CN107037212B - Porcine circovirus 2 type antigen immue quantitative detection reagent box - Google Patents
Porcine circovirus 2 type antigen immue quantitative detection reagent box Download PDFInfo
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Abstract
The present invention provides porcine circovirus 2 type antigen immue quantitative detection reagent box, belongs to field of biological technology detection.Secrete the hybridoma cell strain 1G9 of 2 type monoclonal antibody of resisting porcine circovirus, deposit number are as follows: CCTCC NO:C2016105.Porcine circovirus 2 type antigen immue quantitative detection reagent box, comprising: detection antibody and the ELISA Plate for being coated with the 2 type monoclonal antibody of anti-pig annulus, the detection antibody are the 2 type monoclonal antibody of anti-pig annulus of enzyme label.Porcine circovirus 2 type antigen immue quantitative detection reagent box of the present invention, only with a kind of antibody as being coated with and detecting antibody, preparation method is simple, at low cost, antigen broad spectrum activity is good, high sensitivity, specificity height.
Description
Technical field
The invention belongs to field of biological technology detection, and in particular to a kind of porcine circovirus 2 type antigen quantitative detecting reagent
Box.
Background technique
Porcine circovirus 2 type (Porcine circovirustype2, PCV2) belongs to circovirus section Circovirus,
For the sub-thread minus strand cyclic DNA virus of no cyst membrane, diameter is one of known the smallest animal virus in 17-20nm.According to pig
The encoding gene of circovurus type 2 structural proteins Cap, can be subdivided into Multi-genotype, such as PCV2a, PCV2b, PCV2c,
In play principal causative PCV2b there are multiple strains again.PCV2 infection can cause pmws,
Farrowing sow breeding difficulty, wean pig and fattening porcine respiratory disease, pigskin inflammation and nephritic syndrome, the congenital shake of young age piglet
It the diseases such as quivers.PCV2 infection is found in Canada, the U.S., Europe, Southeast Asia swinery in succession the 1990s.China
Since the report for occurring the first Infection of Porcine circovirus from 2000, PCV2 infection swinery situation is serious, shows as Epidemic Scope
Extensively, positive rate is high, mixed infection, and boar infection rate is high, the serious development for hindering pig breeding industry.
Currently, the prevention of porcine circovirus associated diseases relies primarily on immune PCV2 viral inactivation vaccine and expression
The recombinant vaccine of Porcine circovirus type 2 Cap.Strain used in the inactivated vaccine of domestic each producer's production has the PCV2-SH of a genotype
The PCV2 strain of strain and multiple b gene types.Since China's prevalence circovirus is PCV2b type, prepared using PCV2b strain
Vaccine in control and prevention of disease better effect.
The emphasis of inactivated vaccine quality control first is that the content of antigen, detection method is all based on the immune of antibody technique
Method.Fermentation virus titer is measured using indirect immunofluorescence (IFA method) in production.This method operation cycle is long
(one week), equipment requirement height (fluorescence microscope).Also there is the PCV2 antigen detecting agent developed using sandwich ELISA techniques at present
Box.Kit reported in the literature is all made of monoclonal antibody and polyclonal antibody or two different monoclonal antibody matching sides
Formula building.The former has that the stability between batch is poor, result consistency is poor；In cost, due to reagent preparation
Box needs to produce Multiple Antibodies, and production method is complex, higher cost.The kit of latter approach building is sensitive in detection
Demand is unable to satisfy on degree.
In addition, finding in practical application, commercially available monoclonal antibody is often to the wide of different PCV2 strain used in each producer
Spectrum discrimination is poor, detects the import PCV2 monoclonal antibody or how anti-detection serum of work Price-dependent valuableness.Therefore, overcome available reagent
Box there are the problem of, develop a kind of porcine circovirus 2 type antigen detection kit that can be used in the detection of multiple PCV2 strains, it is right
More stringent requirements are proposed for main material-monoclonal antibody of kit.
Summary of the invention
The object of the present invention is to provide a kind of porcine circovirus 2 type antigen immue quantitative detection reagent box, the detections of the kit
Antibody be will coated antibody carry out enzyme label after obtain, preparation method is simple, at low cost, antigen broad spectrum activity is good, high sensitivity,
It is specific high, reproducible.
The purpose of the present invention adopts the following technical scheme that realization.
The present invention provides a kind of hybridoma cell strain 1G9 for secreting 2 type monoclonal antibody of resisting porcine circovirus, deposit number
Are as follows: CCTCC NO:C2016105.
Also invention provides the 2 type monoclonal antibody of resisting porcine circovirus of the hybridoma cell strain secretion for this.
Also invention provides a kind of porcine circovirus 2 type antigen immue quantitative detection reagent box for this, which includes: detection
Antibody and the ELISA Plate for being coated with the 2 type monoclonal antibody of anti-pig annulus, the detection antibody are the claim 2 of enzyme label
The 2 type monoclonal antibody of anti-pig annulus.
In preferred technical solution, the detection antibody is anti-pig described in the claim 2 of horseradish peroxidase-labeled
2 type monoclonal antibody of annulus.
In preferred technical solution, it is that the ELISA Plate is anti-using the anti-2 type monoclonal of pig annulus of 0.5-1.5 μ g/mL
Body is coated；The concentration of the detection antibody is 400-600ng/mL.
In preferred technical solution, the kit further includes standard items mother liquor, negative control, Sample dilution, termination
Liquid, cleaning solution, horseradish peroxidase enzyme catalytic substrate A liquid and substrate B liquid；The standard items mother liquor is that viral level is
105.5TCID50The PCV2-NJ strain virus culture solution of/mL；The negative control is PK15 cell pyrolysis liquid；The Sample dilution
For MEM culture solution；The horseradish peroxidase enzyme catalytic substrate A liquid is the H that volumetric concentration is 3%2O2Aqueous solution；It is described peppery
Root Catalyzed Synthesis By Peroxidase substrate B liquid is to contain 3,3', the solution of 5,5'- tetramethyl benzidine；The terminate liquid is the sulphur of 2M
Aqueous acid；The cleaning solution is the PBS buffer solution of the pH 7.4 of the Tween 20 added with final concentration of 0.05%, 10mM.
