CN106928351A - A kind of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) and preparation method thereof - Google Patents
A kind of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) and preparation method thereof Download PDFInfo
- Publication number
- CN106928351A CN106928351A CN201710193758.5A CN201710193758A CN106928351A CN 106928351 A CN106928351 A CN 106928351A CN 201710193758 A CN201710193758 A CN 201710193758A CN 106928351 A CN106928351 A CN 106928351A
- Authority
- CN
- China
- Prior art keywords
- sample
- rsv
- liquid
- virus
- cell
- 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.)
- Pending
Links
- 241000725643 Respiratory syncytial virus Species 0.000 title claims abstract description 40
- 108060003951 Immunoglobulin Proteins 0.000 title claims abstract description 35
- 102000018358 immunoglobulin Human genes 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 33
- 238000001990 intravenous administration Methods 0.000 title claims abstract description 24
- 210000002381 plasma Anatomy 0.000 claims abstract description 66
- 238000012360 testing method Methods 0.000 claims abstract description 51
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 28
- 108020005544 Antisense RNA Proteins 0.000 claims abstract description 26
- 238000011330 nucleic acid test Methods 0.000 claims abstract description 26
- 238000001914 filtration Methods 0.000 claims abstract description 21
- 238000012216 screening Methods 0.000 claims abstract description 16
- 238000002156 mixing Methods 0.000 claims abstract description 12
- 238000010998 test method Methods 0.000 claims abstract description 8
- 230000002779 inactivation Effects 0.000 claims abstract description 6
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 6
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 4
- 230000008569 process Effects 0.000 claims abstract description 4
- 238000000605 extraction Methods 0.000 claims abstract description 3
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 3
- 238000000926 separation method Methods 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 88
- 239000007788 liquid Substances 0.000 claims description 63
- 241000700605 Viruses Species 0.000 claims description 60
- 238000012423 maintenance Methods 0.000 claims description 48
- 238000004448 titration Methods 0.000 claims description 31
- 239000012895 dilution Substances 0.000 claims description 27
- 238000010790 dilution Methods 0.000 claims description 27
- 230000003612 virological effect Effects 0.000 claims description 26
- 239000006285 cell suspension Substances 0.000 claims description 21
- 239000013642 negative control Substances 0.000 claims description 21
- 239000000047 product Substances 0.000 claims description 20
- 238000006386 neutralization reaction Methods 0.000 claims description 19
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- 238000001556 precipitation Methods 0.000 claims description 17
- 238000005070 sampling Methods 0.000 claims description 16
- 239000002002 slurry Substances 0.000 claims description 15
- 238000002474 experimental method Methods 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 239000007924 injection Substances 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 230000009514 concussion Effects 0.000 claims description 9
- 230000003902 lesion Effects 0.000 claims description 9
- 239000012898 sample dilution Substances 0.000 claims description 9
- 229960000583 acetic acid Drugs 0.000 claims description 8
- 208000015181 infectious disease Diseases 0.000 claims description 8
- 201000010099 disease Diseases 0.000 claims description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 7
- 239000003643 water by type Substances 0.000 claims description 7
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 6
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 6
- 239000012362 glacial acetic acid Substances 0.000 claims description 6
- 238000012856 packing Methods 0.000 claims description 6
- 239000003507 refrigerant Substances 0.000 claims description 6
- 230000029087 digestion Effects 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 230000003472 neutralizing effect Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000008351 acetate buffer Substances 0.000 claims description 4
- 239000012141 concentrate Substances 0.000 claims description 4
- 238000009472 formulation Methods 0.000 claims description 4
- 229920002271 DEAE-Sepharose Polymers 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 125000000129 anionic group Chemical group 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 239000003223 protective agent Substances 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 102000004506 Blood Proteins Human genes 0.000 claims description 2
- 108010017384 Blood Proteins Proteins 0.000 claims description 2
- 102000006395 Globulins Human genes 0.000 claims description 2
- 108010044091 Globulins Proteins 0.000 claims description 2
- 238000005374 membrane filtration Methods 0.000 claims description 2
- 239000002504 physiological saline solution Substances 0.000 claims description 2
- 239000008215 water for injection Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims 1
- 238000002086 displacement chromatography Methods 0.000 claims 1
- 238000011534 incubation Methods 0.000 claims 1
- 206010061603 Respiratory syncytial virus infection Diseases 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 4
- 208000030925 respiratory syncytial virus infectious disease Diseases 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 206010023825 Laryngeal cancer Diseases 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 206010023841 laryngeal neoplasm Diseases 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 206010057190 Respiratory tract infections Diseases 0.000 description 2
- 239000002120 nanofilm Substances 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 208000002330 Congenital Heart Defects Diseases 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 241000713340 Human immunodeficiency virus 2 Species 0.000 description 1
- 241000711920 Human orthopneumovirus Species 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010035742 Pneumonitis Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 108010065394 aminopherase Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 206010006475 bronchopulmonary dysplasia Diseases 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 208000028831 congenital heart disease Diseases 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 229940117323 privigen Drugs 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Virology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pulmonology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) and preparation method thereof, it is more than or equal to 1 using microneutralization test method screening RSV NATs first：300 raw blood plasma；Then the raw blood plasma that will be filtered out is prepared by mixing into blood plasma test sample, and the RSV NATs titre of the blood plasma test sample is more than or equal to 1：600, by the blood plasma test sample according to Low-temperature Ethanol Processes protein separating method, separation and Extraction immunoglobulin component is dispensed after being incubated inactivation, prepare through press filtration, chromatography, ultrafiltration, low pH and obtains product.Its NAT of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) is not less than 1：3000, purity is more than or equal to 98%, and protein content is 40~60mg/ml, is the active drug for preventing respiratory syncytial virus infection.
