CN109536456A - The monoclonal antibody for identifying PCV2 virus-like particle and its application in qualitative and quantitative detection PCV2 virus-like particle - Google Patents
The monoclonal antibody for identifying PCV2 virus-like particle and its application in qualitative and quantitative detection PCV2 virus-like particle Download PDFInfo
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Abstract
The invention discloses a kind of monoclonal antibody for identifying PCV2 virus-like particle and its applications in qualitative and quantitative detection PCV2 virus-like particle.Wherein, the monoclonal antibody for capableing of specific recognition 2 porcine circovirus virus-like particle is generated by the hybridoma cell strain secretion that deposit number is CGMCC NO.15793.The specificity of obtained monoclonal antibody 3H9 is identified by indirect immunofluorescence experiment, the results showed that the monoclonal antibody can be with specific recognition PCV2 virion, and can react with PCV2 VLPs, without reacting with the linear coated elisa plate of cap albumen.Therefore, the present invention establish it is a kind of can qualitative and quantitative analysis PCV2 VLPs simultaneously short-cut method, solve the problem of PCV2 VLP particle structure detecting instrument is expensive, and operating difficulties and quantitative approach cannot distinguish between nonspecific proteins and broken subunit.
Description
Technical field
The monoclonal antibody of PCV2 virus-like particle is identified the present invention relates to one plant and secretes the miscellaneous of the monoclonal antibody
Oncocyte is handed over, further relates to the monoclonal antibody in qualitative and quantitative detection PCV2 virus-like particle (virus-like
Particles, VLPs) in application.The invention belongs to biomedicine technical fields.
Background technique
It researches and develops, the links of production, such as strain idenfication culture, isolates and purifies, product testing links in vaccine
It is required to carry out qualitative and quantitative analysis detection to vaccine effective component.It ferments in Spawn incubation, protein purification, vaccine formulation mistake
Journey to vaccine effective component VLPs carry out quantitative detecting analysis, rapidly and accurately vaccine is analyzed and characterized, for establish and
Optimize separation purifying technique, improve product quality, reduce production cost, and ensures that vaccine safety effectively all has important meaning
Justice.Compared with other biological molecule, VLP vaccine often molecular weight bigger (molecular weight is up to millions of or even tens million of), structure
More complicated (multiple subunit assemblings), stability are poor, therefore, select suitable analysis method to seem and are even more important, and it is accurate to establish
The difficulty of reliable analysis method is also bigger.
Currently, carrying out qualitative analysis to VLPs, method there are many VLPs structure and the integralities and correctness of assembling is identified,
But be required to special instrument and complete, it operates in process of production relatively difficult.Main method there are several types of:
Circular dichroism (CD) is the method for measuring secondary protein structure being most widely used and main research means
One of, be study protein conformation it is a kind of than faster, simple, accurate method.It can be close to its physiological status
Solution state under measure, it is sensitive to conformation change, be widely used in the conformation research of protein.Albumen forms aggregation
After VLPs, conformation would generally change, and the ratio decline of alpha-helix, β-folding ratio increases.Therefore, in VLP epidemic disease
In the research of seedling, CD is mainly used for the comparison and vaccine construct of secondary structure analysis, vaccine sample and standard items of vaccine etc.
The phenetic analysis of variation.
Transmission electron microscope (Transmission electron microscope, TEM) technology is as quickly detection
One of the conventional means of virus plays abnormal important role in viral diagnosis.The resolution ratio minimum of TEM can achieve
0.1nm, it can be seen that the submicroscopic structure or ultra microstructure that can not be seen clearly under an optical microscope, it is so far can be straight
Connect the main method of observation virus and VLP vaccination particles specific form.
Electron cryo-microscopy and the available high-resolution virion of X-ray crystal diffraction or VLP structure, resolution ratio far dare to
Transmission electron microscope is the method for a kind of finer direct observation virus and VLP particle specific form.
