CN106546747A - A kind of detection method of interferon biological activity - Google Patents
A kind of detection method of interferon biological activity Download PDFInfo
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- CN106546747A CN106546747A CN201610928699.7A CN201610928699A CN106546747A CN 106546747 A CN106546747 A CN 106546747A CN 201610928699 A CN201610928699 A CN 201610928699A CN 106546747 A CN106546747 A CN 106546747A
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- 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
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6866—Interferon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/555—Interferons [IFN]
- G01N2333/57—IFN-gamma
Abstract
The present invention relates to the detection method of interferon biological activity, detects interferon activity as challenge viruses as cell, vesicular stomatitis virus (VSV) are determined using porcine kidney cell (IBRS 2).Observation that what the inventive method was present when can detect pig IFN γ potency in effectively solving existing method when being used for pig IFN γ titrations be difficult, subjective error are big, sensitivity is poor, because activity cannot be detected caused by species specificity the problems such as.But the inventive method is not limited to the titration of pig IFN γ, also suitable for other pig interferon titrations, such as pig IFN α；Also it is applied to non-II type human interferon, such as people IFN α simultaneously.
Description
Technical field
The invention belongs to immunology and microbiological art, are related to a kind of biologic activity detection method of biological product,
Specifically related to a kind of detection method of interferon biological activity.
Background technology
China has long pig history, be the world pig big country, pig year livestock on hand about 1,000,000,000.With pig industry scale
Change and intensive development, swine diseasess are particularly the viral disease of pig and bring serious threat to pig industry, in recent years pig circular ring virus 2
The appearance of the virosiss such as malicious (PCV2) disease and Porcine reproductive and respiratory syndrome (PRRS), what especially these virus infection were caused are exempted from
Epidemic disease suppresses, and causes swinery that the mixed infection of multiple pathogens occurs, and the mortality rate of pig reaches 8%-10% or higher, annual therefore to make
Into huge economic loss.Although China is inoculated with various swine diseasess vaccines at present, due to virus variation soon, new virus and variation disease
Poison continuously emerges, and the control effect of vaccine is barely satisfactory.
Interferon (Interferon, IFN) is the multi-functional secreted protein that a class animal body is produced, with disease-resistant
The biological functions such as poison, antitumor, immunomodulating.Therefore pig interferon is respectively provided with anti-to the infectious disease virus with significant threat
Imperial and inhibitory action.A series of in vitro testses show：Pig IFN-α can effectively suppress the vigor of foot and mouth disease viruses；Infected pigs are infected
Property marcy agent (TGEV) piglet, the pig IFN-α of anti-TGEV can be produced rapidly in gut epithelium tissue；Use porcine IFN γ
The pig macrophage of infection porcine reproductive and respiratory syndrome virus (PRRSV) is processed, PRRSV can be suppressed to breed；Use at IFN-γ
After reason MARC-145 cells, PRRSV street strains and cell adaptation strain propagation can be suppressed；Porcine IFN γ can suppress infection to infect
Virus replication in the pig epithelial cell and pulmonary macrophage of property marcy agent；Porcine IFN γ may also suppress infection swine fever virus
Virus replication of mononuclear cell and pulmonary macrophage etc..When preventing and treating viral disease using interferon, need to utilize genetic engineering
Technology production restructuring pig interferon, sets up the system of efficiently production pig interferon, and which can just pushed away in large-scale cultivation production
It is wide to use, produce good diseases prevention and treatment effect.
At present, the pig interferon for uniquely obtaining Ministry of Agriculture's " novel chiral synthon certificate " and " production approval number " is " pig leucocyte
Interferon ", it is the activated protein mixture for stimulating cell generation by virus, based on pig IFN-α.In its quality standard still
The Determination of biological activity method of human interferon is so continued to use, i.e., determination of activity is carried out using the WISH-VSV systems of pharmacopeia.Specifically
For：Using human amniotic cell (WISH) as cell is determined, vesicular stomatitis virus (VSV) are used as challenge viruses；Human interferon
After acting on WISH cell, can inducing cell set up antiviral state, power and the human interferon of cell anti-virus ability
Active size is directly proportional, and can reflect the active size of human interferon according to cytopathic degree.