The present invention also provides application of the kit in quantitative detection porcine circovirus 2 type antigen.
Kit of the present invention compared with prior art, has the beneficial effect that:
(1) kit of the present invention is suitable for the detection of the PCV2 strain of multiple genotype: the present invention is using natural PCV2 disease
For poison as immunogene and screening antigen, the anti-PCV2 monoclonal antibody 1G9 ' of acquisition and natural PCV2 virus reactivity are good.
The antibody identifies that PCV2 type includes: PCV2-NJ plants, PCV2-ZJ/C plants, PCV2-WH plants, PCV2-DBN-SX07 plants and PCV2-
SH plants.Solve the problems, such as that detection antibody caused by the difference of different vendor's PCV2 seed culture of viruses source is uncurrent.
(2) kit of the present invention is suitable for the detection of recombinant vaccine: utilizing Escherichia coli or baculovirus expression
Porcine circovirus type 2 Cap can simulate natural viral structure, be assembled into virus like particle (VLP).1G9 ' Dan Ke in kit of the present invention
Grand antibody and the PCV2VLP antigen of gene engineering method production still maintain good reactivity.Therefore, kit of the present invention
Detection suitable for PCV2 recombinant vaccine.
(3) kit specificity of the present invention is good, can be used as antidiastole purposes: kit of the present invention and PPV, PEDV,
The pigs common virus no cross reaction such as CSFV, PRRSV, PRV, FMDV, JEV.Therefore it can be used as antidiastole purposes.
(4) kit of the present invention and IFA method coincidence rate are high: the result of kit detection PCV2 viral titer of the present invention with
It is highly relevant that traditional immunofluorescence technique (IFA method) measures malicious valence result, and the operation cycle is shorter, and equipment requirement is low, knot
Fruit determines without interference caused by subjective factors, alternative existing PCV2 poison valence measuring method.
(5) cost of kit of the present invention it is low, batch between it is reproducible: capture antibody in kit of the present invention is monoclonal
Antibody 1G9 ', detection antibody are the monoclonal antibody 1G9 ' of horseradish peroxidase-labeled, therefore in kit of the present invention production
Two or more monoclonal antibody or polyclonal antibody are not needed to prepare, it is only necessary to produce a kind of antibody and mark to it
Note significantly reduces production cost, avoids reagent contamination.With anti-and monoclonal antibody combining form productions more in existing patent
PCV2 antigen detection kit is compared, and kit of the present invention repeatability between criticizing is upper more preferable.Reason is: containing polyclonal antibody
Serum in can specifically bind the antibody of target protein and only account for 0.5%-5% (BradburyA, Pl ü
ckthunA.Reproducibility:Standardize antibodies used in research[J].Nature,
2015,518:27-29.) antibody combination generated is immunized, and every time by immune animal individual difference, immune, antibody purification
Operation etc. the influence of factors and it is never identical.
(6) kit of the present invention overcomes sensitivity caused by two plants of different monoclonal antibody pairings insufficient: entitled " pig
Circovurus type 2 ELISA antigen detection kit and preparation method thereof and its application ", Patent No. CN201210356552.7
Using same strain monoclonal antibody as capture antibody and detection antibody, performance occur seriously is reduced, and is unable to satisfy detection for middle trial
The problem of.Monoclonal antibody 1G9 ' better performances provided by the invention, pairing effect is also preferable, detection performance reached it is more anti-with
The kit level of monoclonal antibody combination.
(7) kit detection of the present invention is accurate, simple and efficient to handle: to detect with currently used IFA and RT-PCR method
PCV2 Antigen Method is compared, and the detection method for the PCV2 antigen immue quantitative detection reagent box that the present invention establishes is at low cost, easy to operate, inspection
Survey the period by foreshorten within one week 2 hours, it is reproducible, quantitatively provided for antigen in PCV2 production of vaccine practical, quick, effective
Detection instrument.
Detailed description of the invention
The His-VHH, wherein M: protein standard sample of Fig. 1 SDS-PAGE electrophoresis detection purifying；4: His-VHH before purification；
1-3: His-VHH after purification.
Fig. 2 SDS-PAGE method detects the purity for the PCV2-NJ strain virus that affinity purification obtains, wherein M: protein standard
Sample；1: the PCV2-NJ strain virus obtained after affinity purification；2:PCV2 virus-culturing fluid.
Fig. 3 immunofluorescence method detects the reactivity of anti-PCV2 monoclonal antibody and PCV2-NJ, and figure left side is labelled with experiment
The monoclonal antibody of use, lower section are labelled with the strain that experiment uses.
The SDS-PAGE analysis chart of Fig. 4 hybridoma cell strain 1G9 ascites after purification, M: protein standard sample；1-9: after purification
Monoclonal antibody 1G9 '；10: the ascites supernatant dilution (30 times of dilution) containing monoclonal antibody 1G9 '.
The standard curve of Fig. 5 kit of the present invention.
The specificity of Fig. 6 kit of the present invention.
Application of Fig. 7 kit of the present invention in detection PCV2 subunit vaccine antigenic, positive control are PCV2-NJ plants of diseases
Venom；Negative control is PK15 cell pyrolysis liquid.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary, and 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 one immunizing antigen of example
In order to keep the natural structure of immunizing antigen (PCV2-NJ plants), purification efficiency is improved, purification cycle is shortened, is used
Nickel column carry recombinant protein His-VHH carries out specific affinity purification to PCV2-NJ strain virus.