Ultra centrifugation techniques are a kind of methods indispensable in the detection of VLP vaccine, mainly include differential centrifugation and
Density-gradient centrifugation method.Meanwhile ultracentrifugation is also a kind of effective ways of small scale purification VLP vaccine, can be concentrated with it is pure
Change VLP vaccine, is characterized for subsequent analysis and other performance.The ultra centrifugation techniques of analytic type can also directly measure VLP vaccine
Buoyant density and and sedimentation coefficient.
Efficient liquid phase size exclusion chromatograph (High Performance Liquid Size Exclusion
Chromatography, HPSEC) it is mainly used for the measurement of VLP Vaccine molecules amount, purity and content.HPSEC passes through standard VLP
Retention volume of the vaccine sample on analytical column can substantially judge the molecular weight of purification of samples.The molecular weight of general VLP vaccine
It is larger with the mass difference of its subunit, easily complete VLP and subunit are separated by HPSEC, and then can be to pure
The integrality for changing vaccination particles in sample is quickly detected.HPSEC high sensitivity simultaneously, can reach the inspection of Gamma Magnitude
It surveys, the standard curve of concentration-peak area (or peak height) is established with the standard sample of various concentration, may be implemented to micro vaccine
Quick, the accurate quantitative analysis of sample.
One of the key technology that the quantitative analysis method reliably and repeated is also vaccine development & production is established, in order to protect
It is reliable to demonstrate,prove analysis method, it is necessary to sufficiently verify to quantitative approach.Mainly consider several points: specificity guarantees exist in sample
In the case where interference component, analysis method can accurately, exclusively measure analyte；Standard curve and quantification range should provide
The linear equation and related coefficient of standard curve；Lower limit of quantitation；Precision, with the within-run and between-run analysis coefficient of quality-control sample
(CV) accuracy of method is investigated；Accuracy, under determining analysis condition, the biological sample concentration and actual concentration that measure
Degree of closeness.
Quantitative to the antigen of subunit vaccine, conventional method has electrophoresis (PAGE), Enzyme-linked Immunosorbent Assay (ELISA), for
The sample composition of purifying can carry out concentration mensuration with based on the quantitative BCA of protein characteristic or ultraviolet spectrophotometry.Additionally
The efficient liquid phase size exclusion chromatograph method for thering is above-mentioned qualitative analysis to mention.
A kind of the most commonly used method, is widely used in antigen or anti-in the current Protein Detection of Enzyme-linked Immunosorbent Assay (ELISA)
The qualitative and quantitative analysis of body.ELISA method have high sensitivity, high specificity, detection it is rapid, easy to operate be easy to automate,
The advantages that can measuring, being applied convenient for amplification in batches.ELISA also has important application in VLP vaccine active determination in vitro,
In measurement, being reacted by inspection sample and enzyme labelled antibody by the antigen or antibody of different step and surface of solid phase carriers.It is logical
It crosses washing and removes extra antigen, finally combine the amount of tested substance in the amount and sample of the enzyme on solid phase carrier at certain ratio
Example.It, can be according to the depth of color reaction to tested substance qualitative or quantitative analysis after the substrate specificity of coloured enzyme is added.By
In the high catalytic efficiency of enzyme, can greatly iodine effect, to make measuring method that there is very high susceptibility.
Electrophoresis (page) can carry out analysis and semi-quantitative analysis to VLP purity.General VLP Vaccine molecules are big, it is difficult to use
Non-reduced electrophoresis is characterized；Reduction electrophoresis joined reducing agent (DTT or mercaptoethanol) in sample treatment, can be by VLP epidemic disease
Seedling is opened, and is reduced into the form of monomer completely.SDS-PAGE electrophoresis, can be in conjunction with the Western Blot of fluorescent marker secondary antibody
Realize the quantitative analysis to viral subgenomic.
