CN109053890A - A kind of anti-CD80 Chimeric antibody and preparation method thereof of 131I label - Google Patents

Abstract

The present invention relates to one kind¹³¹The anti-CD80 Chimeric antibody and preparation method thereof of I label.The labelled antibody is prepared in accordance with the following methods: (1) using 30 μ g Iodogen iodine mark pipes, the 0.04M PB+0.1M NaCl solution of 20 μ L is added, anti- 80 μ g of CD80 Chimeric antibody is continuously added, sodium iodide 55.5MBq is added, is reacted 10 minutes after being mixed under room temperature；(2) it with 0.04M PB+0.1M NaCl solution balance activation PD10 column, all drains off to solution, reaction solution is added to PD10 column, all flow to end to solution, add 0.04M PB+0.1M NaCl elution product, collect efflux.The labelled antibody mark rate and radiochemicsl purity are high, and stability is prominent, are able to maintain higher immunocompetence.The present invention is to lay a good foundation by the radio-immuno-image of target spot and treatment of CD80.

Description

A kind of anti-CD80 Chimeric antibody and preparation method thereof of 131I label

Technical field

The present invention relates to antibody labelling technique fields, specifically, being related to one kind¹³¹The anti-CD80 people-mouse of I label is chimeric Antibody and preparation method thereof.

Background technique

Radio-immuno-image (radioimmunoimaging, RII) is the specific antibody that will be directed to tumor associated antigen With human body is injected after radioisotope labeling, tumor tissues are reached with blood flow, in conjunction with the related antigen of tumour, to make tumour Tissue local radioactivity is dense poly- more than normal tissue, and the positive scintigraphy figure of tumour is then obtained with external imaging technique.Radioactivity Antitumor and its related antigen the antibody of isotope labeling is used as biological missile, is tumor positive imaging agent, referred to as tumour immunity Diagnostic imaging technique can be used as growth viable tumor qualitative examination really, it is also possible to be suitable for different core to screen Element label same antibody makees the patient of radioimmunotherapy.

Radioimmunotherapy (radioimmunotherapy, RIT) is the monoclonal antibody using radioisotope labeling The method for treating tumour.It passes through the specific antigen or tumor associated antigen on antibody specificity tumor cell surface, by Radionuclide emission irridiation injury tumour target cell in conjunction with antibody.Radioimmunotherapy agent is by antibody and radionuclide Two parts composition, antibody are combined as a kind of particularity carrier with radionuclide, the former is passed through in a manner of biological missile The corresponding antigens automatic guide tumor tissues of tumor cell, then carrier itself and radionuclide can be located in swollen simultaneously Tumor tissue, radiobiology effect caused by the β ray using the radionuclide emission concentrated in tumor tissues are broken The structure and function of bad interference tumour target cell, achieve the purpose that kill tumour cell.The influence of this method normal tissue compared with It is small, it opened up a new way for (immune) treatment of radiation in tumour.

Malignant lymphoma is to be primary in the malignant tumour of lymph node and other organ lymphoid tissues, is divided into Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL).It falls ill mainly common with American-European countries, in the U.S., NHL is that number of the infected increase is most fast Malignant tumour.The disease incidence of China NHL is similarly rising, wherein 90% or more lymthoma is NHL.The pathogenic factor of NHL It is also very not clear at present, may be related with infection, aging of population, HIV infection and environmental pollution may be that its disease incidence increases Long reason.

Costimulating factor CD80 plays an important role in immune response, autoimmune disease and tumour immunity.It grinds Study carefully and shows that some malignant Bs high can express CD80 molecule, and the pernicious increasing of the numerator mediated signal and tumour cell It grows and shifts related.Therefore, the expression of cell surface CD80 molecule is detected by radio-immuno-image technology, or is exempted from by radiation Tumour cell is killed in epidemic disease treatment targeting, provides new tool for the diagnosing and treating of related disease.

