CN114634550B

CN114634550B - Non-diagnostic purpose detection method for tetrodotoxin

Info

Publication number: CN114634550B
Application number: CN202210198282.5A
Authority: CN
Inventors: 徐振林; 李亚茹; 罗林; 韦柳娜; 雷红涛; 李向梅; 沈玉栋; 王弘
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-03-01
Filing date: 2022-03-01
Publication date: 2023-08-18
Anticipated expiration: 2042-03-01
Also published as: CN114634550A

Abstract

The invention discloses a detection method for non-diagnostic purpose of tetrodotoxin, which establishes a sensitive and rapid competitive enzyme-linked immunoassay method by using phage of the obtained amino acid sequence such as a mimotope peptide shown as SEQ ID NO. 1, 3, 5, 7, 9 or 11; the method IC ₅₀ The detection range is 9.8ng/mL, and the detection range is 3.4-52.8 ng/mL. Compared with the sensitivity of the traditional hapten-based immunoassay method, the method is more sensitive; the method has good cross reaction to other marine toxins.

Description

Non-diagnostic purpose detection method for tetrodotoxin

Technical Field

The invention relates to the technical field of tetrodotoxin detection, in particular to a non-diagnostic target detection method for tetrodotoxin.

Background

Tetrodotoxin is a non-protein neurotoxin with strong toxicity, and acts on central and peripheral nervous systems, and selectively binds with proteins on sodium ion channels on the surface of cell membranes, so that the sodium ion channels are blocked, and the conduction of excitability of neuromuscular is directly influenced, so that the neuromuscular is in a paralyzed state, and even death is caused. Tetrodotoxin is not only a marine toxin with strong toxicity, but also has very stable properties, and is difficult to destroy by common cooking means, so that a plurality of tetrodotoxin poisoning events are caused, and the detection of tetrodotoxin becomes particularly important. Besides, tetrodotoxin has important application value in medicine, and can be used as a good analgesic and anesthetic drug, a drug for stopping drug addiction and resisting arrhythmia, and the like. The detection of tetrodotoxin is also of great significance for the extraction and identification of toxins.

At present, the detection method of tetrodotoxin mainly adopts a physicochemical analysis method and an immunochemical method, but both methods have certain limitations, wherein the method has the advantage that a standard substance of tetrodotoxin is needed to be used as a competitive antigen, a standard or a competitive tracer. At present, the international tetrodotoxin standard has a low price, belongs to biological agents subject to international forbidden transport, and is extremely toxic and a potential safety hazard for laboratories and operators in the laboratories. Therefore, a nontoxic detection method is established, so that the detection pressure caused by the rarity of toxin standard products can be relieved to a great extent, and the risk of poisoning of laboratory personnel can be reduced.

Phage display technology can insert randomly encoded target genes into filamentous phage gene III and display by fusion with pIII protein, thus forming a peptide library using phage as carrier. And separating specific phage capable of combining with an antibody from the peptide library by utilizing the specific combination effect between proteins, wherein the specific phage is a mimotope of the antibody, namely a short peptide capable of specifically combining with the antibody and competing with a binding site of an analyte through the mimotope. The non-toxic detection method is to replace the interaction of toxin and antibody by using the mimic epitope screened from the phage peptide library, thereby providing an environment-friendly non-toxic analysis method. The method has the advantages of convenient screening, time saving, labor saving, low cost and stable property, and can reduce the risk of exposure of laboratory personnel to toxic environments. In the field of immunodetection of toxic and harmful substances, the mimotope has wide application prospect in replacing antigen for immunoassay.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a non-diagnostic purpose detection method for tetrodotoxin.

It is a first object of the present invention to provide a tetrodotoxin mimotope peptide.

A second object of the present invention is to provide a gene encoding tetrodotoxin mimotope peptide.

It is a third object of the present invention to provide a plasmid.

It is a fourth object of the present invention to provide a recombinant microorganism.

A fifth object of the present invention is to provide a phage

A sixth object of the present invention is to provide a method for detecting tetrodotoxin with non-diagnostic purposes.

The seventh object of the present invention is to provide the use of one or more of said tetrodotoxin mimotope peptide, said gene, said plasmid, said recombinant microorganism, said phage for the establishment of a kit for the detection of tetrodotoxin and/or for the detection of tetrodotoxin for non-diagnostic purposes.

