CN107037212A - 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.The hybridoma cell strain 1G9 of the type monoclonal antibody of resisting porcine circovirus 2 is secreted, preserving number is：CCTCC NO:C2016105.Porcine circovirus 2 type antigen immue quantitative detection reagent box, including:Detection antibody and the ELISA Plate for being coated with the type monoclonal antibody of anti-pig annulus 2, the detection antibody are the type monoclonal antibody of anti-pig annulus 2 that enzyme is marked.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, cost is low, antigen broad spectrum activity is good, sensitivity is high, specific 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 technology
Porcine circovirus 2 type (Porcine circovirustype2, PCV2) belongs to PCV-II section Circovirus,
For the sub-thread minus strand cyclic DNA virus without cyst membrane, diameter, in 17-20nm, is one of known minimum animal virus.According to pig
Circovurus type 2 structural proteins Cap encoding gene, can be subdivided into Multi-genotype, such as PCV2a, PCV2b, PCV2c, its
In play the PCV2b of principal causative and there are multiple strains again.PCV2 infection can cause pmws,
Farrowing sow breeding difficulty, wean and pig and fatten that porcine respiratory disease, pigskin be scorching and the congenital shake of nephritic syndrome, young age piglet
The disease such as quiver.PCV2 infects to be found in Canada, the U.S., Europe, Southeast Asia swinery in succession in 1990s.China
Since the report for the first Infection of Porcine circovirus occur in 2000, PCV2 infection swinery situations are serious, show as Epidemic Scope
Extensively, positive rate is high, mixed infection, and boar infection rate is high, the development of serious obstruction pig industry.
At present, the prevention of porcine circovirus associated diseases relies primarily on immune PCV2 viral inactivation vaccines and expression
The recombinant vaccine of Porcine circovirus type 2 Cap.Strain used in the inactivated vaccine of domestic each manufacturer production has the PCV2-SH of a genotype
Strain and the PCV2 strains of multiple b gene types.Because the popular PCV-II of China is PCV2b types, therefore prepared using PCV2b strains
Vaccine it is better in control and prevention of disease.
One of emphasis of inactivated vaccine quality control is the content of antigen, and detection method is all based on the immune of antibody technique
Method.IIF (IFA methods) is used in production and determines fermentation virus titer.This method operation cycle is long
(one week), equipment requirement height (fluorescence microscope).Also there are the PCV2 antigen detecting agents developed using sandwich ELISA techniques at present
Box.The kit of document report is using the monoclonal antibody monoclonal antibody matching side different from polyclonal antibody or two kinds
Formula is built.The problem of there is less stable between batch in the former, result uniformity is poor；In cost, due to reagent preparation
Box needs to produce Multiple Antibodies, and production method is complex, cost is higher.The kit that latter approach is built is detecting sensitive
Demand can not be met on degree.
In addition, being found in practical application, commercially available monoclonal antibody is often to the wide of the different PCV2 strains used in each producer
Spectrum discrimination is poor, detection work Price-dependent expensive import PCV2 monoclonal antibodies or how anti-detection serum.Therefore, available reagent is overcome
The problem of box is present, develops a kind of porcine circovirus 2 type antigen detection kit that can be used in multiple PCV2 strains detections, right
Main material-monoclonal antibody of kit proposes higher requirement.
The content of the invention
It is an object of the invention to provide a kind of porcine circovirus 2 type antigen immue quantitative detection reagent box, the detection of the kit
Antibody is obtained after coated antibody is carried out into enzyme mark, preparation method is simple, cost is low, antigen broad spectrum activity is good, sensitivity is high,
Specificity is 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 the type monoclonal antibody of resisting porcine circovirus 2, preserving number
For：CCTCC NO:C2016105.
This also invention provides the type monoclonal antibody of resisting porcine circovirus 2 that the hybridoma cell strain is secreted.
This also invention provides a kind of porcine circovirus 2 type antigen immue quantitative detection reagent box, and the detection kit includes:Detection
Antibody and the ELISA Plate for being coated with the type monoclonal antibody of anti-pig annulus 2, the detection antibody are the claim 2 that enzyme is marked
The type monoclonal antibody of anti-pig annulus 2.
It is preferred that technical scheme in, it is described detection antibody for horseradish peroxidase-labeled claim 2 described in anti-pig
The type monoclonal antibody of annulus 2.
It is preferred that technical scheme in, be the ELISA Plate be using the anti-type monoclonals of pig annulus 2 of 0.5-1.5 μ g/mL resist
Body is coated；The concentration of the detection antibody is 400-600ng/mL.
