CN1285611C - Synthetic process for alkaline phosphatase marked free thyroid hormone - Google Patents
Synthetic process for alkaline phosphatase marked free thyroid hormone Download PDFInfo
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- CN1285611C CN1285611C CN 200410097170 CN200410097170A CN1285611C CN 1285611 C CN1285611 C CN 1285611C CN 200410097170 CN200410097170 CN 200410097170 CN 200410097170 A CN200410097170 A CN 200410097170A CN 1285611 C CN1285611 C CN 1285611C
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Abstract
The present invention discloses a synthesis technology for alkaline phosphatase marked free thyroid hormone. A structure of alkaline phosphatase is modified to make the alkaline phosphatase coupled with free thyroid hormone in order to prepare a marked antigen. The technology of the present invention has the advantages of simplicity, convenience and low cost and solves the difficult problem puzzling the chemiluminescence immune assay field all the time that a key constituent alkaline phosphatase marker for detecting a free thyroid hormone kit is difficult of prepare.
Description
Technical field
The present invention relates to a kind of synthesis technique that is applied to the alkaline phosphatase marked free thyroid hormone in the chemiluminescence immunoassay technology.
Background technology
Triiodothyronine comprises thyroxine (Thyroxine, T4) and triiodothyronine (Triiodothyronine, T3), their overwhelming majority in blood plasma exist with the protein binding form, 0.2% the ratio of only being no more than is with free thyroxine (Free Thyroxine, FT4) and free triiodothyronine (Free Triiodothyronine, FT3) form exists, but have only the free form that physiologically active is just arranged, thereby free triiodothyronine (FT3) and free thyroid hormone (FT4) are the important indicators of diagnosis thyroid disease.
Radioimmunoassay (RIA) commonly used at present is to use
125I designate similar thing realizes that its synthesis technique complexity, validity period weak point have certain pollution to environment, and the factor that influences detected result is more.The research of chemiluminescence immunoassay technology (CLIA) has obtained breakthrough over surplus in the of nearly 10 year, no matter be sensitivity, easy fast and multinomial technical indicator such as level of automation all meet or exceed the RIA level, particularly the stability of marker and free from environmental pollution be that RIA is unrivaled.The equal import of domestic big-and-middle-sized hospital at present CLIA automatical analysis system.But instrument and reagent cost an arm and a leg, and particularly the marking process of test kit neutral and alkali Phosphoric acid esterase (Apase) is the highly confidential proprietary technology of external producer, up to now, do not see the report of this technology both at home and abroad as yet.
Summary of the invention
The object of the present invention is to provide the simple and easy to do synthesis technique with alkaline phosphatase polymer marked free thyroid hormone of a kind of technology, its synthetic alkaline phosphatase polymer-free thyroid hormone binding substances is the key ingredient that chemiluminescence immune assay detects the free thyroid hormone test kit.
The inventor is through lot of experiments, according to the molecular structure of free thyroid hormone and the physicochemical property of alkaline phosphatase, structure to alkaline phosphatase is transformed, and with the free thyroid hormone coupling with the preparation labelled antigen, develop a kind of simple and easy to do synthesis technique.
Specifically, the synthesis technique of alkaline phosphatase marked free thyroid hormone of the present invention comprises: alkaline phosphatase polymer and free thyroid hormone reaction are generated alkaline phosphatase polymer-free thyroid hormone binding substances.
Wherein, the reaction of described alkaline phosphatase polymer and free thyroid hormone is linked reaction.
Described linked reaction is alkaline phosphatase polymer and the reaction of free thyroid hormone in the presence of coupling agent, after for example alkaline phosphatase polymer and free thyroid hormone mix, drips coupling agent and react in the solution of pH 6～8.
In described linked reaction, the consumption of each reactant and coupling agent can be the conventional or known consumption in this area.The consumption of alkaline phosphatase polymer and free thyroid hormone also can be the alkaline phosphatase polymer: free thyroid hormone is the ratio of 1nmol: 2～10nmol.The coupling agent consumption can be according to the alkaline phosphatase polymer of the every 1nmol coupling agent with 20～50 μ L.Reaction medium can be conventional or known medium, for example physiological saline of pH 6～8 of this area.
