CN108918870A - The liquid-phase chip detection method of pig parvoviral - Google Patents
The liquid-phase chip detection method of pig parvoviral Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract
The invention discloses a kind of liquid-phase chip detection methods of pig parvoviral, it is desirable to provide a kind of high specificity, high sensitivity, testing cost is low, the liquid-phase chip detection method of PPV antibody easy to operate；Its technical solution includes the following steps：Antigen coat；2) by 2 times of doubling dilutions of serum sample, the microballoon being coated with is diluted to 50/μ L with PBS-TBN, microballoon is resuspended；) into 96 hole elisa plates, the microballoon that 50 μ L have diluted is added in every hole；Serum sample is added；It is incubated for 120min, microballoon is separated, the PBS-TBN cleaning solution in 100 holes μ L/ is added, discards washing lotion, reaction plate is removed from magnetic separator, the goat-anti pig secondary antibody of 100 μ L biotin labelings is added, is incubated for, separates microballoon, discard reaction solution, the Streptavidin 100uL of phycoerythrin label is added in washing lotion, every hole, is incubated for 30min；Microballoon is separated, abandons and 100 μ LPBS-TBN is added, reaction plate is placed in oscillator oscillation 1min；Utilize the ratio for taking median fluorescence and calculate positive MFI value Yu feminine gender MFI value machine-readable in luminex system.
Description
Technical field
The present invention relates to the detection methods of pig parvoviral, in particular to the liquid-phase chip of pig parvoviral is examined
Survey method, belongs to field of biotechnology.
Background technique
Porcine parvovirus infection is the clinical common breeding difficulty of the sow caused by parvovirus (Parvovirus, PPV)
Property disease, the sixties in last century in European first discovery, the whole world is found later.China northern territory and Southern Coast
Province is raised pigs compact district also this popular disease.Reproductive performance is the main production target for investigating sow economic value, sow after infection
Utility value substantially reduces, huge to cultivation field loss.Liquid-phase chip technology is a new fast high-flux detection technique, should
Technology collecting type cell technology, fluorescence-encoded micro-beads, laser, Digital Signal Processing and traditional biochemical technology are one in one
The multi-functional analysis platform of kind.
Chinese patent ZL201510140003.X discloses a kind of pig parvoviral liquid-phase chip detection kit, uses
Monoclonal antibody technology of preparing is prepared for pig parvoviral monoclonal antibody, and is coupled with No. 21 microballoons of carboxylated, and it is micro- to obtain capture monoclonal antibody-
Ball；Rabbit source pig parvoviral polyclonal antibody is prepared for using how anti-technology of preparing, it is mostly anti-to obtain detection through biotinylation；It adopts
The mixed of pig parvoviral structural proteins truncate VP2312 and non-structural protein truncate NS1217 is prepared for recombinant DNA technology
Three recombinant proteins, i.e. VP2312-NS1217-VP2312 are closed, as examination criteria product.What it was detected is pig parvoviral sheet
Body, for example, pork liver infected go to detect this pig after the virus whether have the virus in vivo, detection method is in magnetic bead
It is coated with monoclonal antibody above, goes in the sample for detecting this infection whether have this virus, sample acquisition difficulty is big, and operation is not square
Just.
Chinese patent ZL201410606021.8 discloses a kind of liquid phase genetic chip of synchronous detection five boars virus
It is more to carry out multiple asymmetric nucleic acid amplification joint by extracting the pig viral nucleic acid in measuring samples for nucleic acid and its detection method
Weight liquid phase genetic chip (suspending chip) detection equally exists sampling hardly possible, the problem of operation sequence complexity.
Summary of the invention
In view of the above-mentioned problems, testing cost is low the object of the present invention is to provide a kind of high specificity, high sensitivity, operation
The liquid-phase chip detection method of simple PPV antibody.
