CN108896767A - A kind of PCV2 antibody detection method based on xMAP technology - Google Patents
A kind of PCV2 antibody detection method based on xMAP technology Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
Abstract
The invention discloses a kind of PCV2 antibody detection method based on xMAP technology, the preparation including recombinant antigen；Purify destination protein；Recombinant antigen coating；Carry out xMAP test：Calculate the ratio of positive MFI value and 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.The advantage of the invention is that：The MFI value difference of PCV2 microballoon and other swine disease original serum is different obvious, and no cross reaction can accurately identify PCV2 antibody；Detection limit is low, high sensitivity, reproducible.
Description
Technical field
The present invention relates to a kind of detection technique field of swine disease poison more particularly to the detection technique fields of PCV2.
Background technique
PCV2 is the common viral infectious disease pathogens of pig.PCV2 vaccine popularity rate only has 30% or so at present, once
Morbidity mainly causes development to slow down and the symptoms such as sow breeding difficulty, the death rate etc. reach as high as 25% piglet.2010
The listing of August China 1st generation annulus vaccine.But after 2010, annulus positive rate is still up to 48.8%.Its antibody detection method master
If ELISA kit, higher cost is bad to the early detection effect of virus.And liquid-phase chip technology (flexible
MuLtiple-analyte profiling solve for unknown x, xMAP) as a kind of novel high-throughput detection
Technology has the characteristics that easy to operate, accuracy is high, Multiple detection etc. is better than conventional method, is suitable for the quick of a variety of cause of diseases
Detection.The advantage of Luminex be exactly it is at low cost, tens kinds of antibody can be detected simultaneously, it is high-throughput.This research is dedicated to establishing
A kind of Luminex detecting PCV2 antibody.
Summary of the invention
The purpose of the present invention is to provide a kind of PCV2 antibody detection methods based on xMAP technology, to solve PCV2 antibody
Detection sensitivity, cost, in terms of problem.
The present invention adopts the following technical scheme that in order to achieve the above object：
A kind of PCV2 antibody detection method based on xMAP technology, includes the following steps：
(1) preparation of recombinant protein：Positive bacteria is cultivated first and induces its expression, and then ultrasonication positive bacteria, makes
Cellular lysate is simultaneously centrifuged, and supernatant is precipitated and is separated, respectively takes a certain amount of precipitating lysate of a certain amount of supernatant on albumen respectively
In sample buffer, SDS-PAG analysis is carried out, the expression position of the i.e. recombinant antigen of destination protein is determined according to destination protein stripe size
It sets；
(2) purifying protein operation is carried out, protein sample PCV2 CAP albumen is obtained；
(3) recombinant antigen is coated with：The protein sample PCV2 CAP albumen that step (2) is obtained, using xMAP
Antibody Coupling Kit kit is simultaneously coated with according to its specification, and the microballoon being coated with is obtained；
(4) xMAP test is carried out：Test serum sample carries out 2 times of doubling dilutions；Dilute the microballoon being coated with；To microballoon
It is vibrated, ultrasound, resuspension processing；Into elisa plate, microballoon and test serum sample, oscillation incubation elisa plate is added in every hole；
Elisa plate is placed in magnetic separator, separates microballoon, discards reaction solution, washs, and the goat-anti of biotin labeling is added toward elisa plate
Pig secondary antibody；Oscillation incubation elisa plate, elisa plate are placed in magnetic separator, separate microballoon, discard reaction solution, wash, toward ELISA
SAPE, oscillation incubation elisa plate is added in plate, and elisa plate is placed in magnetic separator, separates microballoon, discards reaction solution, washs, benefit
Median fluorescence is taken with machine-readable in luminex system；
(5) ratio of positive MFI value and feminine gender MFI value, i.e. P/N value are calculated；Result by P/N value more than or equal to 3 defines
For the positive, when test 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, says
Bright coating is effective.
