CN107991485A - For detecting the kit of soluble ST2 albumen - Google Patents
For detecting the kit of soluble ST2 albumen Download PDFInfo
- Publication number
- CN107991485A CN107991485A CN201711318542.3A CN201711318542A CN107991485A CN 107991485 A CN107991485 A CN 107991485A CN 201711318542 A CN201711318542 A CN 201711318542A CN 107991485 A CN107991485 A CN 107991485A
- Authority
- CN
- China
- Prior art keywords
- cgmcc
- hybridoma
- protein
- deposited
- kit
- 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.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a kind of kit for being used to detect soluble ST2 albumen.In particular it relates to soluble ST2 protein detection kits include coupling and have the magnetic bead of the first anti-ST2 protein monoclonal antibodies and the enzyme working solution of the second anti-ST2 protein monoclonal antibodies comprising labeled substance markers.On the other hand, hybridoma that the preserving number that the hybridoma and on October 23rd, 2017 that the preserving number for being deposited in CGMCC the present invention relates on October 23rd, 2017 is CGMCC No.14727 are deposited in CGMCC is CGMCC No.14728, the anti-ST2 protein monoclonal antibodies produced by the hybridoma and the anti-ST2 protein monoclonal antibodies are preparing the purposes in being used to detect the kit of soluble ST2 albumen.
Description
Technical field
The invention belongs to molecular immunology field, and in particular to a kind of Soluble growth stimulates the expression factor 2 (ST2) albumen
Protein monoclonal antibody of detection kit and detection ST2 albumen and application thereof.
Background technology
Heart failure (abbreviation heart failure) is the whole story and severe stage of various heart diseases, and incidence is high, is current important
One of angiocardiopathy.Heart failure is due to cardiac structure or dysfunction causes ventricular filling or penetrates one group that blood ability is damaged
Complex clinical syndrome, its incidence and case fatality rate are high, and 5 annual survival rates are close with malignant tumour.
Aggravated recently as aging of population degree, hypertension, diabetes, incidence of obesity rise year by year and various blood
The increase of pipe hazards, heart failure incidence increasingly rise.Therefore quickly judge patients with heart failure prognosis, take positive pipe in early days
Reason method is of great significance.
ST2 genes found that ST2 albumen is made of 328 amino acid in 1989 first, were Toll/IL-1 (IL-1R) super
One of member of receptor family.ST2 genes are located at No. 2 chromosomes (2ql2), comprising 11 extrons, a proximal promoter and
A Proximal promoter.ST2 albumen mainly has two kinds of primary hypotypes regulated and controled by different promoters, and a kind of soluble protein is (solvable
Property ST2 or sST2) and a kind of cross-film form albumen (ST2L).Soluble ST2 is subject to excessively to pull the mistake being damaged in cardiac muscle
Largely generated in journey, as a kind of Decoy receptor, can by with ST2L competitive binding IL-33, block the signal through ST2L
Transduction, negative regulator is carried out to the effect of IL-33/ST2L signal transduction pathways.Soluble ST2 is a kind of mechanical stress risers egg
In vain, as heart volume load increases at cardiac muscle cell when solubility ST2 concentration lift mechanisms are probably heart failure during heart failure
In extended state, in the expression quantity rise of the cardiac muscle cell and cardiac fibroblast solubility ST2 of machinery and inflammatory stimulus.
Clinically, acute heart failure and Patients with Chronic Heart Failure serum soluble ST2 concentration are significantly raised, and rise
High level is proportionate with the heart failure order of severity.In addition, the horizontal rises of solubility ST2 can be used as heart failure patient badness come-off
The risk prediction factor.Follow-up to patients with heart failure finds that soluble ST2 concentration can be predicted 1 annual death rate of patient and can simultaneously improve
Mortality Prediction efficiency.Existing market main product is the ST2 reaction kits of Critical companies, and total time is 120 minutes；And
It is real for operating personnel and the ST2 kits Main Components of Critical companies need to be diluted or dissolve in advance mostly
Apply relatively complicated, the present invention has that detection speed is fast, easy to operate, high sensitivity, specificity is good, stability is high, can be in batches
The features such as production.
The content of the invention
On the one hand, the present invention provides a kind of solubility ST2 protein detection kits, it includes：
Coupling has the magnetic bead of the first anti-ST2 protein monoclonal antibodies and the second anti-ST2 albumen comprising labeled substance markers
The enzyme working solution of monoclonal antibody, wherein the label be selected from horseradish peroxidase, alkaline phosphatase, radioactive label and
Immunofluorescence label；Wherein described first anti-ST2 protein monoclonal antibodies and the second anti-ST2 protein monoclonal antibodies difference
Selected from the hybridoma for being CGMCC No.14727 by the preserving number for being deposited in CGMCC on October 23rd, 2017 and 2017 10
Monoclonal antibody caused by the hybridoma that months 23 days preserving numbers for being deposited in CGMCC are CGMCC No.14728；Or
The first anti-ST2 protein monoclonal antibodies and the second anti-ST2 protein monoclonal antibodies were respectively selected from by October, 2017
The preserving number for being deposited in CGMCC on the 23rd is the hybridoma of CGMCC No.14728 and is deposited on October 23rd, 2017
The preserving number of CGMCC is monoclonal antibody caused by the hybridoma of CGMCC No.14727.