In order to solve the above technical problems, technical solution provided by the invention is such：
A kind of liquid-phase chip detection method of pig parvoviral, successively includes the following steps：
1) antigen coat
2) xMAP is detected
(1) detection serum sample prepares, by 2 times of doubling dilutions of serum sample；
(2) the PPV microballoon (i.e. fluorescence magnetic nanoscale microsphere) being coated with is diluted to 50/μ L with PBS-TBN；
(3) vortex oscillation and each 10s of ultrasound, are resuspended microballoon；
(4) into 96 hole elisa plates, the microballoon that 50 μ L have diluted is added in every hole；
(5) serum sample that 50 μ L have diluted by a certain percentage is added；(specific ratio)
(6) plate lid is covered, reaction plate is placed in oscillator, room temperature, which is slightly shaken, is incubated for 120min；
(7) reaction plate is placed in magnetic separator 3-5min, separates microballoon, discard reaction solution, 100 holes μ L/ are added
PBS-TBN cleaning solution, discards washing lotion, is repeated once；
(8) reaction plate is removed from magnetic separator, the goat-anti pig secondary antibody of 100 μ L biotin labelings is added；
(9) plate lid is covered, reaction plate is placed in oscillator, room temperature, which is slightly shaken, is incubated for 60min；
(10) reaction plate is placed in magnetic separator 3-5min, separates microballoon, discards reaction solution.100 holes μ L/ are added
PBS-TBN cleaning solution discards washing lotion and is repeated once；
(11) the Streptavidin 100uL of phycoerythrin label is added in every hole, and room temperature, which is slightly shaken, is incubated for 30min；
(12) reaction plate is placed in magnetic separator 3-5min, separates microballoon, discards reaction solution, and the PBS- in 100 holes μ L/ is added
TBN cleaning solution, discards washing lotion, is repeated once；
(13) reaction plate is removed from magnetic separator, 100 μ LPBS-TBN is added, reaction plate is placed in oscillator oscillation
1min；
(14) using the ratio for taking median fluorescence and calculate positive MFI value Yu feminine gender MFI value machine-readable in luminex system,
That is P/N value is defined as the positive more than or equal to 3 result by P/N value, when serum difference dilution and MFI value are in a linear relationship, and
The P/N value of maximum positive reference product is greater than 5, illustrates that coating is effective.
Further, the liquid-phase chip detection method of above-mentioned pig parvoviral, the antigen coat concentration are 20ul/
5x105 magnetic bead.
Further, the liquid-phase chip detection method of above-mentioned pig parvoviral, the revolving speed of the oscillator are
800rpm。
Further, the liquid-phase chip detection method of above-mentioned pig parvoviral, the goat-anti pig secondary antibody working concentration 2
μg/mL。
Further, the work of the liquid-phase chip detection method of above-mentioned pig parvoviral, the SAPE 100uL is dense
Spend 2.5ug/ml.
Further, the liquid-phase chip detection method of above-mentioned pig parvoviral contains 0.01M in the PBS-TBN
PBS, 0.1%BSA, 0.05%NaN3,0.02%Tween-20.
Further, the liquid-phase chip detection method of above-mentioned pig parvoviral, the microballoon are the glimmering of Luminex company
Photomagnetism nanosphere.
Compared with prior art, technical solution provided by the invention has the advantages that：
PPV antibody is enough realized under conditions of very low cost while being detected to technical solution provided by the invention, when detection
Between section, save time cost.
For technical solution provided by the invention by specific detection, Sensitivity comparison, discovery is anti-without intersecting with other antibody
It answers, high sensitivity, a kind of low, the method for Multiple detection that provides new cost for antibody test.
Detailed description of the invention
Fig. 1 is PPV specificity experiments result；
Fig. 2 is the xMAP sensitivity experiment result of PPV.
Specific embodiment
With reference to embodiment, claim of the invention is described in further detail, but do not constituted pair
Any restrictions of the invention, the modification of anyone limited times made in the claims in the present invention protection scope etc, still exist
Within claims of the invention.
1 material and equipment
1.2 viruses, strain, carrier and clinical sample
PPV inactivated vaccine is purchased from Shandong animal health company, product batch number 150042.The purchase of ELISA antibody assay kit
From Ke Qian biotech firm.
1.2 positive and negative control serums and clinical sample
Positive and negative standards' serum comes from ELISA kit.Clinical sample comes from Guangdong pig farm.
