CN104491881A - Fluorescence-coupled specific sentinel node photographic developer and preparation method thereof - Google Patents
Fluorescence-coupled specific sentinel node photographic developer and preparation method thereof Download PDFInfo
- Publication number
- CN104491881A CN104491881A CN201410742313.4A CN201410742313A CN104491881A CN 104491881 A CN104491881 A CN 104491881A CN 201410742313 A CN201410742313 A CN 201410742313A CN 104491881 A CN104491881 A CN 104491881A
- Authority
- CN
- China
- Prior art keywords
- rituximab
- lymph node
- fluorescent material
- purification
- nhs
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention provides a fluorescence-coupled specific sentinel node photographic developer and a preparation method thereof and belongs to the field of preparation of biological medicines. The specific sentinel node photographic developer disclosed by the invention is formed by separating and purifying rituximab coupled with Cy5.5-N-hydroxyl pyrrolidine dione ester (Cy5.5-NHS) which is Cy5.5-SLN-F for short. The photographic developer is convenient to use, stable in performance, as long as 24 months in preservation time, above 95% in labeling rate, high in operability, applicable to noninvasive detection of an infrared-developed sentinel node, low in ingestion by a secondary lymph node, low in retention at an injection point, not limited in development and biopsy time, good in development within 30min to 24 hours after injection, as high as 100% in development rate, higher in safety, low in cost and relatively good in clinical application prospect.
Description
Technical field
The present invention relates to field of biological pharmacy, particularly, relate to specific sentinel lymph node developer of a kind of fluorescence coupling and preparation method thereof.
Background technology
Operation is the Main Means for the treatment of breast carcinoma, because Status of axillary lymph node is the important independence prognostic factor affecting breast cancer disease people living, conventional ALND is as a kind of means of Status of axillary lymph node Diagnosis and Treat clinically, and simple breast excision to add ALND (axillary lymph node dissection, ALND) be the most frequently used modus operandi.Along with examination and the early diagnosis of breast carcinoma, 50% ~ 70% postoperative pathological axillary-node-negative in newfound patient with breast cancer, implements ALND to this some patients and belongs to over-treatment; Therefore the reasonability that all patient with breast cancers all carry out ALND is under suspicion, the urgent axillary fossa technology-sentinel lymph node biopsy in breast cancer (sentinel lymph node biopsy, SLNB) by stages expecting to substitute accurate, the Wicresoft of ALND.SLN is first lymph node of breast carcinoma lymph metastasis, can obtain the diagnostic significance of 95%, few intercurrent disease.
SLNB, owing to can provide comparatively accurately by stages, reducing traditional operation complication and one of focus becoming research, is also be expected to one of standardization operation formula becoming breast cancer treatment.In numerous research, the radioactive indicator chosen be nearly all rely on lymph node phagocytosis to carry out detecting radiocolloid (
99mtc-sulfur colloid or
99mtc-human serum albumin etc.), its granular size is uneven, and Secondary Lymphoid system has development, and each to inject tracer particles sum wayward, and video picture and biopsy time requirement high, otherwise non-SLN all can be caused develop, cause SLNB unsuccessfully.Ratify the new drug-Lymphoseek being used for lymph node location in March, 2013 Bureau of Drugs Supervision of the U.S. (FDA) first.It can with lymphatic reticular endothelial cell surface mannose receptor (mannose receptor, CD206) specific binding, accurately detect primary tumo(u)r (breast carcinoma, melanoma) to lymph metastasis situation.Under the prerequisite of offshore company's patent, technical protection, need Development of Novel specific lymph node location developer badly, to tackle clinical demand.
Summary of the invention
The object of the present invention is to provide a kind of easy to use, stable performance, the workable and specific sentinel lymph node developer of the fluorescence coupling of seedless agent radiation.
Another object of the present invention is to provide the above-mentioned preparation method preparing the specific sentinel lymph node developer of fluorescence coupling.
The specific sentinel lymph node developer of fluorescence coupling provided by the invention is obtained after separation and purification by the Rituximab of fluorescent material coupling.
Particularly, the fluorescent material that the present invention uses is Cy5.5-N-hydroxysuccinimide eater (Cy5.5-NHS) or Cy7-NHS.Cy5.5-NHS is used in the embodiment of the present invention.
