CN106119210B - A kind of Poliovirus I monoclonal antibody and its application - Google Patents
A kind of Poliovirus I monoclonal antibody and its application Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
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- G—PHYSICS
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- G01N2469/10—Detection of antigens from microorganism in sample from host
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The present invention relates to a kind of polio I type viral monoclonal antibodies and its applications, belong to field of immunology and field of vaccinology.More particularly it relates to which deposit number is the application of the Poliovirus I monoclonal antibody hybridoma cell strain of CGMCC No.12292, the monoclonal antibody and the hybridoma cell strain and the monoclonal antibody that are generated by the hybridoma cell strain.
Description
Technical field
The present invention relates to field of immunology and field of vaccinology, and in particular, to a kind of I type of poliomyelitis virus is single
Clonal antibody and generate the antibody hybridoma cell strain and antibody application.
Background technique
Polio is the acute infectious disease that children's health is seriously endangered as caused by poliovirus, the virus
For neurotropic virus, the main motor nerve cells for invading central nervous system, damaged with anterior horn motor neurons based on.
Patient is mostly 1~6 years old children, and cardinal symptom is to generate heat, general malaise, limbs pain when serious, and occurrence and distribution is irregularly and light
The flaccid paralysis that weight does not wait, is commonly called as infantile paralysis.Polio clinical manifestation is varied, including very light non-of degree
Flaccid inability (the paralytic spinal cord of specific lesions, aseptic meningitis (nonparalytic poliomyelitis) and various muscle groups
Poliomyelitis).Polio patient, since anterior horn motor neurons are impaired, muscle associated therewith loses nerve
Adjustment effect and atrophy occurs, while subcutaneous fat, tendon and bone also atrophy makes entire body attenuate.
Known poliovirus has 3 serotypes, and the nucleotide number of this 3 serotypes is 7500 left sides
It is right.Although having 71% or so nucleotide is different nucleotide sequence common to 3 type polioviruses
It is all located in code area, therefore neutralization test no cross reaction between 3 type viruses.Disease can be found in conjunction with test with antigen-antibody
For poison there are two types of antigen, one kind being known as D (densification) antigen, and another kind is known as C (coreless) antigen.The former be present in it is mature, have
It is the neutralization antigen of the virus in infective virion, there is type specificity.C antigen be present in by 56 DEG C inactivation or
It is a kind of heat-resisting antigen composition in the immature hollow virion of person.D antigen and C antigen can be with the antiserums of virus
It is in antigen-antibody combination positive reaction.D antigen is that its content can be well reflected immunogene with respect to an advantage of C antigen
Property effect.
After oral ridge ash attenuated live vaccine is promoted, the whole world eliminates the action of ridge ash and achieves the achievement to attract people's attention.But it is real
The target that ridge ash is eliminated in the existing whole world still has many obstacles and challenge.It after Nigeria's ridge ash is revivable, spreads within 2008
To 8 neighbouring countries.And at 1995~1996 years and 1999, Yunnan Province of China and Qinghai Province have occurred respectively by Burma and print
After local prevention and control of diseases department takes urgent measure secondary case does not just occur for the Ji Hui street strain case of degree input.
Since the mid-50, Salk inactivated vaccine and Sabin attenuated live vaccine have been used always, immune effect is good,
Significantly reduce the disease incidence of polio.Salk vaccine is mixed after formalin-inactivated by 3 type viruses, muscle
Injection can induce body and generate neutralizing antibody.Its advantage is that convenient for saving and transporting, no attenuated strain atavism, and side effect
It is less.Sabin vaccine is made of attenuation variant, using oral, method simplicity, can not only make body generation antibody, moreover it is possible to
It stimulates intestinal wall thick liquid cell to generate secretory IgA, has elimination effect to street strain, so that its propagation in crowd is cut off, thus
The immune effect of Sabin vaccine is more preferable.
Current trivalent vaccine just widely used in the world be through using the Sabin strain of attenuation inactivation, prepare after purification and
At injection type vaccine, not only immune protective effect is good for the vaccine, but also avoids the danger of virulence reversion.Sabin plants of ridges
Marrow poliovirus is the safe strain that WHO is recommended to use, and can be used for the research and clinical detection of vaccine.
