CN105418730A - Antibody purification method based on magnetic bead method - Google Patents
Antibody purification method based on magnetic bead method Download PDFInfo
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- 238000006243 chemical reaction Methods 0.000 claims description 12
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 10
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- 239000012149 elution buffer Substances 0.000 claims description 6
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
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- 239000012190 activator Substances 0.000 claims description 4
- 150000001718 carbodiimides Chemical class 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 3
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 239000012280 lithium aluminium hydride Substances 0.000 claims description 3
- -1 lithium aluminum hydride Chemical group 0.000 claims description 3
- 235000020183 skimmed milk Nutrition 0.000 claims description 3
- 239000012279 sodium borohydride Substances 0.000 claims description 3
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims description 2
- 108010010803 Gelatin Proteins 0.000 claims description 2
- 108010058846 Ovalbumin Proteins 0.000 claims description 2
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 2
- 229910052782 aluminium Inorganic materials 0.000 claims description 2
- ZETCGWYACBNPIH-UHFFFAOYSA-N azane;sulfurous acid Chemical compound N.OS(O)=O ZETCGWYACBNPIH-UHFFFAOYSA-N 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- 229910001448 ferrous ion Inorganic materials 0.000 claims description 2
- 229920000159 gelatin Polymers 0.000 claims description 2
- 239000008273 gelatin Substances 0.000 claims description 2
- 235000019322 gelatine Nutrition 0.000 claims description 2
- 235000011852 gelatine desserts Nutrition 0.000 claims description 2
- 229910000037 hydrogen sulfide Inorganic materials 0.000 claims description 2
- 229940092253 ovalbumin Drugs 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 238000000746 purification Methods 0.000 abstract description 11
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- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 description 10
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- 238000007885 magnetic separation Methods 0.000 description 3
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- 238000001042 affinity chromatography Methods 0.000 description 2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an antibody purification method based on a magnetic bead method. The method includes the steps that protein A magnetic beads are added into a liquid carrying system containing antibodies so that the antibodies can be combined with the magnetic beads, an magnetic field is externally applied after the system is balanced so that the magnetic beads carrying the target antibodies can be fixed by the magnetic field, system liquid is discarded to achieve solid-liquid separation, the externally-applied magnetic field is removed, a washing solution is added to wash the magnetic beads, the magnetic field is externally applied again so that the magnetic beads carrying the target antibodies can be fixed by the magnetic field, liquid is discarded, the externally-applied magnetic field is removed, eluant is added to wash the magnetic beads, the magnetic field is externally applied to remove the magnetic beads, then pure antibodies can be obtained, a preservation buffer solution is added, and the pH and the ion environment needed by the antibodies are balanced to achieve long-term preservation conditions. Through control over the use quantity of the magnetic beads, the method is suitable for purification systems of different sizes and can be suitable for both small-system trial production in laboratories and large-system production of large-scale preparation in factories; the method can also be applied to automatic experimentation and production, operation efficiency can be improved, and manual operation can be reduced.
Description
Technical field
The present invention relates to immunology antibody purification technical field, especially relate to a kind of antibody purification process based on paramagnetic particle method.
Background technology
At present, conventional antibody purification process has based on antibody molecule size and water-soluble different dialysis method, to cross post method based on the affinity chromatography that aglucon is different, based on the different high performance liquid chromatography of moving phase partition ratio.For obtaining purer antibody, above-mentioned three kinds of purification process also can be combined usually, and gained antibody purity is higher, but still has some limitations.Dialysis method needs the operating time longer; Column chromatography system is not easily amplified, is reduced; It is higher that high performance liquid chromatography consumes chromatographic column cost; And above three kinds of methods are all difficult to carry out automated operation, from the lab scale multiplication of system to actual production process of laboratory, have that difficulty is large, the time long, be difficult to automated operation, and the shortcoming of not easily technique for fixing, make gained antibody characteristic difficult parameters with stable.
In recent years, along with the development of magnetic separation technique, the Superparamagnetic beads of albumin A bag quilt is applied to immunodetection and small-scale immunoaffinity purification field more and more.Albumin A bag is by the tomography devices of magnetic beads for purifying technology without the need to complexity, the clarity of sample is not limited, only need simple magnetic absorption step easily can be separated monoclonal antibody quickly from monoclonal antibody expression product, efficiently solve the weak point of traditional chromatographic technique.But because the albumin A bag occurred on the market is at present low by the antibody binding efficiency of magnetic bead, therefore still fail to be applied to antibody purification industry on a large scale.
Summary of the invention
For solving the problem, the object of this invention is to provide a kind of simple operation, the antibody purification process based on paramagnetic particle method rapidly and efficiently.
