CN102175847A - Kit and method for detecting tetrodotoxin with gene engineering single-chain antibody - Google Patents
Kit and method for detecting tetrodotoxin with gene engineering single-chain antibody Download PDFInfo
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Abstract
The invention provides a kit and a method for detecting tetrodotoxin with a gene engineering single-chain antibody. The reagents in the kit comprise a sample extraction solution, a standard reagent, an enzyme-labeled antigen reagent, a cleaning solution, a bovine serum albumin (BSA) reagent, a chromogenic substrate and a stop solution. The content of tetrodotoxin in the sample is calculated through sample treating, reagent equilibrium, cleaning, sample adding, cleaning, developing and stopping. The anti-tetrodotoxin single-chain antibody employed in the kit can be highly and efficiently expressed in colon bacillus and massively produced as well. The invention is simple and feasible in preparation, time-saving, labor-saving and economical. When the kit is used for detecting tetrodotoxin, the contamination of the sample by tetrodotoxin can be determined and the content of tetrodotoxin can be calculated within 1.5-2 hours. The kit is convenient to use and low in cost.
Description
Technical field
The present invention relates to a kind of detection kit of utilizing phage single chain antibody to detect the ocean toxin, related more specifically to utilize phage single chain antibody to detect tetraodotoxin (tetrodotoxin, TTX) kit further relates to the method for using this kit to detect TTX.
Background technology
Tetraodotoxin (TTX) tetraodotoxin (Tetrodotoxin, TTX) be a kind of important marine active substance, because of its initial separation is gained the name, claim circle Puffer element (Spheroidine), salamanderin (Tarichatoxin), globefish toxin (Fugapoison) etc. again in the globe fish body.Tetraodotoxin is a kind of strong neural poison of non-albumen, and its toxicity is big 1000 times than sodium cyanide, belongs to alkaloid, and the local paralysis effect is also stronger 160,000 times than cocaine.Die 0.5mg tetraodotoxin can cause people's death by poisoning, the tetraodotoxin of 1g is enough to kill 3000 people.Because tetraodotoxin is distributed widely in various marine animals, plant, the microbial body, it is difficult to degraded under state of nature, and the product of part degraded also has certain toxicity.Filefish is not only own poisonous, also can pass through the food chain enrichment, and this existence of tetraodotoxin has seriously polluted maritime waters and all kinds of marine product, and there are huge threat in environment and food security.Detection method commonly used has technology such as HPLC method, surface plasma body resonant vibration, vapor-phase chromatography and monoclonal antibody detection method, but more or less there is following problem in these methods: complicated operating process, cost is big, consuming time, changeability is high, sensitivity is low and be subjected to sample quantitative limitation to be checked, can not provide deterministic evidence etc. to particular toxin.Therefore lack simple and effective detection method.
Summary of the invention
The object of the present invention is to provide a kind of simple, effectively utilize kit and method that phage single chain antibody detects tetraodotoxin, make that the testing process of tetraodotoxin is simple and easy to do, save time, laborsaving, economical,
Technical scheme of the present invention is as follows:
The kit that utilizes phage single chain antibody to detect the ocean toxin of the present invention, its reagent comprises:
(1) PBS of 0.02 mol/L of sample extracting solution: pH7.2;
(2) standard reagent: BSA-TTX toxin;
(3) enzyme-labelled antigen reagent: the PBS solution for 0.02 mol/L of the pH7.2 that contains BSA-TTX-HRP coupling protein, be stored in the glycerine of volume ratio 50% ,-20 ℃ of preservations, tiring is 5000; Use the PBS solution dilution during use;
(4) cleansing solution: TNT solution; Formula constituent: volume ratio is 0.05% Tween-20,20 mmol/L Tris-HCl, and 150 mmol/L NaCl, pH 7.4;
(5) the TNT solution of BSA BSA reagent: contain the 5%(weight ratio);
(6) chromogenic substrate: the 10 ml liquid that develops the color; Formula constituent: 250 μ L, 20 mg/mL DAB, 40 μ L, 40 mg/mL NiCl, the 1.0 mol/L Tris-HCl (pH 6.8) of 9.75 ml ,-20 ℃ of preservations; Add 7 μ L 30%(volume ratios during use) H
2O
2
(7) stop buffer: contain in the deionized water: 0.1 mol/L sodium sulphite, 2 mol/L sulfuric acid.