It is preferred that technical scheme in, the kit also include 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
105.5TCID50/ mL PCV2-NJ strain virus nutrient solutions；The negative control is PK15 cell pyrolysis liquids；The Sample dilution
For MEM nutrient solutions；The horseradish peroxidase enzyme catalytic substrate A liquid is the H that volumetric concentration is 3%2O2The aqueous solution；It is described peppery
Root Catalyzed Synthesis By Peroxidase substrate B liquid is to contain 3,3', the solution of 5,5'- tetramethyl benzidines；The terminate liquid is 2M sulphur
Aqueous acid；The cleaning solution is pH 7.4,10mM PBS added with final concentration of 0.05% Tween 20.
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 applied to the detection of the PCV2 strains of multiple genotype：The present invention is using natural PCV2 diseases
Poison is as immunogene and screening antigen, and the anti-PCV2 monoclonal antibodies 1G9 ' of acquisition and natural PCV2 virus reactivity are good.
Antibody identification PCV2 species includes：PCV2-NJ plants, PCV2-ZJ/C plants, PCV2-WH plants, PCV2-DBN-SX07 plants and PCV2-
SH plants.Solve the uncurrent problem of the different caused detection antibody in different vendor's PCV2 seeds culture of viruses source.
(2) kit of the present invention is applied to the detection of recombinant vaccine：Utilize 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
The PCV2VLP antigens that grand antibody is produced with gene engineering method remain in that good reactivity.Therefore, kit of the present invention
Suitable for the detection of PCV2 recombinant vaccines.
(3) kit specificity of the present invention is good, can be used as antidiastole purposes：Kit of the present invention and PPV, PEDV,
The pig common virus no cross reaction such as CSFV, PRRSV, PRV, FMDV, JEV.Therefore antidiastole purposes can be used as.
(4) kit of the present invention and IFA methods coincidence rate are high：The result of kit of the present invention detection PCV2 viral titers with
Traditional immunofluorescence technique (IFA methods) determines malicious valency result height correlation, and the operation cycle is shorter, and equipment requirement is low, knot
Fruit judgement is without interference caused by subjective factors, alternative existing PCV2 poison valency measures 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
During antibody 1G9 ', detection antibody is the monoclonal antibody 1G9 ' of horseradish peroxidase-labeled, therefore kit of the present invention is produced
Two or more monoclonal antibody or polyclonal antibody need not be prepared, it is only necessary to produce a kind of antibody and rower is entered to it
Note, significantly reduces production cost, avoids reagent contamination.Produced with many anti-and monoclonal antibody combining forms in existing patent
PCV2 antigen detection kits are compared, and kit of the present invention is more preferable in repeatability between criticizing.Reason is：Contain 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.), the Antibody Combination produced and is immunized every time by immune animal individual difference, immune, antibody purification
Operation etc. the influence of factor and it is never identical.
(6) sensitivity that kit of the present invention overcomes two plants of different monoclonal antibody pairings to cause is not enough：Entitled " pig
Circovurus type 2 ELISA antigen detection kits and preparation method thereof and its application ", Patent No. CN201210356552.7
Middle trial is using same strain monoclonal antibody as capture antibody with detecting antibody, and performance occur seriously reduces, it is impossible to meet detection
The problem of.Monoclonal antibody 1G9 ' the better performances that the present invention is provided, pairing effect is also preferable, detection performance reached it is many anti-with
The kit level of monoclonal antibody combination.
(7) kit detection of the present invention is accurate, simple and efficient to handle：Detected with conventional at present IFA and RT-PCR method
PCV2 Antigen Methods are compared, and the detection method cost of PCV2 antigen immue quantitative detection reagent boxes that the present invention is set up is low, easy to operate, inspection
The survey cycle foreshortened to 2 hours by one week, reproducible, for PCV2 production of vaccine moderate resistance originally amount provide it is practical, quick, effective
Detection instrument.
Brief description of the drawings
The His-VHH, wherein M of Fig. 1 SDS-PAGE electrophoresis detections purifying：Protein standard sample；4:His-VHH before purification；
1-3：His-VHH after purification.
The purity for the PCV2-NJ strain virus that Fig. 2 SDS-PAGE methods detection affinity purification is obtained, wherein M：Protein standard
Sample；1:The PCV2-NJ strain virus obtained after affinity purification；2：PCV2 virus-culturing fluids.
Fig. 3 immunofluorescence methods detect anti-PCV2 monoclonal antibodies and PCV2-NJ reactivity, and figure left side is labelled with experiment
The monoclonal antibody of use, lower section is labelled with the strain that experiment is used.
The SDS-PAGE analysis charts of Fig. 4 hybridoma cell strain 1G9 ascites after purification, M：Protein standard sample；1-9：After purification
Monoclonal antibody 1G9 '；10：Ascites supernatant dilution (30 times of dilution) containing monoclonal antibody 1G9 '.
The standard curve of Fig. 5 kits of the present invention.
The specificity of Fig. 6 kits of the present invention.
