CN115746015A - Shrimp-magnolia scallop toxin hapten, artificial antigen, antibody and preparation method and application thereof - Google Patents
Shrimp-magnolia scallop toxin hapten, artificial antigen, antibody and preparation method and application thereof Download PDFInfo
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- CN115746015A CN115746015A CN202211300672.5A CN202211300672A CN115746015A CN 115746015 A CN115746015 A CN 115746015A CN 202211300672 A CN202211300672 A CN 202211300672A CN 115746015 A CN115746015 A CN 115746015A
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- patinopecten yessoensis
- artificial antigen
- hapten
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a scallop toxin hapten, an artificial antigen and an antibody of a shrimp magnolia flower as well as a preparation method and application thereof. The structural formula of the patinopecten yessoensis toxin hapten is shown as a formula (I), and the artificial antigen and the antibody for detecting the patinopecten yessoensis toxin are prepared by applying the hapten. The artificial antigen and antibody of Japanese scallop provided by the inventionThe detection sensitivity of the enzyme linked immunosorbent assay kit prepared by the body to the toxin of the Japanese scallop is 0.001ng/mL, IC 50 0.11ng/mL, and a linear range of 0.001-0.75 ng/mL. The enzyme linked immunosorbent assay kit is convenient to use, low in detection cost, rapid, efficient and accurate in detection method, suitable for field control of the toxin residues of the scallop in marine algae and shellfish tissues and screening of a large number of samples, and has good application prospects.
Description
Technical Field
The invention relates to the technical field of food detection, and in particular relates to a scallop toxin hapten, an artificial antigen and an antibody as well as a preparation method and application thereof.
Background
Patinopecten Yessotoxin (YTX) and derivatives thereof are fat-soluble polycyclic polyether compounds containing 2 sulfonyl groups, and the chemical structures of the compounds are very similar, wherein some compounds are generated by marine dinoflagellate, and part of the compounds are obtained by metabolic conversion in vivo of filter-feeding shellfish. Presently, marine organisms such as shellfish containing YTXs in vivo are distributed in the waters around China, japan, new Zealand, norway, italy, canada, and the United states. The biological toxicology research shows that YTX is a cytotoxin with strong toxicity, the acute lethality rate is very high, the lethal dose of mice is about 100 mu g/kg, and the semilethal dose (LC 50) is respectively different along with the difference of organisms, and the value is between 80 and 750000 mu g/kg. Relevant laws and regulations are formulated in the European Union, the edible safety concentration of YTX is regulated to be 1mg/kg of shellfish meat, and the safety limit is regulated to be 3.75mg/kg of shellfish meat in 2013. YTX can reduce the cAMP concentration in cells, induce and activate human neuroblast cancer cell lines, liver cells, liver cancer cells, hela cells and mouse myoblast cell line apoptosis-related caspase family proteins, and is considered as a tumor promoter.
At present, the literature of the patinopecten yessoensis toxin determination method is not reported a lot, and the method comprises a mouse biological method, a Thin Layer Chromatography (TLC), a High Performance Liquid Chromatography (HPLC) and a chromatography-mass spectrometry (LC-MS/MS). The mouse biological method is a traditional method for detecting shellfish toxins, and the mouse method is widely used for detecting marine toxins in China in 1994, can obtain the toxicity of a detected substance, but lacks specificity for YTX detection, has low detection sensitivity, and causes false positive of a test result due to interference of other marine toxins in the process of extracting YTX toxins. From the ethical aspect of animals, the european union has now abandoned the mouse method for the determination of marine toxins. The TLC method is mainly used for separating and purifying YTX toxin and has low detection sensitivity. The HPLC-FLD method has problems in sensitivity and selectivity with fluorescence detection due to co-effluent background interference. The selectivity and sensitivity can be improved by adopting a chromatographic mass spectrometry (LC-MS/MS) for detection, but equipment is expensive and has high requirements on professional technicians, and the method is determined as a standard method for quantitatively detecting YTX in European Union in 2011. The immunoassay detection technology is based on the principle of antigen-antibody reaction, utilizes a method for detecting toxicants by competitively combining the toxicants and labeled toxicants with antibodies, and can be used for screening tests of certain toxicants. The immunoassay method has the characteristics of simple and convenient sample pretreatment, simple operation, rapidness, high sensitivity, high flux and the like, and has wide application prospect in the field of food safety. Therefore, in order to ensure the food safety and the development of export trade, it is necessary to establish a qualitative and quantitative method which is accurate, reliable, highly sensitive, and has low requirements for instruments, equipment and personnel, and is further suitable for field monitoring and large-scale sample screening. The key point of the establishment of the immunoassay method is to design a suitable artificial antigen of the patinopecten yessoensis toxin and obtain an antibody with high sensitivity and strong specificity, but relevant reports on hapten, artificial antigen, antibody and the like of the patinopecten yessoensis toxin are not found at present.
