CN101130765B - Reagent kit for detecting syncytial virus of respiratory passage - Google Patents
Reagent kit for detecting syncytial virus of respiratory passage Download PDFInfo
- Publication number
- CN101130765B CN101130765B CN2006101116509A CN200610111650A CN101130765B CN 101130765 B CN101130765 B CN 101130765B CN 2006101116509 A CN2006101116509 A CN 2006101116509A CN 200610111650 A CN200610111650 A CN 200610111650A CN 101130765 B CN101130765 B CN 101130765B
- Authority
- CN
- China
- Prior art keywords
- syncytial virus
- cell
- antibody
- virus
- respiratory syncytial
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a hybridomas cell strain with preserving number at CGMCC 1546, anti-respiratory syncytial virus N protein monoclone antibody and respiratory syncytial virus detecting agent box (colloidal gold method), which can detects the respiratory syncytial virus with high specificity, sensibility and accuracy to be preserved and transported conveniently for clinical and domestic usage with industrial property.
Description
Technical field
The present invention relates to the anti respiratory syncytial virus monoclonal antibody, produce the hybridoma cell strain of this monoclonal antibody and contain the reagent kit for detecting syncytial virus of respiratory passage of this monoclonal antibody.
Background technology
Respiratory syncytial virus (RSV is called for short syncytial virus, belongs to Paramyxoviridae) was separated in the orangutan body with cold symptoms first in 1956.Be to cause the modal cause of disease of infantile viral pneumonia, because of it can form the warm pathology of special cell in cell cultures, so name.The infection of this virus can cause interstitial pneumonia, and capillary bronchitis.In Beijing, 48% virus pneumonia and 58% capillary bronchitis system cause (1980～1984) by syncytial virus; In Guangzhou, 31.4% of infantile pneumonia and capillary bronchitis causes (1973～1986) by syncytial virus; In the U.S., 20%～25% infantile pneumonia and 50%～75% capillary bronchitis are caused by syncytial virus.
RSV finding and parainfluenza virus under Electronic Speculum is similar, the virion size is about 150nm, slightly little than parainfluenza virus, be RNA viruses, to the ether sensitivity, no hemagglutination forms the distinctive born of the same parents (syncytium) that close in the cultivation of people's epithelium, viral in the endochylema internal breeding, visible intracytoplasmic inclusion.With molecular biology method proof syncytial virus two hypotypes are arranged.
Be 2～8 days (mostly being 4～6 days) latent period of syncytial virus infection.The typical finding of syncytial virus pneumonia is that monocytic matter is soaked into.Mainly show as alveolar septum broadening and ooze out, comprising lymphocyte, plasmocyte and scavenger cell based on matter between monocyte.Alveolar space is full of edematous fluid in addition, and visible pulmonary hyaline membrane forms.In some cases, the lymphocytic infiltration of also visible bronchiole wall.Oedema occurs in pulmonary parenchyma, cause alveolar filling, consolidation and wither with the necrotic area.
Because the generation of preventing infection fully of female biography antibody, whenever the syncytial virus pneumonia all may take place after birth.Be more common in below 3 years old, the man was more than the woman as seen than the grave illness example in 1～6 month.Northern China is more common in winter-spring season, and spring and summer then is more common in Guangdong.Because antibody can not be protected from infection fully, the infection again of syncytial virus is very common, and the someone observed 10 years, and infection rate is up to 65% again.The infectivity of syncytial virus is very strong, has the report kinsfolk to infect in succession, and when taking place within the family, older youngster and adult are generally upper respiratory tract infection.Secondary syncytial virus infection rate is up to 30%～50% in the bibliographical information institute.
Syncytial virus infection is more common in the infant, wherein more than half be 1 years old with interior baby, man more than the woman, its ratio is about 1.5～2: 1.About 4～5 days of latent period.As seen initial stage cough, nasal obstruction.About 2/3 case has high heat, the highest can be to 41 ℃, but heating is not a persistence generally, is more easily brought down a fever by febrifuge, high hot time majority is 1～4 day, minority is 5～8 days.About 1/3 sick child's moderate heating continues 1～4 day more.
The hot journey of most cases is 4～10 days.Light disease case expiratory dyspnea and nervous symptoms are outstanding, in, severe have more significantly have difficulty in breathing, breathe heavily suppress, cyanotic lips, flaring nares and three depressions sign, the minority severe cases also can concurrent heart failure.The chest auscultation has tiny or thick, medium rales more, and percussion does not generally have voiced sound, and minority has hyperresonant note.
Clinical patient is because the disease symptoms that different Respirovirus (as influenza virus, parainfluenza virus, adenovirus etc.) infection causes can be quite similar.This has caused relatively difficulty of popular diagnosis, makes a definite diagnosis and often depends on laboratory diagnosis.The method of quick diagnosis should be that the their early stage in disease just can obtain clear and definite diagnosis, is convenient to stop the infection of disease and changes the popular outburst.