Application of Fig. 7 kits of the present invention in detection PCV2 subunit vaccine antigenics, positive control is PCV2-NJ plants of diseases
Venom；Negative control is PK15 cell pyrolysis liquids.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But embodiment is only exemplary, does not constitute any limitation to the scope of the present invention.Those skilled in the art should
It should be appreciated that, the details and form of technical solution of the present invention can be repaiied without departing from the spirit and scope of the invention
Change or replace, but these modifications and replacement are each fallen within protection scope of the present invention.
The preparation of the immunizing antigen of example one
In order to keep the natural structure of immunizing antigen (PCV2-NJ plants), purification efficiency is improved, shortens purification cycle, is used
Nickel post carry recombinant protein His-VHH, specific affinity purification is carried out to PCV2-NJ strain virus.
His-VHH is the anti-PCV2 nano antibodies with His label proteins.The recombinant protein preparation method is as follows：By patent
Number for ZL201210249621.4, it is entitled " two-humped camel single domain heavy chain antibody of the type of resisting porcine circovirus 2 and preparation method thereof and
Purposes " moderate resistance PCV2 nano antibodies VHH nucleotide sequence (see SEQ ID No.4 in the proprietary sequence table) upstream and downstream adds respectively
Plus Nde I and the restriction enzyme sites of Hind III, the artificial synthesized DNA fragmentation.The synthesis fragment inserting expressioning carrier pET32a (+) (is carried
Body contains His sequence labels) Nde I and the restriction enzyme sites of Hind III between, be then introduced into Escherichia coli induced expression.Express egg
It is white to use nickel post affinity purification, that is, obtain 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.Recombinant protein His-VHH purification effect 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, specific method is as follows：(1) 10mg His-VHH eggs are taken
In vain, diluted with sample-loading buffer (pH7.2,20mM PB buffer solutions) solution after 20 times, with 1mL/min flow velocitys to nickel post loading；
(2) nickel 5 column volumes of post are washed with sample-loading buffer (pH7.2,20mM PB buffer solutions)；(3) PCV2-NJ plants of diseases of 500mL are taken
Malicious nutrient solution (after PK15 cells propagation, cracking, centrifugation, take and obtained after supernatant) with 1mL/min to nickel post loading so that
It is combined with the His-VHH albumen hung on nickel post；(4) nickel post is washed with sample-loading buffer (pH7.2,20mM PB buffer solutions)
5 column volumes；(5) with the sample-loading buffer elution samples of the imidazoles containing 500mM, the PCV2-NJ strains being combined with His-VHH albumen
Virus is eluted together, collects eluent；(6) super filter tube using molecular cut off as 5kD is by the buffer solution in eluent
PBS is replaced into, PCV2-NJ strain antigens after purification have been obtained.(7) electrophoretic analysis purified product.Electrophoresis result is shown in figure
2.As shown in Figure 2, this method efficiently concentrating virus and can remove most of impurity protein.PCV2-NJ strains after purification
Antigen, because purity is higher, as immunizing antigen in use, preferable immune effect can be obtained.
The screening of the monoclonal antibody of example two, identification, preparation and purification
1. mouse immune
The PCV2-NJ strain antigens that example one is obtained (are purchased from as immunizing antigen using BCA protein quantification kits
Pierce companies) detection protein content, concentration is adjusted to 0.5 μ g/ μ L, it is immune 6 weeks by the subcutaneous multi-point injection mode of nape part
Age female Balb/c mouse, the μ g/ of first immunisation dosage 100 only, with 50 μ g/ dosage booster immunizations after three weeks, are hereafter spaced 2 weeks
Booster immunization is carried out again.The immune one week after blood sampling of last time, using non-immune serum as control, using indirect
The serum titer of the immune mouse of ELISA method detection.
Indirect ELISA method is specific as follows：Mice serum makees 10 multiple proportions gradient dilutions using PBST buffer solutions, and dilution factor is
103~107；To the inspection of porcine circovirus 2 type ELISA antibody assay kits (being purchased from Wuhan Ke Qian Biological Co., Ltd.)
Survey the mice serum added in ELISA Plate after dilution, 100 μ L/ holes；37 DEG C of incubation 1h, after washing 3 times, add ELIAS secondary antibody work
(the sheep anti mouse secondary antibody for being marked HRP with the PBST buffer solutions dilution containing 1%BSA makees 1 to liquid:5000 dilutions, the sheep of HRP marks
Anti- mouse secondary antibody is purchased from Jackson ImmunoResearch, and article No. is 115-065-146) 37 DEG C be incubated 1h；Added after washing peppery
Root Catalyzed Synthesis By Peroxidase substrate A liquid and the μ L/ holes of the isometric mixed solution of B liquid (be the same as Example five) 100,37 DEG C of colour developing 10min
Afterwards, terminate liquid (2M H is added2SO4Solution) 100 μ L/ holes.Using ELIASA detection per light absorption value of the hole at 450nm.Selection
PCV2 antibody titers are 105Mouse above is merged, 3 days, every μ g of mouse peritoneal injection 50 before cell fusion is carried out
Immunizing antigen.