However, the qualitative analysis for VLPs, identifies that VLPs structure and the integrality of assembling and the method for correctness are both needed to
It wants special instrument to complete, operates in process of production relatively difficult.What circular dichroism method detected is the second level knot of albumen
Structure needs very high sample purity, and measuring circular dichroism spectrometer price is not suitable for production of vaccine enterprise at million yuan or so
Industry；The problem of transmission electron microscope, electron cryo-microscopy and X-ray diffraction equally exist instrument, and sample preparation is difficult, needs to dye, is fixed,
Electron bombardment or crystal structure are formed under dry, high vacuum, and operation difficulty is larger, is only applied to conceptual phase at present；Hypervelocity
Centrifugation technique needs buoyant density and sedimentation coefficient by measurement to evaluate VLP mass, but the measurement of these indexs is not
Homogeneous operation does not have different to biconditional operation person even same operator, therefore is mainly used in the purifying of VLP, is concentrated；
HPSEC is used for the qualitative and quantitative analysis of VLP vaccine, but needs to make molecular shape and pre-suppose that, complex steps and
Error is larger.Also need the monodisperse phase standard items using one group of known molecular amount make under similarity condition a series of chromatograms and
Calibration curve increases the difficulty of molecular weight determination, extends experimental period, reduce successively as the benchmark of data processing
The reliability of result is analyzed, the error of the molecular weight measured is possible to very big.In addition, during HPSEC, between sample and medium
Non-specific interaction can also reduce precision of analysis.
The shortcomings that quantitative analysis method mainly has: the semidefinite to sample may be implemented in the standard items of electrophoresis control known concentration
Amount, but can only often detect the purity of VLP vaccine and the molecular weight of subunit, be unable to characterize VLP vaccine overall molecule amount and
The integrality and situation of change of structure；Protein content, but the quantification of nonspecific proteins chemical reaction of BCA are measured with BCA method
It is quantitative, equally specific it cannot identify VLPs structural intergrity and its purity, and complicated for operation.Due to VLPs albumen concentration compared with
It will form invertibity sedimentation when big, generate precipitating.VLP protein content is measured with BCA method, it is bigger final that there are extension rates
The higher situation of obtained protein concentration values；The measurement of uv-spectrophotometric value equally cannot specificity identification VLPs structural intergrity
And purity, and the light absorption value of every kind of albumen is widely different, converts.
Therefore, at present there is an urgent need to establish it is a kind of can qualitative and quantitative analysis VLPs simultaneously short-cut method, solve VLP
Particle structure detecting instrument is expensive, and operating difficulties and quantitative approach cannot distinguish between asking for nonspecific proteins and broken subunit
Topic.
Summary of the invention
One of the objects of the present invention is to provide the monoclonal antibodies that one plant is capable of specific recognition PCV2 virus-like particle
And generate the hybridoma cell strain of the monoclonal antibody；
The second object of the present invention is to provide a kind of for qualitative and quantitative detection PCV2 virus-like particle double fastener
Heart ELISA detection kit；
The third object of the present invention is to provide a kind of for qualitative and quantitative detection PCV2 virus-like particle double fastener
Heart ELISA detection method.
In order to achieve the above object, present invention employs following technological means:
The present invention is thin using the PCV2 virus-like particle immune mouse spleen cell and sp2/0 of Bacillus coli expression and assembling
Born of the same parents fusion after subclone screening obtain one plant can be with the monoclonal antibody of specific recognition PCV2 VLPs, which can
With specific recognition PCV2 VLPs comformational epitope, the VLPs for forming whole grain structure and the cap albumen list commonly expressed are distinguished
Body and other nonspecific proteins.Using the monoclonal antibody, we construct a kind of double crush syndrome kit and are used for
The qualitative and quantitative analysis of PCV2 VLPs subunit vaccine carries out quality control to the VLPs correctly assembled and to protein content
Quick and precisely, specific quantitative analysis.It is demonstrated experimentally that detecting PCV2 using double sandwich-ELISA kit of the present invention
VLPs is accurate and reliable, repeats, and it is anti-that only with assembling correct VLP specific antigen-antibody occurs for monoclonal antibody contained therein
It answers, it can be ensured that specificity guarantees in sample that analysis method accurately, can be measured exclusively there are in the case where interference component
Analyte；Secondly, quantitative analysis method standard curve is stablized, 2 > 0.98 of coefficient R；Quality-control sample batch in and batch between become
Different coefficient CV < 5% has good accuracy；Detection range has good linear relationship, most in 0.293-75ug/ml
Low-detection lower limit is 146.48ng/ml.