For achieving the above object, the present invention adopts following technical scheme:
Based on an antibody purification process for paramagnetic particle method, it comprises the following steps:
Step 1, the magnetic bead of albumin A bag quilt to be added in the carrier fluid system containing antibody, stir, incubated at room temperature 5 ~ 60min, magnetic bead is evenly distributed state, in the process magnetic bead and antibodies in the solution; The immunomagnetic beads that the preparation method of magnetic bead a kind of immunomagnetic beads disclosed in the patent ZL201410181299.5 of the application of above-mentioned albumin A bag quilt obtains;
After system balance in step 2, step 1, externally-applied magnetic field, makes magnetic bead carry antibody and is fixed by magnetic field, discard system liquid, carry out solid-liquid separation, makes antibody and magazins' layout purifying;
Step 3, remove externally-applied magnetic field, add lavation buffer solution and rinse magnetic bead 2 ~ 4 times, cleaning antibody surface impurity; Repeating step 2 ~ 3 once;
Step 4, remove externally-applied magnetic field, add elution buffer and rinse magnetic bead 2 ~ 4 times, make antibody and Beads enrichment;
Step 5, externally-applied magnetic field remove magnetic bead, namely obtain the antibody after purifying;
Step 6, add preservation damping fluid to the antibody after purifying, pH needed for balance antibody and ionic environment, reach long-time preservation condition.
Above-mentioned based in the antibody purification process of paramagnetic particle method, the preparation method of the magnetic bead of albumin A bag quilt in its step 1, it comprises the following steps:
S1,1 ~ 2ml immunomagnetic beads is added 800 μ l ~ 2ml concentration of volume percent is in the activator of 5 ~ 50%, lucifuge under room temperature condition, concussion activation 1 ~ 5h, and Magneto separate, abandons supernatant;
S2, to preserve the magnetic bead after buffer solution for cleaning activation with 1 ~ 5ml for several times, then the magnetic bead after activation is dispersed in 400 ~ 800 μ l and preserves in damping fluids, obtain the bead suspension activated;
S3, joined in the bead suspension activated in step S2 containing the Protein A solution of 500ug ~ 1mg albumin A by 100 ~ 200 μ l, make reaction be totally 500ul ~ 1ml, under room temperature condition, concussion reaction is spent the night, and Magneto separate, abandons supernatant liquor;
S4, preserve damping fluid resuspended magnetic bead with 1 ~ 5ml, Magneto separate, abandons supernatant liquor; Repetitive operation once;
S5, add in magnetic bead 1 ~ 5ml preserve damping fluid, under room temperature condition shake 2 ~ 3h, Magneto separate, abandons supernatant liquor;
S6, with the resuspended magnetic bead of 1 ~ 5ml cleaning buffer solution, Magneto separate, abandons supernatant liquor; Repetitive operation twice;
S7, clean resuspended magnetic bead, Magneto separate with the ultrapure water of precooling, abandon supernatant liquor; Repetitive operation once;
S8, concentration preparation suspension containing magnetic beads 1 ~ 5ml according to 10mg/mL, and add the reductive agent that volume is suspension containing magnetic beads 1 ~ 10%, react 2 ~ 3h under 4 ~ 8 DEG C of conditions, mixing magnetic bead, Magneto separate, abandons supernatant;
S9, preserve damping fluid resuspended magnetic bead with 1 ~ 2ml, Magneto separate, abandons supernatant liquor; Repetitive operation twice; Finally magnetic bead is resuspended in magnetic bead by 10mg/ml concentration to preserve in damping fluid, is placed in 2 ~ 8 DEG C of preservations.
Further, above-mentioned cleaning buffer solution is pH7 ~ 8, containing PBST or the Tris-HCl damping fluid of concentration of volume percent 0.01% ~ 0.1%Tween-20 and pH7 ~ 9,0.1 ~ 0.5M, TritonX-100 containing concentration of volume percent 0.1 ~ 3%TritonX-100.
Further, above-mentioned clear de-damping fluid be 0.01M ~ 0.5M, pH2 ~ 3, containing the Gly-HCl of 0.1M ~ 0.2MNaCl or 0.01 ~ 0.3M, pH2 ~ 3, GITC containing 0.1M ~ 0.2MNaCl.
Further, above-mentioned preservation damping fluid is pH7 ~ 8, containing 1 × PBS of concentration of volume percent 0.002% ~ 0.05%NaN3 and 0.5 ~ 5% protein protective agent or 0.5M ~ 1M, Tris-HCl containing concentration of volume percent 0.002% ~ 0.05%NaN3 and 0.5 ~ 5% protein protective agent.
Further, above-mentioned protein protective agent is more than one in bovine serum albumin, ovalbumin, skim-milk, sucrose, gelatin, trehalose.
Further, above-mentioned activator is glutaraldehyde or carbodiimide.