Description
Technical field
The present invention relates to human immunoglobulin(HIg) and preparation method thereof technical field, more particularly to a kind of intravenous respiratory tract is closed
Cellular virus human immunoglobulin(HIg) and preparation method thereof.
Background technology
Respiratory Syncytial Virus(RSV) (respiratory syncytial virus, RSV) is Paramyxoviridae, pneumonitis virus
Category, is tunicary minus-strand RNA virus.RSV viruses are the most important virus causing diseases of childrens respiratory tract infection, and its early stage is felt
Dye symptom is similar with many cause of diseases, serious harm children's health.Rsv infection be winter-spring season infants in hospital main cause it
One.According to WHO report, the annual about 64,000,000 people infection RSV in the whole world, wherein dead 160,000.A newest research shows, often
Year April November to next year, 20% is relevant with rsv infection in the infant being in hospital with internal cause ARI for 5 years old.RSV feels
Hair dyeing disease can outbreak of epidemic in global, locally, it has also become global public health problem.Respiratory Syncytial Virus(RSV) people is immunized
Globulin RSV-IgG clinically mainly reduces the admission rate of people at highest risk, bag because can specifically neutralize Respiratory Syncytial Virus(RSV) with it
Include and with broncho-pulmonary dysplasia or suffer from the children of congenital heart disease less than 6 week old babies, premature within 1 years old, within 2 years old
Youngster, old man and immunosupress patient., up to more than 90%, screening is simultaneously for Respiratory Syncytial Virus(RSV) Natural infection rate in general population
The blood plasma of the IgG antibody of Respiratory Syncytial Virus(RSV) containing high-titer is gathered, respiratory syncystial is prepared using efficient isolation and purification method
Viral human immunoglobulin(HIg) medicine, the severe infection for preventing to cause by Respiratory Syncytial Virus(RSV) has irreplaceable clinic
Application value.
The content of the invention
The present invention provides a kind of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) aiming at above-mentioned defect
And preparation method thereof, can effectively prevent because of the respiratory tract infection of infants that respiratory syncytial virus infection induces.
A kind of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) of the invention and preparation method thereof technical scheme is that this is quiet
Its NAT of note Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) is not less than 1：3000, purity is more than or equal to 98%, albumen
Content is 40~60mg/ml.
The intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) formulation be liquid preparation, formulation specification be 50ml/ bottles or
100ml/ bottles.
A kind of preparation method of described intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg), comprises the following steps：
(1) microneutralization test method screening RSV NATs are more than or equal to 1：300 raw blood plasma；
(2) raw blood plasma that will be filtered out is prepared by mixing into blood plasma test sample, the RSV neutralizing antibodies of the blood plasma test sample
Potency titre is more than or equal to 1：600；
(3) by the blood plasma test sample according to Low-temperature Ethanol Processes protein separating method, separation and Extraction immunoglobulin group
Part, packing obtains product after being incubated inactivation, prepare through press filtration, chromatography, ultrafiltration, low pH.
Microneutralization test method specifically includes following steps described in step (1)：
1. Sample Dilution
Plasma sample is carried out 1 in advance with cell maintenance medium：56 DEG C inactivate 30 minutes after 10 dilutions；In 96 hole depth orifice plates
540ul cell maintenance mediums are added, the plasma sample 60ul after inactivation is subsequently adding, concussion mixes standby；
96 porocyte plates of q.s are opened, viral residual titration plate and sampling test plate is indicated, and in each sample breadboard
The upper numbering for indicating the plate test specimen；
33.3ul cell maintenance mediums are added in the various kinds sample wells of sampling test plate, viral residual titration hole adds with negative control hole
Enter 50ul cell maintenance mediums；
The good sample liquid of beforehand dilution is added separately to the relevant position of corresponding 96 porocyte plates, product 16.7 are loaded per hole
μ l, each sample is parallel to do four holes；
2. challenge virus
Prepare challenge virus liquid：Number of samples according to experiment prepares enough challenge virus liquid with maintaining liquid, will be using disease
Poison is diluted to 100CCID50/0.05ml；In addition to viral residual titration hole and negative control hole, remaining each Kong Zhongjun adds 50 μ l to contain
100CCID50Challenge virus liquid；
Residual titration 100CCID50 challenge virus liquid：Challenge virus liquid is made 10 with cell maintenance medium-1、10-2、10-3Dilution, will
Its challenge virus liquid with its 10-1、10-2、10-3The virus liquid of concentration is added to corresponding virus residual titration with 96 porocyte plates holes,
Each dilution factor adds 10 holes, 50 μ l/ holes；
Negative control：To adding 50 μ l cell maintenance mediums in each hole of negative control；
All 96 porocyte plates are put into 35 DEG C, contain CO2Percentage by volume be 5% incubator in and culture it is 1.5 small
When；
3. the preparation of cell suspension and addition
The preparation of cell suspension：The Hep-2 cells of q.s are taken, is counted after digestion, experiment concentration of cell suspension should be 2
×105Individual/ml, the cell liquid for preparing is used immediately；
Cell is outstanding to be added：Add cell suspension 100 μ l, about 2 × 10 per hole after neutralizing culture4Individual/hole；
4. cultivate and observe statistics
96 porocyte plates of operation place 35 DEG C, contain CO more than completing2Percentage by volume be 5% incubator in train
Support 7 days, the 5th~7 day situation of basis of microscopic observation sample protection cell infection virus, record statistics；
There are holes and the above that lesion person's as high-titer blood plasma does not occur in four repeating sample sample wells.
The cell maintenance medium is the MEM liquid of hyclone that volume ratio is 3%.
Step (2) be learn from else's experience step (1) neutralization test method measure NAT be more than 1：300 human plasma, 2
Melt slurry at~30 DEG C, merging is prepared by mixing into blood plasma test sample.
