CN115746015B - Patinopecten yessoensis toxin hapten, artificial antigen and antibody as well as preparation methods and application thereof - Google Patents
Patinopecten yessoensis toxin hapten, artificial antigen and antibody as well as preparation methods and application thereof Download PDFInfo
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Classifications
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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 patinopecten yessoensis toxin hapten, an artificial antigen and an antibody as well as preparation methods and application thereof. The structural formula of the patinopecten yessoensis toxin hapten is shown as a formula (I), and the hapten is used for preparing artificial antigens and antibodies for detecting patinopecten yessoensis toxin. The enzyme-linked immunosorbent assay kit prepared by adopting the patinopecten yessoensis artificial antigen and the antibody provided by the invention has the detection sensitivity of the patinopecten yessoensis toxin of 0.001ng/mL and IC 50 The linear range is 0.001-0.75 ng/mL. The ELISA kit is convenient to use, low in detection cost, and the detection method is rapid, efficient and accurate, is suitable for field control of patinopecten yessoensis toxin residues 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, in particular to a patinopecten yessoensis toxin hapten, an artificial antigen, an antibody and a preparation method and application thereof.
Background
Patinopecten Yessoensis Toxin (YTX) and derivatives thereof are fat-soluble polycyclic polyether compounds containing 2 sulfonyl groups, the chemical structures of the compounds are very similar, and some compounds are produced by marine dinoflagellate and part of compounds are metabolically converted in filter feeding shellfish bodies. Marine organisms such as shellfish containing YTXs in the body, which have been examined at present, are distributed in the neighborhood of countries such as china, japan, new zealand, norway, italy, canada and the united states. The biological toxicology study shows that YTX is a cytotoxin with stronger toxicity, the acute mortality rate is very high, the lethal dose of mice is about 100 mug/kg, the semi-lethal dose (LC 50) is different according to different organisms, and the numerical value is 80-750000 mug/kg. The European Union has established relevant laws and regulations, 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 intracellular cAMP concentration, induce and activate apoptosis-related Capase family proteins of human neuroblastoma cell lines, liver cells, liver cancer cells, hela cells and mouse myoblast cell lines, and is therefore considered as a tumor promoter.
Currently, there are few reports of patinopecten yessoensis toxin assay methods, including mouse biology, thin Layer Chromatography (TLC), high Performance Liquid Chromatography (HPLC), and chromatography-mass spectrometry (LC-MS/MS). The mouse biological method is a traditional method for detecting shellfish toxins, the mouse method is widely used for detecting marine toxins in China in 1994, the method can obtain the toxicity of detection substances, but the method lacks specificity for YTX detection, the detection sensitivity is low, and false positives of test results are caused by interference of other marine toxins in the process of extracting YTX toxins. From the ethical sector of animals, the European Union has now abolished the use of the mouse method for the determination of marine toxins. The TLC method is mainly used for separation and purification of YTX toxin, and has low detection sensitivity. The HPLC-FLD method has problems in sensitivity and selectivity due to co-effluent background interference using fluorescence detection. The adoption of chromatographic mass spectrometry (LC-MS/MS) detection can improve selectivity and sensitivity, but equipment is expensive, and the requirements on professional technicians are high, and the method is determined as a standard method for quantitatively detecting YTX in 2011 by European Union. The immunoassay detection technology is based on the principle of antigen-antibody reaction, and can be used for screening test of some toxic drugs by using a method for detecting poison by using a poison and a labeled poison competitive binding antibody. The immunoassay detection method has the characteristics of simple sample pretreatment, simple and quick operation, sensitivity, high flux and the like, has wide application prospect in the field of food safety, but for the immunoassay method, the antibody is used as a core raw material, and the effect of the antibody is greatly dependent on the antigen structure which causes the corresponding animal to generate immune reaction. Therefore, in order to ensure food safety and development of export trade, the establishment of accurate and reliable, high sensitivity and low requirements on instruments and personnel on patinopecten yessoensis toxin immunoassay detection technology is necessary, and further a qualitative and quantitative method suitable for on-site monitoring and large-scale sample screening is necessary. The key point of the establishment of the immune detection method is to design a proper patinopecten yessoensis toxin artificial antigen and obtain an antibody with high sensitivity and strong specificity, but no related report on hapten, artificial antigen, antibody and the like of the patinopecten yessoensis toxin is available at present.