Disclosure of Invention
The invention aims to overcome the defects of lack of patinopecten yessoensis toxin hapten, artificial antigen and antibody in the prior art, and provides the patinopecten yessoensis toxin hapten, artificial antigen and antibody, and preparation methods and applications thereof.
The first purpose of the invention is to provide a scallop toxin hapten of the shrimp magnolia flower.
The second purpose of the invention is to provide a preparation method of the patinopecten yessotoxin hapten.
The third purpose of the invention is to provide a scallop toxin artificial antigen of the shrimp magnolia flower.
The fourth purpose of the invention is to provide a preparation method of the patinopecten yessoensis toxin artificial antigen.
The fifth object of the present invention is to provide an antibody against a toxin of scallop adderi flos magnoliae.
The sixth object of the present invention is to provide a kit for detecting patinopecten yessoensis toxin. .
The seventh object of the present invention is to provide an immunoassay method for detecting patinopecten yessoensis toxin.
The above object of the present invention is achieved by the following technical solutions:
a scallop toxin hapten of a shrimp magnolia flower, wherein the structural formula of the hapten is shown as a formula (I):
the-Br group with activity of the patinopecten yessoensis toxin hapten provided by the formula (I) can be combined with amino groups on molecules such as carrier protein, antibody, horseradish peroxidase, alkaline phosphatase and the like to prepare various detection reagents. The patinopecten yessoensis toxin hapten furthest reserves the molecular skeleton structure of the patinopecten yessoensis toxin and is beneficial to the induction of high-specificity antibodies.
The invention also provides a preparation method of the patinopecten yessoensis toxin hapten, which comprises the following steps:
s1, dissolving patinopecten yessoensis toxin in a solvent, adding a buffer solution, adding 1,2-ethanedithiol for reaction, extracting and combining organic phases after the reaction is finished, washing, drying, filtering, removing the solvent, and drying to obtain a yellow solid;
s2, dissolving the yellow solid obtained in the step S1 in a solvent, adding epoxy bromopropane for reaction, removing the solvent and excessive epoxy bromopropane after the reaction is finished to obtain a yellow paste, recrystallizing for multiple times, and drying to obtain a yellow solid, namely the patinopecten yessoensis toxin hapten shown in the formula (I).
Preferably, the solvent is tetrahydrofuran.
Preferably, the buffer is an ammonium phosphate buffer at pH 7.4,0.1M.
Preferably, the reaction described in step S1 is a room temperature reaction for 6h, indicating by TLC whether the reaction is complete.
Preferably, the extractant used for the extraction is ethyl acetate.
Preferably, the washing is performed by using dilute hydrochloric acid, and the drying is performed by using anhydrous sodium sulfate.
Preferably, the solvent removal in step S1 is performed by evaporating the solvent under reduced pressure.
Preferably, the molar ratio of the patinopecten yessotoxin to 1,2-ethanedithiol is 1.
Further preferably, the molar ratio of the patinopecten yessotoxin to 1,2-ethanedithiol is 1; still preferably, the molar ratio of the patinopecten yessotoxin to 1,2-ethanedithiol is 1.
Preferably, the reaction in step S2 is a 40 ℃ isothermal reaction for 3h, and the completion of the reaction is indicated by TLC.
Preferably, the solvent removal in step S2 is performed by rotary evaporation.
Preferably, the recrystallization adopts a mixed solvent of acetone and n-hexane with the volume ratio of 1:1.
Preferably, the molar ratio of the patinopecten yessoensis toxin to the propylene oxide is 1:5-15.
Further preferably, the molar ratio of the patinopecten yessoensis toxin to the propylene bromide oxide is 1:8-12; still preferably, the molar ratio of patinopecten yessoensis toxin to propylene oxide is 1:9-11.
The invention also provides application of the patinopecten yessoensis toxin hapten in preparation of the patinopecten yessoensis toxin artificial antigen.
A patinopecten yessoensis toxin artificial antigen, wherein the structural formula of the patinopecten yessoensis toxin artificial antigen is shown as a formula (II):
preferably, the carrier protein is selected from Bovine Serum Albumin (BSA) or chicken Ovalbumin (OVA).
