CN108802361A - A kind of preparation method of tetraiodothyronine enzyme conjugates - Google Patents
A kind of preparation method of tetraiodothyronine enzyme conjugates Download PDFInfo
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- CN108802361A CN108802361A CN201810550550.9A CN201810550550A CN108802361A CN 108802361 A CN108802361 A CN 108802361A CN 201810550550 A CN201810550550 A CN 201810550550A CN 108802361 A CN108802361 A CN 108802361A
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The present invention relates to a kind of preparation methods of tetraiodothyronine enzyme conjugates.This method uses tetraiodothyronine derivative（T4-NHS）Crosslinking is directly carried out with enzyme to be combined into, and need not additionally add activator and coupling agent.The tetraiodothyronine derivative is the chemical synthesis substance of n-hydroxysuccinimide (NHS) and tetraiodothyronine, or the chemical synthesis substance for the sulfonated HOSu NHSs of N- (Sulfo-NHS) and tetraiodothyronine, this method is easy to operate, of low cost, stability is good.
Description
Technical field
The present invention relates to a kind of tetraiodothyronine enzyme conjugates applied in chemiluminescence immunoassay technology
Preparation method.
Background technology
Tetraiodothyronine（Tetraiodothyronine, T4）It is the major hormone of thyroid gland, molecular weight is
777, half-life period is 6-7 days, is synthesized by the iodate of thyroglobulin tyrosine residue, by enzymolysis from thyroid gland ball
Albumen dissociation enters blood circulation, and the formation and release of T4 is by thyrotropic hormone（Thyroid Stimulating
Hormone, TSH）Adjusting.
Most of trilute in blood plasma（Triiodothyronine, T3）It is to be passed through in peripheral tissues by T4
5 ' deiodinations are metabolized, although T3 is thyroid hormone more more active than T4, the content that T4 is recycled in serum
Considerably beyond T3.T4 and T3 plays a part of adjusting internal various biochemical processes, to normal physiological metabolism and nerveous system
Activity of uniting is necessary.
T4 is most of in recycling in vivo to be combined with protein, and only 0.04% T4 is in free state, it is now recognized that trip
From T4 just have biological activity.Keep dynamic equilibrium, guarantee thyroid just with protein bound T4 and free hormone
Chang Gongneng.T4 binding proteins mainly have thyroid binding globulin（TBG）, prealbumin and albumin have accounted for about respectively
60%, 30% and 10%.The level of T4 can change with the change of TBG concentration, for example, being pregnant or taking contraceptive, long-term hepatitis
The level of TBG can be increased with courage hardening etc., nephrosis and androgen in treating etc. can then reduce.
The concentration of serum/plasma TT4 has close relationship with thyroid function, and the quantitative detection of TT4 is assessment thyroid gland
One important clinical indices of function and pathological state, for ensure thyroid function diagnosis accuracy, TT4 should same TSH,
The hormons such as Free T4, TT3 and Free T3 detect.
For the tetraiodothyronine that dissociates in vivo（FT4）With total tetraiodothyronine（TT4）What is measured is main
Method has radio immunoassay, enzyme linked immunosorbent assay analysis method, chemiluminescence immunoassay etc..Currently used radiation
Immunoassay（RIA）It is to use I125Label tetraiodothyronine haptens is come what is realized, and synthesis technology is complicated, effectively
Phase is short, has certain pollution to environment, the factor for influencing testing result is more.In recent years, chemiluminescence immunoassay technology was sent out
Exhibition is rapid, and sensitivity, specificity and the degree of automation have met or exceeded RIA levels, the especially stability of marker
With it is free from environmental pollution be that RIA methods are incomparable.
Current major Medium Sized Hospitals equal import automatic chemiluminescence immunoassay system, but instrument and reagent price
Costliness, enzyme marker synthesis technology is the highly confidential patented technology of external producer in kit.
Domestic manufacturer's enzyme linked immunosorbent assay analysis method（ELISA）Kit and chemiluminescence immunoassay（CLIA）Examination
The tetraiodothyronine haptens enzyme conjugates that agent box mainly uses is as marker, wherein tetraiodothyronine half
Antigen is tetraiodothyronine derivative（T4-CMO）With bovine serum albumin(BSA)（BSA）Conjugate（T4-CMO-BSA）Or
Chicken ovalbumin（OVA）Conjugate（T4-CMO-OVA）, production process is complex and costly for such enzyme conjugates, is easy
Generate the combination of steric interference tetraiodothyronine specific antibody.