Inventor not finding obvious problem using above-mentioned WISH-VSV system detectios pig IFN-α, but with the system pair
Porcine IFN γ carries out determination of activity and is just no longer suitable for.Show as being difficult observation, subjective error greatly, sensitivity is poor, actual potency is inclined
It is low.Though additionally, there is document report, porcine kidney cell (PK-15)-vesicular stomatitis virus (VSV) system can be imitated as pig interferon
Valency measurement system, but there is also necessarily not to observe, it is difficult to embody the problem of the true potency of interferon.
The content of the invention
Present invention seek to address that being difficult of existing when porcine IFN γ is detected of existing interferon titer detection method observe, it is main
See error big, sensitivity is poor, because activity cannot be detected caused by species specificity the problems such as.
Analyze and literature research through lot of experiments, inventor has found that why there are the problems referred to above is disturbed due to II type
The biological effect of element such as IFN-γ has the species specificity of height, i.e. people's IFN-γ does not have live biology zooblast
Property, porcine IFN γ is to people's cell (such as WISH) also no biologic activity；And interferon type Ⅰ such as IFN-α is without species specificity.Base
In this, the people's cell (WISH) in existing detection system is replaced with pig cell by inventor, is made using porcine kidney cell (IBRS-2)
To determine cell, vesicular stomatitis virus (VSV) detect interferon activity as challenge viruses.
The detection method of above-mentioned interferon biological activity, specifically includes following steps：
(1), cultured IBRS-2 cell monolayers are diluted to cell density 2-3 × 10 in advance5The cell suspension of individual/mL,
Culture；
, etc. (2), after cell attachment, interferon is carried out into 4 times of gradient dilutions, 12 gradients are done；Interferon one after dilution
Secondary to add 96 adherent porocyte plates to carry out changing liquid, 100 μ L/ holes, culture are long to uniform monolayers cell；
(3) supernatant, is abandoned, in every hole, adds VSV virus liquids, cell control well to add maintaining liquid；Culture about 24h；
(4), observation of cell pathological changes, count each dilution factor CPE holes and without CPE hole counts；
(5), pig interferon potency is calculated by Reed-Muench Liang Shi methods.
Said method can be deposited during effectively solving existing method detection porcine IFN γ potency when being used for porcine IFN γ titration
Problem, but be not limited to the titration of porcine IFN γ, also suitable for other pig interferon titrations, such as pig IFN-
α；Also it is applied to non-II type human interferon, such as humanIFN-α simultaneously, but is not suitable for II type human interferon such as people's IFN-γ, this
It is that species specificity is not had based on interferon type Ⅰ, and interferon type Ⅱ has the species specificity of height.
Jing comparative study of the present invention finds, detecting systems of the IBRS-2 with reference to VSV, cytopathy significantly easily observation, can be most
Big degree reduces the error that subjective judgement brings；The pig interferon biologic activity of detection is high, sensitivity is strong, can more reflect that pig does
Disturb the anti-swine diseasess cytotoxic activity of reality of element；The impact of species specificity is eliminated, the anti-swine diseasess poison of reality of true reflection porcine IFN γ is living
Property.Therefore, compared with common WISH-VSV interferon detecting system and PK-15-VSV interferon detecting systems, use
IBRS-2-VSV systems carry out the measure of pig interferon biologic activity, testing result more science, true, effectively, based on this
The activity determination method of system is a kind of method for being more suitable for pig interferon Determination of biological activity.
Description of the drawings
Fig. 1：The cytopathy of IBRS-2-VSV system measurements pig interferon activity is observed, and left figure is：Cell control well, it is right
Figure is：Virus control wells.
Fig. 2：The cytopathy of PK-15-VSV system measurements pig interferon activity is observed, and left figure is：Cell control well, it is right
Figure is：Virus control wells.
Fig. 3：The cytopathy of WISH-VSV system measurements pig interferon activity is observed, and left figure is：Cell control well, right figure
For：Virus control wells.
