CN1591015A - Influenza Virus B colloidal gold quick detection test paper - Google Patents
Influenza Virus B colloidal gold quick detection test paper Download PDFInfo
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Abstract
The present invention discloses hybridoma cell strain whose conservative number is CGMCC0988, anti-fluenza B virus nucleoprotein monoclonal antibody produced by said hybridoma cell strain and influenza B virus colloidal gold fast detection testing paper containing said monoclonal antibody. Said detection testing paper can be used for quickly detecting influenza A virus, and its specificity, sensitivity and accuracy are high, and its storage and transportation are convenient.
Description
Technical field
The present invention relates to anti-influenza B virus nucleoprotein monoclonal antibody, produce the hybridoma cell strain of this monoclonal antibody and contain the influenza B virus colloidal gold fast detecting test paper of this monoclonal antibody.
Background technology
Influenza (influenza) is called for short influenza, is the acute respiratory infectious disease that is caused by influenza virus.Clinical characters is the anxious high heat that rises, Muscular stiffness, weak, or accompany slight respiratory symptom.This disease is short latent period, and infectiousness is strong, propagates rapidly.Influenza virus is divided first, second, the third three types, and first, influenza B threatens maximum.Because the influenza virus pathogenicity is strong, easily morphs, the crowd lacks immunity to variant, easily causes outbreak of epidemic, and influenza is very big to the mankind's harm, the popular reduction that causes crowd's mean lifetime of a field flow sense.Up to now, it is little popular that four big popular and several times had taken place in the world, causes billions of people morbidity, and tens of millions of people's death has had a strong impact on people's life and The development in society and economy.The most serious in the world flu outbreak had more than 2,000 ten thousand people's death in 1918, surpassed total death toll of the World War I, was the maximum infectious disease great outburst that causes death.And at present in the U.S., the annual death toll that causes because of influenza surpasses the death toll that traffic accident and acquired immune deficiency syndrome (AIDS) cause, and has become the No.1 formidable enemy of serious harm human health in China and developing country.
Influenza B virus is a single strand RNA virus, and genome is made up of 8 sections RNA, and encoding influenza virus albumen.Influenza virus is divided into: Flu-A very easily causes worldwide popular; But influenza B is localized epidemics then, and influenza C often be distribute popular.4 big variations have taken place in influenza A virus after being very popular in the world in 1918.Isolated the A0 type in 1933 first, variation is Al (inferior B-mode, nineteen forty-six separates), A2 (Asia A type, nineteen fifty-seven separates), A3 (Asia, Hong Kong A type, nineteen sixty-eight separates) and new A1 type (separating in 1977 years) in succession.Endangering big to the people is first, influenza B, and influenza C harm is little.Because the neuraminidase (NA) on first, influenza B virus surface and hemagglutinin (HA) are escaped the selection of body immune system and very easily morphed, and the nucleoprotein of influenza virus (NP) variability is little, and be relatively very conservative.Therefore etiologic regularty of epidemic that is characterized as influenza of above influenza virus and diagnosis and study on prevention are laid a good foundation.
The influenza four seasons all can fall ill, but are many with winter-spring season.The incidence of disease in 5-20 year is higher, but new subtype is very popular and does not then have remarkable age difference.This sick infectious agent is patient and subclinical infection person, through droplet transmission, and crowd general susceptible, particularly child and the elderly.Outbreak of epidemic easily takes place, is very popular even worldwide being very popular except that distributing in this disease.The popular of influenza lived scattered rural area behind the first city after the first collective often along the traffic lines rapid spread.
During the influenza pandemic,, can make clinical diagnosis according to contact history and colony morbidity history, typical clinical symptom and sign and laboratory examination.Sporadic cases, because of similar to many acute diseases initial stage symptom, diagnosis is difficulty comparatively.As long as comprehensive grasp epidemiology and Clinical symptoms and the laboratory examination (blood leukocytes counting and classification, throat swab cultivation, chest X-ray etc.) that some are necessary, clinical diagnosis is not difficult yet.
Influenza is the main infectious disease of outburst in the winter time that is caused by B-mode and influenza A virus.The infection factor to the clinical diagnosis that infects of great use, this infection is considered to self to resist in healthy population, but then can cause ill even dead for child and the low patient of immunity.Therefore it is very necessary carrying out clear and definite pathological diagnosis with clinicopathologic each stage at the initial stage of a disease.
Clinical patient is because the disease symptoms that different Respirovirus (as influenza virus, Respiratory Syncytial Virus(RSV), parainfluenza, adenovirus etc.) infection causes can be quite similar, and this has caused relatively difficulty of popular diagnosis, makes a definite diagnosis and often depends on laboratory diagnosis.Diagnostic method should be that their early stage in disease just can obtain clear and definite diagnosis fast, is convenient to stop the infection of disease and changes popular outburst.
The detection of influenza virus at present mainly contains following several method:
One, Routine Test Lab detects
1, virus is separated
The goldstandard of laboratory diagnosis influenza is to separate influenza virus.The time of adopting sample 5 days is advisable with morbidity, the sample that nasopharyngeal secretions separates as virus, and positive rate is higher than other samples.Can be with the chicken embryo culture or with the method isolated viral of cellular incubation.Current owing to have " O " appearance of phase strain, when separating influenza virus, should consider to adopt sensitive cells as far as possible, during with the chicken embryo, adopt amniotic inoculation.When separator is carried out the hemagglutination activity inspection, should not adopt chicken red blood cell, and should adopt the red blood cell of people or cavy.Separate influenza B virus and adopt 9-11 day instar chicken embryo more.But these methods have serious defective.Because their not only time-consuming but also efforts needed 2-7 days just can obtain net result usually, at clinicing aspect effective treatment of patient just there is certain limitation like this.