On the basis of the studies above, firstly, being capable of stably excreting specific recognition PCV2 the invention proposes one plant
The hybridoma cell strain of VLPs monoclonal antibody, the hybridoma cell strain are named as 3H9, and classification naming is to secrete anti-PCV2
The monoclonal antibody hybridoma cell of virus-like particle is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms
Center (China General Microbiological Culture Collection Center, CGMCC), address is in north
No. 3 Institute of Microorganism, Academia Sinica, institute of the Chaoyang District Jing Shi North Star West Road 1, deposit number are CGMCC NO.15793, are protected
The hiding time is on June 6th, 2018.
Secondly, the invention also provides by the described hybridoma secretion generate being capable of specific recognition PCV2
The monoclonal antibody of VLPs.And purposes of the monoclonal antibody in detection PCV2 virus-like particle.
Again, the invention also provides a kind of for qualitative and quantitative detection PCV2 virus-like particle double sandwich-ELISA
Detection kit, wherein containing monoclonal antibody of the present invention, rabbit-anti PCV2 VLPs polyclonal antibody and HRP label
Goat-anti rabbit polyclonal antibody.
Wherein, it is preferred that the kit further includes ELISA Plate, coating buffer, confining liquid, dilution, developing solution, washing
Liquid, terminate liquid, HRP conjugate stabilization/diluent and PCV2 VLPs standard items.
Wherein, it is preferred that when for the detection of 2 porcine circovirus virus-like particle, follow the steps below:
(1) monoclonal antibody (3H9) of the present invention is diluted with coating buffer according to 1:12800, and 100 microlitres of every hole adds
Enter to elisa plate, 4 DEG C of coatings are overnight；
(2) elisa plate being coated with is washed 4 times with cleaning solution, 200 microlitres of every hole, 5 minutes every time；It is micro- to be added 200
To elisa plate, 37 DEG C are closed 2 hours liter/hole confining liquid；
It (3) is 600 μ g/ml with PBS adjustment PCV2 VLPs standard concentration, after obtaining gradient dilution after 2 doubling dilutions
PCV2 VLPs standard items；
(4) take each dilution PCV2 VLPs standard items and each 100 microlitres of sample to be tested, be separately added into and closed
Elisa plate, 37 DEG C are incubated for 1 hour；
(5) elisa plate is washed 4 times with PBST solution after being incubated for, 200 microlitres of every hole, and 5 minutes every time；
(6) rabbit-anti PCV2 VLPs polyclonal antibody is diluted according to 1:3200,100 microlitres of every hole addition ELISA plate, and 37 DEG C
It is incubated for 1 hour；
(7) it is washed 4 times with PBST solution, 200 microlitres of every hole, 5 minutes every time；The goat-anti rabbit polyclonal antibody of HRP label
It is diluted with HRP conjugate stabilization/diluent according to 1:10000,100 microlitres of every hole addition elisa plate, 37 DEG C are incubated for 1 hour；
(8) elisa plate is washed 4 times, every time 5 minutes with cleaning solution after antibody incubation；
(9) every hole is added 100 microlitres of developing solution, and 37 DEG C are reacted 10 minutes, and every hole is added 50 microlitres of 2M sulfuric acid and terminates reaction；
(10) microplate reader is set into Detection wavelength as 450nm, elisa plate is read in 10 minutes, measure extinction
Value；It is abscissa (X-axis) by standard concentration logarithm, corresponding OD value is ordinate (Y-axis), draws canonical plotting, will be to
The light absorption value of sample substitutes into equation, calculates protein content.