The invention provides the method for the specific sentinel lymph node developer preparing fluorescence coupling, containing following steps:
(1) purification Rituximab, and allocate its concentration be applicable to fluorescent material coupling;
(2) in PBS buffer solution, make the abundant coupling of Rituximab that fluorescent material and step (1) obtain;
(3) separation and purification conjugate.
In said method of the present invention, the method for step (1) purification is: carry out purification by gel chromatographic columns to rituximab.
In method of the present invention, step (1) adopts the Mabthera monoclonal antibody concentration after PBS buffer solution allotment purification to be 5 ~ 10mg/mL.
Further, described PBS buffer concentration is 0.01 ~ 0.1M, pH is 6.0 ~ 8.0.Preferably, pH is 7.4.
Wherein the fluorescent material of step (2) is Cy5.5-NHS, and its DMF solution concentration is 1 × 10
-2~ 1 × 10
-3mol/mL.
Wherein, step (2) fluorescent material with the method for the abundant coupling of Rituximab is: the two mixed with the volume ratio 1:10 ~ 1:100 of Mabthera monoclonal antibody according to Cy5.5-NHS, and add the PBS buffer solution that volume is Cy5.5-NHS volume 10-100 0.1M pH=6.0-8.0 doubly, with the Na of 1mol/L
2hPO
4solution regulates the pH of reaction system to be 8.0-9.5, fully mixes lucifuge reaction 12-24h under 4 DEG C of conditions.
Preferably, step (2) fluorescent material with the method for the abundant coupling of Rituximab is: the two mixed with the volume ratio 1:10 of Rituximab according to Cy5.5-NHS, and add the PBS buffer solution that volume is the 0.1M pH=7.4 of Cy5.5-NHS volume 100 times, with the Na of 1mol/L
2hPO
4solution regulates the pH of reaction system to be 8.5, fully mixes lucifuge reaction 12h under 4 DEG C of conditions.
In method of the present invention, step (3) adopts PD-10 column separating purification conjugate.
Present invention also offers above-mentioned specific sentinel lymph node developer and prepare the application in breast cancer detection kit.
Specific sentinel lymph node developer provided by the invention has the following advantages: this developer is easy to use, stable performance, and the holding time is long, can reach 24 months, and mark rate is up to more than 95%; Workable, can be used for the noinvasive detection of the sentinel node of infrared video picture, the picked-up of Secondary Lymphoid knot is low, injection point is detained low, video picture and the biopsy time not limited, from injection after 30min-24h can develop very well, visualization ratio reaches close to 100%, without any radioactive radiation, have better safety, cost is low simultaneously, has good potential applicability in clinical practice.
Accompanying drawing explanation
Fig. 1 is the coupling schematic diagram of Cy5.5-SLN-F sentinel lymph node developer.
Fig. 2 is the sentinel node fluorescent imaging figure of Cy5.5-SLN-F monoclonal antibody nude mice, by left and the right side totally 6 pictures, is the mice sentinel node fluorescent imaging figure after subcutaneous injection Cy5.5-SLN-F 30min, 6h, 1d, 2d, 5d, 9d respectively.
Detailed description of the invention
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art; Reagent used in embodiment is commercial goods.
The preparation (1) of the specific sentinel lymph node developer of embodiment 1 fluorescence coupling
1) purification (for Rituximab) of CD20 targeting monoclonal antibody: the monoclonal antibody with CD20 antigen targeting getting 1.0mL 10mg/ml, add in pretreated PD-10 post, then add the PBS solution of 0.01M pH 7.4 wherein, collect the monoclonal antibody of wherein 1mL.And carry out protein quantification by biological method, and add the PBS solution of 0.01M pH 7.4 wherein, be diluted to 5mg/mL stand-by;
2) with the PBS solution of 0.01M pH 7.4 for buffer, getting concentration is 1 × 10
-2the DMF solution 10 μ L of the Cy5.5-NHS of mol/mL mixes with above-mentioned 0.1mL Rituximab solution, adds 1mL 0.1M PBS solution (pH=7.4) wherein, with the Na of 1mol/L
2hPO4 solution regulates the pH of reaction system to be 8.0, fully mixes, lucifuge reaction 12h under 4 DEG C of conditions.By PD-10 column separating purification.Acquisition can carry out the specific sentinel lymph node medicine Cy5.5-SLN-F of infrared video picture.