The eighth object of the invention is to provide a tetrodotoxin detection kit.

In order to achieve the above object, the present invention is realized by the following means:

(1) The purified tetrodotoxin monoclonal antibody is coated on a high-adsorption ELISA plate, the ELISA plate is blocked by 3% bovine serum albumin, then a random cyclic heptapeptide phage display library is added into the ELISA plate for panning, and the panning is carried out according to a combination-elution-amplification panning scheme and passes through 3 rounds of enrichment panning. Wherein, the dosage of antibody coated by the three rounds of elutriation screens and the dosage of tetrodotoxin standard used for competing and eluting phage are sequentially reduced;

(2) After 3 rounds of panning, 40 phage monoclonal were randomly selected for preliminary identification of phage ELISA, and the 12 positive clones obtained were amplified and sequenced to find 6 sequences in total.

According to the invention, phage clones of the mimotope peptide can be displayed by using a competition mode of panning, and phages displaying the mimotope protein compete with an object to be detected (tetrodotoxin) for a monoclonal antibody coated on an ELISA plate (the antibody can specifically recognize tetrodotoxin). The basis for judging positive phage clones is that the reaction well to which the mixture of phage and PBS is added has absorbance, i.e., binds to phage and antibody; meanwhile, the absorbance value of the phage in a control hole (the mixture of phage and PBS diluted analyte is added) is obviously reduced, namely the analyte can compete the phage from the binding site of the antibody, and the phage can display phage clone of the mimotope peptide. The phage takes M13 phage plasmid as a vector, and a gene encoding exogenous mimotope peptide is inserted into gIII gene of membrane protein encoded by phage, so that the exogenous protein can be displayed at N end of phage PIII capsid protein.

The invention therefore claims to be within the following:

a tetrodotoxin mimotope peptide has an amino acid sequence shown in SEQ ID No. 1, 3, 5, 7, 9 or 11.

Preferably, the amino acid sequence is shown in SEQ ID NO. 1.

A gene for coding tetrodotoxin analog epitope peptide, which codes for peptide with amino acid sequence as shown in SEQ ID No. 1, 3, 5, 7, 9 or 11.

Preferably, the peptide has the amino acid sequence shown in SEQ ID NO. 1.

Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO. 2, 4, 6, 8, 10 or 12.

More preferably, the nucleotide sequence of the gene is shown in SEQ ID NO. 2.

A plasmid comprising said gene and/or expressing said tetrodotoxin mimotope peptide.

A recombinant microorganism comprising said gene, and/or expressed tetrodotoxin mimotope peptide.

A phage having a tetrodotoxin mimotope peptide displayed on its surface.

Preferably, the bacterial cells use M13 phage plasmid as vector, the gene is inserted into the phage encoding membrane protein gIII gene.

A non-diagnostic target detection method of tetrodotoxin uses an anti-tetrodotoxin antibody as a coating antibody, and uses the tetrodotoxin mimotope peptide and/or phage as competitive antigens for enzyme-linked immunosorbent assay.

Preferably, the detection method is a competitive enzyme-linked immunoassay.

Preferably, the anti-tetrodotoxin antibody is an anti-tetrodotoxin monoclonal antibody.

Most preferably, the method comprises the steps of

1. Coating an antibody which specifically recognizes tetrodotoxin on a solid-phase carrier;

2、Phage ELISA：

adding phage clone with epitope peptide with amino acid sequence shown as SEQ ID NO. 1 on surface and sample to be tested or tetrodotoxin standard substance diluted in gradient into solid phase carrier coated with antibody, incubating, adding HRP-labeled anti-M13 phage HRP secondary antibody after PBST cleaning, cleaning with PBST again, incubating, adding TMB color development liquid, developing in dark, and 10% (v/v) H ₂ SO ₄ The reaction was terminated. The absorbance at 450nm was read.

3. Interpretation of results

And (3) taking the tetrodotoxin standard substance with each concentration as an abscissa, and the corresponding B as an ordinate (B is the light absorption value of a series of tetrodotoxin concentration holes), fitting a graph, and obtaining the detection range aiming at the tetrodotoxin from the graph. When the sample to be detected is detected, the reading of the light absorption value is obtained through the same reaction, and the corresponding toxin concentration can be found in the graph according to the reading result, namely the toxin concentration of the sample to be detected.

