CN103159842A - Cys-Annexin V kit used for 99mTc labeling and preparation method and application thereof - Google Patents
Cys-Annexin V kit used for 99mTc labeling and preparation method and application thereof Download PDFInfo
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- CN103159842A CN103159842A CN2013100873145A CN201310087314A CN103159842A CN 103159842 A CN103159842 A CN 103159842A CN 2013100873145 A CN2013100873145 A CN 2013100873145A CN 201310087314 A CN201310087314 A CN 201310087314A CN 103159842 A CN103159842 A CN 103159842A
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Abstract
The invention belongs to the development technical field of single photon emission computed tomography (SPECT) and in particular relates to a Cys-Annexin V kit used for 99mTc labeling, a preparation method and application thereof in apoptosis detection. Each kit provided by the invention is prepared as follows: 25-150ug of Cys-Annexin V, 0.5-6mg of edetate, 5-40ug of tin salt and 0.1mmol of pH buffer agent. When the kit provided by the invention carries out 99mTc labeling for Cys-Annexin V, the operation is simple, the labeling rate is stable and larger than 95%, and the labeled product can be obviously distinguished from the impurities possibly appearing during the reaction. Compared with the labeling method in the existing technology, the method provided by the invention has obvious advantages.
Description
Technical field
The invention belongs to single photon emission computed tomography (SPECT) imaging technique field, be specifically related to a kind of for
99mThe Cys-Annexin V medicine box of Tc mark, and compound method with and in the application that detects aspect apoptosis.
Background technology
Apoptosis (Apoptosis) claims again apoptosis (Programmed Cell Death, PCD), is the important component part in cell life cycle, is to be different from the cell initiative death process by gene regulating downright bad and unexpected death.Apoptosis relates to the change of a series of biomolecules and morphocytology, wherein, phosphatidylserine in the Cell membrane lipids bilayer (PS) turn to skin by film inner layer, be exposed to cell surface, being the early stage event of apoptosis, before occurring in the apoptotic morphologic change, is also the primary event of apoptosis cascade reaction, the sign of being identified by phagocytic cell as apoptotic cell, thus the shrinkage of endochylema, chromatinic concentrating and nuclear dna degraded etc. further caused.Therefore, the PS that turns up is present most study and is hopeful most to detect target spot with the apoptosis of application prospect, but the early detection apoptosis has higher ageingly, normal utilizes the medicine with the PS specific binding to detect apoptosis both at home and abroad.
Annexin V (Annexin V claims again Annexin A5 and AnxA5) is a kind of human body endogenous protein, belongs to Ca
2+Dependency phospholipids incorporate protein family is to separate from placenta the earliest, has the effect that some anti-freezings reach anti-inflammatory admittedly, once is used as a kind of antithrombotics research.Chromosomal localization analysis demonstration, Annexin V is by the Annexin V genes encoding on the 4th the karyomit(e) 4q26-q28 that is positioned at the people, contains 319 each amino acid, comprises 70-80 amino acid repeating unit, molecular weight is about 35kD.Studies confirm that Annexin V can with the PS specific binding, avidity is up to 10
-9Mol/L.Therefore, carry out apoptosis imaging in body with radioisotope labeling Annexin V as developer, realize that apoptotic live body is without the wound video picture, with the generation of apoptosis in the Real-Time Monitoring body, become the research emphasis in monitoring active somatic cell apoptosis field, and obtained greater advance.
The synthetic key of Annexin V class developer is the mark of radionuclide, should obtain higher radiochemical purity, and the combination that can not damage again Annexin V simultaneously is active.At present, with
99mTc mark Annexin V most study,
99mThe advantage of Tc is that the transformation period is 6h, in time video picture, and the dosage that patient is subjected to is less, and can be obtained easily by the Mo/Tc producer.Yet,
99mThe direct mark Annexin of Tc V is more difficult, and mark rate is low, and can produce some metaproteins in labeling process, causes the radioactive uptake of liver to increase.Therefore, Annexin V
99mThe Tc mark is take indirect labelling as main, and difunctional intercalating agent commonly used has N-1-imino--4-sulfydryl butyl (Imino), quadrol halfcystine (EC), disulfide dinitrogen (N
2S
2, claim BTAP again) etc., the most common with hydrazino-nicotinamide (HYNIC), mark obtains
99mTc-HYNIC-Annexin V specific activity is higher, and radiochemical purity is greater than 90%, and the good assessment of acquisition in the apoptosis animal model.But these indirect labelling procedure more complicated are strict to technical qualification, and product needs purifying, especially is difficult for making medicine box, and clinical use is subject to certain limitation.
