CN104498576A - Immobilized enzyme digestion based preparation method of Fab antibody and application thereof - Google Patents
Immobilized enzyme digestion based preparation method of Fab antibody and application thereof Download PDFInfo
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Abstract
The invention belongs to the medical field of immunoassay and particularly relates to an immobilized enzyme digestion based preparation method of an Fab antibody and application thereof. The immobilized enzyme digestion based preparation method of the Fab antibody comprises the following steps: (1) preparing an immobilized enzyme: adding activated microspheres into the solution of papain, and coupling the papain with the activated coupling fillers completely; (2) digesting with the immobilized enzyme: weighing 5mg of a 2H4 antibody, mixing the 2H4 antibody with 5ml of an EDTA-Na2 digestion buffer solution containing 100mu l of immobilized papain, and incubating, wherein the concentration of the immobilized papain is 0.05mg/ml; and (3) separating and purifying the Fab antibody after finishing digesting with the immobilized enzyme to obtain the Fab antibody. The immobilized enzyme digestion based preparation method of the Fab antibody has the advantages of high enzyme digestion speed and simple separation and is suitable for popularization at home. In addition, the immobilized enzyme can be reused, no high-cost full-automatic equipment is used, and the reagents are low in cost.
Description
Technical field
The present invention relates to field of immunoassay medicine, particularly relate to the method and uses thereof of a kind of harden monitoring cutting for Fab antibody.
Background technology
Monoclonal antibody technique is that British scientist Milstein and Kohler in 1975 invented, and being widely used in clinical treatment and diagnostic field at present, is the important breakthrough of field of immunology.Mouse monoclonal antibody often needs to excise the false positive interference caused when its Fc section reacts (HAMA) and clinical detection with the people's against murine reduced in immunotherapy process.The position that papoid (papain) can be hydrolyzed IgG is held at the nearly N of 2 heavy chains of hinge area disulfide linkage connection, three fragments can be obtained after cracking, 2 same clip are called Fab (fragment antigen binding, and 1 FC (fragmentcrystallizable, Fc) Fab).
Antibody needs to be separated Fab fragment and Fc fragment after papoid enzyme is cut, and the papoid that removing adds could use.At present, conventional method removes Fc section by Protein G/A to adopt the mode of gel-filtration to be separated by molecular size range afterwards, and the method is more time-consuming, once can only process 1-2ml sample, is unsuitable for extensive preparation.Also have report to carry out quick affinity purification by Protein A and Protein G different affine positions difference to be separated, but the method be not suitable for the mouse monoclonal antibody of different antibodies subclass.
Summary of the invention
The shortcoming of prior art in view of the above, the present invention is by being coupled to the NHS-sepharose microballoon of activation by enzyme, then joined antibody and enzyme to cut microseism in buffering and swing enzyme and cut, enzyme cut and terminate after centrifugation microballoon proteinA/G mono-step both can be separated enzyme cut after Fab antibody, to provide a kind of harden monitoring cutting for method of Fab antibody and uses thereof, for solving the problems of the prior art.
For achieving the above object and other relevant objects, first aspect present invention provides a kind of harden monitoring cutting for the method for Fab antibody, comprises the steps:
1) preparation method of harden monitoring: the microballoon adding activation in papain solution, makes papoid and the abundant coupling of activation coupling filler;
2) harden monitoring enzyme is cut: get 2H4 antibody 5mg, with the EDTA-Na of 5ml containing 100 μ l immobilized papains
2the mixing of digestion damping fluid, at 37 DEG C, hatch 2h, enzyme concn is 0.05mg/ml;
3) enzyme is cut rear Fab antibody separation and purification and is namely obtained Fab antibody.
Preferably, in described step 1, the microballoon of activation is specially NHS-activated Sepharose 4Fast Flow microballoon.
Preferably, after described step 2 enzyme is cut, with Tris-HCl (1.0mol/L, pH 8.0) by antibody digestion products pH regulator to 7.5 ~ 8.0.