Paper " the CD80 mouse-people's chimeric antibody that Chinese periodical " China Immunology Journal " the 2nd periodical of volume 25 in 2009 goes out Expressing cho cell and extracorporeal biology function Primary Study ", the anti-human CD80 mouse-people of successful expression is embedding in Chinese hamster ovary celI Antibody ch4E5 is closed, method particularly includes: expression vector pIRES/ch4E5 of the building containing chimeric weight, light chain gene；Expression vector 293T cell is first transfected, after FCM detects the transient expression of ch4E5 antibody, expression vector again stablize by transfection CHO cell, building Expression cell strain CHO-ch4E5；ProteinG affinity chromatography is purified from CHO-ch4E5 cell non-serum culture supernatant Ch4E5 antibody.Then author has detected identification of the ch4E5 antibody to membranous type CD80 by FCM, and is handled just with mitomycin Ordinary person's peripheral blood mononuclear cells PBMCs is stimulation cell, using allosome human peripheral lymphocytes PBLs as reacting cells, is used Mtt assay analyzes the blocking effect of ch4E5 antibody, it was confirmed that there is the chimeric antibody parental antibody CD80 to be blocked to mediate in vitro Costimulatory signal biological activity.Therefore, anti-human CD80 mouse-people's chimeric antibody ch4E5 can be used for the treatment of tumour.Suzhou The Master's thesis of university 2008 " Primary Study of the building of CD80 mouse-people's chimeric antibody, expression and biological function " is also in detail Ground describes expression and the biological function of ch4E5.

Paper " the Iodogen method that Chinese periodical Nuclear Technology the 5th periodical of volume 32 in 2009 goes out¹²⁵I marks monoclonal anti- Body 10D9 " selects optimum mark condition to carry out using Iodogen method¹²⁵I labeled monoclonal antibody 10D9.Marked product is used Sephadex G-50 is isolated and purified, and trichloroacetic acid (TCA) method measures mark rate and top coal drawing, and to the immunocompetence of marker And stability is analyzed.The results show that¹²⁵It is 10 μ g of Iodogen dosage, 20 μ of 10D9 dosage that I, which marks the optimum condition of 10D9, G, Na is added¹²⁵I22.2MBq, room temperature reaction 8min,¹²⁵The mark rate of I-10D9 is (84.10 ± 3.18) %, and top coal drawing is (98.49 ± 1.14) %, specific activity 933.51MBq/mg；¹²⁵The specific binding rate of I-10D9 and Daudi cell is (23.21 ± 1.14) %；Marker be added in the PBS containing 1%BSA place 3W after radiochemicsl purity still > 90%.The result shows that: Iodogen method¹²⁵I marks 10D9 mark rate and top coal drawing high, and method is easy, and the stability of marker is good and can preferably keep Its immunocompetence.Chinese periodical " Chinese radiation hygiene " the 5th periodical of volume 23 in 2014 go out paper "¹³¹I marks anti-CD80 mono- Clonal antibody is in the intracorporal radio-immuno-image of tumor bearing nude mice ", anti-CD80 monoclonal antibody 4E5 has been carried out using Iodogen method 's¹³¹I label, and immunocompetence to labelled antibody and stability etc. are analyzed.As a result¹³¹I labeled monoclonal antibody 4E5 Mark rate be (78.3 ± 2.4) %, radiochemical purity be (95.7 ± 1.8) %；¹³¹I-4E5 antibody and Raji cell are most Big Percentage bound is (36.1 ± 2.6) %,¹³¹I-4E5 antibody is added to placed 3 days in serum after radiochemicsl purity be still greater than 90%；Lotus The injection of tumor nude mice¹³¹After I-4E5 antibody, extension at any time, tumor locus radioactivity it is dense it is poly- gradually increase, in 72h, tumour is aobvious Shadow is the most clear, T/NT at when up to 3.2.Patent document CN107286243A, publication date 2017.10.24, discloses A kind of radiation iodine labeling PD-L1 monoclonal antibody and the preparation method and application thereof.This method comprises: adding into phosphate buffer Enter PD-L1 monoclonal antibody, Na¹³¹I solution and chloramine-T are reacted, after reaction, with the weighting sulphur with chloramine-T equivalent Sour sodium terminates reaction；Wherein, the PD-L1 monoclonal antibody, the Na¹³¹The consumption proportion of I solution and the chloramine-T is 50 μg:0.58-2.9mci:10-60μg；The Na¹³¹The amount of I solution with¹³¹The radioactive activity measuring meter of I, the results showed that the probe energy Enough significantly target tumor tissues, gained image clearly.Above each document all refers to the radioiodination of antibody.