Disclosure of Invention
The invention aims to overcome the defect and the defect of lack of patinopecten yessoensis toxin hapten, artificial antigen and antibody in the prior art and provides a patinopecten yessoensis toxin hapten, artificial antigen and antibody as well as preparation methods and application thereof.
The first object of the invention is to provide a patinopecten yessoensis toxin hapten.
The second object of the invention is to provide a preparation method of the patinopecten yessoensis toxin hapten.
The third object of the invention is to provide an artificial antigen of patinopecten yessoensis toxin.
The fourth object 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 a patinopecten yessoensis toxin antibody.
The sixth object of the invention is to provide a kit for detecting patinopecten yessoensis toxin. .
The seventh object of the 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 patinopecten yessoensis toxin hapten is shown in a structural formula (I):
the patinopecten yessoensis toxin hapten with the activity-Br group provided by the formula (I) can be combined with amino groups on molecules such as carrier protein, antibodies, horseradish peroxidase, alkaline phosphatase and the like to prepare various detection reagents. The patinopecten yessoensis toxin hapten furthest reserves a molecular skeleton structure of 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 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 performed for 6 hours at room temperature, indicating by TLC whether the reaction is complete.
Preferably, the extractant used for extraction is ethyl acetate.
Preferably, the washing is with dilute hydrochloric acid and the drying is with anhydrous sodium sulfate.
Preferably, the solvent is removed in step S1 by distillation under reduced pressure.
Preferably, the molar ratio of the patinopecten yessoensis toxin to the 1, 2-ethanedithiol is 1:20-100.
Further preferably, the molar ratio of the patinopecten yessoensis toxin to the 1, 2-ethanedithiol is 1:40-80; still preferably, the molar ratio of the patinopecten yessoensis toxin to the 1, 2-ethanedithiol is 1:50-60.
Preferably, the reaction described in step S2 is a constant temperature reaction at 40 ℃ for 3 hours, indicating by TLC whether the reaction is complete.
Preferably, the solvent removal in step S2 is performed by rotary evaporation.
Preferably, the recrystallization adopts a mixed solvent of acetone and normal hexane in a volume ratio of 1:1.
Preferably, the mole ratio of the patinopecten yessoensis toxin to the epibromohydrin is 1:5-15.
Further preferably, the molar ratio of the patinopecten yessoensis toxin to the epibromohydrin is 1:8-12; and preferably, the molar ratio of patinopecten yessoensis toxin to epibromohydrin is 1:9-11.
The invention also provides application of the patinopecten yessoensis toxin hapten in preparing patinopecten yessoensis toxin artificial antigens.
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 comprises the steps of dissolving the patinopecten yessoensis toxin hapten shown in the formula (I) in a solvent, dripping the solution into a buffer solution containing carrier protein for reaction, adding ethanolamine for sealing, regulating the pH value to 7.2-7.5,4-6 ℃ for reaction, and dialyzing and purifying to obtain the patinopecten yessoensis toxin artificial antigen shown in the formula (II).
Preferably, the molar ratio of the patinopecten yessoensis toxin hapten to the carrier protein is 10-70:1.
Further preferably, the molar ratio of the patinopecten yessoensis toxin hapten to the carrier protein is 30-60:1; still preferably, the molar ratio of the patinopecten yessoensis toxin hapten to the carrier protein is 40-50:1.
Preferably, the molar ratio of the patinopecten yessoensis toxin hapten to the ethanolamine is 1:10-60.
Further preferably, the molar ratio of the patinopecten yessoensis toxin hapten to the ethanolamine is 1:20-50; and preferably, the molar ratio of the patinopecten yessoensis toxin hapten to the ethanolamine is 1:30-40. .
As a preferred embodiment, the preparation method of the patinopecten yessoensis toxin artificial antigen comprises the following steps:
dissolving patinopecten yessoensis toxin hapten by using DMF as a solvent, dripping the solution into carrier protein solution with the concentration of 10-20 mg/mL, stirring, adjusting the pH value to 7.4, and reacting for 18 hours at room temperature; adding ethanolamine for sealing, adjusting pH to 7.4, and stirring at 4deg.C overnight; dialyzing with 0.01M phosphate buffer solution with pH of 7.2 for 6 times to obtain patinopecten yessoensis toxin artificial antigen.
