Okadaic acid mimic epitope peptide and application thereof

Abstract

The invention relates to an okadaic acid mimic epitope peptide and application. The amino acid sequence of the okadaic acid mimic epitope peptide is shown as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15. The mimic epitope peptide can be specifically combined with an okadaic acid antibody, and a sensitive and rapid competitive enzyme-linked immunosorbent assay is established by using the phage for displaying the mimic epitope peptide; according to the method, the IC50 is 0.83 ng / mL, the LOD is 0.21 ng / mL, and the detection range is 0.35 to 1.98 ng / mL; the competitive immunoassay method established on the basis of the mimic epitope peptide provided by the invention is more sensitive than the traditional immunoassay method, has good cross reaction to other marine toxins, avoids the use of toxin antigens, and is green and environment-friendly.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and in particular to an okadaic acid mimic epitope peptide and its application. Background Technology

[0002] Okada acid, also known as okada sponge acid, is a small-molecule, lipophilic marine toxin produced by red tide algae such as *Prorocentrum* and *Prorocentrum*. It belongs to the polyether marine biotoxin class and is a major component of diarrhetic shellfish toxins. It is named after the *Okada sponge* from which it was first isolated. It induces protein hyperphosphorylation by inhibiting threonine and serine phosphatase activity. It is of low toxicity and there is no specific antidote. As a highly harmful carcinogen, it damages human liver cells and nerve cells, exhibiting long-term toxic effects; long-term accumulation can lead to birth defects and cancer.

[0003] Okadaic acid does not lose its toxicity under high temperatures. When people accidentally ingest seafood contaminated with okadaic acid, they may experience symptoms such as diarrhea, nausea, and vomiting. Currently, there is no specific antidote. Therefore, monitoring okadaic acid in food is essential.

[0004] Traditional methods for detecting okadaic acid include chromatography, bioassay, and immunological assays. Chromatography requires expensive equipment and complex sample pretreatment, while bioassays are time-consuming and lack sufficient accuracy, failing to meet the requirements for rapid on-site detection. Immunological techniques, however, offer advantages such as high specificity, high sensitivity, and the elimination of complex equipment, making them a commonly used technique for rapid toxin detection. However, the immunological process requires the chemical synthesis of artificial antigens using okadaic acid standards. This synthesis involves cumbersome reaction steps, expensive raw materials, and the use of large quantities of toxic standards and organic solvents, posing a significant health hazard to operators.

[0005] Therefore, researching green and non-toxic small-molecule antigen substitutes is an important direction for developing environmentally friendly immunoassays. In recent years, researchers have successfully replaced small-molecule antigens for immunological detection using phage display random peptide library technology to select highly specific and specific mimic epitopes. Compared with traditional chemically synthesized competitors, mimic epitope proteins have advantages such as rapid and simple acquisition, stable properties, and low cost. Most importantly, they reduce the risk of laboratory personnel being exposed to toxic environments. In the field of immunoassay detection of toxic and hazardous substances, mimic epitopes have broad application prospects in replacing antigens for immunoassay. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an okadaic acid mimic epitope peptide and its application.

[0007] The first objective of this invention is to provide an okadaic acid mimic epitope peptide.

[0008] A second objective of this invention is to provide a biomaterial.

[0009] A third objective of this invention is to provide a coupling.

[0010] A fourth objective of this invention is to provide a method for detecting okadaic acid.

[0011] A fifth objective of this invention is to provide a kit for detecting okadaic acid.

[0012] A sixth objective of the present invention is to provide the use of the okadaic acid mimic epitope peptide, the biomaterial, or the conjugate in the detection of okadaic acid.

[0013] A seventh objective of the present invention is to provide the use of the okadaic acid mimic epitope peptide, the biomaterial, or the conjugate in the preparation of a kit for detecting okadaic acid.