On the one hand, the present invention provides a kind of hybridoma, it was filed in China Microbiological bacterium on October 23rd, 2017
Kind preservation administration committee's common micro-organisms center (CGMCC), address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China
Institute of microbiology of the academy of sciences, postcode：100101；Preserving number is CGMCC No.14727, and Classification And Nomenclature is ST2 hybridomas
Cell line.
On the other hand, the present invention provides a kind of hybridoma, it was filed in China Microbiological on October 23rd, 2017
Culture presevation administration committee common micro-organisms center (CGMCC), address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in
Institute of microbiology of the academy of sciences of state, postcode：100101；Preserving number is CGMCC No.14728, and Classification And Nomenclature hybridizes for ST2
Tumor cell strain.
Some specific embodiments according to the present invention, there is provided anti-ST2 protein monoclonal antibodies according to the present invention exist
Prepare the purposes in diagnostic reagent.Some specific embodiments according to the present invention, there is provided hybridoma of the invention exists
Prepare the purposes in diagnostic reagent.Two kinds of anti-ST2 protein monoclonal antibodies of the present invention can be individually used for preparing diagnostic reagent,
But it is preferred that two kinds of anti-ST2 protein monoclonal antibodies combinations of the present invention are used to prepare diagnostic reagent.In some specific embodiment parties
In case, the diagnostic reagent is soluble ST2 protein assay reagents.Soluble ST2 protein assay reagents refer to be used for it is quantitative or
The reagent of solubility ST2 albumen in qualitative detection human sample.Soluble ST2 protein assay reagents can detect can in human sample
The presence of dissolubility ST2 albumen and/or content.The diagnostic reagent can be any appropriate form in the prior art, including but
It is not limited to ELISA detection reagents, immunoturbidimetry detection reagent, magnetic particle detections reagent, chemiluminescence detection reagent, radio-immunity
Detection reagent, immunofluorescence detection agent.In fact, anti-the ST2 protein monoclonal antibodies or hybridoma of the present invention can be with
It is used to prepare any detection reagent based on antigen-antibody interaction.In some specific embodiments, the diagnostic reagent
It can be presented as liquid form, dry powder, freeze-dried powder form, particle form, multiwell plate format or other forms well known in the art.
Preferably, enzyme is also included in the enzyme working solution of the soluble ST2 protein detection kits provided according to the present invention
Buffer solution, the enzyme buffer solution include disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, calf serum and Proclin-300.
When produced by the hybridoma that the monoclonal antibody on magnetic bead is CGMCC No.14727 as preserving number, then in enzyme working solution
Monoclonal antibody by preserving number be CGMCC No.14728 another strain of hybridoma produce；Vice versa, when on magnetic bead
Monoclonal antibody be CGMCC No.14728 as preserving number hybridoma produced by when, then the monoclonal in enzyme working solution
Antibody is produced by the hybridoma that preserving number is CGMCC No.14727.
In a preferred embodiment, the mass ratio of the magnetic bead and the first anti-ST2 protein monoclonal antibodies
For 10: 1 to 40: 1, preferably 20:1.
In still another preferred embodiment, the enzyme work of the second anti-ST2 protein monoclonal antibodies comprising mark
Make the final concentration of 1mg/L of the second anti-ST2 protein monoclonal antibodies in liquid.
In some specific embodiments, soluble ST2 protein detection kits of the invention can also include as needed
Sample dilution, enzyme reaction substrate, ST2 albumen calibration object and ST2 albumen quality-control products.Sample dilution includes salt, surface-active
Agent, buffer composition, pH is between 5-8, preferably 6-7.In some specific embodiments, Sample dilution includes NaCl, phosphoric acid hydrogen
Disodium, sodium dihydrogen phosphate, lowlenthal serum, polysorbas20, Proclin-300, pH value 6.2-6.6.In some specific embodiments, sample
This dilution can be configured to concentrated type.The enzyme reaction substrate changes according to the enzyme marked, such as when use horseradish mistake
During oxide enzyme, suitable substrate includes but not limited to o-phenylenediamine (OPD), ABTS, tetramethyl benzidine (TMB) or 4 amino
Antipyrine etc..When using alkaline phosphatase, suitable substrate includes but not limited to BCIP/NBT.
In some specific embodiments, ST2 protein detection kits of the invention include：It is coated with one kind of the present invention
Magnetic bead, enzyme working solution, Sample dilution, enzyme reaction substrate, ST2 albumen calibration object and the ST2 eggs of anti-ST2 protein monoclonal antibodies
White quality-control product, wherein enzyme working solution include another ST2 protein monoclonal antibodies of horseradish peroxidase-labeled.