His-VHH is the anti-PCV2 nano antibody with His label protein.The recombinant protein is the preparation method is as follows: by patent
Number for ZL201210249621.4, it is entitled " two-humped camel single domain heavy chain antibody of 2 type of resisting porcine circovirus and preparation method thereof and
Nucleotide sequence (see SEQ ID No.4 in the proprietary sequence table) upstream and downstream of anti-PCV2 nano antibody VHH adds respectively in purposes "
Add III restriction enzyme site of Nde I and Hind, the artificial synthesized DNA fragmentation.The synthesis fragment inserting expressioning carrier pET32a (+) (is carried
Body contains His sequence label) III restriction enzyme site of Nde I and Hind between, be then introduced into Escherichia coli inducing expression.Express egg
It is white to use nickel column affinity purification, i.e. acquisition recombinant protein His-VHH.The recombinant protein can be used for PCV2-NJ plants purifying and
Capture antibody in the detection of hybridoma cell clone supernatant antibody titer.The purification effect of recombinant protein His-VHH is shown in Fig. 1.From
Fig. 1 can see, and the recombinant protein His-VHH that molecular weight is about 15Kd is by successful purification.
PCV2-NJ plants of antigens are purified using recombinant protein His-VHH, the specific method is as follows: (1) taking 10mg His-VHH egg
It is white, after diluting 20 times with sample-loading buffer (the PB buffer of pH7.2,20mM) solution, with 1mL/min flow velocity to nickel column loading；
(2) 5 column volumes of nickel column are washed with sample-loading buffer (the PB buffer of pH7.2,20mM)；(3) PCV2-NJ plants of diseases of 500mL are taken
Malicious culture solution (using after PK15 cell Proliferation, cracking, centrifugation obtains after taking supernatant) with 1mL/min to nickel column loading, thus
It is combined with the His-VHH albumen hung in nickel column；(4) nickel column is washed with sample-loading buffer (the PB buffer of pH7.2,20mM)
5 column volumes；(5) with the sample-loading buffer elution samples of the imidazoles containing 500mM, the PCV2-NJ strain that is combined with His-VHH albumen
Virus is eluted together, collects eluent；(6) with molecular cut off for 5kD super filter tube by the buffer in eluent
It is replaced into PBS buffer solution, has obtained PCV2-NJ strain antigen after purification.(7) electrophoretic analysis purified product.Electrophoresis result is shown in figure
2.As shown in Figure 2, this method can efficiently concentrating virus and remove most of impurity protein.PCV2-NJ strain after purification
Antigen, since purity is higher, as immunizing antigen in use, preferable immune effect can be obtained.
Screening, the identification, preparation and purification of two monoclonal antibody of example
1. mouse immune
The PCV2-NJ strain antigen that example one is obtained (is purchased from as immunizing antigen using BCA protein quantification kit
Pierce company) detection protein content, concentration is adjusted to 0.5 μ g/ μ L, is immunized 6 weeks by the subcutaneous multi-point injection mode of the nape of the neck
Age female Balb/c mouse, 100 μ g/ of first immunisation dosage only, after three weeks with 50 μ g/ dosage booster immunizations, are hereafter spaced 2 weeks
Booster immunization is carried out again.Latter week blood sampling is immunized in last time, using non-immune serum as control, using indirect
ELISA method detects the serum titer of immune mouse.
Indirect ELISA method is specific as follows: mice serum makees 10 multiple proportions gradient dilutions using PBST buffer, and dilution is
103~107；Inspection to porcine circovirus 2 type ELISA antibody assay kit (being purchased from Wuhan Ke Qian Biological Co., Ltd.)
It surveys in ELISA Plate and the mice serum after dilution, 100 holes μ L/ is added；After washing 3 times, ELIAS secondary antibody work is added in 37 DEG C of incubation 1h
The sheep anti mouse secondary antibody that HRP is marked (is made 1:5000 dilution, the sheep of HRP label with the PBST buffer dilution containing 1%BSA by liquid
Anti- mouse secondary antibody is incubated for 1h purchased from 37 DEG C of Jackson ImmunoResearch, article No. 115-065-146)；It is added after washing peppery
100 hole μ L/ of root Catalyzed Synthesis By Peroxidase substrate A liquid and the isometric mixed solution of B liquid (with embodiment five), 37 DEG C of colour developing 10min
Afterwards, the terminate liquid (H of 2M is added2SO4Solution) 100 holes μ L/.Light absorption value of every hole at 450nm is detected using microplate reader.Selection
PCV2 antibody titer is 105Above mouse is merged, and 3 days before carrying out cell fusion, every mouse peritoneal injects 50 μ g
Immunizing antigen.
2. cell fusion
It is sterile to win immune mouse spleen, after being mixed with SP2/0 cell (murine myeloma cell) according to 1:5 ratio, adopt
Conventional chemical fusion is carried out with polyethylene glycol PEG1450.Fused cell carries out selectivity using HAT culture medium (being purchased from sigma)
Culture.
3. the foundation of colony screening detection method
The method for detecting hybridoma cell strain secretory antibody potency: detecting antibody titer using double crush syndrome method,
The recombinant protein His-VHH prepared using in embodiment one (uses PK15 cell Proliferation as capture antibody, PCV2-NJ strain virus
Afterwards, crack, centrifugation obtains after taking supernatant) it is used as sandwich antigen, Hybridoma Cell Culture supernatant is as detection primary antibody, HRP mark
The sheep anti-mouse igg (H+L) (be purchased from Jackson ImmunoResearch) of note is used as secondary antibody, in negative control with centrifugation after
PK15 cell lysate supernatant replaces PCV2-NJ strain virus as sandwich antigen, detects the light absorption value under 450nm.Result judgement:
With portion hybridoma culture supernatant, it is higher than using PCV2-NJ strain virus as the testing result of sandwich antigen and uses PK15 cell
Testing result of the lysate supernatant as sandwich antigen (negative control), can be subcloned.Monoclonal is selected in subclone
Repeat above-mentioned detection.Until the hybridoma cell strain culture supernatant of the monoclonal obtained is not reacted with PK15 cell, with
The reaction of PCV2-NJ strain virus liquid is strong.