Poliovirus antibody refers to animal or IgG, IgM etc. that each type virus of human body poliomyelitis generates
The general name of type antibodies is widely used in determining for poliovirus identification, polio vaccine and each type antigen
In property and quantitative detection.
Polyclonal antibody coating --- the Anti-TNF-α generallyd use due to the detection of current Poliovirus I antigen
The sandwich detection architecture of body, there are biggish cross reactions not only for II, III type for the system, and cannot distinguish D well
Antigen and C antigen, especially when for 3 I type antigen of type polyvalent vaccine Sabin IPV detections, since cross reaction causes
Testing result is unable to the immunogenicity of I type of content and vaccine of I type antigen of actual response, while WHO also recommends using more grams
Grand antibody and the matched mode of monoclonal antibody detect the various antigenic content in trivalent polio vaccine；Still lack in the world at present
The standardized reagent of Sabin IPV D antigen detection, preparation specificity is good, and the monoclonal antibody with neutralization activity can be
The standardization of Sabin IPV vitro efficacy detection method lays the foundation.
Therefore, it is necessary to prepare a kind of I type monoclonal antibody of the polio that type is special, for I type ridge ash antigen, resist
The detection and the identification of virus of body.
Summary of the invention
The purpose of the present invention is to provide a kind of monoclonal antibodies of Poliovirus I, are used for polio I
The detection of type viral antigen content or antibody content.Another object of the present invention is to provide the applications of said monoclonal antibody.
Therefore, in a first aspect, present invention firstly provides a kind of Dan Ke for secreting Sabin plants of Poliovirus Is
Grand antibody hybridoma cell strain J I -112, the cell strain is on May 11st, 2016 in Chinese microorganism strain preservation conservator
It can common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, letter
Claim CGMCC, postcode 100101) preservation, classification naming is the strain of Poliovirus I monoclonal antibody hybridoma cell,
Deposit number is CGMCC No.12292.
In second aspect, the present invention provides the monoclonals of the Sabin plants of Poliovirus Is of secretion of first aspect
Antibody hybridoma cell strain J I -112 is that the hybridoma that deposit number is CGMCC No.12292 is being prepared for diagnosing ridge
Application in the diagnosticum of marrow poliomyelitis.
In the third aspect, the present invention provides the monoclonals of the Sabin plants of Poliovirus Is of secretion of first aspect
Antibody hybridoma cell strain J I -112 be deposit number be CGMCC No.12292 hybridoma preparation for prevent or
Treat the application in the drug of polio.
In fourth aspect, the present invention also provides Poliovirus I monoclonal antibody, which uses Mouse Bone
The preparation of myeloma cells hybridoma technology.
In one embodiment, the preparation may comprise steps of: will exempt from through Poliovirus I stoste
The mouse boosting cell of epidemic disease is merged with myeloma cell, and the miscellaneous of Poliovirus I monoclonal antibody can be secreted by isolating
Oncocyte is handed over, thus which prepares containing specific Poliovirus I monoclonal
The ascites of antibody.
In a further embodiment, the Sabin strain Poliovirus I of the purified inactivation of mouse
Stoste is immune, and the preparation method of the I type poliovirus liquid of Sabin strain of the purifying inactivation is the side of such as following example
Method: the working seed lots seed culture of viruses for taking I type virus to prepare is inoculated with Vero cell by MOI=10~0.05 or other are primary, passage is thin
Born of the same parents, the concentration of cell is 0.1-10 × 10 when virus inoculation6Cell/ml harvests cell conditioned medium, as Sabin after Virus culture
Strain Poliovirus I harvest liquid, after concentration 10 times of (such as passing through ultrafiltration membrane packet) is above, progress sieve chromatography and from
Up to the Sabin strain Poliovirus I stoste of purifying inactivation after sub- displacement chromatography and formalin-inactivated.
In a specific embodiment, the Poliovirus I monoclonal antibody by first aspect secretion
The monoclonal antibody hybridoma cell strain J I -112 of Sabin plants of Poliovirus Is is that deposit number is CGMCC
The secretion of No.12292 hybridoma cell strain obtains.
In one embodiment, the polio I type viral monoclonal antibodies are without any label.