Up-to-date studies show that, Annexin V can be conducive to use in DNA recombinant expression after structural modification
99mTc directly carries out mark.The three-dimensional structure of Annexin V shows, its N-end and C-end all are positioned at the opposite of molecular film binding site, therefore the modification on Annexin V does not affect recombinant protein in the expression of Escherichia colli or folding in regrouping process, does not change the affinity that it is combined with film PS yet.7 amino acid have been added as foreign scholar Tait JF etc. at the N-end, wherein contain 1-2 cysteine residues, three kinds of mutant that obtain, difference called after: Annexin V-116, Annexin V-117 and Annexin V-118, domestic scholars Hua Zichun professors etc. add 10 continuous Histidines, called after His10-Annexin V at the N of Annexin V end, the Annexin V variant of these restructuring is followed the consistent of natural A nnexin V with film in conjunction with activity, and with
99mTc-HYNIC-Annexin V has more similar bio distribution.These show that the protein engineering transformation to Annexin V is feasible.
Chinese patent CN102690345A further discloses again a kind of new Annexin V variant that clone, expression and purifying obtain in intestinal bacteria, and this variant has only added 1 halfcystine (Cys-Annexin V) at the C-end of natural A nnexin V.Experimental result shows, when the SP column purification, with respect to independent Annexin V, Cys-Annexin V is combined more firmly with the SP post, need to just can be eluted with higher ionic strength, this has stronger metal ion binding ability from a side illustration Cys-Annexin V than Annexin V.And adopt direct labelling method that Cys-Annexin V is carried out
99mThe experiment of Tc mark also confirms
99mTc-Cys-Annexin V has the akin performance with Cys-Annexin V, and mark rate is up to 95% left and right.
But the applicant finds under study for action, put down in writing in this patent documentation with in prior art
99mWhen the method for Tc mark is carried out mark to Cys-Annexin V, the marked product that obtains
99mTc-Cys-Annexin V and other radioimpurity radioactive impurities are (as raw material Na
99mTcO
4And by product
99mTc colloid etc.) be difficult to distinguish, affect follow-up research and utilization.
Summary of the invention
For this reason, technical problem to be solved by this invention is in prior art
99mThe problem that in the method for Tc mark Cys-Annexin V, marked product and radioimpurity radioactive impurity are difficult to separate, and then provide that a kind of step is simple, mark rate is high, can be used for
99mThe medicine box of the Cys-Annexin V variant of Tc mark;
Further, the present invention's compound method that above-mentioned medicine box also is provided with and in the application that detects aspect apoptosis.
For solving the problems of the technologies described above, of the present invention for
99mThe Cys-Annexin V medicine box of Tc mark is characterized in that, every described formula comprises:
Cys-Annexin V 25-150μg；
Edetate 0.5-6mg;
Tin salt 5-40 μ g;
The pH buffer reagent is transferred pH6.0-8.0.
Described edetate is sodium ethylene diamine tetracetate or ethylenediamine tetraacetic acid (EDTA) potassium.
Described tin salt is one or more the mixture in tin protochloride, tin protofluoride, stannous tartrate, tin protobromide or stannous citrate.
Described pH buffer reagent buffer reagent is one or more mixture of phosphoric acid salt, Citrate trianion, ammonium salt, acetate, carbonate, borate, barbiturate, phthalate.
The present invention also provides a kind of above-mentioned being used for of preparing
99mThe method of the Cys-Annexin V medicine box of Tc mark, compound method of the present invention can directly be dissolved the reaction system that is hybridly prepared into 1ml according to the content of each component, directly injects the 10ml cillin bottle and carries out lyophilize to powdered form again.But in view of the less solution preparation tolerance range of reaction system problem more rambunctious, compound method of the present invention is selected namely to prepare the reaction system of 100 bottles of described medicine boxs take the 100ml reaction system as the preparation unit, then the mode of carrying out packing.