Specific embodiment
Instrument that the following example is used, material information are as follows：
First, instrument：
| Title | Model | Manufacturer |
| Biohazard Safety Equipment | BSC-1300ⅡA2 | Su Jing is safe and sound |
| CO2Incubator | MCO-20AIC | Sanyo |
| Biological microscope | IX73 | Olympus |
| Hitachi's centrifuge | CF5RE | Hitachi |
| Refrigerator | SC-390 | Haier |
| Autoclave | SX-700 | Tomy |
| Eddy blending machine | IP40 | IKA |
2nd, reagent/reagent：
3rd, material：
| Title | Source |
| 1 age in days SPF hatching egg | Beijing Cimmeria dimension is logical |
| IBRS-2 | Preclinical medicine cell centre |
| WISH | Preclinical medicine cell centre |
| PK-15 | China Veterinery Drug Inspection Office |
| VSV | Beijing animal and veterinary institute |
The culture of 1 VSV of embodiment and malicious valency titration
The embodiment is only used for illustrating, and is not used as limiting the present invention, all conventional VSV cultures and poison valency measures
Method is used equally to the present invention.
(1), the hatching of Embryo Gallus domesticus：
1st, temperature：Most suitable incubation temperature is 37.8 DEG C.
2nd, humidity：Generally 40%～70%.
3rd, place hatching egg should greatly head-up, it is little upside down, it is impossible to keep flat, little head-up can not hatch.Check and approve couveuse
Temperature and humidity, record puts the date and hour of egg.Hatching process is both it is noted that temperature, humidity, will also carry out ventilation and turn over
Egg work (is typically turned over 5 times daily；Egg-turning angle is bent forward and backward each 45 degree with horizontal level and is advisable；During egg-turning action it is light, steady,
Slowly).
4th, transillumination is carried out per daily candler, rejects dead germ.
(2), CEO is for fibroblastic preparation：
1st, instar chicken embryo on the 9th 1 is taken, and chick embryo air sac exochorion is wiped respectively with iodine tincture and cotton ball soaked in alcohol.
1. and 2. 2, two sterilized petri dishes are taken out, and labelling, it is stand-by.
3rd, sterile working is distinguished in RPMI-1640 and Hank ' s liquid and penicillin and streptomycin are added with 1% amount, suction out
10～15mlLHank ' s liquid is to standby in sterilized petri dishes.
4th, big tweezers are rapped at the top of chorion and then carefully remove air chamber exochorion.
5th, carefully tear egg membrane after disinfecting forceps, two people work in coordination to take out Embryo Gallus domesticus and be placed in and fill the plate of Hank ' s liquid 1.
In, carefully decaptitate, internal organs, pawl and mucus and blood cell, be placed in after washing in filling the plate of Hank ' s liquid 2., then fully wash.
6th, take out the piece of tissue cleaned to be placed in big penicillin bottle, be cut into about in flame vicinity Sterile ophthalmic shears
0.5～1mm3Fritter (about 250 times), now volume about 0.5ml；Hank s ' liquid is used to wash twice.
7th, 1% trypsin of 1mL is added, use appropriate Hank ' s liquid adjustment its working concentration to be made for 0.25%, 5.6%
NaHCO3Adjust pH to about 7.3～8.0 (naked eyes are seen as flesh pink, the drop of 1mL suction pipes about 3) and cover bottle stopper, finish writing labelling.
8th, above-mentioned cell bottle is placed in 37 DEG C of water-baths, starts timing, about 15～30min is slowly shaken up therebetween with abundant
Digestion.Digesting the mark for stopping is：Gently rock rear cell to hang, in cloud, assemble agglomerating quickly, show cell viability
It is good.
9th, after digestion terminates, take out, slightly precipitate, slowly suction out supernatant, with carefully washing 2～3 times of Hank ' s liquid, Ke Yijin
Amount reduces tryptic residual quantity, is subsequently adding RPMI-1640 liquid about 5mL, is fully blown and beaten with little head straw, makes cell abundant
Dispersion.Suct clear；Repetitive operation 3～4 times, collects cell conditioned medium.
10th, tetra- layers of sterile gauzes of Jing or 200 mesh rustless steel steel mesh filtering and collecting filter liquids, cell counting, and adjust cell
Density about 1 × 106/mL；
11st, the above-mentioned cell suspension for preparing is evenly distributed in T25 cell bottles, 7mL/ bottles are stoppered screw socket plug, note
Growth faces down, and labelling, including Cell Name, inoculated and cultured time, name etc. are carried out on non-aufwuchsplate.
12nd, cell unification is placed in 37 DEG C that this room is specified, 5%CO2Cultivate in incubator.
(3), malicious valency titration：
1st, after chick embryo fibroblast culture to monolayer, 1:100 dilution VSV kind venom, are diluted with maintaining liquid 5%FBS.