The invention also provides application of any patinopecten yessoensis toxin artificial antigen in preparing patinopecten yessoensis toxin antibodies.
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 an immunogen to immunize animals.
Preferably, the patinopecten toxin antibody is a patinopecten toxin monoclonal antibody.
The invention also provides application of any patinopecten yessoensis toxin artificial antigen and patinopecten yessoensis toxin antibody in preparing a kit for detecting patinopecten yessoensis toxin residues in marine algae and shellfish tissues.
The invention also provides a kit for detecting patinopecten yessoensis toxin, which comprises any patinopecten yessoensis toxin artificial antigen as a coating antigen and any 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 patinopecten yessoensis toxin, which uses any patinopecten yessoensis toxin artificial antigen as a coating antigen and any patinopecten yessoensis toxin antibody as a detection antibody. Such immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensor, immune colloidal gold, and the like.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides patinopecten yessoensis toxin hapten, which has an active-Br group and can be combined with amino groups on molecules such as carrier protein, antibodies, horseradish peroxidase, alkaline phosphatase and the like to prepare various detection reagents. The patinopecten yessoensis toxin hapten furthest reserves a molecular skeleton structure of 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 experimental animal is immunized by the patinopecten yessoensis toxin artificial antigen, the organism generates antibodies against the patinopecten yessoensis toxin. The monoclonal antibody prepared by the patinopecten yessoensis toxin artificial antigen provided by the invention has the characteristics of high titer, strong affinity, low cross reaction rate and the like, does not have cross reaction with other components of patinopecten yessoensis toxin and other shellfish toxins, namely, can not be interfered by structural analogues in the immune detection process, and greatly reduces false positive or false negative in the detection process. The detection sensitivity of the enzyme-linked immunosorbent assay kit for the patinopecten yessoensis toxin prepared by adopting the patinopecten yessoensis antigen antibody provided by the invention is 0.001ng/mL, and the detection sensitivity is IC 50 The linear range is 0.001-0.75 ng/mL. The ELISA kit is convenient to use, low in detection cost, and the detection method is rapid, efficient and accurate, is suitable for field control of patinopecten yessoensis toxin residues in marine algae and shellfish tissues and screening of a large number of samples, and has good application prospects.
Drawings
FIG. 1 is a chart showing the hapten synthesis route of patinopecten yessoensis toxin.
FIG. 2 is a SDS gel electrophoresis chart of artificial antigen of patinopecten yessoensis toxin.
FIG. 3 is a standard ELISA curve of patinopecten yessoensis toxin.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 Synthesis and identification of Patinopecten yessoensis toxin hapten
1. The synthesis of patinopecten yessoensis toxin hapten is shown in figure 1;
(1) Dissolving 50mg of patinopecten yessoensis toxin with 1mL of tetrahydrofuran, dripping 5mL of ammonium phosphate buffer solution with the pH of 7.4 and 0.1M, continuously stirring, dripping 1, 2-ethanedithiol, reacting for 6 hours at room temperature, adding equal volume of ethyl acetate for extraction after TLC shows that the reaction is finished, merging organic phases, washing by dilute hydrochloric acid, drying by anhydrous sodium sulfate, filtering, evaporating a solvent from filtrate under reduced pressure, and drying at 60 ℃ to obtain yellow solid which is the product 1;
(2) Adding epoxy bromopropane into 5mL of tetrahydrofuran dissolution product 1, heating to 40 ℃, reacting for 3h at constant temperature under stirring of a magnet, performing rotary evaporation to remove tetrahydrofuran and excessive epoxy bromopropane after TLC shows that the reaction is finished, obtaining yellow paste, recrystallizing for multiple times by using 2mL of mixed solvent of acetone and n-hexane in a ratio of 1:1, and drying at 60 ℃ to obtain yellow solid which is a product 2, wherein the mole ratio of patinopectoxin to epoxy bromopropane is 1:10.