The preparation method of 100 bottles of Cys-Annexin V medicine boxs is as follows, take pH buffer reagent 10mmol, be dissolved in 90mL distilled water, the edetate that adds 50-600mg, the tin salt solution 0.25-2mL that adds again 2mg/mL adds the Cys-Annexin V solution 0.5-3mL of 5mg/mL at last, then to add distilled water to liquid cumulative volume be 100mL, be sub-packed in the cillin bottle of 10mL specification by the 1.0mL/ bottle again, namely get Cys-Annexin V medicine box after lyophilize.
Further, described lyophilize step is controlled the lyophilize temperature by gradient and described cillin bottle is carried out lyophilize is realized.
Preferably, described lyophilize step is that actual conditions is: control described freezing temp and be-40 ℃, freezing 4h; And vacuumize and make vacuum pressure reach 1-2Pa, more freezing 2h; With extremely-20 ℃ of temperature regulation, freezing 3h; Again with extremely-10 ℃ of temperature regulation, freezing 13h; Subsequently with temperature regulation to 10 ℃, dry 2h; With temperature regulation to 25 ℃, dry 4h namely gets described Cys-Annexin V medicine box after gland again.
Contain 25-150 μ g Cys-Annexin V, the tin salt of 5-40 μ g, the edetate of 0.5-6mg in every medicine box that preparation obtains.After medicine box added 1mL distilled water, after powder dissolution, the pH value scope of whole system was 6.0-8.0.
It is in 1% dilute hydrochloric acid that the present invention selects described tin salt is dissolved in concentration, then adds in reaction system and react.
It is a kind of that the present invention also provides
99mThe method of Tc mark Cys-Annexin V medicine box namely adds the Na of new drip washing in the above-mentioned medicine box
99mTcO
4Solution is put 35-37 ℃ of water-bath 15min and is namely got marked product
99mTc-Cys-Annexin V.
Further, described Na
99mTcO
4The volume that adds of solution is 1ml.
Described Na
99mTcO
4Solution can adopt that in prior art, any means well known to those skilled in the art obtains.
Adopt medicine box of the present invention to carry out mark, not only mark rate is higher, and detect and find to be separated preferably between each mark impurity that may exist and marked product, described detecting step carries out with the HPLC method, and described purification step adopts the HPLC method, and actual conditions comprises: select the TSKGEL gel column, take concentration as 0.05M, the phosphoric acid buffer of pH7.0 is moving phase, flow velocity is 0.8mL/min, and the ultraviolet detection wavelength is 278nm, and is described
99mThe retention time of Tc-Cys-Annexin V is 9.8min.
The present invention also provides a kind of labelled protein, the marked product that is namely obtained by above-mentioned medicine box and marking method.
And a kind of above-mentioned for
99mThe application of the Cys-Annexin V medicine box of Tc mark in detecting apoptosis.
Technique scheme of the present invention has the following advantages compared to existing technology:
1, of the present invention for
99mThe medicine box of the Cys-Annexin V of Tc mark has realized being undertaken that by the ligand exchange method Cys-Annexin V is carried out froze-dried kit direct first
99mThe Tc mark, not only simple to operate, and mark rate higher (reaching more than 95%), and by HPLC method detection display, marked product
99mOther radioimpurity radioactive impurities that may exist in Tc-Cys-annexin V and labeled reactant are (as raw material Na
99mTcO
4And by product
99mTc colloid etc.) obviously distinguish, be conducive to from now in clinical promoting the use of;
2, method steps of the present invention is simple, and the mark visual result is accurate, simultaneously due to labeling effciency up to more than 95%, need not specific purification step can directly use, therefore be easy to prepare medicine box, solved the problem that the marked product that in the prior art, other direct marking methods exist and impurity are easily obscured;
3, the labelled protein and the Cys-annexin V that adopt medicine box mark of the present invention to obtain have akin performance, after mark
99mTc-Cys-annexin V can be used as the apoptosis situation that developer is applied to detect cell, and the rat liver apoptosis experimental results show that, labelled protein of the present invention can intuitively reflect the apoptosis situation of cell, and the video picture result is consistent with the apoptotic result of situ end labeling detection, result is accurate, and medicine box of the present invention can be applicable to apoptotic detection.
Description of drawings
For content of the present invention is more likely to be clearly understood, the below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation, wherein
Fig. 1 is the HPLC color atlas of labelled precursor Cys-Annexin V of the present invention;
Fig. 2 is labelled protein in embodiment 2
99mThe HPLC color atlas of Tc-Cys-Annexin V;
Fig. 3 is in embodiment 3
99mThe HPLC color atlas of Tc-EDTA;
Fig. 4 is in embodiment 4
99mThe HPLC color atlas of Tc colloid;
Fig. 5 is Na in embodiment 4
99mTcO
4The HPLC color atlas;
Fig. 6 is in embodiment 5
99mThe distribution plan of Tc-Cys-Annexin V in the normal mouse body;
Fig. 7 is in embodiment 5
99mThe blood medicine clearance curve of Tc-Cys-Annexin V in the normal mouse body;
Fig. 8 is rat liver Apoptosis Model in embodiment 6
99mTc-Cys-Annexin V video picture figure;
Fig. 9 is the apoptosis situation that embodiment 7 situ end-labellings detect rat apoptosis liver cell;
Figure 10 is that the content of Cys-Annexin V in EXAMPLE Example 8 is on the impact of mark rate;
Figure 11 is the impact of tin salt content on mark rate in embodiment 9;
Figure 12 is that the content of edetate in embodiment 10 (EDTA) is on the impact of mark rate;
Figure 13 is the HPLC figure of reaction solution in embodiment 11.
Embodiment
the concrete process for preparation of the described medicine box of the present embodiment is as follows: take SODIUM PHOSPHATE, MONOBASIC 1.9mmol, Sodium phosphate dibasic 8.1mmol, be dissolved in 90mL distilled water, add the 150mg sodium ethylene diamine tetracetate, tin protochloride dilute hydrochloric acid (1% hydrochloric acid) the solution 1mL that adds 2.00mg/mL, the Cys-Annexin V solution 1.5mL that adds at last 5mg/mL, sterile filtration after mixing, be sub-packed in the 10mL cillin bottle by the 1.00mL/ bottle again, described cillin bottle is put into Freeze Drying Equipment, described Freeze Drying Equipment temperature is set is-40 ℃, carry out freezing 4h, opening subsequently vacuum pump vacuumizes (after the vacuum pump unlatching, whole process is all completed vacuumizing under state, until the medicine box freeze-drying is completed), make vacuum pressure reach 1-2Pa, freezing 2h again, with extremely-20 ℃ of temperature regulation, freezing 3h, subsequently with extremely-10 ℃ of temperature regulation, freezing 13h, again with temperature regulation to 10 ℃, dry 2h, again with temperature regulation to 25 ℃, dry 4h, namely get required froze-dried kit after gland, the material pulverulent solids that is white in color in medicine box.
Every medicine box contains 75.0 μ g Cys-Annexin V, the tin protochloride of 20.0 μ g and the sodium ethylene diamine tetracetate of 1.50mg, adds in medicine box that the pH value is 6.0-8.0 after 1mL distilled water.
Described Cys-Annexin V is provided by Jiangsu Babiao Biopharmaceutical Research Institute Co., Ltd..Using gene engineering technique utilizes escherichia coli cloning, expresses and purifying, adds 1 halfcystine at the C of Annexin V end, uses the metal affinity chromatography purifying, restructuring preparation Cys-Annexin V.Concrete preparation process and feature are seen Chinese patent CN102690345A.
With
99mThe method of Tc mark Cys-Annexin V medicine box
Get the medicine box for preparing in embodiment 1, add the Na of the new drip washing of 1ml in the described medicine box
99mTcO
4Solution is put 35-37 ℃ of water-bath 15min, obtains marked product
99mTc-Cys-Annexin V, described Na
99mTcO
4Solution is provided by clinical of Jiangsu Inst of Atomic Medical Sciences Nuclear Medicine, and exit dose is 18.5-555MBq.
Adopt the HPLC method that described marked product is detected, adopt TSK GEL gel column (SWG2000swxL), take the phosphoric acid buffer of concentration 0.05M, pH7.0 as moving phase, flow velocity 0.8mL/min, ultraviolet detection wavelength 278nm, and be provided with isotope detector.Detect unlabelled Cys-Annexin V with identical testing conditions simultaneously.As shown in Fig. 1-2, record
99mRetention time (the t of Tc-Cys-Annexin V
R) be 9.8min, consistent with the retention time that does not have markd Cys-Annexin V.Analyze by the HPLC color atlas, calculating mark rate is 98.5%.As seen, medicine box of the present invention not only can be realized using
99mTc mark Cys-Annexin V, and the efficient of mark is higher.
According to the described medicine box of the preparation of the method described in embodiment 1, its difference only is in order to incite somebody to action
99mTc-Cys-Annexin V and other radioimpurity radioactive impurities make a distinction, do not add Cys-Annexin V when the described medicine box of preparation, make and do not contain target-marking Cys-Annexin V in the medicine box that obtains, and the operation of other preparations and mark is constant, the marked product that obtains is mainly
99mTc-EDTA carries out HPLC with the testing conditions of HPLC purifying in embodiment 2 and detects, as shown in Figure 3,
99mRetention time (the t of Tc-EDTA
R) be 13.4min.
According to the described medicine box of the preparation of the method described in embodiment 1, its difference only is in order to incite somebody to action
99mTc-Cys-Annexin V and other radioimpurity radioactive impurities make a distinction, do not add Cys-Annexin V and sodium ethylene diamine tetracetate when the described medicine box of preparation, make and do not contain target-marking Cys-Annexin V and sodium ethylene diamine tetracetate in the medicine box that obtains, and the operation of other preparations and mark is constant, and the marked product that obtains is mainly
99mThe Tc colloid carries out HPLC with the testing conditions of HPLC purifying in embodiment 2 and detects, as shown in Figure 4,
99mRetention time (the t of Tc colloid
R) be 12.5min.
Testing conditions with HPLC purifying in embodiment 2 carries out HPLC detection, Na
99mTcO
4Retention time (the t of solution
R) be 15.9min, as shown in Figure 5.
Explanation in Fig. 1-5
99mTc-Cys-Annexin V and other radioimpurity radioactive impurities (as
99mThe Tc colloid,
99mTc-EDTA and Na
99mTcO
4) can separate fully, calculate by HPLC
99mThe mark rate of Tc-Cys-AnnexinV, not only feasible and accurately directly perceived.
By
99mThe bio distribution of Tc-Cys-Annexin V in Mice Body, the biodistribution characteristics that this tagged compound is described meet the requirement as developer.
Get 35 of ICR mouse, be divided at random 7 groups, 5 every group, through tail vein injection 0.2mL(3.7MBq) medicine box described in embodiment 2 carries out the marked product that marking operation obtains
99mTc-Cys-Annexin V is respectively at injecting rear 5,15,30,60,120,180,240min totally 7 time point sacrificed by decapitation.Take out blood, brain, the heart, liver, spleen, lung, kidney, stomach, small intestine, bladder, the internal organs such as muscle and bone are weighed rear with γ calculating instrument measurement radiocounting, calculate every gram internal organs percentage injection dose rate (%ID/g, the radiocounting of every gram internal organs/be injected into the interior gross activity of rat body to count * 100%), as shown in Figure 6, the radioactivity of absorbing in kidney is the highest, next is bladder, liver and lung, and the picked-up in brain is very low.With the match of DAS2.0 software, analyze
99mThe pharmacokinetic curve of Tc-Cys-AnnexinV in the normal mouse body as shown in Figure 7, meets the two-compartment model feature, and medicine for formula is: C=5.972e
-0.123t+ 0.877e
-0.005t, distribution phase transformation period (T
1/2 α) be 5.619min, remove phase transformation period (T
1/2 β) be 129.465min, average blood plasma clearance rate (CL) is 0.084L/min/kg, area (AUC under drug-time curve
0 → ∞Value) be 238.123%ID/g*min.
The rat liver Apoptosis Model
99mTc-Cys-Annexin V video picture
Select 4 of SD rats (Ministry of Health's nuclear medicine emphasis experiment provides), body weight 280-290g, with the standby rat liver Apoptosis Model of the cycloheximide dosimetric system of 5mg/kg, 3 rats are respectively through tail vein injection cycloheximide solution, and another 1 rat injecting normal saline compares.After 3 hours, 4 rats are respectively through tail vein injection 0.2mL(18.5MBq) embodiment 2 acceptance of the bid remembers
99mTc-Cys-AnnexinV, after the injection developer, 3h carries out the static planar imaging in forward and backward position, energy peak 140KeV, energy window 20%, image array 128 * 128, acquisition counter 1000K.As shown in Figure 8, the left side is model mouse, and the arrow place is the video picture of apoptosis liver, and the right side is the normal control mouse, and can be observed the liver position in the static planar imaging figure of the anteposition of left side model mouse has obvious radioactivity dense poly-.
After video picture finishes, rat is put to death, take out immediately liver, a part is fixed with formalin solution, another part is measured radiocounting with the γ calculating instrument, and weigh, calculate every gram internal organs percentage injection dose rate (%ID/g), the radioactivity (ID%/g) of model mouse liver picked-up is 2.83 times of control group.
The situ end labeling (TUNEL) of rat apoptosis liver detects
The TUNEL method is used for unicellular horizontal apoptosis immunohistochemical methods check and quantitative analysis, and its principle is marker DNA fracture position, and detailed step is as follows: fixedly after 24h, cut into slices, every thickness 4-5 μ m by paraffin embedding through formalin solution for (1) rat liver sample; (2) dimethylbenzene dewaxing 30min, after gradient ethanol aquation, PBS rinses, each 1min, (3) drip Proteinase K, 37 ℃ hatch 30min after, PBS washing 3 times, (4) drip TUNEL and detect liquid (available from green skies biotechnology research institute), 37 ℃ of lucifuges are hatched 1h, PBS washing 3 times, (5) with after anti-fluorescent quenching mounting fluid-tight sheet, 100 times of fluorescence microscopy Microscopic observations.As shown in Figure 9, the left side is model mouse, and the right side is control group, and in figure, the arrow indication is fluorescigenic apoptotic body, the visible more fluorescigenic apoptotic body of the liver of model mouse, and in the liver of control group fluorescigenic apoptotic body is just seldom.Described in this result and the embodiment of the present invention 2, the medicine box mark obtains
99mThe result of the rat liver apoptosis imaging of Tc-Cys-Annexin V is consistent, explanation
99mTc-Cys-Annexin V tagged compound can show the apoptosis situation of rat liver as developer.
When following each embodiment studies each component concentration and changes within the specific limits on the situation that affects of medicine box mark rate.
Embodiment 8
Keep constant by in embodiment 1 of other component concentrations, the content that changes Cys-Annexin V in medicine box is 10-200 μ g, press method and the mensuration mark rate of embodiment 2.Result as shown in figure 10, the content of Cys-Annexin V is in 25-150 μ g scope, the medicine box mark rate reaches more than 90%.
Embodiment 9
Keep constant by in embodiment 1 of other component concentrations, change that in medicine box, tin protochloride is tin protobromide, and its content is 1-100 μ g, press the method mensuration mark rate of embodiment 2.Result as shown in figure 11, the content of tin protochloride is in 5-40 μ g scope, the medicine box mark rate reaches more than 90%.
Keep constant by in embodiment 1 of other component concentrations, change that in medicine box, edetate is ethylenediamine tetraacetic acid (EDTA) potassium, and its content is 0.1-10mg, press method mark and the mensuration mark rate of embodiment 2.Result as shown in figure 12, the content of sodium ethylene diamine tetracetate is in the 0.5-6mg scope, the medicine box mark rate reaches more than 90%.
Embodiment 11
With art methods, Cys-Annexin V is carried out mark:
Get the SnCl that makes 3.5mg/ml in the PBS damping fluid that the 3.5mg tin protochloride is dissolved in 10ml pH7.4
2Standby.Under room temperature, get 100 μ L SnCl
2Solution adds the Cys-Annexin V20 μ L of 0.6 μ g/ μ L to react wherein.Add wherein volume less than 0.1ml, exit dose is the Na of 185MBq
99mTcO
4Then solution splash into the static 30min reaction of 10mg/mL Vc10 μ L.With the condition detection reaction liquid of HPLC described in embodiment 2, result as shown in figure 13.As seen, carry out Cys-Annexin V mark with method of the prior art, the impurity that exists in method is difficult to distinguish with labelled protein, easily affects on the one hand the detection of Product Labeling rate, also makes the purity of product be affected simultaneously.