The preparation (2) of the specific sentinel lymph node developer of embodiment 2 fluorescence coupling
1) purification (for Rituximab) of CD20 targeting monoclonal antibody: the monoclonal antibody with CD20 antigen targeting getting 1.0mL 50mg/ml, add in pretreated PD-10 post, then add the PBS solution of 0.01M pH 7.4 wherein, collect wherein Rituximab.And carry out protein quantification by biological method, and add the PBS solution of 0.1M pH 7.4 wherein, be diluted to 10mg/mL stand-by;
2) with the PBS solution of 0.1M pH 7.4 for buffer, getting concentration is 1 × 10
-2the DMF solution 10 μ L of mol/mLCy5.5-NHS mixes with above-mentioned 1.0mL Rituximab solution, adds 1mL 0.1M PBS solution (pH=7.4) wherein, with the Na of 1mol/L
2hPO4 solution regulates the pH of reaction system to be 9.5, fully mixes, lucifuge reaction 24h under 4 DEG C of conditions.By PD-10 column separating purification.Acquisition can carry out the specific sentinel lymph node medicine Cy5.5-SLN-F of infrared video picture.
The preparation (3) of the specific sentinel lymph node developer of embodiment 3 fluorescence coupling
1) purification (for Rituximab) of CD20 targeting monoclonal antibody: the monoclonal antibody with CD20 antigen targeting getting 1.0mL 20mg/ml, add in pretreated PD-10 post, then add the PBS solution of 0.01M pH 7.4 wherein, collect the monoclonal antibody of wherein 0.5-1.5ml.And carry out protein quantification by biological method, and add PBS solution wherein, be diluted to 7mg/mL stand-by;
2) with the PBS solution of 0.05M pH 7.4 for buffer, getting concentration is 1 × 10
-3the above-mentioned Rituximab solution mixing of DMF solution 10 μ L and 1.0mL of the Cy5.5-NNHS of mol/mL, adds 1mL 0.1M PBS solution (pH=7.4), wherein with the Na of 1mol/L
2hPO
4solution regulates the pH of reaction system to be 8.5, fully mixes, lucifuge reaction 18h under 4 DEG C of conditions.By PD-10 column separating purification.Acquisition can carry out the specific sentinel lymph node medicine Cy5.5-SLN-F of infrared video picture.
The effect of the specific sentinel lymph node developer of embodiment 4 fluorescence coupling
Get 30g mice one, at the Cy5.5-SLN-F (embodiment 1 prepares) of its left back sole subcutaneous injection 20 μ L.Rear 30min, 6h, 1d with injection respectively, 2d, 5d, 9d carry out fluorescent imaging with the true toy optical imaging system of smart promise, and merge with the optical image under normal condition, observe the sentinel node picked-up change of mouse hind leg oxter and whole body distribution situation, its fluorescent imaging the results are shown in Figure 1.As shown in Figure 1, after subcutaneous injection Cy5.5-SLN-F, the position at SLN place clearly during 30min, can be shown, and clearly can observe the large body image of mice, at 30min until after 9 days, still can see sentinel node picture rich in detail.In whole videograph process, have no the development of Secondary Lymphoid knot all the time.Nude mice is anaesthetized with the isoflurane of 2% (volume fraction) and oxygen mix, maintains in fluorescent imaging process with the isoflurane of 0.5% ~ 1.5% (volume fraction).Use C rank (12.5cm) visual field in gatherer process, each acquisition time is 1min.Spectrum Living Imaging 4.2 software is adopted to carry out image procossing.In processing procedure, the area-of-interest (region of interest, ROI) chosen adopts photon radiation efficiency (Radiant efficiency) to be unit, and concrete unit is [p/sec/cm2/sr]/[μ W/cm
2], concrete data are: during 30min, and its fluorescent absorption is 3000; During 6h, fluorescent absorption is 8000; During 2d, fluorescent absorption is 6000; During 5d, fluorescent absorption is 5000; During 9d, fluorescent absorption is 4000.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a specific sentinel lymph node developer for fluorescence coupling, is characterized in that, is obtained after separation and purification by the Rituximab of fluorescent material coupling.