The invention also provides a preparation method of the patinopecten yessoensis toxin artificial antigen, which is characterized in that the patinopecten yessoensis toxin hapten shown in the formula (I) is dissolved in a solvent, then is dripped into a buffer solution containing carrier protein for reaction, ethanolamine is added for sealing, the pH is regulated to 7.2-7.5,4-6 ℃ for reaction, and the patinopecten yessoensis toxin artificial antigen shown in the formula (II) is obtained after dialysis and purification.
Preferably, the molar ratio of the patinopecten yessotoxin hapten to the carrier protein is 10-70.
Further preferably, the molar ratio of the patinopecten yessoensis toxin hapten to the carrier protein is 30-60; still preferably, the molar ratio of the patinopecten yessoensis toxin hapten to the carrier protein is 40-50.
Preferably, the molar ratio of the patinopecten yessoensis toxin hapten to the ethanolamine is 1.
Further preferably, the molar ratio of the patinopecten yessoensis toxin hapten to the ethanolamine is 1; still preferably, the molar ratio of the patinopecten yessotoxin hapten to the ethanolamine is 1. .
As a preferable embodiment, the method for preparing the patinopecten yessoensis toxin artificial antigen comprises the steps of:
dissolving patinopecten yessoensis toxin hapten by using DMF as a solvent, dropwise adding the solution into 10-20 mg/mL carrier protein solution, stirring, adjusting the pH to 7.4, and reacting at room temperature for 18h; adding ethanolamine for sealing, adjusting the pH to 7.4,4 ℃, and stirring overnight; dialyzing with 0.01M phosphate buffer solution with pH of 7.2 for 6 times to obtain artificial antigen of Patinopecten yessotoxin.
The invention also provides an application of any one of the patinopecten yessoensis toxin artificial antigens in preparation of the patinopecten yessoensis toxin antibody.
The invention also provides a patinopecten yessoensis toxin antibody which is prepared by taking the patinopecten yessoensis toxin artificial antigen shown in the formula (II) as immunogen immune animals.
Preferably, the patinopecten yessoensis toxin antibody is a patinopecten yessoensis toxin monoclonal antibody.
The invention also provides application of any one of the artificial antigens and the antibodies of the patinopecten yessoensis toxin in preparing a kit for detecting the patinopecten yessoensis toxin residue in marine algae and shellfish tissues.
The invention also provides a kit for detecting the patinopecten yessoensis toxin, which comprises any one of the patinopecten yessoensis toxin artificial antigen as a coating antigen and any one of the patinopecten yessoensis toxin antibody as a detection antibody.
Preferably, the kit is an enzyme linked immunosorbent assay kit.
Further preferably, the kit further comprises an enzyme-linked immunosorbent assay related reagent.
The invention also provides an immunoassay method for detecting the patinopecten yessoensis toxin, which takes any one of the artificial antigens of the patinopecten yessoensis toxin as a coating antigen and takes any one of the antibodies of the patinopecten yessoensis toxin as a detection antibody for detection. Such immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensing, immunocolloidal gold, and the like.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a patinopecten yessoensis toxin hapten, which has an active-Br group and can be combined with amino groups on molecules such as carrier protein, antibody, horseradish peroxidase, alkaline phosphatase and the like to prepare various detection reagents. The patinopecten yessoensis toxin hapten furthest reserves the molecular skeleton structure of the patinopecten yessoensis toxin and is beneficial to the induction of high-specificity antibodies. Meanwhile, the artificial antigen obtained by directly coupling the patinopecten yessoensis toxin hapten with the carrier protein is simple in synthesis method and high in purity and yield. After the Patinopecten yessotoxin artificial antigen is used for immunizing experimental animals, the body can generate antibodies aiming at the Patinopecten yessotoxin. The monoclonal antibody prepared by the artificial antigen of the patinopecten yessoensis toxin provided by the invention has the characteristics of high potency, strong affinity, low cross reaction rate and the like, has no cross reaction with other components of the patinopecten yessoensis toxin and other shellfish toxins, namely avoidsThe detection process of the vaccine is not interfered by the structural analogues, so that the false positive or false negative of the detection process is greatly reduced. The detection sensitivity of the enzyme linked immunosorbent assay kit prepared by the patinopecten yessoensis antigen antibody provided by the invention for the toxin of the patinopecten yessoensis is 0.001ng/mL, IC 50 0.11ng/mL, and a linear range of 0.001-0.75 ng/mL. The enzyme linked immunosorbent assay kit is convenient to use, low in detection cost, rapid, efficient and accurate in detection method, suitable for field control of the toxin residues of the scallop in marine algae and shellfish tissues and screening of a large number of samples, and has good application prospects.
