CN108241058A - A kind of pre-coated detection method of III type D antigens of poliovirus and its detection kit and application - Google Patents
A kind of pre-coated detection method of III type D antigens of poliovirus and its detection kit and application Download PDFInfo
- Publication number
- CN108241058A CN108241058A CN201810032959.1A CN201810032959A CN108241058A CN 108241058 A CN108241058 A CN 108241058A CN 201810032959 A CN201810032959 A CN 201810032959A CN 108241058 A CN108241058 A CN 108241058A
- Authority
- CN
- China
- Prior art keywords
- poliovirus
- iii type
- antigens
- detection
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of pre-coated poliovirus produce in batches（Polio virus）III type D antigen detection methods, using ox（Rabbit, sheep）Anti- III type poliovirus D antigen purifications antibody coating microwell plate, and pass through after protective agent protects coating plate and coated antibody and be prepared into pre-coated plate, while the ox of mating horseradish peroxidase-labeled（Rabbit, sheep）Anti- III type poliovirus enzyme labelled antibody and other reagents.This method（Reagent）The qualitative and quantitative detection of specificity can be carried out to III type D antigens of poliovirus, to III type C antigens of poliovirus, Ith, II type C, D antigen and other enteroviruses are without intersection, it is species specificity height, sensitive height, III 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 III 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 WHO masters of second of infectious disease to be 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
Polio vaccine correlation paralysis (VAPP) case and Vaccine derived strains Polio virus VDPV can be caused), IPV then can be avoided effectively
The generation of VAPP and VDPV.Therefore, utterly destroying Polio virus finally needs 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, D antigens respectively with 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 so as to guard against poliomyelities, though and C antigens have group-specific, immune serum 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 used to after testing find that its coating microwell plate stability is poor, it is impossible to be prepared into pre-coated plate
Finished product can only use preceding coating, and coating is finished 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, and to the behaviour of testing staff
Make skill set requirements height, change operating personnel and be likely to result in personnel's operation deviation appearance, be unfavorable for the stability of vaccine quality.Base
The requirement that demand and production of vaccine in market are examined and determine, applicant have developed it is a kind of can be used for III type IPV virus D antigens qualitative or
The quantitative method quickly detected, can carry out pre-coated, and batch production, whole detection times shorten to 3.5 hours from 3 days, have
Effect improves 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）III type D antigen detection methods, this method can carry out III 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）III type D antigen detection kits.
On the one hand, III 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, III type poliovirus antibody is resisted to be prepared and purified to animal, obtains animal and resist III type polio sick
The antibody purification of malicious D antigens；
S2, the purifying of III type poliovirus D antigens is resisted to resist to animal by sodium periodate method with horseradish peroxidase
Body is marked, and obtains the detection enzyme labelled antibody that animal resists III type poliovirus D antigens, and enzyme labelled antibody use is made
Liquid；
S3, the antibody purification of III type poliovirus D antigens is resisted to carry out the pre-coated processing of enzyme mark detection plate with animal, in advance
Protective agent, which is added in, in coating processing carries out closed protective；The protective agent includes but not limited to following component：BSA, gelatin, junket egg
In vain, it is one or more in albumin, trehalose, phosphate, carbonate, pre-coated carrier, the pre-coated carrier energy of gained is made
Batch making and it can meet long-term storage is realized under testing conditions index；
S4, antigen formation antigen antibody complex to be checked is captured, then combined with enzymic-labelled antibody using pre-coated carrier, passes through bottom
Object catalyzing enzyme develops the color, and the qualitative detection to III type D antigens of poliovirus or quantitative detection are completed according to colour developing result.
