CN108287237A - A kind of pre-coated detection method of II type D antigens of poliovirus and its detection kit and application - Google Patents
A kind of pre-coated detection method of II type D antigens of poliovirus and its detection kit and application Download PDFInfo
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- CN108287237A CN108287237A CN201810032960.4A CN201810032960A CN108287237A CN 108287237 A CN108287237 A CN 108287237A CN 201810032960 A CN201810032960 A CN 201810032960A CN 108287237 A CN108287237 A CN 108287237A
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Abstract
The present invention provides a kind of pre-coated poliovirus produce in batches（Polio virus）II type D antigen detection methods, using ox（Rabbit, sheep）Anti- II type poliovirus D antigen purification antibody is coated with microwell plate, and is prepared into pre-coated plate, while the ox of mating horseradish peroxidase-labeled after being protected to coating plate and coated antibody by protective agent（Rabbit, sheep）Anti- II type poliovirus enzyme labelled antibody and other reagents.This method（Reagent）The qualitative and quantitative detection of specificity can be carried out to II type D antigens of poliovirus, to II type C antigens of poliovirus, I, III type C, D antigen and other enteroviruses are without intersection, it is species specificity height, sensitive height, II easy to use, widely used type poliovirus D antigen detection methods have higher application value in the calibrating of IPV production of vaccine.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of II type D antigens of poliovirus are pre-coated
Detection method and its detection kit and application.
Background technology
Poliovirus (polio virus) belongs to Picornaviridae, enterovirus genus, point 3 serum
Type mainly invades ventricornu grey matter area motor neuron, and Clinical symptoms is flaccid mascular paralysis, mostly occur 5 years old with
Lower children, especially infant, therefore also known as infantile paralysis are the masters of WHO second of infectious disease being eliminated after smallpox
Want pathogen.Do not have specific treatment means after Infected With Polioviruses In Vitro, can only be prevented by vaccine, currently used for
There are two types of the popular vaccines of prevention and control ridge ash：Poliomyelitis vaccine,Sabin (OPV) and inactivated polio vaccine
(IPV), OPV is inoculated with because being easy to, advantage of lower cost and be once widely used, but find in the long-term use, can
Can cause polio vaccine correlation paralysis (VAPP) case and Vaccine derived strains Polio virus VDPV), IPV then can be avoided effectively
The generation of VAPP and VDPV.Therefore, Polio virus is utterly destroyed finally to need all OPV to be replaced to carry out prevention and control with IPV.
Poliovirus shares 3 serotypes, respectively III type of I types of Polio, II types of Polio and Polio, and 3
The virion of a type has 2 kinds of forms, respectively has the D antigens of infective complete virion form and does not have
Infective hollow bead C antigens.C antigens pass through D antigen infection cells process, heating, ultraviolet light irradiation, alkaline denaturation, virulence
It degrades and generates, VP4 disappears without infectivity.D antigens have immunogenicity, can induce body and generate in protectiveness and resist
Body, the generation to guard against poliomyelities, though and C antigens have group-specific, immune serum that cannot still neutralize virus, due to
D antigenic contents and inactivated polio vaccine（IPV body has preferable consistency between generating immune efficacy after) being inoculated with,
Therefore, main component antigen of the D antigens as IPV vaccines, content is the key index of IPV vaccine vitro efficacies, to IPV epidemic diseases
Seedling production Quality Control is of great significance.
The detection of D antigens is used often in IPV production of vaccine, quality control procedure, and monovalent stoste detection, semi-finished product are matched
System, the detection of trivalent semi-finished product, finished product calibrating all relate to the project that Polio virus D antigens detect, since D antigenic contents are IPV
One major criterion of vaccine quality, directly affects the protecting effect of vaccine, thus the quality of D antigen detection methods and it is convenient
The production process and quality standard of vaccine can be directly influenced to a certain extent.
