CN108103002A - Preparation of mdck cell host's residual protein and application thereof - Google Patents
Preparation of mdck cell host's residual protein and application thereof Download PDFInfo
- Publication number
- CN108103002A CN108103002A CN201711242201.2A CN201711242201A CN108103002A CN 108103002 A CN108103002 A CN 108103002A CN 201711242201 A CN201711242201 A CN 201711242201A CN 108103002 A CN108103002 A CN 108103002A
- Authority
- CN
- China
- Prior art keywords
- mdck cell
- culture
- cell
- protein
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- 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
-
- 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
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- General Engineering & Computer Science (AREA)
- Water Supply & Treatment (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of preparations of mdck cell host residual protein and application thereof, and the preparation method of the albumen includes the following steps, (1) collects the culture solution for the mdck cell for stablizing culture；(2) micro-filtration is carried out to the culture solution, collects filter liquor；(3) filter liquor is concentrated by ultrafiltration, obtains concentrate；(4) molecular sieve gel chromatographic purifying is carried out to the concentrate, obtains refined solution；(5) refined solution is concentrated again, obtains technique differential protein (mdck cell host residual protein) solution of the mdck cell of the stable culture.The purposes includes but not limited to prepare antibody or kit using the albumen, for evaluating vaccine quality.
Description
Technical field
The invention belongs to bacterin preparation field, a kind of preparation in particular to mdck cell host residual protein and
Its purposes.
Background technology
MDCK ((Madin-Daby canine kidney cells), by Madin and Darby in 1958 from the U.S.
The renal tissue separation of the female bent frame dogs of Cocker Spaniel, which is cultivated, establishes, and the Epithelial typically grown with adherent manner is thin
Born of the same parents.It since its viral efficiency of infection is high, multiplication is fast, and is not easy to make a variation, mdck cell system (MDCK Cell Lines) is acknowledged as
It is most suitable for one of 3 kinds of cell lines of first, influenza B virus production.
The host cell proteins (host cell protein, HCP) remained in biological products belong to heterologous protein, both
Structural proteins liquid including host cell also includes the somatomedin of host cell (passage cell) secretion.HCP not only has can
It can induce body and generate anti-HCP antibody, body is caused to generate allergic reaction, it is also possible to have " adjuvant effect " and cause body pair
Pharmaceutical grade protein generates antibody, influences medication effect.Therefore HCP contents are ensure vaccine quality one in control vaccine
Important content.1998, WHO was formulated《Biological products regulation is produced using zooblast》, wherein the HCP to passage cell
Residual quantity proposes guiding requirement, i.e., passage cell HCP contents is reduced to acceptable level.At present, most of vaccine
It is by the way that the total protein content of vaccine is controlled to carry out indirect control.
South Korea scholar Kim HyunSu etc. have been reported first in Japanese Encephalitis Vaccine,Live prepared by detection Vero cells
HCP residual content methods, Japanese scholars Keishin Sugawara etc. are thin using enzyme-linked indirect double antibody sandwich method detection Veto
HCP contents in the encephalitis B inactivated vaccine of born of the same parents' production, field, which is won, waits reports to establish Dot-ELISA, but poor repeatability is sensitive
Spend low, the reports such as Wang Hui establish enzyme-linked double antibody sandwich method detection Vero cell residue protein contents.
The method of the enzyme-linked double antibody sandwich method detection Vero cell residue protein contents of the foundation such as Wang Hui is closest to this
The technical solution of invention.Step mainly includes：Standby Veto cell protein antigen reference materials, immunizing rabbit, purification antibody is as bag
It is marked by antibody and with enzyme, establishes enzyme linked immunosorbent detection system.It is bent that quantitative criterion is drawn with Vero cell protein antigens reference material
Line, and indices are verified.
Mdck cell is mainly used as the development of influenza vaccines as cellular matrix, but is there is no in the world with MDCK systems at present
Standby influenza vaccines listing, there are no the detection means of specific mdck cell host protein (HCP).It is also used on the market without selling
In the kit of detection mdck cell HCP residual volumes, main cause is mdck cell technique differential protein and for the albumen
Specific antibody be difficult to prepare.Problem to be solved by this invention is the antigen standard for providing detection mdck cell HCP
And antibody, while corresponding detection method is set up, in case detecting mdck cell HCP residual volumes in production of vaccine and Quality Control.
The content of the invention
(it is that mdck cell host is residual present invention firstly relates to a kind of technique differential protein of the mdck cell of stable culture
Stay albumen) preparation method, described method includes following steps,
(1) culture solution for the mdck cell for stablizing culture is collected；
(2) micro-filtration is carried out to the culture solution, collects filter liquor；
(3) filter liquor is concentrated by ultrafiltration, obtains concentrate；
(4) molecular sieve gel chromatographic purifying is carried out to the concentrate, obtains refined solution；
(5) refined solution is concentrated again, the technique differential protein for obtaining the mdck cell of the stable culture is molten
Liquid.