Preferably, solubility ST2 protein detection kits according to the present invention, wherein the kit further wraps
Containing enzyme reaction substrate, ST2 albumen calibration object and ST2 albumen quality-control products；Wherein
The ST2 albumen calibration object is the serum of known ST2 protein concentrations, and wherein ST2 protein concentrations are respectively 200ng/
Ml, 100ng/ml, 50ng/ml, 20ng/ml, 5ng/ml, 0ng/ml,
The ST2 albumen quality-control product for ST2 protein concentrations 80ng/ml to 120ng/ml and 16ng/ml to 24ng/ml it
Between serum.
In a specific embodiment, the present invention provides a kind of solubility ST2 protein detection kits, it includes：
Coupling has the magnetic bead working solution of the first anti-ST2 protein monoclonal antibodies,
Enzyme working solution,
Calibration object dilution,
Chemical luminescence for liquid A,
Chemical luminescence for liquid B,
Enzyme reaction substrate,
ST2 albumen calibration objects, and
ST2 albumen quality-control products；
Wherein described magnetic bead working solution includes the magnetic microsphere of the pre-coated first anti-ST2 protein monoclonal antibodies, by titration
Concentration, which is used, contains 12 water of disodium hydrogen phosphate：5.8g/L, two water of sodium dihydrogen phosphate：0.59g/L, sodium chloride：9g/L、BSA：10g/
L、Tween-20：1ml/L、Proclin300：1ml/L；
The enzyme working solution include 5.8g/L disodium hydrogen phosphates, 0.59g/L sodium dihydrogen phosphates, 9.0g/L sodium chloride,
200ml/L calf serums, 1.0g/L enzyme stabilizers and 1ml/L Proclin-300；
The calibration object dilution includes 9.0g/L sodium chloride, 12 water of 5.8g/L disodium hydrogen phosphates, 0.59g/L di(2-ethylhexyl)phosphates
Hydrogen sodium, 10g/L bovine serum albumin(BSA)s, 50g/L sucrose, 1ml/L Proclin-300；
The chemical luminescence for liquid A contains luminol and luminescence enhancer, and chemical luminescence for liquid B contains peroxide and luminescence enhancement
Agent；
The enzyme reaction substrate is the substrate of horseradish peroxidase；
The ST2 albumen quality-control product for ST2 protein concentrations 80ng/ml to 120ng/ml and 16ng/ml to 24ng/ml it
Between serum；
Produced by the hybridoma that the first ST2 protein monoclonal antibodies are CGMCC No.14727 as preserving number
When, the 2nd ST2 protein monoclonal antibodies are as produced by preserving number for the hybridoma of CGMCC No.14728.
On the other hand, the present invention provides a kind of anti-ST2 protein monoclonal antibodies, it was deposited in by October 23rd, 2017
The hybridoma that the preserving number of CGMCC is CGMCC No.14727 produces or is deposited in CGMCC's by October 23rd, 2017
The hybridoma that preserving number is CGMCC No.14728 produces.
On the other hand, the present invention provides be CGMCC by the preserving number for being deposited in CGMCC on October 23rd, 2017
The hybridoma of No.14727 and the preserving number for being deposited in CGMCC on October 23rd, 2017 are the hybridization of CGMCC No.14728
The combination of monoclonal antibody caused by oncocyte is preparing the purposes in being used to detect the kit of soluble ST2 albumen.
Preferably, purposes according to the present invention, the kit is selected from ELISA detection kit, immunoturbidimetry is examined
Test agent box, magnetic particle detections kit, chemiluminescence detection kit, immunofluorescence detection agent box and Placenta function
Kit.
On the other hand, the present invention provides a kind of solubility ST2 protein detection kits, it includes：
It is the hybridoma and 2017 of CGMCC No.14727 by the preserving number for being deposited in CGMCC on October 23rd, 2017
On October 23, in, the preserving number for being deposited in CGMCC was monoclonal antibody caused by the hybridoma of CGMCC No.14728.
Preferably, kit according to the present invention is selected from ELISA detection kit, immunoturbidimetry detection kit, magnetic
Grain detection kit, chemiluminescence detection kit, immunofluorescence detection agent box and Placenta function kit.
Brief description of the drawings
The linear standard curve of soluble ST2 protein detection kits alignment product prepared by Fig. 1 present invention.Wherein
Linear curve fit：
Log (Abs)=3.694+1.028Log (Conc)；
Related coefficient=1.00.
Embodiment
In order to make present invention may be readily understood, with reference to specific embodiment, the present invention is further explained.The following provide this
Specific material and its source used in invention embodiment.It is to be understood that what these were merely exemplary,
It is not intended to be limiting of the invention, with the type of following reagent and instrument, model, quality, property or functionally the same or similar material
Material may be incorporated for implementing the present invention.
Embodiment 1：The acquisition of monoclonal antibody
1. animal immune
In order to produce anti-ST2 monoclonal antibodies, 6 week old, the female Balb/c mouse of weight about 20g are chosen.Initial immunity,
Taking ST2 recombinant antigens, (antigen is purchased from Beijing Ze Rui Bioisystech Co., Ltd ST2protein (article No.s：0101512101)20-
50 μ g add the subcutaneous multi-point injection of Split completely, carry out second respectively within the 14th and 28 day and third time immunizing dose is same as above,
Freund Freund's incomplete adjuvant intraperitoneal injection, 3 days booster immunizations before fusion, dosage 20-50 μ g are advisable, and after 3 days, take spleen to merge.