Preferably, in described step 3, the actual conditions of separation and purification comprises the steps:
(1) Tris-HCl (1.0mol/L, pH 8.0) is used by antibody digestion products pH regulator to 7.5 ~ 8.0.
(2) antibody digestion products is collected effluent liquid by blank chromatography column, elimination immobilized enzyme is also reclaimed.
(3) effluent liquid is passed through albumin A post, collect outflow albumen and be designated as albumen P1.
(4) after using PBS damping fluid fully to balance albumin A post, with HCl-glycine (0.1mol/L, Ph 2.7) wash-out pillar, collect eluted protein and be designated as albumen P2, with Tris-HCl (1.0mol/L, pH 8.0) by the pH regulator of P2 to 7.5 ~ 8.0.
(5) gained albumen is dialysed in PBS.
Second aspect present invention provides described harden monitoring cutting for the method for Fab antibody in the purposes of Fab antibody preparation field.
Content of the present invention comprises: the 1) preparation method of harden monitoring; 2) harden monitoring enzyme cuts the buffer system that antibody uses; 3) harden monitoring enzyme cuts the method for antibody; And 4) enzyme cuts the separation of rear Fab antibody.Further, the method for harden monitoring produced according to the present invention and application thereof comprise the following steps: 1) configure enzyme solution and be coupled to the NHS-sepharose microballoon of activation; 2) configure enzyme cutting buffering liquid and add antibody and microballoon; 3) enzyme is cut mixture be placed in 37 DEG C concussion enzymes cut 30-60 minute; And 4) proteinA/G be separated enzyme cut after Fab antibody.
The Fab antibody preparation method that the present invention sets up has that enzyme cutting speed degree is fast, being separated simple, harden monitoring can Reusability, and do not need expensive automatic equipment, the advantages such as reagent cost is cheap, are adapted at domestic penetration and promotion.
Accompanying drawing explanation
Fig. 1 is shown as immobilization method abzyme of the present invention and cuts result figure.
Fig. 2 is shown as immobilized papain coupling electrophoresis result of the present invention.
Fig. 3 is shown as antigen immune affinity column coupling electrophoresis result of the present invention.
Fig. 4 is shown as indirect elisa method and detects Fc section.
Fig. 5 is shown as indirect elisa method and detects Fab sheet fragment immunocompetence.
Embodiment
Below by way of specific specific examples, embodiments of the present invention are described, those skilled in the art the content disclosed by this specification sheets can understand other advantages of the present invention and effect easily.The present invention can also be implemented or be applied by embodiments different in addition, and the every details in this specification sheets also can based on different viewpoints and application, carries out various modification or change not deviating under spirit of the present invention.
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, instead of in order to limit the scope of the invention; In specification sheets of the present invention and claims, unless explicitly pointed out in addition in literary composition, singulative " ", " one " and " this " comprise plural form.
When embodiment provides numerical range, should be understood that except non-invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique understand usually.Except the concrete grammar used in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, any method of prior art that is similar with the method described in the embodiment of the present invention, equipment, material or that be equal to, equipment and material can also be used to realize the present invention.
Unless otherwise indicated, disclosed in the present invention experimental technique, detection method, preparation method all adopt the routine techniques of the molecular biology of the art routine, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and association area.These technology are existing in existing document improves explanation, specifically can see the MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring HarborLaboratory Press, 1989and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; The seriesMETHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODSIN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), AcademicPress, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
The experiment material used in embodiments of the invention is as follows:
The BNP mouse monoclonal antibody 2H4 (subclass is IgG1) that experiment uses and corresponding BNP recombinant protein provide by clinical biochemical teaching and research room of Third Military Medical University, and serum specimen is collected in clinical laboratory of affiliated hospital.
Albumin A/L Sepharose Fast Flow and NHS-activated Sepharose 4Fast Flow activates coupling filler purchased from Amersham company.