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 III type poliovirus D antigens antiserum：Inactivated poliovirus is close by 10-30% sucrose
Gradient centrifugation is spent, collects D antigen layers, is prepared after Electronic Speculum detection confirms for immune serum；Experimental animal ox or rabbit or sheep, it is first
Exempt from, III 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 measure is carried out using microneutralization test；
2）Ox or rabbit or III type poliovirus D antigen purification Antibody preparations of goat-anti：Take albumin A/G affinity columns, room
Temperature places balance 30 minutes, and chromatographic column is balanced with the equilibrium liquid of 5 times of column volumes；Antiserum is compared in equal volume with equilibrium liquid
Loading after mixing elutes foreign protein with the equilibrium liquid of 10 times of column volumes, retains destination protein；It is eluted with 10 times of the conventional of column volume
Liquid elutes destination protein from affinity column, collects eluting peak, spare after desalination；Protein content is measured with lowrry methods,
Ox or rabbit or III type poliovirus D antigen purification antibody of goat-anti are obtained, less than -20 DEG C preserve for use.
Further, the S2 steps include：After horseradish peroxidase is dissolved with water for injection, NaIO4 is added in,
It stands, adds in ethylene glycol solution, stand, then mixed with step S1 gained antibody purifications, dialysed overnight；Add sodium borohydride, stand,
Add saturated ammonium sulfate, centrifuge；Take precipitation dissolve after dialysed overnight；Obtain ox, rabbit, III 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 III 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 50-150 μ l/ holes 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 in the amount in 100-250 μ l/ holes, 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 in after measuring samples are diluted in the detection hole of pre- wrapper sheet, it is synchronous to add in III type poliovirus D antigens ginseng
Product are examined, quantitative sample and content reference material multiple holes sample-adding, 50-150 μ l；Former times of qualitative detection sample 50-150 μ l/ holes add in, 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）50-150 μ l/ holes add in enzyme mark antibody purification using liquid in the enzyme mark detection plate washed, and blank well is not added with, and 37
DEG C be incubated 0.5-3 hours, 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 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 or the detection for calculating quantitative according to antigenic content reference material can be obtained according to cutoff values
As a result.
On the other hand, the present invention also provides a kind of III 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 III type D antigens fast qualitative of poliovirus, quantitative detection sides
Application of the method in poliovirus detection reagent or kit is prepared.
On the other hand, the present invention also provides a kind of III type D antigens fast qualitative of poliovirus, quantitative detections to use
Kit, which is characterized in that including following reagent component：III 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 III type D antigen positives of poliovirus, polio
Virus type iii D antigen-negative controls, III type D antigenic content reference materials of poliovirus.
Further, the preparation method of the III type D antigen detection plates of poliovirus is：
1）The antibody purification of III type poliovirus D antigens of purified ox, rabbit or goat-anti by chessboard is tested and is confirmed
Coating condition, after according to condition diluting antibody to 0.1-15 μ g/ml, 50-150 μ l/ holes are added in enzyme mark dedicated detector board, 2~8 DEG C
Overnight, board-washing；
2）Enzyme mark detection plate is closed：Protective agent is added in the amount in 100-250 μ l/ holes, 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- III type poliovirus D antigen purification antibody, with horseradish peroxidase
Mark ox（Rabbit, sheep）Anti- III type marrow poliovirus D antigen purification antibody makees amplification system, can detect III type marrow with high specificity
Poliovirus D antigens improve detection sensitivity and specificity.
（2）The III 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 operating error, the advantages of detection time is 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 coating of animal acquisition is immunized 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 realizes the pre-coated detection plate that can be preserved for a long time, and the preparation that the detection mode that can be interrupted is reduced in immune detection prepares
Directly using the pre-coated plate for preparing so as to shorten detection time, it is small from 3 days to be shorten to 3-4 by step for general detection time
When, solve the problems, such as Polio virus antibody coating after cannot preserve for a long time, detection reagent is allow to prepare 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 is realized and prepares, detects alienable detection method, another is prominent
It is that can carry out stage Quality Control to detection reagent to go out effect, convenient for it is practical prepare, the picket in detection process for mistake and control,
Be conveniently operated and reduce personal error, not only preparing, improving efficiency in detection, it is ensured that the repeatability of testing result and
The stability of vaccine quality reaches dual controllability and protection to preparing quality, detecting quality.That is pre- wrapper sheet of the invention
Detection reagent can be produced in batches and can be preserved for a long time, can effectively reduce III type polio disease in actual use
Experiment reagent and operating error in malicious D antigens detection, are conducive to improve working efficiency.