There are many iodate labeling method of polypeptide or protein, for example, iodine monochloride method, chloramine-t method, peroxidase method, Iodogen method, electrolytic labeling method, linkage flag method etc..For every kind of method, different flag conditions influences labeling polypeptide or egg Mark rate, immunocompetence, stability of white matter etc..After iodination reaction terminates, then select various biochemical or physical methods will be by The substance and free-iodine of iodate separate, and common method has dialysis, electrophoresis, various chromatographic techniques etc..Isolated effect directly affects Radiochemicsl purity.

The radiation for having excellent mark rate, radiochemical purity, stability and specific cell Percentage bound is had no at present The anti-CD80 Chimeric antibody of property isotope labeling.

Summary of the invention

The purpose of the present invention is aiming at the shortcomings in the prior art, provide one kind¹³¹The anti-CD80 people-mouse inosculating antibody of I label Body.

Another purpose of the invention is to provide described¹³¹The preparation method of the anti-CD80 Chimeric antibody of I label.

To realize above-mentioned first purpose, the technical solution adopted by the present invention is that:

It is a kind of¹³¹The anti-CD80 Chimeric antibody of I label, prepares in accordance with the following methods:

(1) 10-100 μ g Iodogen iodine mark pipe is used, 0.04M PB+0.1M NaCl solution is added, continuously adds anti- CD80 Chimeric antibody 10-160 μ g, be eventually adding iodine (¹³¹I) change sodium 18.5-111.0MBq, reacted after mixing at normal temperature 4-12 minutes, reaction tube is sucked out in reaction solution after reaction；

(2) PD10 column is activated with 0.04M PB+0.1M NaCl solution balance using preceding, is discarded after solution all drains off All effluxes, the reaction solution of step (1) is added on PD10 column, is all flow to end to solution, discards all effluxes, then plus Enter 0.04M PB+0.1M NaCl and elute product to PD10 column, collects all effluxes up to product.

It is described as a preference¹³¹The anti-CD80 Chimeric antibody of I label is prepared in accordance with the following methods:

(1) 30 μ g Iodogen iodine mark pipes are used, 0.04M PB+0.1M NaCl solution is added, continuously adds anti-CD80 80 μ g of Chimeric antibody, be eventually adding iodine (¹³¹I) change sodium 55.5MBq, reacted 10 minutes after mixing at normal temperature, reaction knot Reaction tube is sucked out in reaction solution after beam；

(2) PD10 column is activated with 0.04M PB+0.1M NaCl solution balance using preceding, is discarded after solution all drains off All effluxes, the reaction solution of step (1) is added on PD10 column, is all flow to end to solution, discards all effluxes, then plus Enter 0.04M PB+0.1M NaCl and elute product to PD10 column, collects all effluxes up to product.

It is described as another preference¹³¹The top coal drawing of the anti-CD80 Chimeric antibody of I label is greater than 90%.

To realize above-mentioned second purpose, the technical solution adopted by the present invention is that:

It is a kind of¹³¹The preparation method of the anti-CD80 Chimeric antibody of I label, comprising the following steps:

(1) 10-100 μ g Iodogen iodine mark pipe is used, 0.04M PB+0.1M NaCl solution is added, continuously adds anti- CD80 Chimeric antibody 10-160 μ g, be eventually adding iodine (¹³¹I) change sodium 18.5-111.0MBq, reacted after mixing at normal temperature 4-12 minutes, reaction tube is sucked out in reaction solution after reaction；

(2) PD10 column is activated with 0.04M PB+0.1M NaCl solution balance using preceding, is discarded after solution all drains off All effluxes, the reaction solution of step (1) is added on PD10 column, is all flow to end to solution, discards all effluxes, then plus Enter 0.04M PB+0.1M NaCl and elute product to PD10 column, collects all effluxes up to product.

