CN106866819A - A kind of method of utilization toluene-sodium-sulfonchloramide mark OxLDL Ab - Google Patents
A kind of method of utilization toluene-sodium-sulfonchloramide mark OxLDL Ab Download PDFInfo
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Abstract
The invention belongs to drug labelling technical field, a kind of method of utilization toluene-sodium-sulfonchloramide mark OxLDL Ab is disclosed.The method is comprised the following steps:(1) PBS, NaI solution, OxLDL Ab solution and toluene-sodium-sulfonchloramide are sequentially added in reaction vessel, and in 2~10 seconds inner cap upper bottle covers, reacts 1~5min;(2) Sodium Metabisulfite Na is added in the product for obtaining2S2O5Solution is so that reaction terminating;Wherein Na2S2O5The concentration of solution is 2.5~6mg/ml;Na2S2O5It is 1.2~2.4 with the molar ratio of toluene-sodium-sulfonchloramide;(3) with the posts of PBS drip washing PD 10 until column equilibration;(4) posts of PD 10 crossed the product that step (2) is obtained after the balance obtained in step (3).The method has mark rate high, short mark time, good stability, radiochemical purity are high and are easy to the beneficial effect of standard convention operation.
Description
Technical field
The invention belongs to drug labelling technical field, and in particular to a kind of method that utilization toluene-sodium-sulfonchloramide marks OxLDL-Ab.
Background technology
OxLDL-Ab is the antibody that a class is produced using OxLDL as antigen induced its body immune response, for difference
The OxLDL-Ab that the antigenic determinant in site produces is also different.Academic circles at present generally acknowledges OxLDL ELISA (oxidized
Low-density lipoprotein, OxLDL) played a crucial role in formation and the evolution of atherosclerosis, it is
Most important mark in pulse atherosclerosis early stage and the full course of disease.
The Close relation of OxLDL-Ab and AS (atheroma) has also gained public acceptance, and has been achieved with biological sample
The external accurate quantitative analysis detection of the important factors such as OxLDL-Ab.Usual OxLDL-Ab is IgG types, and molecular weight is in several kDa to tens of
KDa does not wait (Da is dalton), and current Chengdu Hua Shen biotechnologys Co., Ltd have developed a kind of new IgM types
OxLDL-Ab antibody, it is the brand-new antibody of a class, and its molecular weight is 900KDa, belongs to μ type immunoglobulins, is deposited with pentamer
, by disulfide bond, structure relatively stablize.The antibody specificity is high, diagnoses and treats in atherosclerosis radioimmunoassay
There is exploitation future in direction.
But especially IgM types OxLDL-Ab localization diagnosis aspects in vivo still lack specificity to current OxLDL-Ab
The labelled antibody of high, good stability is used for localization diagnosis, also there is no living animal successfully to carry out the report of AS imagings, for radiation
Property image for AS early diagnosis also need to carry out in-depth study.Iodine is medically conventional radionuclide, iodine label
Property than other radioactive metal isotope labeling things closer to precursor biochemical characteristic, and with general crystal scintillation counter just
Greater efficiency can be obtained, as a result accurately.Liu Zihua be equal to 2009《Isotope》Disclosure reports entitled on magazine《125I is marked
Note OxLDL ELISA antibody》Article, the side that OxLDL-Ab is marked using Iodogen iodide processes is disclosed in article
Method, the method exist the mark time it is long (>8min), the defect such as poor, repetitive rate difference of stability, therefore urgent need research and development one kind uses iodine mark
Remember the new method of OxLDL-Ab.
The content of the invention
(1) goal of the invention
According to the problems of prior art, the invention provides a kind of mark rate it is high, the mark time is short, stability
Good, radiochemical purity is high and the method that is easy to the use iodine labeling OxLDL-Ab of standard convention operation.
(2) technical scheme
In order to solve the problems of prior art, the present invention is achieved by the following technical solutions:
A kind of method that utilization toluene-sodium-sulfonchloramide marks OxLDL-Ab, the method is comprised the following steps:
(1) sequentially added in reaction vessel PBS, NaI solution and it is well mixed after, recycle pipette to this
OxLDL-Ab solution is added in solution after mixing, pipette lower end will stretch to 1~3mm in mixed solution liquid level during addition
Place, is well mixed again;Toluene-sodium-sulfonchloramide is subsequently adding, and in 2~10 seconds inner cap upper bottle covers, reacts 1~5min;
The PBS is phosphate buffer;The NaI is Na125I、Na123I or Na131I;The toluene-sodium-sulfonchloramide solution
Concentration be 2~6mg/ml, the concentration of PBS is 0.1~0.4mg/ml, and pH value is 7.0~7.8;OxLDL-Ab solution
It is 30 μ g/28MBq~40 μ g/28MBq with the amount ratio of NaI solution;With molecular weight calculation, the consumption of toluene-sodium-sulfonchloramide and OxLDL-Ab
Than being 3963.6:1.