The method for stablizing culture mdck cell includes：
(1) with 3 × 105A/cm2Inoculating cell is cultivated into reactor；
(2) cell changes the fresh culture without serum into after exponential phase progresses into plateau, continues to train
It supports；
(3) continue culture 2~4 days, be preferably 72h, cell is the stable mdck cell cultivated at this time.
It is described micro-filtration is carried out to culture solution method be：Select 5 inches of pillars of two-stage of 0.8+0.65u, 0.45+0.2u
Filter core forms microfiltration systems, carries out micro-filtration to cell culture supernatant, collects filter liquor.
The method of the ultrafiltration concentration is：Filter liquor is concentrated by ultrafiltration in the film bag of selection MWCO=100KD, step
For：
(1) micro-filtration filter liquor is first concentrated into the 1/10 of original volume to be less than the transmembrane pressure of 0.3MPa；
(2) fully washed thoroughly with the pH7.4 of 8~10 volumes of ultrafiltrate, the PBS of 0.01M,
(3) ultrafiltrate is concentrated into the 1/60~1/80 of original volume.
The method of the molecular sieve gel chromatographic purifying is：Chromatographic purifying is carried out using Sepharose molecular sieve gels,
Level pad and elution buffer select 0.01M, the PBS of pH7.4, linear flow rate 50cm/h, and target eluting peak is the
One eluting peak collects 280nm wavelength absorptions peak >=20mAU elution fractions.
The step of described concentration again is：Refined solution is concentrated into original volume with the ultra-filtration centrifuge tube of MWCO=100KD
1/2~1/3, method for concentration is identical with the ultrafiltration concentration method again.
The invention further relates to a kind of method for the detection antibody for preparing the technique differential protein, the method includes
Following steps：
(1) immune animal is subcutaneously injected using the albumen, the animal is preferably rabbit or cavy；The albumen dosage
For 0.3~0.4mg/kg, immunologic adjuvant is Freund's adjuvant, and adjuvant dosage is 1 with protein solution：1(v/v)；
(2) directional diffusion measure serum titer is immunized and reaches >=1：After 32, put to death animal and take whole blood；
(3) antiserum for selecting potency higher respectively is purified, and obtains anti-mdck cell protein I gG.
The antiserum purification step is：
(1) caprylic acid-ammonium slightly purifies antiserum；
(2) affinity chromatography is carried out with commercially available prepackage Protein-A affinity columns, collects the elution of eluting peak >=30mAU
Liquid part merges eluent and with the ultrafiltration concentration centrifuge tube concentrate eluant of MWCO=10KD into the 1/10 of original volume, simultaneously
Replacement buffer system is 0.01M, the PBS of pH7.4, obtains the IgG antibody of anti-mdck cell albumen.
The invention further relates to the detection kit of the detection technique differential protein of the detection Antibody preparation, preferably
, the kit is the ELISA detection kit based on double-antibody method principle.
It is described the invention further relates to the technique differential protein for stablizing the mdck cell cultivated prepared by the method
The molecular weight ranges of albumen are 13KD, 17KD, 29KD~44.3KD, 44.3KD~66.4KD and 97.2KD.
The invention further relates to the application of the detection antibody or detection kit in vaccine quality is evaluated.
The invention further relates to application of the technique differential protein technique differential protein in vaccine quality is evaluated.
MDCK is established the present invention provides mdck cell technique albumen and preparation method thereof, and using the albumen as immunogene
The detection method of cell HCP and detection testing standard product.Mdck cell technique albumen application range prepared by this method is more
Greatly, H5N1 type influenza vaccines are can be not only used for, can be also used for quality control and analysis in vaccine preparation process, and it is special
It is different in nature strong, high sensitivity, reproducible.
Description of the drawings
Fig. 1, mdck cell technique differential protein preparation method flow chart.
Fig. 2,7.5L reactor cell culture result.
Fig. 3, mdck cell Protein S epharose 4Fast Flow molecular sieve gel thin layer chromatography figures.
Fig. 4, mdck cell technique differential protein standard items SDS-PAGE examine dye figure (left side) and silver staining figure (right side).
Identification and analysis after Fig. 5, two kinds of antibody purifications (left side is western blot figures to examine the dye figure right side).
Fig. 6, MDCK HCP antibody test Trendline.
Fig. 7, the antibody optimum detection range of linearity.
Fig. 8, antibody specificity verification.
The detection Trendline of Fig. 9, vaccine HCP concentration.