2. cell fusion
It is sterile to win immune mouse spleen, with SP2/0 cells (murine myeloma cell) according to 1:After 5 ratios are mixed, adopt
Conventional chemical fusion is carried out with polyethylene glycol PEG1450.Fused cell carries out selectivity using HAT culture mediums (being purchased from sigma)
Culture.
3. the foundation of colony screening detection method
The method for detecting hybridoma cell strain secretory antibody potency：Antibody titer is detected using double crush syndrome method,
The recombinant protein His-VHH prepared using in embodiment one is as capture antibody, and PCV2-NJ strain virus is (using PK15 cells propagation
Afterwards, crack, centrifugation, take and obtained after supernatant) as sandwich antigen, Hybridoma Cell Culture supernatant is used as detection primary antibody, HRP marks
The sheep anti-mouse igg (H+L) (be purchased from Jackson ImmunoResearch) of note as secondary antibody, in negative control with centrifugation after
PK15 cell lysate supernatants replace PCV2-NJ strain virus as sandwich antigen, detect 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 cells
Lysate supernatant as sandwich antigen (negative control) testing result, you 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 cells, 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 is obtained, is subcloned, full positive monoclonal is obtained
After hybridoma cell strain, it is enlarged culture and freezes.The final hybridoma for obtaining the anti-PCV2 monoclonal antibodies of 7 plants of stably excretings
Cell line, is respectively designated as 1G9,1B2,1E7,6H2,4C2,23F5,1F3, and the anti-PCV2 monoclonal antibodies of secretion are named successively
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 strain
Clonal antibody) carry out hypotype identification by the immunoglobulin standard subclass Rapid identification kit operational manual of Sigma companies.
Subgroup identification result shows that monoclonal antibody 1G9 ', 1B2 ', 1E7 ', 6H2 ' are IgG2a types；Monoclonal antibody 4C2 ' is IgG3
Type；Monoclonal antibody 23F5 ', 1F3 ' are IgG2b types.
(2) monoclonal antibody reactive：Found through experiment, monoclonal antibody 1G9 ' is not suitable for detecting in SDS-PAGE
PCV2-NJ plants, and suitable for IFA and ELISA.Because SDS-PAGE is related to denaturing samples processing exposure albumen linear structure, 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
Detect the monoclonal antibody of above-mentioned 7 strain of hybridoma strain secretion to PCV2-NJ plants using IIF
Identification situation.Concrete operation step：PK15 cells are layered in 96 orifice plates, when cell growth to 80% degrees of fusion, are divided into
Two groups：One group of cell is added without PCV2 virus liquids as negative control, and another group of cell is separately added into PCV2 different strains disease
Venom is incubated 24 hours, is then changed the nutrient solution of two groups of cells and is continued to cultivate for the DMEM nutrient solutions containing 2% calf serum
72h.Immunofluorescence experiment step：80% acetone soln of -20 DEG C of precoolings is in 4 DEG C of fixed cell 30min, and PBST washs three bats
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 setting SP2/0 cell culture supernatants as negative control.After PBST is washed 3 times, FITC mark sheep anti-mouse iggs are added
Antibody (is purchased from Wuhan Boster Biological Technology Co., Ltd.), and 37 DEG C are incubated 1h, and PBST is seen after washing 3 times with fluorescence microscope
Examine.As a result show, monoclonal antibody 1B2 ', 1G9 ', 4C2 ' and 23F5 ' can be recognized PCV2-NJ plants (Fig. 3).Therefore, select single
Clonal antibody 1B2 ', 1G9 ', 4C2 ', 23F5 ' carry out following experiment as research object.
6. hybridoma cell strain Detection of Stability
After hybridoma cell strain freezes 12 months, taken out from liquid nitrogen freeze hybridoma cell strain recovery, using containing
Double crush syndrome method detection hybridoma is thin in the present embodiment title 3 after the DMEM medium cultures 24h of 10% hyclone
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
Identical when supporting the potency of supernatant with freezing, antibody-secreting is stable.To the hybridoma cell strain culture supernatant inspection after 50 generations of passage
Survey antibody titer, still with freeze before it is identical, therefore the monoclonal antibody performance of these cell lines secretion is stable.
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) the ascites supernatant containing monoclonal antibody is conventionally prepared：8~10 week old female Balb/c mouse are taken,
Incomplete Freund's adjuvant 0.5mL/ is injected intraperitoneally only, hybridoma cell strain in good condition is resuspended with PBS after 7 days
It is floating, with 5 × 105An individual cell/intraperitoneal injection of mice.Ascites is gathered after about 10 days, centrifugation removes insoluble impurity, takes ascites supernatant
Freeze standby in -70 DEG C.