Further, the concrete operations of the step (1) are：The PCV2-CAP-pet32a plasmid of purchase is transformed into
BL21 competent cell forms positive bacteria；Positive bacterium solution 100uL is taken, is inoculated into the 10ml culture medium of the mycin of benzyl containing ammonia, 37 DEG C
Shaken cultivation 2-3 hours, when OD value is about 0.6,1ml bacterium solution is taken out as normal control, IPTG is added to end in remaining bacterium solution
Concentration is 0.5-1mmol/L, 16 DEG C or 37 DEG C induction 4-16h.Induction bacterium solution 1ml is placed in 5min on ice, 5000g is centrifuged 5min
Thallus is harvested, 500uLPBS has hanged the thallus, and 10000rpm is centrifuged 5min, abandons supernatant；Ultrasound after 1ml PBS has hanged again is added
Thallus is crushed to crack completely；
Bacterium solution after above-mentioned ultrasonication is centrifuged 1min with 12000rpm, separates supernatant precipitating, 500uL PBS hangs
Precipitating respectively takes precipitating, supernatant 40uL to be added in 5 × albumen of 10uL sample-loading buffer, and boiling water boils 5-10min, and 20uL is taken to carry out
SDS-PAGE analysis, the expression position of the i.e. recombinant antigen of destination protein is determined according to destination protein stripe size；
Glue part is concentrated when electrophoresis and uses 80V constant pressure 30min, separation gel uses 110V constant pressure 1h10min, until bromophenol blue is moved
It moves separation gel lowermost end and stops electrophoresis, separation gel is put into coomassie brilliant blue staining liquid and dyes 1h, it is then de- with destainer
Color is thin out to background, and band is clear, saves gel photograph.
Further, the concrete operations of the step (4) are：
(1) prepare test serum sample, by 2 times of doubling dilutions of test serum sample；
(2) microballoon being coated with is diluted to 50/μ L with PBS-TBN；
(3) vortex oscillation and each 10s of ultrasonic treatment, have hanged 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 steps (1) have diluted is added.
(6) plate lid is covered, elisa plate is placed in oscillator, 800rpm room temperature, which is slightly shaken, is incubated for 120min；
(7) elisa plate is placed in magnetic separator 3-5min, separates microballoon, discards reaction solution；Add by the amount in 100 holes μ L/
Enter PBS-TBN cleaning solution, discards washing lotion；It is repeated once；
(8) elisa plate is removed from magnetic separator, the goat-anti pig secondary antibody of 100 μ L biotin labelings, working concentration is added
2μg/mL；
(9) plate lid is covered, reaction plate is placed in oscillator, 800rpm room temperature, which is slightly shaken, is incubated for 60min；
(10) elisa 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) elisa plate is placed in magnetic separator 3-5min, separates microballoon, discards reaction solution；Add by the amount in 100 holes μ L/
Enter PBS-TBN cleaning solution, discards washing lotion；It is repeated once；
(13) reaction plate is removed from magnetic separator, 100 μ LPBS-TBN is added；Elisa plate is placed in oscillator oscillation
1min；
(14) median fluorescence is taken using machine-readable in luminex system.
Further, the best peridium concentration of antigen is 8 μ g recombinant antigens/2.5x 10 in the step (3)5A microballoon.
The advantage of the invention is that：The MFI value difference of PCV2 microballoon and other swine disease original serum is different obvious, no cross reaction,
PCV2 antibody can accurately be identified；Detection limit is low, high sensitivity, reproducible.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes part of this application, not
Inappropriate limitation of the present invention is constituted, in the accompanying drawings：
Fig. 1 is the protein induced expression result chart of PET-32a-PCV2 CAP；
Fig. 2 is that the SDS-PAGE after PCV2 CAP protein purification runs glue result figure；
Fig. 3 is PCV2 antibody test specificity experiments result；
Fig. 4 is the sensitivity experiment result of PCV2 antibody test.
Specific embodiment
Below in conjunction with attached drawing and specific embodiment, the present invention will be described in detail, herein with schematic implementation of the invention
Example and explanation are used to explain the present invention, but not as a limitation of the invention.
18.1 materials and equipment
18.1.1 virus, strain, carrier, positive and negative control serum and test serum sample
PCV2 CAP-pet32a recombinant plasmid is purchased from Guangzhou Yi Weien Biotechnology Co., Ltd, is frozen by this laboratory；
The positive and negative standards' serum of PCV2 is purchased from Spain Ingenasa PC-IFA-PCV2/NC-IFA- respectively
PCV2；
Test serum sample comes from Guangdong pig farm.