Wherein, it is preferred that the carbonate that the coating buffer is 50mM is coated with buffer, pH9.6；The confining liquid is
0.01M PBS solution containing 1w/v%BSA, pH 7.6；The dilution is 0.02M PBS buffer, pH7.0；Described
Cleaning solution is the 0.1mol/L PBS solution containing 0.5v/v%Tween20, and pH 7.6 dilutes 10 times when use；Described is aobvious
Color liquid is TMB developing solution；The terminate liquid is 2M sulfuric acid.
Wherein, it is preferred that the R2 value of canonical plotting should be not less than 0.9800.
This method can be applied to the research and development of PCV2 VLP vaccine, production and quality inspection links, to product quality is improved, reduce
Production cost, it is ensured that vaccine safety effectively has great importance.
Detailed description of the invention
Fig. 1 is the SDS-PAGE result of purpose albumen；
Wherein: 1,2,3 different batches convert bacterial strain, and M is albumen Marker；
Fig. 2 is the antigenic result that western blot (WB) identifies cap albumen；
Fig. 3 is express express target protein small scale purification SDS-PAGE figure；
Wherein: 1-5 is respectively 1000,500,250,125,62.5mg/ml BSA albumen, and 6-10 is respectively doubling dilution
Purifying protein, M are albumen Marker；
Fig. 4 is scanning electron microscopic observation virus-like particle；
Fig. 5 is 3H9 antibody purification SDS-PAGE figure；
Fig. 6 is indirect immunofluorescence experiment；
Wherein: A:3H9；B: negative serum；
Fig. 7 is that two strain antibodies are reacted with linearisation cap albumen western blot (WB)；
Fig. 8 is protein quantification canonical plotting.
Specific embodiment
Further describe the present invention below with reference to specific example, the advantages and features of the present invention will be with description and more
It is clear.But these examples be only it is exemplary, it is not intended to limit the scope of the present invention in any way.Those skilled in the art answer
It should be appreciated that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form repair
Change or replace, but these modifications and replacement are fallen within the protection scope of the present invention.
The preparation of the monoclonal antibody of 1 specific recognition 2 porcine circovirus virus-like particle of embodiment
1, the preparation of PCV2 VLPs
(1) clone's PCV2 cap protein gene is connected in PET-28a expression vector, and digestion identification clone is correctly inserted into
Afterwards, sequencing identification recombinant plasmid sequence is correct.Recombinant plasmid transformed expression bacterium BL21 is subjected to inducing expression, collects thallus through height
It presses homogenizer to break bacterium, is redissolved after ammonium sulfate precipitation, SDS-PAGE electrophoresis result is shown, has apparent band in 27KD size, is accorded with
It closes cap molecular weight of albumen (Fig. 1).
(2) expression albumen being subjected to westernblot identification, mycoprotein is through SDS-PAGE and is transferred on PEDV film,
It identifies there is 27KD size purpose band as the result is shown through his tag antibody, shows that cap albumen is correctly expressed (Fig. 2).
(3) protein purification and identification: the expression bacterium of collection is crushed according to the method described above, cell conditioned medium is through ammonium sulfate repeatedly
Deposition and purification obtains the albumen (Fig. 3) of higher degree.
(4) assembling of PCV2 VLPs: cap albumen can be self-assembled into PCV2 in vitro inside buffer system appropriate
VLPs, buffer composition are as follows: 0.1M NaH2PO4,0.1M Na2HPO4,20mM imidazole, 10 mM Tris base,
300mM NaCl, 50mM KCl, 2mM MgCl2,0.1M ammonium citrate, and 5%glycerol, pH 8.0.It sweeps
It retouches Electronic Speculum and is able to observe that uniform form, circular hollow, size and the similar virus like particle of PCV2 virus size, see Fig. 4.