Further, above-mentioned reductive agent is lithium aluminum hydride, tetrahydrofuran (THF), hydrazine hydrate, ammonium sulfite, hydrogen sulfide, sodium borohydride, aluminum borohydride, POTASSIUM BOROHYDRIDE or ferrous ion reductive agent.
Owing to adopting technical scheme as above, the present invention has following superiority:
The present invention is based on the antibody purification process of paramagnetic particle method, it utilizes immunomagnetic beads to carry out antibody purification, by controlling the magnetic bead consumption used, be applicable to the purification system of different size, both gone for the little system trial-production in laboratory, and gone for again the large system production that factory prepares in a large number, automation experiment and production can also be applied to, improve operation efficiency, save manual operation.
The present invention is based on the antibody purification process of paramagnetic particle method, its paramagnetic particle method adsorb antibodies utilized rapidly and efficiently, usually suitable in system, under mixing sufficient situation, in one minute, namely magnetic bead can combine with antibody, sectional interest can complete combination in 30 seconds, antibodies is on magnetic bead, under the magnetic bead of albumin A bag quilt drives, usual a few second can with other magazins' layout in liquid, discard impurity also very convenient, can by suction or pouring liquid, also can by directly shifting magnetic bead, efficiency improves greatly; Whole purge process by carrying the automation equipment operation of removable magnetic means, both can have been saved artificial, and can avoid again the trickle process variations because human users causes and make antibody specific nature parameters unstable; The magnetic means that solid-liquid separation uses can be manually-operated equipment for magnetic separation, also can be equipment for magnetic separation or the Magneto separate machine of automatization.
The present invention is based on the antibody purification process of paramagnetic particle method, it is simple to operate, realize automated operation, can simple and easy amplification, reduce system, reduce costs, rapidly and efficiently, can be used alone, also can be combined with traditional dialysis method, affinity column chromatography method, high performance liquid chromatography, greatly can improve the purity of object product as pre-treatment step, save the more time when making it to enter downstream procedures, reduce the consumption of chromatographic column, chromatography column simultaneously.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE detected result figure of each step products in the purification process of the embodiment of the present invention 1; Wherein, applied sample amount is 40 μ l; 1st swimming lane is protein standard marker Marker; 2nd, 3,4,5 swimming lanes are the antibody that albumin A magnetic bead reuses purifying after 1 time, 3 times, 5 times, 7 times; 6th swimming lane is pure human IgG standard control; 7th swimming lane is the eluted product of pure human IgG standard control after albumin A magnetic beads for purifying; 8th swimming lane is serum sample;
Fig. 2 is ProteinA antibody purification magnetic bead ultralow non-specific adsorption experimental protein SDS-PAGE detected result figure; Wherein, applied sample amount is 40 μ l; 1st swimming lane is negative; 2nd swimming lane is the antibody after purifying; 3rd swimming lane is the serum sample after dilution;
Fig. 3 is that ProteinA antibody purification magnetic bead reuses experimental result picture.
Embodiment
Can be described in further detail the present invention with reference to following examples; But following examples are only illustrations, and the present invention is not limited to these embodiments.
Embodiment 1
Extraction purification human IgG from a small amount of human serum
First the magnetic bead of albumin A bag quilt is prepared
Fully mixed by immunomagnetic beads suspension before S1, use, 1ml immunomagnetic beads being added 800 μ l concentration of volume percent is in the glutaraldehyde of 10%, lucifuge under room temperature condition, concussion activation 1h on sample mix instrument, and Magneto separate, abandons supernatant;
S2, with 1mlpH7, containing the magnetic bead 3 times after 1 × PBS cleaning activation of matter concentration of volume percent 0.002%NaN3 and 1%BSA, then cleaned magnetic bead is added in new centrifuge tube, magnetic bead after activation is dispersed in 400 μ lpH7,1 × PBS containing mass percent concentration 0.002%NaN3 and 1%BSA, obtains the bead suspension activated;
S3,100 μ l are joined in the bead suspension of activation containing the Protein A solution of 800ug albumin A, make reaction be totally 500ul, under room temperature condition, sample mix instrument shakes reaction and spends the night, Magneto separate, abandon supernatant liquor;
S4, with 1mlpH7, the resuspended magnetic bead of 1 × PBS containing mass percent concentration 0.002%NaN3 and 1%BSA, Magneto separate, abandons supernatant liquor; Repetitive operation once;
S5, in magnetic bead, add 1mlpH7,1 × PBS containing concentration of volume percent 0.002%NaN3 and 1%BSA, under room temperature condition, sample mix instrument shakes 2h, Magneto separate, abandon supernatant liquor;
S6, the resuspended magnetic bead of use 2mlPBST, Magneto separate, abandons supernatant liquor; Repetitive operation twice;
S7, clean resuspended magnetic bead, Magneto separate with the ultrapure water of precooling, abandon supernatant liquor; Repetitive operation once;
S8, suspension containing magnetic beads 3ml according to the concentration preparation precooling of 10mg/mL, and add lithium aluminum hydride 0.15ml, react 3h under 4 DEG C of conditions, every 15 minutes of period put upside down mixing magnetic bead, and as found, in pipe, bubble is too much, venting of can uncapping, and Magneto separate, abandons supernatant;
S9, the resuspended magnetic bead of use 1ml1 × PBS, Magneto separate, abandons supernatant liquor; Repetitive operation twice; Finally magnetic bead is resuspended in 1 × PBS by 10mg/ml concentration, is placed in 3 DEG C of preservations.