2, serology detects
Detect patient's acute stage and convalescent paired sera simultaneously with known influenza antigen, more than 4 times or 4 times diagnostic significance is arranged if anti-influenza B virus antibody titer is higher than the acute stage in the convalescent serum.Because the influenza antigen variation is very complicated, different regions, even areal the antigenicity of institute of commensurate epidemic isolates is not incomplete same, thereby carry out serum antibody when measuring, the used antigen epidemic strain of handy there and then adds representative strain.
2,1 hemagglutination-inhibition test
This method biggest advantage is easy to be reliable.Shortcoming is the disturbing effect that is subject to nonspecific inhibition in the serum, and serum need not handled, and bigger 2mm has diagnostic significance with the top to the haemolysis circle of convalescent serum than the acute stage.
2,2 single haemolysis that expand are measured
The advantage of this method is that susceptibility suppresses to measure height than blood clotting, is not measured the disturbing effect of nonspecific inhibition in the serum, and serum is not treated, just needn't dilute and can do experiment.Shortcoming is need use complement in the experiment.This measures the same hemagglutination-inhibition test of used antigen, and acute stage and convalescent serum should be on same block of plates, and control board is arranged.The haemolysis circle that has only convalescent serum has diagnostic significance than the big 2mm of acute stage with the top.
Two, quick diagnosis
Check that directly viral antigen and viral nucleic acid can reach the quick diagnosis purpose, common method has immunofluorescence technique, immunoenzyme, radioimmunology, immune colloid gold, electron microscopy, nucleic acid hybridization and PCR method etc.
1, immunological method
1, the ultimate principle of 1 immunoenzyme immunoenzyme (EIA), be after utilizing enzyme and antibody or antigen combining, form enzyme conjugates, it neither changes the immunologic competence of antibody or antigen, the enzyme activity that keeps enzyme itself again, the corresponding substrate that adds enzyme then makes original colourless substrate generation hydrolysis, oxidation or other reactions under the catalytic action of enzyme, generate coloured product.
Utilize Directigen influenza B virus quick diagnosis reagent kit (Directigen Flu-A), diagnostic test can be finished in 15 minutes, can directly use Pharyngeal aspirate or the throat swab leachate gathered from patient to detect.This experiment is dyed method based on immuno-enzymatic and is designed.At first extract viral antigen in the various clinical samples, viral antigen is dripped on detection film again with adsorptive power with detergent mixture.The anti-influenza B virus nucleoprotein monoclonal antibody that adds alkali phosphatase enzyme mark then treats that antigen and antibody fully act on the back and adds the substrate colour developing.This method is fast responsive, but can't distinguish the different subtype influenza infection, also can't diagnose the infection of influenza B virus, simultaneously the price height.
Boon A.C.M. etc. utilizes micropore titre cellular enzymes immunization (cell-EIA) to be used for method as area monitoring influenza first.According to different color reactions or two anti-different marks, can be used for detecting the different hypotype of influenza B, the method can detect a large amount of samples in one day.Flu outbreak can be used as the method for effectively cheap fast diagnosis influenza B virus season.Utilize cell-EIA and commercial Directigen Flu-A that the diagnosis of sample is compared, cell-EIA sensitivity (74%) and specificity (91.6%) all are higher than DirectigenFlu-A (65%, 84.6%), but specificity is not high.
Reina J etc. can also directly detect first and influenza B virus antigen simultaneously fast with new dot blot EIA method.They identify clinical 160 samples, utilize the cell method to identify that 74 are the influenza positive, utilize new EIA method that the positive sample of identifying is further identified, 68.9% is identified the positive.Wherein the new EIA method evaluation of 41 routine first influenza utilizations has 34 examples positive; The new EIA method of 33 routine second influenza utilizations is identified 17 examples positive (P＜0.05), and utilizing the EIA method to identify does not have false positive results.Therefore as detect influenza B the result shows and can utilize this method to carry out conventional sense to the first influenza, but influenza B detected weaker,, can utilize other method such as cellular incubation or RT-PCR.
EIA susceptibility height, high specificity, easy and simple to handle, naked eyes can be observed, and are convenient to extensive detection, in influenza virus research, it is mainly used in the quick diagnosis and the monoclonal antibody of influenza and selects, the antigenicity analysis that seldom is used for Strain, not high because of finding its specificity, be prone to cross reaction between the different subtype strain.
1,2 immunofluorescence techniques can directly be looked into influenza antigen with patient's nasopharyngeal secretions cast-off cells smear with immunofluorescence technique.Immunofluorescence is an extremely sensitive and special technology, but it need be in sample a spot of cell and technical professional come explanation results.
1,3 immune colloid gold colloid gold labels are actually macromolecules such as protein and are adsorbed to the bag on colloid gold particle surface by process.Adsorption mechanism may be the collaurum surface negative charge, forms strong bonded with the positive charge group of protein because of Electrostatic Absorption.The immune colloid gold diagnostic techniques of using in the medical test mainly contains two kinds, collaurum fast immune chromatographic method and dot immunogold filtration assay at present.The ultimate principle of these two kinds of methods all is to be carrier with the miillpore filter, bag is by known antigens or antibody, after adding sample to be checked, through the capillary syring effect of filter membrane or transudation the antibody or the antigen of the bag quilt on antigen in the sample or antibody and the film are combined, reach testing goal with collaurum bond mark again.Patterson S etc. utilizes the immune colloid gold method to detect the antigenic determinant of inner albumen of influenza and outside albumen, and can be used for the fast detecting influenza virus, but specificity is not high.
Immune colloid gold quick diagnosis technology has the advantage that diagnostic methods such as radioimmunology, enzyme immunoassay are not replaced: at first, it does not need desired substrate in the euzymelinked immunosorbent assay (ELISA), thereby can save the certain operations step, makes simple to operateization; Secondly.Collaurum is pollution-free, can not endanger operator and contaminated environment; Once more, colloidal gold antibody compound room temperature storage under lyophilised state is stable; In addition collaurum also have detect rapidly, sensitive, do not need complex instrument equipment, product colour developing advantage such as forever not to take off.