The avidin of biotinylation reagent and HRP mark is purchased from Pierrce company; The goat anti-mouse of HRP mark is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge; Calf serum is folium ilicis chinensis Products; Halfcystine, papoid, BSA are purchased from Beijing Ding Guo Bioisystech Co., Ltd; Other reagent are analytical pure, and experimental water is deionization distilled water.
Elisa plate, purchased from Jet Biofit company.
The instrument used in embodiments of the invention is as shown in table 1:
Table 1
The reagent used in embodiments of the invention is as shown in table 1:
Table 2
Digestion damping fluid
1(2 ×): containing the PBS of 0.02mol/L EDTA (non-sodium salt) and 0.02mol/L halfcystine, Fresh, ice puts use in <10 hour.
Digestion damping fluid
2(2 ×): containing 0.02mol/L EDTA-Na
2with the PBS of 0.02mol/L halfcystine, Fresh, ice puts use in <10 hour.
Affinity column balance liquid: 1 × PBS damping fluid (filtration)
PBS damping fluid: 137mmol/L NaCl, 2.7mmol/L KCl, 10mmol/L Na
2hPO
4, 2mmol/L KH
2pO
4be adjusted to pH 7.4 with HCl, high voltage standby is used.
The SDS-PAGE gel electrophoresis of 15%:
A liquid: (Tris 18.15g, is dissolved in suitable quantity of water to 1.5mol/L Tris-HCl damping fluid, adjusts pH 8.8, be settled to 100mL with HCl; 8ml.
B liquid: 30% acrylamide, 0.8%N, N-methylene-bis methene acrylamide; 4ml.
C liquid: 1%SDS (sodium laurylsulfonate); 1.6ml.
D liquid: 10% Tetramethyl Ethylene Diamine; 10 μ l.
E liquid: 10% ammonium persulphate (APS), prepared before use; 100 μ l.
F liquid: (Tris 6.05g's 0.5mol/L Tris-HCl damping fluid is dissolved in water, and adjusts pH 6.8, be settled to 100mL with HCl; 2.25ml.
ddH
2O：2.28ml。
Electrode buffer: Tris 3g, glycine 14.4g, SDS 1g, with water dissolution, adjusts pH 8.3 with HCl, is settled to 1L.Sample buffer: Tris 0.303g, tetrabromophenol sulfonphthalein 2mg, SDS 0.8g, measures HCl 0.189mL, and glycerine 4mL, is diluted with water to 10mL, and this solution is used for non-reduced SDS-PAGE, as reducing SDS-PAGE, then adds beta-mercaptoethanol 2mL again.
Xylene Brilliant Cyanine G dye liquor: Xylene Brilliant Cyanine G R
2501g, adds formaldehyde 200mL, Glacial acetic acid 50mL, and water 250mL mixes.Xylene Brilliant Cyanine G destainer: methyl alcohol 400mL, Glacial acetic acid 100mL, mixes with water 500mL.
ELISA coating buffer: 1.59g Na
2cO
3, 2.93g NaHCO
3, 0.2g NaN
3after adding deionized water dissolving, NaOH regulates pH to 9.6, is settled to 1L, and normal temperature is preserved.
ELISA washings: 8.0g NaCl, 0.2g KCl, 0.2g KH
2pO
4, 2.9g Na
2hPO
412H
2after O, 0.5mL Tween-20 adds deionized water dissolving, regulate pH to 9.4, be settled to 1L, normal temperature is preserved.
Sample diluting liquid: 8.0g NaCl, 0.2g KCl, 0.2g KH
2pO
4, 2.9g Na
2hPO
412H
2after O, Tween201ml add deionized water dissolving, regulate pH to 9.4, be settled to 1L, normal temperature is preserved.
ELISA confining liquid: 5ml BSA, 95ml sample diluting liquid, matching while using.