Specific embodiment
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 III type poliovirus D antigens antiserum of embodiment
Inactivated poliovirus by 10-30% sucrose density gradients 40000rpm is centrifuged 4-8 hours, 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, III type list
Valency 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 measure.
2 Ns of embodiment（Rabbit, sheep）Anti- III type poliovirus antibody purification（Referred to as anti-III type IPV-IgG）System
It is standby
1st, 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；2nd, antiserum and equilibrium liquid are eluted foreign protein with the equilibrium liquid of 10 times of column volumes, left in equal volume than loading after mixing
Destination protein；3rd, destination protein is eluted from affinity column with the conventional eluent of 10 times of column volumes, collects eluting peak, taken off
It is spare after salt；4th, protein content is measured with lowrry methods, obtains ox（Rabbit, sheep）Anti- III type IPV-IgG, less than -20 DEG C preservations are treated
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 with water for injection 1ml, 0.2mlNaIO4 is added in, 0.1-1 hours is stood, adds
Enter 0.5ml ethylene glycol solutions, stand 0.1-1 hours, then mixed with step 2 gained antibody purification 1ml, dialysed overnight；Add
0.5ml sodium borohydrides stand 0.1-1 hours, and 1：1 adds saturated ammonium sulfate, 15000rpm centrifugations；Precipitation is taken to dialyse after dissolving
Night；Obtain ox（Rabbit, sheep）Anti- III 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- III type poliovirus D antigen purification antibody is diluted to 0.1-15 μ g/
Ml is added to by the amount in 50~150 μ l/ holes in ELISA Plate, and 2~8 DEG C overnight, board-washing 3~5 times；
2）ELISA Plate is closed：Protective agent closing is added in by the amount in 100~250 μ l/ holes, 2~8 DEG C overnight, are sealed ELISA Plate
Protection is closed, board-washing 3~5 times is dried, and refrigerator saves backup after vacuum packaging, and the protective agent is included by every 1000ml protective agents
7 ~ 13g BSA, 7 ~ 13 trehaloses, 3 ~ 7g gelatin, 1 ~ 2g caseins, 30 ~ 70mMol phosphate solutions are made；Or protective agent is by packet
It includes：5%BSA, 5% gelatin, 3% casein, 3% albumin, 15% trehalose, 20% ~ 30% phosphate or carbonate are made.
5 III type poliovirus D antigens of embodiment detect
1st, pre- wrapper sheet detection hole will be added in after III 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 50-150 μ l/ holes again, synchronous to add in
III 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；
2nd, enzyme labelled antibody is added in the enzyme mark detection plate washed in step 1 and uses liquid, 50-150 μ l/ holes, blank well is not added with, and 37
DEG C be incubated 0.5-3 hours, board-washing 5 times；
3rd, 100 μ l/ holes of TMB developing solutions are added in the enzyme mark detection plate washed in step 2, mixing is put into wet box, and 37 DEG C incubate
It educates 5-15 minutes；
4th, terminate liquid terminates reaction；
5th, 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；
6th, 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 III type poliovirus D antigen positive samples,
Sample A value ＜ Cutoff values, for feminine gender, which is determined as III 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 above experiment include I, II, III type poliovirus D antigens, C antigens and other enteron aisles
Virus, III 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 III 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 it can be used for vaccine detection.Detection method sensitivity analysis, with the present invention to III type IPV-D antigenic contents reference material into
Row detection, Monitoring lower-cut can measure 0.125 DU/ml.Detection method accuracy is analyzed, and present invention experiment CV is small for 5.6 %
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 III pre-coated quick detection reagent specific test of type poliovirus D antigens of table
The 6 III 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 4 DEG C and less than -20 DEG C long-term stable experiments for doing 6,9,12,18 months；It the results are shown in Table
5；
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 III type poliovirus D antigens of table
Pre-coated -20 DEG C of stability test results of plate quick detection reagent of 4 III type poliovirus D antigens of table
Pre-coated plate prepared by the present invention has preferable stability under 37 DEG C of experiment conditions that accelerate the failure, and testing result meets
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
Test agent is detected 7 samples（Comparative example, table 5）, sample detection value 95% fiducial limit range of fluctuation.Sample size
Testing result fluctuation meets the requirement of ELISA detection reagents in 95% fiducial limit range, and pre- wrapper sheet detection reagent of the invention can be with
It is produced in batches and can be preserved for a long time, the detection of III type poliovirus D antigens can be effectively reduced in actual use
In experiment reagent and operating error, be conducive to improve working efficiency.