(2) Sodium Metabisulfite Na is added in the product that step (1) is obtained2S2O5Solution is so that reaction terminating;Its
Middle Na2S2O5The concentration of solution is 2.5~6mg/ml;Na2S2O5It is 1.2~2.4 with the molar ratio of toluene-sodium-sulfonchloramide;
(3) it is leacheate drip washing PD-10 posts with PBS, after 3~5 column volumes of drip washing, adds pure containing ox blood
The PBS of albumen (BSA) drip washing PD-10 posts again, are subsequently adding PBS drip washing to column equilibration;
(4) the PD-10 posts crossed the product that step (2) is obtained after the balance obtained in step (3), the product
Be using pipette be transferred to PD-10 posts top and pipette can not contact post jamb and cylinder, question response product fully penetrates into post
Behind face, PBS drip washing being added immediately, and collecting leacheate, the leacheate is final product after purification;
The PD-10 posts are cross-link dextran (sephandexG50) gel filtration chromatography post, and flow velocity is 6~10cm/
min;
Preferably, OxLDL-Ab described in step (1) is IgM type OxLDL-Ab, and its molecular weight is 900KDa, with pentamer
In the presence of by disulfide bond.
Preferably, the concentration of step (1) the toluene-sodium-sulfonchloramide solution is 5mg/ml, and the concentration of PBS is 0.2mg/ml,
PH value is 7.3;OxLDL-Ab solution is 30 μ g/28MBq with the amount ratio of NaI solution;Reaction time is 1min.
Preferably, Na in step (2)2S2O5The concentration of solution is 5mg/ml;Na2S2O5It is 1.5 with toluene-sodium-sulfonchloramide molar ratio.
Preferably, in the PBS containing bovine serum albumin(BSA) (BSA) described in step (3), the PBS bufferings
The PH of liquid is 7.4, and concentration is 0.01mol/L;Volume ratio of the bovine serum albumin in PBS is 1%~3%.
Preferably, NaI solution described in step (1) is Na123I solution.
Preferably, reaction temperature is room temperature in step (1).
(3) beneficial effect
Using chloramine-t method, successfully to OxLDL-Ab, especially IgM types OxLDL-Ab carries out iodine labeling to the application, and mark rate exists
More than 80% and reproducible, it is adaptable to which clinic imaging needs.The method has the advantage that specific as follows:
1. it, with the less IgG types antibody of molecular weight, is secretion in immune response that conventional OxLDL-Ab labelled antibodies are mostly
Stage casing antibody, belong to secondary immune response product, antibody producing amount is big, and Stability Analysis of Structures, its molecular weight is small, mostly 150kDa,
And exposed marker site is more (tyrosine and serine residue are more), easy mark, but mark poor repeatability, mark the time
It is long.And the IgM types antibody in the application has essential distinction with traditional antibody, with label stabilization, to environment temperature etc.
It is the antibody secreted first in immune response it is required that low advantage, plays " pioneer is immunized ", with very strong immunocompetence,
Mostly protection antibody, belongs to one-level immune response product.But the antibody production is few and is difficult to extract separation, and molecular weight is
900kDa, and exposed marker site is few, and there is steric hindrance to act on, it is not easy to mark.It is by IgM monomer shapes in structure
Into a pentamer cyclic structure for closing, surface exposure site is few, and is also easily to inhale with a very hydrophobic core
The reason for being attached to chamber wall.
2. the order that adds of the application step (1) reaction solution is different from general labeling method, and most labeling methods are
First plus antibody, afterwards plus radionuclide is reducing influence of the radioactivity to operator.But the application provide labeling method due to
The exposed site of antibody is small, easily adsorbs, and need to first add radionuclide NaI solution, is subsequently adding OxLDL-Ab solution, Cai Nengman
Requirement of the foot to mark rate.