Use the above-mentioned kit that utilizes phage single chain antibody to detect the ocean toxin to detect the method for TTX toxin, may further comprise the steps successively:
(1) sample preparation: add the 4ml sample extracting solution in the 1g sample, liquid nitrogen or mechanical lapping are pulverized, ultrasonic extraction, centrifugal, and supernatant is the extract that contains sample;
The extract that contains sample is got with enzyme-labelled antigen reagent equal-volume evenly mixed, A reagent;
It is mixed evenly with enzyme-labelled antigen reagent equal-volume respectively that standard reagent is got series concentration, the B reagent of series concentration; When using, described enzyme-labelled antigen reagent uses earlier the PBS solution dilution;
(2) reagent balance: with the kit balance to room temperature;
(3) aperture numbering: take out enzyme mark bar and be placed on the reaction plate, negative hole, No. 1 hole of mark, the 2-6 hole is T-2 toxin standard control hole, all the other holes are sample well; Enzyme mark bar has had the anti-TTX toxin of immobilization single-chain antibody in every hole;
(4) washing: add 200～300 μ L cleansing solutions every hole in, cleansing solution must not overfolw hole outside, behind the placement 2min, remove cleansing solution, on thieving paper, pat dry, repeated washing is once;
(5) application of sample: No. 1 the hole adds 50 μ L BSA reagent, and the 2-6 hole adds the B reagent 50 μ L of series concentration respectively, and sample well adds corresponding A reagent 50 μ L, and light shaking makes each hole mixing;
(6) reaction: reaction plate is put into 37 ℃ of constant temperature ovens hatch 30 min;
(7) washing: take out reaction plate, remove reactant liquor, pat dry; Every hole adds 200～300 μ L cleansing solutions, and cleansing solution must not overflow, place 2 min after, remove cleansing solution, on thieving paper, pat dry repeated washing 4 times;
(8) colour developing: every hole adds chromogenic substrate 100 μ L, shakes up, and reaction plate is put into 37 ℃ of constant temperature ovens deposit 15 min;
(9) termination and Instrument measuring: every hole adds stop buffer 50 μ L respectively, measure the light absorption value A in each hole then at 450 nm places with microplate reader, series concentration with B reagent is a horizontal ordinate, with its corresponding light absorption value A is ordinate drawing standard curve, obtain the concentration of TTX in the sample according to typical curve, according to computing formula TTX content (μ g/g)=C*V/m, the concentration of C-TTX wherein, μ g/mL; The volume of V-sample extracting solution, mL; The m-sample quality, g; The content of TTX in the calculation sample.
The preparation method of described anti-TTX toxin single-chain antibody is as follows: use the Trizol kit to extract total RNA from the spleen of immune mouse, reverse transcription becomes cDNA, through the variable region of heavy chain V of pcr amplification cDNA
HGene and variable region of light chain V
LGene, pcr amplification program are 94 ℃ of 5 min; 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 45s, 33 circulations; 72 ℃ are extended 10 min; 1% agarose gel electrophoresis is verified pcr amplification product then; Because the PCR product all has only a band, reclaim V
HAnd V
LThe PCR product.By one section connection peptides (Linker, Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser) with V
HAnd V
LConnect into single-chain antibody (scFv), form single-chain antibody, then it is cloned into carrier (pCANTAB 5E) and goes up and make up phage antibody library, again by biological elutriation with the anti-TTX toxin single-chain antibody of acquisition to TTX toxin tool high-affinity.The amino acid sequence of described anti-TTX toxin single-chain antibody is shown in SEQ ID No.1.
The invention provides a kind of phage single chain antibody, its structure as shown in Figure 1.This single-chain antibody (scFv) is with the antibody heavy chain variable region (V of gene engineering method with 345 bp
H) and the variable region of light chain (V of 325 bp
L) recombinant protein that is formed by connecting by one section connection peptides (Linker); The size of anti-as shown in Figure 2 TTX toxin single-chain antibody (scFv) is 750 bp; This single-chain antibody (scFv) has kept the affine activity of antigen and the functional antibody fragment of specific minimum of parental antibody, can economy of large scale production in bacterium, make that the antibody producing that is used for immune detection is easy and economical, significantly reduced the expense of diagnostic reagent.The minimum detectable activity of kit of the present invention is 50 μ g/Kg.