Specific embodiment
Main material：
Mdck cell is introduced from ATCC (Unite States Standard Culture Collection Center)：MDCK CCL-34, P55, Lot：
59681577；
Rabbit and cavy are provided by limited company of Wuhan Biological Products Inst. animal house；
ELISA Plate is purchased from Costar companies of the U.S.；
Horseradish peroxidase (HRP) is purchased from Aladdin company.
Key instrument equipment：
Device is concentrated by ultrafiltration purchased from Sai Duolisi companies in ultrafilter, ultrafiltration membrane；
Chromatogram purification instrumentChromatographic column, purification media are purchased from GE Healthcare companies.
Embodiment 1, the preparation method of mdck cell technique albumen：
1st, 7.5L bioreactor cultures cell：
The cell bank mdck cell recovery that works is taken, is amplified to 10L rolling bottles through T bottles of recovery passages, culture is arrived certain
Cell concentration, with 3 × 105A/cm2Inoculating cell is cultivated into reactor.Cell enters logarithm quickly through of short duration adjustment
Growth period is simultaneously maintained to 72h, progresses into plateau from 96h cells, cell concentration maintains 2 × 106A/cm2.After cultivating 96h,
PBS thoroughly washes remaining serum, changes the fresh culture without serum into, continues to cultivate.From growth curve, cell
It stops growing, and starts death, quantity is begun to decline, and after 4 days (192h), cell density falls to approximately 3 × 104/cm2, receive at this time
Obtain cell culture supernatant.Temperature stabilization in entire incubation, pH value fluctuates between 7.1~7.5, DO 36%~
It is fluctuated between 42%.Fig. 2 is the growth curve chart for cultivating mdck cell in 7.5L reactors three times, and the trend of curve can from figure
To find out, stablize between cell culture batch.
2nd, supernatant is harvested：
Simulation influenza vaccines connect malicious technique, and after cell growth in reactor is stablized, culture solution is changed into without tire ox blood
Clear culture solution, continuously perfused culture have been free of hyclone (content can be ignored) at this time after 3~5 days in culture solution, receive
Collect cell culture fluid.
3rd, the micro-filtration of mdck cell culture solution：
According to the state for the cell culture supernatant that step 2 is harvested, the two-stage 5 of selection 0.8+0.65u, 0.45+0.2u
Inch pillar filter core forms microfiltration systems, carries out micro-filtration to cell culture supernatant, collects filter liquor (about 6000ml).
The comparison of total protein concentration before and after 1 micro-filtration of table
4th, it is concentrated by ultrafiltration：
It is reported according to influenza virus vaccine preparation process and pertinent literature, selects the film bag of MWCO=100KD to step 3 institute
Filter liquor is obtained to be concentrated by ultrafiltration.The present invention is first filtered out micro-filtration with being less than the transmembrane pressure of 0.3MPa when being concentrated by ultrafiltration
Liquid is concentrated into the 1/10 of original volume, is then fully washed thoroughly with the pH7.4 of 8~10 volumes of ultrafiltrate, the PBS of 0.01M, finally will
Ultrafiltrate is concentrated into the 1/60~1/80 of original volume.Host protein ingredient is remained to greatest extent, is also risen while concentration
Certain purification effect is arrived.Following table is the variation of total protein concentration before and after being concentrated by ultrafiltration, as can be seen from the table, ultrafiltration
Concentration loss is very big, and protein recovery is low, while collected albumen is compared with general crack protein technique specificity higher.
The comparison of mdck cell protein content before and after 2 ultrafiltration of table
4th, the chromatographic purifying of liquid is concentrated by ultrafiltration：
Concentrate carries out chromatographic purifying using Sepharose 4Fast Flow types molecular sieve gel, in purification process, puts down
Weighing apparatus buffer solution and elution buffer select 0.01M, the PBS of pH7.4, linear flow rate 50cm/h, according to the system of influenza vaccines
Standby technique, target eluting peak are the first eluting peak, 280nm wavelength absorptions peak >=20mAU elution fractions are collected, referring to Fig. 3.
The comparison of mdck cell protein content before and after 3 chromatographic purifying of table
5th, concentrate again：Collection liquid is concentrated into 20mL with the ultra-filtration centrifuge tube of 30mL, MWCO=100KD, you can obtain
The special mdck cell albumen of technique.
6th, mdck cell technique differential protein carries out SDS-PAGE electrophoresis, Coomassie blue stain, silver staining (Fig. 4) after purification.
Experimental result：PAGE gel electrophoresis showed, MDCK HCP are typical mixed proteins, are in molecular weight ranges
There is protein molecular distribution at 13KD, 17KD, 29KD~44.3KD, 44.3KD~66.4KD and 97.2KD, can clearly differentiate
Item carry more than 40 items.Although the 3 batches of MDCK HCP total protein concentrations prepared slightly have difference, protein component height one
It causes, illustrates MDCK HCP reference material stable preparation process, there is repeatability
Embodiment 2, the anti-how anti-preparation of mdck cell albumen：
Material source：
The mdck cell technique albumen source of purifying is in embodiment 1.