ELISA stop buffer: 2mol/L H
2sO
4
Conjugate diluent: 0.1M NaHCO
3, 0.5M NaCl, pH 8.3
Affinity column elutriant: 0.1M glycine-HCI PH 2.3
Embodiment 1 immobilized papain enzyme is cut:
1. prepare different concns papain solution:
Accurately take 0.0050g papoid, be dissolved in 0.5ml PBS damping fluid, be made into 10mg/ml papain solution.With damping fluid dilution, be made into 0.1mg/ml, 0.05mg/ml, 0.025mg/ml papain solution.
2. reaction system is set up:
In papain solution, add enough NHS-activated Sepharose 4Fast Flow, concussion, make papoid and the abundant coupling of activation coupling filler, prepare immobilized papain.SDS-PAGE detects coupling effect.It is as shown in table 1 that immobilized enzyme cuts reaction system:
Table 1
37 DEG C of incubators jolt and hatch 3h.Respectively at 1h, 2h, 3h time point from supernatant collection product 40 μ l, use Tris-HCl (pH 8.0) by pH regulator to 7.5-8.0, freezen protective.
By SDS-PAGE electrophoresis, determine optimum reaction conditions: 1. join glue: according to for protein isolate molecular weight, prepare the polyacrylamide gel of 15% concentration.2. prepare: add sample-loading buffer 10 μ l in protein sample, 100 DEG C are boiled 5min.3. electrophoresis: select constant voltage, the voltage of concentrated glue is 80V, and the voltage of separation gel is 120V.4. dye: Xylene Brilliant Cyanine G dye liquor dyes half an hour.5. decolour: the decolouring of Xylene Brilliant Cyanine G destainer is spent the night.Damping fluid kind, the suitableeest enzyme concn, optimal reaction time is selected according to electrophoresis result.
Visible by experiment, after 2H4 abzyme is cut, heavy chain disappears, and light chain of antibody position is identical.When reaction proceeds to 2h, abzyme is cut effect and is substantially completed, and occurs object band.Extend the reaction times, the antibody of two kinds of buffer systems is all cut by excessive enzyme, produces non-specific band.As shown in Figure 1, in Fig. 1, LANE 1 and 5 is immobilization method optimum antibody enzyme tangent condition specific experiment result: complete 2H4 antibody molecule; LANE 2 is: enzyme cuts 1h; LANE 3 is: enzyme cuts 2h; LANE 4 is: enzyme cuts 3h (2-4 is in the non-sodium salt buffer conditions of EDTA); M:Prestained Protein Marker; LANE 6 is: enzyme cuts 1h; LANE 7 is: enzyme cuts 2h; LANE 8 is: enzyme cuts 3h (6-8 is in EDTA-Na2 buffer conditions).
The selection of enzyme tangent condition requires: choose same enzyme cut effect under minimum Papain enzyme concn and the shortest enzyme cut the time, because excessively enzyme cuts the output and activity that can affect Fab antibody.Consider that EDTA-Na2 solvability is good simultaneously, solid mass can be reduced in reaction system on the impact of endonuclease reaction.The enzyme concn of immobilized papain is fixed, and therefore best enzyme tangent condition is decided to be: EDTA-Na2 damping fluid, 0.05mg/ml enzyme concn, 2h reaction times.
After obtaining best enzyme tangent condition, large-scale abzyme can be carried out and cut.Immobilization method enzyme cuts 2H4 antibody 5mg, and concrete optimum condition is: get 2H4 antibody 5mg, with the EDTA-Na of 5ml containing 100 μ l immobilized papains
2the mixing of digestion damping fluid, is placed in 37 DEG C of incubators and hatches 2h.After having hatched, with Tris-HCl (1.0mol/L, pH 8.0) by antibody digestion products pH regulator to 7.5 ~ 8.0.Collect gained digestion products, enter next step separation and purification stage.
