CN1844140A - Process for synthesizing artificial antigen of ciguatoxin - Google Patents
Process for synthesizing artificial antigen of ciguatoxin Download PDFInfo
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- CN1844140A CN1844140A CN 200510101906 CN200510101906A CN1844140A CN 1844140 A CN1844140 A CN 1844140A CN 200510101906 CN200510101906 CN 200510101906 CN 200510101906 A CN200510101906 A CN 200510101906A CN 1844140 A CN1844140 A CN 1844140A
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- ciguatoxin
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Abstract
The invention discloses a process for synthesizing artificial antigen of ciguatoxin by employing carboxyl groups in dichromic acid oxidized ciguatoxin molecules through connecting the carboxyl groups with amido groups on the carrier protein using carbodiimide as the cross linking agent, and carrying out dialytic cryodesiccation.
Description
Technical field
The invention belongs to the immunological technique field, be specifically related to a kind of synthesizing artificial antigen of ciguatoxin.
Background technology
Ciguatoxin (ciguatoxin CTX) is a kind of fat-soluble organic compound, and intramolecularly comprises 13 cyclic ethers that connect continuously, and chemical structural formula is as follows:
Ciguatoxin often is present in fish liver, the flesh of fish and the fish head of coral fish, is topmost a kind of human coral ichtyhotoxisin of poisoning that causes, toxicity is extremely strong, is 2.0ug/kg to the Lethal Dose 50 of primate.Ciguatoxin is eaten marine alga by small fish by food chain, and big fish swallowing little fish gathers layer by layer and accumulate.Ciguatoxin can be by pyrolytic decomposition, so the process of boiling can not be removed toxin.After toxin enters blood, need toxin to be discharged for a long time, the patient if touch ciguatoxin once more, even if eat deal seldom, also can produce toxicity symptom in the future.Toxicity symptom shows as the coral fish after a few hours that contains ciguatoxin on the feed, phenomenons such as stomach or neural system discomfort can appear, main symptom comprises vomiting, diarrhoea, bicker and tetraplegia, cold and hotly feels to put upside down, joint and myalgia etc., and illness can be kept a couple of days to a few weeks longer and not wait.
Though existing accurate to the instrumental analysis such as the method measurement results such as gas-chromatography, liquid chromatography and mass spectrum of ciguatoxin, need expensive instrument, for the pre-treatment complex operation of sample, the requirement of incompatible on-the-spot fast simple and batch samples residue detection.The method (United States Patent 6,174,690) that adopts biological method to measure ciguatoxin content is arranged, but the test process time is long abroad, sensitivity is low, is not extensive use of.Other has patent (the United States Patent 6 that adopts film immunization method mensuration ciguatoxin by the polyclone of preparation ciguatoxin and monoclonal antibody, 770,490 and United StatesPatent 6,770,490), but test process needs repeatedly incubation and washing, and the O-Phenylene Diamine that uses carinogenicity is as chromogenic substrate, complex steps is unsuitable in situ measurement.At present, the mensuration to ciguatoxin mainly adopts U.S. Oceanit TestSystems both at home and abroad, Inc. (
Www.cigua.com) company utilizes U.S. Patent number 6,770, the immunoassay kit of 490 developments, and the cost of a sample of this kit measurement of domestic purchase is more than 500 yuan.
Because the domestic measuring method report of not seeing ciguatoxin as yet, therefore, the necessary scientific research of carrying out this respect has the ciguatoxin method for quick of independent intellectual property right with exploitation.In order to set up the on-the-spot immune analysis method of ciguatoxin, at first must obtain anti-ciguatoxin antibody.And the ciguatoxin molecular weight is little, belongs to haptens, can not produce antibody by the direct immunization animal.Must at first itself and carrier protein coupling be prepared its complete antigen, just can bring out animal and produce antibody.
For this reason, by in the ciguatoxin molecule, producing carboxyl behind the potassium dichromate oxidation, with carbodiimide hydrochloride it is linked to each other with amino group on the protein then, the dialysis postlyophilization promptly gets the ciguatoxin artificial antigen, through gel electrophoresis and immune mouse proof synthetic ciguatoxin artificial antigen is successful, this synthesizing artificial antigen is easy and simple to handle, and is pratical and feasible.
Summary of the invention
The present invention is following realization:
This synthetic method has following steps: be carboxyl with the hydroxyl in the dichromic acid oxidation ciguatoxin molecule earlier, being cross-linking reagent with carbodiimide again links to each other the carboxyl on the ciguatoxin molecule with amino group on the carrier protein, the dialysis postlyophilization promptly gets the ciguatoxin artificial antigen.