Comparative example
Detect sample using conventional III type poliovirus D antigens detection method is synchronous with the present invention, and to testing result into
Row compares, and method is as follows, ox（Rabbit, sheep）Anti- III 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 III 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 is as follows used in the present embodiment：
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 III 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, III type poliovirus antibody is resisted to be prepared and purified to animal, obtains animal and resist III type polio sick
The antibody purification of malicious D antigens；
S2, the purifying of III type poliovirus D antigens is resisted to resist to animal by sodium periodate method with horseradish peroxidase
Body is marked, and obtains the detection enzyme labelled antibody that animal resists III type poliovirus D antigens, and enzyme labelled antibody use is made
Liquid；
S3, the antibody purification of III type poliovirus D antigens is resisted to carry out the pre-coated processing of enzyme mark detection plate with animal, in advance
Protective agent, which is added in, in coating processing carries out closed protective；The protective agent includes but not limited to following component：BSA, gelatin, junket egg
In vain, it is one or more in albumin, trehalose, phosphate, carbonate, pre-coated carrier, the pre-coated carrier energy of gained is made
Batch making and it can meet long-term storage is realized under testing conditions index；
S4, antigen formation antigen antibody complex to be checked is captured, then combined with enzymic-labelled antibody using pre-coated carrier, passes through bottom
Object catalyzing enzyme develops the color, and the qualitative detection to III type D antigens of poliovirus or quantitative detection are completed according to colour developing result.
2. III 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：It is bright that 7 ~ 13g BSA, 7 ~ 13 trehaloses, 3 ~ 7g are included per 1000ml protective agents
Glue, 1 ~ 2g caseins, 30 ~ 70mMol phosphate solutions；The pre-coated carrier use is stored after vacuumizing packaging.
3. III 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 III type poliovirus D antigens antiserum：Inactivated poliovirus is close by 10-30% sucrose
Gradient centrifugation is spent, collects D antigen layers, is prepared after Electronic Speculum detection confirms for immune serum；Experimental animal ox, rabbit or sheep, it is first
Exempt from, III 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 measure is carried out using microneutralization test；
2）Ox or rabbit or III type poliovirus D antigen purification Antibody preparations of goat-anti：Take albumin A/G affinity columns, room
Temperature places balance 30 minutes, and chromatographic column is balanced with the equilibrium liquid of 5 times of column volumes；Antiserum is compared in equal volume with equilibrium liquid
Loading after mixing elutes foreign protein with the equilibrium liquid of 10 times of column volumes, retains destination protein；It is eluted with 10 times of the conventional of column volume
Liquid elutes destination protein from affinity column, collects eluting peak, spare after desalination；Protein content is measured with lowrry methods,
III type poliovirus D antigen purification antibody of ox, rabbit or goat-anti is obtained, less than -20 DEG C preserve for use.
4. III 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 in, is stood, is added in
Ethylene glycol solution is stood, then is mixed with step S1 gained antibody purifications, dialysed overnight；Add sodium borohydride, stand, add saturation sulphur
Sour ammonium, centrifugation；Take precipitation dissolve after dialysed overnight；It obtains ox or rabbit or the detection of III type poliovirus D antigens of goat-anti is used
Enzyme labelled antibody, Cord blood are spare.