In another embodiment, the Poliovirus I monoclonal antibody is marked through biomarker or chemistry
Note.Preferably, the Poliovirus I monoclonal antibody is marked through enzyme.Preferably, the enzyme is horseradish peroxidating
Object enzyme (HRP) or alkaline phosphatase (AP).
At the 5th aspect, the present invention provides the monoclonal antibody of fourth aspect in preparation for detecting polio I
Application in the detection agent of type viral antigen.
At the 6th aspect, the present invention provides the monoclonal antibody of fourth aspect in preparation for detecting polio I
Application in the detection agent of type antiviral antibody.
During preparing the detection agent for detecting Poliovirus I antigen or antibody, above-mentioned four directions
The monoclonal antibody in face, can be by adding through biomarker or chemical labeling when without biomarker or chemical labeling
The secondary antibody of (such as such as the enzyme of horseradish peroxidase (HRP) or alkaline phosphatase (AP) mark) carries out, i.e., with one
The poliomyelitis antiviral antibody of a species is capture antibody with monoclonal antibody of the invention as coated antibody, above
It states through the secondary antibody of biomarker or chemical labeling as detection antibody, for detecting or preparing detection reagent.
At the 7th aspect, the present invention provides a kind of for detecting the kit of Poliovirus I antigen, contains
The monoclonal antibody of fourth aspect.
In eighth aspect, the present invention provides a kind of for detecting the kit of Poliovirus I antibody, contains
The monoclonal antibody of fourth aspect.
The present invention provides a kind of cell strains of transformation that can be used for producing polio therapeutic antibodies, and packet
The gene information of protection antibody contained in cell strain.
Poliovirus I monoclonal antibody provided by the invention has following one or more or whole beneficial
Effect:
1, monoclonal antibody of the invention potency with higher is reactivity, and indirect method potency is up to 105More than.
2, monoclonal antibody of the invention has the preferable ability for neutralizing virus, for Poliovirus I
Neutralization titer is greater than 1:16384.
3, Poliovirus I monoclonal antibody of the invention can specifically distinguish polio I type disease
Poison and II, III type virus are the monoclonal antibodies that can be specifically bound with Poliovirus I not with latter reaction.
4, monoclonal antibody of the invention and hepatitis A virus, enterovirus type 71 viruses, Coxsack A16 virus etc. are without friendship
Fork reaction, there is preferable virus-specific.
5, the present invention provides a kind of technical solutions that technical concept is completely new, the i.e. monoclonal antibody of fourth aspect.
6, monoclonal antibody of the invention is substantially only and as the antigen D antigen-reactive that can produce neutralizing antibody, therefore
It can be used as the effective tool of reflection vaccine protecting effect.
7, other than specific reaction occurs with Sabin plants of Poliovirus Is, monoclonal antibody of the invention
Can also at present at home and abroad it is commercially available by street strain Mahoney plants of Poliovirus I prepare polio
Inactivated vaccine reacts, therefore has comparable broad spectrum activity, can be widely applied.
8, monoclonal antibody of the invention can be widely applied to the detection, identification, screening of Poliovirus I with it is right
In the antigen detection of production of vaccine and epidemiological survey；Detection is I type specific antigen simultaneously, can be more efficiently anti-
The I type antigenic content for reflecting polyvalent vaccine controls the technical study and the quality of production and vaccine product of vaccine and quality is ground
Study carefully and is of great significance.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art；
Reagent used in embodiment is commercial goods.
The preparation of 1 immunogene of embodiment and animal immune
(1) take Vero cell work cell bank recover after in 36.5 ± 0.5 DEG C cultivate, until cell concentration be 0.1-10 ×
106When cell/ml, virus inoculation.
(2) the working seed lots seed culture of viruses for taking Sabin plants of Poliovirus Is to prepare is inoculated with by MOI=5~0.1
Vero cell sets 32.5 ± 0.5 DEG C of cultures.
(3) Virus culture 2~4 days harvest cell conditioned medium, as Sabin plants of I type virus harvest of poliovirus
Liquid.
10 times or more are concentrated with ultrafiltration membrane packet after the clarification of (4) I type virus harvest liquids.