Remarkable advantage of the present invention:
The anti-TTX toxin single-chain antibody that adopts in the kit of the present invention can efficiently express in Escherichia coli, and can be mass-produced, and preparation process is simple and easy to do, time saving and energy saving saving money.Utilize kit of the present invention to detect the TTX toxin, can determine in the 1.5-2 h time whether sample is subjected to the TTX endotoxin contamination, and the amount of contained TTX toxin, convenient to use, with low cost.
Description of drawings
Fig. 1 is a single-chain antibody structural representation of the present invention;
Fig. 2 is the electrophoretogram of single-chain antibody gene scFv amplification of the present invention;
Fig. 3 is single-chain antibody competitive ELISA detection curve figure of the present invention.
Embodiment
Be instantiation of the present invention below, further describe the present invention, but the present invention be not limited only to this.
Make the kit that utilizes phage single chain antibody to detect tetraodotoxin by following prescription:
(1) PBS of 0.02 mol/L of sample extracting solution: pH7.2;
(2) BSA-TTX toxin standard reagent: be respectively and contain 0.1,0. 2, the BSA-TTX toxin sample of 0.4,0.8,1.6 μ g/mL;
(3) enzyme-labelled antigen reagent: the PBS solution for 0.02 mol/L of the pH7.2 that contains BSA-TTX-HRP coupling protein, be stored in the glycerine of volume ratio 50% ,-20 ℃ of preservations, tiring is 5000; Use the PBS solution dilution during use;
(4) cleansing solution: TNT solution; Formula constituent: volume ratio is 0.05% Tween-20,20 mmol/L Tris-HCl, and 150 mmol/L NaCl, pH 7.4;
(5) BSA reagent: the TNT solution that contains 5% BSA;
(6) chromogenic substrate: the 10 ml liquid that develops the color; Formula constituent: 250 μ L, 20 mg/mL DAB, 40 μ L, 40 mg/mL NiCl, the 1.0 mol/L Tris-HCl (pH 6.8) of 9.75 ml ,-20 ℃ of preservations; Add 7 μ L 30%H during use
2O
2
(7) stop buffer: contain in the deionized water: 0.1 mol/L sodium sulphite, 2 mol/L sulfuric acid.
Detect the TTX toxin according to the mentioned reagent box by following program:
(1) sample preparation: adding 4ml sample extracting solution in 1g filefish sample (or flesh of fish samples such as abalone, hairtail, scallop, little yellow croaker), liquid nitrogen or mechanical lapping are pulverized, ultrasonic extraction 10 min, 5000rpm, centrifugal 10 min, supernatant is the extract that contains sample;
100 μ l got by the extract that contains sample and 100 μ l enzyme-labelled antigen reagent are evenly mixed, A reagent;
Each 100 μ L of BSA-TTX toxin standard reagent 0.1,0. 2,0.4,0.8, the 1.6 μ g/mL of series concentration are evenly mixed with 100 μ L enzyme-labelled antigen reagent respectively, the B reagent of series concentration; When described enzyme-labelled antigen reagent uses with PBS solution dilution (adding 1 μ L enzyme-labelled antigen reagent in 5 milliliters of PBS solution);
(2) reagent balance: kit is taken out from-20 ℃ of refrigerators, place more than 15 min, balance is to room temperature;
(3) aperture numbering: take out enzyme mark bar as required and be placed on the reaction plate support.Negative hole, No. 1 hole, 2～No. 6 the hole is BSA-TTX toxin standard control hole, all the other holes are sample well; Enzyme mark bar has had the anti-TTX single-chain antibody of immobilization in every hole; Described anti-TTX single-chain antibody preparation process is: extract total RNA from the spleen of immune mouse, become cDNA through reverse transcription, through pcr amplification antibody heavy chain variable region V
HGene and variable region of light chain V
LGene, by one section connection peptides (Linker) V
HAnd V
LConnect into single-chain antibody (scFv), be cloned into then and make up phage antibody library on the carrier, again by biological elutriation to obtain single-chain antibody to TTX tool high-affinity.