5. III 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：By purified ox or rabbit or the antibody purification of III type poliovirus D antigens of goat-anti
It is tested by chessboard and confirms coating condition, after according to condition diluting antibody to 0.1-15 μ g/ml, it is special that 50-150 μ l/ holes add to enzyme mark
With in detection plate, 2~8 DEG C overnight, board-washing；
2）Enzyme mark detection plate is closed：Protective agent is added in the amount in 100-250 μ l/ holes, 2~8 DEG C overnight, and board-washing is dried, vacuum packet
Refrigerator saves backup after dress.
6. III 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 in after measuring samples are diluted in the detection hole of pre- wrapper sheet, it is synchronous to add in III type poliovirus D antigens ginseng
Product are examined, quantitative sample and content reference material multiple holes sample-adding；Former times of qualitative detection sample 50-150 μ l/ holes add in, if blank well 1
Hole and each 2 hole of antigen-negative controls；37 DEG C are incubated 1-3 hours, board-washing 3-5 times；
2）1）50-150 μ l/ holes add in enzyme labelled antibody using liquid 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 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 or the detection for calculating quantitative according to antigenic content reference material can be obtained according to cutoff values
As a result.
7. a kind of III 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 III type D antigens fast qualitative of poliovirus, quantitative detection side described in claim 1-6 any one
Application of the method in poliovirus detection reagent or kit is prepared.
9. a kind of III type D antigens fast qualitative of poliovirus, the kit of quantitative detection, which is characterized in that including
Following reagent component：III 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 III type D antigen positives of poliovirus, III type D antigens of poliovirus the moon
Property control, III type D antigenic content reference materials of poliovirus.
10. III type D antigens fast qualitative of a kind of poliovirus according to claim 9, the examination of quantitative detection
Agent box, which is characterized in that the preparation method of the III type D antigen detection plates of poliovirus is：
1）The antibody purification of purified ox or rabbit or III type poliovirus D antigens of goat-anti by chessboard is tested and is confirmed
Coating condition, after according to condition diluting antibody to 0.1-15 μ g/ml, 50-150 μ l/ holes are added in enzyme mark dedicated detector board, 2~8 DEG C
Overnight, board-washing；
2）Enzyme mark detection plate is closed：Protective agent is added in the amount in 100-250 μ l/ holes, 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810032959.1A CN108241058A (en) | 2018-01-13 | 2018-01-13 | A kind of pre-coated detection method of III type D antigens of poliovirus and its detection kit and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810032959.1A CN108241058A (en) | 2018-01-13 | 2018-01-13 | A kind of pre-coated detection method of III type D antigens of poliovirus and its detection kit and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108241058A true CN108241058A (en) | 2018-07-03 |
Family
ID=62699603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810032959.1A Pending CN108241058A (en) | 2018-01-13 | 2018-01-13 | A kind of pre-coated detection method of III type D antigens of poliovirus and its detection kit and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108241058A (en) |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1070484A (en) * | 1991-07-29 | 1993-03-31 | 生物化学药物有限公司 | Be used for detecting simultaneously the immunodetection of HIV-1, HIV-2, HTLV-I and HTLV-II antibody |