(1), sample preparation: get anti-body contg and be about the human serum of 0.2mg in 20 μ L centrifuge tubes, with binding buffer liquid 10 times dilution, mix; Binding buffer liquid is 0.05M ~ 0.5MpH7 ~ 8PBS damping fluid;
(2), magnetic bead pre-treatment: the magnetic bead of the albumin A bag quilt of preparation is shaken up, makes it fully resuspended, get 100 μ L (about 1mg) suspension containing magnetic beads and carry out magnetic resolution in new centrifuge tube, make magnetic bead be attracted to tube wall and clarify to solution, abandon supernatant liquor;
(3), to magnetic bead in step (2) add 1ml cleaning buffer solution PBST, after mixing, magnetic resolution 50s, abandons supernatant liquor; Repeating step (2), (3) twice;
(4), antibody absorption: the sample prepared in step (1) is added in the ProteinA magnetic bead in step (3) after cleaning, be placed in upset mixed instrument under room temperature or overturn centrifuge tube gently by hand, jog mixing 10min, reacts complete, magnetic resolution 1min, abandons supernatant;
(5), magnetic bead washing: add 1ml cleaning buffer solution PBST in centrifuge tube, jog mixes, and magnetic resolution 1min, abandons supernatant, repeating step (4), (5) twice;
(6), antibody elution: 500 μ l elution buffer 0.05MpH2Gly-HCl are added in step (5) and is combined with in the ProteinA magnetic particle of human IgG, be placed in upset mixed instrument under room temperature or overturn centrifuge tube gently by hand, jog mixes about 10min, centrifuge tube is placed on magnetic separator, magnetic resolution 1min, supernatant sucking-off is transferred in the centrifuge tube being added with 1MpH9.0Tris-HCl25 μ l in advance, is the antibody-solutions that purifying obtains;
(7), antibody dialysis: because antibody elution damping fluid contains the salinity of higher concentration, user can select suitable neutral low salt solutions (dialyzate) to carry out dialysis treatment to the supernatant liquor that step (6) obtains according to downstream experimental conditions; This step is done according to the choosing of concrete downstream experimental conditions;
(8), magnetic bead aftertreatment: the magnetic bead lavation buffer solution 0.5MpH9Tris-HCl after use carries out resuspended, and then magnetic resolution, abandons supernatant liquor, and repetitive operation once; Add according to the concentration of about 10mg/mL and preserve pH of buffer 71 × PBS, be placed in 3 DEG C of preservations.Human IgG purity is 95%, the rate of recovery 95%.
Human IgG purification effect:
As can be seen from Figure 1, the human IgG of purifying and pure human IgG standard substance electrophoretic band position consistency from human serum, purity is very high, compares and protein content in serum, and after purified, brightness is substantially constant, and the purifying rate of recovery is high.Confirm that the purifying rate of recovery is more than 95% by measuring pure human IgG standard substance and the standard substance IgG after albumin A magnetic beads for purifying in 280nm absorbancy.
As can be seen from Figure 2, through the antibody of albumin A magnetic bead purifying from human serum, purity is high, and the rate of recovery is high, and IgG is free of losses almost.
As can be seen from Figure 2, high by albumin A magnetic bead IgG purity of purifying from human serum, and after reclaiming magnetic bead, reuse repeatedly, degree of purification and efficiency are without degeneration.