Described alkaline phosphatase polymer and free thyroid hormone mix, and can be to prepare the polymeric normal saline solution of alkaline phosphatase earlier, add free thyroid hormone again, mix; Otherwise also can.
Described linked reaction can be to get the alkaline phosphatase polymer to pack in the dialysis tubing, with normal saline dialysis 6～8 hours, liquid is changed 1 time in the centre, get alkaline phosphatase polymer 2nmol and add physiological saline to 1000 μ L, add 5～20nmol free thyroid hormone, 100 μ L, thorough mixing dropwise adds 40～100 μ L coupling agents, room temperature reaction 2～4 hours in mixed solution.
Described coupling agent can be conventional or known coupling agent, for example glutaraldehyde of this area.
Synthesis technique of the present invention also comprises separated product from reaction system, described from reaction system the method for separated product can be this area routine or separation known method, for example adopt sephadex column partition method separated product.
The condition of described employing sephadex column partition method separated product can be that this area is conventional or known, for example reaction solution is added in the SephadexG100 post after the damping fluid balance use pH 6～8 in advance, damping fluid drip washing with pH6～8, flow velocity 0.1～0.4ml/min, collect leacheate, select the leacheate of second absorption peak of ultraviolet 280nm, be required product.
Described alkaline phosphatase polymer is with the polymer of alkaline phosphatase by 4～6 molecules of linked reaction generation, also can adopt the commercially available prod.
Described alkaline phosphatase polymer can be by the conventional or known method preparation in this area.Described alkaline phosphatase polymer can be prepared as follows: the ratio that coupling agent is added 20～50 μ L coupling agents in every milligram of alkaline phosphatase dropwise adds in pH6～8 alkaline phosphatase enzyme solution reacts, and then reaction solution is isolated the alkaline phosphatase polymer with the sephadex column partition method from reaction system.
Preferred, described alkaline phosphatase polymer adopts the preparation of following method: get alkaline phosphatase through normal saline dialysis after 18～24 hours, the ratio that adds 20～50 μ L coupling agents in every milligram of alkaline phosphatase dropwise adds, behind the room temperature reaction 1～2 hour, with 4 ℃ of dialysis of physiological saline 18～24 hours, liquid is changed 1～3 time in the centre, then reaction solution is added in the Sephadex G100 post after the damping fluid balance use pH6～8 in advance, damping fluid drip washing with pH6～8, flow velocity 0.1～0.4ml/min, collect leacheate, middle portion and the molecular weight of getting the absorption peak of ultraviolet 280nm are the alkaline phosphatase polymer liquid of 30～600,000 Da.
Described coupling agent is the conventional or known coupling agent in this area, is preferably glutaraldehyde.
The technical scheme of alkaline phosphatase polymer marked free thyroid hormone synthesis technique the best of the present invention may further comprise the steps:
(1) the polymeric preparation of alkaline phosphatase
Get alkaline phosphatase through normal saline dialysis 18～20 hours, add physiological saline to 5mg/mL, the ratio that adds 20～50 μ L coupling agents in every milligram of alkaline phosphatase dropwise adds coupling agent, behind the room temperature reaction 1～2 hour, reaction solution is packed in the dialysis tubing of diameter 8mm, with 4 ℃ of dialysis of physiological saline 18～20 hours, liquid is changed 2 times in the centre, the reaction solution adding is used in pH 7.0 phosphate buffer solns (PBS) the equilibrated Sephadex G100 post in advance, with pH 7.0PBS drip washing, flow velocity 0.2ml/min, leacheate is in charge of collection, middle portion and the molecular weight of getting the absorption peak of ultraviolet 280nm are the alkaline phosphatase polymer liquid of 30～600,000 Da, add the equivalent glycerol, and 4 ℃ of preservations are standby.
