CN107043752A - Hybridoma cell strain and the anti-CRH antibody N D19 based on hybridoma cell strain and its application of context of detection - Google Patents
Hybridoma cell strain and the anti-CRH antibody N D19 based on hybridoma cell strain and its application of context of detection Download PDFInfo
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Abstract
The present invention relates to biological technical field, anti-CRH antibody N D19 and its application of context of detection of hybridoma cell strain are disclosed.The hybridoma cell strain, China typical culture collection center is preserved on April 13rd, 2016, and Classification And Nomenclature is hybridoma N D19, and deposit number is CCTCC NO.C2015216.The monoclonal antibody N D19 of the anti-Adrenergic beta-antagonists (CRH) produced based on the hybridoma cell strain, and the monoclonal antibody are applied to the kit of detection acute high altitude sickness.The anti-CRH monoclonal antibodies N D19 that the present invention is provided, can recognize CRH N-terminal epitope；With good specific and higher potency, its potency is up to 1：4×106.The detection CRH of present invention double-antibody sandwich elisa kit, with good specific and higher sensitivity.
Description
Technical field
The invention belongs to technical field of bioengineering, more particularly to a kind of hybridoma cell strain, and a kind of anti-adrenal gland
The monoclonal antibody of cortin releasing hormone (CRH), and its kit.
Background technology
According to the annual report of the World Health Organization 1996, it is 1.4 hundred million that height above sea level 2500m above population is lived in the whole world, and
And it is annual there are about 40,000,000 people and come high mountain and highlands, acute high altitude sickness is that the major class into high mountain, highlands is normal
See disease and frequently-occurring disease, human body is radical to be exposed to the various pathologic reactions produced after low-oxygen environment.If prevention or malpractice,
Acute high altitude sickness can also be further development of plateau brain edema, pulmonary edema, to the health care belt of plateau work, tourism and rescue personnel
Carry out very big threat.Therefore, the prediction diagnosis for moving into plateau crowd how is realized, easily ill crowd is diagnosed to be early, to height
The preventing and treating of former disease is significant.
Brain be high altitude anoxia stress most sensitive organ, under low-oxygen environment, for ensure brain oxygen supply, hypothalamus-hang down
Body-hypothalamic pituitary adrenal axis (HPA axis) has played crucial adjustment effect, wherein, Adrenergic beta-antagonists (CRH) triggering
The unlatching of hpa axis.The polypeptide that CRH is made up of 41 amino acid, it is main by the inferior colliculus ventricles of the brain in mammalian brain
The neuron of core parvocellular part is synthesized and secreted.By median eminence, CRH is secreted into hypophysis, and Stimulation of Pituitary Gland secretion promotees on kidney
The secretion of gland cortin with gland cortin (ACTH), and then promotion.
CRH plays important regulative in stressed condition reaction.Now there are some researches show Basal plasma CRH>50pg/ml or
Person's saliva CRH>In 21pg/ml crowd, reach behind plateau, occur AMS, high-caliber CRH and acute height more than 80% individual
Former disease (Acute Mountain Sickness, AMS) has direct correlation.Basic CRH levels detection is acute in prediction
It is significant in the generation of altitude sickness, by prediction, AMS easily ills crowd can be instructed to carry out necessary preventing and treating in advance and arranged
Apply, so that AMS harm be minimized.
The content of the invention
The problem to be solved in the present invention is that there is provided a kind of hybridoma cell strain for CRH antibody and correlation technique not enough.
In order to solve the above technical problems, the technical solution adopted by the present invention is：A kind of hybridoma cell strain, in 2016 4
It is preserved within 13rd China typical culture collection center the moon, address is Wuhan, China Wuhan University, and Classification And Nomenclature is that hybridoma is thin
Born of the same parents, deposit number is CCTCC NO.C2015216.
Present invention simultaneously provides a kind of monoclonal antibody N-D19 of anti-Adrenergic beta-antagonists (CRH), it is special
Levy and be：Produced as hybridoma cell strain described in claim 1.
The monoclonal antibody N-D19 can recognize CRH N-terminal epitope, and CRH N-terminal epitope sequences are SEQ ID NO:In 1
Shown sequence.The monoclonal antibody N-D19 is IgG2a types, and light chain is κ types；
Monoclonal antibody N-D19 of the present invention preparation method, it is characterised in that comprise the following steps：
S01：CRHN epitopes and KLH are coupled, KLH-CRHN coupling peptides is formed, is used as immunogene；
S02：Mixed with KLH-CRHN coupling peptides with immunologic adjuvant, after immune balb/c mice, take mouse boosting cell；
S03：Mouse boosting cell is merged with SP2/0 murine myeloma cells, the hybridoma is obtained, induces in vivo
Or in vitro culture, it is purified, produce.
The present invention also provides described anti-CRH monoclonal antibody C-D17 application, it is characterised in that：Applied to preparation
Detection instrument in terms of detection CRH or the application in terms of preparing detection and CRH and having the reagent of related disorders.
Further, it is described to there are related disorders to be acute high altitude sickness with people CRH.