Since Polio virus antibody is for finding that its coating microwell plate stability is poor after testing, pre-coated plate cannot be prepared into
Finished product can only use preceding coating, coating to finish and need to be detected in time, and otherwise testing result will appear larger difference, therefore mesh
It is preceding that there is no Polio virus D antigen detection kits commercially in the market.Existing D antigen detection methods（Reagent）It can not batch
It prepares, causes detection cycle long using preceding coating every time, a detection could be completed at least 3 days, excessive cycle influences vaccine inspection
Fixed timeliness, and the operative skill of testing staff is required high, it changes operating personnel and is likely to result in personnel's operation deviation
It is existing, it is unfavorable for the stability of vaccine quality.The requirement of demand and production of vaccine calibrating based on market, applicant have developed one kind
It can be used for the method that II type IPV virus D antigens qualitatively or quantitatively quickly detect, pre-coated, batch production can be carried out, all inspection
The survey time shorten to 3.5 hours from 3 days, effectively increased the calibration operation efficiency of vaccine and the stability of testing result.
Invention content
Based on the problems of the above-mentioned prior art and deficiency, the present invention is intended to provide a kind of poliovirus
（Polio virus）II type D antigen detection methods, this method can carry out II type D antigens of poliovirus high special
The accurate qualitative and quantitative detection of property.The present invention also provides a kind of pre-coated polio that can be produced in batches
Virus（Polio virus）II type D antigen detection kits.
On the one hand, II type D antigens fast qualitative of a kind of poliovirus provided by the invention, quantitative detecting method,
Judge purposes for non-clinical medical diagnosis on disease treatment and non-health situation, which is characterized in that include the following steps：
S1, resist II type poliovirus antibody to be prepared and purified to animal, obtain the anti-II type polio disease of animal
The antibody purification of malicious D antigens；
S2, resist the purifying of II type poliovirus D antigens anti-animal by sodium periodate method with horseradish peroxidase
Body is marked, and obtains the detection enzyme labelled antibody that animal resists II type poliovirus D antigens, and enzyme labelled antibody use is made
Liquid；
S3, resisted with animal II type poliovirus D antigens antibody purification carry out enzyme mark detection plate pre-coated processing, in advance
Protective agent is added in coating processing and carries out closed protective；The protective agent includes but not limited to following component：BSA, gelatin, junket egg
In vain, one or more in albumin, trehalose, phosphate, carbonate, pre-coated carrier is made；, the pre-coated carrier energy of gained
Batch making and it can meet and realize long-term storage under testing conditions index；
S4, antigen formation antigen antibody complex to be checked is captured using pre-coated carrier, then is combined using liquid with enzymic-labelled antibody,
It is developed the color, the qualitative detection to II type D antigens of poliovirus is completed according to colour developing result or quantified by substrate catalyzing enzyme
Detection.
Further, protective agent described in step S3 is：Include 7 ~ 13g BSA per 1000ml protective agents, 7 ~ 13 trehaloses,
3 ~ 7g gelatin, 1 ~ 2g caseins, 30 ~ 70mMol phosphate solutions；The pre-coated carrier use is stored after vacuumizing packaging.
Further, the preparation process of antibody purification includes：
1）It is prepared by II type poliovirus D antigen antiserums：Inactivated poliovirus is close by 10-30% sucrose
Gradient centrifugation is spent, D antigen layers are collected, is prepared for immune serum after Electronic Speculum detection confirms；Experimental animal ox, rabbit or sheep, it is first
Exempt from, II type unit price inactivation of viruses D antigens and freund adjuvant equivalent mixing are subcutaneously injected and are immunized, adopted after carrying out 3 booster immunizations
Blood detaches serum, and serum neutralizing antibody titers measurement is carried out using microneutralization test；
2）II type poliovirus D antigen purification Antibody preparations of ox, rabbit or goat-anti：Take albumin A/G affinity columns, room temperature
It places balance 30 minutes, chromatographic column is balanced with the equilibrium liquid of 5 times of column volumes；By antiserum and equilibrium liquid in equal volume than mixed
Loading after conjunction elutes foreign protein with the equilibrium liquid of 10 times of column volumes, retains destination protein；With the conventional eluent of 10 times of column volumes
Destination protein is eluted from affinity column, collects eluting peak, it is spare after desalination；Protein content is measured with lowrry methods, is obtained
II type poliovirus D antigen purification antibody of ox, rabbit or goat-anti, -20 DEG C or less preserve for use.