In general, preparing the monoclonal antibody in Muridae source, refer to《Antibody》Described in handbook method (Harlow and Lane,
Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor NY, pp.726,1988) or Kohler and Milstein description prepare hybridoma technology (Nature, 256:
495-497,1975).
2. cell fusion
Prepare feeder layer：A non-immune Balb/c mouse is taken, 6 weeks, mouse is put to death, is immersed in 75% alcohol
Interior, 5min, cuts off skin, exposure peritonaeum, (forbids to puncture with the nutrient solution of asepsis injector injection 6ml precoolings with sterile scissors
Intestinal tube), rinse repeatedly, suction out flushing liquor, flushing liquor is put into 10ml centrifuge tubes, 1200rpm/ separation 6min, with 20% tire ox blood
The nutrient solution of (FCS) is suspended clearly, adjustment cell number to 1 × 105/ ml, adds 96 orifice plates, 100 μ l/ holes, are put into 37 DEG C of CO2 incubators
Culture.
Prepare immune spleen cell：The Balb/c mouse being immunized are taken, are put to death, it is sterile to take spleen, not exclusively cultivated with 10ml
Liquid is washed once, and spleen is ground, and crosses 200 mesh cell sieves, splenocyte is transferred in 10ml centrifuge tubes, 800rpm centrifugation 10min, carefully
Born of the same parents are washed 2 times with 10ml nutrient solutions, and cell count, takes 1 × 108Splenic lymphocytes suspension is spare.
Prepare myeloma cell SP2/0：The growth myeloma cell that takes the logarithm centrifuges, and is washed 2 times with serum-free medium, counts
Number, obtains 5 × 107Cell is spare.
Cell fusion：Myeloma cell and splenocyte are pressed 1：10 ratio mixes, and is used in 50ml centrifuge tubes
The endless full nutrient solution of serum-free is washed 1 time, centrifugation, 1200rpm, 8min；Abandon supernatant, with suction pipe exhaust residual liquid, so as not to influence
Polyethylene glycol (PEG) concentration.Gently attack centrifuge tube bottom, makes cell precipitation slightly loose dynamic.
Add 1ml 45%PEG (molecular weight 4000) solution of 37 DEG C of pre-temperatures, side edged gentle agitation.37 DEG C of water-bath effects
90s.The endless full nutrient solution of 37 DEG C of pre-temperatures is added to be separately added into 1ml, 2ml, 3ml, 4ml, 5ml every 2min to terminate PEG effects
And 6ml.Centrifugation, 800rpm, 6min.Fill with clearly, be resuspended with the selection nutrient solution containing 20% calf serum HAT.By above-mentioned cell, add
Into 96 orifice plates of existing feeder layer, add 100 μ l per hole.Culture plate is put 37 DEG C, is cultivated in 5%CO2 incubators.
3. the screening of hybridoma
5 days after splenocyte and myeloma cell's fusion, the mixture of various kinds of cell is formed, adds HAT culture medium 100ul,
Change HT medium cultures within 10th day.When hybridoma is covered with 1/5 area of bottom hole, you can detected and cultivated using indirect elisa method
Supernatant, screening positive clone.With 100 μ l/ holes of ST2 recombinant antigens coated elisa plate (5 μ g/ml), 4 DEG C overnight, by ELISA Plate hole
In liquid to the greatest extent, add PBST, repeated washing is three times；Add confining liquid 200 μ l/ holes to be closed, be placed in 37 DEG C, 1 it is small when.
Confining liquid is washed, adds 100 μ l cells and supernatants, positive control selects the immune serum of mouse, and negative control selects SP2/0 to cultivate
Supernatant, blank are cleaning solution, and 2h is stood in 37 DEG C.Add HRP mark sheep anti mouses secondary antibody (1:5000) 100 μ l/ holes are quiet in 37 DEG C
Put 60min.PBST is added, repeated washing is three times；100 μ l/ holes of substrate reactions liquid are added, dark place is put for 37 DEG C and reacts 10 minutes.Add
Enter H2SO4(2mol/L) 50 μ l/ holes, terminate reaction.Microplate reader detects 450nm absorbances.As a result it is immunized with ST2 recombinant antigens
Mouse, successfully obtains the hybridoma cell strain of secretion ST2 monoclonal antibodies.
4. the cloning of hybridoma
Obtained positive colony is screened, using limiting dilution assay to hybridoma clone, feeder cells are prepared within 1 day before clone
Layer, the hybridoma that will be cloned cannot be used up full culture medium and are gently dried up out of culture hole, count.It is thin for 5 to adjust cell
Born of the same parents/ml.The Tissue Culture Plate of the feeder layer of preparation is taken, 100 μ l of diluting cells are added per hole.It is incubated in 37 DEG C, 5%CO2
In incubator.Liquid was changed at the 7th day, changes liquid 1 time within every 3 days later.9 days visible cell Clone formations, ELISA method detection detection antibody effect
Valency.And by two plants of most strong positive colony clonings again, until cell positive rate is up to 100%, you can singling；The cell of singling
Expand culture.