As a preference, comprising the following steps:

(1) 30 μ g Iodogen iodine mark pipes are used, 0.04M PB+0.1M NaCl solution is added, continuously adds anti-CD80 80 μ g of Chimeric antibody, be eventually adding iodine (¹³¹I) change sodium 55.5MBq, reacted 10 minutes after mixing at normal temperature, reaction knot Reaction tube is sucked out in reaction solution after beam；

(2) PD10 column is activated with 0.04M PB+0.1M NaCl solution balance using preceding, is discarded after solution all drains off All effluxes, the reaction solution of step (1) is added on PD10 column, is all flow to end to solution, discards all effluxes, then plus Enter 0.04M PB+0.1M NaCl and elute product to PD10 column, collects all effluxes up to product.

As another preference, preparation¹³¹The top coal drawing of the anti-CD80 Chimeric antibody of I label is greater than 90%.

The invention has the advantages that:

1, the present invention uses¹³¹Anti- CD80 Chimeric antibody is marked in I, has selected suitable flag condition and purifying item Part, mark rate and radiochemicsl purity are high, and the stability of marker is good, and method is easy, is convenient for promoting；¹³¹Antibody after I label It is able to maintain higher immunocompetence.This method is established as laying by the radio-immuno-image of target spot and Therapy study of CD80 Good basis.

2, it is an unexpected discovery of the invention that Iodogen dosage is 30 μ g, anti-CD80 Chimeric antibody dosage is 80 μ g, iodine (¹³¹I) change sodium 55.5MBq, 10min is reacted after mixing at normal temperature, with PD10 column purification, 0.04M PB+0.1M NaCl solution Elution, obtained labelled antibody mark rate, in terms of performance it is very excellent, especially have very high steady It is qualitative, it provides convenience for clinical detection or treatment.

Detailed description of the invention

Fig. 1 radio thin layer chromatographic scan method detects labelled antibody mark rate.Wherein, the flag condition of the labelled antibody Are as follows: Iodogen (20 μ g) iodine mark pipe is used, the 0.04M PB+0.1M NaCl solution of 20 μ L, anti-CD80 people-mouse inosculating antibody is added 80 μ g of body additional amount, be eventually adding iodine (¹³¹I) change sodium 74.0MBq, react 8min after mixing at normal temperature；Purification condition are as follows: PD10 column, the elution of 0.04M PB+0.1M NaCl solution.

Fig. 2 radio thin layer chromatographic scan method detects labelled antibody radiochemicsl purity.Wherein, the slug of the labelled antibody Part are as follows: use Iodogen (30 μ g) iodine mark pipe, the 0.04M PB+0.1M NaCl solution of 20 μ L is added, anti-CD80 people-mouse is chimeric 80 μ g of antibody additional amount, be eventually adding iodine (¹³¹I) change sodium 55.5MBq, react 10min after mixing at normal temperature；Purification condition are as follows: PD10 column, the elution of 0.04M PB+0.1M NaCl solution.

Fig. 3 radio thin layer chromatographic scan method detection free-iodine [¹³¹I] control group radiochemicsl purity.

Specific embodiment

It elaborates with reference to the accompanying drawing to specific embodiment provided by the invention.

Embodiment 1¹³¹I marks the preliminary experiment of anti-CD80 Chimeric antibody

One, experimental method

1 label

Method one:

It is marked using Iodogen method, using Iodogen (50 μ g) iodine mark pipe, anti-CD80 Chimeric antibody is added (present of University Of Suzhou Qiu Yu China laboratory, can also prepare: the .CD80 such as Xu Yaoyu, Hu Lingling, Chen Yong well according to the record of document The Primary Study China Immunology Journal .2009 (25) of mouse-people's chimeric antibody expressing cho cell and extracorporeal biology function: 114-117, hybridoma cell strain 4E5, that is, CN102190729A described in document, the anti-human CD80 of secretion described in 2011.09.21 The cell strain of molecule bivalent antibody, preservation information are as follows: depositary institution: China Committee for Culture Collection of Microorganisms's commonly micro- life Object center；Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；Preservation date: On 01 24th, 2011；Deposit number: CGMCCNo.4571；Classification naming: the cell of anti-human CD80 molecule bivalent antibody is secreted Strain) 100 μ g, add iodine (¹³¹I) change sodium 74MBq, react 8min after mixing at normal temperature.