Further, the S2 steps include：After horseradish peroxidase is dissolved with water for injection, NaIO4 is added,
It stands, ethylene glycol solution is added, stand, then mixed with antibody purification obtained by step S1, dialysed overnight；Add sodium borohydride, stand,
Add saturated ammonium sulfate, centrifuges；Dialysed overnight after taking precipitation to dissolve；Obtain ox, rabbit, II type poliovirus D antigens of goat-anti
Detection enzyme mark antibody purification, Cord blood are spare.
Further, the S3 steps include：
1）Enzyme mark detection plate is coated with：The antibody purification of II type poliovirus D antigens of purified ox, rabbit or goat-anti is led to
It crosses chessboard experiment and confirms coating condition, after according to condition diluting antibody to 0.1-15 μ g/ml, it is special that the holes 50-150 μ l/ add to enzyme mark
In detection plate, 2~8 DEG C overnight, board-washing；
2）Enzyme mark detection plate is closed：Protective agent is added with the amount in the holes 100-250 μ l/, 2~8 DEG C overnight, and board-washing is dried, vacuum packet
Refrigerator saves backup after dress, and the protective agent includes but not limited to following component：BSA, gelatin, casein, albumin, seaweed
It is one or more in sugar, phosphate, carbonate.
Further, qualitatively or quantitatively detection includes the following steps in the S4：
1）It is added after measuring samples are diluted in the detection hole of pre- wrapper sheet, it is synchronous that II type poliovirus D antigens ginseng is added
Product are examined, quantitative sample and content reference material multiple holes sample-adding, 50-150 μ l；Former times of the holes qualitative detection sample 50-150 μ l/ are added, if
1 hole of blank well and each 2 hole of antigen-negative controls；37 DEG C are incubated 1-3 hours, board-washing 3-5 times；
2）1）Enzyme labelled antibody is added using liquid in the holes 50-150 μ l/ in the enzyme mark detection plate washed, and blank well is not added with, and 37 DEG C incubate
0.5-3 hours are educated, board-washing 3-5 times；
3）2）TMB developing solutions are added in the enzyme mark detection plate washed, mixing is put into wet box, and 37 DEG C are incubated 7-10 minutes；
4）It is terminated and is reacted using terminate liquid；
5）Under detection 450nm wavelength, 630 tuning wavelengths, absorbance A value is detected after being returned to zero with blank well, according to yin and yang attribute pair
According to calculating cutoff values, the detection that qualitative results can be obtained according to cutoff values or calculate quantitative according to antigenic content reference material
As a result.
On the other hand, the present invention also provides a kind of II type D antigens fast qualitative of poliovirus, quantitative detection sides
Application of the method in IPV vaccine developments and its quality arbitration.
On the other hand, the present invention also provides a kind of II type D antigens fast qualitative of poliovirus, quantitative detection sides
Application of the method in preparing poliovirus detection reagent or kit.
On the other hand, the present invention also provides a kind of II type D antigens fast qualitative of poliovirus, quantitative detections to use
Kit, which is characterized in that including following reagent component：II type D antigen detection plates of poliovirus, enzymic-labelled antibody
Using liquid, TMB developing solutions A, developing solution B concentrate washing lotion, the control of II type D antigen positives of poliovirus, polio
Virus type II D antigen-negative controls, II type D antigenic content reference materials of poliovirus.
Further, the preparation method of II type D antigen detection plates of the poliovirus is：
1）The antibody purification of II type poliovirus D antigens of purified ox, rabbit or goat-anti is tested by chessboard and is confirmed
Coating condition, after according to condition diluting antibody to 0.1-15 μ g/ml, the holes 50-150 μ l/ add in enzyme mark dedicated detector board, 2~8 DEG C
Overnight, board-washing；
2）Enzyme mark detection plate is closed：Protective agent is added with the amount in the holes 100-250 μ l/, 2~8 DEG C overnight, and board-washing is dried, vacuum packet
Refrigerator saves backup after dress, and the protective agent includes：5%BSA, 5% gelatin, 3% casein, 3% albumin, 15% trehalose, 20% ~
30% phosphate or carbonate.