The detection of respiratory syncytial virus infection at present mainly contains following several method:
One, Routine Test Lab detection-isolation of virus
The gold standard of laboratory diagnosis respiratory syncytial virus infection is viral isolating method.Adopt the time of sample to be advisable in 5 days, get patient's nasopharynx cotton swab or cough up phlegm and carry out viral separation and Culture with premorbid.This virus can not be in the internal breeding of chicken embryo, can only cultivate propagation in the cell strains such as Hela at people and MC such as Hep-2, cultivates approximately 2～3 weeks just to occur the cytopathy that the cell boundary line is unclear, be fused into polykaryocyte etc., and is viral by the release of sprouting.But viral isolating method has serious defective.Because their not only time-consuming but also efforts need the long period just can obtain net result usually, at clinicing aspect effective treatment of patient there is certain limitation like this.
Two, quick diagnosis
Check that directly viral protein antigen and viral nucleic acid can reach the purpose of quick diagnosis, method commonly used has immunofluorescence technique, immunoenzyme, radioimmunology, immune colloid gold method, electron microscopy, nucleic acid hybridization and PCR method etc.
1, immunological method
1, the ultimate principle of 1 immunoenzyme immunoenzyme (EIA) is after utilizing enzyme and antibody and antigen combining, form enzyme conjugates, it neither changes antibody or antigenic immunologic competence, the with a hook at the end enzymic activity of enzyme itself, the corresponding low thing that adds enzyme then, under the katalysis of enzyme, make original colourless low thing generation hydrolysis, oxidation or other reactions, generate coloured product.
EIA method susceptibility height, high specificity, easy and simple to handle, naked eyes can be observed, and are convenient to extensive detection, and in respiratory syncytial virus research, it is mainly used in the screening of the quick diagnosis and the monoclonal antibody of syncytial virus, the less antigenicity analysis that is used for virus strain.
1,2 immunofluorescence techniques can directly be looked into syncytial viral antigens with patient's nasopharyngeal secretions cast-off cells smear with immunofluorescence technique.Immunofluorescence is a kind of 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 polymers such as protein and are adsorbed to the bag on colloid gold particle surface by process.Adsorption mechanism may be the Radioactive colloidal gold surface negative charge, forms mortise with proteinic positive charge group because of electrostatic adhesion.The immune colloid gold diagnostic techniques of using in the medical test mainly contains two kinds, Radioactive colloidal gold fast immune chromatographic method and dot immunogold filtration assay at present.The ultimate principle of these two kinds of methods all is as carrier with millipore filtration, bag is by known antigens or antibody, after adding sample to be checked, through the capillary pipet 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, in the purpose that reaches detection with Radioactive colloidal gold binding substances mark.
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 needed low thing in the euzymelinked immunosorbent assay (ELISA), therefore can save the certain operations step, makes simple to operateization; Secondly, Radioactive colloidal gold is pollution-free, can not endanger operator and contaminate environment; Once more, colloidal gold antibody mixture room temperature storage under lyophilised state is stable; In addition Radioactive colloidal gold 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 advantages such as its convenient, sensitive, safety, low cost, make its popularization rapidly in diagnostic field.Sale and bibliographical information mainly concentrate on women's gestation series, pathogen antigen or antibody test series in the market, drug abuse detects series, disease related protein detection series etc.
2, gene diagnosis
2,1 nucleic acid hybridization
This method is more more special, responsive than methods such as Electronic Speculum, immunoenzyme marks, and can be quantitative.The quick diagnosis that is usually used in respiratory syncytial virus at present.The making nucleic acid molecular hybridization technology can be widely used in the detection of virus sequence in the clinical samples, for no other reason than that the virus in the detection clinical samples that conventional method (as immunological method) just can be successful, 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, and is as the needs certain device, time-consuming.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
Concerning many RNA viruses the RT-PCR diagnostic method separate than traditional virus and the diagnostic antigen method not only soon but also sensitivity.And RT-PCR can diagnose multiple virus easily simultaneously, and other diagnostic methods such as virus are separated and immunofluorescence analysis but can not.
Although technique of gene detection has many good qualities, but weak point is also arranged, and as round pcr, biggest advantage has exactly been brought its biggest obstacle in actual applications, colleges and universities' property of DNA cloning has caused the pollution of denier both false positive can occur, thereby makes distortion as a result.Virus must just can design primer or probe as the invader of outer protogene when illustrating its all or part of nucleotide sequence in addition, carries out making nucleic acid molecular hybridization and PCR and detects.
From the detection method of existing respiratory syncytial virus 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 plant and instrument and certain conditions and shortcoming such as time-consuming, and the sophisticated immune colloid gold diagnostic techniques of developed recently has specificity height, susceptibility height, simple to operate and do not need professional and plant and instrument, has become the new developing direction of syncytial virus diagnosis.