3. the application step (1) add toluene-sodium-sulfonchloramide after, in 2~10 seconds inner cap upper bottle covers.It is that applicant fully takes into account chlorine
Amine T produces hypochlorous acid in aqueous, and hypochlorous acid can make iodine anion oxygen be melted into iodine molecule (free I2) and then divide with antibody
In son there is halogenation in tyrosine and some histidines.Because adding the moment of toluene-sodium-sulfonchloramide that priming reaction can occur, easily produce
The I of raw literization2Steam is, it is necessary to cover rapidly bottle cap, it is to avoid radioactive material contamination, while it also avoid I2The reduction of concentration, carries
Reactivity high, improves mark rate.
When 4. adding OxLDL-Ab solution in the application step (1), it is high that pipette lower end will stretch to mixed solution liquid level
In degree at 1~3mm;Otherwise antibody is easily adsorbed on wall, has a strong impact on mark rate, even results in mark failure, and mark rate<
20%.
5. in the application step (2), reducing agent Na is added2S2O5Carry out terminating reaction, and traditional way is to add Na2S2O5
And free tyrosine carrys out terminating reaction, because the I of activation2It is that substitution reaction occurs with the tyrosine residue on antibody, adds junket
Propylhomoserin can accelerate the iodine molecule in neutralization reaction, then the antibody and iodotyrosine and reducing agent point that will be marked through gel filtration
Leave.And the application is the step for then eliminate, and to (the terminating reaction time terminating reaction time the step for save<30s)
And mark rate is not influenceed, mark rate is more than 80%.
6. NaI solution is preferably Na in the application123I, different from the Na for commonly using125I, this mainly due to123I half-life period
It is 13.2h, the γ photons of 159kev can be launched, is especially suitable for nuclear medicine image and uses.
Brief description of the drawings
Fig. 1 is influence schematic diagram of the reaction time to mark rate.
Specific embodiment
The present invention is further elaborated below in conjunction with specific embodiment and Figure of description.
Embodiment 1
A kind of method that utilization toluene-sodium-sulfonchloramide marks OxLDL-Ab, the method is comprised the following steps:
(1) sequentially added in reaction vessel PBS, NaI solution and it is well mixed after, recycle pipette to this
OxLDL-Ab solution is added in solution after mixing, pipette lower end will be stretched in mixed solution liquid level at 2mm during addition, then
It is secondary well mixed;Toluene-sodium-sulfonchloramide is subsequently adding, and in 2 seconds inner cap upper bottle covers, the reaction time was 1min;
The PBS is phosphate buffer, and concentration is 0.2mg/ml, and pH value is 7.3;The NaI is Na123I;
The concentration of toluene-sodium-sulfonchloramide solution is 5mg/ml;OxLDL-Ab solution and Na123The amount ratio of I solution is 30 μ g/28MBq;Reaction temperature
It is room temperature.
(2) Na of Sodium Metabisulfite 5mg/ml is added in the product that step (1) is obtained2S2O5Solution is so that anti-
Should terminate, Na2S2O5It is 1.5 with toluene-sodium-sulfonchloramide molar ratio.
(3) it is leacheate drip washing PD-10 posts with PBS, after 3~5 column volumes of drip washing, adds pure containing ox blood
The PBS of albumen (BSA) drip washing PD-10 posts again, are subsequently adding PBS drip washing to column equilibration;Institute in step (3)
In the PBS containing bovine serum albumin(BSA) (BSA) stated, the PH of the PBS is 7.4, and concentration is 0.01mol/
L;Volume ratio of the bovine serum albumin in PBS is 1%.
(4) the PD-10 posts crossed the product that step (2) is obtained after the balance obtained in step (3), the product
Be using pipette be transferred to PD-10 posts top and pipette can not contact post jamb and cylinder, question response product fully penetrates into post
Behind face, PBS drip washing being added immediately, and collecting leacheate, the leacheate is final product;
The PD-10 posts are cross-link dextran (sephandexG50) gel filtration chromatography post, and flow velocity is 6~10cm/
min;
Influence applicants studied reaction condition to mark rate:
(1) influence of the reaction time to mark rate
A reaction bulb is taken, the PBS (pH 7.3) of 210 μ L 0.2mol/L, 5 μ L Na is sequentially added123I (28MBq), mixes
After closing uniformly, 85 μ L OxLDL-Ab (30 μ g) are added, shake reaction bulb, be allowed to well mixed, be subsequently adding 6 μ L toluene-sodium-sulfonchloramides
(30 μ g), shakes reaction bulb, is allowed at room temperature fully reaction.Other conditions are consistent, respectively the reaction time be 1min,
When 2min, 3min, 4min and 5min, sampled with capillary, mark rate is determined respectively, add 9 μ L Na2S2O5(45 μ g) terminates anti-
Should.As shown in Figure 1.