Drawings
FIG. 1 is a scheme for synthesis of patinopecten yessoensis toxin hapten.
FIG. 2 is SDS gel electrophoresis of artificial antigens of Patinopecten yessoensis toxin.
FIG. 3 shows a standard curve of Patinopecten toxin ELISA.
Detailed Description
The invention is further described with reference to the drawings and specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 Synthesis and identification of Patinopecten yessotoxin hapten
1. Synthesis of patinopecten yessoensis toxin hapten, wherein the synthetic route is shown in figure 1;
(1) Dissolving 50mg of patinopecten yessoensis toxin by using 1mL of tetrahydrofuran, dripping 5mL of ammonium phosphate buffer solution with pH of 7.4 and 0.1M, continuously stirring, then dripping 1,2-ethanedithiol, wherein the molar ratio of the patinopecten yessoensis toxin to 1:2-ethanedithiol is 1;
(2) Dissolving the product 1 with 5mL of tetrahydrofuran, adding propylene oxide, heating to 40 ℃, reacting at constant temperature for 3h under magnetic stirring, displaying that the reaction is finished by TLC, removing tetrahydrofuran and excessive propylene oxide by rotary evaporation to obtain yellow paste, recrystallizing for many times with 2mL of a mixed solvent of acetone and n-hexane with the proportion of 1:1, and drying at 60 ℃ to obtain a yellow solid as a product 2, wherein the molar ratio of patinopecten yessotoxin to propylene oxide is 1.
2. Identification of patinopecten yessoensis toxin hapten
The product 2 obtained by the preparation is sent to Beijing Zhongkohui Kehuren science and technology limited company for nuclear magnetic identification, and the specific results are as follows: 1 H NMR:δ1.19-2.42(50H,1.28(ddddd,J＝12.8,8.8,6.7,5.5,1.5Hz),1.33(dddd,J＝13.0,6.8,1.6,1.5Hz),1.36(ddddd,J＝12.8,10.3,8.8,5.5,2.7Hz),1.36(ddddd,J＝12.8,5.5,3.1,2.7,1.4Hz),1.40(dddd,J＝12.9,7.4,3.1,2.7Hz),1.41(dddd,J＝13.0,9.5,7.5,6.7Hz),1.41(ddddd,J＝10.1,7.4,6.2,3.7,2.3Hz),1.44(ddddd,J＝12.8,7.5,6.8,5.5,1.4Hz),1.43(ddd,J＝13.2,4.5,4.3Hz),1.47(dddd,J＝12.9,10.3,6.2,2.7Hz),1.48(ddd,J＝13.1,4.6,4.2Hz),1.50(ddd,J＝13.1,10.1,9.9Hz),1.50(dddt,J＝9.6,4.6,3.6,1.8Hz),1.52(ddddd,J＝9.5,4.5,3.7,1.8,1.6Hz),1.52(dt,J＝13.1,10.2Hz),1.55(dddd,J＝13.1,4.5,3.1,1.6Hz),1.58(ddd,J＝13.0,8.1,7.7Hz),1.59(dddd,J＝13.0,8.8,7.1,1.4Hz),1.63(ddddd,J＝10.2,8.1,3.6,2.9,2.5Hz),1.62(ddd,J＝13.1,2.3,1.9Hz),1.65(dddd,J＝12.9,8.7,4.5,2.4Hz),1.67(ddd,J＝13.0,7.9,7.8Hz),1.69(dddd,J＝13.1,8.7,5.9,3.7Hz),1.69(ddd,J＝13.1,2.1,1.8Hz),1.70(ddd,J＝13.1,2.9,2.6Hz),1.71(ddd,J＝13.0,4.7,2.5Hz),1.72(dddd,J＝13.3,10.1,8.8,1.4Hz),1.73(dddd,J＝13.0,6.3,1.7,1.4Hz),1.74(ddd,J＝13.2,2.0,1.8Hz),1.78(ddd,J＝13.0,2.0,,1.8Hz),1.83(dddd,J＝13.3,6.3,1.7,1.4Hz),1.83(dddd,J＝12.9,5.9,3.2,1.6Hz),1.86(ddd,J＝