Summary of the invention
The purpose of this invention is to provide a kind of can be fast, the accurate reagent kit for detecting syncytial virus of respiratory passage (colloidal gold method) of surveyor's respiratory syncytial virus infection.
Another object of the present invention has provided the monoclonal antibody of anti respiratory syncytial virus, and produces the hybridoma cell strain of this monoclonal antibody.
For above-mentioned purpose, the technical scheme that the present invention takes is as follows:
The hybridoma cell strain 2A7 of the respiratory syncytial virus monoclonal antibody that the present invention produced is on November 28th, 2005, and in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is: CGMCC1546.
Anti respiratory syncytial virus monoclonal antibody of the present invention can be the hybridoma cell strain 2A7 generation of CGMCC1546 from preserving number with routine techniques.
Reagent kit for detecting syncytial virus of respiratory passage of the present invention (colloidal gold method), it comprises from preserving number is the anti respiratory syncytial virus monoclonal antibody of the hybridoma cell strain 2A7 generation of CGMCC1546.
Advantage of the present invention is: reagent kit for detecting syncytial virus of respiratory passage of the present invention (colloidal gold method) has following having a few:
1. detect fast: went out the result in 10 minutes;
2. specificity: only respiratory syncytial virus is positive, and to 13 kinds of pathogenic agent such as other Respirovirus and bacterium result that is negative;
3. susceptibility: can detect 3 * 10 to respiratory syncytial virus
4TCID
50/ 0.1ml;
4. accuracy rate height: through eight front three hospital clinicals tests totally 3026 examples, with the virus culture method relatively, total coincidence rate is 95.2%;
5. storage and transport are convenient: at room temperature can preserve 18 months;
6. clinical and household is used very convenient, and has industry.
In order further to understand essence of the present invention, the present invention is described further below in conjunction with embodiment.
Embodiment
Embodiment 1: the anti respiratory syncytial virus 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 Hep-2 cell cultures good after, directly be coated with on the slide ,-30 ℃ of Ultralow Temperature Freezers are preserved, the clone uses for the monitoring anti-cell.
(2) immune parental cell
Immunity is purchased in Inst. of Viruses, China Preventive Medicine Science Academy with respiratory syncytial virus.Strain is seeded in the Hep-2 cell cultivates, treat that cytopathy reaches (+++), puts into-70 ℃ of Ultralow Temperature Freezers immediately and preserves, with this antigen as immune animal.
The preparation of respiratory syncytial viral antigens substrate tablet: respiratory syncytial virus is seeded in the Hep-2 cell cultivates, treat 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 state is fixed, dries up,-30 ℃ of Ultralow Temperature Freezers are preserved, and are equipped with antibody test and use.
After the antigen liquid of preparation takes out dissolving from-70 ℃ of Ultralow 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 antigen 0.15ml at mouse spleen and abdominal cavity.
(3) cytogamy
Fusogen PEG (molecular weight 1500, Japan produces); Nutrient solution: 10% calf serum DMEM.The lymphocyte of the BALB/C mice of SP2/0 cell and immunity is respectively by 2 * 10
7With 1 * 10
8Ratio merges.
(4) monitoring in positive hole
To merge that the hole supernatant liquor is added in respectively on the above-mentioned viroplast sheet and Hep-2 cell sheet on.Put 37 ℃ of thermostat containers interior 30 minutes.Show with immunofluorescence technique sheep anti mouse fluorescent marker.With the fluorescence microscope result, all in viral sheet reactor again with Hep-2 cell sheet reactor, be the anti-cell positive, should discard.Use gene recombination respiratory syncytial virus N protein 10 ug/ml envelope antigen plate simultaneously, add and merge the hole supernatant, and anti-with sheep anti mouse enzyme labelling two, measure with enzyme connection instrument, every positive greater than 2 OD values of blank.
(5) obtain positive hole
Obtain 38 holes, positive hole.
(6) positive hole is carried out enlarged culturing, goes down to posterity and is cloned
With positive hole recover immediately, frozen, the frozen work of going down to posterity and recovering.
4 cell strains that (7) will obtain carry out cloning with limiting dilution assay, cultivate to go down to posterity 5 months.In about 40 generations, every biography generation is carried out one-time detection, and carries out repeatedly liquid nitrogen cryopreservation and recovery.
(8) antibody-secreting stability experiment
More than the 4 strains hybridoma cell strain that produces anti-syncytial virus antibody detect the monoclonal antibody 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 is tired: 1: 6400-1: 12800 (++) IFA, or the indirect enzyme-linked immunosorbent supernatant is tired 1: 25600-1: more than 51200.