Step (3) specifically,
1. it is 4.0 with pH value with the serum protein content in physiological saline regulation blood plasma test sample to 45~50mg/ml
Acetate buffer solution adjusts pH to 6.0~6.2, adds 95% ethanol to adjust concentration of alcohol to 20%, and reaction temperature is -5.5~-
4.5 DEG C, press filtration is separated and obtains FI+II+III precipitations；
2. the precipitation for 1. step being obtained is dissolved with 10 times of low-temperature injection waters of volume, slow with the acetic acid that pH value is 4.0
In fliud flushing regulation pH to 5.2, plus the ethanol of percent by volume 95% to the reaction solution, the final ethanol percent by volume is set to be
14%, reacting liquid temperature to -5.5~-4.5 DEG C is controlled using refrigerant, press filtration is separated goes precipitation, collects supernatant；
3. the supernatant pH to 6.8~7.2 for 2. being collected with 0.1M NaOH regulating steps, adds 95% ethanol regulation ethanol
To 25%, reaction temperature is -7.5~-6.5 DEG C to concentration, and press filtration is separated and obtains FII precipitations；
4. the precipitation for 3. step being obtained is dissolved with 10 times of low-temperature injection waters of volume, and reaction liquid temperature is controlled using refrigerant
To 0~1 DEG C, press filtration is separated goes precipitation to degree, collects supernatant；
5. upper DEAE-Sepharose Fast Flow weak anionic switching layer after the supernatant liquid filtering 4. step collected
Analysis, reaction temperature is 0~1 DEG C, collects efflux；
6. the efflux 5. step collected glacial acetic acid adjusts PH to 3.8~4.4, and 8~10 times of ultrafiltration dialysis are concentrated to give
To concentrate；
7. the concentrate for 6. step being obtained adjusts pH to 3.8~4.0 with glacial acetic acid, adds maltose to make ultimate density extremely
10% used as protective agent, and adding low-temperature injection water makes protein content be 5.5~6.0%, obtains stoste；
8. the stoste for 7. step being obtained is incubated at 23~25 DEG C and put 21 days；
9. prepare：Adjustment product protein content is diluted to 51~55mg/ml with water for injection, while adding maltose extremely
10%, with 0.2mol/L salt acid for adjusting pH to 3.8~4.4.
The step 9. after, carry out degerming packing, it is degerming through 0.2 μm of membrane filtration, control filter pressure be not more than
0.25MPa, by 51~55mg/ml of protein content, NAT is more than or equal to 1：3000,50ml/ bottles or 100ml/ bottles
Specification is dispensed.
Comprised the following steps before step (1)：
A. the method for the epidemiology survey based on microneutralization test extrapolates plasma screening standard (threshold value) substantially：
RSV NATs titre is not less than 1：300；
B. the method that the slurry based on microneutralization test is verified determines the screening criteria of blood plasma：RSV NATs
Titre is not less than 1：300；
The method of epidemiology survey specifically includes following steps described in step A：
1. Sample Dilution：
Plasma sample carries out 1 in advance with cell maintenance medium：56 DEG C inactivate 30 minutes after 4 dilutions；
96 porocyte plates of q.s are opened, viral residual titration plate and sampling test plate is indicated, and in each sample breadboard
The upper numbering for indicating the plate test specimen；
50ul cell maintenance mediums are added in the various kinds sample wells of sampling test plate, then sample is added in A1-H1 and A7-H7
Product, 25ul/ holes, upper and lower holes for repeat, sequentially for first from top to bottom, after from left to right；
The volley of rifle fire dilutes：1 row and 7 the row volley of rifle fire are mixed inhales 25ul to next column, and such dilution is arranged up to 6 row and 12, finally
25ul is discarded；
Viral residual titration hole adds 50ul cell maintenance mediums with negative control hole；
The cell maintenance medium is the MEM liquid of hyclone that volume ratio is 3%；
2. challenge virus
Prepare challenge virus liquid：Number of samples according to experiment prepares enough challenge virus liquid with maintaining liquid, will be using disease
Poison is diluted to 100CCID50/0.05ml；In addition to viral residual titration hole and negative control hole, remaining each Kong Zhongjun adds 50 μ l to contain
100CCID50Challenge virus liquid；
Residual titration 100CCID50 challenge virus liquid：Challenge virus liquid is made 10 with cell maintenance medium-1、10-2、10-3Dilution, will
Its challenge virus liquid with its 10-1、10-2、10-3The virus liquid of concentration is added to corresponding virus residual titration with 96 porocyte plates holes,
Each dilution factor adds 10 holes, 50 μ l/ holes；
Negative control：To adding 50 μ l cell maintenance mediums in each hole of negative control；
All 96 porocyte plates are put into 35 DEG C, contain CO2Percentage by volume be 5% incubator in and culture it is 1.5 small
When；
3. the preparation of cell suspension and addition
The preparation of cell suspension：The Hep-2 cells of q.s are taken, is counted after digestion, experiment concentration of cell suspension should be 2
×105Individual/ml, the cell liquid for preparing is used immediately；
Cell is outstanding to be added：Add cell suspension 100 μ l, about 2 × 10 per hole after neutralizing culture4Individual/hole；
4. cultivate and observe statistics
96 porocyte plates of operation place 35 DEG C, contain CO more than completing2Percentage by volume be 5% incubator in train
Support 7 days, the 5th~7 day situation of basis of microscopic observation sample protection cell infection virus, record statistics；
Screening the pulp standard is calculated by the method for accumulated weights average value.