The present invention has following advantages and effect compared with prior art：
（1）Using pre-coated ox（Rabbit, sheep）Anti- II type poliovirus D antigen purification antibody, with horseradish peroxidase
Mark ox（Rabbit, sheep）Anti- II type marrow poliovirus D antigen purification antibody makees amplification system, can detect II type ridge with high specificity
Marrow poliovirus D antigens improve detection sensitivity and specificity.
（2）The II type poliovirus particle obtained using density gradient centrifugation separation（D antigens do not contain C antigens
Ingredient）, existing specificity is high, sensitivity is good, can prepare in batches, reduce and detect and advantage that operating error, detection time are short.Exempt from
Epidemic disease is with C antigenic components are eliminated in antigen, the antiserum specificity of acquisition is more preferable, the detection plate specificity higher thus prepared.
（3）The present invention solves the problems, such as that cell culture lesion method cannot detect inactivation of viruses, is the research of related vaccines
Exploitation and vaccine quality calibration operation provide strong technical guarantee.
（4）The present invention carries out pre-coated protection using protective agent to coated antibody, and the antibody that animal obtains is immunized and is coated with simultaneously
It is protected through protective agent, the state that script traditional detection process needs can not persistently break is formd to the side that can cut off segmentation step
Formula, realize can with the pre-coated detection plate of long-term preservation, can intermittent detection mode reduce the preparation in immune detection and prepare
It is small from 3 days to be shorten to 3-4 directly using the pre-coated plate for preparing so as to shorten detection time by step for general detection time
When, solve the problems, such as Polio virus antibody coating after be unable to long-term preservation, so that detection reagent is prepared in batches, store it is same
When improve detection efficiency.（5）Pre-coated plate stability prepared by the present invention is good, and testing result meets ELISA experiment demands,
Keep also having good stability -20 DEG C long-term, it is 18 months longest holding times, detection sensitivity, linear（R2）, accuracy
(CV) meet vaccine detection and the requirement of ELISA detection reagents.It realizes and prepares, detects alienable detection method, another
Prominent effect is to carry out stage Quality Control to detection reagent, is convenient for the picket for mistake and control in practical preparation, detection process
System, is conveniently operated and reduces personal error, is not only preparing, is improving efficiency in detection, it is ensured that the repetition of testing result
Property and vaccine quality stability, reach to prepare quality, detect quality dual controllability and protection.I.e. of the invention is pre-
Wrapper sheet detection reagent can carry out producing in batches and can effectively reduce II type gray nucleus in actual use with long-term preservation
Experiment reagent in scorching virus D antigens detection and operating error, are conducive to improve working efficiency.
Specific implementation mode
It is further illustrated the present invention below with embodiment, but the present invention is not intended to be limited thereto.It is not noted in the following example
The experimental method of bright actual conditions, usually according to normal condition, or according to the normal condition proposed by manufacturer.
It is prepared by 1 II type poliovirus D antigen antiserums of embodiment
Inactivated poliovirus is centrifuged 4-8 hours by 10-30% sucrose density gradients 40000rpm, collects D antigens
Layer is prepared after D antigens are confirmed as in Electronic Speculum detection for immune serum；Experimental animal（Ox, rabbit, sheep）, head exempts from, II type list
Valence inactivation of viruses D antigen 1s 0-20ml and Freund's complete adjuvant equivalent mixing, are subcutaneously injected and are immunized, and then carry out 3 booster immunizations
After take a blood sample, detach serum, using microneutralization test carry out serum neutralizing antibody titers measurement.
2 Ns of embodiment（Rabbit, sheep）Anti- II type poliovirus antibody purification（Referred to as resist II type IPV-IgG）System
It is standby
1, albumin A/G affinity columns are taken, balance 30 minutes is placed at room temperature for, chromatographic column is carried out with the equilibrium liquid of 5 times of column volumes
Balance；2, antiserum is left than loading after mixing with the equilibrium liquid elution foreign protein of 10 times of column volumes in equal volume with equilibrium liquid
Destination protein；3, destination protein is eluted with the conventional eluent of 10 times of column volumes from affinity column, collects eluting peak, taken off
It is spare after salt；4, protein content is measured with lowrry methods, obtains ox（Rabbit, sheep）Anti- II type IPV-IgG, -20 DEG C or less preservations wait for
With；The washing lotion, equilibrium liquid are the routine liquid of the prior art.