(10) nuclear cytology feature: it is 87 that above-mentioned hybridoma Metaphase Chromosome is detected; Prove that they are hybridomas of SP2/0 myelomatosis and mouse cell.
(11) choose the hybridoma cell strain called after 2A7 that wherein a strain produces the anti respiratory syncytial virus monoclonal antibody, and on November 28th, 2005 at China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number: CGMCC 1546
Embodiment 2: mouse source virus checking
For the specificity of the anti respiratory syncytial virus monoclonal antibody of identifying preparation, promptly with employed mouse source virus no cross reaction.
Check that mouse source virus comprises: hemorrhagic fever virus (EHFV); Lymphocyte choriomeningitis virus (LCMV); 3 type arc reovirus virus (Reovirus); Sendai virus; Take off the pedopathy poison; Mouse adenovirus (MAV); Mouse pneumonia virus (PVM).
Cell inoculation method: 2A7 hybridoma cell strain secretion supernatant is inoculated in Vero (African green monkey kidney passage cell) respectively; 2BS (human embryo lung (HEL)) diploid cell, inoculum size is 10
7Passed for two generations behind the inoculating cell, whether observation of cell has pathology, and film-making simultaneously detects virus antigen with the IFA method, and is negative.
The animal inoculation pvaccination method: the inoculation of 2A7 hybridoma cell strain is born 24 hours with each 10 of interior suckling mouses; Each 10 of body weight 15-20 gram adult mices; Each 5 of the cavys of body weight 300-350 gram.Every animal intraperitoneal inoculation 10
7Viable cell, survival rate are 85%.
Embodiment 3: the mycoplasma inspection
Culture method: the film-making of 2A7 hybridoma cell strain cell adds mycoplasma monoclonal antibody (purchasing in Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C) effect 30 minutes, and the yin, yang contrast is done in fluorescent dye 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
Immunoglobulins and subclass: use immune double diffusion method, the result: the anti respiratory syncytial virus monoclonal antibody that the 2A7 hybridoma cell strain produces is IgG2b.
Avidity: it is 1.2 * 10 that the anti respiratory syncytial virus monoclonal anti body and function IFA method that the 2A7 hybridoma cell strain produces is measured affinity constant
-6M/L.
Embodiment 5:2A7 monoclonal antibody is the evaluation at respiratory syncytial virus N albumen monoclonal antibody
Adopting indirect ELISA to measure respiratory syncytial virus cell strain 2A7 monoclonal antibody is at the proteic monoclonal antibody of respiratory syncytial virus N, tests as follows:
1. material and method
1.1 antibody respiratory syncytial virus cell strain 2A7 prepares the monoclonal antibody (lot number, 200501,200502) of purifying, is prepared by our company.
1.2 antigen respiratory syncytial virus N recombinant protein antigen is prepared by our company.
1.3 reagent HRP-sheep anti-mouse igg antibody is available from middle mountain Science and Technology Ltd.; NBT stock solution, 0.5g NBT in 10ml DMT, 4 ℃ of preservations; NBT/BCIP solution faces with before joining.
Be diluted to 0.5mg/ml 1.4ELISA measure rN with coating buffer, every hole adds 100 μ l bag by in enzyme reaction plate, and does two multiple holes
↓ 4 ℃ of refrigerator overnight
0.01M PBS gives a baby a bath on the third day after its birth inferior, each 5min adds 3%BSA 100 μ l sealing
↓ room temperature 60min
0.01M PBS gives a baby a bath on the third day after its birth inferior, each 5min adds the mouse-anti human respiratory syncytial virus 2A7 that dilutes at 1: 1000
↓37℃?60min
0.01M PBS gives a baby a bath on the third day after its birth inferior, each 5min adds 3.0 μ g/ml HRP-rabbit anti-mouse iggs, two anti-100 μ l
↓37℃?60min
0.01M PBS gives a baby a bath on the third day after its birth inferior, each 5min adds OPD-H
2O
2Substrate solution 100 μ l
↓37℃?10-20min
Add 2N H
2SO
4Stop buffer 50 μ l survey the OD490 value on microplate reader
2. result
The monoclonal antibody purification (lot number, 200501,200502) that detects the hybridoma 2A7 preparation of respiratory syncytial virus rN antigen and respiratory syncytial virus with indirect ELISA presents high specific combination immunocompetence, the results are shown in Table 1.
3. conclusion
Above result of study confirms that the monoclonal antibody of the hybridoma 2A7 preparation that respiratory syncytial virus Radioactive colloidal gold rapid detection box is selected for use is at the proteic antibody of respiratory syncytial virus N.
Table 1.ELISA measures 2A7 monoclonal antibody and respiratory syncytial virus rN antigen bonded immunocompetence
Embodiment 6: specificity
Blocking test:, confirm that 2A7 is sealed by the heterogenous animal serum of different concns respectively with the anti-2A7 serum sealing of rabbit.