Another object of the present invention is to provide a kind of detection CRH double-antibody sandwich elisa kit, including it is described
Monoclonal antibody N-D19；Also include monoclonal antibody C-D17；
The monoclonal antibody C-D17 is that a kind of hybridoma cell strain is produced, and the hybridoma cell strain was May 31 in 2016
It is preserved in China typical culture collection center day, address is Wuhan, China Wuhan University, and Classification And Nomenclature is hybridoma, is protected
It is CCTCC NO.C2015215 to hide numbering.The monoclonal antibody C-D17 can recognize CRH C-terminal epitope, CRH C-terminal epitope
Sequence is SEQ ID NO:Sequence shown in 2.
Further, the monoclonal of ELISA Plate and HRP marks that the kit includes pre-coated monoclonal antibody N-D19 resists
Body C-D17；Or the monoclonal antibody N-D19 of pre-coated monoclonal antibody C-D17 ELISA Plate and HRP mark.
It is preferred that, the monoclonal of ELISA Plate and HRP marks that the kit includes pre-coated monoclonal antibody C-D17 resists
Body N-D19.
Further, the method for coating of the pre-coated monoclonal antibody N-D19 or C-D17 is：By monoclonal antibody N-D19
Or C-D17 is diluted to 10ug/mL with coating buffer, it is added to by 100uL/ holes in ELISA Plate, 4 DEG C of coatings are stayed overnight, cleaning solution washing,
Confining liquid is added, 2h, cleaning solution washing is closed.
It is preferred that, the coating buffer is 0.05mmol/L carbonate buffer solution (pH 9.6)；The confining liquid be PBS and
Mass fraction is 1% BSA.
Further, the method for HRP the labeled monoclonal antibodies N-D19 or C-D17 are：10mg HRP are weighed first to do
Powder, fully dissolves in 5mL distilled waters, adds the 0.2mol/L NaIO of Fresh4Solution 0.5mL, lucifuge is in 4 DEG C of levels
30min is gently shaken on shaking table, addition has been dialysed to monoclonal antibody N-D19 or the C-D17 antibody of carbonate buffer solution
10mg, after mixing, solution is fitted into the bag filter anticipated that molecular cut off is 50kDa, and its is right
0.05mol/L pH9.6 4 DEG C of dialysis of carbonate buffer solution, a dialyzate is changed per 6h, is dialysed four times；After dialysis terminates,
Liquid in bag filter is taken out, the NaBH that concentration is 5mg/mL is added thereto4100 μ L, 4 DEG C of standing 4h；Addition is isometric to satisfy
And ammonium sulfate, 4 DEG C stand overnight；5000rpm centrifuges 10min, after being resuspended with 1mL 0.05mmol/L PBS (pH7.4),
4 DEG C of dialysis of PBS (pH 7.4) buffer solution to 0.05mmol/L, a dialyzate is changed per 6h, is dialysed four times；Take out after dialysis
Solution, thereto add marking fluid in add isometric glycerine, produce purified HRP and mark to obtain monoclonal antibody N-
D19 or C-D17.
Further, the kit also includes CRH standard items；Standard items or sample diluting liquid：PH buffers for 7.4 PBS
Liquid；Enzyme labelled antibody dilution：PBS and mass fraction are 1%BSA；Cleaning solution：0.02mol/L PBS and mass fraction are
0.05% polysorbas20；Nitrite ion：TMB nitrite ions；Terminate liquid：2mol/L H2SO4。
The application method of kit of the present invention, comprises the following steps：Added into pre-coated ELISA Plate and treat test sample
The monoclonal antibody N-D19 or C-D17 of product, HRP marks, nitrite ion colour developing, terminate liquid are terminated, and detect OD450。
It is preferred that, the concentration of the monoclonal antibody N-D19 or C-D17 of the HRP marks preservation is 1mg/mL, during work
It is 1 to dilute volume multiple:10000.
The present invention has the advantages and positive effects of：The invention provides a kind of hybridoma cell strain, in April, 2016
China typical culture collection center was preserved in 13rd, preservation address is：Wuhan, China university；Classification And Nomenclature is that hybridoma is thin
Born of the same parents, deposit number is CCTCC NO.C2015216.It can be produced with excellent specific anti-CRH based on affiliated hybridoma
Monoclonal antibody N-D19.
The anti-CRH monoclonal antibodies N-D19 that the present invention is provided, can recognize CRH N-terminal epitope；With good special
Property with higher potency, its potency is up to 1：4×106。
The detection CRH of present invention double-antibody sandwich elisa kit, with good specific and higher sensitive
Degree：CRH antigens are detected by double crush syndrome, test limit can reach 15.6pg/ml, be compared with prior art products,
Sensitivity is significantly improved.In addition, this stabilization of kit, can be preserved 2 years very well under the conditions of 4 DEG C.Meanwhile, this detection
Kit material therefor expends few, detects the consumption of sample in a microlitre level, testing cost is relatively low, it is adaptable to Susceptible population it is general
Sieve, is adapted to large-scale promotion.Empirical tests, the kit provided using the present invention is entered to 10 patients with acute high altitude disease serum samples
Row identification, its testing result and expection are completely the same, and accuracy is up to 100%.