13.0,9.6,9.4Hz),1.87(ddddd,J＝10.3,4.3,3.0,2.6,2.0Hz),1.93(ddddd,J＝10.1,7.8,7.6,1.8,1.7Hz),1.95(ddddd,J＝9.9,4.2,3.0,2.1,1.9Hz),1.96(ddtd,J＝7.7,7.1,4.7,1.7Hz),2.01(ddd,J＝14.8,2.0,1.5Hz),2.09(ddd,J＝14.8,5.2,3.9Hz),2.10(ddddd,J＝7.9,7.7,3.7,3.1,2.0Hz),2.10(ddd,J＝13.0,1.8,1.6Hz),2.16(dt,J＝14.5,7.9Hz),2.23(ddd,J＝14.5,4.2,3.9Hz),2.25(ddd,J＝14.8,3.4,2.7Hz),2.28(ddd,J＝14.8,5.7,4.3Hz),2.30(dd,J＝7.1,6.6Hz),2.30(dd,J＝7.1,6.6Hz),2.32(ddd,J＝14.8,1.9,1.4Hz),2.33(ddd,J＝14.8,2.9,2.1Hz),2.35(dd,J＝14.2,5.4Hz)),2.53(1H,dd,J＝14.2,7.8Hz),3.62(1H,dd,J＝4.8,2.8Hz),3.76(1H,ddd,J＝4.8,3.2,2.4Hz),3.88(1H,ddd,J＝5.2,3.9,1.5Hz),4.15-4.41(9H,4.21(ddd,J＝9.4,4.8,1.6Hz),4.23(ddd,J＝3.9,2.2,2.0Hz),4.23(ddd,J＝4.3,2.2,1.9Hz),4.26(ddd,J＝5.7,4.6,1.4Hz),4.29(ddd,J＝7.8,5.4,3.9Hz),4.28(ddd,J＝4.6,3.4,2.1Hz),4.29(td,J＝2.9,2.7Hz),4.32(ddd,J＝7.8,7.7,4.2Hz),4.33(ddd,J＝7.9,7.6,3.9Hz)),4.79-5.11(5H,4.86(dd,J＝10.3,1.7Hz),4.92(t,J＝6.6Hz),5.03(dd,J＝17.4,1.7Hz),5.06(d,J＝1.5Hz),5.06(d,J＝1.5Hz)),5.60(1H,dt,J＝15.7,7.1Hz),6.01(1H,dd,J＝15.7,10.3Hz),6.48(1H,dt,J＝17.4,10.3Hz).C 65 H 100 Br 2 O 15 S 4 ,Exact Mass 1406.43Molecular weight1409.55m/z:1408.43；
the structural formula of the patinopecten yessoensis toxin hapten is shown as the formula (I):
example 2 Synthesis of Patinopecten yessotoxin Artificial antigen
Dissolving 10mg of the patinopecten yessoensis toxin hapten synthesized in example 1 in 0.2mL of DMF, dropwise adding into 1mL of a bovine serum albumin phosphate buffer solution (0.1M, pH 7.4) with a concentration of 13mg/mL, stirring, and reacting at room temperature for 18h; adding 12mg of ethanolamine for sealing, adjusting the pH to 7.4,4 ℃, and stirring overnight; dialyzing with 0.01M phosphate buffer solution with pH of 7.2 for 6 times to obtain artificial antigen YTX-BSA of Patinopecten yessoensis toxin, subpackaging, and storing at-20 deg.C.
SDS-PAGE identification: the artificial antigen is identified by SDS-PAGE gel running, the voltage of the concentrated gel is 90V, the voltage of the separation gel is 120V, and the protein content of each hole is about 2-5 mug during sample loading. After the glue is run, dyeing is carried out for 2h by using a Coomassie brilliant blue dyeing solution, then, decoloring is carried out overnight by using the Coomassie brilliant blue decoloring solution, after the decoloring is completed, a gel imaging system shoots, and the result is stored and analyzed.
As shown in FIG. 2, it can be seen that the electrophoretic bands of the artificial coupling product YTX-BSA increased the molecular weight by about 20000Da compared to the bands of the carrier protein BSA, respectively, indicating that the coupling of YTX hapten and macromolecular carrier protein was successful.