It is prepared by 3 enzymic-labelled antibody of embodiment
After horseradish peroxidase 10mg is dissolved 1ml with water for injection, 0.2ml NaIO4 are added, stands 0.1-1 hours, adds
Enter 0.5ml ethylene glycol solutions, stand 0.1-1 hours, then is mixed with antibody purification 1ml obtained by step 2, dialysed overnight；Add
0.5ml sodium borohydrides stand 0.1-1 hours, and saturated ammonium sulfate, 15000rpm is added to centrifuge 30-60 minutes；After taking precipitation to dissolve thoroughly
Analysis is overnight；Obtain ox（Rabbit, sheep）Anti- II type IPV-IgG-HRP, Cord blood are spare.
The preparation of 4 pre-coated elisa plate of embodiment
1）ELISA Plate is coated with：By ox（Rabbit, sheep）Anti- II type poliovirus D antigen purification antibody is diluted to 0.1-15 μ g/
Ml is added to by the amount in 50~150 holes μ l/ in ELISA Plate, and 2~8 DEG C overnight, board-washing 3~5 times；
2）ELISA Plate is closed：Protective agent closing is added by the amount in 100~250 holes μ l/, 2~8 DEG C overnight, are sealed ELISA Plate
Protection is closed, board-washing 3~5 times is dried, and refrigerator saves backup after vacuumizing packaging, and the protective agent is pressed per 1000ml protective agent packets
Containing 7 ~ 13g BSA, 7 ~ 13 trehaloses, 3 ~ 7g gelatin, 1 ~ 2g caseins, 30 ~ 70mMol phosphate solutions are made；Or protective agent is pressed
Including：5%BSA, 5% gelatin, 3% casein, 3% albumin, 15% trehalose, 20% ~ 30% phosphate or carbonate are made.
5 II type poliovirus D antigens of embodiment detect
1, pre- wrapper sheet detection hole will be added after II type poliovirus D antigen samples to be checked and the dilution of other sample series
In, quantitative sample and content reference material multiple holes sample-adding；Qualitative detection sample original is directly added into the holes 50-150 μ l/ again, synchronous to be added
II type poliovirus D antigens reference material and antigen-negative controls, if 1 hole of blank well, 37 DEG C are incubated 0.5-3 hours, wash
Plate 5 times；
2, enzyme labelled antibody is added in the enzyme mark detection plate that step 1 has been washed uses liquid, the holes 50-150 μ l/, blank well to be not added with, and 37
DEG C be incubated 0.5-3 hours, board-washing 5 times；
3,100 holes μ l/ of TMB developing solutions are added in the enzyme mark detection plate that step 2 has been washed, mixing is put into wet box, and 37 DEG C incubate
It educates 5-15 minutes；
4, terminate liquid terminates reaction；
5, under detection 450nm wavelength, 630 tuning wavelengths, after being returned to zero with blank well absorbance A value is detected to tie to get detection
Fruit；
6, result judgement：
1）Cutoff values=negative control average value × 2.1,（The average A-value of negative control is less than or equal to 0.05, based on actual value
It calculates）.
2）According to sample A values >=Cutoff values, which is determined as II type poliovirus D antigen positive samples,
Sample A value ＜ Cutoff values, for feminine gender, which is determined as II type poliovirus D antigen negative samples.
3）The quantitative detection sample A values being serially diluted are brought into corresponding formula with antigenic content reference material A values to count
It calculates, you can obtain the content of measuring samples.