Invention content
For overcome the deficiencies in the prior art, the invention discloses a kind of easy to operate, of low cost, stability it is good four
Iodine thyronine enzyme conjugates, the enzyme conjugates are the free tetraiodothyronines of chemiluminescence immunoassay detection
The key component of kit and total tetraiodothyronine kit.
A kind of preparation method of tetraiodothyronine enzyme conjugates, which is characterized in that the tetraiodo thyroid gland original ammonia
Sour enzyme conjugates is by tetraiodothyronine derivative（T4-NHS）It is undergone coupling reaction to produce with enzyme, which does not need
Additional addition activator and coupling agent.
Preferably, the tetraiodothyronine derivative is n-hydroxysuccinimide (NHS) and tetraiodo thyroid gland
The chemical synthesis substance of former propylhomoserin, chemical structural formula are：
Or the tetraiodothyronine derivative is the sulfonated HOSu NHSs of N- (Sulfo-NHS) and tetraiodo first
The chemical synthesis substance of shape gland original ammonia acid, chemical structural formula are：
Preferably, the tetraiodothyronine derivative is used is prepared with processing step：
S1. the preparation of tetraiodothyronine-succinate：It takes tetraiodothyronine to be added in distilled water, is added
Copper sulphate, back flow reaction 5 hours in 100 DEG C of water-baths after being cooled to room temperature, are filtered to remove solid impurity, then by filtrate ice
Bath cooling has crystallization to be precipitated for 2 hours, with ethyl alcohol recrystallization, obtains white flaky crystals tetraiodothyronine-succinate.
S2. the preparation of n-hydroxysuccinimide (NHS) and tetraiodothyronine synthetic：Step S1 is taken to obtain
Tetraiodothyronine-succinate be dissolved in citric acid solution（PH4.0）In, n-hydroxysuccinimide and 1- is added
(3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides, 2~8 DEG C of reaction 8h under the conditions of being protected from light, cross chromatographic column and collect N- hydroxyls
The eluent of base succinimide and tetraiodothyronine synthetic after concentration, is recrystallized with absolute ethyl alcohol, obtains white
The tetraiodothyronine derivative of acicular crystal.
Preferably, tetraiodothyronine derivative and enzyme are added in buffer solution, are protected from light, removed not
The tetraiodothyronine derivative that the reaction was complete, obtains tetraiodothyronine enzyme conjugates.
Preferably, the enzyme is alkaline phosphatase, horseradish peroxidase, beta galactosidase, urase, glucose-
One kind in 6- phosphate dehydrogenases, glucose oxidase and malic dehydrogenase, preferably alkaline phosphatase.
Preferably, the tetraiodothyronine derivative and enzyme are to be added in buffer solution according to a certain percentage,
The mass ratio of the tetraiodothyronine derivative and enzyme is 1:5~1:200, preferred 1:20~1:150, most preferably
It is 1:50~1:100.
Preferably, the buffer solution is EDTA buffer solutions, EGTA buffer solutions, citrate buffer system, phosphate delay
Flushing system, acetate salt buffer system, SSC buffer systems, SSPE buffer systems, 2- (N- morpholinoes) ethanesulfonic acids (MES) are slow
Any one of flushing system, piperazine-N, N ' bis- (2-ethanesulfonic acids) (PIPES) buffer system, preferably carbonate buffer solution.
Preferably, the ph value of buffer solution ranging from 7.0~10.0, preferably 8.0~9.0.
Preferably, the temperature being protected from light is 4 DEG C~37 DEG C, and the reaction time being protected from light is 0.5~24
Hour.
Preferably, the method for removing unreacted tetraiodothyronine derivative is ultrafiltration or dialysis or takes off
Salt plug method, preferably ultrafiltration.
The technical effects of the invention are that：
Tetraiodothyronine enzyme conjugates of the present invention is by tetraiodothyronine derivative（T4-NHS）With
Enzyme is undergone coupling reaction to produce, which need not additionally add activator and coupling agent.Other patents that compare or producer use
Be thyroxine-BSA derivatives（T4-CMO-BSA）Or thyroxine-OVA derivatives（T4-CMO-OVA）, this method cost
Lower, technique is simpler, more controllably.There may be the interference of certain steric hindrance for the high molecular weight protein derivative of thyroxine, influence
The accuracy of test result, and tetraiodothyronine derivative of the present invention is similar with thyroxine structure small
Molecular substance, the steric hindrance interference effectively avoided the problem that.
Specific implementation mode
It is further illustrated the present invention with reference to example, the advantages and features of the present invention becomes apparent from what is be described.But
It is to be understood that this example is only a kind of example of the present invention, any restrictions can't be done to the scope of the present invention.