Adopt the IFA method: 2A7 combines with respiratory syncytial virus, is positive.Combine with the Hep-2 cell, reaction is negative.More than prove the specificity of monoclonal antibody and target antigen.
Embodiment 7: cross matching
The reaction that all is negative of the anti respiratory syncytial virus monoclonal antibody that 2A7 produces and influenza virus A, influenza virus B, parainfluenza virus 1,2,3 types, adenovirus 3,7 types, reovirus and SARS virus.
Titration:
Indirect fluorescent method IFA measures and tires: 1: 6400-1: 12800.
Indirect enzyme-linked mensuration is tired: 1: 25600-1: 51200.
Embodiment 8: the generation of the foundation of cell bank and anti respiratory syncytial virus monoclonal antibody
Monoclonal antibody production place should meet the GLP requirement, and is clean pollution-free, and staff's free from infection must not be engaged in other and can cause contagium to pollute monoclonal antibody formulation 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
2A7 hybridoma: the 2A7 hybridoma that produces the anti respiratory syncytial virus monoclonal antibody.
1,2 seed cell storehouses
The 2A7 hybridoma is the frozen cell seed of enlarged culturing, several 20 of the every batch of cell pipe, and the labelled liquid nitrogen container of putting into is preserved.Leave and take for every batch and carry out bacterium, fungi, mycoplasma and virus pollution detection when preserving.
1,3 produces cell bank
Cultivate in a large number by working condition through the 2A7 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 freeze.
2, anti respiratory syncytial virus MONOCLONAL ANTIBODIES SPECIFIC FOR
2,1 mouse ascites method
(1) mouse: SPF level mouse, do not have mouse source virus pollution on inspection, in the ascites production process 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 culturing: get and produce batch 1 recovery of cell pipe, Ensure Liquid liquid carries out enlarged culturing, 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 pristane 0.5ml.Every injected in mice 2A7 hybridoma 1-3 * 10, one week back
5
(5) collection of ascites: behind the injection cell 7-10 days, or mouse once gathers ascites before dying, can also repeatedly gather.Centrifugation goes out supernatant both for slightly carrying antibody.Indicate lot number, gather the date, put-20 ℃ of preservations.
2,2 cell culture methods: available cell cultures flask culture is collected supernatant liquor and is prepared monoclonal antibody.
(1) seed cell: derive from the production cell bank.
(2) substratum: bovine serum substratum.
(3) antibody is collected: but disposable collecting also can collect continuously, the supernatant of collecting after each pipe seed cell enlarged culturing 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 to carry out following calibrating (1) sterility test.(2) mycoplasma inspection.(3) mouse source virus checking.(4) titration; More than detect and be used for purifying antibody after qualified.
2,4 antibody purifications
(1) with the 2A7 monoclonal antibody respectively with 50%, 33%, the SAS of 33% 3 kind of concentration saltouts three times.SAS generally can not cause protein denaturation, can remove unwanted albumin molecule.
(2) the DEAE-52 Mierocrystalline 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 2A7 monoclonal antibody ascites purifying rate of recovery is 100%, and immunoelectrophoresis result shows a precipitation line clearly.
(4) use: the monoclonal antibody of purifying is applied to detect by the 4-8 unit of tiring.
Three batches of continuous production, each batch product must meet the quality inspection requirement, has good repeatability between batch.
1) the ascites calibrating of tiring: 2A7 monoclonal antibody ascites is after take out in the mouse abdominal cavity, carry out titration with FluA antigen substrate respectively immediately, require IFA to tire and reach 1: 6400-1: 12800, or the indirect enzyme-linked immunosorbent supernatant 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: 6400-1: 12800.
2,5 purifying aftertreatments:
(1) deactivation: 56 ℃ of water-bath deactivations in case of necessity 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, removes the part labile protein.
(3) degerming packing: with deactivation antibody 0.22um membrane filtration, after the filtration, every 4-8 unit adds penicillin 10 units, and Streptomycin sulphate 1ug/ml adds 20% an amount of N.F,USP MANNITOL again and carries out packing, every 2ml, and-30 ℃ are spent the night, and carrying out lyophilize next day.
Embodiment 9: the mass production of monoclonal antibody
Adopt inoculation hybridoma in the body, preparation ascites or serum.
(1) solid tumor method: the 2A7 hybridoma of logarithmic phase is pressed 1-3 * 10
7It is subcutaneous that/ml is inoculated in mouse back, every place's injection 0.2ml, 2-4 point altogether.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 abdominal injection 0.5ml Pristane (pristane) or whiteruss in BALB/C mice, 1-2 week pneumoretroperitoneum injection 1 * 10
6Individual hybridoma can produce ascites after inoculating cell 7-10 days, healthy state of close observation animal and ascites sign, treat that ascites is many as far as possible, and before mouse is on the verge of death, put to death mouse, 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 ascites in the ascites, and this is present the most frequently used method, also cell in the ascites can be stored away, and it is fast, maximum that recovery back transferred species mouse peritoneal then produces ascites.