Because immune colloid gold quick diagnosis technology reaches its maturity, and characteristics such as its convenient, sensitive, safety, low cost, make its popularization rapidly in diagnostic field.Sale and each bibliographical information mainly concentrate on pregnant series, pathogen antigen or antibody test series, drug abuse detection series, the disease related protein detection series etc. surveyed of women in the market.
2, gene diagnosis
2, this method of 1 nucleic acid hybridization is more more special, responsive than methods such as Electronic Speculum, immuno-enzymatic marks, and can be quantitatively and somatotype.Be usually used in influenza virus quick diagnosis and virion genetic analysis at present.The making nucleic acid molecular hybridization technology can be widely used in the detection of virus sequence in the clinical samples, and the virus in the detection clinical samples that (as immunological method etc.) just can be successful for no other reason than that the conventional method of using does not clinically just need it to diagnose the illness.On the other hand, the making nucleic acid molecular hybridization technology still has certain difficulty as the instrument that diagnoses the illness, as the needs certain device, and time-consuming length, so still can not use as routine diagnostic method in many laboratories, therefore this method only is used for research at present.
2,2 RT-PCR RT-PCR diagnostic method concerning many RNA viruses separate than traditional virus and the diagnostic antigen method not only soon but also sensitive.In animals such as birds and poultry, detected the influenza first and had 15 kinds of HA and 9 kinds of NA, and H5N1 and H9N2 type influenza virus in Hong Kong discovery animal also can directly be infected to the mankind.Though be the influenza test strong tool virus is separated and immunofluorescence etc., false negative result may occur aspect birds that detect different phenotypes and the domestic animals influenza.Fouchier RA.M etc. utilizes influenza first the 7th gene segment memebrane protein conservative gene sequence to do primer, carries out RT-PCR and detects, and is quick, sensitive, special, and the influenza methyl at present all is suitable for because of the variation kind.
RT-PCR can diagnose multiple virus easily simultaneously, and other diagnostic methods such as virus are separated and immunofluorescence analysis but can not.Though immunofluorescence analysis can utilize the cell detection influenza first and the second of clinical samples, adenovirus, the existence of respiratory syncytial viral antigens is not widespread use to the diagnosis of influenza and adenovirus.Be considered to goldstandard though virus is separated, infect in the time of owing to influenza and adenovirus, can before adenovirus growth obviously, just destroy the cell unimolecular layer, identify these Respiroviruses with regard to being difficult to by the method for cellular incubation like this.
First type, influenza B virus, the A type Type B of Respiratory Syncytial Virus(RSV), adenovirus, multiple virus such as Respiratory Syncytial Virus(RSV) can cause viral respiratory infection, tympanitis, mumps.The infection that is caused by first and second influenza virus can be from slight breathing problem to the lethal pneumonia, and can break out global morbidity and death.Influenza virus C only causes slight breathing problem usually in children and teenager, cause endemic popular.Influenza virus C is difficult to find quick and responsive diagnostic method to be to understand less to its epidemiology behavior.Coiras MT etc. are according to the conservative region of influenza virus nucleoprotein, the antigen-4 fusion protein gene of Respiratory Syncytial Virus(RSV), the hexon gene synthetic pcr primer thing of adenovirus, utilize the method for multiple reverse transcription nest-type PRC can diagnose first and second influenza virus Cs simultaneously, Respiratory Syncytial Virus(RSV) and adenovirus etc. cause the Respirovirus of similar clinical symptoms
[15]Grondahl B etc. utilizes the method for multiple reverse transcription PCR (m-RT-PCR) once diagnosing out fast the influenza first and second type viruses, adenovirus, Respiratory Syncytial Virus(RSV), mycoplasma pneumoniae, Chlamydia pneumoniae, enterovirus, these 9 kinds of pathogen that cause similar ARI of parainfluenza 1,3 types virus in the experiment.With organize unconformable EIA method relatively for more, m-RT-PCR diagnostic result and correct result's coincidence rate reaches more than 95%.
Plakokefalos E etc. compare with ELISA and RT-PCR the diagnosis of first type, influenza B virus, and the susceptibility of ELISA and RT-PCR is respectively 64% and 100%, and specificity is respectively 98% and 97%; Positive diagnosis is respectively 94% and 86%, and negative diagnosis is respectively 93% and 100%.The result shows that RT-PCR has tangible susceptibility than ELISA, still can detect the generation of influenza when the inactivation of virus phenomenon takes place; And the provincialism monitoring management to influenza has diagnostic significance.
Rebelo H. etc. utilize virus to separate to the outburst of influenza, EIA, and RT-PCR, complement fixation test diagnose and compare.The positive diagnosing rate that RT-PCR, EIA, virus are separated in 1685 similar samples of influenza in continuous 7 years is respectively 43.6%, 17.5%, 5%.The result shows that RT-PCR diagnosis is faster, sensitive than other three kinds of diagnostic methods, and and the form of regional flu outbreak good correlativity is arranged.
Although technique of gene detection has many good qualities, but not enough going out also arranged, and as round pcr, biggest advantage has exactly been brought its maximum in actual applications obstacle, the high efficiency of DNA cloning has caused the pollution of denier false positive can occur, thereby makes distortion as a result.In addition, virus must just can design primer or probe as the invador of outer protogene when illustrating its all or part of nucleotide sequence, carries out making nucleic acid molecular hybridization and PCR and detects.
From existing influenza test method as seen, although virus separation, EIA, RT-PCR diagnostic method all have the certain specificity and the advantage of susceptibility, but in operation, need professional and technical personnel, special instrument and equipment and certain conditions and shortcoming such as time-consuming, and the immune colloid gold diagnostic techniques of developed recently maturation has specificity height, susceptibility height, simple to operate and do not need professional and instrument and equipment, has become the developing direction of influenza quick diagnosis.