Brief description of the drawings
Fig. 1 is N-D19 through protein G affinity purification rear electrophoresis figures (M：Marker；1：Reduce electrophoresis；2：Non-reduced electricity
Swimming)
Fig. 2 is KLH-CRHN coupling peptide immune serum bioactivities (M：Marker；1：Reduce electrophoresis；2：It is non-reduced
Electrophoresis)
Fig. 3 is N-D19 specific detections
Fig. 4 is C-D17 through protein G affinity purification electrophoretograms
Fig. 5 is that KLH-CRHC is coupled peptide immune serum potency figure
Fig. 6 is C-D17 specific detections
Fig. 7 is CRH examination criteria curves
Embodiment
Unless otherwise indicated, term used herein is respectively provided with the implication that those skilled in the art routinely understand, this hair
The instrument of bright use is all conventional commercial product, can all be bought in market.For the ease of understanding the present invention, by used herein one
A little terms have carried out following definitions.
Use in the specification and in the claims, odd number type " one " and " this " include plural reference, unless on
Hereafter separately there is clear statement.For example, term " (one) cell " includes the cell of plural number, including its mixture.
All Digital IDs, such as pH, temperature, time, concentration, including scope, are all approximations.It is to be understood that, although
Term " about " is all added before always not describing all Digital IDs clearly.While it will also be understood that, although it is always not clear and definite
Narration, reagent described herein is only example, and its equivalent is known in the art.
The monoclonal antibody N-D19 of the present invention of embodiment 1 preparation and purification.
1st, immunogene is prepared
1.1 prepare CRHN synthetic peptides：CRH N-terminal epitope is synthesized, sequence is SEQ ID NO:Sequence shown in 1, purity is big
In 99%.
1.2KLH-CRHN is coupled the acquisition of peptide：Weigh the m- maleimidobenzoyl-N-hydroxy-succinamide esters of 3mg
(MBS) 200 μ L dimethylformamides (DMF) are dissolved in, the phosphate for taking 1.5mg KLH to be dissolved in 0.5ml 0.05mol/L delays
Fliud flushing (pH 7.0), adds the μ L of MBS solution 70 configured, 30min is stirred at room temperature thereto；KLH/MBS mixed solutions cross PD-
10 desalting columns, 0.05mol/L phosphate buffers (pH 6.0) elution, collect the KLH/MBS for obtaining 3.5mL purifying；Addition
0.5mL deionized waters, 5mg CRHN synthetic peptides are dissolved in 100 μ L DMF, are rapidly added the KLH/MBS of 1mL purifying, quick concussion is simultaneously
Immediately with 2N NaOH regulation pH to 7.0-7.2, by the mixed solution, 4 DEG C stand overnight；Finally add 3mL 0.1mol/L
NH4HCO3, it is freeze-dried and obtains KLH-CRHN coupling peptides, the as immunogene for preparing antibody.
2nd, animal immune and antiserum titre monitoring
Experimental animal：6 week old female Balb/c mouse are purchased from Beijing magnificent experimental animal company of dimension tonneau, immunologic adjuvant
Quick Antibody are purchased from Beijing Kang Bi springs Bioisystech Co., Ltd, and murine myeloma cell sp2/0 is preserved by our unit.
2.1 mouse immune：Take 50 μ gKLH-CRHN to be coupled peptide (the μ L PBS of in 250), blown and beaten with isometric immunologic adjuvant
After mixing, 100 every mouse of μ L, leg muscle multi-point injection；It was immunized once every 10 days.
2.2 bioactivity：Respectively before immune, it is immune every time after take within the 7th day blood, monitor antiserum titre.Step includes：
(1) it is coated with：KLH-CRHN is coupled peptide (or KLH) with 0.05mol/L carbonate buffer solutions (pH9.0) and is diluted to 10
μ g/mL, are added in micro- 96 orifice plate of polystyrene, and per the μ L of hole 100,4 DEG C of coatings are stayed overnight, discard liquid in hole, use washing buffer
Liquid PBST (0.02mol/L PBS include 0.05%Tween-20) is washed 3 times, every time 3 minutes；
(2) close：300 μ L BSA confining liquids are added per hole, 37 DEG C of closing 2h discard liquid, and use lavation buffer solution
PBST (0.02mol/L PBS include 0.05%Tween-20) is washed 3 times, every time 3 minutes；
(3) serum sample of potency to be measured is added to above-mentioned enzyme with 100 μ L with after BSA confining liquid doubling dilutions per hole
In target, each extension rate does three multiple holes, 37 DEG C of incubation 1h, discards liquid and with lavation buffer solution PBST (0.02mol/L
PBS includes 0.05%Tween-20) wash 3 times, every time 3 minutes；
(4) it is every with 100 μ L with enzyme labelled antibody dilution (PBS+1%BSA) by after 5000 times of sheep anti-mouse igg/HRP dilution
Hole is added in above-mentioned ELISA Plate, 37 DEG C of incubation 0.5h, discards liquid and with lavation buffer solution PBST (in 0.02mol/L PBS
Containing 0.05%Tween-20) wash 3 times, every time 3 minutes；
(5) added per hole after 100 μ L tmb substrate nitrite ions, room temperature lucifuge colour developing 10min, 50 μ L terminate liquids are added per hole
(2mol/L H2SO4) terminating reaction, surveys 450nm absorption values, using mice serum before immune as negative control, with measured value/right
Judge immune serum potency according to value >=2.1 are the positive；
Each blood sampling time point, each group mouse carries out interior eye socket blood sampling, passes through above-mentioned ELISA experiment detection mice serums
Potency, after four times are immune, by potency highest, and potency>105 immune mouse arrange cell fusion experiment.
3rd, cell fusion, cell line build strain and Antibody preparation
3.1 Preparatory work of experiment：The previous day is merged, normal mouse one is put to death, goes mouse peritoneal liquid to prepare Mouse feeder thin
Born of the same parents；On the fusion same day, potency is taken to reach (or more than) 105Mouse, after execution, take out mouse spleen under aseptic condition, pulverize simultaneously
Through sterile 200 mesh sieve net filtration, it is placed in sterile saline and carries out cell count after piping and druming washing；Myeloma cell SP2/0
Cell RPMI-1640 medium cultures, wash and count with splenocyte step；
3.2 cell fusion：Ready SP2/0 and splenocyte are pressed 1:10 ratios are sufficiently mixed, and are added in 1min advance
Fusion agent (sterile PEG-4000,50%) 1mL of 37 DEG C of water-bath insulations is placed on, 37 DEG C stand after 1min, in edge pipe in 2min
Wall is slowly added to the endless full nutrient solutions of 5mL RPMI-1640 (37 DEG C of pre-temperatures), while gently rotating centrifuge tube；Then preheating is added
RPMI-1640 liquid terminate and merge to 20mL, with HAT nutrient solutions sedimentation cell is resuspended and by cell point in 800rpm centrifugation 10min
Plant in 96 porocyte culture plates cultures, observe daily and record cell growth state.Measured with HAT nutrient solutions half within the 3rd day after fusion
Liquid is changed, is measured with HT nutrient solutions half within the 7th day, the 10th day and changes liquid, using complete culture solution instead afterwards, (RPMI-1640 includes 15% tire ox
Serum) change liquid culture.
3.3 positive hybridoma cell strains build strain：Reference《The philosophy and technique of in vitro culture》, select within the 10-14 days after fusion
Selecting has cell mass culture supernatant in culture plate carries out ELISA detections, while being coated with KLH is used as negative control.Take the thin of the positive
Cell in hilum, expands behind culture to 48 holes, and potency is detected again, and selection continues as the cell limiting dilution Asia gram of the positive
Grand method is cultivated, and it is still positive cell to select after four subclones, and the cell line is preserved to China General Microbiological bacterium
Preservation administrative center is planted, deposit number is CCTCC NO.C2015216；And with freeze-stored cell after its expansion culture and prepare abdomen
Water.
3.4 prepare ascites：The female Balb/c mouse of 8 week old are taken, 0.5mL atoleines are injected intraperitoneally, after one week, are collected
Expand the hybridoma of culture, every mouse injects 2 × 105 cells, and mouse starts to produce ascites successively from the 6th day, receives
Collect mouse ascites 10,000rpm centrifugation 15min, ascites is diluted and micro- through 0.45 μm with isometric phosphate buffer (pH 7.4)
Hole membrane filtration, in 4 DEG C of standby or -20 DEG C of preservations.
The purifying of 3.5 monoclonal antibodies：Protein G are affine, and filler is purchased from Pharmacia Corp of the U.S., pure with 2mL fillers
Change the ascites of the above-mentioned preservations of 10mL, the antibody of elution determines monoclonal after sephadex G-25 desalting processings with SDS-PAGE
The purity of antibody, N-D19 antibody purities reach more than 90%.N-D19 is shown in Fig. 1 through protein G affinity purification electrophoretograms.
4th, monoclonal antibody N-D19 CHARACTERISTICS IDENTIFICATION
4.1 concentration mensuration：Obtained antibody concentration, N- are purified using Pierce companies BSA protein quantifications kit measurement
D19 is 2.33mg/mL.
4.2 subgroup identification：Using Beijing Wu Kang emerging technologies Co., Ltd Subclass of antibody Rapid identification test paper identification from
The N-D19 monoclonal antibodies of ascites purifying, N-D19 antibody is IgG2a types, and light chain is κ types.
4.3 potency assessments：Surveyed using ELISA method:The potency of monoclonal antibody.First respectively will with coating buffer solution
KLH-CRHN is coupled peptide and is diluted to 10 μ g/mL with KLH, with 100 μ L/ holes coated elisa plates, and 37 DEG C are coated with after 2h, are closed with BSA
After fluid-tight is closed, the N-D19 antibody of doubling dilution is added to each hole, 37 DEG C of incubation 1h, board-washing adds 1:The sheep anti mouse of 5000 dilutions
IgG/HRP, continues to be incubated 30min, abundant board-washing adds TMB colour reagents lucifuge colour developing 10min, with 2mol/L H2SO4 ends
Only chromogenic reaction, reads 450nm light absorption value.As a result Fig. 2 is seen.Fig. 2 results show that the potency of N-D19 antibody can reach 1：4×
106。
4.4 specificity and cross reactivity identification：Using indirect ELISA method detect N-D19 antibody specificity and with
The cross reactivity of KLH, KLH coupling peptide, CRH, interstitialcellstimulating hormone (ICSH) (LH) etc., ELISA results are shown in Fig. 3.As a result show, N-
D19 to being not bound with other albumen in addition to CRH except KLH-CRHN, and N-D19 to KLH-CRHN binding ability with it is right
CRH binding ability does not have notable difference.
The monoclonal antibody C-D17 of the present invention of embodiment 2 preparation and purification.
1st, immunogene is prepared
1.1 prepare CRHC synthetic peptides：CRH C-terminal epitope is synthesized, sequence is SEQ ID NO:Sequence shown in 2, purity is big
In 99%.
1.2KLH-CRHC is coupled the acquisition of peptide：Weigh the m- maleimidobenzoyl-N-hydroxy-succinamide esters of 3mg
(MBS) 200 μ L dimethylformamides (DMF) are dissolved in, the phosphate for taking 1.5mg KLH to be dissolved in 0.5ml 0.05mol/L delays
Fliud flushing (pH 7.0), adds the μ L of MBS solution 70 configured, 30min is stirred at room temperature thereto；KLH/MBS mixed solutions cross PD-
10 desalting columns, 0.05mol/L phosphate buffers (pH 6.0) elution, collect the KLH/MBS for obtaining 3.5mL purifying；Addition
0.5mL deionized waters, 5mg CRHC synthetic peptides are dissolved in 100 μ L DMF, are rapidly added the KLH/MBS of 1mL purifying, quick concussion is simultaneously
Immediately with 2N NaOH regulation pH to 7.0-7.2, by the mixed solution, 4 DEG C stand overnight；Finally add 3mL 0.1mol/L
NH4HCO3, it is freeze-dried and obtains KLH-CRHC coupling peptides, the as immunogene for preparing antibody.
2nd, animal immune and antiserum titre monitoring
Experimental animal：6 week old female Balb/c mouse are purchased from Beijing magnificent experimental animal company of dimension tonneau, immunologic adjuvant
Quick Antibody are purchased from Beijing Kang Bi springs Bioisystech Co., Ltd, and murine myeloma cell sp2/0 is preserved by our unit.
2.1 mouse immune：Take 50 μ gKLH-CRHC to be coupled peptide (the μ L PBS of in 250), blown and beaten with isometric immunologic adjuvant
After mixing, 100 every mouse of μ L, leg muscle multi-point injection；It was immunized once every 10 days.
2.2 bioactivity：Respectively before immune, it is immune every time after take within the 7th day blood, monitor antiserum titre.Step includes：
(1) it is coated with：KLH-CRHC is coupled peptide (or KLH) with 0.05mol/L carbonate buffer solutions (pH9.0) and is diluted to 10
μ g/mL, are added in micro- 96 orifice plate of polystyrene, and per the μ L of hole 100,4 DEG C of coatings are stayed overnight, discard liquid in hole, use washing buffer
Liquid PBST (0.02mol/L PBS include 0.05%Tween-20) is washed 3 times, every time 3 minutes；
(2) close：300 μ L BSA confining liquids are added per hole, 37 DEG C of closing 2h discard liquid, and use lavation buffer solution
PBST (0.02mol/L PBS include 0.05%Tween-20) is washed 3 times, every time 3 minutes；
(3) serum sample of potency to be measured is added to above-mentioned enzyme with 100 μ L with after BSA confining liquid doubling dilutions per hole
In target, each extension rate does three multiple holes, 37 DEG C of incubation 1h, discards liquid and with lavation buffer solution PBST (0.02mol/L
PBS includes 0.05%Tween-20) wash 3 times, every time 3 minutes；
(4) it is every with 100 μ L with enzyme labelled antibody dilution (PBS+1%BSA) by after 5000 times of sheep anti-mouse igg/HRP dilution
Hole is added in above-mentioned ELISA Plate, 37 DEG C of incubation 0.5h, discards liquid and with lavation buffer solution PBST (in 0.02mol/L PBS
Containing 0.05%Tween-20) wash 3 times, every time 3 minutes；
(5) added per hole after 100 μ L tmb substrate nitrite ions, room temperature lucifuge colour developing 10min, 50 μ L terminate liquids are added per hole
(2mol/L H2SO4) terminating reaction, surveys 450nm absorption values, using mice serum before immune as negative control, with measured value/right
Judge immune serum potency according to value >=2.1 are the positive；
Each blood sampling time point, each group mouse carries out interior eye socket blood sampling, passes through above-mentioned ELISA experiment detection mice serums
Potency, after four times are immune, by potency highest, and potency>105Immune mouse arranges cell fusion experiment.
3rd, cell fusion, cell line build strain and Antibody preparation
3.1 Preparatory work of experiment：The previous day is merged, normal mouse one is put to death, goes mouse peritoneal liquid to prepare Mouse feeder thin
Born of the same parents；On the fusion same day, potency is taken to reach (or more than) 105Mouse, after execution, take out mouse spleen under aseptic condition, pulverize simultaneously
Through sterile 200 mesh sieve net filtration, it is placed in sterile saline and carries out cell count after piping and druming washing；Myeloma cell SP2/0
Cell RPMI-1640 medium cultures, wash and count with splenocyte step；
3.2 cell fusion：Ready SP2/0 and splenocyte are pressed 1:10 ratios are sufficiently mixed, and are added in 1min advance
Fusion agent (sterile PEG-4000,50%) 1mL of 37 DEG C of water-bath insulations is placed on, 37 DEG C stand after 1min, in edge pipe in 2min
Wall is slowly added to the endless full nutrient solutions of 5mL RPMI-1640 (37 DEG C of pre-temperatures), while gently rotating centrifuge tube；Then preheating is added
RPMI-1640 liquid terminate and merge to 20mL, with HAT nutrient solutions sedimentation cell is resuspended and by cell point in 800rpm centrifugation 10min
Plant in 96 porocyte culture plates cultures, observe daily and record cell growth state.Measured with HAT nutrient solutions half within the 3rd day after fusion
Liquid is changed, is measured with HT nutrient solutions half within the 7th day, the 10th day and changes liquid, using complete culture solution instead afterwards, (RPMI-1640 includes 15% tire ox
Serum) change liquid culture.
3.3 positive hybridoma cell strains build strain：Reference《The philosophy and technique of in vitro culture》, select within the 10-14 days after fusion
Selecting has cell mass culture supernatant in culture plate carries out ELISA detections, while being coated with KLH is used as negative control.Take the thin of the positive
Cell in hilum, expands behind culture to 48 holes, and potency is detected again, and selection continues as the cell limiting dilution Asia gram of the positive
Grand method is cultivated, and it is still positive cell to select after four subclones, and the cell line is preserved to China General Microbiological bacterium
Preservation administrative center is planted, deposit number is CCTCC NO.C2015215；And with freeze-stored cell after its expansion culture and prepare abdomen
Water.
3.4 prepare ascites：The female Balb/c mouse of 8 week old are taken, 0.5mL atoleines are injected intraperitoneally, after one week, are collected
Expand the hybridoma of culture, every mouse injects 2 × 105 cells, and mouse starts to produce ascites successively from the 6th day, receives
Collect mouse ascites 10,000rpm centrifugation 15min, ascites is diluted and micro- through 0.45 μm with isometric phosphate buffer (pH 7.4)
Hole membrane filtration, in 4 DEG C of standby or -20 DEG C of preservations.
The purifying of 3.5 monoclonal antibodies：Protein G are affine, and filler is purchased from Pharmacia Corp of the U.S., pure with 2mL fillers
Change the ascites of the above-mentioned preservations of 10mL, the antibody of elution determines monoclonal after sephadex G-25 desalting processings with SDS-PAGE
The purity of antibody, C-D17 antibody purities reach more than 90%.C-D17 is shown in Fig. 4 through protein G affinity purification rear electrophoresis figures.
4th, monoclonal antibody C-D17 CHARACTERISTICS IDENTIFICATION
4.1 concentration mensuration：Obtained antibody concentration, C- are purified using Pierce companies BSA protein quantifications kit measurement
D17 is 1.72mg/mL.
4.2 subgroup identification：Using Beijing Wu Kang emerging technologies Co., Ltd Subclass of antibody Rapid identification test paper identification from
The C-D17 monoclonal antibodies of ascites purifying, C-D17 antibody is IgG1 types, and light chain is κ types.
4.3 potency assessments：Surveyed using ELISA method:The potency of monoclonal antibody.First respectively will with coating buffer solution
KLH-CRHC is coupled peptide and is diluted to 10 μ g/mL with KLH, with 100 μ L/ holes coated elisa plates, and 37 DEG C are coated with after 2h, are closed with BSA
After fluid-tight is closed, the N-D19 antibody of doubling dilution is added to each hole, 37 DEG C of incubation 1h, board-washing adds 1:The sheep anti mouse of 5000 dilutions
IgG/HRP, continues to be incubated 30min, abundant board-washing adds TMB colour reagents lucifuge colour developing 10min, with 2mol/L H2SO4 ends
Only chromogenic reaction, reads 450nm light absorption value.As a result Fig. 5 is seen.Fig. 5 results show that the potency of C-D179 antibody can reach 1：4
×106。
4.4 specificity and cross reactivity identification：Using indirect ELISA method detect C-D17 antibody specificity and with
The cross reactivity of KLH, KLH coupling peptide, CRH, interstitialcellstimulating hormone (ICSH) (LH) etc., the antibody conduct of non-immune serum purifying
Compare (Con), ELISA results are shown in Fig. 6.As a result show, C-D17 with other albumen in addition to CRH to not tying except KLH-CRHC
Close, and binding abilities of the C-D17 to KLH-CRHC does not have notable difference with the binding ability to CRH.
The composition and best effort Concentration Testing of the present invention detection of embodiment 3 CRH double-antibody sandwich elisa kit
1st, anti-human CRH the monoclonal antibodies N-D19 or C-D17 of HRP marks are prepared
HRP labeled monoclonal antibodies N-D19 or C-D17 method is：10mg HRP dry powder is weighed first, in the double steamings of 5mL
Fully dissolved in water, add the 0.2mol/L NaIO of Fresh4Solution 0.5mL, lucifuge is gently shaken on 4 DEG C of horizontal shakers
Dynamic 30min, addition has been dialysed to monoclonal antibody C-D17 or N-D19 the antibody 10mg of carbonate buffer solution, after mixing, will
Solution is fitted into the bag filter anticipated that molecular cut off is 50kDa, by its pH9.6 to 0.05mol/L carbon
4 DEG C of dialysis of phthalate buffer, a dialyzate is changed per 6h, is dialysed four times；After dialysis terminates, liquid in bag filter is taken out, to
Wherein add the NaBH that concentration is 5mg/mL4100 μ L, 4 DEG C of standing 4h；Isometric saturated ammonium sulfate solution is added, 4 DEG C put
Put overnight；5000rpm centrifuges 10min, after being resuspended with 1mL0.05mmol/L PBS (pH7.4), to 0.05mmol/L PBS
The 4 DEG C of dialysis of (pH 7.4) buffer solution, a dialyzate is changed per 6h, is dialysed four times；The solution after dialysis is taken out, is added thereto
Isometric glycerine is added in marking fluid, purified HRP is produced and marks to obtain monoclonal antibody N-D19 or C-D17.