Experimental animal：
Rabbit (regular grade) and cavy (cleaning grade) are all from Wuhan Biological Products Inst.'s animal experimental center
Other instruments reagent：
Protein-A affinity columns, goat-anti rabbit-HRP, goat-anti cavy-HRP are purchased from Wuhan doctor's moral company, HRP enzymes
Purchased from Aladdin company, Freund's complete adjuvant, incomplete Freund's adjuvant are purchased from Sigma companies.
1st, immunizing rabbit：
Immune 3 rabbit, every rabbit of initial immunity special mdck cell albumen of technique prepared by 0.6mg examples 1,
Isometric Freund's complete adjuvant is added in, after emulsification fully, dorsal sc injection 6~8 point.It is immunized for second after 2 weeks：0.6mg is real
The mdck cell albumen that the technique of the preparation of example 1 is special adds in isometric incomplete Freund's adjuvant, and after emulsification fully, dorsal sc is more
Point injection.It is immunized for the third time after 2 weeks immune for the second time：The special mdck cell albumen of technique prepared by 1.2mg examples 1, back
Subcutaneous multi-point injection.The 4th time is carried out using third time immunization method to be immunized, adopt after a week most after third time is 10 days immune
Hematometry potency, immune directional diffusion measure serum titer and reach >=1：After 32, take a rabbit whole blood and put to death animal.
4 immunizing rabbit serum titer of table
2nd, immune guinea pig：
Immune 5 cavys, every cavy of initial immunity special mdck cell albumen of technique prepared by 0.3mg examples 1,
Isometric Freund's complete adjuvant is added in, after emulsification fully, dorsal sc injection 6~8 point.It is immunized for second after 2 weeks：0.3mg is real
The mdck cell albumen that the technique of the preparation of example 1 is special adds in isometric incomplete Freund's adjuvant, and after emulsification fully, dorsal sc is more
Point injection.It is immunized for the third time after 2 weeks immune for the second time：The special mdck cell albumen of technique prepared by 0.6mg examples 1, back
Subcutaneous multi-point injection.The 4th time is carried out using third time immunization method to be immunized, adopt after a week most after third time is 10 days immune
Hematometry potency, immune directional diffusion measure serum titer and reach >=1：After 32, take guinea pig whole blood and put to death animal.
5 immune guinea pig serum titer of table
3rd, antiserum purifies：
Two kinds of higher antiserums of animal antiserum potency in above-mentioned steps is selected to be purified respectively.Antiserum is through pungent
Acid-ammonium sulfate method is slightly after purification, and carrying out affinity chromatography with commercially available prepackage Protein-A affinity columns, (purification process refers to GE
Company's prepackage Protein-A affinity columns operation instructions), the eluent part of eluting peak >=30mAU is collected, merges elution
Liquid and with the ultrafiltration concentration centrifuge tube concentrate eluant of MWCO=10KD into the 1/10 of original volume, while replace buffer system and be
The PBS of 0.01M, pH7.4 obtain anti-mdck cell protein I gG.SDS-PAGE electrophoresis, the change of IgG antibody after observation before purification
Change, and measure 280nm and 260nm absorbances.Calculate protein content：C (mg/ml)=1.55 × OD (280nm) -0.76 × OD
(260nm)。
6 purified antibodies content of table
4th, purified antibodies bioactivity：
It is detected using indirect elisa method, the technique for being prepared embodiment 1 with carbonate buffer solution (0.05M, pH9.6) is special
Different mdck cell albumen is diluted to 5ug/ml, and 100ul/ holes coating, 4 DEG C overnight.Next day discards coating buffer, and 3 are washed with PBST
It is secondary, the PBST confining liquids containing 1%BSA, 150ul/ holes are added in, room temperature closing 2h discards confining liquid, and PBST is washed 5 times.By step
Purified anti-mdck cell technique differential protein IgG is diluted to after 1mg/ml again 10 times of dilutions of series respectively in 3, each to dilute
A hole is spent, 100ul/ holes, 37 DEG C are incubated 60 minutes, and PBST is washed 5 times, is patted dry, and 100ul ELIAS secondary antibody (sheep is separately added into per hole
Anti-rabbit-HRP, goat-anti cavy-HRP) 37 DEG C be incubated 1h, PBST is washed 5 times, patted dry.Each hole adds in developing solution A liquid, 37 DEG C of B liquid is incubated
It educates 10 minutes.2mol/L sulfuric acid terminates reaction, the colorimetric in microplate reader, calculating antibody potency.