Summary of the invention
The purpose of this invention is to provide a kind of can be quick, accurately check the influenza B virus colloidal gold fast detecting test paper of influenza B virus.
Another object of the present invention has provided anti-influenza B virus nucleoprotein monoclonal antibody, and produces the hybridoma cell strain of this monoclonal antibody.
For reaching above-mentioned purpose, the technical solution used in the present invention is as follows:
The hybridoma cell strain 3G11 of the used generation influenza B virus of the present invention nucleoprotein monoclonal antibody is on July 30th, 2003, and in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is: CGMCC0988.
The anti-influenza B virus nucleoprotein of the present invention monoclonal antibody can from preserving number be with routine techniques: the hybridoma cell strain 3G11 of CGMCC0988 produces.
Influenza B virus colloidal gold fast detecting test paper of the present invention, it comprises from preserving number and is: the anti-influenza B virus nucleoprotein monoclonal antibody that the hybridoma cell strain 3G11 of CGMCC0988 produces.
Advantage of the present invention is: influenza B virus colloidal gold fast detecting test paper of the present invention has following characteristics:
1. detect fast: went out the result in 10 minutes;
2. specificity: only influenza B virus is positive, and to the pathogen result that is negative more than 12 kinds such as other virus and bacterium;
3. susceptibility: the level that influenza B virus nucleoprotein is detected 20ng.
4. accuracy rate height: through eight front three hospital clinicals tests totally 3000 examples, with immunofluorescence and immuno-enzymatic connection relatively, total coincidence rate is 95.5%～98.8%;
5. storage and transport are convenient: at room temperature can preserve 18 months.
6. be convenient to clinical and the household use, and have industry.
In order further to understand essence of the present invention, the present invention will be further described below in conjunction with embodiment.
Embodiment
Embodiment 1: anti-influenza B virus nucleoprotein MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) myeloma cell
SP2/0 myeloma cell: purchase in Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C.Time spent will be stored in the SP2/0 cell recovery in the liquid nitrogen container, cultivate 48-72 hour in containing 10% calf serum DMEM nutrient solution, treat the cell well-grown, and perfectly round, bright, the big or small homogeneous of cell, marshalling are the logarithm division, prepare to merge.
With the DMCK cellular incubation good after, directly be coated with on the slide ,-30 ℃ of ultra low temperature freezers are preserved, the clone uses for the monitoring anti-cell.
(2) immune parental cell
Influenza B (A/ capital anti-184/93) type virus is purchased in country of Inst. of Viruses, China Preventive Medicine Science Academy influenza center.Strain recovery is seeded in respectively in the DMCK cell cultivates, treat that cytopathy reaches (+++), puts into-70 ℃ of ultra low temperature freezers immediately and preserves, with this antigen as immune animal.
The preparation of influenza B viral antigen substrate tablet: with the various virus inoculation of influenza in the DMCK cellular incubation, when treating that cytopathy reaches (+++), cell is digested from the bottle wall, and the 10 hole slides that the clean collodion of living is coated with are got in centrifugation, sick cell is tiled in the slide hole, dry up with electric fan immediately, acetone fixed dries up again,-30 ℃ of ultra low temperature freezers are preserved, and are equipped with antibody test and use.
After the antigen liquid of preparation takes out dissolving from-70 ℃ of ultra low temperature freezers, splash into 5-6 and drip for respectively BALB/C mice (purchasing animal center) nostril with No. 4 syringe needles, every secondary, 10 days at interval in Military Medical Science Institute.Merged preceding 3 days, and attacked with each 0.15ml of antigen at mouse spleen and abdominal cavity.
(3) Fusion of Cells
Fusion agent PEG (molecular weight 1500, Japan produces); Nutrient solution: 10% calf serum DMEM.The lymphocyte of the BALB/C mouse of SP2/0 cell and immunity is respectively by 2 * 10
7With 2 * 10
8Ratio merges.
(4) monitoring in positive hole
To merge that the hole supernatant is added in respectively on the above-mentioned viroplast sheet and DMCK cell sheet on.Put 37 ℃ of constant temperature ovens interior 30 minutes.Show with immunofluorescence technique sheep anti mouse fluorescent marker.With the fluorescence microscope result, all in viral sheet reactor again in mdck cell sheet reactor, for the positive hole of anti-cell, should discard.Use recombinant expressed influenza B virus nucleoprotein 10 μ g/ml envelope antigens simultaneously, add and merge the hole supernatant, resist, measure combination with enzyme connection instrument with two of sheep anti mouse enzyme labeling, every positive greater than blank 2 these OD values.
(5) obtain positive hole
Influenza B detects positive hole, 45 holes.
(6) positive hole is carried out enlarged culture, goes down to posterity and is cloned
With the strain of influenza B nucleoprotein antibody positive porocyte recover immediately, frozen, the frozen work of going down to posterity and recovering.
(7) 6 of influenza B strains are carried out cloning with limiting dilution assay, cultivate to go down to posterity 5 months.Passed for 40 generations approximately, in 1 generation of every biography, carried out one-time detection, and carry out repeatedly liquid nitrogen cryopreservation and recovery.
(8) antibody-secreting stability experiment
More than the 6 strains hybridoma cell strain FluB-McAb strain that produces influenza B nucleoprotein detect monoclonal antibody (McAb) positive rate through cloning continuously and reach 100%, subculture in vitro separately 5 months, and all can reach the secretory antibody that keeps stable through cryopreservation resuscitation repeatedly, supernatant tires 1: 400-1: 500 (++) IFA.
(9) karyology feature: it is 96 that above-mentioned hybridoma metaphase chromosome is detected influenza B; Prove that they are hybridomas of SP2/0 myeloma and mouse cell.
(10) choose the hybridoma cell strain called after 3G11 that wherein a strain produces anti-influenza B virus, and on July 30th, 2003 at China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number: CGMCC0988.