1.3 enzymes and other reagents
The goat-anti pig secondary antibody of biotin labeling is purchased from Beijing Suo Laibao Science and Technology Ltd, the strepto- parent of phycoerythrin label
Invitrogen company is purchased from plain (SAPE)；Dilution and washing lotion are PBS-TBN (0.01M PBS+0.1%BSA+
0.05%NaN3+0.02%Tween-20)；Fluorescence magnetic nanoscale microsphere, microballoon coupling reagent kit are purchased from U.S. Luminex public affairs
Department；
1.4 instrument
Bio-RAD company of U.S. electrophoresis apparatus, gel imaging system, Tiangeng Tguide nucleic acid automatically extract, Luminex 200
XMAP fluorescence detection platform.
2 methods
2.1 antigen coat
PPV vaccine antigen is coated with according to the specification of xMAPAntibody Coupling Kit kit.
2.2.xMAP (indirect immunoassay) is tested
(1) detection serum sample prepares, by 2 times of doubling dilutions of serum sample.
(2) microballoon being coated with 50/μ L is diluted to PBS-TBN (to be coated with step and say according to microballoon coupling reagent kit
Bright book carries out.
(3) vortex oscillation and each 10s of ultrasound, are resuspended microballoon.
(4) into 96 hole elisa plates, the microballoon that 50 μ L have diluted is added in every hole.
(5) serum sample that 50 μ L have diluted by a certain percentage is added.
(6) plate lid is covered, reaction plate is placed in oscillator (800rpm), room temperature, which is slightly shaken, is incubated for 120min.
(7) reaction plate is placed in magnetic separator 3-5min, separates microballoon, discards reaction solution.100 holes μ L/ are added
PBS-TBN cleaning solution, discards washing lotion.It is repeated once.
(8) reaction plate is removed from magnetic separator, the goat-anti pig secondary antibody (working concentration 2 of 100 μ L biotin labelings is added
μg/mL)。
(9) plate lid is covered, reaction plate is placed in oscillator (800rpm), room temperature, which is slightly shaken, is incubated for 60min.
(10) reaction plate is placed in magnetic separator 3-5min, separates microballoon, discards reaction solution.100 holes μ L/ are added
PBS-TBN cleaning solution, discards washing lotion.It is repeated once.
(11) SAPE 100uL (working concentration 2.5ug/ml) is added in every hole, and room temperature, which is slightly shaken, is incubated for 30min.
(12) reaction plate is placed in magnetic separator 3-5min, separates microballoon, discards reaction solution.The PBS- in 100 holes μ L/ is added
TBN cleaning solution, discards washing lotion.It is repeated once.
(13) reaction plate is removed from magnetic separator, 100 μ LPBS-TBN is added.Reaction plate is placed in oscillator oscillation
1min。
(14) using in luminex system it is machine-readable take median fluorescence (Median Fluorescent Intensity,
MFI)。
And calculate the ratio of positive MFI value Yu feminine gender MFI value, i.e. P/N value.Result by P/N value more than or equal to 3 is defined as
The positive, when serum difference dilution and MFI value are in a linear relationship, and the P/N value of maximum positive reference product is greater than 5, illustrates to be coated with
Effectively.By above-mentioned experiment, finally determines and the serum to be checked of suitable dilution is examined using the magnetic bead of best package amount
It surveys.
The optimization of 2.3 xMAP testing conditions
The best peridium concentration of recombinant antigen determines：By inactivated vaccine measurement concentration be 4.978mg/ml, respectively select 10,
20,40, the recombinant antigen of 105 magnetic bead of 80ul/5x is coated with magnetic bead.By PPV artificial challenge's positive serum (positive
Control) doubling dilution 1:It is detected after 25, while detecting negative control sera (negative control), according to foundation
The detection of xMAP detection method.
2.4 specific test
Using foundation xMAP method to respectively detect swine fever virus (Classical Swine FeverVirus,
CSFV), circovurus type 2 (Porcine Circovirus type II, PCV-2), Pseudorabies virus (Pseudorabies
Virus, PRV), porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory
Syndrome Virus, PRRSV) and parvovirus (Parvovirus, PPV) antibody, to verify the detection side of this experiment foundation
Method whether there is cross reaction.