Example 3 preparation of monoclonal antibody against Patinopecten yessoensis toxin
(1) Patinopecten yessotoxin artificial antigen immune animal
The patinopecten yessoensis toxin artificial antigen YTX-BSA prepared in the embodiment 2 is used as an immune antigen to immunize female BALB/c mice with the age of 6-8 weeks, the immune dose is 50 mu g/mouse, the first immunization is complete Freund adjuvant, then incomplete Freund adjuvant is used, after the fifth immunization is finished, tail vein blood collection is carried out after 10 days, and serum is separated to test the specificity of immune serum. Three days after the last immunization, the spleen of the mouse was harvested and cell fusion was performed.
(2) Cell fusion
Taking immune BALB/c mouse spleen cells and Sp2/0 myeloma cells to mix according to the proportion of 10:1, centrifuging for 15min at 1000rpm, abandoning the supernatant, placing in a water bath at 40 ℃ for preheating, adding 0.6mL of 50% PEG1500 preheated to 38 ℃ within 90s, then adding 30mL of DMEM incomplete medium preheated to 37 ℃ within 5min, standing for 10min at room temperature, abandoning the supernatant, adding 20mL of DMEM incomplete medium with 20% FCS and HAT for resuspension. Adding into 96-well plate with feeder cells, culturing for 7-11 days, observing cell growth, and collecting supernatant for antibody detection.
(3) Screening of antibody hybridoma cell against patinopecten yessoensis toxin
Patinopecten yessotoxin artificial antigen YTX-BSA (protein content 4 mg/mL) is coated with an ELISA plate by 10000-fold dilution with a coating solution of 0.05mol/L pH 9.6 carbonate buffer solution at 100 mu L/hole and is coated for 3h at 37 ℃; PBST washing 3 times, with 300 u L/hole, 5% BSA blocking solution blocking, 37 degrees C placed for 2h; the cell supernatant, 1; washing, adding enzyme-labeled goat anti-mouse IgG diluted by 1 2 O 2 Solutions ofDeveloping in dark for 10min; the reaction was stopped by adding 50. Mu.L of a 2mol/L sulfuric acid solution to each well. OD determination by enzyme-linked immunosorbent assay 450 nm value: zeroing with blank control, when OD of positive reference serum 450 nm value and OD of negative reference serum 450 The ratio of the nm value is more than or equal to 2.1, the detection hole is judged to be positive, and when the OD of the positive reference serum is larger than or equal to the OD of the positive reference serum 450 nm value and OD of negative reference serum 450 The detection holes with the ratio of the nm value less than 1.5 are judged to be negative, and the detection holes with the ratio in the middle are judged to be suspicious.
(4) Subcloning of Positive hybridoma cells by limiting dilution method
Positive wells were subcloned by limiting dilution. After 9 days, cell supernatants were removed and ELISA was performed immediately. Selecting positive monoclonal cells, and performing subcloning for more than 3 times until all cell well supernatants are positive; the hybridoma cell strain which stably secretes the patinopecten yessoensis toxin monoclonal antibody is obtained by expanding culture and freezing the positive cells after multiple times of subcloning culture and is named as 12D-9.
(5) Production of monoclonal antibody against patinopecten yessoensis toxin
BALB/c mice were intraperitoneally inoculated with 0.25mL Freund's incomplete adjuvant, and 3 days later, were intraperitoneally inoculated with 6X 10 5 Hybridoma cells, 13 days later, mice were sacrificed and ascites fluid was aspirated aseptically. Standing at room temperature for 30min,5000rpm, centrifuging for 20min, collecting supernatant, subpackaging, and freezing at-70 deg.C for later use.
Example 4 specificity test of Patinopecten yessoensis toxin monoclonal antibody
(1) Preparation of Patinopecten yessotoxin ELISA Standard Curve
The patinopecten yessoensis toxin artificial antigen YTX-BSA coating antigen is diluted to the concentration of 0.5 mu g/mL by carbonate buffer solution, added into an enzyme label plate and coated overnight at 4 ℃. Serially diluting Patinopecten yessoensis toxin standard (purchased from Dalian inspection Biotechnology limited, product number D-00891) with 0.01M phosphate buffer solution (containing 15% methanol) with pH of 7.2, adding 50 μ L per well, adding appropriate diluted anti-Patinopecten yessoensis toxin monoclonal antibody, bathing at 37 deg.C for 30min, washing plate, adding goat anti-mouse enzyme-labeled secondary antibody for reaction for 30min, washing enzyme-labeled plate, adding TMB-H 2 O 2 And (3) adding 2M sulfuric acid into the substrate after color development to terminate the reaction, and testing the OD value by using an enzyme-labeling instrument. The absorbance value obtained for each concentration of standard solution was divided by the absorbance value (B) of the first standard solution (0 standard) 0 ) And then multiplied by 100% to obtain the percent absorbance value. And drawing a standard curve chart by taking the concentration of the patinopecten yessoensis toxin standard substance (ng/mL) as an X axis and the percentage absorbance value as a Y axis. The resulting standard curve is shown in FIG. 3.