Measuring samples in the above experiment include I, II, III type poliovirus D antigens, C antigens and other enteron aisles
Virus, II type marrow poliovirus D antigenic content reference materials, accuracy reference material etc., and accuracy, line are done to the present embodiment
Property, sensitivity, the detection of repeated performance indicator, the results are shown in Table 1,
The 1 II pre-coated fast detection method performance indicator of type poliovirus D antigens of table
In terms of the performance indicator of detection reagent, average accuracy of the invention, sensitivity and linearly it is satisfied by ELISA detection reagents
It is required that can be used for vaccine detection.Detection method sensitivity analysis, with II type IPV-D antigenic contents reference material of the present invention couple into
Row detection, Monitoring lower-cut can measure 0.125 DU/ml.Detection method accuracy is analyzed, and it is that 5.4 % are small that the present invention, which tests CV,
In 15%, meet ELISA test requirements documents.
Specificity experiments are done to the detection method of the present embodiment, the results are shown in Table 2, is carried out pair with detection method
Various poliovirus D, C antigen and other enteroviruses carry out cross matching, and specificity display occurs without crossing instances.
The 2 II pre-coated quick detection reagent specific test of type poliovirus D antigens of table
The 6 II pre-coated plate correlated performance detection of type poliovirus D antigens of embodiment
The method that step 1. is pressed in embodiment 3 prepares pre-coated plate；Wherein：Protective agent is pressed per 1000ml confining liquid BSA containing 10g,
10g trehaloses, 5g gelatin, 1g caseins, 50mMol phosphate solutions are made；
Pre-coated plate is deposited in the experiment that accelerates the failure in 3,5,8 days of 37 DEG C of works by step 2. respectively；It the results are shown in Table 3；
The pre-coated plate of step 3. is stored in the long-term stable experiment that 4 DEG C and -20 DEG C or less do 6,9,12,18 months；It the results are shown in Table
4；
The pre-coated plate of step 4. stability test brings accuracy reference material, content reference material into after different temperatures placement expires
It is detected according to standard test procedure.
The pre-coated 37 DEG C of stability test results of plate quick detection reagent of 3 II type poliovirus D antigens of table
Pre-coated -20 DEG C of stability test results of plate quick detection reagent of 4 II type poliovirus D antigens of table
Pre-coated plate prepared by the present invention has preferable stability, testing result to meet under 37 DEG C of experiment conditions that accelerate the failure
ELISA tests demand, keeps also having good stability, 18 months longest holding times -20 DEG C long-term（Table 3,4）, detection
It is sensitivity, linear（R2）, accuracy (CV) meet vaccine detection and ELISA detection reagents requirement.With 3 batches of pre-coated inspections
7 samples of test agent pair are detected（Comparative example, table 5）, the fluctuation of sample size testing result in 95% fiducial limit range,
Meet the requirement of ELISA detection reagents, pre- wrapper sheet detection reagent of the invention can carry out producing in batches and can with long-term preservation,
The experiment reagent and operating error in the detection of II type poliovirus D antigens can be effectively reduced in actual use, be conducive to
Improve working efficiency.
Comparative example
Using conventional II type poliovirus D antigens detection method and synchronous detection sample of the invention, and to testing result into
Row compares, and method is as follows, ox（Rabbit, sheep）Anti- II type IPV antibody purification wrapper sheets, absorption is overnight；Board-washing adds confining liquid to be incubated 1 small
When, after board-washing plus measuring samples, absorption is overnight；Board-washing, adds secondary antibody, 2.5 hours；Board-washing, enzyme labeling antibody are incubated 1.5 hours；
Board-washing adds TMB- developing solutions to develop the color, 450nm testing results；By sample A values and antigen reference material A values bring into corresponding formula into
Row calculates, you can obtains the content of measuring samples.Comparing result is as shown in table 5：
The 5 II type pre-coated fast detection method of poliovirus D antigens of table is compared with conventional detection method testing result
It should be understood that after the above for having read the present invention, those skilled in the art can make the present invention various changes
Or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
Solution used in the present embodiment is as follows：
1. washing lotion：Disodium hydrogen phosphate 2.1g, sodium dihydrogen phosphate 0.2g, sodium chloride 8g, potassium chloride 0.2g, tween 0.5ml, injection
With water 1000ml.
2. coating buffer：Natrium carbonicum calcinatum 1.2g, sodium bicarbonate 2. 3g, water for injection 1000ml.
3. terminate liquid：The concentrated sulfuric acid 54 ml, water for injection 445ml.