(2) preparation of alkaline phosphatase polymer-free thyroid hormone
(1) the alkaline phosphatase polymer of getting above-mentioned preparation is packed in the dialysis tubing, with normal saline dialysis 6～8 hours, liquid is changed 1 time in the centre, get alkaline phosphatase polymer 2nmol and add physiological saline to 1000 μ L, add 5～20nmol free thyroid hormone, 100 μ L, thorough mixing dropwise adds 40～100 μ L coupling agents, room temperature reaction 2～4 hours in mixed solution;
(2) reaction solution is added in the SephadexG100 post use in advance after the pH7.5Tris-HCl damping fluid balance, with the drip washing of pH7.5 Tris-HCl damping fluid, flow velocity 0.2ml/min collects leacheate, selects the leacheate of second absorption peak peak value of ultraviolet 280nm;
(3) leacheate of selecting is changed in the clean vial, add MgCl respectively
2And ZnCl
2Making its concentration is 2mmol/L and 0.2mmol/L, adds NaN again
3And bovine serum albumin (BSA) to make its concentration be 0.1% and 10mg/mL, treat that fully the dissolving back adds the equivalent glycerol, fully packing behind the mixing, 4 ℃ of preservations.
In the above-mentioned processing step, described coupling agent preferably is glutaraldehyde.Described reaction system is preferably the physiological saline of pH7.5.
Described alkaline phosphatase can adopt the commercially available prod, for example the product P 5521 of sigma company.The reagent of other uses such as coupling agent and instrument also are the commercially available prod.
The inventive method has solved and has perplexed in the chemiluminescence immune assay field difficult problem that the key ingredient alkali phosphatase enzyme mark thing that detects the free thyroid hormone test kit is difficult to prepare always.It is simple and easy to do, with low cost to adopt the inventive method to prepare alkaline phosphatase polymer-free thyroid hormone binding substances, and alkaline phosphatase links to each other with target molecule by linking agent, and its loss of activity is little.
Embodiment
Below will the invention will be further described by embodiment, these descriptions are not that content of the present invention is done further to limit.One skilled in the art will understand that to be equal to replacement to what technical characterictic of the present invention was done, or corresponding the improvement, still belong within protection scope of the present invention.
Embodiment 1: the polymeric preparation of alkaline phosphatase
(1) gets alkaline phosphatase 5mg (sigma product P 5521) through normal saline dialysis 20 hours, add physiological saline, dropwise add 150 μ L coupling agent glutaraldehyde to 1.0ml, behind the room temperature reaction 1 hour, pack in the diameter 8mm dialysis tubing, with 4 ℃ of dialysis of physiological saline 18 hours, liquid was changed 2 times in the centre.The reaction solution adding is used in 20mmol/L pH7.0PBS equilibrated 1.0 * 35cm SephadexG100 post in advance, with 20mmol/L pH7.0PBS drip washing, flow velocity 0.2ml/min, every 0.5mL collect 1 pipe, collect about 20 pipes altogether, detect each pipe with ultraviolet 280nm, the absorption peak of 280nm is divided into 3 parts, on ultraviolet spectrophotometer, surveys the OD value respectively, calculate 3 parts protein mass separately, every part adds equivalent AR level glycerol, 4 ℃ of preservations.
(2) the learn from else's experience enzymatic polymerization thing liquid of each 200 μ g of isolating 3 parts of post compares the enzymatic polymerization thing molecular weight of 3 parts of mensuration with standard molecular weight albumen at AmershamBiosciences SE400 standard vertical gel electrophoresis system.Record molar mass average＞600,000 Da of first part, about 30～600,000 Da of the molecular weight of second section, the molecular weight of third part is＜30 ten thousand Da approximately.Its polymkeric substance is respectively 6～8 polymers, 4～6 polymers and 2～4 polymers, and with the liquid reservation of second section, being can be for the alkaline phosphatase polymkeric substance of mark.
Embodiment 2-3: the polymeric preparation of alkaline phosphatase
Prepare the alkaline phosphatase polymer according to the method for implementing 1, just adding the coupling agent glutaraldehyde is 100 μ L, 250 μ L, and the room temperature reaction time is 2 hours, and rate of flow in rinse is 0.1ml/min, 0.4ml/min.