Embodiment 2: mouse source virus checking
Check that mouse source virus comprises: hemorrhagic fever viruse (EHFV); Lymphocytic choriomeningitis virus (LCMV); 3 type arc reovirus virus (Reovirus); Sendai virus; Take off pedopathy virus; Mouse adenovirus (MAV); Mouse pneumonia virus (PVM).
Cell inoculation method: CGMCC 0988 3G11 hybridoma cell strain culture supernatant is inoculated in Vero (African green monkey kidney passage cell) respectively; 2BS (human embryo lung (HEL)) diploid cell, inoculum concentration is 107.Passed for two generations behind the inoculating cell, whether observation of cell has pathology, and film-making simultaneously detects viral antigen with the IFA method, and is negative.
The inoculation of animal inoculation pvaccination method: CGMCC 0988 3G11 hybridoma cell strain is born 24 hours with each 10 of interior suckling mouses; Individual 10 of body weight 15-20 gram adult mice; Each 5 of the cavys of body weight 300-350 gram.Every animal intraperitoneal inoculation 107 living cells, survival rate is 85%.
Egg inoculation method: CGMCC 0988 3G11 hybridoma cell strain inoculation SPF chicken (purchasing) the embryo allantoic cavity and the yolk bag of cultivation in Animal Experimental Study center, Beijing, 10 of every kind of approach, every inoculation 106 living cells were hatched 5 days, do HA detection viral hemagglutinin with 1% chicken blood cell and be not aggegation, 80% survival in the chicken embryo on the same group.
Embodiment 3: the mycoplasma inspection:
Cultivation: CGMCC 0988 3G11 hybridoma cell strain cell film-making adds mycoplasma monoclonal antibody (purchasing in Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C) effect 30 minutes, fluorescent dye, do the positive and negative contrast simultaneously, result: negative control result (-); Positive control result (+); Detect the sample result: feminine gender.Culture supernatant wrap by after add monoclonal antibody, detect with the ELISA method, do negative control simultaneously, sample result: feminine gender.
Embodiment 4: the calibrating of monoclonal antibody
The anti-influenza B virus nucleoprotein monoclonal antibody (3G11-McAb) that immunoglobulin class and subclass: use immune double diffusion method, the result: CGMCC 0988 3G11 hybridoma cell strain produces is IgG2a.
It is 1.1 * 10 that the anti-influenza B virus nucleoprotein monoclonal antibody (3G11-McAb) that affinity: CGMCC 0988 3G11 hybridoma cell strain produces is measured affinity constant with the IFA method
-6M/L.
Embodiment 5: specificity
Blocking test:, confirm that 3G11-McAb is sealed by the heterogenous animal serum of variable concentrations respectively with the anti-3G11-McAb serum sealing of rabbit.
Adopt the IFA method: 3G11-McAb combines with influenza B virus, is positive.Combine with mdck cell, reaction is negative.More than prove the specificity of monoclonal antibody and target antigen, adopt western blot test technology identification epitope characteristic simultaneously, the result shows that FluB antigen is after reductive agent is handled, SDS-PAGE 5%-15% gradient glue electrophoresis, can differentiate more than 20 protein band clearly, adopt HRP dyeing after the transfer printing, the combination of the corresponding nucleoprotein antigen specificity of result: 3G11-McAb with 64KD.
Embodiment 6: cross matching
The reaction that all is negative of 3G11-McAb and influenza B virus, parainfluenza virus 1,2,3 types, adenovirus 3,7 types, Respiratory Syncytial Virus(RSV), reovirus and SARS virus.
Titration: 3G11-McAb indirect fluorescent method (IFA method) 1: 6400-1: 128000; It is 1 that indirect enzyme-linked method is measured antibody titer: 12800-1: 25600.
Embodiment 7: the production of the foundation of cell bank and anti-influenza B virus nucleoprotein monoclonal antibody:
GMP compatible is answered in monoclonal antibody production place, and is clean pollution-free, and staff's free from infection must not be engaged in other and can cause the infection sources to pollute the monoclonal antibody preparation work; Unrelated person must not enter in the production area, in same year, must not produce other irrelevant monoclonal antibodies simultaneously.
1, cell bank: set up master cell bank and produce cell bank and seed lot.
1,1 master cell bank:
3G11 hybridoma: the 3G11 hybridoma that produces anti-influenza B virus.
1,2 seed cell storehouses:
The 3G11 hybridoma is the frozen cell seed of enlarged culture, several 20 of the every batch of cell pipe, and labelled side goes in the liquid nitrogen container to preserve.Leave and take for every batch and carry out the detection of bacterium, fungi, mycoplasma and viral pollution when preserving.
1,3 produce cell bank:
Cultivate in a large number by working condition through the 3G11 of assay approval hybridoma, frozen cell pipe number is for producing cell bank.Each is produced cell and criticizes several 10 of cell pipe, gets during each production ascites and produces 1 recovery of pipe, amplification and production, no longer returns and freezes.
2, anti-influenza B virus nucleoprotein MONOCLONAL ANTIBODIES SPECIFIC FOR:
2,1 mouse ascites method
(1) mouse: SPF level mouse, do not have mouse source viral pollution on inspection, in the ascites production run as find that animal is unhealthy, bite, the infected should discard.
(2) animal facility: the above conformity certification of medical faunae secondary that has Beijing medical faunae management committee to issue.
(3) cell enlarged culture: get and produce batch 1 recovery of cell pipe, Ensure Liquid liquid carries out enlarged culture, produces cell for 1 and only uses once, and is no longer frozen.
(4) cell inoculation: preparation ascites all needs to carry out under aseptic condition, before the injection hybridoma, and every mouse peritoneal injection norphytane 0.5ml.Every injected in mice 3G11 hybridoma 1-3 * 10, one week back
6
(5) collection of ascites: behind the injection cell 7-10 days, or mouse once gathers ascites before dying, can also repeatedly gather.Centrifuging goes out supernatant and is and slightly carries antibody.Indicate lot number, gather the date, put-20 ℃ of preservations.2,2 cell culture methods: available Tissue Culture Flask is cultivated the collection supernatant and is prepared monoclonal antibody.