(2) it is monoclonal antibody-purified：1B2 containing monoclonal antibody ', 1G9 ', 4C2 ', 23F5 ' abdomen are prepared as stated above
It is waterborne clear, it is anti-to each monoclonal using GE Hitrap rProteinA FastFlow affinity purifications post (being purchased from GE life sciences)
Body is purified.Monoclonal antibody after purification passes through 10%SDS-PAGE electrophoretic analysis purity (purity>95%), with retention point
Son amount is concentrated for 50kD super filter tube, and is PBS by buffer exchange.Figure 4, it is seen that monoclonal is anti-
Body IG9 ' purity has reached more than 95%.
(3) antibody titer after purification is determined：Monoclonal antibody after purification is diluted to 1mg/mL, from 1:
1000 start 2 doubling dilutions.Antibody titer is determined using double crush syndrome method in the present embodiment title 3,2 are the results are shown in Table.
The potency of the monoclonal antibody of table 2 after purification
| Monoclonal antibody is numbered | Potency |
| 1B2’ | 1.02×106 |
| 1G9’ | 1.02×106 |
| 4C2’ | 2.04×106 |
| 23F5’ | 1.02×106 |
The preparation of the enzyme labelled antibody of example three
1. prepare the anti-PCV2 monoclonal antibodies of horseradish peroxidase-labeled
PCV2 monoclonal antibodies 1B2 ', 1G9 ', 4C2 ', 23F5 ' is resisted respectively using horseradish peroxidase to be marked,
Specific method is as follows：
(1) 5mg HRP (horseradish peroxidase) are dissolved in 1mL deionized waters, plus 0.2ml, 0.1M NaIO4It is molten
Liquid, is mixed, closed, and room temperature lucifuge is reacted 20 minutes.
(2) step (1) resulting solution is fitted into bag filter, using pH4.4,1mM sodium-acetate buffer in 4 DEG C of dialysis
Overnight.
(3) monoclonal antibody 1B2 ', 1G9 ', 4C2 ' or the 23F5 ' of 10mg after purification are used to pH9.5,10mM carbonic acid
Salt buffer dialysed overnight.
(4) take step (2) dialysis to terminate solution in rear bag filter, add 40ul pH9.5,0.2M carbonate buffer solution
PH to 9.0~9.5 is adjusted, the monoclonal antibody then added after (3) are handled is mixed, room temperature lucifuge is reacted 2 hours.
(5) the 4mg/mL sodium borohydride solutions that 0.1mL is newly prepared are added, mixed, 4 DEG C are placed 2 hours.
(6) super filter tube for being 100kD with molecular cut off carries out ultrafiltration to reaction product, removes free HRP, and will be peppery
The anti-PCV2 monoclonal antibodies 1B2 ' of root peroxidase labelling, 1G9 ', 4C2 ' and 23F5 ' buffer exchange into pH7.4,
10mM PBS, adds preservative and isometric glycerine, is placed in -20 DEG C and freezes.
(7) with the anti-PCV2 monoclonal antibodies 1B2 ' of BCA protein quantification kit measurement horseradish peroxidase-labeleds,
1G9 ', 4C2 ' and 23F5 ' concentration, 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 designated as 1B2 ' E, 1G9 ' E, 4C2 ' E, 23F5 ' E.Using double crush syndrome method in the title 3 of embodiment two to each enzyme labelled antibody
Potency is detected.3 are the results are shown in Table, the potency of above-mentioned 4 kinds of Horseradish Peroxidase Conjugates reaches 5.12 × 105And with
On.
The enzyme labelled antibody titration of table 3
| 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 the PCV2 antigen quantitative detecting methods of example four
1. the selection of antibody in double-antibody sandwich pairing
' the E, 4C2 ' E that regard monoclonal antibody 1B2 ', 1G9 ', 4C2 ' and 23F5 ' as coated antibody, 1B2 ' E, 1G9 and
23F5 ' E are split as detecting in antibody, sample detection hole with PCV2-NJ plants as sandwich antigen in negative control hole with PK15 cells
Solve liquid supernatant and substitute PCV2-NJ plants, carry out sandwich pairing property experiment, calculate P/N values, wherein P is the OD in sample detection hole450Value,
N is the OD of negative control hole450Value, selection P/N value highests are paired into optimal Antibody Combination.It the results are shown in Table 4, monoclonal
Antibody 1G9 ' is as coated antibody, and 1G9 ' E are as detection antibody, and effect is best.