4. hybrid tumor cell monoclonal
Limiting dilution assay is used to the hybridoma that step 3 obtains, is subcloned, obtains full positive monoclonal
After hybridoma cell strain, expands culture and freeze.The final hybridoma for obtaining the anti-PCV2 monoclonal antibody of 7 plants of stably excretings
Cell strain, is respectively designated as 1G9,1B2,1E7,6H2,4C2,23F5,1F3, and the anti-PCV2 monoclonal antibody of secretion is successively named
For 1G9 ', 1B2 ', 1E7 ', 6H2 ', 4C2 ', 23F5 ' and 1F3 '.
5. the identification of monoclonal antibody
(1) subgroup identification of monoclonal antibody: by the culture supernatant (list containing its secretion of 7 strain of hybridoma strains
Clonal antibody) by the immunoglobulin standard subclass Rapid identification kit operational manual progress subtype identification of Sigma company.
Monoclonal antibody 1G9 ', 1B2 ', 1E7 ', 6H2 ' are IgG2a type to subgroup identification as the result is shown；Monoclonal antibody 4C2 ' is IgG3
Type；Monoclonal antibody 23F5 ', 1F3 ' are IgG2b type.
(2) monoclonal antibody reactive: it is found through experiment that, monoclonal antibody 1G9 ' is not suitable for detecting in SDS-PAGE
PCV2-NJ plants, and it is suitable for IFA and ELISA.Exposure albumen linear structure is handled since SDS-PAGE is related to denaturing samples, by
This can speculate that monoclonal antibody 1G9 ' is that identification virus protein folds the space conformation to be formed.
(3) application of the monoclonal antibody in Immunofluorescence test
The monoclonal antibody of above-mentioned 7 strain of hybridoma strain secretion is detected to PCV2-NJ plants using indirect immunofluorescence
Identification situation.Concrete operation step: PK15 cell is layered in 96 orifice plates, when cell grows to 80% degrees of fusion, is divided into
Two groups: an one group cells are added without PCV2 virus liquid as negative control, and another group of cell is separately added into the different strains disease of PCV2
Venom is incubated for 24 hours, and the culture solution for then replacing two groups of cells is that the DMEM culture solution containing 2% calf serum continues to cultivate
72h.Immunofluorescence experiment step: 80% acetone soln of -20 DEG C of pre-coolings is clapped three times in 4 DEG C of fixed cell 30min, PBST washing
It is dry, monoclonal antibody 1G9 ', 1B2 ', 1E7 ', 6H2 ', 4C2 ', 23F5 ' and 1F3 ', 37 DEG C of incubations are separately added into every kind of strain
1h, while SP2/0 cell culture supernatant is set as negative control.After PBST is washed 3 times, FITC is added and marks sheep anti-mouse igg
Antibody (be purchased from Wuhan Boster Biological Technology Co., Ltd.), 37 DEG C are seen with fluorescence microscope after incubations 1h, PBST washing 3 times
It examines.The results show that monoclonal antibody 1B2 ', 1G9 ', 4C2 ' and 23F5 ' can identify PCV2-NJ plants (Fig. 3).Therefore, selection is single
Clonal antibody 1B2 ', 1G9 ', 4C2 ', 23F5 ' are used as research object, carry out following experiment.
6. hybridoma cell strain Detection of Stability
After hybridoma cell strain freezes 12 months, it is removed from liquid nitrogen the hybridoma cell strain recovery frozen, using containing
Double crush syndrome method detection hybridoma is thin in the present embodiment title 3 after the DMEM culture medium culture of 10% fetal calf serum for 24 hours
The antibody titer of born of the same parents' strain secretion, to evaluate the stability of hybridoma cell strain.It the results are shown in Table 1, hybridoma cell strain training
The potency for supporting supernatant is identical as when freezing, and antibody-secreting is stablized.To the hybridoma cell strain culture supernatant inspection after 50 generations of passage
Survey antibody titer, still with freeze before it is identical, therefore these cell strains secretion monoclonal antibody performance stablize.
Table 1 shows the antibody titer of hybridoma cell strain 1B2,1G9,4C2,23F5 culture supernatant
7. the preparation and purification of monoclonal antibody:
Be respectively adopted hybridoma cell strain 1B2,1G9,4C2 and 23F5 prepare corresponding monoclonal antibody 1B2 ', 1G9 ',
4C2 ' and 23F5 '.
(1) it conventionally prepares the ascites supernatant containing monoclonal antibody: taking 8~10 week old female Balb/c mouse,
Incomplete Freund's adjuvant 0.5mL/ is injected intraperitoneally only, hybridoma cell strain in good condition is resuspended with PBS buffer solution after 7 days
It is floating, with 5 × 105A cell/intraperitoneal injection of mice.Ascites is acquired after about 10 days, centrifugation removes insoluble impurity, takes ascites supernatant
It freezes spare in -70 DEG C.
(2) monoclonal antibody-purified: to prepare the abdomen of 1B2 containing monoclonal antibody ', 1G9 ', 4C2 ', 23F5 ' according to the above method
It is waterborne clear, it is anti-to each monoclonal using GE Hitrap rProteinA FastFlow affinity purification column (being purchased from GE life science)
Body is purified.Monoclonal antibody after purification passes through 10%SDS-PAGE electrophoretic analysis purity (purity > 95%), with retention point
The super filter tube that son amount is 50kD is concentrated, and is PBS buffer solution by buffer exchange.Figure 4, it is seen that monoclonal is anti-
Body IG9 ' purity has reached 95% or more.