Embodiment 10: 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(antisera), and purification effect is good.By the corresponding purification process of desired degree of purity different mining.General adopt saltout, steps such as gel-filtration and ion exchange chromatography reach the purifying purpose, and the better simply Acid precipitation method of employing is also arranged.The most effective at present monoclonal antibody purification process is an affinity purification, the glucose coccus A albumen or anti-mouse globulin antibody and carrier (the most frequently used Sepharose) used crosslinked more, the preparation affinity column with antibodies after wash-out, the rate of recovery can reach more than 90%.Albumen can combine with IgG1, IgG2a, IgG2b, IgG3, simultaneously also in conjunction with a spot of IgM.Antibody concentration in the elutriant can be used the bigness scale of ultraviolet 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.
Embodiment 11: the preparation of reagent kit for detecting syncytial virus of respiratory passage (colloidal gold method)
(1) chemical reduction method is adopted in the preparation of Radioactive colloidal gold, adds the trisodium citrate reductive agent in the gold trichloride aqueous solution, makes gold ion aggregate into the 40nm colloid gold particle.Elder generation is heated to boiling with 0.01% chlorogold solution 500ml, adds 6.5ml 1% trisodium citrate aqueous solution while stirring rapidly, is heated to occur continuing to boil 7～10 minutes after the redness.Return to room temperature, recover volume to 500ml.
(2) Radioactive colloidal gold-antibody conjugates preparation and purifying
1., get colloid gold particle solution 500ml, under magnetic stirring apparatus, use 0.2M K
2CO
3Regulate PH to 8.2.
2., the anti respiratory syncytial virus monoclonal anti body and function 0.01M PB with the embodiment of the invention 8 or 9 preparations is diluted to 1mg/ml.Get 5 test tubes and respectively add the 1ml colloidal gold solution, add 30ul, 40ul, 45ul, 50ul antibody to be marked respectively at preceding 4 pipes, 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, usage quantity serves as a mark.
3., calculate mark desirable proteins amount, the monoclonal antibody of 1mg/ml is slowly added in the colloidal gold solution, add the proteic time of 10mg to be 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 Radioactive colloidal gold-antibody conjugates stability then: 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%.
5., add 10%PEG, to final concentration be 0.2%, stirring at room 30 minutes.
6., centrifugal 30 minutes of 8500RPM, the careful suction removed supernatant, preserves the liquid precipitation that suspends with the 200ml Radioactive colloidal gold, with 8300RPM centrifugal 30 minutes once more, the careful suction removed supernatant.Preserve the liquid precipitation that suspends with the 50ml Radioactive colloidal gold, it is standby to put 4 ℃ of preservations.
(3) preparation of the plain film of Radioactive colloidal gold-antibody conjugates glass fibre:
Get Radioactive colloidal gold-antibody conjugates solution, evenly be sprayed on the 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 complete drying.It is wide to be cut into 0.6-0.8cm, and the bar that 30cm is long is preserved in the room temperature lucifuge dry environment.
(4) preparation of antibody solid phase nitrocellulose filter
1., the anti respiratory syncytial virus monoclonal anti body and function 0.01M PBS with the embodiment of the invention 8 or 9 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 sheep anti-mouse igg polyclonal antibody is diluted to 2 ± 0.1mg/ml with 0.01M PBS.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 is put dry thorough drying in 37 ℃ of baking boxs.
4., put in the dry environment preserve standby.
(5) assembling test strip
1., paste antibody solid phase nitrocellulose filter in the plastic plate mid-way.
2., on plastic plate the fixing top of nitrocellulose film location, adhere to absorbent pad, low side adheres to the plain film band of golden traget antibody binding substances-glass fibre.Absorbent pad and Radioactive colloidal gold pad should cover about 1 millimeter in nitrocellulose filter edge.
3., Radioactive colloidal gold pad below adheres to sample pad, about 1 millimeter of sample pad covering Radioactive colloidal gold pad.
4., flatten after, on slitting shear machine, be cut into the wide band of 0.4cm.
(6) packing:
(7) finished product warehouse-in
Classify according to verification result, then the reagent kit for detecting syncytial virus of respiratory passage (colloidal gold method) of preparation is put in storage on request.
(8) the assay approval finished product is put under the normal temperature and is preserved.
Reagent kit for detecting syncytial virus of respiratory passage (colloidal gold method) quality-guarantee
The quality-guarantee 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 competence for added value.