5. the preservation of hybridoma
The hybridoma that both monoclonal antibodies can be secreted and on October 23rd, 2017 are deposited in the micro- life of China
Thing culture presevation administration committee common micro-organisms center (CGMCC), preserving number are respectively CGMCC No.14727 and CGMCC
No.14728。
The preparation of 2. monoclonal antibody of embodiment
By two strain of hybridoma of preservation respectively with 1 × 107Cell concentration be injected in BALB/c mouse abdominal cavity, 10
Ascites is collected after it respectively.Purify two plants of monoclonal antibodies respectively by following steps：
1) ascites for collecting gained is centrifuged with 2500rpm, takes supernatant.Add isometric PBS (pH7.4) and 1/2 volume
Saturated ammonium sulfate, 4 DEG C, standing 30min；
2) with 3000rpm, 4 DEG C, 20min is centrifuged；
3) go to precipitate, above reset and add isometric saturated ammonium sulfate, stand 30min；
4) with 3000rpm, 4 DEG C, 20min is centrifuged；
5) precipitation is taken, adds 5ml physiological saline to stand 30min with 5ml saturated ammonium sulfates；
6) with 3000rpm, 4 DEG C, 20min is centrifuged；
7) PBS (PH7.4) of precipitation plus 5ml physiological saline and 5ml 0.02M；
8) the PB buffer solutions balance Protein G gel columns (being purchased from GE) of 8 times of column volumes are added；
9) mixed liquor in the 7th step is added in gel column；
10) using the PB buffer solutions elution foreign protein of 10 times of column volumes；
11) with 0.2M pH2.8 glycine buffers antibody elutions and collect；
12) using regenerated liquid cleaning pillar；
13) plus equilibrium liquid (is recommended) balance pillar by GE.
The preparation of 3. detection reagent of embodiment
1. preparing coupling has the magnetic bead of ST2 first antibodies：
Magnetic bead coupled antibody：Take out and be put into bottle after magnetic bead is shaken up, clean magnetic bead with 0.05MMES, added after cleaning
EDC room temperatures activate 30 minutes.Distinguished according to the amount of magnetic bead according to the ratio of magnetic bead and antibody (mass ratio) 10: 1,20: 1 and 40: 1
Add the coupling of ST2 first antibodies (preserving number is CGMCC No.14727) centrifuge tube room temperature overnight.Magneto separate suctions out reaction solution point
Not Jia Ru PBST clean 2 times, Magneto separate suction out washing lotion, add magnetic bead dilution and magnetic bead working solution (storage liquid) be made, it is spare；
0.05M MES standard recipes：
MES 10.67g
Purified water is settled to 1000ml.
Magnetic bead dilution：
12 water of disodium hydrogen phosphate：5.8g
Two water of sodium dihydrogen phosphate：0.59g
Sodium chloride：9g
BSA：10g
Tween-20：1ml
Proclin300：1ml
Purified water is settled to 1000ml.
Cleaning solution：
12 water of disodium hydrogen phosphate：5.8g
Two water of sodium dihydrogen phosphate：0.59g
Sodium chloride：9g
Tween-20：0.5ml
Purified water is settled to 1000ml.
2. the preparation of Horseradish Peroxidase Conjugates：
(1) 1mg horseradish peroxidases (HRP, purchased from Sigma) are weighed to be dissolved in 300 μ l distilled water.
(2) the 0.1M NaIO4 solution that 0.6 μ l newly match somebody with somebody is added in upper liquid, lucifuge stirs 20 minutes at room temperature.
(3) above-mentioned solution is fitted into bag filter, is dialysed using the sodium-acetate buffer of 1mM PH4.4,4 DEG C overnight.
(4) add 20 μ l 1M PH9.5 carbonate buffer solutions, the PH of HRP is increased to 9.0~9.5, add immediately after
Another strain antibody (CGMCC No.14728) (in 1ml0.01M carbonate buffer solutions) of the 5mg present invention, room temperature lucifuge is gently
Stir 2 it is small when.
(5) the 4mg/ml NaBH4 liquid for adding 0.05ml newly to match somebody with somebody, when mixing, then putting 4 DEG C 2 small.
(6) above-mentioned liquid is fitted into bag filter, dialysed to 0.15M PH7.4PBS, 4 DEG C overnight.
(7) isometric glycerine is added, -20 DEG C of preservations, final horseradish peroxidase labeling antibody concentration is 1mg/ml.