7 purified antibodies potency of table
5th, after purification rabbit and guinea pig antibodies carries out SDS-PAGE identifications and western blotting analyses, as a result such as
Fig. 5, the results showed that, after purified, for the heavy chain of rabbit and guinea pig antibodies IgG near 55kd, the light chain of IgG antibody is attached in 20kd
Closely.Most protein ingredients of purified antibodies and MDCK HCP can be specifically bound.But due to MDCK HCP molecules
Measure in extensive range, the antigen part of small-molecular-weight may be because the loss of antigen site and can not be tied with corresponding antibodies specificity
It closes, so as to cause the loss of partial antibody.
6th, prepared by HRP enzymic-labelled antibodies
The sodium periodate method of improved mistake is selected, respectively the highest anti-mdck cell technique of antibody titer in selecting step 3
Differential protein IgG (1# rabbit-antis/3# rabbit-antis, 3# cavys resist/5# cavys resist) mark horseradish peroxidases (HRP).
The anti-mdck cell technique differential protein IgG of table 8HRP-
Embodiment 3：The foundation of ELISA double antibody sandwich methods
1st, coated antibody working concentration and HRP enzyme labelled antibody working concentrations determine ----square formation titration
(1) coated antibody is diluted to four concentration by antibody diluent：10μg/mL、5μg/mL、2.5μg/mL、1.25μg/
ML, each dilution factor are coated with 3 row ELISA Plates, and per 100 μ L of hole, 2h or 4 DEG C of coating of 37 DEG C of coatings is overnight；
(2) close：Coating buffer is discarded, PBST board-washings 5 times pat dry, PBSTs of the 150 μ L of addition containing 1%BSA per hole, 37 DEG C
Close 1h；
(3) confining liquid is discarded, PBST board-washings 3 times pat dry；MDCK HCP are diluted to seven concentration with carbonate buffer solution：
10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, 0.625 μ g/mL, 0.312 μ g/mL, 0.155 μ g/mL and a moon
Property control (carbonate buffer solution), each concentration sets a row, per 100 μ L of hole, 37 DEG C of incubation 1h；
(4) liquid is discarded, PBST board-washings 3 times pat dry；Enzyme labelled antibody is diluted to three concentration：1：100、1：500、1：
1500, in the concentration of each coated antibody, each enzyme labelled antibody dilution factor sets a line, per hole 100 μ L, 37 DEG C of incubation 1h；
(5) antibody diluent is discarded, PBST board-washings 5 times pat dry；Substrate colour developing A liquid and each 50 μ of B liquid are sequentially added per hole
L, 37 DEG C of 10~15min of isothermal reaction；
(6) 50 μ L terminate liquids are added in per hole and terminate reaction, microplate reader reads the light absorption value A450 at 450nm, records result
And it analyzes.
It is more than 1.0 according to positive A values, negative control A values are minimum, the larger principle of P/N values, and through Checkerboard titration, coating is anti-
The working concentration of body is 5 μ g/mL, and enzyme labelled antibody working concentration is 1：500 (being shown in Table 9)
The selection of 9 coated antibody of table and enzyme labelled antibody working concentration
2nd, the MDCK HCP protein quantifications standard curve range of linearity and minimum detectability determine
According to the coated antibody for the double antibody sandwich ELISA having determined and the working concentration of enzyme labelled antibody, by MDCK
HCP reference materials are diluted to 12 concentration：20μg/mL、10μg/mL、5μg/mL、2.5μg/mL、1.25μg/mL、0.625μg/mL、
It is bent to establish reaction by 0.312 μ g/mL, 0.155 μ g/mL, 0.065 μ g/mL, 0.032 μ g/mL, 0.016 μ g/mL, 0.008 μ g/mL
Line, while negative control (carbonate buffer solution) is set, do 10 multiple holes.Select linear coefficient R2It is more than 0.99 and positive
Property A values larger correspondence MDCK HCP reference materials dilution ranges be the range of linearity, with negative control A values (Aneg) average statistical
Boundary is detected with the conduct of the sum of 2 times of standard deviations, determines the sensitivity of detection.
It is the μ g/mL of 20 μ g/mL~0.008 by MDCK HCP reference materials two-fold dilution, selects coated antibody and enzyme labelled antibody
To working concentration, it is detected.Using HCP concentration as abscissa, A450Value establishes response curve as ordinate (see Fig. 6).Knot
Fruit finds that MDCK HCP are in 0.32~5 μ g/mL scopes, R2More than 0.99, linear dependence is fine.Expand when scope
To after 0.32~10 μ g/mL, R2It is lower than 0.99.Therefore, 0.32~5 μ g/mL can be set to the detection range of linearity (see figure
7)。
3rd, the confirmation of the range of linearity is detected
In the range of linearity, by MDCK HCP reference materials two-fold dilutions into 5 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, 0.625 μ
G/mL, 0.312 μ g/mL, do response curve linear regression, are repeated 4 times, the linearly dependent coefficient R of the straight line of gained fitting2For：
0.9967th, 0.9983,0.9971,0.9939, all greater than 0.99, average 0.9965.