2, the preparation of monoclonal antibody
It is immunized three times with PCV2 VLPs after purification according to 100ug/ mouse, measurement antibody titer can achieve
After 1:12800 booster immunization 4 days, mouse spleen is taken to be merged with the SP2/0 cell that logarithmic phase is grown, it is indirect with foundation
ELISA method is screened, obtain one plant can efficiently, stably excreting express the hybridoma of anti-PCV2 VLPs monoclonal antibody
Cell.4 subclones are carried out to the hybridoma, and carry out continuous passage, obtain to stablize the anti-PCV2 VLPs's of expression
Cell line 3H9.The cell strain is deposited in China General Microbiological culture presevation administrative center (CGMCC), deposit number CGMCC
NO.15793, preservation time are on June 6th, 2018.
3H9 hybridoma is injected into Mice Body, largely prepares ascites, and pure by the progress of protein G pillar
Change, has obtained the monoclonal antibody (Fig. 5) of high-purity.Antibody titer is detected, potency reaches 1:6400；Albumen is dense
Degree is 1291ug/ml.
3, the specificity identification of monoclonal antibody
The specificity of obtained monoclonal antibody 3H9 is identified by indirect immunofluorescence experiment, indirect immunofluorescence is real
It tests the result shows that the monoclonal antibody can be with specific recognition PCV2 virion (Fig. 6), and the monoclonal antibody specificity
It is reacted with PCV2 VLPs, without reacting with the linear coated elisa plate of cap albumen, and control antibodies 2G8 is (with the same batch of 3H9
Screening obtains) it can identify VLPs and linearisation cap (table 1)；WB experiment shows that its epitope is comformational epitope, cannot identify warp
Cross the cap albumen (Fig. 7) of SDS-PAGE denaturation.Result above proves that monoclonal antibody 3H9 can specificity and PCV2-VLPs
Reaction, and the cap albumen that cannot be expressed with normal linear reacts.
1 antibody of table and cap albumen and VLPs albumen indirect ELISA
| cap | VLPs | |
| 3H9 | 0.403 | 3.15 |
| 3H9 | 0.199 | 3.202 |
| 3H9 | 0.628 | 3.09 |
| 3H9 | 0.458 | 3.179 |
| HIS | 3.54 | 0.808 |
| 2G8 | 3.331 | 3.793 |
| BSA | 0.72 | 0.492 |
The preparation of 2 double crush syndrome quantification kit of embodiment
1, use 3H9 monoclonal antibody as coated antibody, the sheep of rabbit-anti PCV2 VLPs polyclonal antibody and HRP label
Anti-rabbit polyclonal antibody is established double sandwich-ELISA detection method, is analyzed VLPs protein concentration as detection antibody.
2, the operating process of double crush syndrome quantification kit
(1) 3H9 monoclonal antibody is dilute according to 1:12800 with coating buffer (carbonate of 50mM is coated with buffer, pH9.6)
It releases, 100 microlitres of every hole is added to elisa plate.4 DEG C of coatings are overnight；
(2) to the elisa plate being coated with, with PBST solution, (the 0.1mol/L PBS containing 0.5v/v%Tween20 is molten
Liquid, pH 7.6 dilute 10 times when use, similarly hereinafter) washing 4 times, 200 microlitres of every hole, 5 minutes every time.200 microlitres of confining liquids are added
(PBS solution 0.01M, pH 7.6 containing 1w/v%BSA) arrives elisa plate, and 37 DEG C are closed 2 hours；
(3) PCV2 VLPs protein content is detected with BCA method, is 600 μ g/ml with PBS adjustment PCV2 VLPs concentration.
PCV2 VLPs is dispensed, is frozen spare.PCV2 VLPs standard items are used as after 2 doubling dilutions.