3. the preparation of antigen immune affinity column:
Operation steps in strict accordance with NHS-activated Sepharose 4Fast Flow coupling filler specification sheets is carried out: 2.5mg BNP recombinant protein is dissolved in 10ml coupling buffer (0.2mol/L NaHCO3 by (1) completely, 0.5mol/L NaCl, pH 8.3) in.(2) get 5ml NHS-activated Sepharose 4Fast Flow Activation filling and be placed in chromatography pipe, rinse filler with the dilute hydrochloric acid that ice-cold concentration is 1mmol/L, remove protection solvent.(3) solvent BNP recombinant protein solution is added filler, sealing, spends the night in 4 DEG C.(4) next day, collect and flow out protein liquid, be used alternatingly buffering A (0.5mol/L thanomin, 0.5mol/LNaCl, pH 8.3) and buffering B (0.1mol/l acetic acid, 0.5mol/L NaCl, pH4.0) repeatedly filler is rinsed, at least five times.(5) filler is stored in PBS for subsequent use.(6) SDS-PAGE detects coupling effect.
The coupling effect of antigen immune affinity column: 2.5mg BNP recombinant protein joins 5ml NHS-activated Sepharose 4Fast Flow, is fully combined with Activation filling.BNP recombinant protein relative molecular mass is about 25 × 10
3, SDS-PAGE result shows, 25 × 10 before coupling
3there is BNP recombinant protein band at place, and only containing minute quantity BNP recombinant protein in the effluent liquid collected after coupling, be about 0.1mg, Conjugate ratio is 96%.Coupling efficiency is high, is applicable to large scale purification preparation work.Concrete detected result as shown in Figure 3.In Fig. 3, LANE 1 is: BNP recombinant protein before coupling; LANE 2 is: BNP recombinant protein after coupling; M:Prestained Protein Marker.
Add enough NHS-activated Sepharose 4Fast Flow in papain solution, papoid can with activation coupling filler abundant coupling.Relative molecular mass 23 × 103, the SDS-PAGE result display of papoid, 23 × 10 before coupling
3there is papoid band at place, and not containing papoid in the outflow protein liquid collected after coupling, Conjugate ratio is close to 100%.Immobilized papain coupling effect is concrete as shown in Figure 2, and LANE 1 and 2 is: before coupling; LANE 3 and 4 is: after coupling; M:Prestained Protein Marker.
4. product purification is separated:
(1) Tris-HCl (1.0mol/L, pH 8.0) is used by antibody digestion products pH regulator to 7.5 ~ 8.0.
(2) antibody digestion products is collected effluent liquid by blank chromatography column, elimination immobilized enzyme is also reclaimed.
(3) effluent liquid is passed through albumin A post, collect outflow albumen and be designated as albumen P1.
(4) after using PBS damping fluid fully to balance albumin A post, with HCl-glycine (0.1mol/L, Ph 2.7) wash-out pillar, collect eluted protein and be designated as albumen P2, with Tris-HCl (1.0mol/L, pH 8.0) by the pH regulator of P2 to 7.5 ~ 8.0.
(5) gained albumen is dialysed in PBS.
Immobilized papain enzyme cuts 2H4 antibody, and gained digestion products, by blank chromatography column, is collected effluent liquid, can be removed immobilized papain completely.Effluent liquid, by protein A column purification, is collected and is flowed out albumen P1 and eluted protein P2.P1 is final Fab antibody.The main component of P2 is: Fc section and the whole antibody do not cut.
The qualification of 5.Fab antibody fragment:
Comprise two aspects to the qualification of Fab antibody fragment, one is qualification Fc section excision effect, and two is the immunocompetences identifying Fab antibody, and both all can adopt ELISA method to measure.
(1) Fc section excision effect is identified
1) enzyme plate pre-treatment: will newly purchase enzyme plate distilled water soaked overnight, dry rear for subsequent use;
2) bag quilt: dilute antigen (BNP recombinant protein) for best effort concentration 5 μ g/ml with coating buffer, every hole adds 100 μ l antigen liquids, and after 37 DEG C of incubation 1h, tape seal, spends the night in 4 DEG C.Wash 5 times next day, empty dry.