The method of the slurry checking described in step B specifically includes following steps：
A. Sample Dilution
Plasma sample carries out 1 in advance with cell maintenance medium：56 DEG C inactivate 30 minutes after 10 dilutions；
Plasma sample more than the screening the pulp standard through calculating of appropriate volume is taken respectively in 10ml apyrogeneity pipes, and concussion is mixed
It is even to be designated as high-titer pooled plasma；
Whole plasma samples of appropriate volume are taken respectively in 10ml apyrogeneity pipes, and concussion mixing is designated as complete mixed-blood slurry；
96 porocyte plates of q.s are opened, viral residual titration plate and sampling test plate is indicated, and in each sample breadboard
The upper numbering for indicating the plate test specimen；
50ul cell maintenance mediums are added in the various kinds sample wells of sampling test plate, then sample, 50ul/ is added in A1-A12
Hole；
The volley of rifle fire dilutes：The volley of rifle fire is mixed and inhales 50ul to next column from top to bottom, and so dilution is until H rows, finally abandon 50ul
Fall；
Viral residual titration hole adds 50ul cell maintenance mediums with negative control hole；
The cell maintenance medium is the MEM liquid of hyclone that volume ratio is 3%；
The condition of culture of following challenge virus, the preparation of cell suspension and addition and cell is with epidemic disease described in step A
The method for learning investigation.
A kind of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) of the invention and preparation method thereof has the beneficial effect that：
(1) the Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) that the present invention is provided, RSV NATs are more than or equal to 1:
3000, purity is more than or equal to 98%, can targetedly neutralize Respiratory Syncytial Virus(RSV), is prevention respiratory syncytial virus infection
Active drug, with larger Social benefit and economic benefit.
(2) present invention in propose first using epidemiology survey method calculate blood plasma screening criteria (threshold value) with
And the method for slurry checking, make the more standby scientific and reliability of experiment；
(3) propose that plasma sample detection uses 4 repetitions in the present invention first in microneutralization test, it is to avoid in the past
The excessive deviation that 2 repetitions bring in neutralization test, makes the accuracy of result further improve；
(4) using (Long plants) of the Respiratory Syncytial Virus(RSV) strain of the existing standard of country as detection carrier, filtering out has
Compared with the specific raw blood plasma of high titre, its standard meets《Pharmacopoeia of People's Republic of China》" biological products are produced in 2015 editions
With blood plasma " regulation, prepare exhaling for high titre, high specific and high-purity through cold ethanol filter-pressing process column chromatographic purifying
Inhale road syncytial virus human immunoglobulin(HIg).
Brief description of the drawings：
Fig. 1 show the preparation flow figure of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) of the present invention.
Specific embodiment：
For a better understanding of the present invention, technical scheme is described in detail with instantiation below, but originally
Invention is not limited thereto.
Embodiment 1
1st, the plasma screening collection of Plasma donors：
Selection blood plasma, selected blood plasma should meet following condition：RSV NATs are not less than 1：300, alanine
Aminopherase (reitman-frankel method monitoring) is not higher than 35 units；Hepatitis B surface antibody is negative, and syphilis is negative, HIV-1/HIV-2
The qualified raw blood plasmas such as negative antibody, HCV negative antibodies, cryogenic freezing storage, storage life is no more than 2 years.
Epidemiologic surveys comprise the following steps：
(1) using human laryngeal cancer epithelial cell Hep-2 as indicator cells, nutrient solution is containing 10% (V/V) hyclone
MEM；
(2) by 500 parts of plasma samples with cell maintenance medium (the as MEM liquid of the hyclone of percent by volume 3%, under
1 is carried out together)：After 4 dilutions, 56 DEG C inactivate 30 minutes.
65 96 porocyte plates are opened, viral residual titration plate and sampling test plate is indicated, and in each sample breadboard subscript
Show the numbering of the plate test specimen；
50ul cell maintenance mediums are added in the various kinds sample wells of sampling test plate, then sample is added in A1-H1 and A7-H7
Product, 25ul/ holes, upper and lower holes for repeat, sequentially for first from top to bottom, after from left to right；
The volley of rifle fire dilutes：1 row and 7 the row volley of rifle fire are mixed inhales 25ul to next column, and such dilution is arranged up to 6 row and 12, finally
25ul is discarded；
Viral residual titration hole adds 50ul cell maintenance mediums with negative control hole.
(3) take Respiratory Syncytial Virus(RSV) RSV (long plants) with cell maintenance medium be diluted to experiment concentration (i.e. 100 ×
CCID50/0.05ml) in equivalent addition test sample plate hole, and Positive control wells, negative control hole and viral residual titration are set simultaneously
Hole.
Viral (positive) control wells：Cell maintenance medium 50ul/ holes are first added in plate hole, is subsequently adding and is applied virus liquid
50ul/ holes, mix；
Cell (feminine gender) control wells：It is directly added into cell maintenance medium 100ul/ holes；
Viral residual titration hole：Cell maintenance medium 50ul/ holes are first added in plate hole, then 10 multiple proportions will be carried out using virus liquid
After dilution, 10 are taken respectively0~10-3The virus liquid of concentration is added in corresponding virus residual titration hole, 50ul/ holes；
(4) neutralization reaction：By 35 DEG C in each plate rearmounted CO2gas incubator of mixing, 5%CO2 is incubated and carries out neutralization reaction
1.5 hours.
(5) cell：The preparation of cell suspension：The Hep-2 cells of q.s are taken, is counted after digestion, adjusted with cell maintenance medium
Section experiment concentration of cell suspension should be 2 × 105Individual/ml, the cell liquid for preparing immediately using or put 4 DEG C and save backup.
Cell is outstanding to be added：After the neutralization reaction time is full, cell suspension 100 μ l, about 2 × 10 are added per hole4Individual/hole.
(6) cultivate：The cell plates operated more than completing place 35 DEG C, 5%CO2Cultivated 7 days in incubator.5th~7 day
The situation of basis of microscopic observation sample protection cell infection virus.