| WO2007131946A1 (en) * | 2006-05-15 | 2007-11-22 | Glaxosmithkline Biologicals S.A. | Detection method and kit |
| WO2011074006A2 (en) * | 2009-12-16 | 2011-06-23 | Serum Institute Of India Ltd. | Vaccine composition |
| CN102331494A (en) * | 2011-06-16 | 2012-01-25 | 广州艺佳生物科技有限公司 | Sealing and stabilizing agent for microporous board |
| WO2012028315A1 (en) * | 2010-09-02 | 2012-03-08 | Sanofi Pasteur Sa | A stabilizer for the preparation of a dry polio injectable vaccine composition |
| CN102382904A (en) * | 2011-10-25 | 2012-03-21 | 上海大学 | Method for detecting poliovirus |
| EP2491947A2 (en) * | 2006-09-07 | 2012-08-29 | GlaxoSmithKline Biologicals S.A. | Vaccine |
| CN102879567A (en) * | 2012-09-29 | 2013-01-16 | 同昕生物技术（北京）有限公司 | Alpha fetoprotein heteroplasmon isolation kit for liver cancer diagnosis, composition reagents of kit and application |
| CN103226146A (en) * | 2012-01-30 | 2013-07-31 | 广州瑞森生物科技有限公司 | Clenbuterol, ractopamine and salbutamol three-in-one euzyme linked immunosorbent assay, special kit and kit detection method |
| CN105603049A (en) * | 2016-01-18 | 2016-05-25 | 沈阳百创特生物科技有限公司 | Compound stabilizer and kit for in vitro diagnosis reagents |
| CN105759033A (en) * | 2016-03-29 | 2016-07-13 | 中国医学科学院医学生物学研究所 | Identification detecting method for Cox A16 and EV71 viral antigens |
| CN106290886A (en) * | 2016-07-28 | 2017-01-04 | 北京科兴生物制品有限公司 | A kind of method of detection III type poliovirus D antigenic content |
| CN107015004A (en) * | 2017-03-21 | 2017-08-04 | 深圳市汇松科技发展有限公司 | A kind of serum amyloid A protein determines kit and preparation method thereof |
-
2018
- 2018-01-13 CN CN201810032959.1A patent/CN108241058A/en active Pending
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1070484A (en) * | 1991-07-29 | 1993-03-31 | 生物化学药物有限公司 | Be used for detecting simultaneously the immunodetection of HIV-1, HIV-2, HTLV-I and HTLV-II antibody |
| WO2007131946A1 (en) * | 2006-05-15 | 2007-11-22 | Glaxosmithkline Biologicals S.A. | Detection method and kit |
| EP2491947A2 (en) * | 2006-09-07 | 2012-08-29 | GlaxoSmithKline Biologicals S.A. | Vaccine |
| WO2011074006A2 (en) * | 2009-12-16 | 2011-06-23 | Serum Institute Of India Ltd. | Vaccine composition |
| WO2012028315A1 (en) * | 2010-09-02 | 2012-03-08 | Sanofi Pasteur Sa | A stabilizer for the preparation of a dry polio injectable vaccine composition |
| CN102331494A (en) * | 2011-06-16 | 2012-01-25 | 广州艺佳生物科技有限公司 | Sealing and stabilizing agent for microporous board |
| CN102382904A (en) * | 2011-10-25 | 2012-03-21 | 上海大学 | Method for detecting poliovirus |
| CN103226146A (en) * | 2012-01-30 | 2013-07-31 | 广州瑞森生物科技有限公司 | Clenbuterol, ractopamine and salbutamol three-in-one euzyme linked immunosorbent assay, special kit and kit detection method |
| CN102879567A (en) * | 2012-09-29 | 2013-01-16 | 同昕生物技术（北京）有限公司 | Alpha fetoprotein heteroplasmon isolation kit for liver cancer diagnosis, composition reagents of kit and application |
| CN105603049A (en) * | 2016-01-18 | 2016-05-25 | 沈阳百创特生物科技有限公司 | Compound stabilizer and kit for in vitro diagnosis reagents |
| CN105759033A (en) * | 2016-03-29 | 2016-07-13 | 中国医学科学院医学生物学研究所 | Identification detecting method for Cox A16 and EV71 viral antigens |
| CN106290886A (en) * | 2016-07-28 | 2017-01-04 | 北京科兴生物制品有限公司 | A kind of method of detection III type poliovirus D antigenic content |
| CN107015004A (en) * | 2017-03-21 | 2017-08-04 | 深圳市汇松科技发展有限公司 | A kind of serum amyloid A protein determines kit and preparation method thereof |
Non-Patent Citations (9)
| Title |
|---|
| D. J. WOOD等: "A WHO Collaborative study on assays of the antigenic content of inactivated poliovirus vaccines", 《BIOLOGICALS》 * |
| GENNADY REZAPKIN等: "Improved ELISA test for determination of potency of Inactivated Poliovirus Vaccine (IPV)", 《BIOLOGICALS》 * |