(1) seed cell: derive from the production cell bank.
(2) nutrient culture media: cow's serum nutrient culture media.
(3) antibody is collected: but disposable collecting also can collect continuously, the supernatant of collecting after each pipe seed cell enlarged culture is a lot number.
2,3 rough detection of antibodies:
No matter be the mouse ascites or the monoclonal antibody of cell culture method preparation, all need carry out following calibrating
(1) sterility test.
(2) mycoplasma inspection.
(3) mouse source virus checking.
(4) titration: measure or indirect enzyme-linked method mensuration the qualified purifying antibody that is used for the IFA method.
2,4 antibody purifications
(1) with 3G11-McAb respectively with 50%, 33%, the SAS of 33% 3 kind of concentration saltouts three times.SAS generally can not cause protein denaturation, can go out to remove unwanted albumin molecule.
(2) the DEAE-52 cellulose carries out wash-out to the monoclonal antibody of saltouing, and as seen antibody activity peak and protein peak is overlapping.
(3) result:
The 3G11-McAb ascites purifying recovery is respectively 100%, and immunoelectrophoresis result shows a precipitation line clearly.
(4) use: the monoclonal antibody of purifying is detected by being applied to of tiring.
Produce three batches continuously, each batch product must meet the quality inspection requirement, has good repeatability between batch.
1) the ascites calibrating of tiring: 3G11-McAb ascites is carried out titration with FluB antigen substrate respectively immediately after take out in the abdominal cavity, require the IFA method to tire and reach 1: 6400-1: 12800; Indirect enzyme-linked method is measured to tire and is reached 1: 12800-1: 25600; The enzyme connection is tired and is reached 1: 25600-1: 51200.
2) ascites purifying: measure tiring behind the purifying at any time, require each measure (IFA) to tire and to reach 1: 1200-1: 10000 (++).
2,5 purifying aftertreatments:
(1) deactivation: 56 ℃ of water-bath deactivations 30 minutes, centrifuging and taking supernatant.
(2) merge: place more than one month at 2-8 ℃ the qualified back of different inferior batch qualified products, goes out except that the part labile protein.
(3) degerming packing: with deactivation monoclonal antibody 0.22um membrane filtration, after the filtration, add penicillin 10 units, streptomysin 1ug/ml adds 20% an amount of sweet mellow wine again and carries out packing, every 2ml, and-30 ℃ are spent the night, and carry out freeze-drying next day again.
Embodiment 8: a large amount of productions of monoclonal antibody
Adopt inoculation hybridoma in the body, preparation ascites or serum.
(1) solid tumor method: the 3G11 hybridoma of exponential phase is by 1～3 * 10
7It is subcutaneous that/ml is inoculated in mouse back, every place's injection 0.2ml, totally 2～4 points.Treat that tumour reaches a certain size back (general 10～20 days) and then can take a blood sample, the content that obtains monoclonal antibody from serum can reach 1～10mg/ml.But blood sampling volume is limited.
(2) preparation of ascites: routine be first lumbar injection 0.5ml Pristane (norphytane) or whiteruss in the BALB/C mouse, 1～2 all pneumoretroperitoneum injections 1 * 10
6Individual hybridoma, inoculating cell can produce ascites after 7～10 days, health status of close observation animal and ascites sign, treat that ascites is many as far as possible, and mouse is put to death mouse frequency domain before the death, with dropper ascites is sucked in the test tube, a general mouse can be obtained 5～10ml ascites.Also available syringe extracting ascites can be collected for several times repeatedly.The monoclonal anti body burden can reach 5～10mg/ml in the ascites, and this is present the most frequently used method, also cell cryopreservation in the ascites can be got up, and recovery back transferred species mouse peritoneal produces then that ascites is fast, amount is many.
Embodiment 9: a large amount of Purification of Monoclonal Antibodies:
The Purification of Monoclonal Antibodies method is with the purifying of polyclonal antibody, and the concentration of ascites specific antibody is than the polyclonal antibody height in the antiserum, and purification effect is good.By the corresponding purification process of desired degree of purity different mining.Adopt step purifying such as ammonium sulfate precipitation, gel filtration and ion-exchange chromatography earlier, the better simply sour intermediate processing of employing is also arranged.How crosslinked with staphylococcal protein A or anti-mouse globulin antibody and carrier (the most frequently used Sepharose) the most effective at present monoclonal antibody purification process is an affinity purification,, the preparation affinity column with antibodies after wash-out, the recovery can reach more than 90%.Albumen can combine with IgG1, IgG2a, IgG2b and IgG3, simultaneously also in conjunction with a spot of IgM.Antibody concentration in the eluent can be used the bigness scale of UV Absorption method, and mouse IgG monoclonal anti liquid solution is when A280nm, and 1.44 (absorbance units) are equivalent to 1mg/ml.Behind low pH wash-out, in collection tube, preset neutralizer or speed and add neutralizer keeping the active most important of antibody purification.
Embodiment 10: the preparation of influenza B virus colloidal gold fast detecting test paper
(1) chemical reduction method is adopted in the preparation of collaurum, adds the trisodium citrate reductive agent in the chlorauride aqueous solution, makes gold ion aggregate into the 40nm colloid gold particle.Elder generation is heated to boiling with 0.01% HAuCl4 solution 500ml, adds 6.5ml 1% trisodium citrate aqueous solution rapidly, is heated to occur continuing to boil 7～10min after the redness.Recover volume to 500ml.
(2) collaurum-antibody conjugates preparation and purifying
1., get colloid gold particle solution 500ml, under magnetic stirring apparatus, reconcile PH to 8.2 with 0.2MK2Co3.