2. Identification of patinopecten yessoensis toxin hapten
The product 2 obtained by the preparation is sent to Beijing Zhongke Hui ren technology Co., ltd 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 yessoensis toxin Artificial antigen
10mg of patinopecten yessoensis toxin hapten synthesized in example 1 is dissolved by 0.2mL of DMF, and is added dropwise into 1mL of bovine serum albumin phosphate buffer (0.1M, pH 7.4) with the concentration of 13mg/mL, and the mixture is stirred and reacted for 18h at room temperature; adding 12mg of ethanolamine for sealing, adjusting pH to 7.4, and stirring at 4 ℃ 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 preserving at-20deg.C.
SDS-PAGE identification: the artificial antigen is identified by SDS-PAGE running gel, 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 running the gel, the gel is stained with coomassie brilliant blue staining solution for 2 hours, and then decolorized with coomassie brilliant blue decolorizing solution overnight, and after complete decolorization, the gel imaging system photographs, and the results are stored and analyzed.
As a result, as shown in FIG. 2, it can be seen that the electrophoresis bands of the artificial conjugate product YTX-BSA have increased molecular weight by about 20000Da as compared with the bands of the carrier protein BSA, respectively, indicating successful coupling of YTX hapten and macromolecular carrier protein.
EXAMPLE 3 preparation of Patinopecten toxin monoclonal antibody
(1) Patinopecten yessoensis toxin artificial antigen immune animal
The patinopectoxin artificial antigen YTX-BSA prepared in example 2 is used as an immune antigen, female BALB/c mice of 6-8 weeks old are immunized with 50 mug/mouse, the first immunization is Freund's complete adjuvant, freund's incomplete adjuvant is used, after the fifth immunization is finished, tail vein blood is collected after 10 days, and serum is separated to test the specificity of immune serum. Three days after the last immunization, the spleens of the mice were taken out for cell fusion.
(2) Cell fusion
The immunized BALB/c mouse spleen cells and Sp2/0 myeloma cells are mixed according to the proportion of 10:1, the mixture is centrifuged at 1000rpm for 15min, the supernatant is discarded, the mixture is preheated in a water bath at 40 ℃, 0.6mL of 50% PEG1500 preheated to 38 ℃ is added in 90s, 30mL of DMEM incomplete culture medium preheated to 37 ℃ is added in 5min, the mixture is stood for 10min at room temperature, the supernatant is discarded, and 20mL of DMEM culture medium with 20% FCS and HAT is added for resuspension. After the cells were cultured in a 96-well plate containing feeder cells for 7 to 11 days, the growth of the cells was observed, and the supernatant was used for antibody detection.
(3) Screening of anti-patinopecten yessoensis toxin antibody hybridoma cells
Coating an ELISA plate with coating solution of 0.05mol/L pH 9.6 carbonate buffer solution and 10000 times dilution, coating 100 mu L/hole, and coating at 37 ℃ for 3h; PBST was washed 3 times, blocked with 300. Mu.L/well, 5% BSA blocking solution, and left at 37℃for 2h; adding cell supernatant, positive reference serum diluted 1:500 and negative reference serum diluted 1:500 into corresponding holes, and incubating at 37 ℃ for 40min; washing, adding 1:10000 diluted enzyme-labeled goat anti-mouse IgG,100 μl/well, standing at 37deg.C for 30min, washing, adding 100 μl/substrate TMB-H 2 O 2 The solution is developed for 10min in dark; the reaction was terminated by adding 50. Mu.L of 2mol/L sulfuric acid solution to each well. OD determination by means of an ELISA reader 450 nm value: zeroing with blank control, when OD of positive reference serum 450 OD of nm value and negative reference serum 450 The ratio of nm value is more than or equal to 2.1, the detection hole is judged to be positive, and the OD of positive reference serum 450 OD of nm value and negative reference serum 450 The detection holes with the nm value ratio less than 1.5 are judged as negative, and the ratio is judged as suspicious in the middle.
(4) Subcloning positive hybridoma cells by limiting dilution
Positive wells were subcloned by limiting dilution. Cell supernatants were taken 9 days later and ELISA was performed in time. Selecting positive monoclonal cells, and performing the same subcloning for more than 3 times until all cell hole supernatant liquid detection is positive; and (3) performing expansion culture and freezing storage on the positive cells subjected to multiple subcloning culture to obtain a hybridoma cell strain which stably secretes the patinopecten yessoensis toxin monoclonal antibody, wherein the hybridoma cell strain is named as 12D-9.