In the range of linearity, choose low (1.0 μ g/mL), in (2.0 μ g/mL) and height (4.0 μ g/mL) three concentration, with MDCK
HCP reference materials concentration makes equation of linear regression, respectively these three concentration is repeated to examine as ordinate, A values as abscissa
It surveys 4 times, from the results, it was seen that the coefficient of variation of low middle high three concentration is respectively less than 5%, in linear detection range, the party
Method accurate can detect the concentration of sample.
10.1 reference material standard curve of table
The confirmation of 10.2 range of linearity of table
Embodiment 4：The ELISA double antibody sandwich method methodology validations of foundation
1st, precision ----repeatability verification
MDCK HCP reference materials are diluted to basic, normal, high (1.0 μ g/mL, 2.0 μ g/mL, 4 μ g/mL) three concentration, each
Concentration measures 4 holes in same ELISA Plate, and the detected value of each concentration is gone out by standard curve regression equation calculation, is calculated respectively
Average, standard deviation, the coefficient of variation and the rate of recovery of each concentration, as a result (being shown in Table 11) display, three concentration MDCK HCP references
The CV of product examine measured value is respectively 10.94%, 4.37%, 4.11%, and the rate of recovery illustrates accurate in this method plate more than 90%
Degree and repeatability are good.
Precision and repeatability measure in 11 plate of table
In addition, taking three pieces of ELISA Plates, basic, normal, high three kinds of MDCK HCP reference materials are detected three times in same plate, three pieces of enzyme marks
Plate is completed in three different time sections, as a result following (being shown in Table 12), it can be seen that basic, normal, high three concentration detects between plate
The coefficient of variation for 11.0%, 5.6%, 4.37%, within the acceptable range, meanwhile, the rate of recovery of three concentration is also all
More than 90%, as a result illustrate that this method has good repeatability.
Precision and repeatability measure between 12 plate of table
2nd, specificity verification
It is thin that the ELISA method that this experiment is established is mainly used to detect host in the intermediate products in mdck cell production of vaccine
Born of the same parents' protein content, the other compositions in addition to host protein (HCP) that these intermediate products may contain should not be generated to this detection
Interference, these ingredients mainly have：Hyclone, cell culture fluid, BSA, pancreatin, influenza virus protein, resisiting influenza virus egg
- IgG in vain.
Therefore, with PBST, hyclone, cell culture fluid, 1%BSA, influenza virus protein (1：4 dilutions, 2.0ug/
Ml), resisiting influenza virus albumen-IgG, 0.25% pancreatin, influenza vaccines semi-finished product are as sample, and each sample detection 5 times calculates
A450It is worth average and standard deviation, judges the specificity of the detection method, as a result see the table below (table 13 and Fig. 8).As a result it can be seen that,
1st, it is negative to be had significant difference (P ＜ 0.05) by inspection product and positive control (antigen reference material) testing result；2nd, it is positive by inspection product
(influenza vaccines semi-finished product) there was no significant difference with positive control testing result (P ＞ 0.05), as a result illustrates the inspection that problem is established
Survey method specificity is good,
13 specificity verification of table
3rd, applicability is verified
Preparation process according to mdck cell matrix influenza vaccines prepares H5N1 type influenza virus vaccines semi-finished product 4 batches, even
It is continuous to detect 5 times, mdck cell HCP content results such as following table (being shown in Table 14, Fig. 9), it can be seen that testing result repeats between vaccine batch
Good, the coefficient of variation 6.78% of property, less than 10%.Testing result confidence interval is (1318.1 ± 178.96) between vaccine batch
Ng/mL, although testing result slightly fluctuates between criticizing, this method can be perfectly suitable for mdck cell matrix influenza vaccines
Detection.
14 influenza vaccines semi-finished product mdck cell HCP contents of table
4th, stability is verified
The MDCK HCP reference materials of basic, normal, high (1.0 μ g/mL, 2.0 μ g/mL, 4 μ g/mL) three concentration are placed in 37 DEG C
Next week Concentration Testings are carried out to three concentration, calculate the average value of each concentration detection value, standard deviation, the coefficient of variation and returned
Yield the results are shown in Table 15.The result shows that after 37 DEG C of Acceleration studies, basic, normal, high three reference materials of MDCK HCP reference materials
The rate of recovery of concentration under the conditions of 4 DEG C the same as that compared with by inspection concentration, in the range of ± 30%, can receive.It is therefore contemplated that
It is stable that MDCK HCP reference materials preserve one-year age under 4 DEG C of environment.