Embodiment 1
100ug alkaline phosphatases are added in 1mL10mM sodium carbonate buffers (pH8.0), 1ug tetraiodo thyroid gland original ammonia is added
Acid derivative（T4-NHS）, after 37 DEG C is reacted 4 hours, enzyme labelled antibody is purified with ProteinG affinity columns (GE companies), obtains four
Iodine thyronine enzyme conjugates.
Embodiment 2
100ug alkaline phosphatases are added in 1mL10mM sodium carbonate buffers (pH8.0), 2ug tetraiodo thyroid gland original ammonia is added
Acid derivative（T4-NHS）, after 4 DEG C are reacted 24 hours, with the super filter tube of 15mL 30KD molecular cut offs, (Millipore is public
Department), ultrafiltration purification enzyme conjugates obtains tetraiodothyronine enzyme conjugates.
Embodiment 3
100ug alkaline phosphatases are added in 1mL10mM sodium carbonate buffers (pH8.0), 1ug tetraiodo thyroid gland original ammonia is added
Acid derivative（T4-NHS）, after 25 DEG C is reacted 8 hours, enzyme labelled antibody is purified with ProteinG affinity columns (GE companies), obtains four
Iodine thyronine enzyme conjugates.
Embodiment 4
100ug alkaline phosphatases are added in 1mL10mM phosphate buffers (pH8.0), 1ug tetraiodo thyroid gland original ammonia is added
Acid derivative（T4-NHS）, after 37 DEG C are reacted 4 hours, with the super filter tube of 15mL 30KD molecular cut offs, (Millipore is public
Department), ultrafiltration purification enzyme conjugates obtains tetraiodothyronine enzyme conjugates.
Tetraiodothyronine enzyme conjugates application experiment：The tetraiodothyronine enzyme that Examples 1 to 4 is generated
Rb in the tetraiodothyronine kit that conjugate is produced with certain import firm（Tetraiodothyronine enzyme conjugates）
Component is respectively applied to carry out contrast experiment in tetraiodothyronine chemical luminescence immune analysis reagent box, as a result as follows：
It can be seen that by above result：The tetraiodothyronine enzyme conjugates and foreign same type product of this technique synthesis
Applied in chemical luminescence immune analysis reagent box, believe it is dry than and stability test result very close to reaching good results, this hair
Bright tetraiodothyronine enzyme conjugates preparation method has good applicability and advance.
Embodiment described above is only the preferred embodiment of the present invention.It should be pointed out that dilution ratio of the present invention
Refer to that ratio diluted in mass ratio is not departing from the technology of the present invention side for those skilled in the art
Under the premise of case, some improvements and modifications can also be made, these improvement and modification also should be regarded as protection scope of the present invention, this
The available prior art of each component part being not known in embodiment is realized.
Claims (10)
1. a kind of preparation method of tetraiodothyronine enzyme conjugates, it is characterised in that：The tetraiodothyronine
Enzyme conjugates is by tetraiodothyronine derivative（T4-NHS）It is undergone coupling reaction to produce with enzyme, which does not need volume
Outer addition activator and coupling agent.
2. a kind of preparation method of tetraiodothyronine enzyme conjugates according to claim 1, it is characterised in that：Institute
State the chemical synthesis that tetraiodothyronine derivative is n-hydroxysuccinimide (NHS) and tetraiodothyronine
Substance, chemical structural formula are：
Or the tetraiodothyronine derivative is the sulfonated HOSu NHSs of N- (Sulfo-NHS) and tetraiodo first
The chemical synthesis substance of shape gland original ammonia acid, chemical structural formula are：
。
3. a kind of preparation method of tetraiodothyronine enzyme conjugates according to claim 1 or 2, feature exist
In：The trilute derivative is used to be prepared with processing step：
S1. the preparation of tetraiodothyronine-succinate：It takes tetraiodothyronine to be added in distilled water, is added
Copper sulphate, back flow reaction 5 hours in 100 DEG C of water-baths after being cooled to room temperature, are filtered to remove solid impurity, then by filtrate ice
Bath cooling has crystallization to be precipitated for 2 hours, with ethyl alcohol recrystallization, obtains white flaky crystals tetraiodothyronine-succinate；
S2. the preparation of n-hydroxysuccinimide (NHS) and tetraiodothyronine synthetic：Take four that step S1 is obtained
Iodine thyronine-succinate is dissolved in citric acid solution（PH4.0）In, n-hydroxysuccinimide and 1- (3- is added
Dimethylamino-propyl) -3- ethyl-carbodiimide hydrochlorides, react 8h for 2~8 DEG C under the conditions of being protected from light, cross chromatographic column and collect N- hydroxyls
The eluent of succinimide and tetraiodothyronine synthetic after concentration, is recrystallized with absolute ethyl alcohol, obtains white needle
The tetraiodothyronine derivative of shape crystallization.