[0014] To achieve the above objectives, the present invention is implemented through the following technical solution: (1) The purified okadaic acid monoclonal antibody was coated onto a high-adsorption ELISA plate, and the plate was blocked with 3% skim milk powder. Then, a random linear dodecapeptide phage display library was added to the ELISA plate for panning, following a binding-elution-amplification panning protocol, and three rounds of enrichment and panning were performed. The amount of antibody used for coating and the amount of okadaic acid standard used for competitive phage elution decreased sequentially in the three rounds of panning. (2) After three rounds of screening, 95 phage single clones were randomly selected for preliminary identification by phage ELISA. The 24 positive clones were amplified and sequenced to find a total of 8 sequences. This invention utilizes a competitive screening method to display phage clones of mimic epitope peptides. The phage displaying the mimic epitope protein competes with the analyte (okadaic acid) on a ELISA plate with a monoclonal antibody (this antibody specifically recognizes okadaic acid). A positive phage clone is determined by the absorbance of the reaction well containing the phage and PBS mixture, indicating phage binding to the antibody; simultaneously, the absorbance of the control well (containing a mixture of phage and PBS diluted with the analyte) is significantly reduced, indicating the analyte can displace the phage from the antibody binding site. This phage then displays the mimic epitope peptide phage clone. The phage uses the M13 phage plasmid as a vector, with the gene encoding the exogenous mimic epitope peptide inserted into the gⅢ gene of the phage-encoded membrane protein, allowing the exogenous protein to be displayed at the N-terminus of the phage PⅢ capsid protein.

[0015] Therefore, this invention claims protection for an okadaic acid mimic epitope peptide, the amino acid sequence of which is shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15. A biological material, comprising one or more of the following: (1) The gene encoding the okadaic acid mimic epitope peptide described above; (2) An expression cassette containing the gene described in (1); (3) A recombinant vector containing the gene described in (1) or the expression cassette described in (2); (4) A recombinant microorganism containing the gene described in (1), the expression cassette described in (2), or the recombinant vector described in (3); (5) A cell line containing the gene described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

[0016] Preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16.

[0017] Preferably, the recombinant microorganism is a bacteriophage expressing the okadaic acid mimic epitope peptide on its surface.

[0018] More preferably, the okadaic acid mimic epitope peptide is displayed at the N-terminus of the phage PⅢ capsid protein.

[0019] More preferably, the phage uses the M13 phage plasmid as a vector, and the gene is inserted into the gⅢ gene of the membrane protein encoded by the phage.

[0020] A conjugate comprising the aforementioned okadaic acid mimic epitope peptide conjugated to a carrier protein.

[0021] Preferably, the carrier protein is OVA or BSA.

[0022] Most preferably, the carrier protein is BSA.

[0023] A method for detecting okadaic acid includes the following steps: Enzyme-linked immunosorbent assay (ELISA) was performed using anti-okadaic acid antibody as the coating antibody and any one of the okadaic acid mimic epitope peptide, the bacteriophage, or the conjugate as a competitive antigen.

[0024] Preferably, the anti-okadaic acid antibody is a monoclonal antibody obtained by immunizing mice with okadaic acid conjugated with bovine serum albumin as an immunogen and then screening.

[0025] As a specific implementation method, the following steps are included: 1. Antibody coating Anti-Okada acid antibody was diluted with PBS and coated onto an ELISA plate, then incubated overnight; the next day, the plate was washed, blocked, and dried with PBST.

[0026] 2. Phage ELISA The phage clone, along with the test sample or serially diluted okadaic acid standard, was added to the wells of an antibody-coated ELISA plate. After incubation, the plate was washed with PBST, and then diluted HRP-labeled anti-M13 phage HRP secondary antibody was added. The plate was washed again with PBST, and TMB chromogenic buffer was added. The reaction was incubated in the dark, and terminated with 10% (v / v) H2SO4. The absorbance was read at 450 nm. 3. Result Interpretation Plot a standard curve: plot the logarithm of the series of okadaic acid concentrations on the x-axis and the corresponding B / B0 on ​​the y-axis (B0 is the absorbance value when the okadaic acid concentration is 0, and B is the absorbance value of other series of okadaic acid concentrations). When testing the sample, obtain the absorbance value reading through the same reaction. Based on the reading, the corresponding toxin concentration can be found in the graph, which is the toxin concentration of the sample to be tested.