(2) immune parental generation respiratory syncytial virus is purchased in Inst. of Viruses, China Preventive Medicine Science Academy, is the cell strain 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: detected result antibody-secreting stability positive rate is 100%, and the nuclear cytology feature meets the nuclear of hybridoma and learns feature, and mouse source virus checking is negative, and sterility test is negative.
The quality-guarantee of 2 monoclonal antibodies
(1) immunoglobulins and subclass: use immune double diffusion method, the result meets the requirements.
(2) avidity: 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%.
(4) cross matching: 2A7 monoclonal antibody and influenza virus A, Type B, parainfluenza virus 1,2,3 types, adenovirus 3,7 types, the SARS virus reaction that all is negative.
(5) titration:, be not less than 1: 6400-1 with indirect fluorescent method (IFA): 12800, meet the requirements.
3 rough detection of antibodies
No matter be the mouse ascites or the monoclonal antibody of cell culture method preparation, all need to carry out following calibrating.
(1) sterility test: be asepsis growth, feminine gender.
(2) mycoplasma inspection: negative.
(3) mouse source virus checking: negative.
(4) titration: measure or indirect enzyme-linked immunosorbent method mensuration with the IFA method, meet purifying and want ball.
4 one-tenth quality establishments of standard
(1) visual inspection
Test strip width 4mm ± 0.2mm.
The test paper surfacing, the edge is neat.
Each component of test strip is pasted firmly, is connected closely, with moving continuously of liquid behind the assurance application of sample.
(2) liquid translational speed
Extract mobile 2cm time on nitrocellulose filter was not less than 15 seconds.
Experiment: the detected result evaluation of reagent kit for detecting syncytial virus of respiratory passage of the present invention (colloidal gold method)
Experiment 1: and the reaction of respiratory syncytial virus
Be positive with two strains (A and B) of respiratory syncytial virus.
Experiment 2: cross reaction
With influenza virus A, Type B no cross reaction; Parainfluenza virus 1,2,3 type no cross reactions; With adenovirus 3,7 type no cross reactions; With 4 strain SARS virus no cross reactions.
Experiment 3: the detection verity of reagent kit for detecting syncytial virus of respiratory passage (colloidal gold method) is estimated.
Detect 1108 parts of patient specimens altogether in three different hospitals, wherein detect positive 504 parts in 512 parts of positive, detect negative 592 parts in 596 parts of negative samples.
Sensitivity: detect positive 504 parts in 512 parts of positive, sensitivity is 98.4%.
Specific degree: detect negative 592 parts in 596 parts of negative samples, specific degree is 99.3%.
Claims (4)
1. preserving number is the hybridoma cell strain of CGMCC 1546.
2. anti respiratory syncytial virus monoclonal antibody, described antibody are that the hybridoma cell strain of CGMCC 1546 produces by preserving number.
3. reagent kit for detecting syncytial virus of respiratory passage, it is characterized in that: it comprises the described anti respiratory syncytial virus monoclonal antibody of claim 2.
4. reagent kit for detecting syncytial virus of respiratory passage as claimed in claim 3 is characterized in that: the attached Radioactive colloidal gold of the described anti respiratory syncytial virus monoclonal antibody of claim 2 bag, described test kit is used for the Radioactive colloidal gold detection method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006101116509A CN101130765B (en) | 2006-08-21 | 2006-08-21 | Reagent kit for detecting syncytial virus of respiratory passage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006101116509A CN101130765B (en) | 2006-08-21 | 2006-08-21 | Reagent kit for detecting syncytial virus of respiratory passage |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101130765A CN101130765A (en) | 2008-02-27 |
| CN101130765B true CN101130765B (en) | 2011-04-06 |
Family
ID=39128171
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2006101116509A Expired - Fee Related CN101130765B (en) | 2006-08-21 | 2006-08-21 | Reagent kit for detecting syncytial virus of respiratory passage |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101130765B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102253205A (en) * | 2011-06-16 | 2011-11-23 | 昆明倍尔遵生科技有限公司 | Colloidal gold test paper strip for detecting respiratory syncytial virus antibody and preparation method thereof |
| CN103048450B (en) * | 2011-10-13 | 2015-09-30 | 苏州药明康德新药开发股份有限公司 | The quantitative assay of high-throughout rsv protein content and detection kit thereof |
| CL2011003002A1 (en) | 2011-11-25 | 2012-05-25 | Univ Pontificia Catolica Chile | Monoclonal antibody or a fragment thereof that binds to the m2-1 protein of the human respiratory syncytial virus (RSV); nucleotide sequences; pharmaceutical composition; method of diagnosis of infection produced by vrs; kit; and use of said antibody to prepare a medicament. |