| LEIGH A. SAWYER等: "Quantitation of D Antigen Content in Inactivated Poliovirus Vaccine Derived from Wild-type or Sabin Strains", 《BIOLOGICALS》 * |
| MORAG FERGUSON等: "Antigenic structure of poliovirus in inactivated vaccines", 《JOURNAL OF GENERAL VIROLOGY》 * |
| 党小军等: "《临床免疫学检验技术》", 31 July 2014, 科学技术文献出版社 * |
| 张春梅等: "脊髓灰质炎病毒2型和3型D抗原单克隆抗体的制备及鉴定", 《细胞与分子免疫学杂志》 * |
| 李树香等: "脊髓灰质炎灭活疫苗抗原含量检测方法的研究进展", 《国际生物制品学杂志》 * |
| 杨怀德: "脊髓灰质炎病毒Sabin株D抗原定量及其中和抗体检测的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 杨怀德等: "脊髓灰质炎病毒Sabin株D抗原定量及其中和抗体检测的研究", 《云南大学学报（自然科学版）》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102072957B (en) | Hepatitis C virus antibody diagnostic kit and preparation method thereof | |
| CN108107220A (en) | A kind of fluorescence immunoassay magnetic particle quantitatively detects O-shaped foot-and-mouth disease antibody kit | |
| CN101419238B (en) | Hepatitis C virus core antigen chemiluminescence ELISA detection kit | |
| CN107741495A (en) | A kind of double crush syndrome kit and its application for Senecan virus antigen detection | |
| CN105675866B (en) | For detecting that the solid phase of O-shaped FMDV antibody blocks ELISA kit | |
| CN102175853A (en) | ELISA (enzyme linked immunosorbent assay) kit for detecting pig progenitive and respiratory syndrome (PRRS) antibody | |
| CN105759033A (en) | Identification detecting method for Cox A16 and EV71 viral antigens | |
| CN104155449A (en) | Method and kit for detecting TORCH IgM antibodies and preparation method of kit | |
| CN107202898A (en) | Kit for Shanghai irregular antibody | |
| CN101539576A (en) | Hepatitis B virus pre S1 antigen chemiluminscence immunoassay kit and preparation method thereof | |
| CN108303533A (en) | A kind of pre-coated detection method of I type D antigens of poliovirus and its detection kit and application | |
| CN101750493A (en) | Enzymatic chemiluminescence immunoassay qualitative diagnostic reagent kit for simultaneously testing communicable disease projects | |
| CN108287237A (en) | A kind of pre-coated detection method of II type D antigens of poliovirus and its detection kit and application | |
| CN101738476B (en) | Rapid diagnosis kit for pre-S1 antigens of hepatitis B viruses and method for preparing same | |
| CN106383226A (en) | A quantitative detection method for the titer of a swine fever neutralizing antibody in swine serum and a detection kit | |
| CN106771121A (en) | A kind of foot and mouth disease virus colloidal gold strip, cause of disease quick detection kit and preparation method thereof | |
| CN108241058A (en) | A kind of pre-coated detection method of III type D antigens of poliovirus and its detection kit and application | |
| CN101655495A (en) | Detection method of EV71 virus antigen | |
| CN109613249A (en) | A kind of russian spring-summer encephalitis virus IgM antibody ELISA detection kit and preparation method thereof | |
| CN107064488B (en) | A kind of total antibody solid phase of swine fever virus serum blocks the preparation method of antigen used in ELISA kit | |
| CN108387726A (en) | I, II, III type D antigens of poliovirus simultaneously and rapidly differentiate, quantitative detecting method and its detection kit and application | |
| CN106771134B (en) | A kind of carp herpesvirusⅡtype sandwich ELISA detection kit and detection method | |
| CN104678104A (en) | ELISA immunoassay kit for detecting American-type PRRS antibodies | |
| CN109799342A (en) | A kind of O-shaped antibodies against foot-and-mouth disease virus competitive ELISA detection kit | |
| CN108387725A (en) | A kind of I type of poliovirus, II type, the pre-coated associated detecting method of III type D antigens and its detection kit and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180703 |
|
| WD01 | Invention patent application deemed withdrawn after publication |