(5) sieve chromatography and ion-exchange chromatography are then carried out, monitoring wavelength is 280nm, collect respectively eluent with
Liquid is flowed through to get refined solution, up to the Sabin strain polio I type virus stock solution used of purifying inactivation after formalin-inactivated.
(6) by the Sabin strain polio I type virus stock solution used of purifying inactivation, (first immunisation is Freund with Freund's adjuvant
Freund's complete adjuvant, later period are immunized as incomplete Freund's adjuvant) exempt from after isometric mixing and emulsifying in the 0th, 14,28 day dorsal sc multiple spot
Epidemic disease BALB/c mouse, 0.2ml/ is only.
(7) the Sabin strain polio I type virus stock solution used intravenous injection into mice that inactivation was purified in the 39th day, after 3 days
Mouse spleen is taken, cell fusion is carried out.
2 cell fusion of embodiment and build strain
(1) recovery culture SP2/0 cell strain before cell fusion, the fusion culture of expansion in first 3 days, merges first 1 day removal RPMI
1640 cell culture fluids (Gibco) add culture solution again, prepare SP2/0 cell.
(2) immune mouse is put to death, prepares mouse boosting cell suspension according to a conventional method.
(3) it is separately added into appropriate not exclusively IMDM culture solution (Gibco) according to splenocyte and SP2/0 cell counts,
SP2/0 cell, which shakes, to be mixed, and splenocyte is blown and beaten uniform with pipette.Then splenocyte is mixed with SP2/0 cell by 1:2~10:1
Together in 50ml centrifuge tube, mix.
(4) plus incomplete IMDM culture solution is to 50ml, is centrifuged 5-10 minutes, evacuation supernatant.Bottom of fusion pipe is tapped, makes to precipitate
Cell is loosely uniform, and centrifuge tube sets 37 DEG C of water-baths, prepares fusion.
(5) by 37 DEG C preheat 50% PEG4000 1ml be slowly dropped into cell mixing pipe with dropper, while drop while rotate from
Heart pipe makes cell be stored in mixing state.
(6) the IMDM culture medium (Gibco) (37 DEG C) of 15ml serum-free is slowly added to after standing 90 seconds immediately, is then centrifuged for
It 5-10 minutes, discards supernatant.
(7) IMDM complete culture solution (Gibco) is added, mixes, suspension is added to respectively in 96 porocyte culture plates,
100 holes μ l/, in 37 DEG C, 5%CO2Culture in incubator.
Add 100 holes μ l/ HAT culture solution (IMDM (Sigma) containing 1*HAT) to cell plates within (8) the 2nd days.
(9) a HAT culture solution is changed within every 2-3 days, sees whether hybridoma occur, is changed to HT culture medium (IMDM after two weeks
Containing 1*HT (Sigma)), observe fused cell upgrowth situation.
(10) growing state for starting observation hybridoma for the 7th day after cell fusion, to its length to hole floor space 1/10
Supernatant is sucked out when above and carries out antibody ELISA detection.Positive hole cell is transferred to 24 orifice plates and expands culture, is subcloned in time.
(11) 28 cell lines for capableing of stably excreting antibody, composite antibody potency and specificity are obtained through 3 subclones
(test method sees below) two indices, the cell line that we select one of them best are named as J I -112, freeze this
Cell.Hybridoma cell strain is subjected to preservation, deposit number is CGMCC No.12292, classification naming are as follows: I type of polio
Viral monoclonal antibodies hybridoma cell strain, preservation time: on May 11st, 2016；Depositary institution: China General Microbiological strain
Preservation administrative center (China General Microbiological Culture Collection Center, CGMCC),
Address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101.