Embodiment 2
Therefrom measure extraction purification rabbit igg in rabbit anteserum
First the magnetic bead of albumin A bag quilt is prepared
Fully mixed by immunomagnetic beads suspension before S1, use, 1.5ml immunomagnetic beads being added 1ml concentration of volume percent is in the glutaraldehyde of 15%, lucifuge under room temperature condition, concussion activation 3h on sample mix instrument, and Magneto separate, abandons supernatant;
S2, with 2ml0.5M containing the magnetic bead 3 times after the Tris-HCl cleaning activation of concentration of volume percent 0.002%NaN3 and 1%OVA, then cleaned magnetic bead is added in new centrifuge tube, magnetic bead after activation is dispersed in 500 μ lTris-HCl, obtains the bead suspension activated;
S3,100 μ l are joined in the bead suspension of activation containing the Protein A solution of 600ug albumin A, make reaction be totally 900ul, under room temperature condition, sample mix instrument shakes reaction and spends the night, Magneto separate, abandon supernatant liquor;
S4, the resuspended magnetic bead of use 1.5mlTris-HCl, Magneto separate, abandons supernatant liquor; Repetitive operation once;
S5, in magnetic bead, add 2mlTris-HCl, under room temperature condition, sample mix instrument shakes 2h, Magneto separate, abandon supernatant liquor;
S6, the resuspended magnetic bead of use 3mlTris-HCl damping fluid, Magneto separate, abandons supernatant liquor; Repetitive operation twice;
S7, clean resuspended magnetic bead, Magneto separate with the ultrapure water of precooling, abandon supernatant liquor; Repetitive operation once;
S8, suspension containing magnetic beads 5ml according to the concentration preparation precooling of 10mg/mL, and add tetrahydrofuran (THF) 0.5ml, react 2h under 5 DEG C of conditions, every 15 minutes of period put upside down mixing magnetic bead, and as found, in pipe, bubble is too much, venting of can uncapping, and Magneto separate, abandons supernatant;
S9, the resuspended magnetic bead of use 1mlTris-HCl, Magneto separate, abandons supernatant liquor; Repetitive operation twice; Finally magnetic bead is resuspended in Tris-HCl by 10mg/ml concentration, is placed in 5 DEG C of preservations.
(1), sample preparation: get anti-body contg and be about 2mg rabbit anteserum 200 μ L in 5ml centrifuge tube, with the 10 times of dilutions of binding buffer liquid 0.1MpH7.5 ~ 8.5Tris-HCl damping fluid, mix;
(2), magnetic bead pre-treatment: the magnetic bead of albumin A bag quilt is shaken up, makes it fully resuspended, get 1ml (about 10mg) suspension containing magnetic beads in new 2ml centrifuge tube and carry out magnetic resolution, make magnetic bead be attracted to tube wall and clarify to solution, abandon supernatant liquor;
(3), add 1.5mlTris-HCl damping fluid add TritonX-100 to magnetic bead in step (2), after mixing, magnetic resolution 1min, abandons supernatant liquor; Repeating step (2), (3) twice;
(4), antibody absorption: the sample prepared in step (1) is added in the ProteinA magnetic bead in step (3) after cleaning, be placed in upset mixed instrument under room temperature or overturn centrifuge tube gently by hand, jog mixing 10min, reacts complete, magnetic resolution 1min, abandons supernatant;
(5), magnetic bead washing: add 4mlTris-HCl damping fluid and add TritonX-100 in centrifuge tube, jog mixes, and magnetic resolution 1min, abandons supernatant, repeating step (4), (5) twice;
(6), antibody elution: 1ml0.01MpH2GITC elution buffer is added in step (5) and be combined with in the ProteinA magnetic particle of rabbit igg, be placed in upset mixed instrument under room temperature or overturn centrifuge tube gently by hand, jog mixing 10min; Centrifuge tube is placed on magnetic separator, magnetic resolution 1min, supernatant sucking-off is transferred in the centrifuge tube being added with 1MpH9.0Tris-HCl250 μ l in advance, be the antibody-solutions that purifying obtains;
(7), antibody dialysis: because antibody elution damping fluid contains the salinity of higher concentration, user can select suitable neutral low salt solutions (dialyzate) to carry out dialysis treatment to the supernatant liquor that step (6) obtains according to downstream experimental conditions; This step is done according to the choosing of concrete downstream experimental conditions;
(8), magnetic bead aftertreatment: the magnetic bead lavation buffer solution after use carries out resuspended, and then magnetic resolution, abandons supernatant liquor, and repetitive operation once; Add according to the concentration of about 10mg/mL and preserve damping fluid 0.6MTris-HCl, be placed in 5 DEG C of preservations.Rabbit igg purity is 93%, the rate of recovery 96%.