18.1.2 enzyme and other reagents
His label protein purification kit is purchased from green skies Bioisystech Co., Ltd；
Fluorescence magnetic nanoscale microsphere, microballoon coupling reagent kit are purchased from Luminex company of the U.S.；
The goat-anti pig secondary antibody of biotin labeling is purchased from Beijing Suo Laibao Science and Technology Ltd；
The Streptavidin (SAPE) of phycoerythrin label is purchased from Invitrogen company；
Dilution and washing lotion are PBS-TBN (0.01M PBS+0.1%BSA+0.05%NaN3+0.02%Tween-
20)；
ELISA antibody assay kit is purchased from Spain INgezim company.
18.1.3 instrument
Bio-RAD company of U.S. electrophoresis apparatus, 200 xMAP fluorescence detection platform of Luminex.
18.2 methods
18.2.1 prepared by recombinant protein
18.2.1.1 the culture and inducing expression of bacterium
The PCV2-CAP-pet32a plasmid of purchase is transformed into BL21 competent cell, takes positive bacterium solution 100uL, is inoculated with
Into the 10ml culture medium of the mycin of benzyl containing ammonia, 37 DEG C shaken cultivation 2-3 hours, when OD value is about 0.6, take out 1ml bacterium solution make
For the normal control for not adding IPTG, IPTG to final concentration of 0.5-1mmol/L, 16 or 37 DEG C of induction 4-16h is added in remaining bacterium solution.
Induction bacterium solution 1ml is placed in 5min on ice, 5000g is centrifuged 5min and harvests thallus.500uLPBS hangs, 10000rpm centrifugation
5min abandons supernatant.Ultrasonication after 1ml PBS has hanged again is added to crack completely to thallus.
18.2.1.2 SDS-PAG is analyzed
Bacterium solution after above-mentioned ultrasonication is centrifuged 1min with 12000rpm, goes supernatant to precipitate respectively, 500uL PBS is outstanding
Play precipitating.Respectively take precipitating, supernatant 40uL be added 10uL 5x albumen sample-loading buffer in, boiling water boils 5-10min, take 20uL into
Row SDS-PAGE analysis.Glue part is concentrated when electrophoresis and uses 80V constant pressure 30min, separation gel uses 110V constant pressure 1h10min, until
Bromophenol blue is moved to separation gel lowermost end and stops electrophoresis, and separation gel is put into coomassie brilliant blue staining liquid and dyes 1h, is then used
Destainer decolourizes thin out to background, and band is clear, saves gel photograph.Determine that albumen recombinates according to destination protein stripe size
The expression position of antigen is in supernatant.
18.2.1.3 protein purification
Remaining supernatant is purified according to the His tag purification kit specification of green skies company, obtains albumen
Sample P CV2 CAP albumen.
18.2.2 xMAP detection method is established
18.2.2.1 antigen coat
The protein sample PCV2 CAP albumen that will be obtained, according to saying for xMAP Antibody Coupling Kit kit
Bright book is coated with, and the fluorescence magnetic nanoscale microsphere being coated with, this specification abbreviation microballoon are obtained.
18.2.2.2 xMAP tests (indirect immunoassay)
(1) prepare test serum sample, by 2 times of doubling dilutions of test serum sample.
(2) 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) serum sample that 50 μ L steps (1) have diluted 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 microballoon of best package amount
It surveys.
18.2.3xMAP the optimization of testing conditions
The best peridium concentration of recombinant antigen and CUT OFF value (determining positive or negative critical value) determine：It will recombination
Antigen diluent, every 2.5x 105A microballoon selects recombinant antigen (the i.e. purified purpose of 0.8,2,8,20,40 μ g respectively
Albumen, that is, PCV2CAP albumen) it is coated with, positive standard serum (positive control) is diluted 400 times, is pressed
It is detected according to the xMAP detection method of above-mentioned foundation, while being diluted according to by negative control sera (negative control)
It 400 times, is detected according to the xMAP detection method of above-mentioned foundation.
18.2.4 specific test
Using above-mentioned foundation xMAP method to respectively detect swine fever virus (Classical Swine Fever Virus,
CSFV), circovurus type 2 (Porcine Circovirus type II, PCV2), 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.
18.2.5 sensitivity tests
Positive standard serum is made to 2 multiple proportions respectively to be serially diluted, the xMAP method established using this experiment is to dilute serum
Do sensitivity Detection.