(4) each dilution standard items and each 100 microlitres of sample to be tested are taken, the ELISA plate closed is separately added into.37
DEG C be incubated for 1 hour.
(5) elisa plate is washed 4 times with PBST solution after being incubated for, 200 microlitres of every hole, and 5 minutes every time.
(6) rabbit-anti PCV2 VLPs polyclonal antibody is diluted according to 1:3200,100 microlitres of every hole addition ELISA plate, and 37 DEG C
It is incubated for 1 hour.
(7) it is washed 4 times with PBST solution, 200 microlitres of every hole, 5 minutes every time.The goat-anti rabbit polyclonal antibody of HRP label
According to 1:10000 HRP conjugate stabilization/dilution dilution agent, 100 microlitres of every hole addition elisa plate, 37 DEG C are incubated for 1 hour.
(8) elisa plate is washed 4 times, every time 5 minutes with PBST solution after antibody incubation.
(9) every 100 microlitres of the hole of tmb substrate is added, 37 DEG C are reacted 10 minutes, and every hole is added 50 microlitres of 2M sulfuric acid and terminates instead
It answers.
(10) microplate reader is set into Detection wavelength as 450nm, elisa plate is read in 10 minutes, measure extinction
Value.It is abscissa (X-axis) by standard concentration logarithm, corresponding OD value is ordinate (Y-axis), draws canonical plotting (R2 value
It should be not less than 0.9800).The light absorption value of sample to be tested is substituted into equation, calculates protein content.
3, result
Testing result proves that the kit can be 146.48ng/ml with specific detection VLPs, lowest detection lower limit, quick
It is perceptual strong；Antigen concentration has good linear relationship in 0.293-75ug/ml, is repeatedly measured linear relationship curve difference not
Greatly, y=0.2233x-1.0056,2 > 0.99 (Fig. 8) of coefficient R.
4, degree of conformity is tested
We choose the height containing 90,50,4ug/ml PCV2 VLPs, in, the sample of low three concentration carries out coincidence rate
Detection.For sample concentration by quantitatively calculating after BCA kit quantification through standard curve, mean concentration is respectively as follows: the coefficient of variation
It is respectively as follows: 86.28；49.523；4.23, make a variation equal < 5%, illustrates that kit of the invention has preferable accuracy.Meanwhile
The quantitative analysis of SDS-PAGE gray scale is passed through to purifying protein, using the BSA albumen of known concentration as standard items, the results showed that gray scale
The concentration 785.4673mg/ml measured is analyzed, the calculated protein content of double sandwich-ELISA method of the present invention is 815.1mg/
Ml, degree of conformity are very high.
5, the assembling of kit
ELISA Plate: 96 hole brand Czech
Coated antibody reagent: 3H9 monoclonal antibody
Coating buffer: the carbonate of 50mM is coated with buffer, pH9.6
Confining liquid: the PBS solution 0.01M, pH 7.6 containing 1w/v%BSA
Detect antibody reagent: the goat-anti rabbit polyclonal antibody of rabbit-anti PCV2 VLPs polyclonal antibody and HRP label
Dilution: 0.02M PBS buffer solution, pH7.0；
10 × cleaning solution: the 0.1mol/L PBS solution (PBST solution) containing 0.5v/v%Tween20, pH 7.6 make
Used time dilutes 10 times；
Developing solution: TMB developing solution；
Terminate liquid: 2M sulfuric acid；
Protein stabilized agent solution: Huzhou English creates HRP conjugate stabilization/diluent I article No. HRP-SD-001
PCV2 VLPs standard items: 600 μ g/ml.
Claims (8)
1. one plant being capable of stably excreting specific recognition porcine circovirus 2 type (Porcine circovirus type 2, PCV2)
The hybridoma cell strain of virus-like particle (virus-like particles, VLPs) monoclonal antibody, the hybridoma are thin
Born of the same parents' strain is named as 3H9, is deposited in China General Microbiological culture presevation administrative center, and deposit number is CGMCC NO.15793,
The preservation time is on June 6th, 2018.