Each 25cm2Cell bottle adds the virus liquid after 7mL dilutions.
2nd, 37 DEG C are put, 5%CO2In incubator cultivate, appear above cytopathy to 90%, seal, by cell bottle freeze in-
20℃。
3rd, naturally to thaw is taken out after freezing, is shaken in course of defrosting for several times, so that cell comes off from bottle wall safely.
4th, collect virus liquid in centrifuge tube, 3000rpm, 4 DEG C, 10min, remove cell debriss, collect supernatant (i.e. virus
Liquid).Subpackage cryopreservation is standby.
5th, about 2-3 × 10 are diluted to growth-promoting media after IBRS-2/WISH/PK-15 cell dissociations5Individual/96 orifice plate of mL bed boards,
100uL/ holes, 37 DEG C, 5%CO2Cultivate to cell and grow up to monolayer.
6th, virus liquid is made into continuous 10 times of dilutions in centrifuge tube, from 10-1～10-10。
7th, by the poison disease vaccination for having diluted in 96 porocyte plates, each dilution factor is inoculated with 8 holes, 100uL/ holes.
8th, two tandem of normal cell controls is set, that is, is replaced by 5%FBS culture fluid (diluent).
9th, observe day by day and record result, about observe 3～5d.Lesion degree " ++ " and the above are judged to CPE holes, are otherwise judged to
Without CPE holes.Lesion degree is shown with following symbol table："-" represents cell without pathological changes；"+" represents that less than 25% cell has pathological changes；
" ++ " represents that the cell of 25%-50% has pathological changes；" +++ " represents that the cell of 50%-75% has pathological changes；" ++++" represent 75%-
100% cell has pathological changes.
10th, TCID is calculated by Reed-Muench methods50
It is attached：Reed-Muench Liang Shi methods (it is listed in Table without CPE hole counts, occur CPE hole counts numerical value be only this example explanation
Random value, not test concrete numerical value)
CPE hole counts during the specific dilution factor of virus liquid：It is specific that the CPE hole counts occurred from minimum dilution factor are added to this one by one
CPE hole counts during dilution factor, such as dilution factor are 10-1CPE hole counts be dilution factor be 10-6、10-5、10-4、10-3、10-2、10-1When
CPE hole counts add up one by one, i.e. 0+1+3+7+8+8=27；Dilution factor is 10-2CPE hole counts be dilution factor be 10-6、10-5、
10-4、10-3、10-2When CPE hole counts add up one by one, 0+1+3+7+8=19.
During the specific dilution factor of virus liquid without CPE hole counts：This is one by one added to without CPE hole counts from highest dilution specific dilute
During degree of releasing without CPE hole counts, such as dilution factor is 10-2Without CPE hole counts be dilution factor be 10-1、10-2When it is tired without CPE hole counts
Plus, i.e. 0+0=0；If dilution factor is 10-3Without CPE hole counts be dilution factor be 10-1、10-2、10-3When it is tired without CPE hole counts
Plus, i.e. 0+0+1=1.
CPE：Cytopathic effect cytopathys
Distance proportion=(higher than the percent -50% of 50% pathological changes rate)/(higher than the percent-be less than of 50% pathological changes rate
The percent of 50% pathological changes rate)=(91.6-50)/(91.6-40)=0.8
lgTCID50Difference between=distance proportion × dilution factor logarithm+higher than 50% pathological changes rate dilution logarithm=
0.8 × (- 1)+(- 3)=- 3.8
TCID50=10-3.8/0.1ml.Implication：By the viral dilution 103.8100 μ L of inoculation occur can 50% cell again
Pathological changes.
2 pig interferon sample of embodiment Determination of biological activity in different cell line detecting systems：
(1), method
1st, advance cultured IBRS-2/WISH/PK-15 cell monolayers, with 10% after being digested with 0.05%Trypsin
FBS MEM/DMEM (IBRS-2/WISH MEM culture medium, PK-15 DMEM culture medium) are diluted to suitable cell density (about
2-3×105Individual/mL) cell suspension, paving 96 porocyte plates 100 μ L/ holes, 37 DEG C of 5%CO2Culture 4h or so, treats which is adherent.