Embodiment 3
Therefrom measure extraction purification sheep IgG in sheep blood serum
First the magnetic bead of albumin A bag quilt is prepared
Fully mixed by immunomagnetic beads suspension before S1, use, 2ml immunomagnetic beads being added 2ml concentration of volume percent is in the carbodiimide of 30%, lucifuge under room temperature condition, concussion activation 5h on sample mix instrument, and Magneto separate, abandons supernatant;
S2, with 2ml0.5M containing the magnetic bead after the Tris-HCl cleaning activation of concentration of volume percent 0.002%NaN3 and 3% skim-milk, 2% sucrose 3 times, then cleaned magnetic bead is added in new centrifuge tube, magnetic bead after activation is dispersed in 500 μ lTris-HCl, obtains the bead suspension activated;
S3,150 μ l are joined in the bead suspension of activation containing the Protein A solution of 0.2mg albumin A, make reaction be totally 0.5ml, under room temperature condition, sample mix instrument shakes reaction and spends the night, Magneto separate, abandon supernatant liquor;
S4, the resuspended magnetic bead of use 2mlTris-HCl, Magneto separate, abandons supernatant liquor; Repetitive operation once;
S5, in magnetic bead, add 3mlTris-HCl, under room temperature condition, sample mix instrument shakes 2.5h, Magneto separate, abandon supernatant liquor;
S6, the resuspended magnetic bead of use 3mlTris-HCl damping fluid, Magneto separate, abandons supernatant liquor; Repetitive operation twice;
S7, clean resuspended magnetic bead, Magneto separate with the ultrapure water of precooling, abandon supernatant liquor; Repetitive operation once;
S8, suspension containing magnetic beads 3ml according to the concentration preparation precooling of 10mg/mL, and add hydrazine hydrate 0.15ml, react 1.5h under 6 DEG C of conditions, every 15 minutes of period put upside down mixing magnetic bead, and as found, in pipe, bubble is too much, venting of can uncapping, and Magneto separate, abandons supernatant;
S9, the resuspended magnetic bead of use 1.5mlTris-HCl, Magneto separate, abandons supernatant liquor; Repetitive operation twice; Finally magnetic bead is resuspended in Tris-HCl by 10mg/ml concentration, is placed in 6 DEG C of preservations.
(1), sample preparation: get antibody and be about 5mg sheep blood serum 500 μ L in 10ml centrifuge tube, with binding buffer liquid 10 times dilution, mix; Binding buffer liquid is 0.05M ~ 0.5MpH7 ~ 8PBS damping fluid;
(2), magnetic bead pre-treatment: the magnetic bead of albumin A bag quilt is shaken up, makes it fully resuspended, get 3ml (about 30mg) suspension containing magnetic beads in new 5ml centrifuge tube and carry out magnetic resolution, make magnetic bead be attracted to tube wall and clarify to solution, abandon supernatant liquor;
(3), to magnetic bead in step (2) add 3ml cleaning buffer solution PBST, after mixing, magnetic resolution 1min, abandons supernatant liquor; Repeating step (2), (3) twice;
(4), antibody absorption: the sample prepared in step (1) is added in the ProteinA magnetic bead in step (3) after cleaning, be placed in upset mixed instrument under room temperature or overturn centrifuge tube gently by hand, jog mixing 20min, reacts complete, magnetic resolution 1min, abandons supernatant;
(5), magnetic bead washing: add 9ml cleaning buffer solution PBST in centrifuge tube, jog mixes, and magnetic resolution 2min, abandons supernatant, repeating step (4), (5) twice;
(6), antibody elution: 10ml elution buffer 0.2MpH2.5Gly-HCl is added in step (5) and is combined with in the ProteinA magnetic particle of sheep IgG, be placed in upset mixed instrument under room temperature or overturn centrifuge tube gently by hand, jog mixes about 10min, centrifuge tube is placed on magnetic separator, magnetic resolution 1min, supernatant sucking-off is transferred in the centrifuge tube being added with 1MpH9.0Tris-HCl25 μ l in advance, is the antibody-solutions that purifying obtains;
(7), antibody dialysis: because antibody elution damping fluid contains the salinity of higher concentration, user can select suitable neutral low salt solutions (dialyzate) to carry out dialysis treatment to the supernatant liquor that step (6) obtains according to downstream experimental conditions; This step is done according to the choosing of concrete downstream experimental conditions;
(8), magnetic bead aftertreatment: the magnetic bead lavation buffer solution 0.6MpH9Tris-HCl after use carries out resuspended, and then magnetic resolution, abandons supernatant liquor, and repetitive operation once; Add according to the concentration of about 10mg/mL and preserve pH of buffer 7.51 × PBS, be placed in 6 DEG C of preservations.Sheep IgG purity is 93%, the rate of recovery 96%.