Experiment every time must have virus control and cell controls, and lesion must occur in virus control, and cell controls must be just
Often.Result judgement：It is neutralization titer with the test sample dilution factor that 50% cell can be protected to occur without lesion.With more than a certain neutralization
The accumulated weights value of the neutralization titer of all of above blood plasma of antibody titer is more than 6 times of whole blood plasma accumulated weights values, then should
NAT is screening the pulp standard (threshold value), as shown in table 1.
The epidemiology survey result of table 1.
Note：Potency accumulated weights average value described in table 1:The potency of all samples more than corresponding valence value is average
Value；
Slurry verification method comprises the following steps：
Using human laryngeal cancer epithelial cell Hep-2 as indicator cells, nutrient solution is containing 10% (V/V) hyclone MEM to A；
B plasma samples carry out 1 in advance with cell maintenance medium：56 DEG C inactivate 30 minutes after 10 dilutions；
100ul neutralization titers are taken respectively 1：In 10ml apyrogeneity pipes, concussion mixing is designated as more than 300 plasma sample
High-titer pooled plasma；
Whole plasma samples of 10ul are taken respectively in 10ml apyrogeneity pipes, and concussion mixing is designated as complete mixed-blood slurry；
96 porocyte plates of 4 plates are opened, viral residual titration plate and sampling test plate is indicated, and in each sample breadboard subscript
Show the numbering of the plate test specimen；
50ul cell maintenance mediums are added in the various kinds sample wells of sampling test plate, then sample, 50ul/ is added in A1-A12
Hole；
The volley of rifle fire dilutes：The volley of rifle fire is mixed and inhales 50ul to next column from top to bottom, and so dilution is until H rows, finally abandon 50ul
Fall；
Viral residual titration hole adds 50ul cell maintenance mediums with negative control hole
Following viral addition, neutralization and the addition of cell and condition of culture are identical with epidemiology investigation method；
Experiment every time must have virus control and cell controls, and lesion must occur in virus control, and cell controls must be just
Often.Result judgement：It is neutralization titer with the test sample dilution factor that 50% cell can be protected to occur without lesion.High-titer slurry is neutralized
Antibody titer is that more than the 6 times screening the pulp standards (threshold value) for then calculating of whole slurries are set up, as shown in table 2.
The slurry the result of table 2.
Neutralization test assay method comprises the following steps：
A (), using human laryngeal cancer epithelial cell Hep-2 as indicator cells, nutrient solution is containing 10% (V/V) hyclone
MEM；
B () enters plasma sample with cell maintenance medium (as the MEM liquid of the hyclone of percent by volume 3%, similarly hereinafter)
Row 1：After 10 dilutions, 56 DEG C inactivate 30 minutes.
540ul cell maintenance mediums are added in 96 hole depth orifice plates, the plasma sample 60ul after inactivation is subsequently adding, concussion is mixed
It is even standby；
The cell plates of q.s are taken, 33.3ul/ holes cell maintenance medium is added in sample panel, be subsequently adding corresponding 1：
100 prediluted each sample liquid 16.7ul/ holes, per parallel 4 hole of sample.
Following viral addition, neutralization and the addition of cell and condition of culture are identical with epidemiology investigation method；
Experiment every time must have virus control and cell controls, and lesion must occur in virus control, and cell controls must be just
Often.Result judgement：It is neutralization titer with the test sample dilution factor that 50% cell can be protected to occur without lesion.4 repeating sample sample wells
In have more than holes and holes and lesion does not occur be qualified blood plasma, statistics.
Specific plasma containing high titre carries out hybrid detection before operation, and antibody titer is not less than 1: 600, continuous three batches
Pooled plasma testing result is shown in Table 3.
3. continuous three batches of pooled plasma testing results of table
| Detection project | 2014S01 | 2014S02 | 2014S03 |
| Protein content | 56g/L | 56g/L | 57g/L |
| HRSV NATs | 1∶724 | 1∶675 | 1∶675 |
2nd, the preparation of Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg)：
(1) the 300 person-portion blood plasma by NAT more than 1: 300, melt slurry at 2~30 DEG C, merge mixing, after mixing
Volume is 180L, detects that neutralization titer is 1: 675 with neutralization test method；
(2) 18kg normal saline dilution blood plasma is used, pH to 6.01 is adjusted with the acetate buffer solution 6kg that pH value is 4.0, added
To in retort, regulation concentration of alcohol to 20%, reaction temperature is -4.5 DEG C to 95% ethanol 54.4kg, and press filtration is separated and obtains 20kg
FI+II+III；
(3) precipitation for obtaining step (2) 200kg low-temperature injection waters dissolve, with the acetate buffer solution that pH value is 4.0
In 2.1kg regulations pH to 5.2, plus the ethanol of 35kg 95% to the reaction solution, make final ethanol percent by volume be 14%, make
Reacting liquid temperature to -4.5 DEG C is controlled with refrigerant, press filtration is separated goes precipitation, collects supernatant 280kg；
(4) the supernatant pH to 7.05 collected with 18kg 0.1M NaOH regulating steps (3), adds the second of 46.8kg 95%
Alcohol adjusts concentration of alcohol to 25%, and reaction temperature is -7.0 DEG C, and press filtration is separated and obtains FII precipitations 5.2kg；
(5) precipitation for obtaining step (4) low-temperature injection water of 52kg is dissolved, and reacting liquid temperature is controlled using refrigerant
To 0.1 DEG C, press filtration is separated goes precipitation, collects supernatant 72kg；
(6) upper DEAE-Sepharose Fast Flow weak anionics are exchanged after the supernatant liquid filtering for collecting step (5)
Chromatography, reaction temperature is 0.6 DEG C, collects efflux 75kg；
(7) the liquid glacial acetic acid that flows through that step (6) is collected is adjusted into PH to 3.93,10 times of ultrafiltration dialysis are concentrated into 13kg
(protein content 8.1%, pH：4.25)；
(8) concentrate for obtaining step (7) adjusts pH to 3.93 with 5ml glacial acetic acid, adds 1.8kg maltose to make final dense
Degree makes protein content be 5.65% to 3.3kg low-temperature injection waters 10% as protective agent, are added, and final volume is 18.5kg；
(9) stoste that step (8) is obtained is incubated at 23~25 DEG C and remove virus with nano-film filtration after putting 21 days；
(10) prepare：Stock protein content is 5.6% after determining nano-film filtration, is diluted with 1.6kg waters for injection and adjusted
Product protein content is to 52mg/ml, while 0.15kg maltose to 10% is added, with 20ml 0.2mol/L salt acid for adjusting pH extremely
4.05。
3rd, degerming packing after preparing, protein content is 52mg/ml, and NAT is 1：3300, it is divided into and fills 375 bottles of systems
Product, take 30 bottle products and send quality arbitration portion to examine and determine.