(3) antibody titer measurement after purification: being diluted to 1mg/mL for monoclonal antibody after purification, from 1:
1000 start 2 doubling dilutions.Antibody titer is measured using double crush syndrome method in the present embodiment title 3, the results are shown in Table 2.
The potency of the monoclonal antibody of table 2 after purification
| Monoclonal antibody number | Potency |
| 1B2’ | 1.02×106 |
| 1G9’ | 1.02×106 |
| 4C2’ | 2.04×106 |
| 23F5’ | 1.02×106 |
The preparation of three enzyme labelled antibody of example
1. preparing the anti-PCV2 monoclonal antibody of horseradish peroxidase-labeled
PCV2 monoclonal antibody 1B2 ', 1G9 ', 4C2 ', 23F5 ' is fought respectively using horseradish peroxidase to be marked,
The specific method is as follows:
(1) 5mg HRP (horseradish peroxidase) is dissolved in 1mL deionized water, adds the NaIO of 0.2ml, 0.1M4It is molten
Liquid mixes, and closed, room temperature is protected from light 20 minutes.
(2) step (1) acquired solution is fitted into bag filter, is dialysed using the sodium-acetate buffer of pH4.4,1mM at 4 DEG C
Overnight.
(3) monoclonal antibody 1B2 ', 1G9 ', 4C2 ' or the 23F5 ' of 10mg after purification are used to the carbonic acid of pH9.5,10mM
Salt buffer dialysed overnight.
(4) solution in bag filter, is added the carbonate buffer solution of 40ul pH9.5,0.2M after taking step (2) to dialyse
PH to 9.0~9.5 is adjusted, is then added by (3) treated monoclonal antibody, mixes, room temperature is protected from light 2 hours.
(5) the 4mg/mL sodium borohydride solution that 0.1mL is newly prepared is added, mixes, 4 DEG C are placed 2 hours.
(6) ultrafiltration is carried out to reaction product with the super filter tube that molecular cut off is 100kD, removes free HRP, and will be peppery
The anti-PCV2 monoclonal antibody 1B2 ' of root peroxidase labelling, 1G9 ', 4C2 ' and 23F5 ' buffer exchange at pH7.4,
The PBS buffer solution of 10mM is added preservative and isometric glycerol, is placed in -20 DEG C and freezes.
(7) with the anti-PCV2 monoclonal antibody 1B2 ' of BCA protein quantification kit measurement horseradish peroxidase-labeled,
The concentration of 1G9 ', 4C2 ' and 23F5 ', adjustment concentration to 4mg/mL.
2. the antibody titer of horseradish peroxidase-labeled
Monoclonal antibody 1B2 ', 1G9 ', 4C2 ' and the 23F5 ' of horseradish peroxidase-labeled are diluted to 1mg/mL, respectively
It is denoted as 1B2 ' E, 1G9 ' E, 4C2 ' E, 23F5 ' E.Using double crush syndrome method in two title 3 of embodiment to each enzyme labelled antibody
Potency is detected.It the results are shown in Table 3, the potency of above-mentioned 4 kinds of Horseradish Peroxidase Conjugates reaches 5.12 × 105And with
On.
3 enzyme labelled antibody titration of table
| Enzyme labelled antibody | 1G9’E | 1B2’E | 4C2’E | 23F5’E |
| Antibody titer | 5.12×105 | 1.02×106 | 2.05×106 | 5.12×105 |
The foundation of four PCV2 antigen quantitative detecting method of example
1. the selection that double-antibody sandwich matches centering antibody
By monoclonal antibody 1B2 ', 1G9 ', 4C2 ' and 23F5 ' be used as coated antibody, 1B2 ' E, 1G9 ' E, 4C2 ' E and
23F5 ' E, which is used as, detects antibody, is split in sample detection hole with PCV2-NJ plants for sandwich antigen in negative control hole with PK15 cell
It solves liquid supernatant and substitutes PCV2-NJ plants, carry out sandwich pairing property experiment, calculate P/N value, wherein P is the OD in sample detection hole450Value,
N is the OD of negative control hole450Value, P/N value is highest is paired into optimal antibody combination for selection.It the results are shown in Table 4, monoclonal
Antibody 1G9 ' is used as coated antibody, and for 1G9 ' E as detection antibody, effect is best.
