CN106706582B - A kind of method of high-throughput quickly detection hantaan virus neutralize antibody titers - Google Patents
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Abstract
The invention discloses a kind of methods of high-throughput quickly detection hantaan virus neutralize antibody titers.The present invention is characterized in that using In-cell Western (ICW) method simplicity, intuitively detecting the cell infected by HTNV.This method detection sensitivity high (destination protein of detectable 5g), period short (72 hours), result interpretation be objective, favorable repeatability, high throughput, method can standardize, convenient for laboratory monitoring popularization, it can especially be directed to cell-free its appeal of pathological effect viral diagnosis.The present invention discloses the above method simultaneously and measures (TCID in HTNV appeal50), HTNV neutralize antibody titers measurement, anti-HTNV molecule screening and identification, people's HTNV vaccine immunity protectiveness evaluation etc. application.
Description
Technical field
The invention belongs to hantaan virus detection technique fields, are related to a kind of high-throughput quickly detection hantaan virus neutralizing antibody
The method of potency.
Background technique
Hantaan virus (Hantaan virus, HTNV) is under the jurisdiction of bunyaviridae (Bunyaviridae) Hantaan virus
Belong to (Hantavirus), be sub-thread minus-stranded rna virus, genome is made of tri- segments of L, M, S, be separately encoded viral RNA according to
Bad RNA polymerase (RdRp), envelope glycoprotein (GP) and nucleocapsid protein (NP).The host animal and the infection sources of the virus are equal
For rodent, is mainly polluted by animal excrements and be broadcast to via approach such as respiratory tract, alimentary canal or the skins of breakage
The mankind cause a kind of disease of natural focus, i.e. hemorrhagic fever with renal syndrome (Hemorrhagic fever with renal
Syndrome, HFRS).The disease clinical manifestation is fever, bleeding and acute renal function injury, morbidity people wide with Epidemic Scope
The features such as number is more, case fatality rate is high.
China is the country of HFRS epidemic situation most serious in the world, and annual number of the infected accounts for the world and always falls ill 90% or more number,
The death rate is 0.1-15%.According to the literature, between 1950-2007,1,557,622 HFRS occur altogether for China, wherein dead
46,427, the death rate is about 3%；It counting according to another Chinese Center for Disease Control and Prevention, China occurs 10 whole year altogether within 2015,
812 HFRS, dead 63.Since the host of HTNV is extensive, route of transmission is more, and clinically still shortage specifically has so far
The therapeutic agent of effect, therefore its prevention and treatment task is very arduous.It is worth noting that, HTNV or the world " forbid biological weapons public
Biological warfare agent listed by about " constitutes potential threaten to future military activity.
The most fundamental means that HTNV preventative vaccine is considered as HFRS caused by preventing HTNV infection is developed in exploitation, and pre-
The key of anti-property vaccine success or not is that can it induce effective humoral immunity.High titre HTNV neutralizing antibody is effective against
The infection of external HTNV, therefore establish and stablize, effective vaccine evaluation system, the protecting effect of detection vaccine immunity reaction is right
It is of great significance in the development and Quality Control of vaccine.
Neutralization test (neutralization test) refers to that virus in conjunction with corresponding antibody, makes in antibody with virus
It loses the appeal to sensitive cells, which can detect antibody from serum to be checked, or disease is detected from sick sample sheet
Poison can also measure antiviral serum neutralization titer thus diagnosis of viral infectious disease, or to new isolated viral carry out identification and
Parting.Its principle is the antiviral immunity serum (neutralizing antibody) and virus function of specificity, is able to suppress virus to sensitive thin
The absorption of born of the same parents is penetrated and is shelled, to prevent the breeding of virus, loses infection ability.It is worth noting that, the reaction is not
It is showed only as the aspect of matter, i.e., a kind of virus can only be neutralized by corresponding immune serum；And the aspect for the amount of being also manifested in, i.e., in
With the appeal of a certain amount of virus, the antibody of certain potency is needed.
Detecting virucidin's potency by neutralization test is to evaluate the common method of vaccine protecting effect.In common
It include simple qualitative method, the viral method of fixed sample dilution, fixed virus diluted sample this law and plaque-reduction method with test.Plaque subtracts
Few method is " goldstandard ", i.e., with the known titer viral suspension (100TCID for the patients serum and equivalent being serially diluted50) mixing,
Inoculation sensitive cells is cultivated after acting on a period of time at room temperature, half cell culture well can be protected not generate cytopathy
Become the serum highest dilution of (CPE) as endpoint titers.
Summary of the invention
Present invention solves the problem in that a kind of method of high-throughput quickly detection hantaan virus neutralize antibody titers is provided,
The virus nucleocapsid albumen in HTNV infection cell is detected by In-cell Western (ICW) technology, and based on this measurement
HTNV titre, the advantage for having survey high sensitivity, period short.