2. by hybridoma described in claim 1 secretion generate being capable of specific recognition porcine circovirus 2 type virus-like
The monoclonal antibody of particle.
3. purposes of the monoclonal antibody as claimed in claim 2 in detection 2 porcine circovirus virus-like particle.
4. a kind of for qualitative and quantitative detection PCV2 virus-like particle double sandwich-ELISA detection kit, which is characterized in that
Goat-anti rabbit polyclonal containing monoclonal antibody as claimed in claim 2, rabbit-anti PCV2VLPs polyclonal antibody and HRP label
Antibody.
5. kit as claimed in claim 4, which is characterized in that the kit further includes ELISA Plate, coating buffer, closing
Liquid, dilution, developing solution, cleaning solution, terminate liquid, HRP conjugate stabilization/diluent and PCV2VLPs standard items.
6. kit as described in claim 4 or 5, which is characterized in that detected for 2 porcine circovirus virus-like particle
When, it follows the steps below:
(1) monoclonal antibody as claimed in claim 2 is diluted with coating buffer according to 1:12800, and 100 microlitres of every hole is added to
Elisa plate, 4 DEG C of coatings are overnight；
(2) elisa plate being coated with is washed 4 times with cleaning solution, 200 microlitres of every hole, 5 minutes every time；200 microlitres/hole is added
To elisa plate, 37 DEG C are closed 2 hours confining liquid；
(3) it is 600 μ g/ml with PBS adjustment PCV2VLPs standard concentration, the PCV2VLPs mark of gradient is obtained after 2 doubling dilutions
Quasi- product；
(4) take each dilution PCV2VLPs standard items and each 100 microlitres of sample to be tested, be separately added into the ELISA closed
Plate, 37 DEG C are incubated for 1 hour；
(5) elisa plate is washed 4 times with PBST solution after being incubated for, 200 microlitres of every hole, and 5 minutes every time；
(6) rabbit-anti PCV2VLPs polyclonal antibody is diluted according to 1:3200,100 microlitres of every hole addition elisa plate, and 37 DEG C are incubated for 1
Hour；
(7) it is washed 4 times with PBST solution, 200 microlitres of every hole, 5 minutes every time；The goat-anti rabbit polyclonal antibody HRP of HRP label
Conjugate stabilization/diluent is diluted according to 1:10000,100 microlitres of every hole addition elisa plate, and 37 DEG C are incubated for 1 hour；
(8) elisa plate is washed 4 times, every time 5 minutes with cleaning solution after antibody incubation；
(9) every hole is added 100 microlitres of developing solution, and 37 DEG C are reacted 10 minutes, and every hole is added 50 microlitres of 2M sulfuric acid and terminates reaction；
(10) microplate reader is set into Detection wavelength as 450nm, elisa plate is read in 10 minutes, measure light absorption value；It will
Standard concentration logarithm is abscissa (X-axis), and corresponding OD value is ordinate (Y-axis), canonical plotting is drawn, by sample to be tested
Light absorption value substitute into equation, calculate protein content.
7. such as kit described in claim 5 or 6, which is characterized in that the carbonate coating that the coating buffer is 50mM is slow
Fliud flushing, pH9.6；The confining liquid is the 0.01M PBS solution containing 1w/v%BSA, pH7.6；The dilution is 0.02M
PBS buffer solution, pH7.0；The cleaning solution is the 0.1mol/L PBS solution containing 0.5v/v%Tween20, and pH 7.6 makes
Used time dilutes 10 times；The developing solution is TMB developing solution；The terminate liquid is 2M sulfuric acid.
8. kit as claimed in claim 6, which is characterized in that the R2 value of canonical plotting should be not less than 0.9800.
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