When carrying out crosslinking reaction in the aforesaid method, getting the ciguatoxin solution 4.0mL that is oxidized to behind the carboxyl adds in 8.0～12.0mL carrier proteins liquid, add 1.0～3.0mL carbodiimide aqueous solution again, magnetic agitation, reaction is 24 hours under 15～25 ℃ of conditions, water intaking layer is packed in the dialysis tubing of dialysis membrane preparation of molecular weight 10,000 then, dialysis tubing is suspended from the PBS damping fluid of 2000mL beaker, after changing water under 4 ℃ of conditions in per 4 hours and changing water 8 times for 1 time altogether, the liquid freezing drying in the dialysis tubing is synthetic ciguatoxin artificial antigen.
Above-mentioned ciguatoxin is dissolved in the methylene dichloride, and concentration is 0.01～0.06mg/mL; Dichromic acid is dissolved in the pyridine, forms pyridinium dichromate, and concentration is 0.20～0.50mg/mL; In the methylene dichloride of 5.00mL ciguatoxin, add 0.20～0.80mL pyridinium dichromate then, reacted 1 hour.
Above-mentioned carrier protein can be used bovine serum albumin (BSA) or egg white protein (OVA), is dissolved in respectively in the distilled water, and concentration is 0.50～3.00mg/mL; Difunctional cross-linking reagent carbodiimide is made into 0.10～0.20mg/mL with distilled water.
Embodiment
Embodiment 1
Getting the 0.10mg ciguatoxin is dissolved in the 5.00mL methylene dichloride, adding 0.60mL concentration then is 0.40mg/mL pyridinium dichromate solution, react after 1 hour, getting wherein 4.00mL, to join 8.0mL concentration be in the 1.00mg/mL carrier bovine serum albumin liquid, adding 2.0mL concentration again is the carbodiimide aqueous solution of 0.10mg/mL, reaction is 24 hours under 25 ℃ of conditions of magnetic agitation, water intaking layer is packed in the dialysis tubing of dialysis membrane preparation of molecular weight 10,000 then, dialysis tubing is suspended from the PBS damping fluid of 2000mL beaker, after changing water under 4 ℃ of cold condition in per 4 hours and changing water 8 times for 1 time altogether, the liquid freezing drying in the dialysis tubing is synthetic ciguatoxin artificial immunization antigen (being called for short BSA-CTX).
Embodiment 2
Getting the 0.15mg ciguatoxin is dissolved in the 5.00mL methylene dichloride, adding 0.80mL concentration then is 0.50mg/mL pyridinium dichromate solution, react after 1 hour, getting wherein 4.00mL, to join 8.0mL concentration be in the 1.00mg/mL carrier ovalbumin liquid, adding 2.0mL concentration again is the carbodiimide aqueous solution of 0.10mg/mL, reaction is 24 hours under 25 ℃ of conditions of magnetic agitation, water intaking layer is packed in the dialysis tubing of dialysis membrane preparation of molecular weight 10,000 then, dialysis tubing is suspended from the PBS damping fluid of 2000mL beaker, after changing water under 4 ℃ of cold condition in per 4 hours and changing water 8 times for 1 time altogether, the liquid freezing drying in the dialysis tubing is synthetic ciguatoxin envelope antigen (being called for short OVA-CTX).
The evaluation of artificial antigen:
1 usefulness gel electrophoresis method is measured the coupling rate of artificial antigen, and the coupling rate of BSA-CTX is 13, and the artificial antigen coupling rate of OVA-CTX is 11.
2 immune animals proof has produced the antibody of anti-ciguatoxin: the experiment of (1) mouse immune is made into above-mentioned synthetic BSA-CTX with 0.8% physiological saline the solution of 0.5mg/mL, immune for the first time with 0.6mL complete Freund's adjuvant and the above-mentioned BSA-CTX balanced mix for preparing, after fully emulsified, every mouse (BaLb/c mouse, 6 ages in week) abdominal injection 0.2mL, be equivalent to 50 μ g protein, altogether 4 mouse of immunity.Every 2 weeks from the 2nd immunity with use the same method of incomplete Freund's adjuvant booster immunization 3 times more later on instead.The 4th immunity was got blood from mouse tail in back 15 days, the preparation antiserum(antisera).(2) ELISA competition inhibition test every hole in enzyme mark bar adds the OVA-CTX envelope antigen of 100 μ L with the carbonate buffer solution dilution of pH9.6, includes 1 μ g OVA-CTX, after 37 ℃ of constant temperature are hatched 1h, and 4 ℃ of refrigerator overnight of dislocation.Taking-up is given a baby a bath on the third day after its birth time with PBST, and (PBST:PBS 20nmoL/L, 0.05%Tween-20 pH7.4), dry.Therefrom getting three is placed on the enzyme plate, add (the PBS preparation of 50 μ L, two anti-diluents in every preceding 1-2 hole (totally 6 holes), include 0.1% gelatin), the 3-4 hole adds the standard ciguatoxin liquid 50 μ L with dimethyl sulfoxide (DMSO) and two anti-diluent preparing, include 0.05 μ g standard specimen ciguatoxin, the 5-6 hole is with the 3-4 hole, include 0.5 μ g standard specimen ciguatoxin, the 7-8 hole includes 5.0 μ g standard specimen ciguatoxin, the 9-10 hole includes 50 μ g standard specimen ciguatoxin, the 11-12 hole also adds 50 μ L, two anti-diluents, every preceding 10 holes all add 1: 2000 above-mentioned antiserum(antisera) 50 μ L with two anti-diluent preparing then, the 11-12 hole still adds 50 μ L, two anti-diluents, be placed on 37 ℃ of constant temperature water tanks after adding and hatch abundant reaction 2h, take out, it is inferior to give a baby a bath on the third day after its birth with PBST, every then hole adds 1: 5000 the sheep anti mouse ELIAS secondary antibody of 100 μ L with two anti-diluent preparing, 37 ℃ of constant temperature water tanks are hatched 1h, taking-up is given a baby a bath on the third day after its birth inferior with PBST, add O-Phenylene Diamine and the hydrogen peroxide substrate solution of 100 μ L with pH5.0 phosphoric acid-citric acid preparation, after 15min is placed in 37 ℃ of constant temperature water tank sealings, every hole adds 50 μ L, 10% vitriol oil, read the OD value with the 492nm wavelength then on microplate reader, it is as follows to get 6 hole average computation results:
Competition suppresses the ELISA experimental result
| Hole count | 1-2 | 3-4 | 5-6 | 7-8 | 9-10 | 11-12 |
| The OD value | 1.4231 | 1.115 | 0.943 | 0.753 | 0.321 | 0.079 |
Competition suppresses ELISA and experimental results show that mouse has produced the specific antibody of anti-ciguatoxin, proves that further artificial antigen synthesizes successfully.
Claims (4)
- The synthesizing artificial antigen of 1 one kinds of ciguatoxin, it is characterized in that this synthetic method has following steps: be carboxyl with the hydroxyl in the dichromic acid oxidation ciguatoxin molecule earlier, being cross-linking reagent with carbodiimide again links to each other the carboxyl on the ciguatoxin molecule with amino group on the carrier protein, the dialysis postlyophilization promptly gets the ciguatoxin artificial antigen.
- 2. the synthesizing artificial antigen of ciguatoxin according to claim 1, it is characterized in that: when carrying out crosslinking reaction in the aforesaid method, getting the ciguatoxin solution 4.0mL that is oxidized to behind the carboxyl adds in 8.0～12.0mL carrier proteins liquid, add 1.0～3.0mL carbodiimide aqueous solution again, magnetic agitation, reaction is 24 hours under 15～25 ℃ of conditions, water intaking layer is packed in the dialysis tubing of dialysis membrane preparation of molecular weight 10,000 then, dialysis tubing is suspended from the PBS damping fluid of 2000mL beaker, after changing water under 4 ℃ of conditions in per 4 hours and changing water 8 times for 1 time altogether, the liquid freezing drying in the dialysis tubing is synthetic ciguatoxin artificial antigen.
- 3. the synthesizing artificial antigen of ciguatoxin according to claim 2, it is characterized in that: above-mentioned ciguatoxin is dissolved in the methylene dichloride, and concentration is 0.01～0.06mg/mL; Dichromic acid is dissolved in the pyridine, forms pyridinium dichromate, and concentration is 0.20～0.50mg/mL; In the methylene dichloride of 5.00mL ciguatoxin, add 0.20～0.80mL pyridinium dichromate then, reacted 1 hour.
- 4. the synthesizing artificial antigen of ciguatoxin according to claim 2, it is characterized in that: above-mentioned carrier protein can be used bovine serum albumin (BSA) or egg white protein (OVA), is dissolved in respectively in the distilled water, and concentration is 0.50～3.00mg/mL; Difunctional cross-linking reagent carbodiimide is made into 0.10～0.20mg/mL with distilled water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510101906 CN1844140A (en) | 2005-12-02 | 2005-12-02 | Process for synthesizing artificial antigen of ciguatoxin |
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| CN 200510101906 CN1844140A (en) | 2005-12-02 | 2005-12-02 | Process for synthesizing artificial antigen of ciguatoxin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101963614A (en) * | 2010-09-03 | 2011-02-02 | 青岛科技大学 | The capillary electrophoresis electrochemical enzyme-linked immuno assay detects the method for ciguatoxin |
| CN105301253A (en) * | 2015-09-07 | 2016-02-03 | 北京勤邦生物技术有限公司 | T-2 toxin-enriched immunomagnetic bead as well as preparation method and application thereof |
-
2005
- 2005-12-02 CN CN 200510101906 patent/CN1844140A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101963614A (en) * | 2010-09-03 | 2011-02-02 | 青岛科技大学 | The capillary electrophoresis electrochemical enzyme-linked immuno assay detects the method for ciguatoxin |
| CN105301253A (en) * | 2015-09-07 | 2016-02-03 | 北京勤邦生物技术有限公司 | T-2 toxin-enriched immunomagnetic bead as well as preparation method and application thereof |
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