The preparation of 3 cell strain of monoclonal antibody ascites of embodiment and antibody ELISA method bioactivity
The hybridoma that the embodiment 2 frozen obtains is recovered according to a conventional method, is cultivated, covers 25ml to cell
50% or more Tissue Culture Flask bottom of bottle when can conventionally intraperitoneal inoculation BALB/c mouse, periodically collect ascites J I-
112。
The Sabin strain Poliovirus I of purifying inactivation is diluted using 0.01M PBS 1:200,100 holes μ l/
Coated elisa plate, 2-8 DEG C overnight, and then 1:10 is added in 100 holes μ l/2Originate the negative control and J I -112 of 10 times of gradient dilutions
Monoclonal antibody ascites, 37 DEG C are reacted 1 hour, the sheep anti-Mouse secondary antibody of the diluted HRP label of 100 holes μ l/ addition 1:4000, and 37
DEG C board-washing, colour developing, termination after reaction 1 hour, reads OD450nm, the spinal cord for thus detecting hybridoma of the present invention secretion is grey
The indirect elisa method antibody titer of I type virus ascites J I -112 of matter inflammation, antibody titer is up to 105More than, potency is higher.As a result see
Table 1.
1 antibody indirect ELISA method bioactivity result of table
The detection of 4 cell strain of monoclonal antibody ascites neutralize antibody titers of embodiment
I -112 monoclonal antibody ascites of J, feminine gender and positive serum controls are subjected to 2 times of gradient dilutions, then 50 hole μ l/
Cell plates are added, each dilution adds 2 holes in parallel, then the I type Polio virus of every hole addition 100CCID50/0.05ml, and 35.0
100 hole μ l/ 0.2-1.6 × 10 are added after being incubated in ± 0.5 DEG C of incubator5Vero cell suspension, continue culture 5~7 days determine
As a result, the neutralize antibody titers of the Poliovirus I ascites J I -112 of detection hybridoma secretion of the present invention.It neutralizes
Antibody titer reaches 1:16384 or more, and potency is higher.
5 anitibody type specific detection result of embodiment
II type of Sabin strain polio of purifying inactivation and III type virus stock solution used is dilute using 0.01M PBS 1:10
It releases, 100 hole μ l/ coated elisa plates, 2-8 DEG C overnight, the Poliovirus I monoclonal antibody that detection embodiment 3 obtains
The antibody titer of ascites J I -112, while negative and positive control is set, it is feminine gender.It the results are shown in Table 2.
2 indirect elisa method anitibody type specific detection result of table
Positive control has preferable reactivity in the present invention, and negative control is reactionless, and test is set up.I -112 cell strain of J
The monoclonal antibody (J I -112) of secretion is not reacted with II type of polio and III type virus, illustrates that monoclonal of the invention is anti-
Body can distinguish I type of poliovirus and II type, III type virus.The list of I -112 hybridoma of J secretion of the invention
Clonal antibody can in the antigenic content detection for polio vaccine (sample of I type, II type and the mixing of III type virus)
Specifically to identify I type antigen of polio, without being interfered by other amphitypys, there is preferable type specificity and apply valence
Value.
Testing result of 6 antibody of embodiment to D antigen-reactive specificity
By Sabin plants of I types of polio solid viral (D antigen), hollow viral (C antigen), 56 DEG C of D antigen heating
30min restrovirus (heat treatment C antigen) is with 100 hole μ l/ coated elisa plates after 0.01M PBS 1:200 times dilution, 2-8 DEG C of mistake
Night；By the monoclonal antibody (J I -112) of the cell strain secretion in the present invention with 1:102Originate 100 μ l/ after 10 times of gradient dilutions
ELISA Plate is added in hole, while doing negative control.Thereafter test procedure is same as Example 3, detects hybridoma point of the present invention
The potency and specificity for I -112 pair of above-mentioned antigen of Poliovirus I ascites J secreted, the results are shown in Table 3.
3 antibody of table is to D antigen-reactive specific detection result
As a result as it can be seen that Poliovirus I ascites J I -112 only it is solid to I type virus have stronger reactivity,
Potency about 105, hollow virus and heat treatment virus are not reacted.Document shows, only solid viral (D antigen) can be with
Generate the protection antibody for the grey I type virus of ridge.Originally experiments have shown that, which is only capable of detection Effective Antigens i.e. D antigen, for
It can be used for Effective Antigens epitope and content by invalid antigen anergy, the recognition capability with preferable Effective Antigens
Detection, it is significant.