The present invention is to be achieved through the following technical solutions:
A kind of method of high-throughput quickly detection hantaan virus neutralize antibody titers, including following operation:
1) A549 cell is uniformly inoculated in ICW microwell plate, is cultivated to 80~90% cell confluency degree；
2) it when cell confluency degree reaches 70~80% in step 1), is mixed using the method for fixed virus diluted sample
Close the preparation of liquid: by sample to be tested etc. than gradient dilution, the sample to be tested of each dilution ratio is mixed with isometric HTNV respectively
It closes, is incubated for 2h in 37 DEG C of incubators, is during which gently mixed by inversion for several times；
3) culture solution abandoned in step 1) microwell plate is inhaled, every hole is added DPBS and washs 1-2 times, then will incubate in step (2)
Microwell plate is added in the mixed liquor for educating completion, and each dilution gradient sets multiple repeating samples, and Loading sequence according to carrying out from low to high；Together
When viral negative control group, Isotype control group, virus positive control group, blank control group are set；
4) microwell plate in step (3) is put into 37 DEG C of incubators and is incubated for after 2.5h and inhale abandoning mixed liquor, washing 1-2 is added in every hole
It is secondary, then the DMEM containing 2%FBS is added to every hole；
5) microwell plate in step 4) is put into taking-up after 37 DEG C of incubators are incubated for 2d and carries out ICW detection；
6) ICW detects the NP in HTNV infection cell: inhaling the culture solution abandoned in microwell plate first, PBS washing is added；Then
The fixation buffer that 4 DEG C of pre-coolings are added into every hole is fixed, and exempts from shake and is incubated at room temperature；It inhales and abandons fixed buffering
Liquid elutes cell with permeable membrane buffer；Inhale abandon permeable membrane buffer, along tube wall to every hole be added blockade liquid, on shaking table slowly on
Shake is incubated at room temperature；It inhales to abandon and blockades liquid, primary antibody is added to every hole along tube wall, slowly shakes incubation at room temperature；It inhales and abandons primary antibody, add
Enter washing buffer, washing is multiple；It inhales and abandons washing buffer, secondary antibody is added to every hole along tube wall, is protected from light slowly shakes at room temperature,
It is incubated for；Washing buffer is added, is protected from light slowly shakes at room temperature, washing is multiple；After elution terminates, eluent is completely removed；
Finally, the microwell plate after elution is scanned using the double-colored fluoroscopic imaging systems of near-infrared, it is logical using 700nm and 800nm two
Road scans simultaneously, to obtain the IR fluorescence intensity of HTNV NP；
7) HTNV neutralize antibody titers are calculated:
Every hole is obtained in the IR fluorescence intensity in the channel 700nm and 800nm, it is infrared glimmering then to calculate HTNV NP in every hole
Luminous intensity compares NIR, NIR=NP fluorescent value/internal reference protein fluorescence value；It recycles Karber method to calculate and obtains sample to be tested
Dilution factor, i.e. neutralize antibody titers.
It is described to calculate sample to be tested dilution factor with Karber method specifically:
1) it calculates each viral dilution positive number of perforations P and negative hole number N, the discrimination standard in positive hole is, if the hole
Multiplicity of infection >=1.5 HTNV, then otherwise it is feminine gender that the hole, which is the positive,；
Wherein, NIR background values is the arithmetic mean of instantaneous value of four hole NIR of background group, the positive controls HTNV sense of microwell plate
Dye coefficient >=1.5 are otherwise to repeat this hardened fruit using this group of testing result；
2) it calculates and neutralizes the sum of ratio, neutralization ratio=negative hole/multiple holes sum of each dilution gradient
Neutralize the sum of ratio=all dilution gradients the sum of infection ratio
3) calculate dilution group away from, by gradient dilution ratiometric conversion at 10 index, using the tolerance of index equal-difference arrangement as
Dilution group away from；
4) dilution factor is calculated, measures LgPD50 (half protective number) and neutralization titer LgPD50=respectively with Karber method
L+D × (S-0.5)；
Neutralization titer=PD50；Wherein, L is the logarithm of minimum dilution；D be dilution group away from；S is to neutralize the sum of ratio.
The primary antibody includes that anti-HTNV NP mouse monoclonal antibody and anti-β-actin rabbit are mostly anti-；The secondary antibody includes Goat
anti-rabbit IRDyeTM680 and Goat anti-mouse IRDyeTM800CW, respectively to detect 700nm and
The fluorescence intensity in the channel 800nm.
The progress ICW detection in second day after A549 cell infection HTNV.
The infective dose of the A549 cell infection HTNV is MOI=0.1.
Compared with prior art, the invention has the following beneficial technical effects:
Present invention optimizes the optimum time of ICW detection HTNV infection cell, the dose,optimum of virus used, screenings
Antiviral protein antibody used in ICW, and standardized.The present invention can be used for measuring (the histocyte culture of HTNV titre
Median infective dose TCID50), detection HTNV neutralize antibody titers, screening and the anti-HTNV molecule of identification, appraiser's HTNV vaccine immunity
Protectiveness etc..In addition to HTNV, other coherent detections without CPE or without obvious CPE viral (such as Hepatitis C Virus) see also
The present invention is implemented, but antibody used should be depending on surveying virus.