(4) washing: every hole adds 250 μ L cleansing solutions, and washing lotion must not be overflowed, and behind the placement 2min, removes cleansing solution, pats dry on thieving paper, repeats to wash plate once;
(5) application of sample: No. 1 the hole adds 50 μ L BSA reagent, and 2～No. 6 holes add the B reagent 50 μ L of series concentration respectively, and sample well adds corresponding A reagent 50 μ L, and light shaking makes each hole mixing;
(6) reaction: reaction plate is put into 37 ℃ of constant temperature ovens hatch 30 min;
(7) washing: take out reaction plate, remove reactant liquor, pat dry.Every hole adds 250 μ L cleansing solutions, and washing lotion must not be overflowed, place 2 min after, remove cleansing solution, on thieving paper, pat dry, repeat to wash plate 4 times;
(8) colour developing: every hole adds chromogenic substrate 100 μ L, shakes up, and reaction plate is put into 37 ℃ of constant temperature ovens deposit 15 min;
(9) stop and Instrument measuring: every hole adds stop buffer 50 μ L respectively, then with the light absorption value A of microplate reader in each hole of mensuration, 450 nm places, is horizontal ordinate with the series concentration of B reagent, is ordinate drawing standard curve with its corresponding light absorption value A.
To resist TTX single-chain antibody and the anti-E-tag antibodies that is fixed on the enzyme mark bar by the E-tag on the anti-TTX single-chain antibody, form insolubilized antibody; Add testing sample and enzyme-labelled antigen reagent, antigen in the sample and enzyme-labelled antigen competition combine with insolubilized antibody, antigenic content is high more in the testing sample, what then combine with insolubilized antibody is many more, the chance that makes enzyme-labelled antigen combine with insolubilized antibody is just few more, even the combination of having no chance, adding like this and just do not develop the color or develop the color behind the substrate very shallow, the dark person that develops the color is negative.As shown in table 1, along with the progressively rising of the concentration of the normal concentration BSA-TTX toxin that is added, corresponding light absorption value OD value gradually reduces.The BSA-TTX toxin normal concentration curve of drawing according to this data as shown in Figure 3, the concentration of OD450 nm place light absorption value and testing sample is inverse relation, but so according to the OD450 nm place light absorption value of testing sample concentration with regard to the TTX that contains in the judgement sample.
According to TTX content in the computing formula sample (μ g/g)=C*V/m, the concentration of TTX in the C-sample wherein, μ g/mL; The volume of V-sample extracting solution, mL; The m-sample quality, g; The content of TTX in the calculation sample.When light absorption value is 0.993, TTX content: 0.2*4/1=0.8 μ g/g.
The light absorption value that the competitive ELISA of table 1 single-chain antibody of the present invention detects
Annotate: when concentration is 0ng/mL, be enzyme-labelled antigen reagent and isopyknic PBS, other sample wells also add isopyknic BSA-TTX toxin standard reagent (concentration is respectively 0.1,0. 2,0.4,0.8,1.6 μ g/mL) respectively except enzyme-labelled antigen reagent.