By reactant SephadexG-50 column chromatography, is eluted with the PBS of 0.05M pH7.4, it is every to collect eluent Pipe 0.5ml measures the radiocounting of each pipe, until the second peak is eluted to background, merges first radioactivity peak solution i.e. For product peak.

Method two:

It is marked using Iodogen method, using Iodogen (50 μ g) iodine mark pipe, the 0.04M PB+0.1M of 20 μ L is added NaCl solution continuously adds anti-100 μ g of CD80 Chimeric antibody, be eventually adding iodine (¹³¹I) change sodium 74MBq, mix at normal temperature 8min is reacted after conjunction, and reaction tube is sucked out in reaction solution after reaction.

PD10 column is activated with 0.03M PB+0.05M NaCl solution 35mL balance using preceding first, is all drained off to solution After discard all effluxes, above-mentioned reaction solution is added on PD10 column, is all flow to end to solution, all effluxes are discarded, then 2mL 0.03M PB+0.05M NaCl is added and elutes product to PD10 column, collects all effluxes up to product.

Method three:

It is marked using Iodogen method, using Iodogen (50 μ g) iodine mark pipe, the 0.04M PB+0.1M of 20 μ L is added NaCl solution continuously adds anti-100 μ g of CD80 Chimeric antibody, be eventually adding iodine (¹³¹I) change sodium 74MBq, mix at normal temperature 8min is reacted after conjunction, and reaction tube is sucked out in reaction solution after reaction.

PD10 column is activated with 0.04M PB+0.1M NaCl solution 35mL balance using preceding first, after solution all drains off All effluxes are discarded, above-mentioned reaction solution is added on PD10 column, are all flow to end to solution, discard all effluxes, then plus Enter 2mL 0.04M PB+0.1M NaCl and elute product to PD10 column, collects all effluxes up to product.

Method four:

It is marked using Iodogen method, using Iodogen (50 μ g) iodine mark pipe, the 0.04M PB+0.1M of 20 μ L is added NaCl solution continuously adds anti-100 μ g of CD80 Chimeric antibody, be eventually adding iodine (¹³¹I) change sodium 74MBq, mix at normal temperature 8min is reacted after conjunction, and reaction tube is sucked out in reaction solution after reaction.

PD10 column is activated with 0.05M PB+0.15M NaCl solution 35mL balance using preceding first, is all drained off to solution After discard all effluxes, above-mentioned reaction solution is added on PD10 column, is all flow to end to solution, all effluxes are discarded, then 2mL 0.05M PB+0.15M NaCl is added and elutes product to PD10 column, collects all effluxes up to product.

The detection of 2 radio-chemical purities

By on the above-mentioned product point sample to Radio-iTLC paper being collected into, the ascending development in 85% methanol system, use is thin Layer radioactive scanning instrument scanning；By on radio-iodidesodium stoste point sample to Radio-iTLC paper, in 85% methanol system on Row expansion as a control group, calculates R_fValue, R_fIt is as follows to be worth calculation formula:

Wherein 0.15min is point sample origin, and 0.78min is solvent front.Retention time is less than the R of 0.15min_fValue is fixed Justice is 0, and retention time is greater than the R of 0.78min_fValue is defined as 1.It is required that target product R_f=0~0.1, radio-iodidesodium stoste R_f=0.8~1；Product radioactivity chemical purity is greater than 90%.

The stability observing of 3 labelled antibodies

Marker is added separately in serum and PBS, under the conditions of setting 37 DEG C, the top coal drawing of marker is measured after 5 days, Observe the stability of labelled antibody.