The invention has the following advantages that 1, directly detection, easy to operate；2, detection cycle is short (72 hours)；3, sensitivity
Height, the surveyed fluorescence intensity of 5pg-100ng protein I CW are positively correlated with protein content；4, favorable repeatability；5, result interpretation is objective；
6, high-throughput；7, testing cost is low；8, method can standardize, and promote convenient for laboratory monitoring.
Detailed description of the invention
Fig. 1 is the selection for the optimum time that ICW detects HTNV infection cell；
Fig. 2 is the selection for the most suitable virus titer that ICW detects HTNV infection cell；
Fig. 3 is antiviral protein antibody used in screening ICW；
Fig. 4 is present invention detection HTNV titre and its comparison with classic ELISA method；
Fig. 5 is present invention detection patients serum's neutralize antibody titers and its comparison with classic ELISA method；
Fig. 6 is appraiser HTNV vaccine immunity protectiveness of the present invention and its comparison with classic ELISA method；
Fig. 7 is the HTNV neutralize antibody titers that the present invention detects and its comparison with classic ELISA method；
Fig. 8 is present invention screening and identifies anti-HTNV molecule.
Specific embodiment
Below with reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
HTNV infection does not generate apparent CPE, therefore is difficult to reduce testing inspection neutralize antibody titers with traditional plaque,
Meanwhile also the difficult generation by CPE measures HTNV virulence and titre.The method of evaluation HTNV neutralize antibody titers is mainly at present
Micro-CPE neutralization test neutralization test based on elisa technique, the disadvantages of the method are as follows cumbersome, length experimental period (about 11~
12 days), result show that neutralizing antibody that is not intuitive, being difficult to use in detection larger scale clinical sample is horizontal.Therefore, exploitation one is needed
Kind high sensitivity, specificity is good, the period is short and is able to achieve the high-throughput method detected.
In recent years, In-cell Western (ICW) technology gradually attracts attention and applies, basic principle be immunized it is glimmering
Light technology is similar, accurate using the double-colored fluoroscopic imaging systems of near-infrared, quick, special, high-throughput to detect certain intracellular hatching egg
White content.
In view of this, the present invention provides a kind of high-flux detection method of HTNV neutralize antibody titers.This method passes through ICW
Technology detects the virus protein (i.e. nucleocapsid protein NP) of HTNV in infection cell, and measurement E6 amplification is viral first based on this
Titre, HTNV neutralize antibody titers are detected by the method for fixed virus diluted sample later.This method is easy to operate, detects
Period short (about 3 days), visual result, interpretation be objective, favorable repeatability, high throughput, method can standardize, and pushes away convenient for laboratory monitoring
Extensively.
The present invention provides a kind of high-throughput quickly detection HTNV neutralize antibody titers method, includes the following steps:
Step 1: according to the cell confluency degree of standard cell incubation step culture A549 cell to 80~90%.A549 is thin
Born of the same parents are uniformly inoculated in ICW Special micro porous plate (specifically by taking 96 orifice plates as an example), and inoculating cell number is (2-4) × 104/ hole, inoculation
Hole count=number of awaiting test sample × dilution number × multiple holes number (this specification is by taking 4 multiple holes as an example).Note: to keep cell adherent more
Step up it is close can handle microwell plate with poly-D-lysine or collagen in advance, then inoculate cell.
Step 2: when cell confluency degree reaches 70~80% in step 1, using fixed virus diluted sample method into
Row microneutralization test, it may be assumed that
Step (1) is by sample to be tested etc. than gradient dilution, and e.g., serum sample is according to 1:10,1:20,1:40,1:80,1:
160,1:320 ratios are diluted using PBS, and each dilution ratio is arranged 4 biology and repeats, and each dilution ratio finally dilutes body
Product is 400ul.As shown in table 1.
Table 1: sample to be tested dilution process
Step (2) by the 400ul sample to be tested of each dilution ratio in step (1) respectively with 400ul (100TCID50)
HTNV mixing, is incubated for 2h in 37 DEG C of incubators, is during which gently mixed by inversion for several times every 0.5h.
Step (3) inhales the culture solution abandoned in step 1 microwell plate, and every hole is added 200ul DPBS and washs 1-2 times, by step
(2) it is incubated for the mixed liquor completed in, microwell plate is added, mixed liquor 200ul is added in every hole, and each dilution gradient is equipped with 4 biology
It repeats, Loading sequence is according to 1:10 to 1:320.Viral negative control group (hole DPBS 200ul/ is added in every hole), homotype pair are set
According to group (sample to be tested stoste 100ul+DPBS 100ul is added in every hole), (every hole is added virus positive control group
100TCID50HTNV 100ul+DPBS 100ul), blank control group (not spreading cell, any liquid is not added).Note: inhaling and abandon liquid
Body, filling operation can be used the volley of rifle fire to reduce the operating time, while the bottom of pipette tips contact microwell plate being avoided to prevent from scratching cell.
Every piece of microwell plate sample to be tested distribution situation such as Fig. 1 (for 96 orifice plates).