4.TMB developing solutions A：TMB 50mg, dimethyl sulfoxide (DMSO) 50ml add water for injection 50ml.
5. TMB developing solutions B：Sodium acetate 4.55g, glacial acetic acid 0.15ml, hydrogen peroxide, 2.5ml are injected water to
500ml。
6. confining liquid：1000ml confining liquids BSA containing 10g, 10g trehalose, 5g gelatin, 1g caseins, 50mMol phosphate
Solution.
Claims (10)
1. a kind of II type D antigens fast qualitative of poliovirus, quantitative detecting method are treated for non-clinical medical diagnosis on disease
Judge purposes with non-health situation, which is characterized in that include the following steps：
S1, resist II type poliovirus antibody to be prepared and purified to animal, obtain the anti-II type polio disease of animal
The antibody purification of malicious D antigens；
S2, resist the purifying of II type poliovirus D antigens anti-animal by sodium periodate method with horseradish peroxidase
Body is marked, and obtains the detection enzyme labelled antibody that animal resists II type poliovirus D antigens, and enzyme labelled antibody use is made
Liquid；
S3, resisted with animal II type poliovirus D antigens antibody purification carry out enzyme mark detection plate pre-coated processing, in advance
Protective agent is added in coating processing and carries out closed protective；The protective agent includes but not limited to following component：BSA, gelatin, junket egg
In vain, one or more in albumin, trehalose, phosphate, carbonate, pre-coated carrier is made；, the pre-coated carrier energy of gained
Batch making and it can meet and realize long-term storage under testing conditions index；
S4, antigen formation antigen antibody complex to be checked is captured using pre-coated carrier, then is combined using liquid with enzymic-labelled antibody,
It is developed the color, the qualitative detection to II type D antigens of poliovirus is completed according to colour developing result or quantified by substrate catalyzing enzyme
Detection.
2. II type D antigens fast qualitative of a kind of poliovirus according to claim 1, quantitative detecting method,
It is characterized in that, protective agent is described in step S3：Include 7 ~ 13g BSA, 7 ~ 13 trehaloses, 3 ~ 7g bright per 1000ml protective agents
Glue, 1 ~ 2g caseins, 30 ~ 70mMol phosphate solutions；The pre-coated carrier use is stored after vacuumizing packaging.
3. II type D antigens fast qualitative of a kind of poliovirus according to claim 2, quantitative detecting method,
It is characterized in that, the preparation process of antibody purification includes：
1）It is prepared by II type poliovirus D antigen antiserums：Inactivated poliovirus is close by 10-30% sucrose
Gradient centrifugation is spent, D antigen layers are collected, is prepared for immune serum after II type D antigens are confirmed as in Electronic Speculum detection；Experimental animal
Ox, rabbit or sheep, head exempt from, and II type unit price inactivation of viruses D antigens and freund adjuvant equivalent mixing are subcutaneously injected and are immunized, and carry out 3 times and add
It takes a blood sample after being immunized by force, detaches serum, serum neutralizing antibody titers measurement is carried out using microneutralization test；
2）II type poliovirus D antigen purification Antibody preparations of ox, rabbit or goat-anti：Take albumin A/G affinity columns, room temperature
It places balance 30 minutes, chromatographic column is balanced with the equilibrium liquid of 5 times of column volumes；By antiserum and equilibrium liquid in equal volume than mixed
Loading after conjunction elutes foreign protein with the equilibrium liquid of 10 times of column volumes, retains destination protein；With the conventional eluent of 10 times of column volumes
Destination protein is eluted from affinity column, collects eluting peak, it is spare after desalination；Protein content is measured with lowrry methods, is obtained
II type poliovirus D antigen purification antibody of ox, rabbit or goat-anti, -20 DEG C or less preserve for use.
4. II type D antigens fast qualitative of a kind of poliovirus according to claim 1, quantitative detecting method,
It is characterized in that, the S2 steps include：After horseradish peroxidase is dissolved with water for injection, NaIO4 is added, stands, is added
Ethylene glycol solution is stood, then is mixed with antibody purification obtained by step S1, dialysed overnight；Add sodium borohydride, stands, add saturation sulphur
Sour ammonium, centrifugation；Dialysed overnight after taking precipitation to dissolve；It obtains ox or rabbit or the detection of II type poliovirus D antigens of goat-anti is used
Enzyme labelled antibody, Cord blood are spare.