The measurement of 7 Subclass of antibody of embodiment
By the Sabin strain Poliovirus I stoste of purifying inactivation using 100 μ after 0.01M PBS1:10 times dilution
The hole l/ coated elisa plate, 2-8 DEG C overnight, then using Sigma company ISO2-1KT mouse monoclonal antibody grouping reagents according to list
Clonal antibody subclass reagent specification is tested, and the rabbit-anti sheep secondary antibody for being eventually adding HRP label carries out monoclonal antibody subclass
Identification.Monoclonal antibody of the invention is IgG1 type as the result is shown.
8 immunoblotting of embodiment (Western blotting) experiment
The Sabin strain Poliovirus I stoste of purifying inactivation is turned equipment using the electrophoresis electricity of BIO-RAD to use
12% SDS-PAGE running gel carries out electrophoresis, and electricity goes to nitrocellulose filter again later, then with hybridoma of the present invention
Ascites is primary antibody (1:2000), AP- sheep anti-mouse igg is that secondary antibody carries out Western blotting identification.The results show that hybridoma
The monoclonal antibody that cell J I -112 secretes is not reacted with Sabin plants of polio type virus VP 1s, VP2, VP3, VP4, table
The bright monoclonal antibody cannot be generated with the antigen after denaturation reacts, and illustrates the Dan Ke that hybridoma J I -112 of the present invention secretes
Grand antibody J I -112 cannot identify linear epitope, be conforma-tional monoclonal antibody, identify space structure.
The measurement of 9 affinity costant of embodiment
Protein content is detected after purification in the monoclonal antibody J I -112 that hybridoma cell strain of the invention is secreted.Using
The Sabin strain Poliovirus I transverse direction coated elisa plate of the diluted various concentration of 1:5,1:10,1:20,100 holes μ l/,
2-8 DEG C of coating is overnight.It closes 2 hours for 37 DEG C, pats dry stand-by after second day board-washing.By bis- times of ladders of monoclonal antibody purification JI-112
Degree dilution, longitudinal ELISA Plate being added after coating, using the OD value of indirect elisa method detection antigen-antibody reaction.With each antigen
The OD value of plateau section under concentration is calculated as 100%, calculates 50%OD value, and it is anti-to investigate the corresponding monoclonal put of 50%OD value
Bulk concentration [Ab] t, further according to the available monoclonal antibody of the present invention of affinity costant calculation formula affinity costant be 0.996 ×
109M-1。
The Detection of Stability of 10 hybridoma cell line secrete monoclonal antibody of embodiment
Respectively behind 3 months and 12 months, it is removed from liquid nitrogen I -112 hybridoma cell strain of J frozen and is recovered, expanded
After big culture, ascites is prepared, indirect ELISA is carried out and detects antibody titer, the ascites with preparation early period is that control is carried out while being examined
It surveys.As a result, monoclonal antibody titer of ascites prepared by hybridoma cell strain of the invention reaches 105More than, it is imitated with ascites early period
Valence indifference shows that the potency of the ascites prepared after cyropreservation does not decline.Therefore monoclonal cell strain secretory antibody of the present invention
Activity do not reduce, have good stability.
The PRELIMINARY RESULTS of 11 antigenic content detection kit of embodiment
Poliovirus I rabbit polyclonal antibody is diluted using 0.05M carbonate buffer solution 1:4000,100 μ l/
Hole coated elisa plate, 2-8 DEG C overnight.37 DEG C of confining liquid are closed 2 hours, and I type antigen reference product of polio and 3 are separately added into
Criticize poliomyelitis vaccine,Salk (poliomyelitis vaccine,Salk includes I, II and III type inactivation of viruses), 37 DEG C of reactions 1
Hour, the Poliovirus I monoclonal antibody J I -112 of 1:8000 is added after board-washing, 37 DEG C are reacted 1 hour, after board-washing
It is added the sheep anti mouse secondary antibody of 1:4000 diluted HRP label, 37 DEG C of board-washings after reaction 1 hour are developed the color, terminated, read, and are examined
Survey I type antigenic content of poliomyelitis vaccine,Salk.It the results are shown in Table 4.
The application of 4 antigenic content detection method of table
It is anti-that I type in 3 batches of poliomyelitis vaccine,Salks is had detected using the antigenic content detection method that the present invention establishes
Former content, detected value relative to theoretical value the rate of recovery between 103%-126%, meet the requirement of quality standard, enzyme-linked
Within the error range that immunization allows, detection method is accurate and reliable.The present invention has preferable application effect.