4th, the sample obtained by being prepared through extensive trial production, this product manufacture revised according to enterprise and vertification regulation and
《Pharmacopoeia of People's Republic of China》The pertinent regulations of 2010 editions three carry out the Respiratory Syncytial Virus(RSV) obtained by calibrating above-mentioned steps
Human immunoglobulin(HIg).The results are shown in Table 4.
The Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) survey report of table 4.
Conclusion：As a result this product meets rule by the manufacture vertification regulation inspection of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg)
It is fixed.
Embodiment 2
Intravenous Respiratory Syncytial Virus(RSV) immunoglobulin of the invention is existing with Shandong Taibang Biological Product Co., Ltd. quiet
The comparative study of the anti-RSV NATs of the third product and external quiet third product
Three batches of quiet third Respiratory Syncytial Virus(RSV) immunoglobulins prepared by the inventive method have with the safe nation's biological products in Shandong
Existing quiet third product of limit company and quiet third products (CLARIFY) of France LFB and the quiet third product Privigen of CSL carry out anti-RSV
The comparative study of NAT.The measure of NAT is determined in the method for above-mentioned microneutralization test.
The neutralization titer comparative study of table 5.
Conclusion：RSV-IVIG NATs prepared by the present invention are existing quiet Shandong Taibang Biological Product Co., Ltd.
Third and external quiet third (conversion is 6 times or so of 5% quiet third).Ability with neutralization Respiratory Syncytial Virus(RSV) very high, be
Prevent the active drug of respiratory syncytial virus infection, with larger Social benefit and economic benefit.
Embodiment described above is only that the preferred embodiment of the present invention is described, not to model of the invention
Enclose and be defined, on the premise of design spirit of the present invention is not departed from, those of ordinary skill in the art are to technical side of the invention
Various modifications and improvement that case is made, all should fall into the protection domain of claims of the present invention determination.
Claims (10)
1. a kind of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg), it is characterised in that intravenous Respiratory Syncytial Virus(RSV) people exempts from
Its NAT of epidemic disease globulin is not less than 1：3000, purity is more than or equal to 98%, and protein content is 40~60mg/ml.
2. a kind of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) according to claim 1, it is characterised in that formulation is
Liquid preparation, formulation specification is 50ml/ bottles or 100ml/ bottles.
3. a kind of preparation method of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) as claimed in claim 1, its feature exists
In comprising the following steps：
(1) microneutralization test method screening RSV NATs are more than or equal to 1：300 raw blood plasma；
(2) raw blood plasma that will be filtered out is prepared by mixing into blood plasma test sample, the RSV NATs of the blood plasma test sample
Titre is more than or equal to 1：600；
(3) by the blood plasma test sample according to Low-temperature Ethanol Processes protein separating method, separation and Extraction immunoglobulin component, warp
Packing obtains product after press filtration, chromatography, ultrafiltration, low pH incubations inactivation, preparation.
4. a kind of preparation method of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) according to claim 3, its feature
It is that microneutralization test method specifically includes following steps described in step (1)：
1. Sample Dilution
Plasma sample is carried out 1 in advance with cell maintenance medium：56 DEG C inactivate 30 minutes after 10 dilutions；Added in 96 hole depth orifice plates
540ul cell maintenance mediums, are subsequently adding the plasma sample 60ul after inactivation, and concussion mixes standby；
96 porocyte plates of q.s are opened, viral residual titration plate and sampling test plate is indicated, and in each sample breadboard subscript
Show the numbering of the plate test specimen；
33.3ul cell maintenance mediums are added in the various kinds sample wells of sampling test plate, viral residual titration hole adds with negative control hole
50ul cell maintenance mediums；
The good sample liquid of beforehand dilution is added separately to the relevant position of corresponding 96 porocyte plates, the μ l of product 16.7 is loaded per hole,
Each sample is parallel to do four holes；
2. challenge virus
Prepare challenge virus liquid：Number of samples according to experiment prepares enough challenge virus liquid with maintaining liquid, will be dilute using virus
Release 100CCID50/0.05ml；In addition to viral residual titration hole and negative control hole, remaining each Kong Zhongjun adds 50 μ l to contain
100CCID50Challenge virus liquid；
Residual titration 100CCID50 challenge virus liquid：Challenge virus liquid is made 10 with cell maintenance medium-1、10-2、10-3Dilution, is attacked
Hit virus liquid with its 10-1、10-2、10-3The virus liquid of concentration is added to corresponding virus residual titration with 96 porocyte plates holes, each
Dilution factor adds 10 holes, 50 μ l/ holes；Negative control：To adding 50 μ l cell maintenance mediums in each hole of negative control；
All 96 porocyte plates are put into 35 DEG C, contain CO2Percentage by volume be 5% incubator in and culture 1.5 hours；
3. the preparation of cell suspension and addition
The preparation of cell suspension：The Hep-2 cells of q.s are taken, is counted after digestion, experiment concentration of cell suspension should be 2 × 105
Individual/ml, the cell liquid for preparing is used immediately；
Cell is outstanding to be added：Add cell suspension 100 μ l, about 2 × 10 per hole after neutralizing culture4Individual/hole；
4. cultivate and observe statistics
96 porocyte plates of operation place 35 DEG C, contain CO more than completing2Percentage by volume be 5% incubator in cultivate 7
My god, the 5th~7 day situation of basis of microscopic observation sample protection cell infection virus, record statistics；
There are holes and the above that lesion person's as high-titer blood plasma does not occur in four repeating sample sample wells.