Microwell plate in step (3) is put into 37 DEG C of incubators and be incubated for after 2.5h by step (4) inhales abandoning mixed liquor, and every hole is added
200ul DPBS is washed 1-2 times, then the DMEM200ul containing 2%FBS is added to every hole.
Microwell plate in step (4) is put into taking-up after 37 DEG C of incubators are incubated for 2d and carries out ICW experiment by step (5).
Step 3:ICW detects the NP in HTNV infection cell, it may be assumed that
Step (1) --- washing: inhaling the culture solution abandoned in microwell plate, and every hole is added 200ul PBS and washs 2 times.Note: ICW
Owning " inhale and abandon " step in the process " gently can also overturn microwell plate to discard liquid in hole ".
Step (2) --- fixed: fixation buffer (4% paraformaldehyde) 100ul that 4 DEG C of pre-coolings are added into every hole is carried out
It is fixed, exempt from shake and is incubated for 20min at room temperature.When being fixed buffer, carefully it is added, is avoided along tube wall with pipettor
Separate cell.
Step (3) --- permeable membrane: inhaling and abandon fixed buffer, with the permeable membrane buffer hole 100ul/ (1%Triton X-100) point
3 elution cells, each 5min, to guarantee complete permeation cell.Note: elution carries out preferably on shaking table every time, slowly shakes
It moves, under room temperature 5min.It is sure not to allow Kong Biangan in elution process, rapidly joins permeable membrane buffer after operation every time.
Step (4) --- closing: inhaling and abandon permeable membrane buffer, and 150ul is added to every hole along tube wall and blockades liquid, delays on shaking table
Slow upper shake is incubated for 1.5h at room temperature.
Step (5) --- it is incubated for primary antibody:
It calculates each liquid volume: 1. calculating primary antibody total volume, primary antibody total volume=hole count × 50 to be measured (ul)；2. calculating institute
Need HTNV NP mouse monoclonal antibody (1A8 is prepared and is named as according to monoclonal antibody standard method, after purification concentration constant volume to 10ug/ml, 1:
3000 dilutions) volume, required 1A8 monoclonal antibody volume=primary antibody total volume/3000 (ul)；3. anti-β-actin rabbit needed for calculating is mostly anti-
(three hawk of Wuhan, CatalogNo:23660-1-AP, 1:300 dilution) volume, required anti-β-actin rabbit multispecific antibody product=primary antibody are total
Volume/500 (ul).4. calculating dilution volume=primary antibody total volume -1A8 monoclonal antibody volume-anti-β-actin rabbit multispecific antibody product；Institute
It is to blockade liquid described in (4) with dilution.
Above-mentioned dilution, HTNV NP mouse monoclonal antibody, anti-β-actin rabbit is mostly anti-uniformly mixed.It inhales to abandon and blockades liquid, along tube wall
50ul primary antibody is added to every hole.2h or 4 DEG C of incubation is slowly shaken at room temperature does not shake overnight incubation.Note: being incubated overnight
When, to prevent cell drying from can cover microwell plate with preservative film.
Step (6) --- washing: inhaling and abandon primary antibody, and washing buffer (0.1%Tween-20) is added to every hole along tube wall,
The hole 300ul/, 5min is slowly shaken at room temperature, wash 5 times or more.
Step (7) --- it is incubated for secondary antibody:
It calculates each liquid volume: 1. calculating secondary antibody total volume, primary antibody total volume=hole count × 50 to be measured (ul)；2. calculating institute
Need Goat anti-rabbit IRDyeTM 680 (LI-COR Cat#926-32221) volume, required Goat anti-rabbit
680 volumes of IRDyeTM=primary antibody total volume/5000 (ul)；3. Goat anti-mouse IRDyeTM 800CW needed for calculating
(LI-COR Cat#926-32210) volume, required Goat anti-mouse IRDyeTM 800CW volume=primary antibody total volume/
5000(ul).4. calculating dilution volume=primary antibody total volume-Goat anti-mouse IRDyeTM 800CW volume-Goat
680 volume volume of anti-rabbit IRDyeTM；Used diluent is to blockade liquid described in (4).
By above-mentioned dilution, Goat anti-mouse IRDyeTM 800CW, Goat anti-mouse IRDyeTM
800CW is uniformly mixed.It inhales and abandons washing buffer, secondary antibody 50ul is added to every hole along tube wall.It is protected from light and slowly shakes at room temperature, be incubated for
1h。
Step (8) --- washing: washing buffer 200ul is added to every hole along tube wall, is protected from light slowly shakes at room temperature, wash
De- 5min.Washing 5 times or more.
Step (9) --- sweep prepare before plate/class of sweeping after save: after elution terminates, eluent is completely removed out of hole.It turns over
It is bottom-up to turn microwell plate, is gently beaten with paper handkerchief.As a result it can be kept in dark place at room temperature at least one moon.
Step (10) --- upper machine sweeps plate: by microwell plate in step (9) using the double-colored fluorescence imaging system of Odyssey near-infrared
System is scanned, and sweeps film simultaneously using two channels 700nm and 800nm, selects mean quality, 169um resolution ratio, 3.0nm burnt
Away from, brightness 5.One Odyssey scanner can scan 8 piece of 96 orifice plate simultaneously.