[0027] A kit for detecting okadaic acid, comprising one or more of the okadaic acid mimic epitope peptide, the bacteriophage, or the conjugate.

[0028] Preferably, it also contains antibodies against okadaic acid.

[0029] More preferably, the antibody is a monoclonal antibody.

[0030] This invention also claims protection for the following applications: The application of the okadaic acid mimic epitope peptide, the bacteriophage, or the conjugate in the detection of okadaic acid.

[0031] The application of the okadaic acid mimic epitope peptide, the bacteriophage, or the conjugate in the preparation of a kit for detecting okadaic acid.

[0032] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the first-ever acquisition of an okadaic acid mimic epitope peptide, which specifically binds to okadaic acid antibodies. A sensitive and rapid competitive enzyme-linked immunosorbent assay (ELISA) was established using the phage displaying the mimic epitope peptide described in this invention. This method has an IC50 assay capability of [missing information]. 50 The concentration of the active ingredient was 0.83 ng / mL, the limit of concentration (LOD) was 0.21 ng / mL, and the detection range was 0.35–1.98 ng / mL. The competitive immunoassay method based on the simulated epitope peptide provided by this invention is more sensitive than traditional immunoassay methods, exhibits good cross-reactivity with other marine toxins, avoids the use of toxin antigens, and is green and environmentally friendly. Attached Figure Description

[0033] Figure 1 A schematic diagram of phage display of simulated epitope peptide screening.

[0034] Figure 2Phage-ELISA screening results for phage-mimicked epitope peptides.

[0035] Figure 3 To establish a standard curve for the detection of okadaic acid based on phage display of mimic epitope peptides. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0037] The anti-okadaic acid antibodies used in the following examples are all: Monoclonal antibodies against okadaic acid, obtained by immunizing mice with bovine serum albumin-conjugated okadaic acid as an immunogen and then screening, were prepared by the Guangdong Provincial Key Laboratory of Food Safety, College of Food Science, South China Agricultural University, and purified using Protein G column chromatography. The okadaic acid, which is coupled with a carrier protein (bovine serum albumin), is prepared by coupling the carrier protein (bovine serum albumin) to the terminal carboxyl group of okadaic acid via an active esterification method. The reaction formula for the preparation method is as follows: , The reaction product was confirmed to have been successfully coupled by ultraviolet spectroscopy, and its structural formula is as follows: .

[0038] The phage display linear dodecapeptide library was purchased from NEB.

[0039] LB liquid medium: 1g peptone, 0.5g yeast extract, 1g NaCl, add 100mL of tertiary water, autoclave, and store at room temperature.

[0040] Top medium: 1g peptone, 0.5g yeast extract, 0.5g NaCl, 0.7g agar, add 100mL of tertiary water, autoclave, and store at room temperature.

[0041] IPTG+Xgal: 1.25g IPTG, 1g Xgal, dissolved in 25ml LDM and sterilized through an organic membrane.

[0042] LB / IPTG / Xgal plates: Sterilize 1L LB medium with 15g / L agar, cool to below 70°C, add 1ml IPTG / Xgal mixture, mix well, and pour into plates. This plate medium should be stored in the dark at 4°C.

[0043] Tet: 20 mg / mL, dissolved in anhydrous ethanol: water = 1:1.

[0044] 20% PEG8000 / NaCl: 80g PEG8000, 58.44g NaCl, diluted to 400mL with grade III water, autoclaved, and stored at room temperature.