3. the working concentration of optimal coated antibody and enzymic-labelled antibody is determined using chessboard method (matrix method).Use step 1
The middle magnetic bead being coupled and coating magnetic bead of the antibody ratios for 10: 1,20: 1 and 40: 1, the enzyme labelled antibody (working concentration of step 2
Respectively：1:250、1:500 and 1:1000 dilutions) tested.It is 20 to determine final magnetic bead and antibody coupling ratio：1, coating
Magnetic bead concentration is 0.2mg/ml (equivalent to the anti-human ST2 monoclonal antibodies in 500ng/ holes), enzyme labelled antibody working concentration is 1:
500。
4. the preparation of Horseradish Peroxidase Conjugates working solution：
By the monoclonal antibody of the horseradish peroxidase prepared in 2 steps, with 1：500 ratio is dissolved in enzyme buffer liquid
In.Enzyme buffer liquid formula is：5.8g/L disodium hydrogen phosphates, 0.59g/L sodium dihydrogen phosphates, 9.0g/L sodium chloride, 200ml/L calves
Serum, 1.0g/L enzyme stabilizers and 1ml/L Proclin-300.The final concentration of the monoclonal antibody of horseradish peroxidase-labeled
For 1mg/L.
5. the preparation of calibration object dilution：
1000ml calibration objects dilution includes 9.0g sodium chloride, 12 water of 5.8g disodium hydrogen phosphates, 0.59g biphosphates
Sodium, 10g bovine serum albumin(BSA)s, 50g sucrose, 1ml Proclin-300.
6. chemical luminescence for liquid A contains luminol and luminescence enhancer, chemical luminescence for liquid B contains peroxide and luminescence enhancement
Agent.
7. assignment and the preparation of calibration object：
Operate, calibrated with working calibration product, to the ST2 high level people of calibration object diluted by contrast agent box specification
Source sample is detected.Under the conditions of different experiments room, different operating person, independently measure 20 times are repeated, draw 20 measurement knots
The RLU values (luminous intensity values) of fruit.The people source sample of RLU value stabilizations is selected to calculate its average value as product calibration objectRoot
The school Qu Fangcheng that the working calibration product prepared according to sterling are arranged, willCorresponding RLU values are brought into above-mentioned equation, obtain correspondence
Concentration value, be product calibration object target value.Calibration object is prepared according to assigned result with calibration object dilution：200ng/ml、
100ng/ml、50ng/ml、20ng/ml、5ng/ml、0ng/ml。
9. the preparation of quality-control product：
People's sample (serum or blood plasma) of collector's ST2 clinical detection high level, with calibration object diluted high level people's blood
Clearly extremely in desired concentration range.High level Quality Control：80ng/ml to 120ng/ml；Low value Quality Control：16ng/ml to 24ng/ml.2-8
DEG C save backup.
10. above-mentioned each reagent is assembled into kit, operation instructions, anti-can also be incorporated in kit as needed
Answer the instruments such as cup, supporting magnet stand.
Test case：
The application method of 1. disclosure people's ST2 chemical luminescence reagent kits of test case
1. prepare before experiment
Kit is taken out from 4 DEG C of refrigerators, room temperature (18-25 DEG C) should all be equilibrated to.
2. test method
2.1 sample-addings incubate：Take sufficient amount of reaction plate or reaction cup, be fixed on frame, respectively set calibration sample wells,
Quality Control sample wells and sample to be tested hole, record each hole site.The sample to be tested of 10 μ l is separately added into sample aperture in order, in school
10 μ l of calibration object are added in quasi- sample wells, 10 μ l of quality-control product are added in Quality Control sample wells, are separately added into 50 μ l coating magnetic bead works again per hole
Make liquid and 50 μ l enzyme working solutions, fully mix, 10min is incubated at 37 DEG C.
2.2 cleaning：
Cleaning by hand：Reaction plate or reaction cup are placed on staticaccelerator adsorption 2 minutes on supporting magnet stand, suction out reaction solution, is moved
Magnet stand is removed, 300 μ l cleaning solutions are added per hole, magnetic bead is mixed, reaction plate or reaction cup is placed on staticaccelerator adsorption 2 on supporting magnet stand
Minute, cleaning solution is suctioned out, repeats to rinse 3 times；
Necessary instrument cleans：Illustrate according to instrumentation, set program, magnetic bead is placed on corresponding magnet stand, static suction
Attached sucking-off reaction solution, leaves magnet stand, and 400 μ l cleaning solutions are added per hole, magnetic bead is placed in static suction on magnet stand after concussion mixing
It is attached, suction out cleaning solution, repetitive operation 3 times.
2.3 add luminescent solution：50 μ l chemical luminescence for liquid A and 50 μ l chemical luminescence for liquid B are added per hole, are fully mixed.
2.4 measure：The reaction plate for having added luminescent solution or reaction cup are put into measure RLU values under Chemiluminescence Apparatus.
2.5 calculate：According to the concentration of calibration object and corresponding RLU, with calibration object S0 holes zeroing (including calibration object S1-
S5, quality-control product and sample to be detected).Using double-log linear fit mode (log (X)-log (Y)), (S0 is not involved in result of calculation
Calculate).
Linear equation is log (RLU)=2.694+1.028log (concentration)；R=1.
Following table represents ST2 magnetic microparticle chemiluminescence kits calibration curve detection RLU values.