Step 4: calculating HTNV neutralize antibody titers, i.e.,
Step (1) handled using Odyssey CLx Image Studio software sweep film as a result, obtain every hole in 700nm and
The IR fluorescence intensity in the channel 800nm.
Step (2) calculates HTNV NP IR fluorescence intensity in every hole and compares (Intensity Ratio, NIR), IR=
NP fluorescent value/internal reference protein fluorescence value
Step (3) is calculated with Karber method and obtains sample to be tested dilution factor, i.e. neutralize antibody titers.
It is described to calculate sample to be tested dilution factor with Karber method specifically:
1. step calculates each viral dilution positive number of perforations (P) and negative number of perforations (N), the discrimination standard in positive hole
For if hole multiplicity of infection >=1.5 HTNV, i.e.,
Then the hole is the positive, is otherwise feminine gender.
Note: NIRBackground valuesFor the arithmetic mean of instantaneous value of four hole NIR of background group.The positive controls HTNV infection system of microwell plate
>=1.5 (multiplicity of infection is obtained according to being fitted after comparing with standard ELISA assay) of number, then prove that the plate reaction system is good；It is no
It then needs to repeat this hardened fruit.
2. step calculates neutralizes the sum of ratio, i.e.,
Neutralization ratio=negative hole of each dilution gradient/multiple holes sum
Neutralize the sum of ratio=all dilution gradients the sum of infection ratio
3. step calculates dilution group away from that is,
By gradient dilution ratiometric conversion at 10 index (arithmetic progression) calculate dilution group away from,
Such as: 1:10=10-1, 1:20=10-1.3, 1:40=10-1.6, 1:80=10-1.9, 1:160=10-2.2, 1:320=
10-2.5, dilution group is away from=0.3.
4. step calculates dilution factor, i.e.,
LgPD50 (half protective number) and neutralization titer are measured respectively with Karber method
LgPD50=L+D × (S-0.5)；
Neutralization titer=PD50
Note: L: the logarithm of minimum dilution；D: group away from；S: the sum of ratio (being detailed in example 5) is neutralized.
Detailed calculation method is shown in example 5.
In some specific embodiments of the invention, in high-flux detection method " ICW Special micro porous plate " described in step 1
For the microwell plate for using clarinet wall transparent pipe bottom；It is sure not using white microwell plate, because the autofluorescence of white tube wall surface can produce
Raw significant noise signal.ICW Special micro porous plate includes the product of following several specifications:
96well format NuncTM(Part Number 161093,165305)；
96well format FalconTM(Part Number 353075,353948)；
384well format NuncTM(Part Number 164688,164730)；
384well format FalconTM(Part Number 353961,353962)。
In some specific embodiments of the invention, " sample to be tested " can described in step 2 in high-flux detection method
To be the serum of HFRS patient or HTNV vaccine immunity subject, HTNV neutralization/non-neutralization of R&D institution's preparation can also be
Property antibody.
In some specific embodiments of the invention, in high-flux detection method described in step 2-(2)
“(100TCID50) HTNV ", HTNV amplification method is as follows: taking the KM nest mouse in 1~2d age, puts to death male mouse, retain female mice.It is testing
Platform spreads one piece and is soaked with 3~5% lysol liquid gauzes, and left hand pinches two ear of suckling mouse and neck makes it lie on one's side on gauze, alcohol disinfecting eye
Ear line midpoint, right hand syringes are pierced under the slightly biased ear side endocranium in eye ear line midpoint, and 2~3mm of inserting needle injects HTNV
0.02~0.03ml of viral suspension, injection, which finishes, disinfects part in alcohol；Before the suckling mouse injected is put back to cage, need to use wine
Smart cotton balls wipes female rat nose, and to destroy its smell, in order to avoid female rat eats up suckling mouse, the suckling mouse after inoculation feeds still together with female rat
It supports.Day by day it observes, died is nonspecific death in 48 hours.Suckling mouse is put to death after virus inoculation 7 days and takes mouse brain, according to 1g
The ratio that 10ml RPMI 1640 is added in mouse brain grinds mouse brain, the filtering of 0.22um filter.The Vero E6 of T-75 culture is until converge
The HTNV virus (250ul/ bottles) for being inoculated with the amplification of mouse brain at 70% or so is spent, discards supernatant, is used instead containing 2%FBS after infecting 2h
Culture medium (DMEM)；T-75 continues culture backsight cell culture density passage in 3~4 days and is seeded to T-175 or T-300 culture bottle
In, the culture medium (DMEM) containing 2%FBS is continued to use, culture bottle cap is tightened；After continuing 7~10d of culture, culture bottle is frozen
It is to be processed to enter -80 DEG C of refrigerators；Three times (37 DEG C/- 80 DEG C) by the Vero E6 cell multigelation in T-175 or T-300 culture bottle
Supernatant is transferred to 50ml high speed centrifugation pipe, 13000g centrifugation × 10min afterwards.Supernatant can freeze spare into -80 DEG C of refrigerators.Gradient is dilute
The HTNV that E6 is expanded is released, the A549 cell in ICW Special micro porous plate is infected, is detected into the cell after infection 2 days using ICW method
Virus protein calculates TCID using Reed-Munch formula50。
In some specific embodiments of the invention, " blockading liquid " described in step 3-(4) is in high-flux detection method
LI-COR Odyssey blockades liquid or 3%BSA.