(5) Production of anti-patinopecten yessoensis toxin monoclonal antibodies
BALB/c mice were inoculated intraperitoneally with 0.25mL of Freund's incomplete adjuvant and, after 3 days, with 6X 10 intraperitoneally 5 After 13 days, the mice were sacrificed and ascites were aspirated under sterile conditions. Standing at room temperature for 30min, centrifuging at 5000rpm for 20min, collecting supernatant, packaging, and freezing at-70deg.C.
EXAMPLE 4 specificity test of Patinopecten toxin monoclonal antibody
(1) Preparation of patinopecten yessoensis toxin ELISA standard curve
The patinopecten yessoensis toxin artificial antigen YTX-BSA coating antigen is diluted to the concentration of 0.5 mug/mL by carbonate buffer solution, added into an ELISA plate and coated overnight at 4 ℃. Serial dilution of patinopecten yessoensis toxin standard (purchased from Dalian fine detection Biotechnology Co., ltd., product No. D-00891) with 0.01M phosphate buffer (containing 15% methanol) pH7.2, adding 50 μl of the solution into each well, adding properly diluted anti-patinopecten yessoensis toxin monoclonal antibody, warm-bathing at 37deg.C for 30min, washing the plate, adding goat anti-mouse enzyme-labeled secondary antibody for 30min, washing the ELISA plate, and adding TMB-H 2 O 2 The substrate, after color development, 2M sulfuric acid is added to stop the reaction, and an enzyme-labeled instrument is used for testing OD value. Dividing the absorbance value by the absorbance value (B) of the first standard solution (0 standard) by the absorbance value of each concentration of standard solution 0 ) Multiplying by 100% to give a percent absorbance value. And drawing a standard curve graph by taking the concentration of patinopecten yessoensis toxin standard substance concentration (ng/mL) as an X axis and the percentage absorbance value as a Y axis. The standard curve obtained 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 competition ELISA, serially diluting other medicines, and replacing the patinopecten yessoensis toxin tested in the step (1) by 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 mug/mL by carbonate buffer solution, added into an ELISA plate and coated overnight at 4 ℃. The patinopecten yessoensis toxin structural analogue standard is serially diluted by using 0.01M phosphate buffer solution (containing 10% methanol) with pH of 7.2, 50 mu L of the patinopecten yessoensis toxin monoclonal antibody is added into each hole, then the patinopecten yessoensis toxin structural analogue standard is added into the patinopecten yessoensis toxin structural analogue standard, the patinopecten yessoensis toxin structural analogue standard is subjected to a temperature bath at 37 ℃ for 30min, a plate is washed, a goat anti-mouse enzyme-labeled secondary antibody is added for 30min, after the enzyme-labeled plate is washed, TMB substrate is added, 2M sulfuric acid is added after color development to stop reaction, and an OD value is tested by an enzyme-labeled instrument.
Plotting concentration-inhibition rate curve according to detection result, calculating IC of each competitor 50 The cross-reactivity of each competitor with patinopecten yessoensis toxin monoclonal antibody was calculated using the following formula.
Cross reaction Rate= [ IC 50 (YTX)/IC 50 (YTX structurally similar competitors)]×100％
The cross-reactivity of patinopecten toxin monoclonal antibodies with patinopecten toxin analogs and other shellfish toxins is shown in table 1. As can be seen from Table 1, the patinopecten yessoensis toxin and the patinopecten yessoensis toxin analogues have no cross reaction, and are not influenced by the patinopecten yessoensis toxin analogues or other shellfish toxins in the detection process, so that the patinopecten yessoensis toxin monoclonal antibody prepared by the invention has strong specificity, is not interfered by the structure analogues in the immune detection process, and greatly reduces the false positive or false negative in the detection process.
TABLE 1 Cross-reactivity of Patinopecten yessoensis toxin monoclonal antibody (12D-9)
Example 5 accuracy test of Patinopecten yessoensis toxin ELISA kit
Pretreatment of patinopecten yessoensis sample: the patinopecten yessoensis sample is cleaned and shelled, 5g of patinopecten yessoensis stem tissue is accurately weighed, 20mL of 80% methanol aqueous solution is added, 40000 turns, homogenization is carried out for 2min, the volume is fixed to 25mL, centrifugation is carried out at 10000rpm for 20min, precipitation is removed, 100 mu L of supernatant is taken, 900 mu L of sample diluent is added, and the patinopecten yessoensis sample can be used for enzyme-linked immunoassay, and the sample dilution multiple is 50. The ELISA plate prepared in example 4 was subjected to additive recovery tests with three concentrations of patinopecten yessoensis toxin standard solutions of 1.0 μg/kg, 2.5 μg/kg and 5.0 μg/kg, respectively, and the recovery rates were calculated in parallel for each concentration of 3.