Embodiment 4: the preparation of alkaline phosphatase polymer-free thyroid hormone
(1) gets in embodiment 1 gained alkaline phosphatase polymer 1mg (the being 2nmol) dialysis tubing of packing into, to normal saline dialysis 6 hours, liquid is changed once in the centre, add physiological saline to 1000 μ L, add 10nmol free thyroid hormone 100 μ L, thorough mixing dropwise adds 60 μ L coupling agent glutaraldehyde, room temperature reaction 2.5 hours in mixed solution;
(2) reaction solution is added in the 1.0 * 35cm Sephadex G100 post use in advance after the 0.1mol/L pH7.5Tris-HCl damping fluid balance, with the drip washing of 0.1mol/L pH7.5Tris-HCl damping fluid, flow velocity 0.2ml/min, collect the every 0.5mLl pipe of leacheate, select the peak value leacheate of second absorption peak of ultraviolet 280nm;
(3) leacheate of collecting is changed in the clean vial, add MgCl respectively
2And ZnCl
2Making its concentration is 2mmol/L and 0.2mmol/L, adds NaN again
3And bovine serum albumin (BSA) makes its concentration be respectively 0.1% and 10mg/mL, and fully the dissolving back adds the equivalent glycerol, and fully packing 100 μ L/ prop up behind the mixing, and 4 ℃ of preservations are put in sealing.
Embodiment 5-7: the preparation of alkaline phosphatase polymer-free thyroid hormone
Prepare alkaline phosphatase polymer-free thyroid hormone according to the method for implementing 4, just add 5nmol, 15nmol, 20nmol free thyroid hormone, adding the coupling agent glutaraldehyde is 40 μ L, 80 μ L, 100 μ L, room temperature reaction 2 hours, 3 hours, 4 hours.
Embodiment 8: alkaline phosphatase polymer-free thyroid hormone binding substances application test
With embodiment 4 synthetic alkaline phosphatase polymers-free thyroid hormone binding substances (Apase-FT3, Apase-FT4) and the Apase-FT3 and the Apase-FT4 binding substances of gondola ADALTIS company be applied to respectively compare experiment in FT3 and the FT4 chemical luminescence immune analysis reagent box, comparing result sees Table 1:
The mass parameter comparing result of table 1 test kit
| Mass parameter | FT3 | FT4 | ||
| Product of the present invention | ADALTIS company | Product of the present invention | ADALTIS company | |
| Sensitivity | 0.17pmol/L | 0.15pmol/L | 0.08pmol/L | 0.06pmol/L |
| CV in crowd | 10.2％ | 11.3％ | 9.1％ | 8.5％ |
| The rate of recovery | 95.6％ | 96.3％ | 102.6％ | 97.0％ |
| Linear r value | 0.9976 | 0.9984 | 0.9981 | 0.9985 |
Respectively 216 routine serum samples are detected simultaneously, the result is as shown in table 2 below:
Table 2 serum sample detected result
| Project | Normal group (example) | The low group of first (example) | Hyperthyroid group (example) | Total coincidence rate | |||
| Product of the present invention | ADALTIS company | Product of the present invention | ADALTIS company | Product of the present invention | ADALTIS company | ||
| FT3 | 115 | 117 | 14 | 11 | 13 | 14 | 95.8％ |
| FT4 | 117 | 116 | 15 | 16 | 13 | 13 | 98.6％ |
Can find out from above contrast and experiment: the synthetic alkaline phosphatase condensate of this technique-free thyroid hormone bond and external like product are applied to the chemical luminescence immune analysis reagent box, that the mass parameter of kit or the result who detects clinical blood serum sample are very approaching, reach excellent results, alkaline phosphatase marked free thyroid hormone new technique for synthesizing of the present invention has fine applicability and advance.
Claims (10)
1, a kind of synthesis technique of alkaline phosphatase marked free thyroid hormone is characterized in that this technology comprises: alkaline phosphatase polymer and free thyroid hormone generate alkaline phosphatase polymer-free thyroid hormone binding substances by linked reaction.
2, synthesis technique according to claim 1 is characterized in that described linked reaction is after alkaline phosphatase polymer and free thyroid hormone mix, and drips coupling agent and reacts in the solution of pH 6～8.