5. a kind of preparation method of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) according to claim 4, its feature
It is that the cell maintenance medium is the MEM liquid of hyclone that volume ratio is 3%.
6. a kind of preparation method of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) according to claim 3, its feature
Be, step (2) be learn from else's experience step (1) neutralization test method measure NAT be more than 1：300 human plasma, 2~30
Melt slurry at DEG C, merging is prepared by mixing into blood plasma test sample.
7. a kind of preparation method of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) according to claim 3, its feature
It is that step (3) is,
1. with the serum protein content in physiological saline regulation blood plasma test sample to 45~50mg/ml, with the acetic acid that pH value is 4.0
Buffer solution adjusts pH to 6.0~6.2, adds 95% ethanol to adjust concentration of alcohol to 20%, and reaction temperature is -5.5~-4.5 DEG C,
Press filtration is separated and obtains FI+II+III precipitations；
2. the precipitation for 1. step being obtained is dissolved with 10 times of low-temperature injection waters of volume, with the acetate buffer solution that pH value is 4.0
In regulation pH to 5.2, plus the ethanol of percent by volume 95% to the reaction solution, make final ethanol percent by volume be 14%, make
Reacting liquid temperature to -5.5~-4.5 DEG C is controlled with refrigerant, press filtration is separated goes precipitation, collects supernatant；
3. the supernatant pH to 6.8~7.2 for 2. being collected with 0.1M NaOH regulating steps, adds 95% ethanol regulation concentration of alcohol
To 25%, reaction temperature is -7.5~-6.5 DEG C, and press filtration is separated and obtains FII precipitations；
4. the precipitation for 3. step being obtained is dissolved with 10 times of low-temperature injection waters of volume, and reacting liquid temperature is controlled extremely using refrigerant
0~1 DEG C, press filtration is separated goes precipitation, collects supernatant；
5. upper DEAE-Sepharose Fast Flow weak anionic displacement chromatographies after the supernatant liquid filtering 4. step collected, instead
Answer temperature for 0~1 DEG C, collect efflux；
6. the efflux 5. step collected glacial acetic acid adjusts PH to 3.8~4.4, and 8~10 times of ultrafiltration dialysis are concentrated to give dense
Contracting liquid；
7. the concentrate for 6. step being obtained adjusts pH to 3.8~4.0 with glacial acetic acid, adds maltose to make ultimate density to 10% work
It is protective agent, adding low-temperature injection water makes protein content be 5.5~6.0%, obtains stoste；
8. the stoste for 7. step being obtained is incubated at 23~25 DEG C and put 21 days；
9. prepare：Adjustment product protein content is diluted to 51~55mg/ml with water for injection, while maltose to 10% is added,
With 0.2mol/L salt acid for adjusting pH to 3.8~4.4.
8. a kind of preparation method of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) according to claim 7, its feature
Be, the step 9. after, carry out degerming packing, it is degerming through 0.2 μm of membrane filtration, control filter pressure be not more than 0.25MPa,
By 51~55mg/ml of protein content, NAT is more than or equal to 1：3000,50ml/ bottles or 100ml/ bottles of specification packing.
9. a kind of preparation method of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) according to claim 3, its feature
It is to be comprised the following steps before step (1)：
A. the method for the epidemiology survey based on microneutralization test extrapolates plasma screening standard (threshold value) substantially：RSV
NAT titre is not less than 1：300；
B. the method that the slurry based on microneutralization test is verified determines the screening criteria of blood plasma：RSV NAT titres
Not less than 1：300.
10. a kind of preparation method of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) according to claim 9, its feature
It is that the method for epidemiology survey specifically includes following steps described in step A：
1. Sample Dilution：
Plasma sample carries out 1 in advance with cell maintenance medium：56 DEG C inactivate 30 minutes after 4 dilutions；
96 porocyte plates of q.s are opened, viral residual titration plate and sampling test plate is indicated, and in each sample breadboard subscript
Show the numbering of the plate test specimen；
50ul cell maintenance mediums are added in the various kinds sample wells of sampling test plate, then sample is added in A1-H1 and A7-H7,
25ul/ holes, upper and lower holes for repeat, sequentially for first from top to bottom, after from left to right；
The volley of rifle fire dilutes：1 row and the 7 row volley of rifle fire are mixed and inhale 25ul to next column, and so dilution will be until 6 row and 12 row, finally will
25ul is discarded；
Viral residual titration hole adds 50ul cell maintenance mediums with negative control hole；
The cell maintenance medium is the MEM liquid of hyclone that volume ratio is 3%；
2. challenge virus
Prepare challenge virus liquid：Number of samples according to experiment prepares enough challenge virus liquid with maintaining liquid, will be dilute using virus
Release 100CCID50/0.05ml；In addition to viral residual titration hole and negative control hole, remaining each Kong Zhongjun adds 50 μ l to contain
100CCID50Challenge virus liquid；
Residual titration 100CCID50 challenge virus liquid：Challenge virus liquid is made 10 with cell maintenance medium-1、10-2、10-3Dilution, is attacked
Hit virus liquid with its 10-1、10-2、10-3The virus liquid of concentration is added to corresponding virus residual titration with 96 porocyte plates holes, each
Dilution factor adds 10 holes, 50 μ l/ holes；Negative control：To adding 50 μ l cell maintenance mediums in each hole of negative control；
All 96 porocyte plates are put into 35 DEG C, contain CO2Percentage by volume be 5% incubator in and culture 1.5 hours；
3. the preparation of cell suspension and addition
The preparation of cell suspension：The Hep-2 cells of q.s are taken, is counted after digestion, experiment concentration of cell suspension should be 2 × 105
Individual/ml, the cell liquid for preparing is used immediately；
Cell is outstanding to be added：Add cell suspension 100 μ l, about 2 × 10 per hole after neutralizing culture4Individual/hole；
4. cultivate and observe statistics
96 porocyte plates of operation place 35 DEG C, contain CO more than completing2Percentage by volume be 5% incubator in cultivate 7
My god, the 5th~7 day situation of basis of microscopic observation sample protection cell infection virus, record statistics；
Screening the pulp standard is calculated by the method for accumulated weights average value.