＜110〉University Of Agriculture and Forestry In Fujian
＜120〉a kind of phage single chain antibody detects the kit and the method for tetraodotoxin
<160>?1
<210>?1
<211>?247
<212>?PRT
＜213〉BABA/c mouse (Mus musculus)
<400>1
Met?Ala?Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Ala?Glu?Leu?Ala?Arg?Pro
1 5 10 15
Gly?Ala?Ser?Val?Lys?Met?Ser?Cys?Lys?Asp?Gly?Ala?Tyr?Phe?Thr?Asn
20 25 30
Asn?Asn?Thr?Trp?Met?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Glu
35 40 45
Trp?Ile?Ser?Gly?Ser?Tyr?Thr?Thr?Tyr?Leu?Pro?Asp?Tyr?Gln?Phe?Lys
50 55 60
Asp?Phe?Lys?Asp?Lys?Ala?Thr?Leu?Thr?Ala?Asp?Glu?Ser?Ser?Ser?Thr
65 70 75 80
Ala?Phe?Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr
85 90 95
Tyr?Cys?Ile?Tyr?Phe?Ser?Asn?Ala?His?Pro?Trp?Asp?Leu?Ser?Val?Arg
100 105 110
Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser
115 120 125
Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln
130 135 140
Ser?Pro?Thr?Ile?Met?Ser?Ala?Ser?Leu?Gly?Glu?Lys?Val?Thr?Asn?Ala
145 150 155 160
Ser?Thr?Cys?Ser?Ser?Ser?Glu?Tyr?Leu?Tyr?Met?Arg?His?Trp?Tyr?Gln
165 170 175
Gln?Lys?Ser?Gly?Ala?Ser?Pro?Lys?Leu?Trp?Ile?Ala?Thr?Tyr?Pro?Gln
180 185 190
Tyr?Ser?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr
195 200 205
Ser?Tyr?Ser?Leu?Thr?Ile?Ser?Ser?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr
210 215 220
Tyr?Tyr?Cys?His?Arg?Thr?Tyr?Phe?Val?His?Ser?Ser?Arg?Thr?Gly?Gly
225 230 235 240
Thr?Lys?Leu?Glu?Ile?Lys?Arg
245
Claims (3)
1. a phage single chain antibody detects the kit of tetraodotoxin, and it is characterized in that: its reagent comprises:
(1) PBS of 0.02 mol/L of sample extracting solution: pH7.2;
(2) standard reagent: BSA-TTX toxin;
(3) enzyme-labelled antigen reagent: the PBS solution for 0.02 mol/L of the pH7.2 that contains BSA-TTX-HRP coupling protein, be stored in the glycerine of volume ratio 50% ,-20 ℃ of preservations, tiring is 5000; Use the PBS solution dilution during use;
(4) cleansing solution: TNT solution; Formula constituent: volume ratio is 0.05% Tween-20,20 mmol/L Tris-HCl, and 150 mmol/L NaCl, pH 7.4;
(5) the TNT solution of BSA BSA reagent: contain the 5%(weight ratio);
(6) chromogenic substrate: the 10 ml liquid that develops the color; Formula constituent: 250 μ L, 20 mg/mL DAB, 40 μ L, 40 mg/mL NiCl, the 1.0 mol/L Tris-HCl (pH 6.8) of 9.75 ml ,-20 ℃ of preservations; Add 7 μ L 30%(volume ratios during use) H
2O
2
(7) stop buffer: contain in the deionized water: 0.1 mol/L sodium sulphite, 2 mol/L sulfuric acid.
2. method of using phage single chain antibody to detect the kit of tetraodotoxin is characterized in that: may further comprise the steps successively:
(1) sample preparation: add the 4ml sample extracting solution in the 1g sample, liquid nitrogen or mechanical lapping are pulverized, ultrasonic extraction, centrifugal, and supernatant is the extract that contains sample;
The extract that contains sample is got with enzyme-labelled antigen reagent equal-volume evenly mixed, A reagent;
It is mixed evenly with enzyme-labelled antigen reagent equal-volume respectively that standard reagent is got series concentration, the B reagent of series concentration; When using, described enzyme-labelled antigen reagent uses earlier the PBS solution dilution;
(2) reagent balance: with the kit balance to room temperature;
(3) aperture numbering: take out enzyme mark bar and be placed on the reaction plate, negative hole, No. 1 hole of mark, the 2-6 hole is T-2 toxin standard control hole, all the other holes are sample well; Enzyme mark bar has had the anti-TTX toxin of immobilization single-chain antibody in every hole;
(4) washing: add 200～300 μ L cleansing solutions every hole in, cleansing solution must not overfolw hole outside, behind the placement 2min, remove cleansing solution, on thieving paper, pat dry, repeated washing is once;
(5) application of sample: No. 1 the hole adds 50 μ L BSA reagent, and the 2-6 hole adds the B reagent 50 μ L of series concentration respectively, and sample well adds corresponding A reagent 50 μ L, and light shaking makes each hole mixing;
(6) reaction: reaction plate is put into 37 ℃ of constant temperature ovens hatch 30 min;
(7) washing: take out reaction plate, remove reactant liquor, pat dry; Every hole adds 200～300 μ L cleansing solutions, and cleansing solution must not overflow, place 2 min after, remove cleansing solution, on thieving paper, pat dry repeated washing 4 times;
(8) colour developing: every hole adds chromogenic substrate 100 μ L, shakes up, and reaction plate is put into 37 ℃ of constant temperature ovens deposit 15 min;
(9) termination and Instrument measuring: every hole adds stop buffer 50 μ L respectively, measure the light absorption value A in each hole then at 450 nm places with microplate reader, series concentration with B reagent is a horizontal ordinate, with its corresponding light absorption value A is ordinate drawing standard curve, obtain the concentration of TTX in the sample according to typical curve, according to computing formula TTX content (μ g/g)=C*V/m, the concentration of C-TTX wherein, μ g/mL; The volume of V-sample extracting solution, mL; The m-sample quality, g; The content of TTX in the calculation sample.