[0045] TBS: First, prepare Tris HCl (pH = 7.5): Add 15.764g Tris to 200mL of grade III water, and adjust the pH to 7.5 with HCl; dissolve 17.532g NaCl in 150mL of Tris HCl, and then bring the volume to 200mL. Autoclave and store at room temperature.

[0046] Gly-HCl (glycine eluent): 0.2M, prepare 50mL, M=75.07 m(Gly)=0.7507g, adjust pH to 2.2 with concentrated hydrochloric acid.

[0047] Tris-HCl (alkaline neutralization solution): 1M, prepare 50mL, M=121.135 m(Tris)=6.057g, adjust pH to 9.1 with concentrated hydrochloric acid.

[0048] Example 1: Panning of mimic epitope peptides that specifically bind to okadaic acid antibodies I. Experimental Methods 1. Phage screening and amplification (technical routes such as...) Figure 1 (As shown) (1) Dilute the purified anti-okadaic acid antibody to 40 μg / ml with 0.01 mol / L PBS, and take 100 μL into a high-adsorption microplate (3 replicates), and coat overnight at 4°C; then wash twice with PBST (300 μL / well), and incubate with 3% (w / v) skim milk powder at 37°C for 1 hour; then spin dry, and add 100 μL of 10% PBST to each well of the microplate. 11 Phage display of a linear dodecapeptide library (diluted with 5% skim milk powder) at pfu / mL was performed as one input round, incubated at 4°C for 2 hours. After washing 10 times with pre-cooled 0.1% PBST, 100 μL of glycine elution buffer was added, and the mixture was incubated at room temperature for 10 min. The supernatant was collected, and the pH was neutralized with alkaline neutralizing solution to approximately 7–7.5, which yielded the eluent after competition. A small amount was taken to measure the titer, which was the output round. (2) The eluent after competition was mixed with 20 mL of Escherichia coli ER2738 (OD2000) 600The phage was cultured in a 250 mL Erlenmeyer flask at 37 °C and 250 rpm for 4.5 h using a shaker. The culture was then transferred to a 50 mL centrifuge tube and centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was collected. One-sixth of the supernatant volume of 20% PEG8000 / NaCl was added, and the mixture was thoroughly mixed and incubated overnight at 4 °C on ice. The next day, the phage was centrifuged at 12000 rpm for 10 min at 4 °C, the supernatant was discarded, and the precipitate was resuspended in 1 mL of TBS. The precipitate was then resuspended in 350 μL of TBS (this is the next input). (3) The obtained 350 μL TBS resuspension was screened in the second round according to the methods in steps (1) and (2), the difference being that the concentration of anti-okadaic acid antibody was changed to 20 μg / mL of anti-okadaic acid antibody, and the glycine elution solution was changed to competitive elution of okadaic acid drug (1 μg / mL) (no need to add alkaline neutralization solution for neutralization), wherein the concentration of okadaic acid standard was 1 μg / mL; (4) The obtained 350 μL TBS resuspension was screened for the third time according to the methods in steps (1) and (2), except that the concentration of anti-okadaic acid antibody was changed to 5 μg / mL; the glycine elution solution was changed to competitive elution of okadaic acid drug (500 ng / mL) (no need to add alkaline neutralization solution for neutralization), wherein the concentration of okadaic acid standard was 500 ng / mL.

[0049] The method for determining the phage titer is as follows: (1) Take 10 mL of LB liquid medium, add 0.1% tetracycline, inoculate with Escherichia coli ER2738, and incubate at 37℃ and 250 rpm until OD. 600 It is ~0.5; (2) Place LB / IPTG / Xgal plates in a 37°C oven to preheat for at least 1 hour, preheating the Top medium and keeping it at around 45°C. (3) Dilute the phage solution to be tested to the appropriate multiple. Generally, the elution buffer is diluted 10-10 times. 3 The amplified phage was diluted 10 times. 8 ~10 10 times; (4) Take 10 μL of the phage at the corresponding dilution factor and add it to 200 μL of the prepared OD. 600 The sample was 0.5g of Escherichia coli ER2738. After mixing, it was added to 3mL of the prepared Top medium, mixed, spread evenly on the plate prepared in step (2), cooled for 10min, and incubated upside down in a 37℃ incubator overnight. (5) Record the blue phage spots on the plate to calculate the titer of the phage. Phage titer calculation formula: Phage titer (pfu / mL) = Number of phage spots (pfu) / (Dilution × Dilution volume (mL)).

[0050] II. Experimental Results The results are shown in Table 1. The results show that enrichment begins from the second round of screening, and the output is highest in the third round. According to the instructions of the phage display library, screening should only be performed in three rounds. Based on this result, a number of clones were picked from the titer assay plate of the third round output for screening positive clones.

[0051] Table 1

[0052] Example 2: Identification of mimic epitope peptides that specifically bind to anti-okadaic acid antibodies I. Experimental Methods In Example 1, after the third round of screening, the titer of the eluent was determined on a plate. From a plate containing fewer than 100 blue phages, 95 blue phage plaques were randomly selected. Each plaque was then inoculated onto a plate containing 1 mL of ER2738 (OD2000) solution. 600 In a 4 mL centrifuge tube containing 0.01 to 0.05 phage, incubate at 37°C and 250 rpm for 4.5 h on a shaker. Centrifuge the culture medium at 12000 rpm for 10 min at 4°C and retain the supernatant (containing the phage) for subsequent identification of positive clones using the phage enzyme-linked immunosorbent assay (ELISA) method.

[0053] The specific method for phage ELISA is as follows: 1. Dilute the anti-Okada antibody to 1 μg / ml with PBS, take 100 μL and put it into a high-adsorption ELISA plate, coat it overnight at 4℃; the next day, wash the ELISA plate twice with PBST (300 μL / well), add 3% (w / v) skim milk powder, and incubate at 37℃ for 1 hour.

[0054] 2. Take the supernatant containing the phage and mix it with equal volumes of (1) 100 ng / mL okadaic acid, (2) PBS or (3) 1 μg / mL BSA / OVA / LF (a mixture of BSA, OVA and LF at 1 μg / mL), and add it to the enzyme label well.

[0055] 3. After incubating at room temperature for 1 hour, wash 7 times with PBST. Dilute 100 μL of anti-M13 phage antibody-HRP 5000 times (v / v) with PBST and add it to the well. Incubate at 37°C for 30 minutes.

[0056] 4. Wash 5 times again, add 100 μL of TMB liquid substrate buffer to each well, and incubate at 37°C for 10 minutes. Finally, stop the reaction with 50 μL of 10% (v / v) H2SO4 and read the absorbance (450 nm).

[0057] 5. The criteria for selecting a positive clone are that the following conditions must be met simultaneously: In step 2, the absorbance of the pore containing the phage supernatant mixed with an equal volume of 100 ng / mL okadaic acid was less than 0.5. In step 2, the absorbance of the well containing the phage supernatant mixed with PBS was higher than 1.5. In step 2, the absorbance of the well containing the phage supernatant mixed with 1 μg / mL BSA / OVA / LF was less than 0.2. The identified positive clones were sequenced using sequencing primer 96gIII to obtain the gene sequence. The sequence of sequencing primer 96gIII is: TTTTGAAATCTAGCAATGCGATTGATACTCCCG.

[0058] II. Experimental Results The results are as follows Figure 2 As shown, 24 of the 95 selected clones were identified as positive clones by phage ELISA and subsequently sequenced. Sequencing revealed eight different mimic epitope peptide sequences.

[0059] The sequencing results are shown in Table 2.

[0060] Table 2. Phage display mimic epitope peptide sequencing results

[0061] Example 3: Application of mimic epitope peptides that specifically bind to okadaic acid antibodies as competitive antigens in enzyme-linked immunosorbent assays (ELISA). I. Experimental Methods 1. Antibody coating Dilute the anti-okadaic acid antibody to 1 μg / mL with PBS buffer, add 100 μL to each well of the microplate, and incubate overnight at 4°C. The next day, wash twice with PBST, and block with 3% skim milk powder at 37°C for 1 hour. After drying, store at 4°C for subsequent experiments.

[0062] 2. Establishment of the standard curve Although all eight phages screened in Example 2 could mimic the binding of okadaic acid epitope peptides to their okadaic acid antibodies, and their sensitivity was not significantly different, the titer determination results of the phages showed certain differences in affinity when the absorbance value was between 1 and 1.2 when binding to okadaic acid antibodies. Among them, the mimic epitope peptide displayed on the surface of phage N.8 (amino acid sequence as shown in SEQ ID NO:15, and its encoding gene sequence as shown in SEQ ID NO:16) had the strongest affinity and the best stability.

[0063] In summary, a standard curve was constructed using the bacteriophage with the highest affinity, numbered N.8 (which displays an epitope peptide with an amino acid sequence as shown in SEQ ID NO:15).

[0064] Phage ELISA: 50 μL of clone N.8 phage (10 12 Add 50 μL of PBS or gradient concentrations (100, 20, 4, 0.8, 0.16, 0.032, 0.0064 ng / mL) of okadaic acid to the microwells coated with the antibody. Incubate at 37°C for 45 min. After washing 7 times with PBST, add 100 μL of 5000-fold (v / v) diluted HRP-labeled anti-M13 phage HRP (secondary antibody). Wash 5 times again with PBST. Add 100 μL of LMB chromogenic solution and develop in the dark for 10 min. Terminate the reaction with 50 μL of 10% (v / v) H2SO4 and read the absorbance at 450 nm.

[0065] The logarithm of the concentration of each okadaic acid standard is used as the x-axis, and the corresponding B / B0 is used as the y-axis (B0 is the absorbance value when the concentration of okadaic acid is 0, and B is the absorbance value of the series of okadaic acid concentrations).

[0066] II. Experimental Results Standard curve such as Figure 3 , This method detects the LOD (IC50) of okadaic acid. 10 The concentration was 0.21 ng / mL, and the detection range was 0.35–1.98 ng / mL.

[0067] Example 4: Okada acid antibody-specifically bound mimic epitope peptides as competitive antigens in enzyme-linked immunosorbent assay (ELISA) 50 μL of phage clone N.8 (10 12Add the HRP-labeled anti-M13 phage HRP (secondary antibody) along with the test sample to the microwell coated with the antibody. Incubate at 37°C for 45 min. After washing 7 times with PBST, add 100 μL of 5000-fold (v / v) diluted HRP-labeled anti-M13 phage HRP (secondary antibody). Wash 5 times again with PBST. Add 100 μL of LMB chromogenic solution and develop in the dark for 10 min. Terminate the reaction with 50 μL of 10% (v / v) H2SO4 and read the absorbance at 450 nm.

[0068] Substitute the absorbance value y obtained from the sample to be tested into the standard curve established above. ), calculate the Okada acid in the sample to be tested. The content of.

[0069] Example 5: Specificity of the method for detecting okadaic acid I. Experimental Methods With the cross-reactivity rate (CR) of okadaic acid as 100%, the cross-reactivity rates of five marine toxins were determined using the method of Example 4: okadaic acid derivatives fin algae toxins (DTX1, DTX2), paralytic shellfish toxins such as scimitar toxin (STX), tetrodotoxin (TTX), and microcystin toxin (MC-LR).