As seen from Figure 1, reaction carries out reachable (81.4 ± 2.6) % of mark rate during 2min, with the extension in reaction time
Mark rate is declined slightly, and mark rate is (68.5 ± 1.9) % after reaction 5min, and this is probably with the extension chlorine in reaction time
The oxidation of amine-T causes part label that the de- iodine of dissociation occurs, and because the oxidisability of toluene-sodium-sulfonchloramide is stronger, increasing the reaction time can
The immunocompetence of energy meeting antagonist produces certain influence.Therefore, it is optimum reacting time that 2min is reacted under this experiment condition, but
It is to consider the factors such as the active influence of mark time antagonist, the reaction time in embodiment 1 is 1min.
(2) reacting liquid pH value pair is marked123The influence of I-OxLDL-Ab mark rates
When other conditions are constant, when reaction system pH is 7.0, mark rate is only (62.5 ± 1.7) %, and mark rate is with pH
Increase and raise, mark rate highest (82.1 ± 2.1) % as reaction system pH 7.3, be then further added by pH, mark rate is anti-
And decline.Therefore this experiment determines that pH 7.3 is optimum reaction condition.
In addition, applicant also uses tlc determination mark rate and radiochemical purity, chromatographic solution used is 85%
Methyl alcohol/15% physiological saline.Result shows that mark rate is more than 80%, and top coal drawing is 95%, and specific activity is 3.29 × 1010Bq/g。
Chloramine-t method mark rate is high, reproducible, and reagent is cheap and easily-available, it is adaptable to which clinic imaging needs, and is to use at present most
Many labeling methods.The amount of the labelled antibody once obtained using Iodogen methods mark is less, is not suitable for Animal imaging experiment,
When lot of antibodies is marked, mark unstable result, mainly due to Iodogen pipes prime coats in bottom, reaction cross-section compared with
It is small, when more radioiodine is added, it is impossible to which reaction is complete, cause mark rate to decline or mark failure.In the present embodiment, adopt
OxLDL-Ab, mark rate is marked to be more than 80% with toluene-sodium-sulfonchloramide, flag condition stabilization, success rate is high.
Embodiment 2
As different from Example 1, the OxLDL-Ab is IgM type OxLDL-Ab, and its molecular weight is 900KDa, poly- with five
Body is present, by disulfide bond.Using addition OxLDL-Ab solution in pipette solution in step (1), during addition under pipette
End will be stretched in mixed solution liquid level at 1mm, and the NaI is Na125I;The concentration of the toluene-sodium-sulfonchloramide solution is 2mg/ml, PBS
The concentration of buffer solution is 0.1mg/ml, and pH value is 7.0;OxLDL-Ab solution is 35 μ g/28MBq with the amount ratio of NaI solution;Instead
It is 2min between seasonable.
Na in step (2)2S2O5The concentration of solution is 2.5mg/ml;Na2S2O5It is 1.2 with the molar ratio of toluene-sodium-sulfonchloramide;
Volume ratio of the bovine serum albumin in PBS is 2% in step (3).
Embodiment 3
As different from Example 1, pipette is to addition OxLDL-Ab solution, liquid relief during addition in the solution after the mixing
Pipe lower end will be stretched in mixed solution liquid level at 3mm;The NaI is Na131I;The concentration of the toluene-sodium-sulfonchloramide solution is 6mg/ml,
The concentration of PBS is 0.4mg/ml, and pH value is 7.8;OxLDL-Ab solution is 40 μ g/ with the amount ratio of NaI solution
28MBq;;Reaction time is 5min.
Na in step (2)2S2O5The concentration of solution is 6mg/ml;Na2S2O5It is 2.4 with the molar ratio of toluene-sodium-sulfonchloramide;
Volume ratio of the haemocyanin in PBS is 3% in step (3).