Table 1
| Concentration (ng/ml) | RLU values 1 | RLU values 2 | RLU (average) |
| 0 | 164 | 149 | 157 |
| 5 | 2524 | 2525 | 2525 |
| 20 | 10987 | 11430 | 11209 |
| 50 | 27431 | 28485 | 27958 |
| 100 | 53161 | 54284 | 53723 |
| 200 | 112130 | 120033 | 116082 |
| Low value Quality Control | 10236 | 10234 | 10235 |
| High level Quality Control | 55423 | 54657 | 55040 |
The ST2 detection kit general reaction times using invention are 10 minutes, and mainstream product in the market Critical is public
The ST2 reaction total times of department are 120 minutes；And the ST2 kits Main Components of Critical companies need mostly in advance into
Row dilution or dissolving, implement relatively complicated for operating personnel.
The methodology index of 2. disclosure people's ST2 chemical luminescence reagent kits of test case
1. accuracy：The rate of recovery should be between 85% to 115%；
2. blank detection limit：1ng/ml should be not higher than；
3. measuring system is linear：Linearly dependent coefficient r >=0.9900 in the range of 1ng/ml to 200ng/ml.
4. repeatability：CV crowdes of planted agents of variation within batch coefficient are no more than 8%；
5. difference between batch：10% should be no more than between interassay coefficient of variation CV batches.
Table 2 shows accuracy, minimum detectability, system linear, repeated testing result.
Table 2
6. analysis specificity：
People's ST2 negative samples N1, positive sample P1 samples are detected respectively.
Add the cross antigen of three concentration gradients respectively in N1, P1 sample, detection cross antigen is to ST2 antibody
The cross reaction situation of detection：
Human cardiac troponin C (cTnC) concentration (1000ng/ml, 500ng/ml, 0ng/ml)；
Cardiac troponin T (cTnT) concentration (1000ng/ml, 500ng/ml, 0ng/ml)；
Human Skeletal Muscle Troponin I (cTnI) concentration (1000ng/ml, 500ng/ml, 0ng/ml).
Cross antigen is typically to have higher homology, the sequence similar or with approx imately-detecting meaning to test substance
The albumen of justice.If it is envisioned that there are during these cross antigens in sample, antibody may identify and with reference to these intersection
Reaction is former, causes false positive.Therefore, the cross reactivity of diagnostic kit is an important index.This is for clinical effectiveness
Reliability it is extremely important.
As seen from Table 5, when human cardiac troponin C is not more than no more than 1000ng/ml, people's Cardiac troponin T
When 1000ng/ml, Human Skeletal Muscle Troponin I are not more than 1000ng/ml, the testing result of people ST2 is had not significant impact.Exhibition
The excellent anti-cross reactivity of disclosure kit is shown.
7. stability
Parameter detecting the results are shown in Table 3 after 7.1 kit each components are stored 3 days, 6 days, 8 days under the conditions of 37 DEG C.
Table 3：37 DEG C of shelf stability experimental results
Parameter detecting the results are shown in Table 4 after 7.2 kit each components are stored 6 months, 12 months, 14 months under the conditions of 4 DEG C.
Table 4：4 DEG C of shelf stability experimental results
R-H, R-L represent high level accuracy reference material, low value accuracy reference material respectively.Kit storage stability is good
It is good, it can store 8 days under the conditions of 37 DEG C, can be stored 12 months under the conditions of 4 DEG C.
8. interfering material is analyzed
8.1 hemoglobins, experimental result are shown in Table 6.
Conclusion：According to as a result, in hemoglobin≤200mg/ml, people's ST2 antigen testing results are had no significant effect.
8.2 triglycerides, experimental result are shown in Table 7.
Conclusion：When triglyceride concentration is in 4000mg/dl, do not have a significant impact to people's ST2 antigens testing result.
8.3 bilirubin, experimental result are shown in Table 8.
Interpretation of result：When bilirubin concentration is not more than 20mg/dl, people's ST2 antigen testing results are had no significant effect.
8.4 human IgGs, experimental result are shown in Table 9.
Interpretation of result：People's ST2 antigen testing results are had no significant effect when human IgG concentration is not more than 20mg/ml.
Claims (10)
1. a kind of solubility ST2 protein detection kits, it includes：
Coupling has the magnetic bead of the first anti-ST2 protein monoclonal antibodies and the second anti-ST2 albumen Dan Ke comprising labeled substance markers
The enzyme working solution of grand antibody, wherein the label is selected from horseradish peroxidase, alkaline phosphatase, radioactive label and is immunized
Fluorescent marker；Wherein described first anti-ST2 protein monoclonal antibodies and the second anti-ST2 protein monoclonal antibodies are respectively selected from
The hybridoma for being CGMCC No.14727 by the preserving number for being deposited in CGMCC on October 23rd, 2017 and October 23 in 2017
The preserving number for being deposited in CGMCC days is monoclonal antibody caused by the hybridoma of CGMCC No.14728；It is or described
First anti-ST2 protein monoclonal antibodies and the second anti-ST2 protein monoclonal antibodies were respectively selected from by October 23rd, 2017
The preserving number for being deposited in CGMCC is the hybridoma of CGMCC No.14728 and is deposited in CGMCC's on October 23rd, 2017
Preserving number is monoclonal antibody caused by the hybridoma of CGMCC No.14727.