The selection of the sandwich pairing antibody of table 4
Hybridoma cell strain 1G9 preservation information is as follows：
Classification And Nomenclature：Hybridoma cell strain 1G9, preservation date is on May 23rd, 2016, and depositary institution's full name is Chinese allusion quotation
Type culture collection, abbreviation CCTCC, depositary institution address：Wuhan University, deposit number is：CCTCC NO：
C2016105。
2. monoclonal antibody is coated with the determination of concentration and enzyme labelled antibody working concentration
Monoclonal antibody 1G9 ' coating concentration and 1G9 ' E working concentration are determined using square formation titration.Specific side
Method is as follows：With coating buffer (0.05M, pH 9.6 carbonate buffer solution) by monoclonal antibody 1G9 ' gradient dilutions, 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 solutions
(concentration expressed in percentage by volume) Tween 20 PBS (pH 7.4, concentration are 10mM)) washing, pat dry；With containing 1% (quality hundred
Point concentration) BSA PBST buffer solutions close 1h under the conditions of 37 DEG C, and wash, pat dry；100 μ L PCV2-NJ is added in sample well
Strain virus liquid (obtains P values), is acted under the conditions of 37 DEG C in 1h times, negative hole with PK15 cell pyrolysis liquids replacement PCV2 viruses
(obtaining N values)；Washed, patted dry using PBST buffer solutions；1G9 ' the E for being 4mg/mL by concentration with the PBST buffer solutions containing 1%BSA
Be diluted to 2000,1000,500, tetra- concentration of 250ng/mL, be separately added into each concentration monoclonal antibody 1G9 ' hole
Enzyme labelled antibody 1G9 ' the E of 100 μ L various concentrations, 1h is acted under the conditions of 37 DEG C；Horseradish peroxidase enzyme catalytic bottom is added per hole
Thing A liquid and the isometric mixed solution of B liquid (be the same as Example five) 100 μ L, 37 DEG C of colour developing 15min；It is eventually adding 100 μ L 2M sulphur
Acid solution terminating reaction, detects OD values under 450nm wavelength.Selected P/N values are maximum, positive hole OD450Value is more than corresponding to 1.0
Capture antibody (i.e. coated antibody) coating concentration and enzyme labelled antibody concentration be optimum condition.It can be seen from data in table 5
Monoclonal antibody 1G9 ' most preferably coating concentration is 1 μ g/mL, and the best use of concentration of enzyme labelled antibody is 500ng/mL.
The coated antibody concentration of table 5 is determined with enzyme labelled antibody working concentration
3. the determination of antigenic action time
ELISA antigen detections are carried out with the control of known yin and yang attribute, are reacted by the program having determined, antigen is anti-with coating
Body reacts 30min, 60min, 90min, 120min respectively at 37 DEG C, in the case of other conditions and response procedures identical, enters
Row ELISA antigens detect that it is optimum reacting time in 2.0 or so action time to take maximum, the positive values of P/N.The number in table 6
According to it is 60min in 37 DEG C of the best use of time with coated antibody to determine antigen.
The antigen of table 6 is determined with coated antibody action time
| 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 times
By the reaction condition having had determined that, ELISA antigen detections, 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.Reaction time when selecting P/N maximum is enzyme
The best effort time of labeling antibody.According to data in table 7, the best effort time for determining enzyme labelled antibody 1G9 ' E is 60min.
Enzyme labelled antibody 1G9 ' the E action times of table 7 determine
| 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 detections, developing time difference are carried out with the control of known yin and yang attribute
For 10min, 15min, 20min.Time when selecting P/N maximum is developing time.The data in table 8, determine that nitrite ion is optimal
Action time is 15min.
The determination of the developing time of table 8
| 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 scope
By standard items mother liquor, (viral level is 105.5TCID50/ mL PCV2-NJ strain virus nutrient solutions, preparation method is shown in reality
Apply example five) carry out doubling dilution, make its PCV2 content for 100,200,400,800,1600,3200,6400,12800,25600,
51200、102400TCID50/ mL, is reacted according to set ELISA, prepares standard curve, selects R2PCV2 more than 0.99
Content range is standard curve range.The corresponding PCV2 contents of standard curve are 200-3200TCID50/mL.It is shown in Table 9 and Fig. 5.
The standard curve range of table 9
The assembling of the kit of example five and application method
1. porcine circovirus 2 type antigen immue quantitative detection reagent box of the present invention includes：
(1) detection plate：Anti- PCV2 monoclonal antibodies 1G9 ' is diluted using 0.05M, pH 9.6 carbonate buffer solution (to implement
In example two prepared by the method for title 7) to 1 μ g/mL, ELISA Plate is added with 100 μ L/ holes, 16-18h is coated with the conditions of 4 DEG C, is used
Cleaning solution (with (9) in the present embodiment title 1) is washed, and is patted dry；With the cleaning solution containing 1% (mass percentage concentration) BSA (with this reality
Apply in a title 1 (9)) 1h is closed under the conditions of 37 DEG C, washing, dry, Vacuum Package obtain detection plate；
(2) the anti-PCV2 monoclonal antibodies 1G9 ' (being abbreviated as 1G9 ' E) of horseradish peroxidase-labeled：1G9 ' E (embodiments
In three method prepare) concentration be 500ng/mL.