Claims (7)
1. a kind of method that utilization toluene-sodium-sulfonchloramide marks OxLDL-Ab, it is characterised in that the method is comprised the following steps:
(1) sequentially added in reaction vessel PBS, NaI solution and it is well mixed after, recycle pipette to the mixing
OxLDL-Ab solution is added in solution afterwards, pipette lower end will be stretched in mixed solution liquid level at 1~3mm during addition, plus
It is well mixed again after entering OxLDL-Ab solution;Toluene-sodium-sulfonchloramide is subsequently adding, and in 2~10 seconds inner cap upper bottle covers, reacts 1~5min;
The PBS is phosphate buffer;The NaI is Na125I、Na123I or Na131I;The toluene-sodium-sulfonchloramide solution it is dense
It is 2~6mg/ml to spend, and the concentration of PBS is 0.1~0.4mg/ml, and pH value is 7.0~7.8;OxLDL-Ab solution and NaI
The amount ratio of solution is 30 μ g/28MBq~40 μ g/28MBq;
(2) Sodium Metabisulfite Na is added in the product that step (1) is obtained2S2O5Solution is so that reaction terminating;Wherein
Na2S2O5The concentration of solution is 2.5~6mg/ml;Na2S2O5It is 1.2~2.4 with the molar ratio of toluene-sodium-sulfonchloramide;
(3) it is leacheate drip washing PD-10 posts with PBS, after 3~5 column volumes of drip washing, addition contains bovine serum albumin(BSA)
PBS drip washing PD-10 posts again, be subsequently adding PBS drip washing to column equilibration;
(4) the PD-10 posts crossed the product that step (2) is obtained after the balance obtained in step (3), the product is profit
PD-10 posts top is transferred to pipette and pipette can not contact post jamb and cylinder, after question response product fully penetrates into cylinder,
PBS drip washing being added immediately, and collecting leacheate, the leacheate is final product after purification;
The PD-10 posts are sephadex filtering chromatogram post, and flow velocity is 6~10cm/min.
2. the method that a kind of utilization toluene-sodium-sulfonchloramide according to claim 1 marks OxLDL-Ab, it is characterised in that in step (1)
The OxLDL-Ab is IgM type OxLDL-Ab, and its molecular weight is 900KDa, is existed with pentamer, by disulfide bond.
3. the method that a kind of utilization toluene-sodium-sulfonchloramide according to claim 1 marks OxLDL-Ab, it is characterised in that step (1) institute
The concentration of toluene-sodium-sulfonchloramide solution is stated for 5mg/ml, the concentration of PBS is 0.2mg/ml, and pH value is 7.3;OxLDL-Ab solution with
The amount ratio of NaI solution is 30 μ g/28MBq;Reaction time is 1min.
4. the method that a kind of utilization toluene-sodium-sulfonchloramide according to claim 1 marks OxLDL-Ab, it is characterised in that in step (2)
The Na2S2O5The concentration of solution is 5mg/ml;Na2S2O5It is 1.5 with toluene-sodium-sulfonchloramide molar ratio.
5. the method that a kind of utilization toluene-sodium-sulfonchloramide according to claim 1 marks OxLDL-Ab, it is characterised in that in step (3)
In the described PBS containing bovine serum albumin(BSA), the PH of the buffer solution is 7.4, and concentration is 0.01mol/L;Cow's serum
Volume ratio of the albumen in PBS is 1%~3%.
6. the method that a kind of utilization toluene-sodium-sulfonchloramide according to claim 1 marks OxLDL-Ab, it is characterised in that in step (1)
The NaI solution is Na123I solution.
7. the method that a kind of utilization toluene-sodium-sulfonchloramide according to claim 1 marks OxLDL-Ab, it is characterised in that in step (1)
Reaction temperature is room temperature.
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CN113385118A (en) * | 2020-03-12 | 2021-09-14 | 益诺思生物技术海门有限公司 | A kind of125I medicament marking and purifying device and method thereof |
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CN107286243A (en) * | 2017-07-27 | 2017-10-24 | 北京大学第医院 | Radiate iodine labeling PD L1 monoclonal antibodies and preparation method and application |
CN110237277A (en) * | 2019-07-26 | 2019-09-17 | 北京健平金星生物科技有限公司 | A kind of new Tumor label method |
CN113385118A (en) * | 2020-03-12 | 2021-09-14 | 益诺思生物技术海门有限公司 | A kind of125I medicament marking and purifying device and method thereof |
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