The results are shown in table 2, and the addition recovery rate of patinopecten yessoensis toxin in patinopecten yessoensis samples is between 85.7% and 92.7%, which indicates that the patinopecten yessoensis toxin detection kit prepared by patinopecten yessoensis antigens and antibodies provided by the invention has higher accuracy.
TABLE 2 patinopecten toxin ELISA additive recovery test
Example 6 precision test of Patinopecten yessoensis toxin ELISA kit
From the ELISA plates prepared in example 4, 10 microwells were each extracted, and absorbance values (OD values) of 1ng/mL standard solutions were measured, and the measurement was repeated 3 times to calculate a coefficient of variation CV (Table 3).
Table 3 shows that the variation coefficient in the batch ranges from 12.3 to 15.6 percent, meets the rule that the variation coefficient is less than 20 percent, and shows that the precision of the standard substance of the kit reaches the standard.
TABLE 3 patinopecten yessoensis toxin ELISA kit precision test
Experimental example 7 stability test of Patinopecten yessoensis toxin enzyme-linked kit
The preservation condition of the kit is 2-8 ℃, and the maximum absorbance value (zero standard), 50% inhibition concentration and the actual measurement value of patinopecten yessoensis toxin addition of the kit are all within the normal range after 6 months of measurement. The results of the accelerated aging test at 37 ℃ show that various indexes of the kit meet the requirements.
The description of the exemplary embodiments presented above is merely illustrative of the technical solution of the present 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 of ordinary skill in the art. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable others skilled in the art to understand, make 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. The patinopecten yessoensis toxin hapten is characterized in that the structural formula of the hapten is shown as a formula (I):
。
formula (I)
2. The method for preparing the patinopecten yessoensis toxin hapten according to claim 1, which is characterized by comprising 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 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 a patinopecten yessoensis toxin hapten as claimed in claim 1 in the preparation of a patinopecten yessoensis toxin artificial antigen.
4. The patinopecten yessoensis toxin artificial antigen is characterized in that the patinopecten yessoensis toxin artificial antigen has a structural formula shown as a formula (II):
。
formula (II)
5. Artificial patinopectoxin antigen according to claim 4, characterized in that said carrier protein is selected from bovine serum albumin or chicken ovalbumin.
6. The preparation method of the patinopecten yessoensis toxin artificial antigen according to claim 4 or 5 is characterized in that the patinopecten yessoensis toxin hapten according to claim 1 is dissolved in a solvent, then is dripped into a buffer solution containing carrier protein for reaction, is then added with ethanolamine for sealing, is adjusted to pH 7.2-7.5,4-6 ℃ for reaction, and is subjected to dialysis and purification, thus obtaining the patinopecten yessoensis toxin artificial antigen shown in the formula (II).
7. Use of the artificial patinopecten yessoensis toxin antigen as claimed in claim 4 or 5 in the preparation of patinopecten yessoensis toxin antibodies.
8. An antibody against patinopecten yessoensis toxin, characterized in that it is prepared by immunizing an animal with the patinopecten yessoensis toxin artificial antigen as defined in claim 4 or 5 as an immunogen.
9. A kit for detecting patinopecten yessoensis toxin, comprising the patinopecten yessoensis toxin artificial antigen of claim 4 or 5 and the patinopecten yessoensis toxin antibody of claim 8.
10. An immunoassay method for detecting patinopecten yessoensis toxin for the purpose of non-disease diagnosis, which is characterized in that patinopecten yessoensis toxin artificial antigen as claimed in claim 4 or 5 is used as a coating antigen, and patinopecten yessoensis toxin antibody as claimed in claim 8 is used as a detection antibody for detection.
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| 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 |
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| Title |
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| Enzyme-Linked Immunosorbent Assay for the Detection of Yessotoxin and Its Analogues;Lyn R. Briggs et al.;J. Agric. Food Chem.;第52卷;5836-5842 * |
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