Step 3-(5) is described in some specific embodiments of the invention, in high-flux detection method also can be used
Anti- more than GAPDH rabbit mostly anti-(three hawk company of Wuhan, CatalogNo:10494-1-AP) or Lamin A rabbit (three hawk of Wuhan,
CatalogNo:10298-1-AP it) is used as internal reference, tests the dilution proportion according to immunofluorescence (IFA) in antibody specification
(GAPDH, 1:200；Lamin A, 1:500).
Method provided by the invention is based on HTNV being blocked to feel for people HTNV specificity neutralizing antibody in sample to be tested
It contaminates cell and establishes.Before underway and test, HTNV is expanded first with E6 cell and titrates virus drop using ICW
Degree.Gradient dilution sample to be tested and with isometric 100TCID50HTNV be incubated for postoperative infection A549 altogether, by ICW detect by HTNV
The cell of infection.If the HTNV specificity neutralizing antibody contained in test serum, can in conjunction with virus surface corresponding epitope, from
And blocking virus enters into the cell, leading to cell, infected or infection cell quantity does not reduce.It is negative by software interpretation, positive
Hole is calculated by Karber method according to testing result, available sample neutralizing antibody titers.
In a kind of method of high-throughput quickly detection hantaan virus neutralize antibody titers provided by the invention it is raw materials used and
Reagent is available on the market.
Specific embodiment is given below
Embodiment 1.ICW detects the selection (Fig. 1) of the optimum time of HTNV infection cell.
A549 cell infection HTNV (MOI=1) detects virus protein N P in different infection time point cells by ICW afterwards
Expression.Left figure is scanning result, and right figure is that software is analyzed as a result, found by Student t check analysis, and is uninfected by
Group is compared, and can be detected out the expression of virus protein within second day (2dpi) after infection.(*P<0.05,**P<0.01,***P<
0.001)
Embodiment 2.ICW detects the selection (attached drawing 2) of the most suitable virus titer of HTNV infection cell.
After A549 cell infects HTNV according to different MOI, the expression feelings of virus protein N P in born of the same parents are detected by ICW in 2dpi
Condition.Left figure is scanning result, and right figure is that software is analyzed as a result, found by Student t check analysis, and is uninfected by a group phase
Than infective dose can obviously detect the expression of virus protein when being MOI=0.1.(*P<0.05,**P<0.01,***P<
0.001)
Embodiment 3. screens antiviral protein antibody (Fig. 3) used in ICW.
Prepare mouse the monoclonal antibody 3D8 and 3G1 of the mouse monoclonal antibody 1A8 and anti-HTNV GP of anti-HTNV NP.Antibody preparation is according to standard
Monoclonal antibody preparation method；Antibody purification process is ammonium sulfate precipitation method, the specific steps are as follows:
1, saturated ammonium sulfate solution (SAS) is prepared
767g (NH4) 2SO4 is slowly added in 1L distilled water while stirring.It is 7.0 with ammonium hydroxide or sulfuric acid tune pH value.This
I.e. saturation degree be 100% ammonium sulfate (4.1mol/L, 25 DEG C).
2, it precipitates
(1) sample (such as ascites) 20000g is centrifuged 30min, removes cell fragment；
(2) retain supernatant and measure volume；
(3) it is slowly added into isometric SAS while stirring into supernatant, final concentration of 1:1；
(4) solution is placed on magnetic stirring apparatus and stirs 6 hours or is stirred overnight (4 DEG C), precipitate protein sufficiently.
3, it dialyses
(1) protein solution 10000g is centrifuged 30min (4 DEG C), abandons supernatant and retains precipitating；
(2) precipitating is dissolved in a small amount of 5ml PBS-0.2g/L Sodium azide, is put into bag filter to PBS-- after precipitating dissolution
0.2g/L Sodium azide dialysed overnight, it is primary to change elution buffer every 3-6 hours, thoroughly to remove ammonium sulfate；
(3) dialyzate is centrifuged, and BCA method measures protein content in supernatant.
Purified antibodies concentration dilution is to 10mg/ml.In Fig. 3, upper figure is to be carried out using different extension rate 1A8 monoclonal antibodies
ICW experiment, test object are that HTNV infects (MOI=1) A549 cell after 2 days, the following figure be using different extension rate 3D8 or
3G1 monoclonal antibody carries out ICW experiment, test object is that HTNV infects (MOI=1) A549 cell after 2 days, as the result is shown low concentration
1A8 monoclonal antibody (0.25 × 10-3Mg/ml virus N P) still can be effectively detected, and ICW experiment effectively detection GP needs the 3D8 of higher concentration
(10-2) or 3G1 (0.5 × 10 mg/ml-2Mg/ml) monoclonal antibody.