18.2.6 repetitive test
Three times to same a sample Parallel testing, three repetitions every time calculate crowd interior and interassay coefficient of variation CV%.
18.2.7 the detection of clinical sample
Using this experiment establish xMAP method 29 parts of clinical samples being collected into are detected, and with purchase
ELISA detection antibody kit result is compared.
18.3 results
18.3.1 prepared by recombinant protein
18.3.1.1 recombinant protein SDS-PAGE analyzes result
Bacterium solution is after IPTG inducing expression, ultrasonic Separation supernatant and precipitating.The results show that as shown in Figure 1, destination protein
20KD or so is expressed in supernatant.
18.3.1.2 recombinant protein SDS-PAGE analyzes result
SDS-PAGE after column purification is run shown in cementing fruit Fig. 2.
18.3.2 the determination of the best peridium concentration of recombinant antigen and Cutoff value
Recombinant antigen is diluted, 0.8,2,8,20,40 μ g recombinant antigens/2.5x 10 is selected5The recombinant antigen of microballoon is coated with
Microballoon detects yin by dilute 400 times of positive standard serum (positive control), while according to the xMAP detection method of foundation
Property control serum (negative control).Above machine-readable to take MFI value, testing result is as shown in table 105, when the packet of recombinant antigen
P/N value is maximum when being measured as 8 μ g, it is thus determined that the best peridium concentration of antigen is 8 μ g recombinant antigens/2.5x 105A microballoon.Together
When, xMAP detection is carried out to 14 parts of pig negative serum samples, MFI value is read and calculates its average and standard deviation, as a result such as table
Shown in 106, the MFI value average value+3SD of 14 parts of pig negative serums measurement is primarily determined as the Pathogen test threshold value (Cutoff
Value), therefore the Cutoff value of substance PCV2 xMAP detection is 1285.29.With the threshold of the multiple antibody test of last clinical sample
Value is different, and the cut off value of latter experiment need to measure every time.Because experiment condition for example serum dilution, incubation time, secondary antibody and
The differences such as SAPE concentration can influence cut off value significant.
The best peridium concentration definitive result of 15 PCV2 antigen of table
2 PCV2 xMAP method of table detects 14 parts of clinical serums
18.3.3 specific detection
It is verified, as shown in figure 3, the PCV2 microballoon (microballoon of coating recombinant antigen) of the technical program preparation and other pigs
The MFI value difference of cause of disease serum is different obvious, and no cross reaction can accurately identify PCV2 antibody.
PCV2 positive standard serum is detected as after 2 times of multiple proportions gradient dilutions by 18.3.2 step.Experimental result such as Fig. 4
It is shown, it is the positive greater than cut off value, this cut off value is tested with best package amount.The product sun of the technical program preparation
Property lowest detection limit be 1：400.ELISA lowest detection limit is 1:200.
3 PCV2 xMAP sensitivity experiment result of table
In upper table, 4164,0.97,3953.5,0.57,1652.5 represent the positive；
18.3.4 the remolding sensitivity pair of xMAP detection method and ELISA detection method
PCV2 xMAP detection is carried out to 29 parts of samples, serum dilution is unified for 1:800.And when primary antibody and secondary antibody incubation
Between be unified for 1h, secondary antibody concentration and SAPE concentration are unified for 2ug/ml, 4ug/ml respectively.Because negative control quantity is relatively
Few, the differences such as reaction condition and serum dilution, each clinical sample should measure a cut off value when detecting.
It is the positive that final criterion, which is set to MFI value greater than cut off value, and the calculation formula of cut off value is：Negative average value is (flat
Mean value computation need to remove the biggish numerical value of deviation)+3 times of sample standard deviations.
4 PCV2 xMAP of table and the sample comparative analysis of ELISA test experience animal clinical
In upper table, 2608,2413.5,2309,1986,3098,2400.5,2091,3454,2242,2771.5,3259,
2957,2167.5,2573.5,2562 be to show positive numerical value.