Table 15MDCK HCP reference material stability tests
Finally it should be noted that above example only helps skilled in the art to understand the essence of the present invention, do not have to
Do limiting the scope of the present invention.
Claims (10)
1. a kind of preparation method of the technique differential protein of the mdck cell of stable culture, described method includes following steps,
(1) culture solution for the mdck cell for stablizing culture is collected；
(2) micro-filtration is carried out to the culture solution, collects filter liquor；
(3) filter liquor is concentrated by ultrafiltration, obtains concentrate；
(4) molecular sieve gel chromatographic purifying is carried out to the concentrate, obtains refined solution；
(5) refined solution is concentrated again, obtains the technique differential protein solution of the mdck cell of the stable culture.
2. according to the method described in claim 1, it is characterized in that, the method for stablizing culture mdck cell includes：
(1) with 3 × 105A/cm2Inoculating cell is cultivated into reactor；
(2) cell changes the fresh culture without serum into after exponential phase progresses into plateau, continues to cultivate；
(3) culture is continued 2~4 days, cell is the stable mdck cell cultivated at this time.
3. method according to claim 1 or 2, which is characterized in that the side that micro-filtration is carried out to culture solution described in step (2)
Method is：5 inches of pillar filter cores of two-stage of 0.8+0.65u, 0.45+0.2u is selected to form microfiltration systems, to cell culture supernatant
Micro-filtration is carried out, collects filter liquor.
4. method according to claim 1 or 2, which is characterized in that the method for the ultrafiltration concentration described in step (3) is：Choosing
Filter liquor is concentrated by ultrafiltration in the film bag for selecting MWCO=100KD, and step is：
1) micro-filtration filter liquor is first concentrated into the 1/10 of original volume to be less than the transmembrane pressure of 0.3MPa；
2) fully washed thoroughly with the pH7.4 of 8~10 volumes of ultrafiltrate, the PBS of 0.01M,
3) ultrafiltrate is concentrated into the 1/60~1/80 of original volume.
5. method according to claim 1 or 2, which is characterized in that molecular sieve gel chromatographic purifying described in step (4)
Method is：Chromatographic purifying is carried out using Sepharose molecular sieve gels, level pad and elution buffer select 0.01M,
The PBS of pH7.4, linear flow rate 50cm/h, target eluting peak are the first eluting peak, collect 280nm wavelength absorptions peak >=20mAU
Elution fractions.
6. method according to claim 1 or 2, which is characterized in that the step of described concentration again is：Refined solution is used
The ultra-filtration centrifuge tube of MWCO=100KD is concentrated into the 1/2~1/3 of original volume, again method for concentration and the ultrafiltration concentration method
It is identical.
7. a kind of method of the detection antibody of technique differential protein prepared described in claim 1~6, the method are included such as
Lower step：
(1) the technique differential protein injecting immune animal that 1~6 any method of usage right requirement prepares, it is described
Animal is preferably rabbit or cavy；The albumen dosage is 0.3~0.4mg/kg, and immunologic adjuvant is Freund's adjuvant, adjuvant dosage
It is 1 with protein solution：1(v/v)；
(2) directional diffusion measure serum titer is immunized and reaches >=1：After 32, put to death animal and take whole blood；
(3) antiserum for selecting potency higher is purified, and obtains anti-mdck cell protein I gG antibody；
Wherein, the antiserum purification step described in step (3) is：
1) caprylic acid-ammonium slightly purifies antiserum；
2) affinity chromatography is carried out with Protein-A affinity columns, collects the eluent part of eluting peak >=30mAU, merging is washed
De- liquid and with the ultrafiltration concentration centrifuge tube concentrate eluant of MWCO=10KD into the 1/10 of original volume, while replace buffer system and be
The PBS of 0.01M, pH7.4 obtain the IgG antibody of anti-mdck cell albumen.
8. the detection kit of the detection technique differential protein of Antibody preparation is obtained with claim 7 the method, preferably
, the kit is the ELISA detection kit based on double-antibody method principle.
9. the technique differential protein for the mdck cell that any the methods of claim 1-6 prepare.