The application of one or more of the tetrodotoxin mimotope peptide, the gene, the plasmid, the recombinant microorganism and the phage in the establishment of a kit for detecting tetrodotoxin and/or for detecting tetrodotoxin for non-diagnostic purposes also belongs to the protection scope of the invention.

A tetrodotoxin detection kit is characterized by comprising one or more of tetrodotoxin mimotope peptide, gene, plasmid, recombinant microorganism and phage.

Preferably, the phage and anti-tetrodotoxin antibodies are contained.

Compared with the prior art, the invention has the following beneficial effects:

the invention establishes a sensitive and rapid competitive enzyme-linked immunoassay method by utilizing the phage of the obtained mimotope peptide; the IC50 of the method is 9.8ng/mL, and the detection range is 3.4-52.8 ng/mL. Compared with the sensitivity of the traditional hapten-based immunoassay method, the method is more sensitive; the method has good cross reaction to other marine toxins.

Drawings

FIG. 1 is a schematic of a phage display mimotope peptide panning.

FIG. 2 is the establishment of a standard curve for tetrodotoxin detection based on phage display mimotope peptides.

FIG. 3 is a graph showing the cross-reactivity of the phage and antibodies to other marine toxins.

Detailed Description

The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

1. Main experimental materials:

anti-tetrodotoxin monoclonal antibodies were purchased from Saint Jiekang Biotechnology Co., ltd. Without tin, and phage display heptacyclic peptides were purchased from NEB Co., ltd.)

2. The main reagent comprises:

peptone, yeast extract, agar, IPTG, xgal, PEG8000, horseradish peroxidase-labeled anti-M13 monoclonal antibody (Sino Biological)

3. The main reagent formula comprises:

LB liquid medium: 10g peptone, 5g yeast extract, 10g NaCl, and 1L tertiary water were added, autoclaved and stored at room temperature.

Top medium: 10g peptone, 5g yeast extract, 5g NaCl,7g agar, and tertiary water 1L were added, autoclaved and stored at room temperature.

Iptg+xgal:1.25g IPTG,1g Xgal, was dissolved in 25 mM MF and sterilized by organic membrane.

LB/IPTG/Xgal plates: 1L LB medium was added with 15g/L agar, sterilized, cooled to 70℃or below, and 1ml of an IPTG/Xgal mixture was added and mixed well, followed by pouring into a plate. The plate medium was stored (in a black bag) protected from light at 4 degrees.

Tet:20mg/mL, dissolved in absolute ethanol: water=1:1

20% PEG8000/NaCl:80g PEG8000, 58.44g NaCl, tertiary water to 400mL, autoclaving and storage at room temperature.

TBS: tris-HCl (ph=7.5) was first formulated: 15.764g Tris, tertiary water to 200mL, pH adjusted to 7.5 with HCl; 150mL of Tris-HCl (pH=7.5) was taken to dissolve 17.532g of NaCl and the volume was then fixed to 200mL. Sterilizing under high pressure and storing at room temperature.

Example 1 screening of mimotope peptides specifically binding to tetrodotoxin antibodies

1. Experimental method

1. Phage selection and amplification (technical route is shown in FIG. 1)

(1) Panning: with 0.1M NaHCO pH 8.6 ₃ The purified anti-tetrodotoxin antibody was diluted to 100. Mu.g/ml, 150. Mu.L was plated in a high-adsorption ELISA plate (3 replicates) and coated overnight at 4 ℃.

(2) The liquid in the ELISA plate of step (1) was aspirated, dried, and 5mg/ml BSA was added, and BSA was dissolved in 0.1M NaHCO3 at pH 8.6. The mixture was left at 4℃for 2 hours.

(3) After the blocking solution of the ELISA plate of step (2) was aspirated, the plate was washed 6 times with 0.1% TBST (25 ml TBST+25. Mu.L Tween). 100. Mu.L of 10 was added to each well in the ELISA plate ¹¹ The pfu/mL phage display heptapeptide library was incubated for 1 hour at room temperature.

(4) Wash 10 times with 0.1% tbst, add 100 μl of acid wash and incubate at room temperature for 10min.

(5) Collecting the supernatant of step (4), adding the competitive eluate (with a small amount of phage titer) into E.coli ER2738 (OD) containing 20mL ₆₀₀ 0.01 to 0.05) was incubated in a 250mL Erlenmeyer flask at 37℃with a shaker at 250rpm for 4.5h.

(6) The amplified phage were transferred to a 50mL centrifuge tube, and centrifuged at 12000rpm at 4℃for 10min to obtain the supernatant.

(7) Adding 20% PEG8000/NaCl 1/6 by volume into the supernatant of step (6), mixing thoroughly, and ice-bathing at 4deg.C overnight

(8) The solution from step (7) was centrifuged at 12000rpm at 4℃for 10min, the supernatant was removed, 1mL TBS was added to resuspend the pellet, and the same conditions were repeated once more.

(9) 350. Mu.L of TBS was resuspended in the pellet from step (8) for the next round of screening.

(10) Steps (1) to (9) are one round of screening and amplification, and (1) to (9) are repeated twice as a second round and a third round of screening and amplification, wherein the antibody concentration used in step (1) in the second round and the third round of screening and amplification is 100 mug/mL and 100 mug/mL, and the tetrodotoxin standard is 100 mug/mL and 50 mug/mL in step (4) in the second round and the third round of screening.

2. Determination of phage titer after eluent or amplification

(1) Taking 10mL of LB liquid medium, adding 0.1% of tetracycline, inoculating escherichia coli ER2738, culturing at 37 ℃ and rotating at 250rpm until OD600 is 0.5;

(2) The LB/IPTG/Xgal plates were oven-preheated for at least 1h at 37℃and the Top medium was preheated to a temperature of about 45 ℃.

(3) Diluting the eluent or amplified phage to a corresponding multiple, and diluting the eluent by 10-10 times ^-3 Double, phage dilution after amplification 10 ^-8 ～10 ^-10 Multiple times. Since the phage is enriched in a large amount, the eluent in the last round is diluted to 10 ^-3 ～10 ^-5 Multiple times.

(4) mu.L of phage of the corresponding dilution was added to 200. Mu.L of prepared E.coli ER2738 with OD600 of 0.5, and mixed well. Adding into 3mL of prepared Top culture medium, mixing, spreading onto the plate prepared in step (2), cooling for 10min, and culturing in an incubator at 37deg.C overnight.

(5) Blue phage spots on the plates were recorded and used to calculate the titer of phage tested.

2. Experimental results

The results are shown in Table 1 below.

Table 1 results of panning tetrodotoxin mimotopes using phage library:

the results showed that the panning was enriched from round 2, round 3 output was highest, and according to phage display library instructions, panning was only performed for 3 rounds, and according to this result several clones were picked from the titer plate of round 3 output for screening of positive clones.

Example 2 identification of mimotope peptides that specifically bind to tetrodotoxin monoclonal antibodies

1. Experimental method

(1) Example 1 following the third round of panning, the eluate was subjected to titer determination, and less than 100 blue phages were selected from the culture substrates, 40 blue plaques were randomly picked from the culture substrates, amplified and identified.

(2) The blue plaque amplification step is similar to the eluate amplification step. Single plaques were inoculated into a pellet containing 1mL ER2738 (OD ₆₀₀ 0.01 to 0.05), shaking table at 37℃at 250rpm, 4.5h.

(3) The culture broth was centrifuged at 12000rpm at 4℃for 10min, and the supernatant was taken for later positive cloning applications for identification and sequencing based on phage ELISA.

(4) The specific method for identification comprises the following steps:

anti-tetrodotoxin monoclonal antibody was diluted to 1. Mu.g/mL with PBS, 100. Mu.L was coated on a high-adsorption ELISA plate at 4℃overnight. The next day the elisa plate was washed twice with PBST (300 μl/well) and incubated with fish gelatin protein for 2 hours at 37 ℃.

50. Mu.L of the supernatant of (3) was mixed with an equal volume of 1. Mu.g/mL tetrodotoxin or PBS, respectively, and added to the microplate wells, while the nonspecific binding ability was detected using 1. Mu.g/mL BSA as a control. After 1h incubation at room temperature and 7 washes with PBST, 100. Mu.L of anti-M13 phage antibody-HRP was diluted 5000-fold (v/v) with PBST and added to the wells and incubated at 37℃for 30 min. The wells were washed 5 more times, 100. Mu.L of TMB liquid substrate buffer was added to each well and incubated at 37℃for 10 minutes. Finally, 50. Mu.L of 10% (v/v) H was used again ₂ SO ₄ The absorbance (450 nm) was read after termination of the reaction.

The criteria for selecting positive clones were: has a bindable antibody (absorbance higher than 1.5) and can be inhibited by tetrodotoxin (absorbance lower than 0.5), and has weak binding ability to BSA (absorbance lower than 0.2).

(5) Sequencing

The selected positive clone samples were sent to the Anshengda Biotechnology Co., ltd, sequenced using 96gIII primers, and the result was not detected, so that the target fragment was obtained by PCR.

Primers in the PCR procedure were designed based on the original sequence of M13KE at the web site neb.com, as shown in Table 2. PCR was performed using phage as template using the system and procedure shown in tables 3 and 4, and after verification by running the nucleic acid gel, the samples were sent to sequencing company to measure the sequence of the phage screened.

TABLE 2 primers designed when phage sequences were obtained

TABLE 3 PCR System used in obtaining phage sequences

TABLE 4 PCR System used in obtaining phage sequences

2. Experimental results

Of the 40 clones selected, 12 positive clones were selected and subsequently sequenced. After sequencing identification, 6 different mimotope peptide sequences were found.

The sequencing results are shown in Table 5

Table 5:

example 3 use of mimotope peptides specifically binding to tetrodotoxin antibodies as competitor antigens in ELISA methods

1. Experimental method

1. Antibody coating

Anti-tetrodotoxin monoclonal antibodies were diluted to 1. Mu.g/mL with PBS buffer, 100. Mu.L per well was added to the ELISA plate and incubated overnight at 4 ℃. The next day, the fish gelatin protein was blocked at 37℃for 2h by washing with PBST 2 times. After spin-drying, the dried product can be stored at 4 ℃ for subsequent experiments.

2. Establishment of a Standard Curve

Although 6 phages screened in example 2 can all simulate the binding of the mimic epitope peptide of tetrodotoxin and the monoclonal antibody thereof, and the sensitivity is not obviously different, the titer measurement result of the phages shows that certain difference exists in affinity according to the binding of the mimic epitope peptide of tetrodotoxin and the monoclonal antibody of tetrodotoxin when the absorbance value is taken to be 1-1.2, wherein the mimic epitope peptide of phage N.1 (the amino acid sequence is shown as SEQ ID NO:1, the encoding gene sequence is shown as SEQ ID NO: 2) has the strongest affinity and the best stability. In summary, a standard curve was constructed using phage N.1 with highest affinity (surface displaying epitope peptide with amino acid sequence shown in SEQ ID NO: 1).

Phage ELISA: 50. Mu.L of N.1 phage clone (surface displaying epitope peptide with amino acid sequence shown as SEQ ID NO: 1) was added to antibody-coated microwells together with 50. Mu.L of PBS or tetrodotoxin of the above series, incubated at 37℃for 45min, PBST washed 7 times, 100. Mu.L of 5000-fold (v/v) dilution of HRP-labeled anti-M13 phage HRP secondary antibody was added, washed 5 times again with PBST, 100. Mu.L of TMB color development liquid was added, light-shielding development was performed for 10min, 50. Mu.L of 10% (v/v) H ₂ SO ₄ The reaction was terminated and the absorbance at a wavelength of 450nm was read.

And fitting a graph line by taking the numerical value corresponding to each tetrodotoxin standard substance concentration as an abscissa and taking the corresponding B as an ordinate (B is the light absorption value of a series of tetrodotoxin concentration holes), and obtaining the detection range for the tetrodotoxin from the graph line.

2. Experimental results

The standard curve is shown in FIG. 2, and the IC for detecting tetrodotoxin ₅₀ The detection range is 9.8ng/mL, and the detection range is 3.4-52.8 ng/mL.

Example 4 evaluation of epitope peptide specificity that can specifically bind to tetrodotoxin antibody

1. Experimental method

3 marine toxins were measured with tetrodotoxin cross-reactivity (CR) as 100%: cross-reactivity of Saxitoxin (STX) among paralytic shellfish toxins, moxatoxin (GTX 2) among paralytic shellfish toxins, and microcystin (MC-LR).

2. Detection method

(1) 50. Mu.L of the N.1 phage clone (surface displaying epitope peptide with amino acid sequence shown in SEQ ID NO: 1) obtained by panning in example 2 was added to the antibody-coated microwells together with 50. Mu.L of the gradient-diluted test substance, incubated at 37℃for 40min, PBST was washed 7 times, 100. Mu.L of 10000-fold (v/v) diluted HRP-labeled anti-M13 phage HRP secondary antibody was added, incubated at 37℃for 30min, washed again with PBST 5 times, 100. Mu.L of TMB color development liquid was added, light-protected development was performed for 10min, 50. Mu.L of 10% H ₂ SO ₄ The reaction was terminated. Reading absorbance at 450nm wavelength with an ELISA readerAnd processes the data.

3. Experimental results

The results are shown in FIG. 3, and the cross-reactivity rate of the paralytic shellfish toxins and microcystins in the more common marine toxins is less than 0.1%. The obtained mimic epitope peptide (the amino acid sequence of which is shown as SEQ ID NO:1, and the encoding gene sequence of which is shown as SEQ ID NO: 2) specifically combined with tetrodotoxin antibody has good specificity, and can realize rapid detection of tetrodotoxin without interference.

Example 5A method for detecting tetrodotoxin

1. Antibody coating

Anti-tetrodotoxin monoclonal antibody was diluted to 1. Mu.g/mL with PBS, 100. Mu.L per well in an ELISA plate, and incubated overnight at 4 ℃. The next day, the fish gelatin protein was blocked at 37℃for 2h by washing with PBST 2 times. After spin-drying, the dried product can be stored at 4 ℃ for subsequent experiments.

2、Phage ELISA

50 μl of the N.1 phage clone having the epitope peptide having the amino acid sequence shown in SEQ ID NO:1 on the surface thereof was added to antibody-coated microwells together with 50 μl of tetrodotoxin standard (or sample to be tested), incubated at 37deg.C for 40min, PBST was washed 7 times, 100 μl of 5000-fold (v/v) dilution HRP-labeled anti-M13 phage HRP secondary antibody was added, washed 5 times with PBST again, incubated at 37deg.C for 3min, 100 μl of TMB color development liquid was added, light-shielding development was performed for 10min,50 μl of 10% (v/v) H ₂ SO ₄ The reaction was terminated. The absorbance at 450nm was read.

4. Interpretation of results

Example 6A kit for detecting tetrodotoxin

1. Composition of the composition

EXAMPLE 2 washing of N.1 phage displaying epitope peptide with amino acid sequence shown in SEQ ID NO:1, tetrodotoxin monoclonal antibody, horseradish peroxidase-labeled anti-M13 monoclonal antibody, PBST, TMB color development liquid, 10% (v/v) H ₂ SO ₄ 。

2. Application method

Same as in example 5.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

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Claims

1. A tetrodotoxin mimotope peptide is characterized in that the amino acid sequence of the tetrodotoxin mimotope peptide is shown as SEQ ID NO. 1.

2. A gene for coding tetrodotoxin analog epitope peptide is characterized in that the coded amino acid sequence is shown as SEQ ID NO. 1.

3. The gene according to claim 2, wherein the nucleotide sequence is shown in SEQ ID NO. 2.

4. A plasmid comprising the gene according to claim 2.

5. A recombinant microorganism comprising the gene of claim 2 and/or expressing the tetrodotoxin mimotope peptide of claim 1.

6. A phage, characterized in that the surface displays the tetrodotoxin mimotope peptide of claim 1.

7. A method for detecting the non-diagnostic purpose of tetrodotoxin, which is characterized in that an anti-tetrodotoxin antibody is used as a coating antibody, and the tetrodotoxin mimotope peptide according to claim 1 and/or the phage according to claim 5 are used as competing antigens for enzyme-linked immunosorbent assay.

8. Use of one or more of the tetrodotoxin mimotope peptides of claim 1, the genes of claim 2, the plasmids of claim 4, the recombinant microorganisms of claim 5, the phages of claim 6 for the establishment of a kit for the detection of tetrodotoxin and/or for the detection of tetrodotoxin for non-diagnostic purposes.

9. A kit for detecting tetrodotoxin, comprising one or more of the tetrodotoxin mimotope peptide of claim 1, the gene of claim 2, the plasmid of claim 4, the recombinant microorganism of claim 5, and the phage of claim 6.

10. The test kit according to claim 9, comprising the phage of claim 6 and an anti-tetrodotoxin antibody.