4. present invention detection HTNV titre of embodiment and its comparison (Fig. 4) with classic ELISA method.
ICW (preceding to have described) and classic ELISA method detection TCID are utilized respectively with a batch E6 amplification virus50(according to
Reed-Munch formula calculate) comparison two methods correlation, Fig. 4 A be ICW detect HTNV TCID50/ ml result (2.43 ×
104), Fig. 4 B is that ELISA is detected with a batch HTNV TCID50/ ml result (2.57 × 104), Fig. 4 C is 7 two methods detections
As a result, result prompts two methods, there are good regression relations for the linear regression of Comparative result.(*P<0.05,**P<
0.01,***P<0.001)
5. present invention detection patients serum's neutralize antibody titers of embodiment and its comparison (Fig. 5) with classic ELISA method.
Fig. 5 A is that ICW detects patients serum's neutralize antibody titers as a result, patient 1,2 serum neutralize antibody titers are respectively 1/
56 and 1/40, by taking patient 1 as an example, calculation method is as follows:
1. result judgement is as follows:
Annotation: serum doubling dilution-1: 10, four hole HTNV multiplicity of infection are respectively less than 1.5 (such as Fig. 5-Patient1---
For the first vertical setting of types of ICW as a result, right side corresponds in relative intensity of fluorescence statistical chart, dotted line position is that multiplicity of infection >=1.5 HTNV are sun
The critical value in property hole), i.e. four holes are negative hole (the Positive fluorescence value that HTNV-NP is not detected), and four hole viruses are complete
Complete to neutralize, neutralizing ratio is 4/4.Subsequent neutralization ratio value calculating method is same as above.Neutralize the sum of ratio=4/4+4/4+4/4+0/4+
0/4=3.The potency calculation method of the surveyed neutralizing antibody of ICW, ELISA carries out in this way.
2. measuring LgPD50 (half protective number) and neutralization titer respectively with Karber method
LgPD50=L+D × (S-0.5)；
Neutralization titer=PD50
Note: L: the logarithm of minimum dilution；D: group away from；S: the sum of ratio is neutralized
In this example:
LgPD50=L+D × (S-0.5)=- 1+-0.3 × (3-0.5)=- 1.75；
Neutralization titer=PD50=10-1.75=1/56；
Illustrate that the test serum neutralization titer is 1/56.
Fig. 5 B is that ELISA detects patients serum's neutralize antibody titers as a result, patient 1,2 serum neutralize antibody titers are respectively
1/56 and 1/33, it is as a result almost the same with ICW.
The appraiser HTNV vaccine immunity protectiveness of the present invention of embodiment 6. and its comparison (Fig. 6) with classic ELISA method.
Fig. 6 A is that ICW detects experimenter's serum HTNV neutralize antibody titers after people HTNV vaccine immunity, and wherein subject 1 exempts from
Epidemic disease strategy is that dosage injects HTNV inactivated vaccine according to the instructions provided, enhances once after 1 year, takes blood within 1 week after enhancing；It is tested
2 immunization strategy of person is that dosage injects HTNV inactivated vaccine according to the instructions provided, does not enhance after 1 year and directly takes blood.Subject 1,2
Serum neutralize antibody titers are respectively 1/20 and 1/10；Fig. 6 B is that ELISA detects patients serum's neutralize antibody titers as a result, patient
1,2 serum neutralize antibody titers are respectively 1/20 and 1/10, as a result consistent with ICW.
7. present invention detection HTNV neutralize antibody titers of embodiment and its comparison (Fig. 7) with classic ELISA method.
Fig. 7 A is the neutralization titer that ICW detects the homemade HTNV neutralizing antibody 3D8 and 3G1 of this department, and potency is respectively 1/
1334 and 1/2240；Fig. 7 B be ELISA detection with a collection of neutralize antibody titers as a result, potency is respectively 1/1334 and 1/2240,
As a result consistent with ICW.
8. present invention of embodiment screens and identifies anti-HTNV molecule (Fig. 8).
The DExD/H family molecules such as the ISG such as Mx1 molecule (Fig. 8 A) and DDX3 (figure is expressed in A549 by slow virus system
8B) (A549 cell, which is spread, infects corresponding slow virus according to MOI=100 after ICW Special micro porous plate, and infection carries out subsequent after 1 day
Experiment), HTNV is infected according to MOI=1 later, 2dpi detects the expression of intracellular HTNV NP by ICW.Fig. 8 A display Mx1,
The molecules such as Mx2, IFIT2 have the function of anti-HTNV infection, and Fig. 8 B shows that the molecules such as DDX3 and DDX21 have anti-HTNV infection
Effect, and the molecules such as DDX17 and DDX50 have the function of promoting HTNV infection.(*P<0.05,**P<0.01,***P<0.001)
Example given above is to realize the present invention preferably example, and the present invention is not limited to the above embodiments.This field
Technical staff's technical solution according to the present invention technical characteristic any nonessential addition, the replacement made, belong to this
The protection scope of invention.