5. II type D antigens fast qualitative of a kind of poliovirus according to claim 1, quantitative detecting method,
It is characterized in that, the S3 steps include：
1）Enzyme mark detection plate is coated with：The antibody purification of II type poliovirus D antigens of purified ox, rabbit or goat-anti is led to
It crosses chessboard experiment and confirms coating condition, after according to condition diluting antibody to 0.1-15 μ g/ml, it is special that the holes 50-150 μ l/ add to enzyme mark
In detection plate, 2~8 DEG C overnight, board-washing；
2）Enzyme mark detection plate is closed：Protective agent is added with the amount in the holes 100-250 μ l/, 2~8 DEG C overnight, and board-washing is dried, vacuum packet
Refrigerator saves backup after dress.
6. II type D antigens fast qualitative of a kind of poliovirus according to claim 1, quantitative detecting method,
It is characterized in that, qualitatively or quantitatively detection includes the following steps in the S4：
1）It is added after measuring samples are diluted in the detection hole of pre- wrapper sheet, it is synchronous that II type poliovirus D antigens ginseng is added
Product are examined, quantitative sample and content reference material multiple holes sample-adding, the holes 50-150 μ l/；The holes qualitative detection sample 50-150 μ l/ original is extraordinarily
Enter, if 1 hole of blank well and each 2 hole of antigen-negative controls；37 DEG C are incubated 1-3 hours, board-washing 3-5 times；
2）1）Enzyme labelled antibody is added using liquid in the holes 50-150 μ l/ in the enzyme mark detection plate washed, and blank well is not added with, and 37 DEG C incubate
0.5-3 hours are educated, board-washing 3-5 times；
3）2）TMB developing solutions are added in the enzyme mark detection plate washed, mixing is put into wet box, and 37 DEG C are incubated 5-15 minutes；
4）It is terminated and is reacted using terminate liquid；
5）Under detection 450nm wavelength, 630 tuning wavelengths, absorbance A value is detected after being returned to zero with blank well, according to yin and yang attribute pair
According to cutoff values are calculated, qualitative results can be obtained according to cutoff values, or the inspection for calculating quantitative according to antigenic content reference material
Survey result.
7. a kind of II type D antigens fast qualitative of poliovirus, quantitative detection side described in claim 1-6 any one
Application of the method in IPV vaccine developments and its quality arbitration.
8. a kind of II type D antigens fast qualitative of poliovirus, quantitative detection side described in claim 1-6 any one
Application of the method in preparing poliovirus detection reagent or kit.
9. the kit of a kind of II type D antigens fast qualitative of poliovirus, quantitative detection, which is characterized in that including
Following reagent component：II type D antigen detection plates of poliovirus, enzymic-labelled antibody use liquid, protective agent, TMB developing solutions
A, developing solution B concentrates washing lotion, the control of II type D antigen positives of poliovirus, II type D antigen the moon of poliovirus
Property control, II type D antigenic content reference materials of poliovirus.
10. the examination of II type D antigens fast qualitative of a kind of poliovirus according to claim 9, quantitative detection
Agent box, which is characterized in that the preparation method of II type D antigen detection plates of the poliovirus is：
1）The antibody purification of II type poliovirus D antigens of purified ox, rabbit or goat-anti is tested by chessboard and is confirmed
Coating condition, after according to condition diluting antibody to 0.1-15 μ g/ml, the holes 50-150 μ l/ add in enzyme mark dedicated detector board, 2~8 DEG C
Overnight, board-washing；
2）Enzyme mark detection plate is closed：Protective agent is added with the amount in the holes 100-250 μ l/, 2~8 DEG C overnight, and board-washing is dried, vacuum packet
Refrigerator saves backup after dress；The protective agent includes：5%BSA, 5% gelatin, 3% casein, 3% albumin, 15% trehalose, 20% ~
30% phosphate or carbonate.
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