The special Journal of Sex Research of 12 antigenic content detection kit of embodiment
Poliovirus I rabbit is how anti-using 0.05M carbonate buffer solution 1:4000 dilution, 100 holes μ l/ coating
2-8 DEG C of ELISA Plate overnight.Then 37 DEG C close 2 hours, be separately added into I, II, III type virus stock solution used of Sabin plants of polio,
Hav inactivated vaccine stoste, Coxsack A16 (CA16) inactivated vaccine stoste, CA16 harvest liquid, enterovirns type 71 (EV71) go out
Live vaccine stoste, EV71 harvest liquid, 37 DEG C are reacted 1 hour, and the Poliovirus I monoclonal of 1:8000 is added after board-washing
Antibody J I -112,37 DEG C are reacted 1 hour, and the sheep anti mouse secondary antibody of the diluted HRP label of 1:4000,37 DEG C of reactions 1 are added after board-washing
Board-washing is developed the color, is terminated, is read after hour, and I type antigenic content of test sample the results are shown in Table 5.
The monoclonal antibody specificity of the invention of table 5 (OD value)
Monoclonal antibody of the invention and the Sabin plants of type of polio II and III virus stock solution useds, hav inactivated vaccine are former
Liquid, CA16 inactivated vaccine stoste, CA16 harvest liquid, EV71 inactivated vaccine stoste, EV71 harvest liquid do not react, show the present invention
Monoclonal antibody effectively polio I type virus can be distinguished with above-mentioned virus.It is established simultaneously using the present invention
System and culture medium and Vero cell culture cross reaction is not present, therefore monoclonal antibody of the invention can be effective
I type antigen of polio is detected, the specificity with height.Since antibody of the invention has as differentiation polio
I, in II, III type polyvalent vaccine various antigen most critical index specificity, therefore monoclonal antibody of the invention can be to spinal cord
I, II, III type polyvalent vaccine of poliomyelitis is effectively distinguished: when detecting I type antigen, monoclonal antibody of the invention is not with II
Type and III type antigenic cross-reaction, therefore can effectively detect I type antigenic content of polio.
Detection of the 13 antigenic content detection kit of embodiment to D antigen
Poliovirus I rabbit polyclonal antibody is diluted using 0.05M carbonate buffer solution 1:4000,100 μ l/
Hole coated elisa plate, 2-8 DEG C overnight.37 DEG C of confining liquid are closed 2 hours；By Sabin plants of Poliovirus Is (D antigen)
And 56 DEG C of heating 30min of poliomyelitis vaccine,Salk, ELISA Plate then is added in sample.Follow-up test with embodiment 12,
Detection kit is investigated for the detection effect of D antigen in the sample before heating and after heating.It the results are shown in Table 6.
Testing result of the monoclonal antibody of the invention of table 6 to I type virus D antigen
According to the record and the first research of the present inventor in existing literature, poliovirus stoste and inactivation epidemic disease
Animal is immunized in seedling after 56 DEG C of heating 30min, and the neutralization titer of the immune serum of animal is extremely low, shows thermally treated sample
In poliovirus no longer there is immunogenicity.And in this application, according to table 6, monoclonal antibody of the invention with
Sabin plants of Poliovirus I stostes and inactivated vaccine have good reaction, but have with the above-mentioned sample after heating
There is poor reaction, be below 3%, this shows that the testing result of antigenic content and the immunogenicity of existing animal experiment have
Good consistency.
Additionally, it is known that after polyclonal antibody-polyclonal antibody polio antigen content detection system and heating
Poliovirus still has the immunogenicity result of stronger reaction and existing animal experiment inconsistent.Therefore, originally
The detection system of invention monoclonal antibody can preferably detect D antigen, preferably reflect the protecting effect of vaccine, testing result
More preferably, it is particularly suited for the quality control of the intermediate products and finished product of vaccine.
Monoclonal antibody of the invention can be used as double-antibody sandwich elisa detection examination when for antigenic content detection
The coated antibody of agent box can also carry out biomarker or chemical labeling to it, as double-antibody sandwich elisa detection kit
Sandwich enzyme labelled antibody；It is used it is also possible to which the polyclonal antibody of it and another species is matched, by the way that sandwich antibody is added
The method of second enzyme labelled antibody prepares antigenic content detection kit.