The method of the slurry checking described in step B specifically includes following steps：
A. Sample Dilution
Plasma sample carries out 1 in advance with cell maintenance medium：56 DEG C inactivate 30 minutes after 10 dilutions；
Plasma sample more than the screening the pulp standard through calculating of appropriate volume is taken respectively in 10ml apyrogeneity pipes, and concussion mixes note
It is high-titer pooled plasma；
Whole plasma samples of appropriate volume are taken respectively in 10ml apyrogeneity pipes, and concussion mixing is designated as complete mixed-blood slurry；
96 porocyte plates of q.s are opened, viral residual titration plate and sampling test plate is indicated, and in each sample breadboard subscript
Show the numbering of the plate test specimen；
50ul cell maintenance mediums are added in the various kinds sample wells of sampling test plate, then sample, 50ul/ holes is added in A1-A12；
The volley of rifle fire dilutes：The volley of rifle fire is mixed and inhales 50ul to next column from top to bottom, and so dilution is until H rows, finally discard 50ul；
Viral residual titration hole adds 50ul cell maintenance mediums with negative control hole；
The cell maintenance medium is the MEM liquid of hyclone that volume ratio is 3%；
Following challenge virus, the preparation of cell suspension are adjusted with the condition of culture of addition and cell with epidemiology described in step A
The method looked into.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710193758.5A CN106928351A (en) | 2017-03-28 | 2017-03-28 | A kind of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710193758.5A CN106928351A (en) | 2017-03-28 | 2017-03-28 | A kind of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106928351A true CN106928351A (en) | 2017-07-07 |
Family
ID=59424852
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710193758.5A Pending CN106928351A (en) | 2017-03-28 | 2017-03-28 | A kind of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106928351A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105601736A (en) * | 2016-01-28 | 2016-05-25 | 哈尔滨派斯菲科生物制药股份有限公司 | Respiratory syncytial virus resistance human immune globulin and preparation method thereof |
-
2017
- 2017-03-28 CN CN201710193758.5A patent/CN106928351A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105601736A (en) * | 2016-01-28 | 2016-05-25 | 哈尔滨派斯菲科生物制药股份有限公司 | Respiratory syncytial virus resistance human immune globulin and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101695570B (en) | Univalent and bivalent inactivated vaccine for hand-foot-and-mouth disease and preparation method thereof | |
| CN105601736B (en) | A kind of anti respiratory syncytial virus human immunoglobulin(HIg) and preparation method thereof | |
| CN101455736B (en) | Use of wild jujube seeds extract | |
| CN111499736B (en) | Preparation method of intravenous injection COVID-19 human immunoglobulin | |
| CN105963692B (en) | Combined vaccine for preventing hand-foot-and-mouth disease | |
| Mukherjee et al. | Rhabdomyolysis in a patient with coronavirus disease 2019 | |
| CN111938230B (en) | Respiratory tract transmitted pathogen enrichment spinning membrane, sampling mask paster and sampling mask | |
| CN102012430B (en) | Avian influenza H5N1 subtype Re-5 strain hemagglutination inhibition antigen standard substance and preparation method | |
| CN106075423A (en) | A kind of combined vaccine preventing hand-foot-mouth disease | |
| CN106928351A (en) | A kind of intravenous Respiratory Syncytial Virus(RSV) human immunoglobulin(HIg) and preparation method thereof | |
| CN105999256A (en) | Combined vaccine for preventing hand-foot-and-mouth disease | |
| CN105669860B (en) | A kind of hand-foot-and-mouth disease resistant human immunoglobulin and preparation method thereof | |
| CN102004151B (en) | Avian influenza virus H9 subtype hemagglutination inhibition antigen standard substance and preparation method thereof | |
| CN104353063A (en) | Biological preparation for preventing and controlling human respiratory syncytial virus infection and preparation method | |
| CN101695511B (en) | Pomegranate rind extract and production method and application thereof | |
| CN103554252B (en) | A kind of Giant cell human immunoglobulin and preparation method thereof | |
| CN108949700A (en) | A kind of JS14-2 plants of goat haemadsorption virus 1 and its application | |
| CN116615234A (en) | Combined vaccine for preventing hand-foot-mouth disease and preparation method and application thereof | |
| Fattal et al. | Enterovirus types in Israel sewage | |
| CN112717128A (en) | Combined vaccine for preventing hand-foot-and-mouth disease and preparation method and application thereof | |
| CN109810952A (en) | The method of human respiratory syncytial virus's large-scale culture | |
| CN106676113A (en) | Method for preparing monoclonal antibody to respiratory syncytial virus | |
| CN106267412B (en) | AIDS immunologic purging instrument | |
| CN106110422B (en) | AIDS immunization therapy absorber | |
| CN111053898A (en) | Vaccine composition 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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170707 |
|
| RJ01 | Rejection of invention patent application after publication |