the sodium nitrite solution when the test paper becomes blue gray, and continuously dripping the sodium nitrite solution when the test paper is unchanged.

Preferably, the concentration of the HCl solution in the step (1) is 0.1M, the concentration of the sodium nitrite solution is 0.01M, the reaction condition is 4 ℃ rotary mixing reaction, the mass volume ratio of the STX solution to the HCl solution is 10 mu mol/1 mL, and before the dropwise addition reaction, the STX solution and the sodium nitrite solution are both required to be precooled to 4 ℃.

Preferably, the low temperature in the step (2) is 4 ℃, and the concentration of the solution B is 2mg/mL.

Preferably, in the step (3), certain conditions refer to 4 ℃ and continuous stirring, the pH is 9-10, certain reaction time refers to 2-4 hours, and the volume ratio of the added 1 XPBS to the reaction solution is 1:1.

Preferably, the dialysis purification conditions in step (4) are: the dialysate is 1 XPBS buffer, dialyzed for 1-3d at 4deg.C, and replaced 3 times per day with an interval of no less than 4 hr.

A method for preparing saxitoxin monoclonal antibody by using the hapten comprises the following steps:

s1, preparing saxitoxin hapten by adopting the method;

s2, taking the synthesized hapten as an immunogen to immunize a mouse;

s3, taking spleen cells of the immunized mice to fuse with myeloma cells of the mice;

s4, screening to obtain hybridoma cells with positive reaction;

s5, inoculating the positive hybridoma cells into a mouse body, and collecting and purifying the ascites antibody to obtain the monoclonal antibody of the anti-saxitoxin.

A saxitoxin monoclonal antibody prepared by the above method.

An application of the saxitoxin monoclonal antibody in preparing an ELISA kit for detecting STX.

Therefore, the saxitoxin monoclonal antibody, the preparation method and the application thereof provided by the application have the following technical effects:

1. the monoclonal antibody of the saxitoxin is successfully prepared through the synthesized artificial hapten of the saxitoxin, and the prepared monoclonal antibody has the advantages of high titer, strong specificity and high sensitivity, can effectively avoid cross reaction, and has good application prospect in the inspection of the saxitoxin;

2. the preparation method of the saxitoxin hapten and the saxitoxin monoclonal antibody provided by the application is safe, effective, nontoxic and harmless, and does not need special instruments and equipment and harsh and complex reaction conditions.

The technical scheme of the application is further described in detail through the drawings and the embodiments.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments of the present application will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic illustration of a preparation scheme for a saxitoxin conjugated hapten of the present application;

FIG. 2 is a graph showing the effect of STX-VLP hapten prepared according to the first embodiment of the present application;

FIG. 3 shows the detection result of absorption wavelength of STX-VLP hapten prepared in example I of the present application;

FIG. 4 shows the SDS-PAGE detection result of the monoclonal antibody prepared in example five of the present application;

FIG. 5 is a standard curve of the competitive ELISA for saxitoxin detection in example six of the application.

Detailed Description

The technical scheme of the application is further described below through the attached drawings and the embodiments.

In order to make the objects, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present application will be clearly and completely described below through the accompanying drawings and examples. The following detailed description is of embodiments, and is intended to provide further details of the application. Unless defined otherwise, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Saxitoxin STX standard (chromatographic purity ∈95%) was purchased from beijing ibpurus technology development limited; VLP superantigen proteins were purchased from biotechnology (Shanghai) limited; mouse myeloma cells SP2/0 were purchased from the cell bank of the national academy of sciences; female BALB/c mice were purchased from Shanghai Laek laboratory animal Co., ltd; the chemical reagents for preparing PBS and CBS are all purchased from Sigma company; the BCA kit is purchased from the Simer Feichi technology company, and the enzyme-labeled secondary antibody is purchased from the Abcam company; UV-2450 continuous spectrophotometry is manufactured by Shimadzu corporation, and Thermo Multiskan FC enzyme-labeled instrument is manufactured by Simer Feishul technology corporation; the dialysis bag was 3500D dialysis bag purchased from beijing solebao technologies.

Example 1

A hapten (STX-VLP) of Saxitoxin (STX) was prepared as follows:

(1) STX purchased was dissolved in dd water as described, 10. Mu. Mol of STX was accurately aspirated, dissolved in 1mL of 0.1M HCl, and cooled to 4 ℃. Then, a pre-cooled 0.01M sodium nitrite solution (ready-to-use) at 4℃was gradually added dropwise, the reaction was monitored with a starch potassium iodide test paper, and the dropwise addition was stopped when the test paper became blue-gray. After rotating and mixing reaction for 30min at 4 ℃, detecting again by using starch potassium iodide test paper, dropwise adding sodium nitrite solution dropwise without changing the test paper, keeping an acidic condition, stopping dropwise adding when the test paper becomes blue-gray, and continuing the reaction for 1h to obtain solution A.

(2) 1mg of VLP was weighed out accurately, added to 1mL of 1 XPBS at 4℃and stirred to dissolve completely, designated as solution B.

(3) Dropwise adding the solution A into the solution B at the temperature of 4 ℃ under the condition of continuous stirring, simultaneously adjusting the pH with a 1M NaOH solution, keeping the pH at 9, stirring for 3 hours at the temperature of 4 ℃, and finally, fixing the volume of the solution to 3mL by using 1 XPBS to obtain the STX-VLP hapten stock solution.

(4) And (3) filling the STX-VLP hapten stock solution prepared in the step (3) into a dialysis bag, dialyzing with 1 XPBS buffer solution at 4 ℃ for 2d (3 times of buffer solution is replaced every day, and each time is separated by not less than 4 h), so as to obtain purified STX-VLP hapten, wherein the preparation process is shown in figure 1, and the effect diagram of the prepared STX-VLP hapten is shown in figure 2.

(5) Detecting the purified STX-VLP hapten prepared in the step (4) by using an ultraviolet spectrophotometer, confirming that a small molecule absorption peak and a VLP absorption peak are not overlapped before detection, and measuring the characteristic absorption wavelength of carrier protein VLP, wherein the concentration is 1.0mg/mL; measuring the characteristic absorption wavelength of STX, and the concentration is 0.1mg/mL; the characteristic absorption wavelength of STX-VLP antigen was measured at a concentration of 0.1mg/mL, and the results are shown in FIG. 3.

Example two

An STX-VLP hapten was prepared in exactly the same way as in example one, except that the pH in step (3) was 10, the reaction time was 4h, and the dialysis was 3d in step (4).

Example III

An STX-VLP hapten was prepared in exactly the same way as in example one, except that the pH in step (3) was 10, the reaction time was 2h, and the dialysis was 1d in step (4).

Example IV

Mouse serum titer detection ELISA method:

(1) Coating: the dilution was coated with STX-VLP antigen (50 ng per well), 100. Mu.L/well, refrigerator overnight at 4 ℃ (about 16-18 h); the liquid in the wells was discarded, washed 4 times with PBST, 3 min/time, and patted dry.

(2) Closing: add 5% PBS-BSA blocking solution, 300. Mu.L/well, incubate at 37℃for 2h, discard the intra-well liquid, wash 4 times with PBST, 3 min/time, and pat dry.

(3) Sample adding: serum was diluted with antibody dilution at a multiple ratio of 100. Mu.L/well, incubated at 37℃for 1h, the intra-well liquid was discarded, PBST was washed 3 times, 3 min/time, and the wells were pat dried.

(4) And (2) secondary antibody: HRP-goat anti-mouse IgG was diluted according to the dilution fold written on the reagent instructions, added to each well at 100 μl/well, incubated for 1h at 37 ℃, the liquid in the well was discarded, PBST washed 3 times, 3 min/time, and patted dry.

(5) Color development: 100 mu L of TMB color reagent is added to each hole, and the mixture is incubated for 30min in a dark place.

(6) And (3) terminating: incubation for lh, the reaction was terminated.

(7) And (3) detection: and (5) detecting the wavelength of 450nm by using an enzyme-labeled instrument.

Example five

Preparing a monoclonal antibody against saxitoxin, comprising the steps of:

s1, immunization of mice. 3 female BALB/c mice of 8 weeks old were immunized with the purified immunizing antigen obtained in example one.

The immune antigen is mixed with equivalent Freund's complete adjuvant, primary immunization is carried out by adopting a intraperitoneal and subcutaneous combined multipoint injection mode, and 50 mug of the immune antigen is injected into each mouse. After 2 weeks of primary immunization, boost was performed with the same amount of Freund's complete adjuvant mixed with the immunization antigen at the same dose and route as the primary immunization. Boosting was performed 1 time every 2 weeks, and after 3 times total boosting, 50 μg antigen was directly injected intraperitoneally for boosting. Antibody production in mice was detected by ELISA detection as described in example IV at 7d after each immunization.

S2, culturing SP2/0 myeloma cells. One SP2/0 myeloma cell frozen in liquid nitrogen is taken out, immediately transferred into a constant temperature water bath kettle at 37 ℃, the frozen storage tube is gently rocked from time to time, and taken out when the cell is melted to a semi-ice crystal state. And (3) operating in a sterile environment, transferring SP2/0 cells in the cryopreservation tube into a 50mL sterile centrifuge tube, taking 10mL of preheated 1640 complete culture medium, slowly dripping into the centrifuge tube, centrifuging at 1000r/min for 5min, and discarding the supernatant. The cell pellet was gently broken up, 5mL of medium was taken to resuspend the cells and transferred to T75 cell flasks. In addition, 5mL of culture medium is added, and the cells are rocked in the cross directionBottle, put in 5% CO ₂ The cells were cultured in a cell incubator at 37 ℃. The cell status was observed under a microscope, and SP2/0 cells were subcultured at a density of about 80%.

S3, preparing spleen cells of the immunized mice. On day 3 after the boost immunization by intraperitoneal injection of 50 μg antigen, 3 immunized mice were individually serum-tested by serum ELISA using the method described in example four, the immunized mice with the highest serum ELISA test titers were selected, the outer skin of the mice were cut off in a biosafety cabinet using sterilized scissors and forceps, the abdominal cavity of the mice was cut off by replacing a new set of sterilized scissors forceps, the spleen was carefully removed by a set of sterilized scissors and forceps, and the excess fat was cut off. A sterile 15mL centrifuge tube was prepared, 10mL DMEM medium was added, the spleen was placed in the tube, and after wetting the spleen, the excess medium was carefully discarded. Then 10mL of DMEM culture medium is sucked and placed in a sterile plate, the spleen is ground by a ground glass plate to prepare single cell suspension, the single cell suspension is filtered into a sterile centrifuge tube through a 200-mesh nylon net, 30mL of DMEM is added into a 50mL sterile centrifuge tube, the nylon net is washed by a suction tube, the centrifuge tube filled with the spleen cell suspension is centrifuged at 1500rpm for 5min, the supernatant is discarded, the cell mass is gently dispersed by hands, 30mL of DMEM culture medium is added for re-suspension, the supernatant is discarded, the cell mass is gently dispersed by hands, and 10mL of DMEM culture medium is added for re-suspension.

S4, cell fusion. 1000rpm, centrifuging and collecting the SP2/0 cells well grown in the step S2 in a 50mL centrifuge tube for 5min, lightly scattering SP2/0 cell mass, adding 30mL of DMEM culture medium for resuspension, centrifuging once again, adding 10mL of DMEM culture medium for resuspension, mixing spleen cell suspension with SP2/0 cell suspension, centrifuging at 1000rpm for 5min, discarding the supernatant, and lightly scattering cell mass. Placing in a 37 ℃ water bath environment, sucking 1mL PEG1500 fusion agent, dripping into a centrifuge tube, and adding 1mL within 1min, wherein the cell state is red homogeneous quicksand, and the rotating tube wall feels like frosted glass.

S5, terminating the fusion. 9mL of DMEM medium was aspirated to terminate the fusion, and the fusion was divided into three phases. 1mL is added dropwise in the first stage in the first 1min, 1mL is added dropwise in the second stage in the first 1min, and the rest 7mL of culture medium is added dropwise in the third stage in the third 3 min. Then, after standing in a water bath at 37℃for 5min, the mixture was centrifuged at 800rpm for 5min.

S6, selective culture of fusion cells. The HAT medium resuspended confluent cells were added to pre-prepared 96-well plates containing feeder cells at 100. Mu.L/well. Placing 5% CO ₂ In the cell culture chamber of (C), at 37℃and with NS-1 and spleen cells as controls. 1/2 of the culture medium in the wells was replaced with fresh HAT medium every 3 days, and after all of the control cells died, HT medium was replaced for continued culture. Positive clones were screened for 18d after fusion by indirect ELISA in the same manner as described in example four. Supernatants from fused cell growth wells were collected separately and tested using VLP as negative control and PBS as blank control.

S7, cloning hybridoma cells by a limiting dilution method. Hybridoma cell densities were adjusted to 3-10/mL and added to 96-well culture plates containing feeder cells at 0.1mL per well. When the cell colony at the bottom of the well is 1-2mm in size, the supernatant of the growing well of the hybridoma cell is sucked out. Antibodies in the supernatant were detected by antibody screening methods. The antibody positive in-well cells were transferred to a 24-well plate for 2-4 days of expansion culture. Repeating the above steps, cloning the positive cells after the expansion culture for 2-3 times again until the positive porosity of the hybridoma cells reaches 100%, namely, the positive cells are regarded as monoclonal, namely, the cell strain with successful strain establishment. Enlarging culture of the screened positive monoclonal according to 10 ⁶ The individual cell/tube format was frozen.

S8, preparing ascites. Collecting positive hybridoma cells obtained in the step S7, inoculating the positive hybridoma cells to abdominal cavities of female BALB/c mice, injecting 0.5mL of the positive hybridoma cells into each mouse, observing the growth state of the mice, collecting ascites after 10 days when the abdomen of the mice is enlarged, collecting the ascites of the mice, centrifuging the ascites of the mice in a centrifuge tube at 8000r/min for 20min, and sucking an intermediate abdominal water layer to detect whether the ascites is successfully prepared by the ELISA detection method described in the fourth embodiment.

S9, purifying and preparing the successful ascites. Purifying by protein G affinity chromatography, balancing the column with 10mmol/L PBS, taking ascites, passing through the column, washing the column with 10mmol/L PBS until the OD value is about 0, eluting with 0.1mol/L glycine-hydrochloric acid solution, collecting eluate, determining the OD value of each collecting tube, and detecting antibody concentration by using BCA protein quantification kit.

Example six

Competitive ELISA detection method for establishing STX

(1) The number of the coated plates is designed according to the experiment requirement, and marks are made on the battens

(2) The saxitoxin-VLP conjugate was diluted to 1. Mu.g/mL with PBS coating, mixed and added to the strip at 100. Mu.L per well overnight at 4 ℃.

(3) After the coating, the coating solution was discarded, the plate was washed 3 times, 200. Mu.L of a blocking solution was added to each well, and the culture was performed in a incubator at 37℃for 1 hour. And taking out the ELISA plate, discarding the internal liquid, and washing the plate for 1 time.

(4) Using STX of 1. Mu.g/mL as the initial concentration, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39ng/mL solutions of 100. Mu.L/well were obtained by continuous dilution with 1 XPBS, and then 100. Mu.L of the STX monoclonal antibody prepared in example five was added to each well, and a blank control and a negative control were set, and then the ELISA plate was incubated at 37℃in an incubator for 1 hour. Wherein, the blank control is 200. Mu.L PBS, and the negative control is 100. Mu.L LPBS+100. Mu.L antibody.

(5) Taking out the ELISA plate, discarding the inner liquid, washing the plate for 3 times, adding 100 mu L of diluted goat anti-mouse ELISA secondary antibody into each hole, diluting 1:10000 according to the proportion of the instruction, and culturing for 1h in a 37 ℃ incubator.

(6) The ELISA plate was removed, the inner solution was discarded, the plate was washed 4 times, 100. Mu.L of TMB developing solution was added to each well, and incubated at 37℃for 30min.

(7) After the color reading was completed, 100. Mu.L of 1M HCl solution was added to each well to terminate the reaction. Immediately read at the wavelength of 450nm of the microplate reader. The test results of each well are shown in Table 1, and the prepared standard curve is shown in FIG. 5.

TABLE 1 Competition ELISA results

Analysis of results

1. Analysis of the hapten prepared

The maximum absorption wavelength of the STX-VLP is between the maximum absorption wavelengths of the STX and VLP because the conjugate contains both the light absorbing group of the STX and the light absorbing group of the carrier protein. As can be seen from FIG. 3, the STX standard (0.1 mg/mL) exhibited peaks at 234nm and 265nm, and the absorbance was 1.17 and 0.68, respectively; VLPs (1 mg/mL) peak at 278nm and at 346nm, with absorbance values of 0.91 and 0.11, respectively; STX-VLPs appeared to peak at 266nm, absorbance at 0.15 and concentration at 68.4. Mu.g/mL. The hapten prepared in example one has the maximum absorption wavelength between STX and VLP, and is expected to be successfully prepared and can be used for subsequent immune reaction.

2. Identification of monoclonal antibodies against STX

(1) Potency identification

The STX-VLP conjugate prepared in example one was used as coating antigen, diluted to 1. Mu.g/mL with CBS buffer and added to 96-well ELISA plates at 100. Mu.L per well overnight at 4 ℃. Discarding residual liquid, washing for three times, beating, adding 300 mu L of sealing liquid into each hole of the 96-hole ELISA plate, incubating for 3h at 37 ℃, and preserving the sealed 96-hole ELISA plate at 4 ℃ for several weeks for later use. During measurement, removing the sealing solution, washing the plate for three times, beating, adding 1:4K,1:8K,1:16K,1:32K,1:64K,1:128K and 1:256K to dilute the antibody, and incubating for 1h at 37 ℃ in 100 mu L of each well; taking out the ELISA plate from the incubator, washing the plate, beating to dry, adding the ELISA anti-mouse secondary antibody diluted properly, incubating for 45min at 37 ℃ at 100 mu L per hole; washing the plate, beating to dry, adding 100 mu L of TMB substrate solution into each well, incubating for 15min at 37 ℃, adding 50 mu L of stop solution into each well to stop the reaction, measuring the OD value of each well at the wavelength of 450nm, and determining the titer of the prepared monoclonal antibody, wherein the antibody titer is defined as the ratio (P/N) of the OD450 value of the sample detection well to the OD450 value of the negative control well to be more than or equal to 2.1, and the result is shown in Table 2.

TABLE 2 monoclonal antibody titre identification results

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

As can be seen from Table 2, both the monoclonal antibodies raised against saxitoxin and the saxitoxin hapten were successful, with an antibody titer of 256K.

(2) SDS-PAGE electrophoresis identification

The IgG antibody in the ascites of the mouse is purified by a ProteinAFF affinity chromatographic column, and a protein peak can be washed out at pH4.2, namely the antibody protein. Two protein bands are obtained through SDS-PAGE identification, one of the two protein bands is darker, the other protein band is lighter, the sizes of the two protein bands are respectively 50kD and 25kD, the two protein bands are consistent with the actual sizes of the heavy chain and the light chain of IgG, and the result is shown in figure 4.

3. Evaluation of the STX competitive ELISA assay established in example six

As can be seen from table 1 and fig. 5, the competitive ELISA assay showed a better linear fit in the range of 1.5ng/mL to 25ng/mL, and the calculated fit curve was determined to be y= -0.252ln (x) +1.0096, indicating that the monoclonal antibody can specifically recognize saxitoxin.

4. Evaluation of accuracy of the STX competitive ELISA assay established in example six

Diluting the STX standard substance with 1 XPBS to obtain a solution with the concentration of 50ng/mL, and marking the solution as a marked sample; the negative samples were 1 XPBS. Detecting OD values of a 50ng/mL negative sample, a 50ng/mL standard substance and a labeled sample on an enzyme labeling instrument at a wavelength of 450nm respectively, and according to the formula: p=benchmarked measurement value/benchmarked amount×100%, and recovery was calculated, and the results are shown in table 3.

TABLE 3 recovery rate detection

As can be seen from Table 3, the recovery rate is between 85 and 115 percent, which meets the requirements of Chinese pharmacopoeia regulations.

Therefore, the prepared saxitoxin monoclonal antibody has the advantages of high purity, strong specificity and high titer, and can avoid the serological cross reaction between different cells and microorganism species or strains, thereby greatly improving the detection specificity and sensitivity of the saxitoxin and having good application prospect; the preparation method of the saxitoxin hapten and the monoclonal antibody is safe, effective, nontoxic and harmless, and does not need special instruments and equipment and harsh and complex reaction conditions.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application and not for limiting it, and although the present application has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the application can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the application.

Claims (10)

1. A saxitoxin hapten, characterized in that VLP is used as a carrier, and the saxitoxin hapten has the structural formula:

2. a method of preparing a saxitoxin hapten according to claim 1, comprising the steps of:

(1) Dissolving STX in HCl solution, dropwise adding sodium nitrite solution at low temperature, monitoring the reaction process by using starch potassium iodide test paper, and obtaining solution A after the reaction is complete;

(2) VLP carrier was dissolved in 1 XPBS under low temperature conditions and designated as solution B;

(3) Dropwise adding the solution A into the solution B under a certain condition, reacting for a certain time, and adding 1 XPBS to obtain STX-VLP hapten stock solution;

(4) And dialyzing and purifying the STX-VLP hapten stock solution to obtain a pure STX-VLP hapten product.

3. The method for preparing saxitoxin hapten according to claim 2, wherein the method comprises the following steps: the step (1) of monitoring the reaction process by using the starch potassium iodide test paper means that the dropping of the sodium nitrite solution is stopped when the test paper becomes blue gray, and the dropping of the sodium nitrite solution is continued when the test paper is unchanged.

4. The method for preparing saxitoxin hapten according to claim 2, wherein the method comprises the following steps: the concentration of the HCl solution in the step (1) is 0.1M, the concentration of the sodium nitrite solution is 0.01M, the reaction condition is 4 ℃ rotary mixing reaction, the mass volume ratio of the STX solution to the HCl solution is 10 mu mol and is 1mL, and before the dropwise addition reaction, the STX solution and the sodium nitrite solution are both required to be precooled to 4 ℃.

5. The method for preparing saxitoxin hapten according to claim 2, wherein the method comprises the following steps: the low temperature in the step (2) is 4 ℃, and the concentration of the solution B is 2mg/mL.

6. The method for preparing saxitoxin hapten according to claim 2, wherein the method comprises the following steps: the certain condition in the step (3) means that the temperature is 4 ℃, the stirring is continuous, the pH is 9-10, the reaction is carried out for a certain time means that the reaction is carried out for 2-4 hours, and the volume ratio of the added 1 XPBS to the reaction liquid is 1:1.

7. The method for preparing saxitoxin hapten according to claim 2, wherein the dialysis and purification conditions in step (4) are as follows: the dialysate is 1 XPBS buffer, dialyzed for 1-3d at 4deg.C, and replaced 3 times per day with an interval of no less than 4 hr.

8. A method for preparing saxitoxin monoclonal antibodies using the hapten of claim 1, comprising the steps of:

s1, preparing a saxitoxin hapten by adopting the method as set forth in any one of claims 2 to 7;

s2, taking the synthesized hapten as an immunogen to immunize a mouse;

s3, taking spleen cells of the immunized mice to fuse with myeloma cells of the mice;

s4, screening to obtain hybridoma cells with positive reaction;

s5, inoculating the positive hybridoma cells into a mouse body, and collecting and purifying the ascites antibody to obtain the monoclonal antibody of the anti-saxitoxin.

9. A saxitoxin monoclonal antibody produced by the method of claim 8.

10. Use of a saxitoxin monoclonal antibody according to claim 9 for the preparation of an ELISA kit for detection of STX.