The consistency analysis of table 5 PCV2 xMAP and ELISA method
* refer to number identical with ELISA experiment Parallel testing result in xMAP experiment
*The identical number of the detection resuLts between xMAP and ELISA
18.3.5 repeated experiment result
Repetitive test in batch is the results show that the maximum Cv of PCV2 detection is 8.37%.Repetitive test result is aobvious between batch
Show, the Cv value of PCV2 detection is 8.82%.Result above is respectively less than 10%, shows that the detection method has preferable repeatability.
6 PCV2 repeated experiment result of table
18.4 brief summaries
The technical program organic combination fluorescence-encoded micro-beads technologies, laser analysis technology, Flow Cytometry, high speed number
The multinomial scientific and technological achievement such as word signal processing technology, computer algorithm has the characteristics that high-throughput, low cost, high sensitivity,
The developing direction that represent life science basic research and medical diagnostic techniqu is evaluated as clinical diagnosis by international industry experts
One of tendency technology.Technical principle includes following 4 points：
(1) fluorescent microsphere is dyed different fluorescence by two kinds of color classification fluorescent dyes of coding microball different ratio
Color, to obtain up to 100 kinds of fluorescence-encoded microballoons；
(2) coating is coupled specific proteins, that is, destination protein by different determinands are directed to namely with the side of covalent coupling
Formula is integrated on specific coding microball；
(3) association reaction mixes fluorescence-encoded micro-beads with the sample containing test antibodies, forms compound (coating recombination
Antigen microballoon) association reaction occurs with mark fluorescent element (Streptavidin of phycoerythrin label) again；
(4) laser analysis microballoon passes sequentially through red green two beams laser in the drive of the flowing sheath fluid column that place an order, red laser to
Determine the fluorescence-encoded of microballoon, green laser reports the fluorescence intensity of molecule to measure on microballoon.
Compared with currently used ELISA kit, liquid-phase chip technology can be realized same under conditions of very low cost
When detect a variety of antigens, also save time cost.This research has carried out specific detection and Sensitivity comparison to this method,
It was found that remolding sensitivity ELISA method is high with other swine diseases Antigen positive hybridomas serum no cross reaction.This research is also to biotin labeling
The use concentration of the streptavidin (SAPE) of goat-anti pig secondary antibody and phycoerythrin label is optimized, and finds most suitable work
Make concentration, when secondary antibody concentration is 2ug/ml, and the use concentration of streptavidin is 2.5ug/ml, P/N value is maximum.To sum up institute
It states, this research has been successfully established a kind of liquid-phase chip detection method of new detection PCV2 antibody.
It is provided for the embodiments of the invention technical solution above to be described in detail, specific case used herein
The principle and embodiment of the embodiment of the present invention are expounded, the explanation of above embodiments is only applicable to help to understand this
The principle of inventive embodiments；At the same time, for those skilled in the art, according to an embodiment of the present invention, in specific embodiment party
There will be changes in formula and application range, in conclusion the contents of this specification are not to be construed as limiting the invention.
Claims (4)
1. a kind of PCV2 antibody detection method based on xMAP technology, it is characterised in that：
Include the following steps：
(1) preparation of recombinant antigen：Positive bacteria is cultivated first and induces its expression, and then ultrasonication positive bacteria, makes thallus
It cracks and is centrifuged, supernatant is precipitated and is separated, respectively take a certain amount of precipitating lysate of a certain amount of supernatant slow in albumen loading respectively
It is mixed in fliud flushing, carries out SDS-PAG analysis, the expression position of the i.e. recombinant antigen of destination protein is determined according to destination protein stripe size
It sets in precipitating or in supernatant；
(2) purifying protein operation is carried out, protein sample PCV2 CAP albumen is obtained；
(3) recombinant antigen is coated with, the protein sample PCV2 CAP albumen that step (2) is obtained, using xMAP Antibody
Coupling Kit kit is simultaneously coated with according to its specification, and the microballoon being coated with is obtained；
(4) xMAP test is carried out：Test serum sample carries out 2 times of doubling dilutions；Dilute the microballoon being coated with；Microballoon is carried out
Oscillation, ultrasound, resuspension processing；Into elisa plate, microballoon and test serum sample, oscillation incubation elisa plate is added in every hole；
Elisa plate is placed in magnetic separator, separates microballoon, discards reaction solution, washs, and the goat-anti of biotin labeling is added toward elisa plate
Pig secondary antibody；Oscillation incubation elisa plate, elisa plate are placed in magnetic separator, separate microballoon, discard reaction solution, wash, toward ELISA
SAPE, oscillation incubation elisa plate is added in plate, and elisa plate is placed in magnetic separator, separates microballoon, discards reaction solution, washs, benefit
Median fluorescence is taken with machine-readable in luminex system；
(5) ratio of positive MFI value and feminine gender MFI value, i.e. P/N value are calculated；Result by P/N value more than or equal to 3 is defined as sun
Property, when test 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 wrap
It is effective.