10. described in the technique differential protein for the mdck cell that any the methods of claim 1-6 prepare, claim 7
Application of the kit in vaccine quality is evaluated described in method acquisition antibody or claim 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711242201.2A CN108103002B (en) | 2017-11-30 | 2017-11-30 | Preparation and application of MDCK cell host residual protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711242201.2A CN108103002B (en) | 2017-11-30 | 2017-11-30 | Preparation and application of MDCK cell host residual protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108103002A true CN108103002A (en) | 2018-06-01 |
| CN108103002B CN108103002B (en) | 2021-09-24 |
Family
ID=62208819
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711242201.2A Active CN108103002B (en) | 2017-11-30 | 2017-11-30 | Preparation and application of MDCK cell host residual protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108103002B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113848321A (en) * | 2021-09-26 | 2021-12-28 | 武汉生物制品研究所有限责任公司 | Influenza vaccine TPCK-pancreatin residual quantity detection kit |
| CN115894677A (en) * | 2022-12-01 | 2023-04-04 | 湖州申科生物技术股份有限公司 | Antibody combination for improving HCP detection antibody coverage rate and application thereof |
| CN117192107A (en) * | 2023-09-11 | 2023-12-08 | 福建基诺厚普生物科技有限公司 | Detection method and kit for process-specific host cell protein residues |
-
2017
- 2017-11-30 CN CN201711242201.2A patent/CN108103002B/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113848321A (en) * | 2021-09-26 | 2021-12-28 | 武汉生物制品研究所有限责任公司 | Influenza vaccine TPCK-pancreatin residual quantity detection kit |
| CN113848321B (en) * | 2021-09-26 | 2023-12-05 | 武汉生物制品研究所有限责任公司 | Influenza vaccine TPCK-pancreatin residue detection kit |
| CN115894677A (en) * | 2022-12-01 | 2023-04-04 | 湖州申科生物技术股份有限公司 | Antibody combination for improving HCP detection antibody coverage rate and application thereof |
| CN117192107A (en) * | 2023-09-11 | 2023-12-08 | 福建基诺厚普生物科技有限公司 | Detection method and kit for process-specific host cell protein residues |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108103002B (en) | 2021-09-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111303254A (en) | Novel coronavirus (SARS-CoV-2) antigen detection kit | |
| CN101735319B (en) | Monoclonal antibody against GP73 protein, preparation method and application thereof | |
| CN108103002A (en) | Preparation of mdck cell host's residual protein and application thereof | |
| CN103627677A (en) | Lp(a) (Lipoprotein(a)) resisting monoclonal antibody and latex-enhanced immunonephelometric detection kit for Lp(a) | |
| CN101915835B (en) | Lily mottle virus double-antibody sandwich enzyme-linked immunosorbent assay kit and preparation method | |
| CN104931710A (en) | Double antibody sandwich method for quantitatively detecting cow milk beta-lactoglobulin | |
| CN105950563A (en) | Hybridoma cell strain 7E3, monoclonal antibody secreted by hybridoma cell strain 7E3 and resistant to FMD type A virus, and application | |
| CN111484552A (en) | Monoclonal antibody against SpA5 protein, application thereof and kit containing monoclonal antibody | |
| CN103333864B (en) | Monoclonal antibody of toxoplasma gondii resistant MIC3 protein and application monoclonal antibody | |
| CN108251381B (en) | Paraquat monoclonal antibody hybridoma cell strain and application thereof | |
| CN106405093A (en) | Detection kit for detecting infectious bovine rhinotracheitis virus antibody | |
| CN106771181A (en) | A kind of bird flu H7N9 hemagglutinin HA antigens detect ELISA kits and detection method | |
| CN101362801A (en) | Rapid detection test strip for detecting campylobacter jejuni specific antigen | |
| CN103454424B (en) | Neomycin phosphotransferase (NPT II) double-antibody sandwich elisa quantitative detecting method in genetically modified crops | |
| CN107389920B (en) | A kind of method and its dedicated kit detecting fowl leukocyte interleukin 17a content | |
| CN104062430B (en) | A kind of kit for detecting influenza virus in sample and detection method thereof and application | |
| CN103641913B (en) | A kind of Hansenula yeast HCP preparation method for antibody, antibody and its application is made | |
| CN110527668A (en) | A kind of resisting toxoplasmosis studies on rhoptry proteins 4(ROP4) monoclonal antibody and the preparation method and application thereof | |
| CN111500546A (en) | Cell strain secreting four subtype antibodies against aflatoxin, antibody secreted by cell strain and immunochromatography detection card | |
| CN110940810A (en) | Plastic package colloidal gold detection card for detecting salmonella toxin | |
| CN104749371B (en) | People's nephroblastoma overepressed gene encoding proteins enzyme linked immunological kit | |
| CN106520702A (en) | Hybridoma cell strain, secreted monoclonal antibody and application thereof | |
| CN109613249A (en) | A kind of russian spring-summer encephalitis virus IgM antibody ELISA detection kit and preparation method thereof | |
| CN115851610A (en) | Hybridoma cell strain for generating anti-human interleukin-6 monoclonal antibody, preparation method and application | |
| CN101570566B (en) | Vero cell cracked protein, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |