CN103499695A - Preparation method of mouse Mucin 5 Subtype B rat monoclonal antibody and enzyme-linked immunosorbent assay kit - Google Patents
Preparation method of mouse Mucin 5 Subtype B rat monoclonal antibody and enzyme-linked immunosorbent assay kit Download PDFInfo
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Abstract
The invention discloses a preparation method of a mouse Mucin 5 Subtype B rat monoclonal antibody and enzyme-linked immunosorbent assay kit. The preparation method is characterized in that the mouse Mucin 5 Subtype B kit comprises Mucin 5 Subtype B, and a rate monoclonal antibody fixation, a biotinylation Mucin 5 Subtype B polyclonal antibody, HRP (horse reddish peroxidase)-labeled avidin, substrate TMB (tetramethylbenzidine) and a stop buffer connected to a solid phase vector. Mouse Mucin 5 Subtype B recombinant proteins good in stability are obtained through a molecular cloning technique, and a monoclonal antibody high in specificity and strong in affinity is obtained through a rat monoclonal antibody preparation technique, and used for preparing the mouse Mucin 5 Subtype B enzyme-linked immunosorbent assay kit.
Description
The invention belongs to biological technical field, in particular to the preparation method's of a kind of mouse mucin 5B rat monoclonal antibody and enzyme-linked immunosorbent assay kit preparation method.
Background technology
Mucin 5B, Mucin5SubtypeB (MUC5B), be one of high-molecular-weight protein family member of high glycosylation modification.Core protein and O-sugar chain, consist of, molecular weight is about more than 400,000, expresses in lachrymal gland and secretes, and is one of main glutinous protein ingredient in saliva and tear.
Mucin 5B is gel-forming type mucin; the monomer whose polymerization of can tangling forms linear polymer; thereby form gel and be anchored on the eye table, it makes slime layer have rheological properties, and when eyelid and eyeball quick relative movement, protective epithelium is avoided the destruction of shearing force.Simultaneously, it can produce nonspecific interaction with various particulates, chemical substance, microorganism etc.Can form little gel around foreign matter, non-viable non-apoptotic cell etc., through nasolacrimal duct, the rubbish of eye table be transported.Mucin 5B is the requisite composition of tear film, plays an important role maintaining aspect tear film stability, detects the mucinous content of eye table and can be used as a kind of method of estimating tear film function.Simultaneously, it also plays lubricity and viscoelastic effect in full saliva, normal lung mucus and cervical mucus.And this albumen has been found to be in some human diseasess, comprise sinus mucosa chronic nasosinusitis (CRS) and nasal polyp CRS, during raising, chronic obstructive pulmonary disease (COPD) stomach trouble relevant with helicobacter pylori plays an important role.Therefore mucinous detection all has certain value for the diagnosis of the diseases such as xerophthalmia, nasosinusitis, lung cancer, gastric ulcer, the assessment of curative effect of medication etc.
Traditional mucin method for quantitatively determining mainly contains high performance liquid chromatography, Western-blotting, RT-PCR, immunohistochemistry and enzyme-linked immunosorbent assay at present.
Wherein Western blot (Western-blotting) is to be applied at present the commonplace method that mucin 5B quantitatively detects, this method can Preliminary Determination mucin 5B content, but what it recorded is the above content of μ g/mL, simple to operate, but need to do the SDS-PAGE electrophoresis and with coomassie brilliant blue staining and decolouring, pilot process is various, influence factor is also a lot, less stable, and finally can only be by the estimation of target stripe gray-scale value quantitatively, result is not very accurate, and disposablely can only detect a few sample, uses clinically less; And although the use staining examine mucin 5B of immuning tissue also can record the above content of μ g/mL, but owing in immuning tissue's dyeing now, also existing some problems, such as organize fixation problem, antigen retrieval problem and positive control problem etc., these problems have also seriously restricted the development of immuning tissue's colouring method and have popularized; RT-PCR mainly predicts the amount of mucin 5B by detecting mRNA, need to use quantitative real time PCR Instrument, and one-time investment is large, expensive, is not suitable for common laboratory and uses.
That high performance liquid chromatography (HPLC) has is highly sensitive, accuracy and the advantage such as reproducible, but needs expensive instrument and equipment, and testing process is loaded down with trivial details, the technical requirement of Sample pretreatment is very high, is difficult to operation.The testing staff need to possess professional detection technical ability and abundant experiment experience.
Enzyme linked immunosorbent assay (ELISA) (ELISA) detection technique is a kind of nonradioactive labeling's immuno analytical method grown up on the basis of RIA basic theories.Utilize the target molecule in specific immune response identification biofluid between Ag-Ab, and utilize the content of target molecule in the quantitative reaction sample to be tested of the chromogenic reaction of enzyme-substrate.Due to its high specificity, sensitivity, the advantages such as not needing large-scale instrument that reaches easy and simple to handle, now be widely used in the quantitative test experience of biological specimen.Key factor in the enzyme linked immunosorbent assay (ELISA) detection kit is the monoclonal antibody that obtains high specific, high-affinity.Mouse is the most frequently used animal used as test in scientific experiment chamber at present, but because mucin 5B derives from mouse, so the preparation method of conventional mouse monoclonal antibody is here impracticable.The preparation of rat monoclonal antibody can address this problem, and rat antibody has the advantages that some mouse antibodies do not possess simultaneously, and the light chain 95% of large mouse antibody molecule is the κ type.Therefore can utilize the mouse-anti body Ig κ of the mouse Chinese People's Anti-Japanese Military and Political College-1a antibody, the monoclonal antibody of Ig κ-1a that high degree of specificity ground produces in conjunction with hybridoma, then reach the purpose of the non-specific Ig κ that removes the host rat and produce-1b antibody, thereby separation and purification obtains highly purified antibody.Therefore, the application utilizes the Rat hybridoma technology to prepare the monoclonal antibody of the albumen of mouse and other species, then for the ELISA kit, researches and develops.
Summary of the invention
The object of the invention is to overcome the defect of prior art, a kind of mouse mucin 5B rat monoclonal antibody and enzyme-linked immunosorbent assay kit and preparation method thereof are provided.In order to realize purpose of the present invention, intend adopting following technical scheme:
One aspect of the present invention relates to the preparation method of a kind of mouse mucin 5B rat monoclonal antibody and enzyme-linked immunosorbent assay kit, it is characterized in that described kit comprises mucin 5B and is connected in the antibody combination on solid phase carrier, biotinylated mucin 5B polyclonal antibody, the Avidin of HRP mark, substrate TMB and stop buffer, described preparation method comprises the steps:
The antibody of mucin 5B on being connected in solid phase carrier is combined, add biotinylated mucin 5B polyclonal antibody after washing plate, after unconjugated biotinylated antibody is cleaned, the Avidin that adds horseradish peroxidase HRP mark, again by Avidin horseradish peroxidase (HRP) amplifying signal, with 3,3 ', 5,5 '-tetramethyl benzidine (TMB) and sulfuric acid is respectively as substrate and stop buffer.
One preferred embodiment in, described kit is usingd polystyrene agent plate (PS) as solid phase carrier, first is placed under ultraviolet light irradiation 2 hours, makes the activation of PS plate.
Swollen in another preferred embodiment of the present invention, the preparation method of described biotinylated mucin 5B antigen comprises the steps:
(1) preparation of mucin 5B antigen: adopt molecule clone technology, design primer, enzyme are cut, are transformed, expression and SDS-PAGE electrophoresis are identified and obtained mouse mucin 5B recombinant protein;
Swollen in another preferred embodiment of the present invention, the preparation method of described biotinylated mucin 5B polyclonal antibody comprises the steps:
(2) preparation of anti-mouse mucin mucin 5B rabbit polyclonal antibody: the mouse 5B albumen of restructuring is adjusted to 0.5-3mg/ml, get 0.2-4ml albumen, add that isopyknic Freund's complete adjuvant is fully emulsified to be mixed, rabbit metapedes pad, the subcutaneous multiple spot immunity in back, every 100-1000ul, after 14d, add isopyknic incomplete Freunds adjuvant with mouse mucin 5B recombinant protein solution and carry out booster immunization, dosage is identical with fundamental immunity dosage, once, immunity is four times altogether, latter 14 days of the 4th immunity in immunity in every two weeks, get whole blood from arteria carotis, measure antibody titer;
Swollen in another preferred embodiment of the present invention, the preparation method of described biotinylated mucin 5B rat monoclonal antibody comprises the steps:
(3) preparation of anti-mouse mucin 5B rat monoclonal antibody:
1) cell is cultivated
Mouse SP2/0 myeloma cell cell is incubated in the RPMIl640 nutrient solution of 5-25% NBCS, and condition of culture is: 1-20%CO
2, 25-45 ℃.
2) immune animal
Select female nude rat in March, getting 0.3-1ml the mouse mucin 5B recombinant protein solution that is 0.5-3mg/ml from concentration mixes with isopyknic complete Freund ' s adjuvant, 0,15 and the vola immunity of 28d rat hindleg, after 10d, with the agarose double diffusion test, detect, when antibody concentration reaches 0.5-3mg/mL by the time, complete immunity.Getting the last time inguinal lymph nodes B cell and murine myeloma cell after immune latter 3 days merges.
3) reagent
HAT selectivity nutrient solution, Hypoxanthine-aminopterin-thymidine (HAT) (50X),
HyPoxanthine-thymidine(HT)(50X)。
The PEG-4000 of PEG fusion agent: 5g is dissolved in the EMEM nutrient solution of 7ml, 56 ℃ of 10min, and 120 ℃ of 15min degerming, add lmlDMSO, through the Millipore filtration sterilization of 0.22 μ M, is distributed into every pipe 2ml, in 37 ℃ of preservations.
Trophocyte: before merging, 2d gets rat peritoneal macrophages, and concentration is 2.0 * 10
5cell/ml, every hole 0.1ml is inoculated in 96 orifice plates.
4) Fusion of Cells
The SP2/0 cell of taking the logarithm growth period and immune rat B cell, with merging liquid EMEM, wash twice, counting cells, ratio cell mixing with SP2/0 cell and B cell 1:1-1:20, centrifugal 1000rpm, 5-20min, remove supernatant, beat gently loose cell mass, add the PEG of 0.5-3ml in 37 ℃, 90 seconds, through 30 seconds, shake, the fusion liquid that dropwise added lml in 90 seconds, finally add 20mlEMEM nutrient solution dilution PEG, the centrifugal 5min of 800rpm, re-suspended cell is in selectivity HAT nutrient solution, and regulating cell density is 10
6splenocyte/ml, every hole 0.lml is inoculated in 96 orifice plates that are covered with trophocyte.
5) screening and anti-preparation of stopping:
14d after merging, visible clone forms, when the clone grows up to suitable 1/3 hole, detect the culture supernatant that hybridoma is arranged with ELISA, positive hole is carried out the limiting dilution assay of twice and is cloned, obtain the anti-hybridoma cell strain of stopping of stably excreting, and it is subcutaneous that hybridoma is inoculated in to the armpit of nude rat, grows up to a 2 * 2cm after 3 weeks
3above reality is stopped knurl, grinds and makes the hybridoma suspension, is inoculated in the nude rat rat abdominal cavity, produces and contains the ascites that monoclonal anti is stopped.
6) preparation of immune affinity column:
Resist with the thick bill of lading of ammonium sulfate precipitation method, obtain the antibody of the anti-mouse mucin 5B that specificity is high by the affinity chromatography purifies and separates.Adopt the specific antibody in affinity chromatography purification of rat ascites.Above-mentioned recombinant protein is coupled on affinity column, is prepared into the affinity column of energy separating mouse mucin 5B antibody.
The purifying concrete steps of mouse mucin 5B antibody are as follows: by 200-300ml rat ascites sample, directly from the chromatographic column injection port loading of mouse mucin 5B antigen coupling, flow velocity is 1ml/min.With after 0.1mol/LpH8.0 phosphate buffer (30-40ml) wash-out foreign protein, then use 0.01mol/LpH8.0 phosphate buffer (20-30ml) wash-out, flow velocity is 2ml/min.Finally use 0.1mol/LGly-HCl elution buffer wash-out, flow velocity is 1.5ml/min, collects the sample eluent, standby after dialysis is concentrated.Adopt indirect elisa method to be identified, antibody titer is 1:1000000-1:2500000.
The present invention obtains the mouse 5B recombinant protein of good stability by molecule clone technology, and obtains by the rat monoclonal antibody technology of preparing monoclonal antibody that specificity is high and affinity is strong, is used for preparing mouse mucin 5B enzyme-linked immunosorbent assay kit.By the comparison in the HPLC method, the aspects such as its precision, the recovery, accuracy and batch detection sample all are better than the HPLC method.The ELISA method is more simply too much than HPLC method sample pre-treatments, and specificity is good, concentrated without purifying.In addition, the ELISA method is shorter than the HPLC method running time, once can process a large amount of samples, has saved a large amount of human and material resources, has improved work efficiency.And the ELISA method is less than the investment of HPLC method, the use of instrument does not need the professional training technician, is more suitable for grass-roots unit and uses.
Embodiment
Embodiment 1:
Main technology path is as follows:
1. the preparation method of mouse mucin 5B enzyme linked immunological kit raw material, its concrete steps are
The preparation of a, mouse mucin 5B antigen:
Adopt molecule clone technology, design primer, enzyme are cut, are transformed, expression and SDS-PAGE electrophoresis are identified and obtained mouse mucin 5B recombinant protein.
The preparation of b, anti-mouse mucin 5B rabbit polyclonal antibody:
1 of healthy male new zealand white rabbit, with the immunity of mouse mucin 5B recombinant protein.The mouse mucin 5B albumen of restructuring is adjusted to 1mg/ml, gets 1ml albumen, add that isopyknic Freund's complete adjuvant is fully emulsified to be mixed.Rabbit metapedes pad, the subcutaneous multiple spot immunity in back, every 500ul.After 14d, add isopyknic incomplete Freunds adjuvant with mouse mucin 5B recombinant protein solution and carry out booster immunization, dosage is identical with fundamental immunity dosage.Once, immunity is four times altogether in immunity in every two weeks, and latter 14 days of the 4th immunity, get whole blood from arteria carotis, measures antibody titer.
The preparation of c, anti-mouse mucin 5B rat monoclonal antibody:
1) cell is cultivated
Mouse SP2/0 myeloma cell cell is incubated in the RPMIl640 nutrient solution of 10% NBCS, and condition of culture is: 5%CO
2, 37 ℃.
2) immune animal
Select female nude rat in March, getting 0.4ml the 5B recombinant protein solution that is 1mg/ml from concentration mixes with isopyknic complete Freund ' s adjuvant, 0,15 and the vola immunity of 28d rat hindleg, after 10d, with the agarose double diffusion test, detect, when antibody concentration reaches 1mg/mL by the time, complete immunity.Getting the last time inguinal lymph nodes B cell and murine myeloma cell after immune latter 3 days merges.
3) reagent
HAT selectivity nutrient solution, Hypoxanthine-aminopterin-thymidine (HAT) (50X),
HyPoxanthine-thymidine(HT)(50X)。
The PEG-4000 of PEG fusion agent: 5g is dissolved in the EMEM nutrient solution of 7ml, 56 ℃ of 10min, and 120 ℃ of 15min degerming, add lmlDMSO, through the Millipore filtration sterilization of 0.22 μ M, is distributed into every pipe 2ml, in 37 ℃ of preservations.
Trophocyte: before merging, 2d gets rat peritoneal macrophages, and concentration is 2.0 * 10
5cell/ml, every hole 0.1ml is inoculated in 96 orifice plates.
4) Fusion of Cells
The SP2/0 cell of taking the logarithm growth period and immune rat B cell, with merging liquid EMEM, wash twice, counting cells, ratio cell mixing with 1 SP2/0 cell and 5 B cells, centrifugal 1000rpm, 5min, remove supernatant, beat gently loose cell mass, add the PEG of lml in 37 ℃, 90 seconds, through 30 seconds, shake, the fusion liquid that dropwise added lml in 90 seconds, finally add 20mlEMEM nutrient solution dilution PEG, the centrifugal 5min of 800rpm, re-suspended cell is in selectivity HAT nutrient solution, and regulating cell density is 10
6splenocyte/ml, every hole 0.lml is inoculated in 96 orifice plates that are covered with trophocyte.
5) screening and anti-preparation of stopping:
14d after merging, visible clone forms, when the clone grows up to suitable 1/3 hole, detect the culture supernatant that hybridoma is arranged with ELISA, positive hole is carried out the limiting dilution assay of twice and is cloned, obtain the anti-hybridoma cell strain of stopping of stably excreting, and it is subcutaneous that hybridoma is inoculated in to the armpit of nude rat, grows up to a 2 * 2cm after 3 weeks
3above reality is stopped knurl, grinds and makes the hybridoma suspension, is inoculated in the nude rat rat abdominal cavity, produces and contains the ascites that monoclonal anti is stopped.
6) preparation of immune affinity column:
Resist with the thick bill of lading of ammonium sulfate precipitation method, obtain the antibody of the anti-mouse mucin 5B that specificity is high by the affinity chromatography purifies and separates.Adopt the specific antibody in affinity chromatography purification of rat ascites.Above-mentioned recombinant protein is coupled on affinity column, is prepared into the affinity column of energy separating mouse mucin 5B antibody.
The purifying concrete steps of mouse mucin 5B antibody are as follows: by 200-300ml rat ascites sample, directly from the chromatographic column injection port loading of mouse mucin 5B antigen coupling, flow velocity is 1ml/min.With after 0.1mol/LpH8.0 phosphate buffer (30-40ml) wash-out foreign protein, then use 0.01mol/LpH8.0 phosphate buffer (20-30ml) wash-out, flow velocity is 2ml/min.Finally use 0.1mol/LGly-HCl elution buffer wash-out, flow velocity is 1.5ml/min, collects the sample eluent, standby after dialysis is concentrated.Adopt indirect elisa method to be identified, antibody titer is 1:1000000-1:2500000.
1, the development of ELISA kit:
This kit application DASELISA immunoassay is measured the level of mucin 5B in mice serum or plasma sample.Preparation process is as follows: use 96 hole polystyrene agent plate (PS) as solid phase carrier, first be placed on irradiation 2h under ultraviolet light, make the activation of PS plate.Be coated with in advance certain density anti-mouse mucin 5B rat monoclonal antibody on the agent plate capillary strip, 4 ℃ are spent the night, and through washing after plate and bovine serum albumin(BSA) sealing are processed, make the ELISA Plate be coated with.During detection, add successively standard items and sample in micropore, the antibody of mouse mucin 5B on being connected in solid phase carrier wherein is combined, add biotinylated mouse mucin 5B polyclonal antibody after washing plate, after unconjugated biotinylated antibody is cleaned, the Avidin that adds horseradish peroxidase HRP mark, again by Avidin horseradish peroxidase (HRP) amplifying signal, with 3,3 ', 5,5 '-tetramethyl benzidine (TMB) and sulfuric acid is respectively as substrate and stop buffer, completes the preparation of the enzyme connection determining adsorption kit of mouse mucin 5B.
2, through a series of quality index of kit is detected, comprise sensitivity, repeatability, specificity, the recovery and linear determination, show this detection system maturation, this kit indices testing result:
A. typical curve
B. sensitivity: when the ELISA method detects the mice serum sample, lowest detection is limited to 6.7pg/ml;
C. the recovery: add respectively the numeraire product of different amounts with same serum sample, make recovery test (n=5).Average recovery rate 95.5%(table 2).
Table 2ELISA method is measured the recovery test of oxytocins
| Numeraire product (pg/ml) | The recovery (%) |
| 600 | 95.83 |
| 250 | 96.42 |
| 30 | 94.25 |
| Average recovery rate | 95.5 |
[0068]technique effect
Contrasted ELISA method (the mensuration kit that the present invention is prepared) and HPLC standard measure and detected, visible ELISA method has characteristics simple to operate, quick, that experimentation cost is low.
| ? | The ELISA method | The HPLC method |
| Testing process | Simply | Loaded down with trivial details |
| Running time | Short (4.5 hours) | Long |
| Operating personnel | Common laboratory technician | The professional training technician |
| Sample mensuration quantity/time | Many | Single |
| Equipment (reagent) price | Cheap | Expensive |
| Sensitivity | 6.7pg/ml | 4.57ng/ml |
Table 3ELISA method and HPLC method are relatively
1. Precision Experiment:
By two methods, high, medium and low value quality-control product is carried out respectively to Precision Experiment, experimental technique is for using respectively three definite value sample 600pg/ml of these two kit measurements, 250pg/ml, 30pg/ml, each sample replication 20 times, in table 4, the visible ELISA method precision of result is better.
Table 4ELISA method and the test of HPLC method precision
2. recovery experiment:
Get the standard solution (mucin 5B concentration is 600pg/ml, 250pg/ml, 30pg/ml) of variable concentrations, add respectively definite value serum (125pg/ml), by HPLC method and ELISA method, detect, and comparative measurements value and desired value are recycled rate (in Table 5).
| ? | ELISA method (%) | HPLC method (%) |
| 600pg/ml | 95.83 | 82.34 |
| 250pg/ml | 96.42 | 87.68 |
| 30pg/ml | 94.25 | 78.45 |
[0079]table 5ELISA method and the test of the HPLC method recovery
The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited to this, and any variation of expecting without creative work or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.
Claims (5)
1. the preparation method of a mouse mucin 5B enzyme-linked immunosorbent assay kit, it is characterized in that described kit comprises mouse mucin 5B and is connected in the antibody combination on solid phase carrier, biotinylated mouse mucin 5B polyclonal antibody, the Avidin of HRP mark, substrate TMB and stop buffer, described preparation method comprises the steps:
The anti-mouse mucin 5B rat monoclonal antibody of mouse mucin 5B on being connected in solid phase carrier is combined, add biotinylated 5B polyclonal antibody after washing plate, after unconjugated biotinylated antibody is cleaned, the Avidin that adds the HRP mark, again by Avidin horseradish peroxidase (HRP) amplifying signal, with 3,3 ', 5,5 '-tetramethyl benzidine (TMB) and sulfuric acid is respectively as substrate and stop buffer.
2. the preparation method claimed in claim 1 that follows up, described kit is usingd polystyrene agent plate (PS) as solid phase carrier, first is placed under ultraviolet light irradiation 2 hours, makes the activation of PS plate.
3. preparation method according to claim 1 and 2, the preparation method of described biotinylated mouse mucin 5B antigen comprises the steps:
The preparation of mouse mucin antigen: adopt molecule clone technology, design primer, enzyme are cut, are transformed, expression and SDS-PAGE electrophoresis are identified and obtained mouse mucin 5B recombinant protein.
4. preparation method according to claim 1 and 2, the preparation method of described biotinylated mouse mucin 5B polyclonal antibody comprises the steps:
The preparation of anti-mouse mucin 5B rabbit polyclonal antibody: the mouse mucin 5B albumen of restructuring is adjusted to 0.5-3mg/ml, get 0.2-4ml albumen, add that isopyknic Freund's complete adjuvant is fully emulsified to be mixed, rabbit metapedes pad, the subcutaneous multiple spot immunity in back, every 100-1000ul, after 14d, add isopyknic incomplete Freunds adjuvant with mouse mucin 5B recombinant protein solution and carry out booster immunization, dosage is identical with fundamental immunity dosage, once, immunity is four times altogether, latter 14 days of the 4th immunity in immunity in every two weeks, get whole blood from arteria carotis, measure antibody titer.
5. preparation method according to claim 1 and 2, the preparation method of described biotinylated mouse mucin 5B rat monoclonal antibody comprises the steps:
The preparation of anti-mouse mucin 5B rat monoclonal antibody:
1) cell is cultivated
Mouse SP2/0 myeloma cell cell is incubated in the RPMIl640 nutrient solution of 5-25% NBCS, and condition of culture is: 1-20%CO
2, 25-45 ℃.
2) immune animal
Select female nude rat in March, getting 0.3-1ml the mouse mucin 5B recombinant protein solution that is 0.5-3mg/ml from concentration mixes with isopyknic complete Freund ' s adjuvant, 0,15 and the vola immunity of 28d rat hindleg, after 10d, with the agarose double diffusion test, detect, when antibody concentration reaches 1mg/mL by the time, complete immunity.Getting the last time inguinal lymph nodes B cell and murine myeloma cell after immune latter 3 days merges;
3) reagent
HAT selectivity nutrient solution, Hypoxanthine-aminopterin-thymidine (HAT) (50X),
HyPoxanthine-thymidine(HT)(50X)；
The PEG-4000 of PEG fusion agent: 5g is dissolved in the EMEM nutrient solution of 7ml, 56 ℃ of 10min, and 120 ℃ of 15min degerming, add lmlDMSO, through the Millipore filtration sterilization of 0.22 μ M, is distributed into every pipe 2ml, in 37 ℃ of preservations;
Trophocyte: before merging, 2d gets rat peritoneal macrophages, and concentration is 2.0 * 10
5cell/ml, every hole 0.1ml is inoculated in 96 orifice plates;
4) Fusion of Cells
The SP2/0 cell of taking the logarithm growth period and immune rat B cell, with merging liquid EMEM, wash twice, counting cells, ratio cell mixing with 1 SP2/0 cell and 5 B cells, centrifugal 1000rpm, 5min, remove supernatant, beat gently loose cell mass, add the PEG of lml in 37 ℃, 90 seconds, through 30 seconds, shake, the fusion liquid that dropwise added lml in 90 seconds, finally add 20mlEMEM nutrient solution dilution PEG, the centrifugal 5min of 800rpm, re-suspended cell is in selectivity HAT nutrient solution, and regulating cell density is 10
6splenocyte/ml, every hole 0.lml is inoculated in 96 orifice plates that are covered with trophocyte;
5) screening and anti-preparation of stopping:
14d after merging, visible clone forms, when the clone grows up to suitable 1/3 hole, detect the culture supernatant that hybridoma is arranged with ELISA, positive hole is carried out the limiting dilution assay of twice and is cloned, obtain the anti-hybridoma cell strain of stopping of stably excreting, and it is subcutaneous that hybridoma is inoculated in to the armpit of nude rat, grows up to a 2 * 2cm after 3 weeks
3above reality is stopped knurl, grinds and makes the hybridoma suspension, is inoculated in the nude rat rat abdominal cavity, produces and contains the ascites that monoclonal anti is stopped;
6) preparation of immune affinity column:
Resist with the thick bill of lading of ammonium sulfate precipitation method, obtain the antibody of the anti-mouse mucin 5B that specificity is high by the affinity chromatography purifies and separates; Adopt the specific antibody in affinity chromatography purification of rat ascites.Above-mentioned recombinant protein is coupled on affinity column, is prepared into the affinity column of energy separating mouse mucin 5B antibody;
The purifying concrete steps of mouse mucin 5B antibody are as follows: by 200-300ml rat ascites sample, directly from the chromatographic column injection port loading of mouse mucin 5B antigen coupling, flow velocity is 1ml/min.With after 0.1mol/LpH8.0 phosphate buffer (30-40ml) wash-out foreign protein, then use 0.01mol/LpH8.0 phosphate buffer (20-30ml) wash-out, flow velocity is 2ml/min.Finally use 0.1mol/LGly-HCl elution buffer wash-out, flow velocity is 1.5ml/min, collects the sample eluent, standby after dialysis is concentrated.Adopt indirect elisa method to be identified, antibody titer is 1:1000000-1:2500000.
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| CN109988241A (en) * | 2017-12-29 | 2019-07-09 | 博阳生物科技（上海）有限公司 | A kind of antibody and its preparation method and application of specific recognition people immune complex |
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| CN109988241A (en) * | 2017-12-29 | 2019-07-09 | 博阳生物科技（上海）有限公司 | A kind of antibody and its preparation method and application of specific recognition people immune complex |
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