3) close: every hole adds confining liquid 350 μ l, hatches 1h for 37 DEG C, washs 5 times, empty dry, seal latter 4 DEG C and save backup.
4) dilute antibody: the whole antibody cut non-enzyme and Fab antibody 1 μ l, add in the PBST antibody diluent of 999 μ l 0.1% respectively, mixing, and carry out 5 × doubling dilution.
5) get bag by good enzyme plate, every hole adds the antibody 100 μ l of dilution, and the PBST antibody diluent of 0.1% does blank.Hatch 1h for 37 DEG C, wash 5 times, empty dry.
6) goat anti-mouse igg that horseradish peroxidase HRP marks is diluted to 1:3000, every hole adds 100 μ l, hatches 1h for 37 DEG C, washs 5 times, empty dry.
7) every hole adds 100 μ l tmb substrate nitrite ions, room temperature lucifuge reaction 10min ~ 15min.
8) every hole adds 100 μ l stop buffers, measures OD value immediately on enzyme-linked immunosorbent assay instrument with 450nm wavelength.
9) result judges.
As shown in Figure 4, raw data makes the separating effect of Fc section between bar graph visual representation three kinds of purification process.As seen from Figure 4, multiple proportions increases extension rate, the colour developing of full IgG antibodies molecule is consistent, the Fab antibody that three kinds of purification process obtain is under high antibody titer condition, the goat anti-mouse still do not marked with HRP is reacted, the colour developing level of three approximates blank, proves that the Fc section in Fab antibody is almost completely separated.Wherein with antigen affinity chromatography purification effect for the best.
(2) immunocompetence of Fab antibody is identified
1) biotinylation of antibody
Antibody mass, between 1-10mg, when volume is between 0.5-2ml, uses the biotin reagent of 20 times of volumetric molar concentrations.Each antibody molecule can connect 4-6 vitamin H group.
1. the required mmole number of biotin reagent and the volume of 10mM concentration biotin reagent is calculated.
1ml IgG×2mg/ml IgG×(1mmol IgG/150000mg IgG)×(20mmol Biotin/1mmol IgG)＝0.000266mmol Biotin
0.000266mmol Biotin×(1000000μl/1L)×(1L/10mmol)＝26.6μl Biotin Reagent
2. get whole antibody and each 2mg of Fab antibody, be dissolved in PBS respectively.
3. 360 μ l ultrapure waters add in 2mg biotin reagent, preparation 10mM concentration biotin reagent (matching while using).
4. getting 10mM concentration biotin reagent 26.6 μ l joins in antibody protein solution, incubated at room temperature 30min.
5. after having hatched, the dialysis unnecessary biotin reagent of removing or antibody.
2) ELISA detects
1. quilt is wrapped: dilute antigen (BNP recombinant protein) for best effort concentration 5ug/ml with coating buffer, every hole adds 100 μ l antigen liquids, tape seal, spends the night in 4 DEG C.Next day, wash 2 times, empty dry.
2. close: every hole adds confining liquid 350 μ l, hatches 1h for 37 DEG C, washs 5 times, empty dry, seal latter 4 DEG C and save backup.
3. dilute antibody: get biotinylated whole antibody and Fab antibody 1 μ l respectively, add in the PBST antibody diluent of 999 μ l 0.1%, mixing, and carry out 5 × doubling dilution.
4. get bag by good enzyme plate, every hole adds the antibody 100 μ l of dilution, and the PBST antibody diluent of 0.1% does blank.Hatch 1h for 37 DEG C, wash 5 times, empty dry.
5. the avidin that horseradish peroxidase HRP marks is diluted to 1:3000, every hole adds 100 μ l, hatches 1h for 37 DEG C, washs 5 times, empty dry.
6. every hole adds 100 μ l tmb substrate nitrite ions, room temperature lucifuge reaction 10min ~ 15min.
7. every hole adds 100 μ l stop buffers, measures OD value immediately with enzyme-linked immunosorbent assay instrument at 450nm wavelength.