Embodiment 4
Extraction purification sheep IgG from a large amount of sheep blood serum
First the magnetic bead of albumin A bag quilt is prepared
Fully mixed by immunomagnetic beads suspension before S1, use, 2ml immunomagnetic beads being added 2ml concentration of volume percent is in the carbodiimide of 50%, lucifuge under room temperature condition, concussion activation 5h on sample mix instrument, and Magneto separate, abandons supernatant;
S2, with 5ml0.5M containing the magnetic bead 3 times after the Tris-HCl cleaning activation of concentration of volume percent 0.03%NaN3 and 2%BSA, 2% sucrose, then cleaned magnetic bead is added in new centrifuge tube, magnetic bead after activation is dispersed in 800 μ lTris-HCl, obtains the bead suspension activated;
S3,200 μ l are joined in the bead suspension of activation containing the Protein A solution of 1mg albumin A, make reaction be totally 1ml, under room temperature condition, sample mix instrument shakes reaction and spends the night, Magneto separate, abandon supernatant liquor;
S4, the resuspended magnetic bead of use 5mlTris-HCl, Magneto separate, abandons supernatant liquor; Repetitive operation once;
S5, in magnetic bead, add 5mlTris-HCl, under room temperature condition, sample mix instrument shakes 2h, Magneto separate, abandon supernatant liquor;
S6, the resuspended magnetic bead of use 5mlTris-HCl damping fluid, Magneto separate, abandons supernatant liquor; Repetitive operation twice;
S7, clean resuspended magnetic bead, Magneto separate with the ultrapure water of precooling, abandon supernatant liquor; Repetitive operation once;
S8, suspension containing magnetic beads 5ml according to the concentration preparation precooling of 10mg/mL, and add sodium borohydride 0.5ml, react 1.5h under 8 DEG C of conditions, every 15 minutes of period put upside down mixing magnetic bead, and as found, in pipe, bubble is too much, venting of can uncapping, and Magneto separate, abandons supernatant;
S9, the resuspended magnetic bead of use 2mlTris-HCl, Magneto separate, abandons supernatant liquor; Repetitive operation twice; Finally magnetic bead is resuspended in Tris-HCl by 10mg/ml concentration, is placed in 8 DEG C of preservations.
(1), sample preparation: get anti-body contg and be about 50mg sheep blood serum 5ml in 100ml wide-mouth reagent bottle, with the 10 times of dilutions of binding buffer liquid 0.1MpH7.5 ~ 8.5Tris-HCl damping fluid, mix;
(2), magnetic bead pre-treatment: the magnetic bead of albumin A bag quilt is shaken up, makes it fully resuspended, get 30ml (about 300mg) suspension containing magnetic beads in new 50ml centrifuge tube and carry out magnetic resolution, make magnetic bead be attracted to tube wall and clarify to solution, abandon supernatant liquor;
(3), add 30mlTris-HCl damping fluid add TritonX-100 to magnetic bead in step (2), after mixing, magnetic resolution 2min, abandons supernatant liquor; Repeating step (2), (3) twice;
(4), antibody absorption: the sample prepared in step (1) is added in the ProteinA magnetic bead in step (3) after cleaning, be placed in upset mixed instrument under room temperature or overturn centrifuge tube gently by hand, jog mixing 30min, reacts complete, magnetic resolution 12min, abandons supernatant;
(5), magnetic bead washing: add 20mlTris-HCl damping fluid and add TritonX-100 in centrifuge tube, jog mixes, and magnetic resolution 2min, abandons supernatant, repeating step (4), (5) twice;
(6), antibody elution: 20ml0.3MpH3GITC elution buffer is added in step (5) and be combined with in the ProteinA magnetic particle of sheep IgG, be placed in upset mixed instrument under room temperature or overturn centrifuge tube gently by hand, jog mixing 10min; Centrifuge tube is placed on magnetic separator, magnetic resolution 2min, supernatant sucking-off is transferred to and is added with in the reagent bottle of 1MpH9.0Tris-HCl50ml in advance, be the antibody-solutions that purifying obtains;
(7), antibody dialysis: because antibody elution damping fluid contains the salinity of higher concentration, user can select suitable neutral low salt solutions (dialyzate) to carry out dialysis treatment to the supernatant liquor that step (6) obtains according to downstream experimental conditions; This step is done according to the choosing of concrete downstream experimental conditions;
(8), magnetic bead aftertreatment: the magnetic bead lavation buffer solution after use carries out resuspended, and then magnetic resolution, abandons supernatant liquor, and repetitive operation once; Add according to the concentration of about 10mg/mL and preserve damping fluid 1MTris-HCl, be placed in 8 DEG C of preservations.Sheep IgG purity is 92%, the rate of recovery 95%.
Claims (8)
1. based on an antibody purification process for paramagnetic particle method, it is characterized in that: it comprises the following steps:
Step 1, the magnetic bead of albumin A bag quilt to be added in the carrier fluid system containing antibody, stir, incubated at room temperature 5 ~ 60min, magnetic bead is evenly distributed state, in the process magnetic bead and antibodies in the solution; The magnetic bead of above-mentioned albumin A bag quilt is immunomagnetic beads;
After system balance in step 2, step 1, externally-applied magnetic field, makes magnetic bead carry antibody and is fixed by magnetic field, discard system liquid, carry out solid-liquid separation, makes antibody and magazins' layout purifying;
Step 3, remove externally-applied magnetic field, add lavation buffer solution and rinse magnetic bead 2 ~ 4 times, cleaning antibody surface impurity; Repeating step 2 ~ 3 once;
Step 4, remove externally-applied magnetic field, add elution buffer and rinse magnetic bead 2 ~ 4 times, make antibody and Beads enrichment;
Step 5, externally-applied magnetic field remove magnetic bead, namely obtain the antibody after purifying;
Step 6, add preservation damping fluid to the antibody after purifying, pH needed for balance antibody and ionic environment, reach long-time preservation condition.