The determination of activity of 4 labelled antibodies

Preparing cell number is respectively 1 × 10⁵、1×10⁶、1×10⁷、5×10⁷、1×10⁸The Raji cell suspension of a/mL, 100 μ L cell suspensions are respectively taken to be added in centrifuge tube, each concentration point does 3 pipes；Every pipe be added 20 μ L labelled antibody (~5 × 10⁴Counts/min), mix, 37 DEG C of culture 2h；Then plus the 500 μ L of PBS of 0.05M pH7.4, shake up, 3000rpm/min, Supernatant is sucked out in 4 DEG C of low-temperature centrifugation 10min, washs precipitating 2 times with PBS, surveys the radiocounting of precipitating, calculate labelled antibody Cell combination rate；It does non-specific binding control tube simultaneously: taking each 100 μ L of concentrations of cells suspension to be added in centrifuge tube, be all provided with 3 Multiple pipe, every pipe be added it is excessive (about 1000 times, according to addition¹³¹I-CD80 antibody protein amount, which calculates, is added unmarked antibody Amount) unmarked antibody, after 37 DEG C of culture 1h, labelled antibody (additional amount and combine pipe unanimously) is added, remaining step is same as above.It is special Anisotropic Percentage bound (%)=total cell Percentage bound (%)-non-specific cell Percentage bound (%).

Standard pipe: pick-and-place exempts from pipe 3, and labelled antibody (with combining pipe consistent) is added thereto and is used as standard pipe, carries out γ It counts.

Two, experimental result

1 mark rate and top coal drawing

The labelled antibody radio-chemical purity obtained with SephadexG-50 column chromatography is below 90%, is unsatisfactory for It is required that.

Method two, method three and method four use PD10 column purification, and the mark rate of obtained labelled antibody is respectively (73.6 ± 2.1) %, (76.0 ± 1.9) %, (77.4 ± 2.5) %, radiochemical purity respectively (95.4 ± 1.8) %, (95.7 ± 1.7) %, (96.3 ± 2.2) %, being added to the radiochemicsl purity after placing 5d in serum is respectively (48.6 ± 1.5) %, (68.9 ± 1.8) %, (42.4 ± 1.3) %, being added to the radiochemicsl purity after placing 5d in PBS is respectively (45.3 ± 1.6) %, (67.2 ± 1.9) %, (39.7 ± 1.2) %, specific cell Percentage bound be respectively (27.1 ± 2.7) %, (28.5 ± 2.6) %, (23.2 ± 1.9) %.

Therefore PD10 column purification is used, the elution of 0.04M PB+0.1M NaCl solution can satisfy labelled antibody radioactivity The requirement of purity is learned, while being conducive to labelled antibody stability and active maintenance.

Embodiment 2¹³¹I marks the condition optimizing of anti-CD80 Chimeric antibody

One, experimental method

1 labeling method

It is marked using Iodogen method, using Iodogen iodine mark pipe, 0.04M PB+0.1M NaCl solution is added, after It is continuous that anti-CD80 Chimeric antibody is added, be eventually adding iodine (¹³¹I) change sodium, reacted after mixing at normal temperature, after reaction will Reaction tube is sucked out in reaction solution.

2 isolate and purify

PD10 column is activated with 0.04M PB+0.1M NaCl solution 35mL balance using preceding first, after solution all drains off All effluxes are discarded, above-mentioned reaction solution is added on PD10 column, are all flow to end to solution, discard all effluxes, then plus Enter 2mL 0.04M PB+0.1M NaCl and elute product to PD10 column, collects all effluxes up to product.

The detection of 3 radio-chemical purities

By on the above-mentioned product point sample to Radio-iTLC paper being collected into, the ascending development in 85% methanol system, use is thin Layer radioactive scanning instrument scanning；By on radio-iodidesodium stoste point sample to Radio-iTLC paper, in 85% methanol system on Row expansion as a control group, calculates R_fValue, R_fIt is as follows to be worth calculation formula:

Wherein 0.15min is point sample origin, and 0.78min is solvent front.Retention time is less than the R of 0.15min_fValue is fixed Justice is 0, and retention time is greater than the R of 0.78min_fValue is defined as 1.It is required that target product R_f=0~0.1, radio-iodidesodium stoste R_f=0.8~1；Product radioactivity chemical purity is greater than 90%.

The stability observing of 4 labelled antibodies

Marker is added separately in serum and PBS, under the conditions of setting 37 DEG C, the top coal drawing of marker is measured after 5 days, Observe the stability of labelled antibody.