3, synthesis technique according to claim 2, the ratio that it is characterized in that described linked reaction neutral and alkali Phosphoric acid esterase polymer and free thyroid hormone is the alkaline phosphatase polymer: free thyroid hormone is 1nmol: 2～10nmol, and the coupling agent consumption is that the alkaline phosphatase polymer of every nmol is with 20～50 μ L.
4, synthesis technique according to claim 1, it is characterized in that described linked reaction packs in the dialysis tubing for getting the alkaline phosphatase polymer, with normal saline dialysis 6～8 hours, liquid is changed 1 time in the centre, get alkaline phosphatase polymer 2nmol and add physiological saline to 1000 μ L, add 5～20nmol free thyroid hormone, 100 μ L, thorough mixing, in mixed solution, dropwise add 40～100 μ L glutaraldehyde, room temperature reaction 2～4 hours.
5, synthesis technique according to claim 1 is characterized in that also comprising with sephadex column partition method separated product from reaction system.
6, synthesis technique according to claim 5, it is characterized in that described sephadex column partition method separated product is that reaction solution is added in the Sephadex G100 post after the damping fluid balance use pH 6～8 in advance, damping fluid drip washing with pH 6～8, flow velocity 0.1～0.4ml/min, collect leacheate, selecting the leacheate of second absorption peak of ultraviolet 280nm is required product.
7,, it is characterized in that described alkaline phosphatase polymer is the polymer that generates 4～6 molecules with alkaline phosphatase by linked reaction according to the described arbitrary synthesis technique of claim 1-4.
8, synthesis technique according to claim 7, it is characterized in that described alkaline phosphatase polymer adopts following method to make: the ratio that coupling agent is added 20～50 μ L coupling agents in every milligram of alkaline phosphatase adds in the alkaline phosphatase enzyme solution of pH 6～8 reacts, and then reaction solution is isolated the alkaline phosphatase polymer with the sephadex column partition method from reaction system.
9, synthesis technique according to claim 7, it is characterized in that described alkaline phosphatase polymer adopts following method to make: get alkaline phosphatase through normal saline dialysis 18～24 hours, the ratio that adds 20～50 μ L coupling agents in every milligram of alkaline phosphatase dropwise adds glutaraldehyde, behind the room temperature reaction 1～2 hour, with 4 ℃ of dialysis of physiological saline 18～24 hours, liquid is changed 1～3 time in the centre, then reaction solution is added in the Sephadex G100 post after the damping fluid balance use pH 6～8 in advance, damping fluid drip washing with pH 6～8, flow velocity 0.1～0.4ml/min, collect leacheate, middle portion and the molecular weight of getting the absorption peak of ultraviolet 280nm are the alkaline phosphatase polymer liquid of 30～600,000 Da.
10, according to claim 3,8 arbitrary described synthesis technique, it is characterized in that described coupling agent is a glutaraldehyde.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200410097170 CN1285611C (en) | 2003-12-17 | 2004-12-13 | Synthetic process for alkaline phosphatase marked free thyroid hormone |
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|---|---|---|---|
| CN200310123659.8 | 2003-12-17 | ||
| CN200310123659 | 2003-12-17 | ||
| CN 200410097170 CN1285611C (en) | 2003-12-17 | 2004-12-13 | Synthetic process for alkaline phosphatase marked free thyroid hormone |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102115737B (en) | 2009-12-31 | 2015-06-03 | 深圳迈瑞生物医疗电子股份有限公司 | Reagent and method for stabilizing alkaline phosphatase or marker of alkaline phosphatase |
| CN102269762B (en) | 2010-06-04 | 2014-12-10 | 深圳迈瑞生物医疗电子股份有限公司 | Preparation method of conjugate and relative kit |
| CN102269761A (en) | 2010-06-04 | 2011-12-07 | 深圳迈瑞生物医疗电子股份有限公司 | Synthesis process for alkaline phosphatase conjugate |
| CN113214380B (en) * | 2021-05-06 | 2023-05-09 | 三诺生物传感股份有限公司 | Preparation method of alkaline phosphatase labeled thyroxine |
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