2. specific sentinel lymph node developer as claimed in claim 1, it is characterized in that, described fluorescent material is Cy5.5-N-hydroxysuccinimide eater or Cy7-N-hydroxysuccinimide eater.
3. prepare the method for specific sentinel lymph node developer according to claim 1, it is characterized in that, containing following steps:
(1) purification Rituximab, and allocate its concentration be applicable to fluorescent material coupling;
(2) in PBS buffer solution, make the abundant coupling of Rituximab that fluorescent material and step (1) obtain;
(3) separation and purification conjugate.
4. method as claimed in claim 3, it is characterized in that, the method for step (1) purification is: carry out purification by gel chromatographic columns to Rituximab.
5. method as claimed in claim 3, is characterized in that, step (1) adopts the Rituximab concentration after PBS buffer solution allotment purification to be 5 ~ 10mg/mL.
6. method as claimed in claim 3, it is characterized in that, the PBS buffer concentration of step (2) is 0.01M ~ 0.1M, pH 6.0-8.0.
7. method as claimed in claim 3, it is characterized in that, the fluorescent material of step (2) is Cy5.5-NHS, and its DMF solution concentration is 1 × 10
-2~ 1 × 10
-3mol/mL.
8. the method as described in as arbitrary in claim 3-7, it is characterized in that, the method of step (2) fluorescent material and the abundant coupling of Rituximab: with the volume ratio 1:10 ~ 1:100 of Rituximab, the two is mixed according to Cy5.5-NHS, and to add volume be Cy5.5-NHS volume 10-100 pH is doubly 0.01M ~ 0.1M PBS buffer solution of 6.0-8.0, with Na
2hPO
4solution regulates the pH of reaction system to be 8.0-9.5, fully mixes lucifuge reaction 12-24h under 4 DEG C of conditions.
9. method as claimed in claim 8, it is characterized in that, step (2) fluorescent material with the method for the abundant coupling of Rituximab is: the two mixed with the volume ratio 1:10 of Rituximab according to Cy5.5-NHS, and add the PBS buffer solution that volume is the 0.1M pH=7.4 of Cy5.5-NHS volume 100 times, with the Na of 1mol/L
2hPO
4solution regulates the pH of reaction system to be 8.5, fully mixes lucifuge reaction 12h under 4 DEG C of conditions.
10. the specific sentinel lymph node developer that the arbitrary described specific sentinel lymph node developer of claim 1-2 or the arbitrary described method of claim 3-11 obtain is preparing the application in breast cancer detection kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410742313.4A CN104491881A (en) | 2014-12-05 | 2014-12-05 | Fluorescence-coupled specific sentinel node photographic developer and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410742313.4A CN104491881A (en) | 2014-12-05 | 2014-12-05 | Fluorescence-coupled specific sentinel node photographic developer and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104491881A true CN104491881A (en) | 2015-04-08 |
Family
ID=52933392
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410742313.4A Pending CN104491881A (en) | 2014-12-05 | 2014-12-05 | Fluorescence-coupled specific sentinel node photographic developer and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104491881A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111789967A (en) * | 2020-07-17 | 2020-10-20 | 王永胜 | Breast cancer sentinel lymph node metastasis in-vivo fluorescence targeting tracer and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101080240A (en) * | 2004-12-17 | 2007-11-28 | 皇家飞利浦电子股份有限公司 | Targeting contrast agents or targeting therapeutic agents for molecular imaging and therapy |
| CN101927009A (en) * | 2009-06-19 | 2010-12-29 | 北京肿瘤医院 | Specific sentinel lymph node developer drug and preparation method thereof |
| CN103675262A (en) * | 2013-12-27 | 2014-03-26 | 步荣发 | Method of stable fluorescence labeling of living cells of oral squamous cell carcinoma |
-
2014
- 2014-12-05 CN CN201410742313.4A patent/CN104491881A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101080240A (en) * | 2004-12-17 | 2007-11-28 | 皇家飞利浦电子股份有限公司 | Targeting contrast agents or targeting therapeutic agents for molecular imaging and therapy |