The selection of the sandwich pairing antibody of table 4
Hybridoma cell strain 1G9 preservation information is as follows:
Classification naming: hybridoma cell strain 1G9, the deposit date is on May 23rd, 2016, depositary institution's full name was Chinese allusion quotation
Type culture collection, abbreviation CCTCC, depositary institution address: Wuhan University, deposit number are as follows: CCTCC NO:
C2016105。
2. the determination of monoclonal antibody peridium concentration and enzyme labelled antibody working concentration
The peridium concentration of monoclonal antibody 1G9 ' and the working concentration of 1G9 ' E are measured using square matrix titration.Specific side
Method is as follows: with coating buffer (carbonate buffer solution of 0.05M, pH 9.6) by monoclonal antibody 1G9 ' gradient dilution, in 4 μ g/mL
4 concentration are taken into 0.5 μ g/mL, each concentration is coated with 16-18h under the conditions of 4 DEG C；(contain 0.05% using PBST buffer
The PBS buffer solution (pH 7.4, concentration 10mM) of (concentration expressed in percentage by volume) Tween 20) washing, it pats dry；With containing 1% (quality hundred
Point concentration) the PBST buffer of BSA closes 1h under the conditions of 37 DEG C, and it washs, pats dry；The PCV2-NJ of 100 μ L is added in sample well
Strain virus liquid (obtains P value), effect 1h time under the conditions of 37 DEG C, with PK15 cell pyrolysis liquid substitution PCV2 virus in negative hole
(obtaining N value)；It is washed, is patted dry using PBST buffer；1G9 ' the E for being 4mg/mL by concentration with the PBST buffer containing 1%BSA
2000,1000, tetra- 500,250ng/mL concentration are diluted to, are separately added into the hole of each concentration monoclonal antibody 1G9 '
Enzyme labelled antibody 1G9 ' the E of 100 μ L various concentrations, acts on 1h under the conditions of 37 DEG C；Horseradish peroxidase enzyme catalytic bottom is added in every hole
Object A liquid and the isometric mixed solution of B liquid (with embodiment five) 100 μ L, 37 DEG C of colour developing 15min；It is eventually adding the sulphur of 100 μ L 2M
Acid solution terminates reaction, and OD value is detected under 450nm wavelength.Selected P/N value is maximum, positive hole OD450Value is greater than corresponding to 1.0
Capture antibody (i.e. coated antibody) peridium concentration and enzyme labelled antibody concentration be optimum condition.It can be seen from data in table 5
The best peridium concentration of monoclonal antibody 1G9 ' is 1 μ g/mL, and the best use concentration of enzyme labelled antibody is 500ng/mL.
5 coated antibody concentration of table and enzyme labelled antibody working concentration determine
3. the determination of antigenic action time
ELISA antigen detection is carried out with the control of known yin and yang attribute, is reacted by the program having determined, antigen and coating are anti-
Body reacts 30min, 60min, 90min, 120min at 37 DEG C respectively, under other conditions and the identical situation of response procedures, into
The detection of row ELISA antigen, taking maximum, the positive value of P/N in 2.0 or so action time is optimum reacting time.By number in table 6
According to, determine antigen and coated antibody 37 DEG C the best use time be 60min.
6 antigen of table and coated antibody action time determine
| The antigenic action time | 30min | 60min | 90min | 120min |
| PCV2(P) | 1.135 | 2.073 | 2.254 | 2.536 |
| PK15(N) | 0.045 | 0.079 | 0.103 | 0.126 |
| P/N | 25.2 | 26.2 | 21.9 | 20.1 |
4. the determination of enzyme labelled antibody 1G9 ' E action time
By the reaction condition having had determined that, ELISA antigen detection, enzyme labelled antibody 1G9 ' are carried out with the control of known yin and yang attribute
Reaction time of the E at 37 DEG C is respectively 30min, 45min, 60min, 90min.Selecting reaction time when P/N maximum is enzyme
The best effort time of labeling antibody.According to data in table 7, determine that the best effort time of enzyme labelled antibody 1G9 ' E is 60min.
7 enzyme labelled antibody 1G9 ' E action time of table determines
| Action time | 30min | 45min | 60min | 90min |
| PCV2(P) | 1.135 | 1.637 | 2.054 | 2.136 |
| PK15(N) | 0.045 | 0.069 | 0.081 | 0.126 |
| P/N | 25.2 | 23.7 | 25.4 | 17.0 |
5. the determination of developing time
By the reaction condition having had determined that, ELISA antigen detection, developing time difference are carried out with the control of known yin and yang attribute
For 10min, 15min, 20min.Selecting time when P/N maximum is developing time.By data in table 8, determine that developing solution is best
Action time is 15min.
The determination of 8 developing time of table
| Developing time | 10min | 15min | 20min |
| PCV2(P) | 1.305 | 1.932 | 2.154 |
| PK15(N) | 0.055 | 0.079 | 0.121 |
| P/N | 23.7 | 24.5 | 17.8 |
6. standard curve line range
By standard items mother liquor (viral level 105.5TCID50The PCV2-NJ strain virus culture solution of/mL, preparation method are shown in reality
Apply example five) carry out doubling dilution, make its PCV2 content 100,200,400,800,1600,3200,6400,12800,25600,
51200、102400TCID50/ mL is reacted according to set ELISA, prepares standard curve, selects R2PCV2 greater than 0.99
Content range is standard curve range.The corresponding PCV2 content of standard curve is 200-3200TCID50/mL.It is shown in Table 9 and Fig. 5.
9 standard curve range of table
The assembling and application method of five kit of example
1. porcine circovirus 2 type antigen immue quantitative detection reagent box of the present invention includes:
(1) detection plate: anti-PCV2 monoclonal antibody 1G9 ' is diluted using the carbonate buffer solution of 0.05M, pH 9.6 and (is implemented
In example two prepared by 7 method of title) to 1 μ g/mL, ELISA Plate is added with 100 holes μ L/, is coated with 16-18h under the conditions of 4 DEG C, uses
Cleaning solution (with (9) in the present embodiment title 1) washing, pats dry；With the cleaning solution containing 1% (mass percentage concentration) BSA (with this reality
(9) are applied in a title 1) 1h is closed under the conditions of 37 DEG C, washing, dry, Vacuum Package obtain detection plate；
(2) the anti-PCV2 monoclonal antibody 1G9 ' (being abbreviated as 1G9 ' E) of horseradish peroxidase-labeled: 1G9 ' E (embodiment
In three method prepare) concentration be 500ng/mL.
(3) standard items mother liquor: viral level 105.5TCID50The PCV2-NJ strain virus culture solution of/mL.PCV2-NJ plants of diseases
Poison culture liquid and preparation method thereof: the MEM culture medium culture PK15 cell for containing 10% (v/v) calf serum is used, to cell fusion degree
When reaching 90%, be inoculated with PCV2-NJ plant, 37 DEG C culture 72 hours, by viral cultures multigelation 3 times, 4 DEG C, 12000r/
Min is centrifuged 20min, removes cell fragment, takes supernatant as PCV2-NJ strain virus culture solution.