2nd, the mark checking of enzyme labelled antibody
N-D19/HRP and C-D17/HRP mark effect is detected using ELISA method.By C-D17 (or the N- after dilution
D19) (1 μ g/mL) is coated on ELISA Plate (4 DEG C overnight), with 37 DEG C of closing 2h of confining liquid, sequentially adds the CRH of doubling dilution
Antigen, N-D19/HRP (C-D17/HRP), continue to be incubated after 1h, abundant board-washing, are reacted through TMB colour developings and terminate liquid color development stopping
Afterwards, OD450 (being shown in Table 1) is read.C-D17 antibody is coated with, and N-D19 is best of breed as detection antibody, to reduce the shadow of background
Ring, detection antibody concentration is：1:10000.
Table 1CRH double crush syndromes detect (OD450)
3rd, kit test limit and detection range
Detection sensitivity 15.6pg/ml, 15.6~1000pg/ml of optimum detection scope.
4th, kit forms and detection method
Kit mainly includes：Pre-coated C-D19 ELISA Plate, PBST lavation buffer solutions, N-D19/HRP detections antibody,
Enzyme labelled antibody dilution, TMB nitrite ions, terminate liquid.
The application method of kit of the present invention, comprises the following steps：
The serum that (1) twice of dilution is collected by centrifugation, adds pre-coated ELISA Plate, 37 DEG C of placement 2h；
(2) PBST is washed 3 times；
(3) with enzyme labelled antibody dilution (PBS+1%BSA) by after 10000 times of HRP labelled antibodies N-D19 dilution, with 100
μ L are added in above-mentioned ELISA Plate per hole, 37 DEG C of incubation 1h, are discarded liquid and are washed with lavation buffer solution PBST 3 times, every time 3 points
Clock；
(4) added per hole after 100 μ L tmb substrate nitrite ions, room temperature lucifuge colour developing 10min, 50 μ L terminate liquids are added per hole
(2mol/L H2SO4) terminating reaction, surveys 450nm absorption values.
The detection human serum sample 1 of the present invention detection of embodiment 4 CRH double-antibody sandwich elisa kit, blood serum sample
Collect
10 patients with acute high altitude disease serum samples are collected, 10 normal controls are collected in addition.
2nd, standard curve is drawn
Using OD450 as ordinate, with the CRH concentration (pg/ml) of doubling dilution for abscissa, standard curve is drawn, and build
Vertical regression equation.Paint standard curve as shown in fig. 7, its regression equation is：Y=0.001x+0.427, wherein R2=0.993 are accorded with
The criterion of zygonema sexual intercourse.
3rd, the detection method according to embodiment 3 is detected to blood serum sample
According to standard curve, CRH contents in each blood serum sample are read, as a result as shown in table 2：
| Plateau patient | CRH contents (pg/ml) | Healthy People | CRH contents (pg/ml) |
| 1 | 87.2 | 1 | 55.9 |
| 2 | 91.5 | 2 | 56.3 |
| 3 | 99.4 | 3 | 47.8 |
| 4 | 81.8 | 4 | 53.5 |
| 5 | 80.7 | 5 | 55.5 |
| 6 | 96.9 | 6 | 61.3 |
| 7 | 92.6 | 7 | 49.8 |
| 8 | 84.7 | 8 | 54.3 |
| 9 | 82.3 | 9 | 63.4 |
| 10 | 78.7 | 10 | 52.7 |
| Average value | 87.58 | Average value | 55.05 |
| Standard deviation | 7.17 | Standard deviation | 4.72 |
As a result show：CRH contents average content is 55.05pg/ml in Healthy Human Serum, CRH contents during plateau patient is clear
Average content is that CRH contents are higher than CRH contents in Healthy Human Serum in 87.58pg/ml, patients serum.It is soft using SPSS13.0
Part is analyzed, and difference has statistical significance (P<0.01).It can be seen that：The kit provided using the present invention, is existed according to the CRH measured
Content can accurately judge whether patient has altitude sickness in serum.And the present embodiment is used as 10 plateau sufferers of typical case's citing
The content of CRH albumen is all higher than CRH contents in the serum of 10 Healthy Peoples in the serum of person.Show the method tool that the present invention is provided
There is good accuracy, can be completely the same with expected results, accuracy rate is up to 100%.
Embodiments of the invention are described in detail above, but the content is only presently preferred embodiments of the present invention,
It is not to be regarded as the practical range for limiting the present invention.All equivalent changes made according to the scope of the invention and improvement etc., all should
Still belong within this patent covering scope.
Claims (10)
1. a kind of hybridoma cell strain, was preserved in China typical culture collection center, Classification And Nomenclature is on April 13rd, 2016
Hybridoma, deposit number is CCTCC NO.C2015216.