8. result judges.
As shown in Figure 5, compare the immunocompetence of the Fab antibody fragment that three kinds of purification process prepare, visible, the immunocompetence of three is substantially identical compared with full IgG antibodies molecule.Wherein, with the purification effect of antigen affinity chromatography for the best, antigen affinity column can be specific in conjunction with corresponding antibodies, except the assorted antibody be mixed in the antibody fragment of de-inactivation and ascites preparation process, reach secondarily purified object, the purity of the Fab antibody prepared by guarantee and immunocompetence.
In sum, the present invention effectively overcomes various shortcoming of the prior art and tool high industrial utilization.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all without prejudice under spirit of the present invention and category, can modify above-described embodiment or changes.Therefore, such as have in art usually know the knowledgeable do not depart from complete under disclosed spirit and technological thought all equivalence modify or change, must be contained by claim of the present invention.
Claims (6)
1. harden monitoring cutting is for a method for Fab antibody, comprises the steps:
1) preparation method of harden monitoring: the microballoon adding activation in papain solution, makes papoid and the abundant coupling of activation coupling filler;
2) harden monitoring enzyme is cut: get 2H4 antibody 5mg, with the EDTA-Na of 5ml containing 100 μ l immobilized papains
2digestion damping fluid mixes, hatches, and enzyme concn is 0.05mg/ml;
3) enzyme is cut rear Fab antibody separation and purification and is namely obtained Fab antibody.
2. a kind of harden monitoring cutting as claimed in claim 1 is for the method for Fab antibody, it is characterized in that, in described step 1, the microballoon of activation is specially NHS-activated Sepharose 4Fast Flow microballoon.
3. a kind of harden monitoring cutting as claimed in claim 1 is for the method for Fab antibody, it is characterized in that, after described step 2 enzyme is cut, with Tris-HCl (1.0mol/L, pH 8.0) by antibody digestion products pH regulator to 7.5 ~ 8.0.
4. a kind of harden monitoring cutting as claimed in claim 1 is for the method for Fab antibody, it is characterized in that, in described step 2, concrete incubation conditions is: at 37 DEG C, hatch 2h.
5. a kind of harden monitoring cutting as claimed in claim 1 is for the method for Fab antibody, and it is characterized in that, in described step 3, the actual conditions of separation and purification comprises the steps:
(1) Tris-HCl (1.0mol/L, pH 8.0) is used by antibody digestion products pH regulator to 7.5 ~ 8.0.
(2) antibody digestion products is collected effluent liquid by blank chromatography column, elimination immobilized enzyme is also reclaimed.
(3) effluent liquid is passed through albumin A post, collect outflow albumen and be designated as albumen P1.
(4) after using PBS damping fluid fully to balance albumin A post, with HCl-glycine (0.1mol/L, Ph 2.7) wash-out pillar, collect eluted protein and be designated as albumen P2, with Tris-HCl (1.0mol/L, pH 8.0) by the pH regulator of P2 to 7.5 ~ 8.0.
(5) gained albumen is dialysed in PBS.
6. the harden monitoring cutting as described in claim as arbitrary in claim 1-4 for the method for Fab antibody in the purposes of Fab antibody preparation field.
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| CN108085358A (en) * | 2018-02-08 | 2018-05-29 | 保定冀中药业有限公司 | A kind of preparation method of canine parvovirus monoclonal antibody IgG2b-Fab segments |
| CN113030461A (en) * | 2021-03-12 | 2021-06-25 | 北京华科泰生物技术股份有限公司 | IgG antibody marked by amino group or carboxyl group-containing substance, human IgG magnetic particle chemical detection kit and preparation method thereof |
| CN113030461B (en) * | 2021-03-12 | 2022-04-22 | 北京华科泰生物技术股份有限公司 | IgG antibody marked by amino group or carboxyl group-containing substance, human IgG magnetic particle chemical detection kit and preparation method thereof |
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