2.5 sensitivity tests
PPV positive serum is made 2 multiple proportions to be serially diluted respectively, sensitivity Detection is done to dilute serum using xMAP method.
2.6 repetitive test
Three times to same a sample Parallel testing, three repetitions every time calculate crowd interior and interassay coefficient of variation CV%.
The detection of 2.7 clinical samples
30 parts of clinical samples being collected into are detected using xMAP method, and are compared with ELISA testing result.
3 results
The determination of 3.1 recombinant antigens best peridium concentration and Cutoff value
It is 4.978mg/ml by inactivated vaccine measurement concentration, selects 10,20,40,80ul/5x 10 respectively5The recombination of magnetic bead
Antigen coat magnetic bead.By PPV artificial challenge positive serum (positive control) doubling dilution 1:It detects after 25, examines simultaneously
It surveys negative control sera (negative control), is detected according to the xMAP detection method of foundation.It is above machine-readable to take MFI value, inspection
The results are shown in Table 1 for survey, and when the package amount of recombinant antigen is 20ul, P/N value is larger, and reaches plateau, but P/N is opposite
Stablize, it is thus determined that the best peridium concentration of antigen is 20ul/5x 105Magnetic bead.Meanwhile 16 parts of pig negative serum samples are carried out
XMAP detection reads MFI value and calculates its average and standard deviation, and the results are shown in Table 2, what 16 parts of pig negative serums measured
MFI value average value+3SD primarily determines as the Pathogen test threshold value (Cutoff value), therefore the Cut off value of PPV is 613.79.
1 PPVxMAP method of table detects 16 parts of clinical serums
The best peridium concentration definitive result of 2 PPV antigen of table
The detection of 3.2 specificity
Specificity experiments the results show that PPV microballoon only and PPV antibody response, with other antibody no cross reactions.PPV is micro-
The specificity that ball detects the xMAP method of PPV antibody is good.
The remolding sensitivity pair of 3.3xMAP detection method and ELISA detection method
Positive serum doubling dilution 1:MFI value is detected after 25 and is mapped, and is Positive judgement standards, sun by cut off value
Property lowest detection limit be 1:200.And the sensitivity of ELISA is 1:25.
The xMAP sensitivity experiment result of 3 PPV of table
3.4 repeated experiment results
It takes with a sample extension rate 1:25, in triplicate, three repetitions every time.Repetitive test result is aobvious in batch
Show, the maximum Cv of PPV detection is 9.25%.Repetitive test between batch is the results show that the Cv value of PPV detection is 6.88%.More than
As a result it is respectively less than 10%, shows that the detection method has preferable repeatability.
4 PPV repeated experiment result of table
3.5 clinical detections are compared with ELISA
29 parts of samples are carried out with the 6 weight xMAP inspections of six kinds of antigens such as PCV-2, TGEV, PRV-GE, PRRSV, CSFV, PPV
It surveys, serum dilution is unified for 1:800.It is the positive that final criterion, which is set to greater than cut of value, and the calculating of cut off value is public
Formula is：Negative+3 times of sample standard deviations of average value (mean value calculation need to remove the biggish numerical value of deviation).
5 PPV xMAP of table and the sample comparative analysis of ELISA test experience animal clinical
The consistency analysis of table 6 PPV xMAP and ELISA method
* refer to number identical with ELISA experiment Parallel testing result in xMAP experiment
By above-mentioned analysis it can be found that compared with currently used ELISA kit, liquid-phase chip technology be can be realized
A variety of antigens are detected simultaneously under conditions of very low cost, also save time cost.This method has been carried out specifically herein
Property detection, Sensitivity comparison, discovery with other antibody no cross reactions, high sensitivity.
Use vaccine totivirus as antigen coat magnetic bead.Determine that the best package amount of PPV is outside 20uL, also to biotin mark
The secondary antibody of note and the final of streptavidin are optimized using concentration, and discovery is 2ug/ml when secondary antibody concentration, and chain enzyme parent
When use concentration with element is 2.5ug/ml, P/N value is maximum.In conclusion the application establishes the liquid phase core of detection PPV antibody
Piece method, a kind of low, the method for Multiple detection that provides new cost for antibody test.