Percent absorbance value% = B/B 0 ×100％
(2) Cross-reactivity of anti-patinopecten yessoensis toxin antibodies
And (3) testing the cross reaction rate of the antibody by adopting indirect competitive ELISA, diluting other medicines in series, and replacing the patinopecten yessoensis toxin tested in the step (1) with the medicine to be tested. The method comprises the following specific steps:
the patinopecten yessoensis toxin artificial antigen YTX-BSA coating antigen is diluted to the concentration of 0.25 mu g/mL by carbonate buffer solution, added into an enzyme label plate and coated overnight at 4 ℃. Diluting the patinopecten yessoensis toxin structural analogue standard substance with 0.01M phosphate buffer solution (containing 10% methanol) with pH7.2, adding 50 μ L into each well, adding the patinopecten yessoensis toxin monoclonal antibody diluted properly, bathing at 37 deg.C for 30min, washing the plate, adding goat anti-mouse enzyme-labeled secondary antibody for reaction for 30min, washing the enzyme-labeled plate, adding TMB substrate, developing, adding 2M sulfuric acid for termination reaction, and testing OD value with an enzyme-labeled meter.
Drawing a concentration-inhibition rate curve according to the detection result, and calculating the IC of each competitor 50 The cross-reactivity of each competitor with the monoclonal antibody against patinopecten yessoensis toxin was also calculated using the following formula.
Cross reaction rate = [ IC = 50 (YTX)/IC 50 (YTX structurally similar competitors)]×100％
The cross-reactivity of the monoclonal antibody against the toxin analogs of Patinopecten yessoensis and other shellfish toxins is shown in Table 1. As can be seen from Table 1, there is no cross reaction between the toxin of Patinopecten yessoensis and the toxin of Patinopecten yessoensis, and the detection process is not affected by the toxin of Patinopecten yessoensis or other shellfish toxins, which indicates that the monoclonal antibody of Patinopecten yessoensis prepared by the invention has strong specificity, and is not interfered by these structural analogs in the immunoassay process, thereby greatly reducing the false positive or false negative in the detection process.
TABLE 1 Cross-reactivity ratio of Patinopecten yessotoxin monoclonal antibody (12D-9)
Example 5 accuracy test of Patinopecten yessotoxin enzyme-linked reagent kit
Pretreatment of a patinopecten yessoensis post sample: washing Japanese scallop sample, removing shell, accurately weighing 5g Japanese scallop shell column tissue, adding 20mL,80% methanol water solution, 40000 r, homogenizing for 2min, diluting to 25ml, centrifuging at 10000rpm for 20min, removing precipitate, collecting 100 μ L supernatant, adding 900 μ L sample diluent, and making into enzyme linked immunosorbent assay with sample dilution factor of 50. With the ELISA plate prepared in example 4, three concentrations of patinopecten yessoensis toxin standard solutions, 1.0. Mu.g/kg, 2.5. Mu.g/kg and 5.0. Mu.g/kg, were taken, and the addition recovery tests were performed on the samples, with 3 replicates per concentration, respectively, and the recovery rates were calculated.
The results are shown in table 2, the adding recovery rate of the patinopecten yessoensis toxin in the patinopecten yessoensis sample is between 85.7% and 92.7%, which shows that the patinopecten yessoensis toxin detection kit prepared by the patinopecten yessoensis antigen and antibody provided by the invention has higher accuracy.
TABLE 2 Patinopecten yessotoxin ELISA addition recovery test
Example 6 precision test of Patinopecten yessotoxin enzyme-linked reagent kit
From each batch of the microplate prepared in example 4, 10 wells were extracted, and the absorbance value (OD value) of 1ng/mL of the standard solution was measured and repeated 3 times to calculate the coefficient of variation CV% (Table 3).
As shown in Table 3, the variation coefficient range in batch is between 12.3% and 15.6%, which meets the requirement that the variation coefficient is less than 20%, and the precision of the standard product of the kit reaches the standard.