When the present invention is used for antibody assay kit, it can be used as coated antibody or enzyme mark competition antibody, can also make
To compete antibody and the method that anti-mouse ELIAS secondary antibody is added is detected.
Detection of the 14 antigenic content detection kit of embodiment to commercially available each strain Poliovirus I
Poliovirus I rabbit polyclonal antibody is diluted using 0.05M carbonate buffer solution 1:4000,100 μ l/
Then Sabin plants of I type antigen reference product of polio and its spinal cord ash is added in hole coated elisa plate, 2-8 DEG C of overnight rear enclosed
Matter inflammation inactivated vaccine and Mahoney plants of international standard substances and its poliomyelitis vaccine,Salk (the polio reference
Product, standard items, inactivated vaccine include I, II and III type inactivation of viruses), 37 DEG C are reacted 1 hour, and the ridge of 1:8000 is added after board-washing
I type viral monoclonal antibodies J I -112 of marrow poliomyelitis, 37 DEG C are reacted 1 hour, and the diluted HRP label of 1:4000 is added after board-washing
Sheep anti mouse secondary antibody, 37 DEG C reaction 1 hour after board-washing developed the color, terminated, read, detect I type of poliomyelitis vaccine,Salk
Antigenic content.It the results are shown in Table 7.
The application of 7 antigenic content detection method of table
The I type virus of listing poliomyelitis vaccine,Salk sold extensively in the world at present include Sabin plants with
Two kinds of Mahoney strain can effectively prevent to infect due to I type poliovirus bring.Using cell strain of the invention
The double-antibody sandwich detection system that the odd contradictive hydroperitoneum J I -112 of preparation is established, for Sabin plants and two kinds of spinal cord ashes of Mahoney strain
The testing result rate of recovery of I type D antigen in matter inflammation inactivated vaccine is between 90%-110%, in the mistake that enzyme-linked immunization allows
Within poor range, the rate of recovery is preferable, and detection method is accurate and reliable.Show effectively detect in commercial available vaccines using the monoclonal antibody
I type D antigenic content, there is wide spectrum applicability to the product quality research of commercial available vaccines and quality control.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. a kind of Poliovirus I monoclonal antibody hybridoma cell strain, deposit number is CGMCC No.12292.
2. hybridoma cell strain described in claim 1 is preparing the application in the diagnosticum for diagnosing polio.
3. the monoclonal antibody that the hybridoma cell strain as described in claim 1 generates.
4. monoclonal antibody according to claim 3, which is characterized in that the monoclonal antibody is through biomarker or change
Learn the monoclonal antibody of label.
5. monoclonal antibody according to claim 4, which is characterized in that the monoclonal antibody is the monoclonal of enzyme label
Antibody.
6. the described in any item monoclonal antibodies of claim 3-5 are in preparation for detecting Poliovirus I antigen
Application in detection agent.
7. the described in any item monoclonal antibodies of claim 3-5 are in preparation for detecting Poliovirus I antibody
Application in detection agent.
8. a kind of for detecting the kit of Poliovirus I antigen or antibody, which is characterized in that contain claim
The described in any item monoclonal antibodies of 3-5.
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| US7138274B2 (en) * | 2004-08-26 | 2006-11-21 | Creagene, Inc. | Advanced oral poliovirus vaccine (Adv-OPV) deprived of the possibility of vaccine associated paralytic poliomyelities (VAPP) |
| US9107906B1 (en) * | 2014-10-28 | 2015-08-18 | Adma Biologics, Inc. | Compositions and methods for the treatment of immunodeficiency |
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| US7138274B2 (en) * | 2004-08-26 | 2006-11-21 | Creagene, Inc. | Advanced oral poliovirus vaccine (Adv-OPV) deprived of the possibility of vaccine associated paralytic poliomyelities (VAPP) |
| US9107906B1 (en) * | 2014-10-28 | 2015-08-18 | Adma Biologics, Inc. | Compositions and methods for the treatment of immunodeficiency |
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