(3) standard items mother liquor：Viral level is 105.5TCID50/ mL PCV2-NJ strain virus nutrient solutions.PCV2-NJ plants of diseases
Poison culture liquid and preparation method thereof：With the MEM medium culture PK15 cells containing 10% (v/v) calf serum, cell fusion degree is treated
When reaching 90%, PCV2-NJ plant are inoculated with, in 37 DEG C of cultures 72 hours, by viral cultures multigelation 3 times, 4 DEG C, 12000r/
Min centrifuges 20min, removes cell fragment, takes supernatant as PCV2-NJ strain virus nutrient solutions.
(4) negative control：PK15 cell pyrolysis liquids.With the MEM medium cultures PK15 containing 10% (v/v) calf serum
Cell, when cell fusion degree reaches 90%, is placed in multigelation 3 times under -20 DEG C and room temperature condition, then by cell culture fluid
Freeze thawing liquid is collected, 12000g/min centrifugation 10min take supernatant as PK15 cell pyrolysis liquids.
(5) Sample dilution：MEM culture mediums (are purchased from GIBCO).
(6) horseradish peroxidase enzyme catalytic substrate A liquid：Volumetric concentration is 3% H2O2The aqueous solution.
(7) horseradish peroxidase enzyme catalytic substrate B liquid：By the 3,3' that 1mL concentration is 10mg/mL, 5,5'- tetramethyl biphenyls
It is to be made into the 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, pH are the compound method of 6.0 phosphate buffer：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：2M aqueous sulfuric acid.
(9) cleaning solution：Final concentration of 0.05% (concentration expressed in percentage by volume) is added with pH 7.4,10mM PBS
Tween 20.PH 7.4,10mM PBS formula：NaCl 8g、KCl 0.2g、KH2PO40.27g、Na2HPO4·
12H2O 3.54g are dissolved in water, are then settled to 1L.
2. the application method of porcine circovirus 2 type antigen immue quantitative detection reagent box of the present invention：
(1) standard items/sample is added：Standard items mother liquor is diluted to standard curve range using Sample dilution.By sample
Appropriate multiple is diluted using Sample dilution.The dilution of standard items mother liquor or sample is added into detection plate with 100 μ L/ holes
1h is incubated in liquid, 37 DEG C of insulating boxs；Negative control (PK15 cell pyrolysis liquids) is added in negative control hole.Use cleaning solution detersive enzyme
Target 3 times.
(2) 1G9 ' E are added：1G9 ' E, 37 DEG C of incubation 1h are added into ELISA Plate with 100 μ L/ holes；Use cleaning solution detersive enzyme
Target 3 times.
(3) develop the color：Horseradish peroxidase enzyme catalytic substrate solution A liquid and substrate solution B liquid are mixed in equal volume, with 100 μ L/ holes
Add in ELISA Plate, 37 DEG C of lucifuges colour developing 10-15min；Terminate liquid is added with 100 μ L/ holes.
(4) determine：ELISA Plate gently vibrates reading immediately after mixing, is determined under wavelength 450nm per hole light absorption value.According to
Standard curve, calculates the concentration of PCV2 antigens 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,
12800TCID50Negative control (PK15 cell pyrolysis liquids) is added in/mL, negative control hole, is carried out using method in embodiment five
Detection.Detect the light absorption value of sample well (standard items mother liquor dilution) and negative control hole at 450nm wavelength.By P/N>2.1
When minimum PCV2 contents be set to kit sensitivity.As a result the sensitivity of visualizingre agent box is 200TCID50/ mL, is shown in Table 10.
The kit sensitivity of table 10
2. kit is specific
Using kit and detection method in embodiment five, to CSFV (CSFV), pig blue-ear disease malicious (PRRSV), pig
Parvovirus (PPV), PRV (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 more than 2.1 according to P/N values, to judge whether kit and sample have friendship
Fork.As seen from Figure 6, other swine diseases virus and kit no cross reaction of the present invention in addition to PCV2, illustrate kit of the present invention
With good specificity.
3. kit of the present invention is detected to the broad spectrum activity of PCV2 different genes hypotypes
Investigate kit in embodiment five (being labeled as kit first) and commercial kitPCV2Ag
Capture (Synbiotics being purchased from, 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 detects what is obtained to 6 kinds of different strains
OD450Value, as a result shows：Kit of the present invention can be detected to 6 kinds of PCV2 strains, with 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
Prepare according to ascites → horseradish peroxidase-labeled → kit manufacture of antibody purification → monoclonal antibody
Order three batch kits of independent production, and carry out differences between batches measure.Different virus is detected using three batch kits simultaneously
PCV2 virus liquids (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 the kit batch of table 12450Value)
5. kit of the present invention and the correlation of IFA methods (immunofluorescence technique)
In order to verify the uniformity of kit of the present invention and IFA method testing results, 10 parts of PCV2 virus liquids are have chosen same
Shi Caiyong IFA methods 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 illustrate kit of the present invention to being no more than 8.13% with a pattern detection result difference
The detected value no significant difference of detected value and IFA.