Claims (7)
1. a kind of liquid-phase chip detection method of pig parvoviral, which is characterized in that successively include the following steps：
1) antigen coat
2) xMAP is detected
(1) detection serum sample prepares, by 2 times of doubling dilutions of serum sample；
(2) the PPV microballoon being coated with is diluted to 50/μ L with PBS-TBN；
(3) vortex oscillation and each 10s of ultrasound, are resuspended microballoon；
(4) into 96 hole elisa plates, the microballoon that 50 μ L have diluted is added in every hole；
(5) 50 μ L diluted serum, dilution 1 is added:800.
(6) plate lid is covered, reaction plate is placed in oscillator, room temperature, which is slightly shaken, is incubated for 120min；
(7) reaction plate is placed in magnetic separator 3-5min, separates microballoon, discard reaction solution, the PBS-TBN in 100 holes μ L/ is added
Cleaning solution discards washing lotion, is repeated once；
(8) reaction plate is removed from magnetic separator, the goat-anti pig secondary antibody of 100 μ L biotin labelings is added；
(9) plate lid is covered, reaction plate is placed in oscillator, room temperature, which is slightly shaken, is incubated for 60min；
(10) reaction plate is placed in magnetic separator 3-5min, separates microballoon, discard reaction solution, the PBS- in 100 holes μ L/ is added
TBN cleaning solution discards washing lotion and is repeated once；
(11) the Streptavidin 100uL of phycoerythrin label is added in every hole, and room temperature, which is slightly shaken, is incubated for 30min；
(12) reaction plate is placed in magnetic separator 3-5min, separates microballoon, discards reaction solution, and the PBS-TBN in 100 holes μ L/ is added
Cleaning solution discards washing lotion, is repeated once；
(13) reaction plate is removed from magnetic separator, 100 μ LPBS-TBN is added, reaction plate is placed in oscillator oscillation 1min；
(14) ratio for taking median fluorescence and calculate positive MFI value Yu feminine gender MFI value machine-readable in luminex system, i.e. P/N are utilized
P/N value is defined as the positive more than or equal to 3 result by value, when serum difference dilution and MFI value are in a linear relationship, and maximum
The P/N value of positive reference product is greater than 5, illustrates that coating is effective.
2. the liquid-phase chip detection method of pig parvoviral according to claim 1, which is characterized in that the antigen packet
It is 20ul/5x 10 by concentration5Magnetic bead.
3. the liquid-phase chip detection method of pig parvoviral according to claim 1, which is characterized in that the oscillator
Revolving speed be 800rpm.
4. the liquid-phase chip detection method of pig parvoviral according to claim 1, which is characterized in that the goat-anti pig
2 μ g/mL of secondary antibody working concentration.
5. the liquid-phase chip detection method of pig parvoviral according to claim 1, which is characterized in that described
The working concentration 2.5ug/ml of SAPE100uL.
6. the liquid-phase chip detection method of pig parvoviral according to claim 1, which is characterized in that the PBS-
PBS containing 0.01M, 0.1%BSA, 0.05%NaN3,0.02%Tween-20 in TBN.
7. the liquid-phase chip detection method of pig parvoviral according to claim 1, which is characterized in that the microballoon is
The fluorescence magnetic nanoscale microsphere of Luminex company.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111551737A (en) * | 2020-04-14 | 2020-08-18 | 苏州药明康德新药开发有限公司 | Method for detecting multiple cytokines by flow type microsphere matrix method |
| CN112485419A (en) * | 2020-11-25 | 2021-03-12 | 广州市进德生物科技有限公司 | Zinc transporter 8 antibody detection kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111551737A (en) * | 2020-04-14 | 2020-08-18 | 苏州药明康德新药开发有限公司 | Method for detecting multiple cytokines by flow type microsphere matrix method |
| CN112485419A (en) * | 2020-11-25 | 2021-03-12 | 广州市进德生物科技有限公司 | Zinc transporter 8 antibody detection kit |
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Application publication date: 20181130 |