Obviously, above-described embodiment is only for example clearly is described, and is not the restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here need not also can't give all embodiments exhaustive.And the apparent variation of being extended out thus or change still are among the protection domain of the invention.
Claims (11)
1. one kind is used for
99mThe Cys-Annexin V medicine box of Tc mark is characterized in that, every described medicine box formula comprises:
Cys-Annexin V 25-150μg；
Edetate 0.5-6mg;
Tin salt 5-40 μ g;
The pH buffer reagent is transferred pH6.0-8.0.
According to claim 1 for
99mThe Cys-Annexin V medicine box of Tc mark is characterized in that:
Described edetate is sodium ethylene diamine tetracetate or ethylenediamine tetraacetic acid (EDTA) potassium.
According to claim 1 and 2 for
99mThe Cys-Annexin V medicine box of Tc mark is characterized in that:
Described tin salt is one or more the mixture in tin protochloride, tin protofluoride, stannous tartrate, tin protobromide or stannous citrate.
According to claim 3 for
99mThe Cys-Annexin V medicine box of Tc mark is characterized in that:
Described pH buffer reagent is one or more mixture of phosphoric acid salt, Citrate trianion, ammonium salt, acetate, carbonate, borate, barbiturate, phthalate.
A preparation claim 1-4 arbitrary described for
99mThe method of the Cys-Annexin V medicine box of Tc mark, it is characterized in that: take pH buffer reagent 10mmol, be dissolved in 90mL distilled water, the edetate that adds 50-600mg, the tin salt solution 0.25-2mL that adds again 2mg/mL, the Cys-Annexin V solution 0.5-3mL that adds at last 5mg/mL, mixing and add distilled water to cumulative volume is 100mL, be sub-packed in by the 1.0mL/ bottle in the cillin bottle of 10mL specification, and it is Powdered that described cillin bottle lyophilize to its inner material is white in color, and namely gets described Cys-Annexin V medicine box.
6. preparation according to claim 5 is used for
99mThe method of the Cys-Annexin V medicine box of Tc mark is characterized in that, described lyophilize step is controlled the lyophilize temperature by gradient described cillin bottle is carried out the lyophilize realization.
7. according to claim 5 or 6 described preparations are used for
99mThe method of the Cys-Annexin V medicine box of Tc mark is characterized in that, described lyophilize step is that actual conditions is: control described freezing temp and be-40 ℃, freezing 4h; And vacuumize and make vacuum pressure reach 1-2Pa, more freezing 2h; With extremely-20 ℃ of temperature regulation, freezing 3h; Again with extremely-10 ℃ of temperature regulation, freezing 13h; Subsequently with temperature regulation to 10 ℃, dry 2h; With temperature regulation to 25 ℃, dry 4h namely gets described Cys-Annexin V medicine box after gland again.
8. according to claim 5 or 6 or 7 described preparations are used for
99mThe method of the Cys-AnnexinV medicine box of Tc mark is characterized in that: take concentration as the described tin salt of 1% diluted hydrochloric acid dissolution.
9. one kind
99mThe method of Tc mark Cys-Annexin V is characterized in that: the Na that adds new drip washing in the arbitrary described medicine box of claim 1-4
99mTcO
4Solution is put 35-37 ℃ of water-bath 15min, namely gets marked product
99mTc-Cys-Annexin V.
10. a labelled protein, is characterized in that, the marked product that obtains for marking method claimed in claim 9.
11. a claim 1-4 arbitrary described for
99mThe application of the Cys-Annexin V medicine box of Tc mark in detecting apoptosis.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115429904A (en) * | 2022-09-23 | 2022-12-06 | 江苏省原子医学研究所 | 99m Tc marked freeze-dried product medicine box and its preparation method and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105669855A (en) * | 2016-02-04 | 2016-06-15 | 江苏省原子医学研究所 | Method for rapidly marking Cys-Annexin V through <18>F and application of method |
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| CN115429904A (en) * | 2022-09-23 | 2022-12-06 | 江苏省原子医学研究所 | 99m Tc marked freeze-dried product medicine box and its preparation method and use |
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