(4) negative control: PK15 cell pyrolysis liquid.With the MEM culture medium culture PK15 for containing 10% (v/v) calf serum
Cell culture fluid is placed in -20 DEG C and under room temperature multigelation 3 times, then when cell fusion degree reaches 90% by cell
Freeze thawing liquid is collected, 12000g/min is centrifuged 10min, takes supernatant as PK15 cell pyrolysis liquid.
(5) Sample dilution: MEM culture medium (is purchased from GIBCO).
(6) horseradish peroxidase enzyme catalytic substrate A liquid: the H that volumetric concentration is 3%2O2Aqueous solution.
(7) horseradish peroxidase enzyme catalytic substrate B liquid: the 3,3' for being 10mg/mL by 1mL concentration, 5,5'- tetramethyl biphenyl
It is to be made into phosphate buffer that 0.1mol/L, pH are 6.0 that amine (TMB) solution, which is added to 100mL concentration,.Concentration is 0.1mol/
L, the preparation method for the phosphate buffer that pH is 6.0: 0.1mol/L biphosphate sodium water solution 87.7mL and 0.1mol/L phosphoric acid
Disodium hydrogen aqueous solution 12.3mL mixing.
(8) terminate liquid: the aqueous sulfuric acid of 2M.
(9) cleaning solution: pH 7.4,10mM PBS buffer solution in be added with final concentration of 0.05% (concentration expressed in percentage by volume)
Tween 20.The PBS buffer solution formula of pH 7.4,10mM: NaCl 8g, KCl 0.2g, KH2PO40.27g、Na2HPO4·
12H2O 3.54g is dissolved in water, is then settled to 1L.
2. the application method of porcine circovirus 2 type antigen immue quantitative detection reagent box of the present invention:
(1) add standard items/sample: standard items mother liquor is diluted to standard curve range using Sample dilution.By sample
Multiple appropriate is diluted using Sample dilution.The dilution of standard items mother liquor or sample is added into detection plate with 100 holes μ L/
Liquid is incubated for 1h in 37 DEG C of insulating boxs；Negative control (PK15 cell pyrolysis liquid) is added in negative control hole.With cleaning solution detersive enzyme
Target 3 times.
(2) 1G9 ' E is added: 1G9 ' E, 37 DEG C of incubation 1h being added into ELISA Plate with 100 holes μ L/；With cleaning solution detersive enzyme
Target 3 times.
(3) it develops the color: horseradish peroxidase enzyme catalytic substrate solution A liquid and substrate solution B liquid being mixed in equal volume, with 100 holes μ L/
It is added in ELISA Plate, 37 DEG C are protected from light colour developing 10-15min；Terminate liquid is added with 100 holes μ L/.
(4) measure: ELISA Plate is read immediately after gently vibrating mixing, and every hole light absorption value is measured at wavelength 450nm.According to
Standard curve calculates the concentration of PCV2 antigen in sample.
Sensitivity, specificity, broad spectrum activity and the repeatability of the kit of the present invention of embodiment six
Investigate sensitivity, specificity, broad spectrum activity and the repeatability of kit (kit of the present invention) prepared by embodiment five.
1. kit sensitivity
Use Sample dilution doubling dilution for 100 in standard items mother liquor, 200,400,800,1600,3200,6400,
12800TCID50/ mL is added negative control (PK15 cell pyrolysis liquid) in negative control hole, is carried out using method in embodiment five
Detection.The light absorption value of test sample hole (standard items mother liquor dilution) and negative control hole at 450nm wavelength.By P/N > 2.1
When minimum PCV2 content be set to kit sensitivity.The sensitivity of kit is 200TCID as the result is shown50/ mL, is shown in Table 10.
10 kit sensitivity of table
2. kit is specific
Using kit and detection method in embodiment five, to swine fever virus (CSFV), pig blue-ear disease poison (PRRSV), pig
Parvovirus (PPV), porcine pseudorabies virus (PRV), swine foot-and-mouth disease virus (FMDV), diarrhea of pigs viral (PEDV), pig encephalitis disease
The cell culture fluid of malicious (JEV) is detected.Whether it is greater than 2.1 according to P/N value, to judge whether kit and sample have friendship
Fork.As seen from Figure 6, other swine disease viruses in addition to PCV2 and kit no cross reaction of the present invention, illustrate kit of the present invention
With good specificity.
3. kit of the present invention detects the broad spectrum activity of PCV2 different genes hypotype
Investigate kit in embodiment five (being labeled as kit first) and commercial kitPCV2Ag
Capture (being purchased from Synbiotics, be labeled as kit second) is to the broad spectrum activity of 6 kinds of difference PCV2 strains.Except PCV2-NJ plants
Outside, remaining virus after being demulsified to commercial available vaccines by obtaining.Table 11 is that kit of the present invention is detected to 6 kinds of different strains
OD450Value, as the result is shown: kit of the present invention can detect 6 kinds of PCV2 strains, have preferable broad spectrum activity.
Testing result of the kit of the present invention of table 11 to 6 kinds of strains
4. repeatability between kit batch
According to ascites preparation → antibody purification → monoclonal antibody horseradish peroxidase-labeled → kit manufacture
Sequence three batch kits of independent production, and carry out differences between batches measurement.Different virus is detected simultaneously using three batch kits
PCV2 virus liquid (the 6400TCID of content50/mL、3200TCID50/mL、1600TCID50/mL、800TCID50/mL、
400TCID50/mL、0TCID50/ mL), compare the difference of three batch kits.As seen from Table 12, kit of the present invention batch between
Difference is no more than 4.04%, illustrates that kit repeatability of the present invention is very good.