| CN105753981B (en) * | 2016-03-08 | 2019-10-08 | 湖北工业大学 | The immune chromatography reagent kit of anti-human Respiratory Syncytial Virus(RSV) N protein antibody and the application antibody |
| CN106520541B (en) * | 2016-12-16 | 2017-08-25 | 常州市环境监测中心 | A kind of kit of zebrafish embryo acute toxicity testing |
| CN109115764B (en) * | 2018-07-30 | 2021-06-15 | 深圳瑞达生物股份有限公司 | Environment-friendly urine hydroxyphenyl derivative detection reagent and preparation method thereof |
| CA3111336A1 (en) | 2018-09-03 | 2020-03-12 | Pontificia Universidad Catolica De Chile | Specific monoclonal antibody against the n antigen of human respiratory syncytial virus (hrsv) useful for treating infection, detection thereof and diagnosis |
| CN113249333B (en) * | 2021-03-16 | 2023-06-02 | 贵州省人民医院 | Hybridoma cell strain RSVN4C3 secreting anti-respiratory syncytial virus monoclonal antibody |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1264425A (en) * | 1997-07-17 | 2000-08-23 | 皮埃尔法博赫药品公司 | Syncytial respiratory virus epitopes and antibodies comprising them, useful in diagnosis and therapy |
-
2006
- 2006-08-21 CN CN2006101116509A patent/CN101130765B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1264425A (en) * | 1997-07-17 | 2000-08-23 | 皮埃尔法博赫药品公司 | Syncytial respiratory virus epitopes and antibodies comprising them, useful in diagnosis and therapy |
Non-Patent Citations (1)
| Title |
|---|
| 田慕贞等.抗呼吸道合胞病毒(RSV)单克隆抗体的制备及其对RSV病毒株抗原性的分析.《中国病毒学》.1992,第7卷(第2期),181-185页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101130765A (en) | 2008-02-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101130765B (en) | Reagent kit for detecting syncytial virus of respiratory passage | |
| CN104007261B (en) | Fowl three kinds of breathing problem three quick detection kit and application | |
| CN104407137B (en) | A kind of CSFV velogen strain and low virulent strain differentiate Test paper | |
| CN108169492A (en) | It is a kind of to be used to detect colloidal gold immuno-chromatography test paper strip of bovine rota and its preparation method and application | |
| CN104459144B (en) | A kind of PRV velogen strain and vaccine strain differentiate Test paper | |
| CN106226518A (en) | Canine distemper virus colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof | |
| CN105950563A (en) | Hybridoma cell strain 7E3, monoclonal antibody secreted by hybridoma cell strain 7E3 and resistant to FMD type A virus, and application | |
| CN105753981B (en) | The immune chromatography reagent kit of anti-human Respiratory Syncytial Virus(RSV) N protein antibody and the application antibody | |
| CN101382549A (en) | Mycobacterium tuberculosis rapid detecting system and method | |
| CN107312088A (en) | Porcine epidemic diarrhea virus specificity SIgA ELISA detection kits and its application | |
| CN104459142A (en) | Test paper for distinguishing virulent strain from attenuated strain of newcastle disease virus | |
| CN106636004A (en) | TMV-CMV-PVY three-virus (tobacco mosaic virus, cucumber mosaic virus and potato virus) colloidal gold rapid test strip | |
| CN106596931B (en) | A kind of Tobacco-growing areas in Guangdong's cucumber mosaic virus colloidal gold fast detecting test paper strip | |
| CN100503821C (en) | Influenza Virus A colloidal gold quick detection test paper | |
| CN108918875A (en) | Pigeon with newcastle disease monoclonal antibody and the application in preparation diagnosis and detection kit | |
| CN109975541B (en) | Detection card for rapidly detecting canine distemper virus antigen and preparation method thereof | |
| CN100503822C (en) | Influenza Virus B colloidal gold quick detection test paper | |
| CN109374886A (en) | Infectious bovine rhinotrachetis virus antibody assay kit and its application | |
| CN102633878A (en) | H1-subtype influenza A virus double-antibody sandwich ELISA kit and application | |
| CN105585635B (en) | The immune chromatography reagent kit of anti-human mycoplasma pneumoniae p1 protein antibody and the application antibody | |
| CN105753982B (en) | The immune chromatography reagent kit of anti-human streptococcus pneumonia fam1 family PspA protein antibodies and the application antibody | |
| CN101000347A (en) | Immunological chromatographic test paper for testing francisella tularensis and its prpearation method | |
| CN103146651B (en) | Test strip capable of testing infectious bovine rhinotracheitis virus (IBRV) by monoclonal antibody mediated nano gold spot infiltration method | |
| CN103509759B (en) | Anti-IHNV (Infectious Hematopoietic Necrosis Virus) monoclonal antibody 5H3 and application thereof | |
| CN106279408A (en) | The monoclonal antibody of resistant to foot and mouth disease O type virus and Antibody Combination and its application in this virus antigen, antibody test |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110406 Termination date: 20140821 |
|
| EXPY | Termination of patent right or utility model |