2nd, etc., after cell attachment, porcine interferon alpha/γ samples is carried out into 4 times of gradient dilutions first, 12 gradients are done, each ladder
Degree repeats 3 holes, dilution 7%FBS MEM/DMEM culture medium.Pig interferon sample after dilution is once added adherent
96 porocyte plates carry out changing liquid, 100 μ L/ holes, cell controls and virus control wells 100 μ L diluents of each addition, 37 DEG C of 5%CO2
Incubated overnight 18-24h is long to uniform monolayers cell.
3rd, supernatant is abandoned, 100 μ L VSV virus liquids (about 1-2 × 10 is added in every hole2TCID50/ hole, a certain cell detection
System carries out specific virus liquid dilution according to the VSV poison valencys determined on corresponding cell), cell control well adds 100 μ L to maintain
Liquid (3%FBS MEM/DMEM culture medium), VSV dilution 3%FBS MEM/DMEM culture medium.Add rearmounted 37 DEG C of 5%CO2Training
Support about 24h.
4th, about 24h after counteracting toxic substances, the observation of cell pathological changes under inverted microscope count each dilution factor CPE holes and without CPE holes
Number.When there are pathological changes (" ++++") in more than 75% cell of virus control wells, result of determination.Lesion degree " ++ " and the above are judged to
CPE holes, are otherwise judged to without CPE holes.Lesion degree is shown with following symbol table："-" represents cell without pathological changes；"+" represent 25% with
Under cell have pathological changes；" ++ " represents that the cell of 25%-50% has pathological changes；" +++ " represents that the cell of 50%-75% has pathological changes；
" ++++" represent that the cell of 75%-100% there are pathological changes.
5th, pig interferon potency is calculated by Reed-Muench Liang Shi methods, pig interferon active unit is represented with U.Can protection
Half cell be immune against attacks viral subversive pig interferon highest dilution inverse, as pig interferon potency.
Citing：It is (listed in Table without CPE hole counts, appearance that Reed-Muench Liang Shi methods calculate testing sample pig interferon potency
The numerical value of CPE hole counts is only the random value of this example explanation, not tests concrete numerical value)
Distance is than=(higher than the percent -50% of 50% pathological changes rate)/(higher than the percent-be less than of 50% pathological changes rate
The percent of 50% pathological changes rate)=(75-50)/(75-25)=0.5
Log4(X)=distance is than the difference between × dilution factor logarithm+higher than dilution logarithm=0.5 of 50% pathological changes rate
× (- 1)+(- 6)=- 6.5
Highest dilution (X)=4-6.5.Implication：Pig interferon dilution factor is to 4-6.5When, half cell can be protected to be immune against attacks
Viral subversive.The potency [pig interferon active unit (U) number] of the dilution 1/X reciprocal, as 0.1mL testing samples.This
In example, the potency of testing sample is 46.5× 10=8.19 × 104U/mL。
(2), measurement result compares：
1st, cytopathy significance is compared with easily observation property
From in terms of cytopathic significance and easily observation property comparative result, IBRS-2 ＞ PK-15 >=K-15.As shown in Figure 1：
The cytopathy of IBRS-2 significantly, cell circle contracting, and can form significant cytopathy plaque, the cell controls with uniform ground
Easily distinguish；As shown in Figure 2：The cytopathy of PK-15 is more significant, and cell shrinkage forms dead cell agglomeration and gathers, but cell controls
Also have the aggregation of a certain degree of living cells, therefore when there is the critical hole of CPE, the aggregation of the pathological changes that are not easily distinguishable dead cell with it is normal
Living cells are assembled, and easily bring subjective error；As shown in Figure 3：The cytopathy of WISH cell is slight, only forms less cell
Gather speckle, be difficult to distinguish with the Normal aggregation of living cells when when there is the critical hole of CPE, CPE aggregation speckles are less, easily
Bring subjective error.Therefore, using IBRS-2 system detectios, cytopathy is significantly easy to difference, and subjective error is little, as a result trueer
It is real reliable.
2nd, sample biologic activity results contrast
From in terms of the potency comparative result that different system (different cells, be VSV viral) determines same porcine interferon alpha sample
(table 1), IBRS-2 ＞ PK-15 ＞ WISH, illustrates that IBRS-2 cells are most strong to the sensitivity of porcine interferon alpha, in theory potency inspection
Result is surveyed closest to actual potency.Porcine kidney cell line IBRS-2 is more suitable for the Determination of biological activity of porcine interferon alpha.
1. different system of table detect the potency ratio of same porcine interferon alpha sample compared with
Determine in terms of the potency comparative result of same Porcine interferon-gamma sample (table 2) from different system, IBRS-2 ＞ PK-15,
WISH cell does not detect Porcine interferon-gamma potency.IBRS-2 cells are higher to the sensitivity of Porcine interferon-gamma, in theory potency
Closer to actual potency, for comparing PK-15, IBRS-2 is more suitable for the bioactivity of Porcine interferon-gamma to testing result.
2. different system of table detect the potency ratio of same Porcine interferon-gamma sample compared with
Different detecting systems detect human interferon and pig interferon sample activity respectively, the results are shown in Table 3.Human interferon sample
Activity is measured in IBRS-2-VSV systems compare the activity that WISH-VSV systems measure and only improve 16 times, but pig interferon sample
Activity is measured in IBRS-2-VSV systems and compares 90 times of the activity raising that WISH-VSV systems are measured, pig interferon sample exists
The active increase rate measured in IBRS-2-VSV systems is significantly higher than human interferon sample, shows pig interferon IBRS-2-
The sensitivity of VSV system detectios is higher, and bioactivity result is closer to its actual potency in theory.
The different detecting systems of table 3. detect respectively the potency ratio of human interferon and pig interferon sample compared with
3rd, species specificity compares
Determine in terms of the potency comparative result of same Porcine interferon-gamma sample (table 2) from different system, IBRS-2 ＞ PK-15,
WISH cell does not detect Porcine interferon-gamma potency.As interferon gamma has strict species specificity, so people's cell pair
Pig interferon is insensitive, does not measure the potency of Porcine interferon-gamma with WISH cell inspection.The result shows, biological for pig interferon
Learn the measure of activity, the especially determination of activity of Porcine interferon-gamma, it is impossible to continue to use the activity monitor system (WISH- of human interferon
VSV).Equally be pig cell, because cellular sensitivity it is stronger, bioactivity result closer to its actual potency, so comparing PK-15
For, IBRS-2 is more suitable for the bioactivity of Porcine interferon-gamma.As can be seen here, IBRS-2-VSV detecting systems are equally also suitable
In non-II type human interferon, the such as titration of humanIFN-α.
In view of the degree of recognition of interferon activity measurement system, in order to keep consistent with human interferon determination of activity system
Property and comparability, so using VSV as interferon activity determine with virus it is more suitable；And VSV equally energy infected pigs, it is
Cause the important virus of swine pox, therefore VSV is more suitable for the measure of pig interferon activity.
In sum, whether from cytopathic significance and easily observation property, cellular sensitivity, or species specificity
Angle considers that the system that porcine kidney cell line IBRS-2 is combined with VSV is all more suitable for the biologic activity detection of pig interferon.
Claims (5)
1. the detection method of interferon biological activity, it is characterised in that using porcine kidney cell IBRS-2 as determining cell, water
Bubble property Stomatoviruses VSV detects interferon activity as challenge viruses.
2. the detection method of the interferon biological activity described in claim 1, it is characterised in that the interferon is pig IFN-
α, pig IFN-β, porcine IFN γ.
3. the detection method of the interferon biological activity described in claim 2, it is characterised in that the interferon is pig IFN-
γ。
4. the detection method of the interferon biological activity described in claim 1, it is characterised in that the interferon is non-II type
Human interferon.
5. the detection method of the interferon biological activity described in claim 1, comprises the following steps：
(1), cultured IBRS-2 cell monolayers are diluted to cell density 2-3 × 10 in advance5The cell suspension of individual/mL, culture；
, etc. (2), after cell attachment, interferon is carried out into 4 times of gradient dilutions, 12 gradients are done；Interferon after dilution once adds
Entering 96 adherent porocyte plates carries out changing liquid, and 100 μ L/ holes, culture are long to uniform monolayers cell；
(3) supernatant, is abandoned, in every hole, adds VSV virus liquids, cell control well to add maintaining liquid；Culture about 24h；
(4), observation of cell pathological changes, count each dilution factor CPE holes and without CPE hole counts；
(5), pig interferon potency is calculated by Reed-Muench Liang Shi methods.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114164304A (en) * | 2021-12-28 | 2022-03-11 | 天津瑞普生物技术股份有限公司 | Method for detecting activity of cat interferon |
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