The determination of activity of 5 labelled antibodies

Preparing cell number is respectively 1 × 10⁵、1×10⁶、1×10⁷、5×10⁷、1×10⁸The Raji cell suspension of a/mL, 100 μ L cell suspensions are respectively taken to be added in centrifuge tube, each concentration point does 3 pipes；Every pipe be added 20 μ L labelled antibody (~5 × 10⁴Counts/min), mix, 37 DEG C of culture 2h；Then plus the 500 μ L of PBS of 0.05M pH7.4, shake up, 3000rpm/min, Supernatant is sucked out in 4 DEG C of low-temperature centrifugation 10min, washs precipitating 2 times with PBS, surveys the radiocounting of precipitating, calculate labelled antibody Cell combination rate；It does non-specific binding control tube simultaneously: taking each 100 μ L of concentrations of cells suspension to be added in centrifuge tube, be all provided with 3 Multiple pipe, every pipe be added it is excessive (about 1000 times, according to addition¹³¹I-CD80 antibody protein amount, which calculates, is added unmarked antibody Amount) unmarked antibody, after 37 DEG C of culture 1h, labelled antibody (additional amount and combine pipe unanimously) is added, remaining step is same as above.It is special Anisotropic Percentage bound (%)=total cell Percentage bound (%)-non-specific cell Percentage bound (%).

Standard pipe: pick-and-place exempts from pipe 3, and labelled antibody (with combining pipe consistent) is added thereto and is used as standard pipe, carries out γ It counts.

6 parameter settings

Change the amount of Iodogen, respectively 10,20,30,50,100 μ g, remaining ingredient immobilizes；Change antibody Amount, respectively 10,20,40,80,160 μ g, remaining ingredient immobilize；Change¹³¹The activity of I, respectively 18.5,37.0, 55.5,74.0,111.0MBq, remaining ingredient immobilize；The change reaction time, respectively 4,6,8,10,12min, remaining Ingredient immobilize；Each 3 pipe of amount repeats, and gropes the effect marked at different conditions, finds an optimum mark condition.

The verifying of 7 optimum mark conditions

According to the optimum mark condition preparation obtained¹³¹I marks anti-CD80 Chimeric antibody, measure its mark rate, Top coal drawing, stability and activity.

Two, experimental result

1 mark rate and top coal drawing

It is isolated and purified, is obtained pure after the completion of label¹³¹I-CD80 Chimeric antibody, radio thin layer's chromatographic scan Method testing result shows that the mark rate of each processing and top coal drawing are as shown in table 1.

The mark rate and top coal drawing of antibody under the different flag conditions of table 1

2 stability

Labelled antibody is added in serum and PBS after placement 5d, and the stability respectively handled is as shown in table 2.

The stability of antibody under the different flag conditions of table 2

3 labelled antibody cell combination rates

The labelled antibody maximum specific cell Percentage bound respectively handled is as shown in table 3.

The maximum specific cell Percentage bound of labelled antibody under the different flag conditions of table 3

It can thus be seen that¹³¹It is 20 μ of Iodogen dosage that I, which marks the optimum mark condition of anti-CD80 Chimeric antibody, Na is added in g, 40 μ g of antibody dosage¹³¹I 74MBq reacts at room temperature time 8min.

The verifying of 4 optimum mark conditions

Further verifying show that the labelled antibody mark rate under the conditions of the above optimum mark is (82.5 ± 2.3) %, radiation Chemical purity be (96.4 ± 1.3) %, be added in serum and PBS place 5d after radiochemicsl purity be respectively (80.3 ± 1.6) % and (74.4 ± 1.5) %, maximum specific cell Percentage bound are (36.3 ± 2.0) %.Thus it is clear that its antibody mark rate and Top coal drawing is horizontal preferably, and stability is fine.

Inventor sets new flag condition, Iodogen dosage 30 μ g, 80 μ of antibody dosage by further analysis Na is added in g¹³¹I 55.5MBq reacts at room temperature time 10min.The labelled antibody mark rate being surprised to find that under the flag condition For (84.2 ± 2.4) %, radiochemical purity is (100.0 ± 0.1) % (see Fig. 2), is added to after placing 5d in serum and PBS Radiochemicsl purity be respectively (94.8 ± 0.3) % and (91.3 ± 1.3) % (being shown in Table 4), the Percentage bound with Raji cell is with cell Several increases and increase, when cell number be 1 × 10⁷Percentage bound reaches highest level when a, and specific cell combines under this condition Rate is (38.2 ± 2.3) %, and various aspects result is very excellent.