2. the antibody purification process based on paramagnetic particle method according to claim 1, is characterized in that: the preparation method of the magnetic bead of albumin A bag quilt in its step 1, and it comprises the following steps:
S1,1 ~ 2ml immunomagnetic beads is added 800 μ l ~ 2ml concentration of volume percent is in the activator of 5 ~ 50%, lucifuge under room temperature condition, concussion activation 1 ~ 5h, and Magneto separate, abandons supernatant;
S2, to preserve the magnetic bead after buffer solution for cleaning activation with 1 ~ 5ml for several times, then the magnetic bead after activation is dispersed in 400 ~ 800 μ l and preserves in damping fluids, obtain the bead suspension activated;
S3, joined in the bead suspension activated in step S2 containing the Protein A solution of 500ug ~ 1mg albumin A by 100 ~ 200 μ l, make reaction be totally 500ul ~ 1ml, under room temperature condition, concussion reaction is spent the night, and Magneto separate, abandons supernatant liquor;
S4, preserve damping fluid resuspended magnetic bead with 1 ~ 5ml, Magneto separate, abandons supernatant liquor; Repetitive operation once;
S5, add in magnetic bead 1 ~ 5ml preserve damping fluid, under room temperature condition shake 2 ~ 3h, Magneto separate, abandons supernatant liquor;
S6, with the resuspended magnetic bead of 1 ~ 5ml cleaning buffer solution, Magneto separate, abandons supernatant liquor; Repetitive operation twice;
S7, clean resuspended magnetic bead, Magneto separate with the ultrapure water of precooling, abandon supernatant liquor; Repetitive operation once;
S8, concentration preparation suspension containing magnetic beads 1 ~ 5ml according to 10mg/mL, and add the reductive agent that volume is suspension containing magnetic beads 1 ~ 10%, react 2 ~ 3h under 4 ~ 8 DEG C of conditions, mixing magnetic bead, Magneto separate, abandons supernatant;
S9, preserve damping fluid resuspended magnetic bead with 1 ~ 2ml, Magneto separate, abandons supernatant liquor; Repetitive operation twice; Finally magnetic bead is resuspended in magnetic bead by 10mg/ml concentration to preserve in damping fluid, is placed in 2 ~ 8 DEG C of preservations.
3. the antibody purification process based on paramagnetic particle method according to claim 1 and 2, is characterized in that: its cleaning buffer solution is pH7 ~ 8, containing PBST or the Tris-HCl damping fluid of concentration of volume percent 0.01% ~ 0.1%Tween-20 and pH7 ~ 9,0.1 ~ 0.5M, TritonX-100 containing concentration of volume percent 0.1 ~ 3%TritonX-100.
4. the antibody purification process based on paramagnetic particle method according to claim 1, is characterized in that: its de-clearly damping fluid be 0.01M ~ 0.5M, pH2 ~ 3, containing the Gly-HCl of 0.1M ~ 0.2MNaCl or 0.01 ~ 0.3M, pH2 ~ 3, GITC containing 0.1M ~ 0.2MNaCl.
5. the antibody purification process based on paramagnetic particle method according to claim 1 and 2, is characterized in that: its preserve damping fluid be pH7 ~ 8, containing 1 × PBS of concentration of volume percent 0.002% ~ 0.05%NaN3 and 0.5 ~ 5% protein protective agent or 0.5M ~ 1M, Tris-HCl containing concentration of volume percent 0.002% ~ 0.05%NaN3 and 0.5 ~ 5% protein protective agent.
6. the antibody purification process based on paramagnetic particle method according to claim 5, is characterized in that: its protein protective agent is more than one in bovine serum albumin, ovalbumin, skim-milk, sucrose, gelatin, trehalose.
7. the antibody purification process based on paramagnetic particle method according to claim 2, is characterized in that: its activator is glutaraldehyde or carbodiimide.
8. the antibody purification process based on paramagnetic particle method according to claim 2, is characterized in that: reductive agent is lithium aluminum hydride, tetrahydrofuran (THF), hydrazine hydrate, ammonium sulfite, hydrogen sulfide, sodium borohydride, aluminum borohydride, POTASSIUM BOROHYDRIDE or ferrous ion reductive agent.
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