2. a kind of monoclonal antibody N-D19 of anti-Adrenergic beta-antagonists (CRH), it is characterised in that：Will by right
Hybridoma cell strain described in 1 is asked to produce.
3. monoclonal antibody N-D19 according to claim 2, it is characterised in that：The monoclonal antibody N-D19 can be recognized
CRH N-terminal epitope (CRHN), CRH N-terminal epitope sequences are SEQ ID NO:Sequence shown in 1.
4. monoclonal antibody N-D19 preparation method described in claim 2, it is characterised in that comprise the following steps：
S01：CRHN albumen and KLH are coupled, KLH-CRHN coupling peptides is formed, is used as immunogene；
S02：Mixed with KLH-CRHN coupling peptides with immunologic adjuvant, after immune balb/c mice, take mouse boosting cell；
S03：Mouse boosting cell is merged with SP2/0 murine myeloma cells, the hybridoma is obtained, induces in vivo or body
Outer culture, it is purified, produce.
5. anti-CRH according to claim 2 monoclonal antibody N-D19 application, it is characterised in that：Examined applied to preparing
Detection instrument in terms of survey CRH or the application in terms of preparing detection and CRH and having the reagent of related disorders.
6. monoclonal antibody N-D19 according to claim 3 application, it is characterised in that：It is described to have related disorders with people CRH
For acute high altitude sickness.
7. a kind of detection CRH double-antibody sandwich elisa kit, it is characterised in that：Including the monoclonal described in claim 3
Antibody N-D19；Also include monoclonal antibody C-D17；
The monoclonal antibody C-D17 is that the hybridoma cell strain that deposit number is CCTCC NO.C2015215 is produced, and can be recognized
CRH C-terminal epitope, CRH C-terminal epitope sequences are SEQ ID NO:Sequence shown in 2.
8. kit according to claim 7, it is characterised in that：The kit includes pre-coated monoclonal antibody N-
The monoclonal antibody C-D17 of D19 ELISA Plate and HRP mark；Or pre-coated monoclonal antibody C-D17 ELISA Plate and HRP mark
The monoclonal antibody N-D19 of note.
9. kit according to claim 8, it is characterised in that：The kit also includes CRH standard items；Standard items or
Sample diluting liquid：PH is 7.4 PBS；Enzyme labelled antibody dilution：PBS and mass fraction are 1%BSA；Cleaning solution：
0.02mol/L PBS and mass fraction are 0.05% polysorbas20；Nitrite ion：TMB nitrite ions；Terminate liquid：2mol/L H2SO4.
10. the application method of any one of the claim 7-9 kits, it is characterised in that comprise the following steps：To pre-coated
ELISA Plate in add testing sample, HRP mark monoclonal antibody C-D17 or N-D19, nitrite ion colour developing, terminate liquid terminate,
Detect OD450。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108267577A (en) * | 2018-01-15 | 2018-07-10 | 广州市妇女儿童医疗中心 | A kind of EV71 viruses IgA antibody test strip |
| JP2021527081A (en) * | 2018-06-11 | 2021-10-11 | ユニバーシティ オブ フロリダ リサーチ ファンデーション インコーポレーティッド | Materials and methods for treating stress-related disorders and cancer |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1157620A (en) * | 1994-07-15 | 1997-08-20 | 纽罗克里恩生物科学有限公司 | Corticotropin-releasing factor-binding-protein inhibitors and their use |
-
2016
- 2016-12-07 CN CN201611112449.2A patent/CN107043752A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1157620A (en) * | 1994-07-15 | 1997-08-20 | 纽罗克里恩生物科学有限公司 | Corticotropin-releasing factor-binding-protein inhibitors and their use |
Non-Patent Citations (6)
| Title |
|---|
| HAGAN P, ET AL.: ""Development of a two-site immunoradiometric assay Development of a two-site immunoradiometric assay"", 《JOURNAL OF IMMUNOLOGICAL METHODS》 * |
| ZYH4085: ""CRF elisa试剂盒"", 《豆丁网,HTTPS://WWW.DOCIN.COM/P-1612542492.HTML》 * |
| 刘新民 等: "《临床药物学》", 31 January 2008, 北京：军事医学科学出版社 * |
| 孔繁平: ""神经肽CRH作为急性高原低氧应激损伤的生物标志分子研究"", 《中国博士学位论文全文数据库》 * |
| 柳钟辉 等: ""抗CRF单克隆抗体制备及鉴定"", 《白求恩医科大学学报》 * |
| 段小莉 等: ""CRF单克隆抗体和多克隆抗体制备及其在睡眠剥夺模型大鼠脑内的变化"", 《细胞与分子免疫学杂质》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108267577A (en) * | 2018-01-15 | 2018-07-10 | 广州市妇女儿童医疗中心 | A kind of EV71 viruses IgA antibody test strip |
| JP2021527081A (en) * | 2018-06-11 | 2021-10-11 | ユニバーシティ オブ フロリダ リサーチ ファンデーション インコーポレーティッド | Materials and methods for treating stress-related disorders and cancer |
| JP7405434B2 (en) | 2018-06-11 | 2023-12-26 | ユニバーシティ オブ フロリダ リサーチ ファンデーション インコーポレーティッド | Materials and methods for treating stress-related disorders and cancer |
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