4. a kind of preparation method of tetraiodothyronine enzyme conjugates according to claim 1, it is characterised in that：It will
Tetraiodothyronine derivative is added to enzyme in buffer solution, is protected from light, and the complete tetraiodo first of unreacted is removed
Shape gland original ammonia acid derivative, obtains tetraiodothyronine enzyme conjugates.
5. a kind of preparation method of tetraiodothyronine enzyme conjugates according to claim 4, it is characterised in that：Institute
The enzyme stated is alkaline phosphatase, horseradish peroxidase, beta galactosidase, urase, glucose-6-phosphate dehydrogenase (G6PD), grape
One kind in carbohydrate oxidase and malic dehydrogenase, preferably alkaline phosphatase.
6. a kind of preparation method of tetraiodothyronine enzyme conjugates according to claim 4, it is characterised in that：Institute
It is to be added in buffer solution according to a certain percentage that tetraiodothyronine derivative, which is stated, with enzyme, the tetraiodo thyroid gland original ammonia
The mass ratio of acid derivative and enzyme is 1:5~1:200, preferred 1:20~1:150, most preferably 1:50~1:100.
7. a kind of preparation method of tetraiodothyronine enzyme conjugates according to claim 4, it is characterised in that：Institute
It is EDTA buffer solutions, EGTA buffer solutions, citrate buffer system, phosphatebuffer buffer system, acetate salt buffer to state buffer solution
System, SSC buffer systems, SSPE buffer systems, 2- (N- morpholinoes) ethanesulfonic acid (MES) buffer system, piperazine-N, N ' are double
Any one of (2-ethanesulfonic acid) (PIPES) buffer system, preferably carbonate buffer solution.
8. a kind of preparation method of tetraiodothyronine enzyme conjugates according to claim 7, it is characterised in that：Institute
State ph value of buffer solution ranging from 7.0~10.0, preferably 8.0~9.0.
9. a kind of preparation method of tetraiodothyronine enzyme conjugates according to claim 4, it is characterised in that：Institute
It is 4 DEG C~37 DEG C to state the temperature being protected from light, and the reaction time being protected from light is 0.5~24 hour.
10. a kind of preparation method of tetraiodothyronine enzyme conjugates according to claim 4, it is characterised in that：
The method for removing unreacted tetraiodothyronine derivative is ultrafiltration or dialysis or desalting column method, is preferably surpassed
Filter method.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113214380A (en) * | 2021-05-06 | 2021-08-06 | 三诺生物传感股份有限公司 | Preparation method of alkaline phosphatase-labeled thyroxine |
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| CN102426255A (en) * | 2011-08-31 | 2012-04-25 | 内蒙古科慧生物科技有限责任公司 | Quantitative detection kit and detection method of free tetraiodothyronine (FT4) |
| CN104402003A (en) * | 2014-09-24 | 2015-03-11 | 海狸纳米科技（苏州）有限公司 | Magnetic microsphere for coupling biological ligand containing primary amino group and preparation method thereof |
| CN106053791A (en) * | 2016-06-30 | 2016-10-26 | 深圳市亚辉龙生物科技股份有限公司 | Anti-mullerian hormone chemiluminescence immunoassay kit and preparation method and application thereof |
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2018
- 2018-05-31 CN CN201810550550.9A patent/CN108802361A/en active Pending
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| EP0639272B1 (en) * | 1992-05-06 | 1996-09-04 | B.R.A.H.M.S Diagnostica GmbH | Method for the determination of the amount of a thyroid hormone ligand in a biological fluid and kit for carrying out such a method |
| CN102426255A (en) * | 2011-08-31 | 2012-04-25 | 内蒙古科慧生物科技有限责任公司 | Quantitative detection kit and detection method of free tetraiodothyronine (FT4) |
| CN104402003A (en) * | 2014-09-24 | 2015-03-11 | 海狸纳米科技（苏州）有限公司 | Magnetic microsphere for coupling biological ligand containing primary amino group and preparation method thereof |
| CN106053791A (en) * | 2016-06-30 | 2016-10-26 | 深圳市亚辉龙生物科技股份有限公司 | Anti-mullerian hormone chemiluminescence immunoassay kit and preparation method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113214380A (en) * | 2021-05-06 | 2021-08-06 | 三诺生物传感股份有限公司 | Preparation method of alkaline phosphatase-labeled thyroxine |
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Application publication date: 20181113 |