2. a kind of PCV2 antibody detection method based on xMAP technology according to claim 1, it is characterised in that：
The concrete operations of the step (1) are：The PCV2-CAP-pet32a plasmid of purchase is transformed into BL21 competent cell,
Form positive bacteria；Positive bacterium solution 100uL is taken, is inoculated into the 10ml culture medium of the mycin of benzyl containing ammonia, 37 DEG C of shaken cultivation 2-3 are small
When, when OD value is about 0.6,1ml bacterium solution is taken out as normal control, IPTG to final concentration of 0.5- is added in remaining bacterium solution
Induction bacterium solution 1ml is placed in 5min on ice by 1mmol/L, 16 DEG C or 37 DEG C induction 4-16h, and 5000g is centrifuged 5min and harvests thallus,
500uLPBS has hanged the thallus, and 10000rpm is centrifuged 5min, abandons supernatant；1ml PBS is added and has hanged rear ultrasonication again to bacterium
Body cracks completely；
Bacterium solution after above-mentioned ultrasonication is centrifuged 1min with 12000rpm, separates supernatant precipitating, 500uL PBS has hanged heavy
It forms sediment, respectively takes precipitating, supernatant 40uL to be added in 5 × albumen of 10uL sample-loading buffer, boiling water boils 5-10min, and 20uL is taken to carry out
SDS-PAGE analysis, the expression position of the i.e. recombinant antigen of destination protein is determined according to destination protein stripe size；
Glue part is concentrated when electrophoresis and uses 80V constant pressure 30min, separation gel uses 110V constant pressure 1h10min, until bromophenol blue is moved to
Separation gel lowermost end stop electrophoresis, separation gel is put into coomassie brilliant blue staining liquid and dyes 1h, then with destainer decolourize to
Background is thin out, and band is clear, saves gel photograph.
3. a kind of PCV2 antibody detection method based on xMAP technology according to claim 1, it is characterised in that：
The concrete operations of the step (4) are：
(1) prepare test serum sample, by 2 times of doubling dilutions of test serum sample；
(2) microballoon being coated with is diluted to 50/μ L with PBS-TBN；
(3) vortex oscillation and each 10s of ultrasonic treatment, have hanged 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 steps (1) have diluted is added.
(6) plate lid is covered, elisa plate is placed in oscillator, 800rpm room temperature, which is slightly shaken, is incubated for 120min；
(7) elisa plate is placed in magnetic separator 3-5min, separates microballoon, discards reaction solution；It is added by the amount in 100 holes μ L/
PBS-TBN cleaning solution, discards washing lotion；It is repeated once；
(8) elisa plate is removed from magnetic separator, the goat-anti pig secondary antibody of 100 μ L biotin labelings, 2 μ g/ of working concentration is added
mL；
(9) plate lid is covered, reaction plate is placed in oscillator, 800rpm room temperature, which is slightly shaken, is incubated for 60min；
(10) elisa 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；
(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) elisa plate is placed in magnetic separator 3-5min, separates microballoon, discards reaction solution；It is added by the amount in 100 holes μ L/
PBS-TBN cleaning solution, discards washing lotion；It is repeated once；
(13) reaction plate is removed from magnetic separator, 100 μ LPBS-TBN is added；Elisa plate is placed in oscillator oscillation
1min；
(14) median fluorescence is taken using machine-readable in luminex system.
4. a kind of PCV2 antibody detection method based on xMAP technology according to claim 1, it is characterised in that：
Antigen coat concentration is 8 μ g recombinant antigens/2.5x 10 in the step (3)5A microballoon.
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| CN109777890A (en) * | 2019-01-23 | 2019-05-21 | 华南农业大学 | A kind of PCV2, CSFV IgG antibody double fluorescent microballoon immunological detection method |
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