3. detect the method for the kit of ocean toxin according to the described use phage single chain antibody of claim 2, it is characterized in that: the amino acid sequence of the described anti-TTX toxin single-chain antibody of step (3) is shown in SEQ ID No.1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102721818A (en) * | 2012-06-28 | 2012-10-10 | 广州瑞博奥生物科技有限公司 | Competitive inhibitory enzyme-linked immunoassay kit of visfatin and method |
| CN104133063A (en) * | 2014-06-09 | 2014-11-05 | 浙江省海洋水产研究所 | Tetrodotoxin detection kit |
| CN104531568A (en) * | 2014-12-12 | 2015-04-22 | 福建省水产研究所 | Anti-tetrodotoxin single-chain antibody and preparation method thereof |
| CN114634550A (en) * | 2022-03-01 | 2022-06-17 | 华南农业大学 | Non-diagnosis purpose detection method of tetrodotoxin |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001017196A (en) * | 1999-07-06 | 2001-01-23 | Japan Science & Technology Corp | Measurement of paralytic shellfish toxin |
| CN101781365A (en) * | 2010-01-29 | 2010-07-21 | 华南农业大学 | Artificial antigen of tetraodotoxin and corresponding specific antibody and preparation method and application thereof |
| CN101881771A (en) * | 2010-05-15 | 2010-11-10 | 福建农林大学 | Kit and method for detecting aflatoxin (AF)B1 of gene engineering single chain antibody |
-
2011
- 2011-01-26 CN CN 201110027079 patent/CN102175847B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001017196A (en) * | 1999-07-06 | 2001-01-23 | Japan Science & Technology Corp | Measurement of paralytic shellfish toxin |
| CN101781365A (en) * | 2010-01-29 | 2010-07-21 | 华南农业大学 | Artificial antigen of tetraodotoxin and corresponding specific antibody and preparation method and application thereof |
| CN101881771A (en) * | 2010-05-15 | 2010-11-10 | 福建农林大学 | Kit and method for detecting aflatoxin (AF)B1 of gene engineering single chain antibody |
Non-Patent Citations (2)
| Title |
|---|
| 孟宪梅: "河豚毒素免疫学检测方法的建立及三种海洋生物毒素融合单链抗体的初步研究", 《中国博士学位论文全文数据库 工程科技I辑》, 15 August 2010 (2010-08-15), pages 4 * |
| 孟宪梅等: "抗河豚毒素单链抗体基因的构建、表达及活性分析", 《中国卫生检验杂志》, vol. 19, no. 01, 10 January 2009 (2009-01-10), pages 14 - 16 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102721818A (en) * | 2012-06-28 | 2012-10-10 | 广州瑞博奥生物科技有限公司 | Competitive inhibitory enzyme-linked immunoassay kit of visfatin and method |
| CN104133063A (en) * | 2014-06-09 | 2014-11-05 | 浙江省海洋水产研究所 | Tetrodotoxin detection kit |
| CN104133063B (en) * | 2014-06-09 | 2016-01-06 | 浙江省海洋水产研究所 | A kind of tetraodotoxin detection kit |
| CN104531568A (en) * | 2014-12-12 | 2015-04-22 | 福建省水产研究所 | Anti-tetrodotoxin single-chain antibody and preparation method thereof |
| CN114634550A (en) * | 2022-03-01 | 2022-06-17 | 华南农业大学 | Non-diagnosis purpose detection method of tetrodotoxin |
| CN114634550B (en) * | 2022-03-01 | 2023-08-18 | 华南农业大学 | Non-diagnostic purpose detection method for tetrodotoxin |
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| CN102175847B (en) | 2013-10-23 |
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