The kit of the present invention of table 13 and the correlation of IFA method testing results
6. application of the kit of the present invention in detection PCV2 subunit vaccine antigenics
Using kit of the present invention and its application method, respectively to Escherichia coli and the PCV2Cap of baculovirus expression
The virus like particle assembled (be shown in by preparation method：1st, Zhao Xiaoyun, Qiao Xuwen, Chen Jin, Li Pengcheng, Yu Xiaoming, Zhu Guoqiang, Zheng
It rises, and marquis utilizes E.coli expression carrying Cap gene of porcine circovirus type 2 production virus sample particle vaccines [J] Chinese agricultures after ripple
Science, 2015, (05):976-986；2nd, the recombinant virus like-particles of 2 type porcine circovirus nucleocapsid protein Cap genes are expressed,
Patent No. CN200810024644.9) detected.Check sample is that Escherichia coli empty bacterium crushes liquid and baculoviral crushes liquid.
Testing result is shown in Fig. 7.As seen from Figure 7 the PCV2Cap assemblings of kit of the present invention and Escherichia coli and baculovirus expression and
Into virus like particle reactivity worth it is good, and do not reacted with corresponding host cell, therefore can be used for detection PCV2 subunits epidemic disease
Seedling antigen.
Claims (7)
1. a kind of hybridoma cell strain 1G9 for secreting the type monoclonal antibody of resisting porcine circovirus 2, preserving number is：CCTCC NO:
C2016105。
2. the type monoclonal antibody of resisting porcine circovirus 2 that hybridoma cell strain described in claim 1 is secreted.
3. a kind of porcine circovirus 2 type antigen immue quantitative detection reagent box, the detection kit includes:Detection antibody and coating are had the right
Profit requires the ELISA Plate of the anti-type monoclonal antibody of pig annulus 2 described in 2, and the detection antibody is described in the claim 2 that enzyme is marked
The anti-type monoclonal antibody of pig annulus 2.
4. porcine circovirus 2 type antigen immue quantitative detection reagent box according to claim 3, it is characterised in that the detection antibody
The anti-type monoclonal antibody of pig annulus 2 described in the claim 2 of horseradish peroxidase-labeled.
5. the porcine circovirus 2 type antigen immue quantitative detection reagent box according to claim 3 or 4, it is characterised in that the enzyme mark
Plate is coated using the anti-type monoclonal antibodies of pig annulus 2 of 0.5-1.5 μ g/mL；The concentration of the detection antibody is 400-
600ng/mL。
6. porcine circovirus 2 type antigen immue quantitative detection reagent box according to claim 5, 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 105.5TCID50/ mL PCV2-NJ strain virus nutrient solutions；The feminine gender
Compare as PK15 cell pyrolysis liquids；The Sample dilution is MEM nutrient solutions；The horseradish peroxidase enzyme catalytic substrate A liquid
The H for being 3% for volumetric concentration2O2The aqueous solution；The horseradish peroxidase enzyme catalytic substrate B liquid is to contain 3,3', 5,5'- tetramethyls
The solution of base benzidine；The terminate liquid is 2M aqueous sulfuric acid；The cleaning solution is added with final concentration of 0.05%
Tween 20 pH 7.4,10mM PBS.
7. application of the kit described in claim 3 in quantitative detection porcine circovirus 2 type antigen.
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| CN109709330A (en) * | 2018-12-25 | 2019-05-03 | 内蒙古必威安泰生物科技有限公司 | A kind of foot and mouth disease virus competitive ELISA detection kit |
| CN109799351A (en) * | 2018-09-19 | 2019-05-24 | 天津瑞普生物技术股份有限公司 | Porcine circovirus 2 type double-antibody sandwich elisa kit and its application |
| CN110794148A (en) * | 2019-11-06 | 2020-02-14 | 海南省农业科学院畜牧兽医研究所 | Double-antibody sandwich ELISA quantitative detection kit for porcine inflammasome NLRP3 and application thereof |
| CN111471102A (en) * | 2020-04-23 | 2020-07-31 | 西北农林科技大学 | Coding gene, vector, anti-idiotype nano antibody, preparation method and application thereof |
| CN114958776A (en) * | 2022-06-30 | 2022-08-30 | 天康制药(苏州)有限公司 | PCV2 monoclonal antibody and hybridoma cell strain 1A6 secreting same |
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