4 labelled antibody stability observing of table (37 DEG C, top coal drawing %)

The present invention successfully uses¹³¹Anti- CD80 Chimeric antibody is marked in I, especially obtains optimum mark condition, shows Work improves labelled antibody stability, lays the foundation for clinical application.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as Protection scope of the present invention.

Claims (6)

1. a kind of¹³¹The anti-CD80 Chimeric antibody of I label, which is characterized in that it is prepared in accordance with the following methods:

(1) 10-100 μ g Iodogen iodine mark pipe is used, 0.04M PB+0.1M NaCl solution is added, continuously adds anti-CD80 Chimeric antibody 10-160 μ g, be eventually adding iodine (¹³¹I) change sodium 18.5-111.0MBq, react 4-12 after mixing at normal temperature Minute, reaction tube is sucked out in reaction solution after reaction；

(2) PD10 column is activated with 0.04M PB+0.1M NaCl solution balance using preceding, is discarded after solution all drains off all Efflux, the reaction solution of step (1) is added on PD10 column, is all flow to end to solution, is discarded all effluxes, add 0.04M PB+0.1M NaCl elutes product to PD10 column, collects all effluxes up to product.

2. according to claim 1¹³¹The anti-CD80 Chimeric antibody of I label, which is characterized in that it is according to lower section Method preparation:

(1) 30 μ g Iodogen iodine mark pipes are used, 0.04M PB+0.1M NaCl solution is added, continuously adds anti-CD80 people-mouse 80 μ g of chimeric antibody, be eventually adding iodine (¹³¹I) change sodium 55.5MBq, react after mixing at normal temperature 10 minutes, after reaction will Reaction tube is sucked out in reaction solution；

(2) PD10 column is activated with 0.04M PB+0.1M NaCl solution balance using preceding, is discarded after solution all drains off all Efflux, the reaction solution of step (1) is added on PD10 column, is all flow to end to solution, is discarded all effluxes, add 0.04M PB+0.1M NaCl elutes product to PD10 column, collects all effluxes up to product.

3. according to claim 1 or 2¹³¹The anti-CD80 Chimeric antibody of I label, which is characterized in that described¹³¹I The top coal drawing of the anti-CD80 Chimeric antibody of label is greater than 90%.

4. a kind of¹³¹The preparation method of the anti-CD80 Chimeric antibody of I label, which comprises the following steps:

(1) 10-100 μ g Iodogen iodine mark pipe is used, 0.04M PB+0.1M NaCl solution is added, continuously adds anti-CD80 Chimeric antibody 10-160 μ g, be eventually adding iodine (¹³¹I) change sodium 18.5-111.0MBq, react 4-12 after mixing at normal temperature Minute, reaction tube is sucked out in reaction solution after reaction；

(2) PD10 column is activated with 0.04M PB+0.1M NaCl solution balance using preceding, is discarded after solution all drains off all Efflux, the reaction solution of step (1) is added on PD10 column, is all flow to end to solution, is discarded all effluxes, add 0.04M PB+0.1M NaCl elutes product to PD10 column, collects all effluxes up to product.

5. the preparation method according to claim 4, which comprises the following steps:

(1) 30 μ g Iodogen iodine mark pipes are used, 0.04M PB+0.1M NaCl solution is added, continuously adds anti-CD80 people-mouse 80 μ g of chimeric antibody, be eventually adding iodine (¹³¹I) change sodium 55.5MBq, react after mixing at normal temperature 10 minutes, after reaction will Reaction tube is sucked out in reaction solution；

(2) PD10 column is activated with 0.04M PB+0.1M NaCl solution balance using preceding, is discarded after solution all drains off all Efflux, the reaction solution of step (1) is added on PD10 column, is all flow to end to solution, is discarded all effluxes, add 0.04M PB+0.1M NaCl elutes product to PD10 column, collects all effluxes up to product.

6. preparation method according to claim 4 or 5, which is characterized in that preparation¹³¹The anti-CD80 people-mouse of I label is embedding The top coal drawing for closing antibody is greater than 90%.