TABLE 3 Japanese scallop toxin ELISA kit precision test
Experimental example 7 stability test of Patinopecten yessotoxin enzyme-linked reagent kit
The storage condition of the kit is 2-8 ℃, and the maximum absorbance value (zero standard), the 50% inhibition concentration and the actual measurement value of the addition of the patinopecten yessoensis toxin are all within the normal range after 6 months of measurement. The results of the 37 ℃ accelerated aging experiments show that all indexes of the kit meet the requirements.
The above description of exemplary embodiments has been presented only to illustrate the technical solution of the invention and is not intended to be exhaustive or to limit the invention to the precise form described. Obviously, many modifications and variations are possible in light of the above teaching to those skilled in the art. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to thereby enable others skilled in the art to understand, implement and utilize the invention in various exemplary embodiments and with various alternatives and modifications. It is intended that the scope of the invention be defined by the following claims and their equivalents.
Claims (10)
1. A scallop toxin hapten of a shrimp magnolia flower is characterized in that the structural formula of the hapten is shown as a formula (I):
2. the method for preparing a patinopecten yessoensis toxin hapten as claimed in claim 1, comprising the steps of:
s1, dissolving patinopecten yessoensis toxin in a solvent, adding a buffer solution, adding 1,2-ethanedithiol for reaction, extracting and combining organic phases after the reaction is finished, washing, drying, filtering, removing the solvent, and drying to obtain a yellow solid;
s2, dissolving the yellow solid obtained in the step S1 in a solvent, adding epoxy bromopropane for reaction, removing the solvent and excessive epoxy bromopropane after the reaction is finished to obtain a yellow paste, recrystallizing for multiple times, and drying to obtain a yellow solid, namely the patinopecten yessoensis toxin hapten shown in the formula (I).
3. Use of the patinopecten yessoensis toxin hapten as claimed in claim 1 for the preparation of a patinopecten yessoensis toxin artificial antigen.
4. The artificial antigen of the patinopecten yessoensis toxin is characterized in that the structural formula of the artificial antigen of the patinopecten yessoensis toxin is shown as the formula (II):
5. the patinopecten yessotoxin artificial antigen of claim 4, wherein the carrier protein is selected from bovine serum albumin or chicken ovalbumin.
6. The method for preparing the patinopecten yessoensis toxin artificial antigen as claimed in claim 4 or 5, characterized in that the patinopecten yessoensis toxin hapten as claimed in claim 1 is dissolved in a solvent, and then is dripped into a buffer solution containing carrier protein for reaction, and then ethanolamine is added for sealing, the pH is adjusted to 7.2-7.5,4-6 ℃ for reaction, and the patinopecten yessoensis toxin artificial antigen shown in formula (II) is obtained after dialysis purification.
7. Use of the artificial antigen of Japanese scallop toxin according to claim 4 or 5 for the preparation of antibodies against Japanese scallop toxin.
8. An antibody against Japanese scallop toxin, which is produced by immunizing an animal with the artificial antigen of Japanese scallop toxin according to claim 4 or 5 as an immunogen.
9. A kit for detecting Japanese scallop toxin, comprising the Japanese scallop toxin artificial antigen of claim 4 or 5 and the Japanese scallop toxin antibody of claim 8.
10. An immunoassay method for detecting a patinopecten yessoensis toxin, characterized in that the patinopecten yessoensis toxin artificial antigen of claim 4 or 5 is used as a coating antigen, and the patinopecten yessoensis toxin antibody of claim 8 is used as a detection antibody.
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| CN1979169A (en) * | 2005-12-05 | 2007-06-13 | 曹际娟 | Diarrhea sheufish-poison competitive enzyme-linked immune quantitative detection reagent box, its preparation and use |
| CN111273015A (en) * | 2020-04-13 | 2020-06-12 | 北京维德维康生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting Gymnodinium breve toxin and preparation and application thereof |
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| CN1979169A (en) * | 2005-12-05 | 2007-06-13 | 曹际娟 | Diarrhea sheufish-poison competitive enzyme-linked immune quantitative detection reagent box, its preparation and use |
| CN111273015A (en) * | 2020-04-13 | 2020-06-12 | 北京维德维康生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting Gymnodinium breve toxin and preparation and application thereof |
Non-Patent Citations (1)
| Title |
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| LYN R. BRIGGS ET AL.: "Enzyme-Linked Immunosorbent Assay for the Detection of Yessotoxin and Its Analogues", J. AGRIC. FOOD CHEM., vol. 52, pages 5836 - 5842, XP055013466, DOI: 10.1021/jf049395m * |
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