2., the anti-influenza B virus nucleoprotein monoclonal anti body and function 0.01M PB that the embodiment of the invention 7 or 8 is made is diluted to 1mg/ml.Get 5 test tubes and respectively add the 1ml colloidal gold solution, be added in preceding 4 pipe 30ul, 40ul, 45,50ul antibody to be marked respectively, last is managed in contrast.Room temperature was placed 5 minutes behind the mixing, respectively added 100ul concentration in preceding 4 pipes and be 10% NaCl.Room temperature was placed 10 minutes and the control tube contrast behind the mixing, not become antibody amount that a blue pipe added as the optimum protein labelled amount.Add 10% on this basis, use amount serves as a mark.
3., calculate mark desirable proteins amount, the monoclonal antibody of 1mg/ml is slowly added in the colloidal gold solution, the time that adds 10mg albumen is no more than 5 minutes according to the practical amount of volume and mark.Stirred 30 minutes under the room temperature.Carry out the calibrating of collaurum-antibody conjugates stability: takes out 2 test tubes, add the colloidal gold solution that 1ml has added antibody in every pipe, wherein add 100ul concentration in the pipe and be 10% NaCl, the room temperature placement is 10 minutes behind the mixing.Contrast the color of two pipes, answer no change.
4., add 10%BSA, to final concentration be 1%, stirring at room 5 minutes.
5., add 10%PEG, to final concentration be 0.2%, stirring at room 5 minutes.
6., centrifugal 30 minutes of 8000rpm/min, the careful suction removed supernatant, preserves the liquid precipitation that suspends with the 200ml collaurum, with 8300RPM centrifugal 30 minutes once more, the careful suction removed supernatant.Preserve liquid suspension precipitation with the 50ml collaurum.It is standby to put 4 ℃ of preservations.
(3) preparation of the plain film of collaurum-antibody conjugates glass fibre:
Get collaurum-antibody conjugates solution, evenly be sprayed on the plain film of glass fibre, be placed on the smooth plastic plate, freezing then 1 hour, put on the freeze drier freeze-drying and spend the night, to bone dry.Be cut into the wide bar of 0.6 ~ 0.8cm, preserve in the dry environment.
(4) preparation of antibody solid phase nitrocellulose filter:
1., the influenza B virus core protein monoclonal anti body and function 0.01MPBS with the embodiment of the invention 7 or 8 preparations is diluted to 3.5 ± 0.1mg/ml.With Membrane jetter above antibody is sprayed on (detection line) on the nitrocellulose filter with the speed of 1ul/cm.
2., the dilution of sheep anti-mouse igg polyclonal antibody is diluted to 2 ± 0.1mg/ml with 0.01MPBS.With Membrane jetter above antibody is sprayed on (control line) on the nitrocellulose filter with the speed of 1ul/cm.The 0.5cm that is spaced apart with detection line.
3., the nitrocellulose filter that is fixed with antibody was put in 37 ℃ of baking boxs dry 30 minutes.Fully dry.
4., put in 2 ℃ ~ 8 ℃ dry environments preserve standby.
(5) assembling test paper:
1., paste antibody solid phase nitrocellulose filter in the plastic plate centre position.
2., on plastic plate the fixing top of cellulose nitrate film location, adhere to adsorptive pads, low side adheres to the plain film band of golden labelled antibody bond-glass fibre.Adsorptive pads and collaurum pad should cover about 1 millimeter in nitrocellulose filter edge.
3., collaurum pad below adheres to sample pad, about 1 millimeter of sample pad covering collaurum pad.
4., on cutting cutter, be cut into the wide band of 0.4cm.
(6) packing:
(7) finished product warehouse-in:
Classify according to verification result, then the influenza B gold mark quick detection test paper of preparation is put in storage on request.
(8) the assay approval finished product is put under the normal temperature and is preserved.
The quality assurance of influenza B virus colloidal gold fast detecting test paper
The quality assurance of 1 hybridoma cell strain
(1) parent myeloma cell purchases in Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C, and this cell has genetic stability and unlimited multiplication capacity.
(2) immune parental generation influenza strain cell is purchased in country of Inst. of Viruses, China Preventive Medicine Science Academy influenza center, is the cell line that meets quality standard through technical test.
(3) immune mouse Balb/C purchases the animal center in Military Medical Science Institute, and this center possesses SPF level raising farm, and through national authentication, mouse meets national standard through calibrating.
(4) set up the monoclonal cell calibration method: testing result antibody-secreting stability positive rate is 100%, and the karyology feature meets the nuclear of hybridoma and learns feature, and mouse source virus checking is negative, and mycoplasma is checked negative, and sterility test is negative.
The quality assurance of 2 monoclonal antibodies:
(1) immunoglobulin class and subclass: use immune double diffusion method, the result meets the requirements.
(2) affinity: measuring affinity constant with the IFA method is 1.1 * 10
-6M/L meets the requirements.
(3) specificity: adopt the IFA method, specificity is 100%, and it is 100% that indirect enzyme-linked immunosorbent is measured specificity.
(4) reaction that all is negative of cross matching: 3G11-McAb and influenza Alphavirus, parainfluenza virus 1,2,3 types, adenovirus 3,7 types, Respiratory Syncytial Virus(RSV), reovirus and SARS virus.
(5) titration: with indirect fluorescent method (IFA method), be not less than 1: 6400, indirect enzyme-linked method was not less than 1: 12800, met the requirements.
3 rough detection of antibodies
No matter be the mouse ascites or the B-mode monoclonal antibody of cell culture method preparation, all need carry out following calibrating
(1) sterility test: no bacterial growth, feminine gender.
(2) mycoplasma inspection: negative.
(3) mouse source virus checking: negative.
(4) titration: measure with IFA method or indirect enzyme-linked method, meet the purifying requirement.
The foundation of finished product quality control standard
1, finished product visual examination
Test strips width 4mm ± 0.2mm
The test paper surfacing, the edge is neat.