Repeatability (OD between 12 kit of table batch450Value)
5. the correlation of kit of the present invention and IFA method (immunofluorescence technique)
In order to verify the consistency of kit of the present invention Yu IFA method testing result, it is same to have chosen 10 parts of PCV2 virus liquids
Shi Caiyong IFA method and kit of the present invention are detected, and calculate the coefficient of variation CV of two methods testing result.As a result see
Table 13, it can be seen that two methods are no more than 8.13% to same a pattern detection result difference, illustrate kit of the present invention
The detected value no significant difference of detected value and IFA.
The correlation of the kit of the present invention of table 13 and IFA method testing result
6. application of the kit of the present invention in detection PCV2 subunit vaccine antigenic
Using kit of the present invention and its application method, respectively to the PCV2Cap of Escherichia coli and baculovirus expression
The virus like particle assembled (be shown in: 1, Zhao Xiaoyun, Qiao Xuwen, Chen Jin, Li Pengcheng, Yu Xiaoming, Zhu Guoqiang, Zheng by preparation method
It rises, and Hou Jibo produces virus sample particle vaccines [J] Chinese agriculture using E.coli expression carrying Cap gene of porcine circovirus type 2
Science, 2015, (05): 976-986；2, the recombinant virus like-particles of 2 type porcine circovirus nucleocapsid protein Cap genes are expressed,
Patent No. CN200810024644.9) it is detected.Check sample is that Escherichia coli empty bacterium is crushed liquid and baculoviral is crushed liquid.
Testing result is shown in Fig. 7.As seen from Figure 7 the PCV2Cap of kit of the present invention and Escherichia coli and baculovirus expression assembling and
At virus like particle reactivity worth it is good, and do not reacted with corresponding host cell, therefore can be used for detecting PCV2 subunit epidemic disease
Seedling antigen.
Claims (5)
1. a kind of porcine circovirus 2 type antigen immue quantitative detection reagent box, which includes: detection antibody and is coated with anti-
The ELISA Plate of 2 type monoclonal antibody of pig annulus, the anti-2 type monoclonal antibody of pig annulus and detection antibody are by deposit number are as follows:
Secreted by the hybridoma cell strain 1G9 of CCTCC NO:C2016105.
2. porcine circovirus 2 type antigen immue quantitative detection reagent box according to claim 1, it is characterised in that the detection antibody
Anti- 2 type monoclonal antibody of pig annulus described in claim 1 for horseradish peroxidase-labeled.
3. porcine circovirus 2 type antigen immue quantitative detection reagent box according to claim 1 or claim 2, it is characterised in that the enzyme mark
Plate is coated using the anti-2 type monoclonal antibody of pig annulus of 0.5-1.5 μ g/mL；The concentration of the detection antibody is 400-
600ng/mL。
4. porcine circovirus 2 type antigen immue quantitative detection reagent box according to claim 3, it is characterised in that the kit is also
Including standard items mother liquor, negative control, Sample dilution, terminate liquid, cleaning solution, horseradish peroxidase enzyme catalytic substrate A liquid and
Substrate B liquid；The standard items mother liquor is that viral level is 105.5TCID50The PCV2-NJ strain virus culture solution of/mL；The feminine gender
Control is PK15 cell pyrolysis liquid；The Sample dilution is MEM culture solution；The horseradish peroxidase enzyme catalytic substrate A liquid
The H for being 3% for volumetric concentration2O2Aqueous solution；The horseradish peroxidase enzyme catalytic substrate B liquid is to contain 3,3', 5,5'- tetramethyl
The solution of base benzidine；The terminate liquid is the aqueous sulfuric acid of 2M；The cleaning solution is added with final concentration of 0.05%
The PBS buffer solution of the pH 7.4 of Tween 20,10mM.
5. application of the kit described in claim 4 in quantitative detection porcine circovirus 2 type antigen.
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| CN109709330B (en) * | 2018-12-25 | 2021-11-09 | 内蒙古必威安泰生物科技有限公司 | Foot-and-mouth disease virus competition ELISA detection kit |
| CN110794148B (en) * | 2019-11-06 | 2023-04-25 | 海南省农业科学院畜牧兽医研究所 | Double-antibody sandwich ELISA quantitative detection kit for pig inflammation small NLRP3 and application thereof |
| CN111471102B (en) * | 2020-04-23 | 2021-08-31 | 西北农林科技大学 | Coding gene, vector, anti-idiotype nano antibody, preparation method and application thereof |
| CN114958776B (en) * | 2022-06-30 | 2024-02-06 | 天康制药股份有限公司 | PCV2 monoclonal antibody and hybridoma cell strain 1A6 secreting same |
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| CN1584597A (en) * | 2004-06-15 | 2005-02-23 | 浙江大学 | ELISA reagent box for determining antibody and antigen of swine circular virus II |
| CN104749361A (en) * | 2015-01-21 | 2015-07-01 | 中国农业科学院哈尔滨兽医研究所 | Porcine circovirus type 2 antigen capture ELISA kit |
| CN105004856A (en) * | 2015-07-10 | 2015-10-28 | 青岛易邦生物工程有限公司 | Porcine circovirus II antibody detection ELISA kit |
| CN105542000A (en) * | 2016-01-05 | 2016-05-04 | 洛阳普莱柯万泰生物技术有限公司 | Monoclonal antibody and application thereof |
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| CN1584597A (en) * | 2004-06-15 | 2005-02-23 | 浙江大学 | ELISA reagent box for determining antibody and antigen of swine circular virus II |
| CN104749361A (en) * | 2015-01-21 | 2015-07-01 | 中国农业科学院哈尔滨兽医研究所 | Porcine circovirus type 2 antigen capture ELISA kit |
| CN105004856A (en) * | 2015-07-10 | 2015-10-28 | 青岛易邦生物工程有限公司 | Porcine circovirus II antibody detection ELISA kit |
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