[0070] The specific method is as follows: 50 μL of N.8 phage clone and 50 μL of serially diluted marine toxin standards were added to the wells coated with the antibody. The mixture was incubated at 37°C for 40 min. After washing seven times with PBST, 100 μL of a 5000-fold diluted HRP-labeled anti-M13 phage HRP secondary antibody was added. The mixture was incubated at 37°C for 30 min, washed five more times with PBST, and then 100 μL of TMB chromogenic buffer was added. The mixture was incubated in the dark for 10 min, and the reaction was terminated with 50 μL of 10% H2SO4. The absorbance at 450 nm was read using a microplate reader, and the data were processed.

[0071] Plot a standard curve with the logarithm of each concentration of marine toxin on the x-axis and the corresponding B / B0 ratio on the y-axis (B0 is the absorbance at 0 concentration of marine toxin, and B is the absorbance at a series of marine toxin concentrations). Simultaneously, calculate the cross-reactivity ratio (CR) using the following formula: .

[0072] III. Experimental Results The results are shown in Table 3. The cross-reactivity rate with its derivative, fucoidotoxin (DTX1), was 11.5%, and with its derivative, fucoidotoxin (DTX2), it was 12.65%. The cross-reactivity rates with common marine toxins such as tetrodotoxin, pufferfish toxin, and microcystin were all less than 0.1%. This indicates that the mimic epitope peptide (amino acid sequence shown in SEQ ID NO:15, encoding gene sequence shown in SEQ ID NO:16) that specifically binds to the okadaic acid antibody has good specificity and is expected to enable rapid detection of okadaic acid with low interference.

[0073] Table 3 shows the cross-reactions of phage-simulated epitope peptides with other marine toxins.

[0074] Example 6: Kit for detecting okadaic acid 1. Composition Anti-Okada acid antibody, clone of phage numbered N.8 obtained from Example 2, PBS, PBST, HRP-labeled anti-M13 phage HRP secondary antibody, TMB chromogenic solution, 10% (v / v) H2SO4.

[0075] 2. Instructions for use Same as Example 4.

Claims

1. An okadaic acid mimic epitope peptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15.

2. A biomaterial, characterized in that, One or more of the following: (1) A gene encoding the okadaic acid mimic epitope peptide of claim 1; (2) An expression cassette containing the gene described in (1); (3) A recombinant vector containing the gene described in (1) or the expression cassette described in (2); (4) A recombinant microorganism containing the gene described in (1), the expression cassette described in (2), or the recombinant vector described in (3); (5) A cell line containing the gene described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

3. The biomaterial according to claim 2, characterized in that, The recombinant microorganism is a bacteriophage whose surface expresses the okadaic acid mimic epitope peptide as described in claim 1.

4. A coupling agent, characterized in that, It is the okadaic acid mimic epitope peptide of claim 1 coupled with a carrier protein.

5. A method for detecting okadaic acid for non-diagnostic purposes, characterized in that, Includes the following steps: Enzyme-linked immunosorbent assay (ELISA) was performed using an anti-okadaic acid antibody as the coating antibody and any one of the okadaic acid mimic epitope peptide described in claim 1, the bacteriophage described in claim 3, or the conjugate described in claim 4 as a competing antigen.

6. The method according to claim 5, characterized in that, The anti-okadaic acid antibody is a monoclonal antibody obtained by immunizing mice with okadaic acid conjugated with bovine serum albumin as an immunogen and then screening them.

7. A kit for detecting okadaic acid, characterized in that, It contains any one or more of the following: the okadaic acid mimic epitope peptide of claim 1, the bacteriophage of claim 3, or the conjugate of claim 4.

8. The reagent kit according to claim 7, characterized in that, It also contains antibodies against okadaic acid.

9. The application of the okadaic acid mimic epitope peptide of claim 1, the biomaterial of claim 2, or the conjugate of claim 4 in the detection of okadaic acid.

10. The use of the okadaic acid mimic epitope peptide of claim 1, the biomaterial of claim 2, or the conjugate of claim 4 in the preparation of a kit for detecting okadaic acid.

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