| CN101927009A (en) * | 2009-06-19 | 2010-12-29 | 北京肿瘤医院 | Specific sentinel lymph node developer drug and preparation method thereof |
| CN103675262A (en) * | 2013-12-27 | 2014-03-26 | 步荣发 | Method of stable fluorescence labeling of living cells of oral squamous cell carcinoma |
Non-Patent Citations (2)
| Title |
|---|
| XIAOFANG JIN等: ""A Generalized Kinetic Model For Amine modification of Proteins with Application to Phage Display"", 《SHORT TECHNICAL REPORTS》 * |
| 林新峰等: ""CD20靶向Cy7-Rituximab分子探针的制备及在小鼠活体荧光成像中的应用"", 《高等学校化学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111789967A (en) * | 2020-07-17 | 2020-10-20 | 王永胜 | Breast cancer sentinel lymph node metastasis in-vivo fluorescence targeting tracer and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bhushan et al. | Current state of breast cancer diagnosis, treatment, and theranostics | |
| Vento et al. | PD-L1 detection using 89 Zr-atezolizumab immuno-PET in renal cell carcinoma tumorgrafts from a patient with favorable nivolumab response | |
| Nagengast et al. | VEGF-SPECT with 111In-bevacizumab in stage III/IV melanoma patients | |
| Thorek et al. | Positron lymphography: multimodal, high-resolution, dynamic mapping and resection of lymph nodes after intradermal injection of 18F-FDG | |
| Soodgupta et al. | Very late antigen-4 (α4β1 integrin) targeted PET imaging of multiple myeloma | |
| CN104168923A (en) | Dynamic contrast enhanced MRI method and agents for the assessment of the macromolecular transport within pathologic tissues | |
| Valliant | A bridge not too far: linking disciplines through molecular imaging probes | |
| US20240081759A1 (en) | Methods for quantitative and enhanced-contrast molecular medical imaging using cross-modality correction for differing tracer kinetics | |
| Pandya et al. | Imaging of fibroblast activation protein alpha expression in a preclinical mouse model of glioma using positron emission tomography | |
| WO2011049405A2 (en) | Optical imaging contrast agent, use and device thereof | |
| WO2016007734A1 (en) | Methods for quantitative and enhanced-contrast molecular medical imaging using cross-modality correction for differing tracer kinetics | |
| Huang et al. | Evaluation of 124I-JS001 for hPD1 immuno-PET imaging using sarcoma cell homografts in humanized mice | |
| Green et al. | Estimation of radiation dosimetry for 68Ga-HBED-CC (PSMA-11) in patients with suspected recurrence of prostate cancer | |
| Hadebe et al. | The role of PET/CT in breast cancer | |
| Rijs et al. | Introducing fluorescence-guided surgery for pediatric Ewing, osteo-, and rhabdomyosarcomas: a literature review | |
| Wen et al. | Pd-l1-targeted radionuclide therapy combined with αpd-l1 antibody immunotherapy synergistically improves the antitumor effect | |
| Oroujeni et al. | The Use of a Non-Conventional Long-Lived Gallium Radioisotope 66Ga Improves Imaging Contrast of EGFR Expression in Malignant Tumours Using DFO-ZEGFR: 2377 Affibody Molecule | |
| Klain et al. | Advances in functional imaging of differentiated thyroid cancer | |
| Frigerio et al. | Anti-PSMA 124 I-scFvD2B as a new immuno-PET tool for prostate cancer: preclinical proof of principle | |
| Kroiss et al. | [68Ga] Ga-DOTA-TOC PET/CT in the localization of head and neck paraganglioma compared with [18F] FDOPA PET/CT and [123I] MIBG SPECT/CT | |
| Moustapha et al. | Technetium-labeled danofloxacin complex as a model for infection imaging | |
| CN104491881A (en) | Fluorescence-coupled specific sentinel node photographic developer and preparation method thereof | |
| CN108434469A (en) | A kind of HER2 affinities body68Ga markers and preparation method thereof, application | |
| Deblock et al. | Dual-Modality Hafnium Oxide Nanocrystals for in Vivo Computed Tomography and Fluorescence Imaging of Sentinel Lymph Nodes | |
| Kairemo | Immunolymphoscintigraphy with 99mTc-labeled monoclonal antibody (BW 431/26) reacting with carcinoembryonic antigen in breast cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150408 |
|
| RJ01 | Rejection of invention patent application after publication |