A test strips component is pasted firmly, is connected closely, with moving continuously of liquid behind the assurance application of sample.
2, the liquid speed of dividing a word with a hyphen at the end of a line
Extract mobile 2cm time on nitrocellulose filter was not less than 15 seconds.
Experiment: the testing result evaluation of the B-mode colloidal gold fast detecting test paper of influenza virus of the present invention
Experiment 1: the reaction of influenza B virus different subtype
The different influenza B virus chicken of 10 strains embryo culture virus supernatant all is positive.
Experiment 2: cross reaction
With the Respiratory Syncytial Virus(RSV) no cross reaction; With parainfluenza virus 1,2,3 type no cross reactions; With adenovirus 3,7 type no cross reactions; With reovirus and SARS virus no cross reaction.
Experiment 3: the authenticity evaluation of the B-mode colloidal gold fast detecting test paper of influenza virus.
Sensitivity: detect positive 489 parts in 500 parts of positive, sensitivity is 97.8%.
Specificity: 500 parts of negative sample kinds detect negative 495 parts, and specificity is 99.0%.
Experiment 5: contrast experiment
Choose four kinds of influenza B virus chicken embryo culture virus supernatants.
These four kinds of influenza B virus chicken embryo cultures virus supernatants are done the dilution of variable concentrations with extract, do contrast with U.S.'s Quidel company product and our company's product then and detect.The result is as follows:
| Dilutability | Stoste | 1∶10 | ?1∶100 | ?1∶500 | ?1∶800 | ?1∶1000 | ??1∶1200 |
| Quidel product testing result | + | + | ?+ | ?+ | ?+ | ?+/- | ??- |
| Our company's product testing result | + | + | ?+ | ?+ | ?+ | ?- | ??- |
| Dilutability | Stoste | 1∶10 | ?1∶100 | ?1∶500 | ?1∶800 | ?1∶1000 | ????1∶1200 |
| Quidel product testing result | + | + | ?+ | ?+ | ?+ | ?- | ????- |
| Our company's product testing result | + | + | ?+ | ?+ | ?+ | ??+ | ????- |
| Dilutability | Stoste | 1∶10 | ?1∶100 | ?1∶500 | ?1∶800 | ?1∶1000 | ????1∶1200 |
| Quidel product testing result | + | + | + | ?+ | ?+ | ?+ | ????- |
| Our company's product testing result | + | + | + | ?+ | ?+ | ?+ | ????+/- |
| Dilutability | Stoste | 1∶10 | ?1∶100 | ?1∶500 | ?1∶800 | ?1∶1000 | ????1∶1200 |
| Quidel product testing result | + | + | ?+ | ?+ | ?+ | ?+ | ????- |
| Our company's product testing result | + | + | ?+ | ?+ | ?+ | ?+ | ????+/- |
Claims (4)
1, has the hybridoma cell strain that preserving number is CGMCC 0988.
2, anti-influenza B virus nucleoprotein monoclonal antibody can be from having the hybridoma cell strain generation that preserving number is CGMCC 0988.
3, influenza B virus colloidal gold fast detecting test paper is characterized in that: it comprises the described anti-influenza B virus nucleoprotein monoclonal antibody of claim 2.
4, influenza B virus colloidal gold fast detecting test paper as claimed in claim 3 is characterized in that: the attached collaurum of the described anti-influenza B virus nucleoprotein monoclonal antibody bag of claim 2.
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| CN100503822C CN100503822C (en) | 2009-06-24 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104245731A (en) * | 2012-01-31 | 2014-12-24 | 株式会社医学生物学研究所 | Human monoclonal antibodies broadly protective against influenza b virus and methods of using the same |
| CN105683756A (en) * | 2013-09-10 | 2016-06-15 | 电化生研株式会社 | Method for measuring influenza b virus |
| WO2022062789A1 (en) * | 2020-09-27 | 2022-03-31 | 东莞市朋志生物科技有限公司 | Antibody and detection reagent kit for influenza b virus |
| CN116715760A (en) * | 2023-07-31 | 2023-09-08 | 南京佰抗生物科技有限公司 | Monoclonal antibody of anti-influenza B virus nucleocapsid protein and application thereof |
| WO2024131964A1 (en) * | 2022-12-24 | 2024-06-27 | 菲鹏生物股份有限公司 | Influenza b virus np antibody |
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2003
- 2003-09-03 CN CNB031565247A patent/CN100503822C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104245731A (en) * | 2012-01-31 | 2014-12-24 | 株式会社医学生物学研究所 | Human monoclonal antibodies broadly protective against influenza b virus and methods of using the same |
| CN104245731B (en) * | 2012-01-31 | 2017-04-05 | 株式会社医学生物学研究所 | The extensive protectiveness human monoclonal antibodies of anti-Influenza B viruss and its using method |
| CN105683756A (en) * | 2013-09-10 | 2016-06-15 | 电化生研株式会社 | Method for measuring influenza b virus |
| CN105683756B (en) * | 2013-09-10 | 2017-10-24 | 电化生研株式会社 | The assay method of influenza B virus |
| WO2022062789A1 (en) * | 2020-09-27 | 2022-03-31 | 东莞市朋志生物科技有限公司 | Antibody and detection reagent kit for influenza b virus |
| WO2024131964A1 (en) * | 2022-12-24 | 2024-06-27 | 菲鹏生物股份有限公司 | Influenza b virus np antibody |
| CN116715760A (en) * | 2023-07-31 | 2023-09-08 | 南京佰抗生物科技有限公司 | Monoclonal antibody of anti-influenza B virus nucleocapsid protein and application thereof |
| CN116715760B (en) * | 2023-07-31 | 2023-10-03 | 南京佰抗生物科技有限公司 | Monoclonal antibody composition for resisting influenza B virus nucleocapsid protein and application thereof |
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| CN100503822C (en) | 2009-06-24 |
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