Claims (5)
1. a kind of method of high-throughput quickly detection hantaan virus neutralize antibody titers, which is characterized in that including following operation:
1) A549 cell is uniformly inoculated in ICW microwell plate, inoculating cell number is (2-4) × 104/ hole, inoculation hole count=to
Sample number × dilution number × multiple holes number is surveyed, is cultivated to 80~90% cell confluency degree；
2) when cell confluency degree reaches 70~80% in step 1), mixed liquor is carried out using the method for fixed virus diluted sample
Preparation: by sample to be tested etc. than gradient dilution, the sample to be tested of each dilution ratio is mixed with isometric HTNV respectively, in
It is incubated for 2h in 37 DEG C of incubators, is during which gently mixed by inversion for several times；
3) culture solution abandoned in step 1) microwell plate is inhaled, every hole is added DPBS and washs 1-2 times, then will be incubated in step (2)
At mixed liquor microwell plate is added, each dilution gradient sets multiple repeating samples, and Loading sequence according to carrying out from low to high；It sets simultaneously
Set viral negative control group, Isotype control group, virus positive control group, blank control group；
4) microwell plate in step (3) is put into 37 DEG C of incubators and is incubated for after 2.5h and inhale abandoning mixed liquor, washing DPBS1-2 is added in every hole
It is secondary, then the DMEM containing 2%FBS is added to every hole；
5) microwell plate in step 4) is put into taking-up after 37 DEG C of incubators are incubated for 2d and carries out ICW detection；
6) ICW detects the NP in HTNV infection cell: inhaling the culture solution abandoned in microwell plate first, PBS washing is added；Then to every
The fixation buffer that 4 DEG C of pre-coolings are added in hole is fixed, and exempts from shake and is incubated at room temperature；It inhales and abandons fixed buffer, use
Permeable membrane buffer elutes cell；It inhales and abandons permeable membrane buffer, be added along tube wall to every hole and blockade liquid, it is slowly upper on shaking table to shake room
Temperature is lower to be incubated for；It inhales to abandon and blockades liquid, primary antibody is added to every hole along tube wall, slowly shakes incubation at room temperature；It inhales and abandons primary antibody, washing is added
Buffer, washing are multiple；It inhales and abandons washing buffer, secondary antibody is added to every hole along tube wall, is protected from light slowly shakes at room temperature, be incubated for；
Washing buffer is added, is protected from light slowly shakes at room temperature, washing is multiple；After elution terminates, eluent is completely removed；Finally,
Microwell plate after elution is scanned using the double-colored fluoroscopic imaging systems of near-infrared, simultaneously using two channels 700nm and 800nm
Scanning, to obtain the IR fluorescence intensity of HTNV NP；
7) HTNV neutralize antibody titers are calculated:
Every hole is obtained in the IR fluorescence intensity in the channel 700nm and 800nm, it is strong then to calculate HTNV NP IR fluorescence in every hole
Spend the NIR that compares, NIR=NP fluorescent value/internal reference protein fluorescence value；It recycles Karber method to calculate and obtains sample to be tested neutralization
Titre, i.e. neutralize antibody titers.
2. the method for high-throughput quickly detection hantaan virus neutralize antibody titers as described in claim 1, which is characterized in that institute
It states and calculates sample to be tested dilution factor with Karber method specifically:
1) it calculates each viral dilution positive number of perforations P and negative hole number N, the discrimination standard in positive hole is, if hole HTNV
Multiplicity of infection >=1.5, then otherwise it is feminine gender that the hole, which is the positive,；
Wherein, NIR background values is the arithmetic mean of instantaneous value of four hole NIR of background group, the positive controls HTNV infection system of microwell plate
Number >=1.5 is otherwise to repeat this hardened fruit using this group of testing result；
2) it calculates and neutralizes the sum of ratio, neutralization ratio=negative hole/multiple holes sum of each dilution gradient,
Neutralize the sum of ratio=all dilution gradients the sum of infection ratio；
3) calculate dilution group away from, by gradient dilution ratiometric conversion at 10 index, using the tolerance of index equal-difference arrangement as dilute
Group away from；
4) dilution factor is calculated, measures LgPD50 and neutralization titer, LgPD50=L+D × (S-0.5) respectively with Karber method；
Neutralization titer=PD50；Wherein, L is the logarithm of minimum dilution；D be dilution group away from；S is to neutralize the sum of ratio.
3. the method for high-throughput quickly detection hantaan virus neutralize antibody titers as described in claim 1, which is characterized in that institute
The primary antibody stated includes that anti-HTNV NP mouse monoclonal antibody and anti-β-actin rabbit are mostly anti-；The secondary antibody includes Goat anti-
rabbit IRDyeTM680 and Goat anti-mouse IRDyeTM800CW, respectively to detect the channel 700nm and 800nm
Fluorescence intensity.
4. the method for high-throughput quickly detection hantaan virus neutralize antibody titers as described in claim 1, which is characterized in that
Progress ICW detection in second day after A549 cell infection HTNV.
5. the method for high-throughput quickly detection hantaan virus neutralize antibody titers as described in claim 1, which is characterized in that institute
The infective dose for stating A549 cell infection HTNV is MOI=0.1.
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