2. solubility ST2 protein detection kits according to claim 1, wherein the magnetic bead and the first anti-ST2
The mass ratio of protein monoclonal antibody is 10: 1 to 40: 1, preferably 20:1.
3. solubility ST2 protein detection kits according to claim 1 or 2, wherein described include labeled substance markers
The second anti-ST2 protein monoclonal antibodies enzyme working solution in the second anti-ST2 protein monoclonal antibodies final concentration of 1mg/L.
4. solubility ST2 protein detection kits according to claim 3, wherein to further include enzyme anti-for the kit
Answer substrate, ST2 albumen calibration object and ST2 albumen quality-control products；Wherein
The ST2 albumen calibration object be known ST2 protein concentrations serum, wherein ST2 protein concentrations be respectively 200ng/ml,
100ng/ml, 50ng/ml, 20ng/ml, 5ng/ml, 0ng/ml,
The ST2 albumen quality-control product is for ST2 protein concentrations in 80ng/ml to 120ng/ml and 16ng/ml between 24ng/ml
Serum.
5. a kind of anti-ST2 protein monoclonal antibodies, it is selected from the preserving number for being deposited in CGMCC by October 23rd, 2017
The hybridoma of CGMCC No.14727 and the preserving number for being deposited in CGMCC on October 23rd, 2017 are CGMCC No.14728
Hybridoma caused by monoclonal antibody.
6. a kind of hybridoma, it is CGMCC No.14727 that it, which is selected from the preserving number for being deposited in CGMCC on October 23rd, 2017,
Hybridoma and on October 23rd, 2017 preserving number for being deposited in CGMCC be CGMCC No.14728 hybridoma.
7. the hybridoma for being CGMCC No.14727 by the preserving number for being deposited in CGMCC on October 23rd, 2017 and 2017
October 23, the preserving number for being deposited in CGMCC was the group of monoclonal antibody caused by the hybridoma of CGMCC No.14728
Close and preparing the purposes in being used to detect the kit of soluble ST2 albumen.
8. purposes according to claim 7, the kit is selected from ELISA detection kit, immunoturbidimetry detection reagent
Box, magnetic particle detections kit, chemiluminescence detection kit, immunofluorescence detection agent box and radioimmunoassay test agent
Box.
9. a kind of solubility ST2 protein detection kits, it includes：
It is the hybridoma of CGMCC No.14727 and 2017 10 by the preserving number for being deposited in CGMCC on October 23rd, 2017
Monoclonal antibody caused by the hybridoma that months 23 days preserving numbers for being deposited in CGMCC are CGMCC No.14728.
10. ST2 protein detection kits according to claim 9, the kit is selected from ELISA detection kit, exempts from
Epidemic disease is than turbid detection kit, magnetic particle detections kit, chemiluminescence detection kit, immunofluorescence detection agent box and radiation
Immunity detection reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711318542.3A CN107991485A (en) | 2017-12-12 | 2017-12-12 | For detecting the kit of soluble ST2 albumen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711318542.3A CN107991485A (en) | 2017-12-12 | 2017-12-12 | For detecting the kit of soluble ST2 albumen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107991485A true CN107991485A (en) | 2018-05-04 |
Family
ID=62037412
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711318542.3A Pending CN107991485A (en) | 2017-12-12 | 2017-12-12 | For detecting the kit of soluble ST2 albumen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107991485A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108982871A (en) * | 2018-07-19 | 2018-12-11 | 北京市心肺血管疾病研究所 | Application of the serum sST2 in Children with Dilated Cardiomyopathy prognosis |
| CN110208549A (en) * | 2019-07-01 | 2019-09-06 | 北京利德曼生化股份有限公司 | A kind of Soluble growth stimulation 2 albumen sST2 luminescence reagent box of expressing gene |
| CN112710841A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | ST2 protein detection kit and detection method |
| WO2021164109A1 (en) * | 2020-02-21 | 2021-08-26 | 苏州旭光科星抗体生物科技有限公司 | Enzyme-linked immunoassay kit for detecting content of soluble st2, and use thereof |
| CN113777324A (en) * | 2021-09-10 | 2021-12-10 | 普十生物科技(北京)有限公司 | Growth stimulation expression factor 2 instant detection kit, preparation method and application thereof |
| CN113846066A (en) * | 2021-09-15 | 2021-12-28 | 海珂分子（北京）科技有限责任公司 | Hybridoma cell strain secreting ST2 antibody and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102830234A (en) * | 2012-08-10 | 2012-12-19 | 杭州华得森生物技术有限公司 | Rapid diagnostic kit for detecting novel marker ST2 of human heart failure |
| CN103487586A (en) * | 2013-09-04 | 2014-01-01 | 石家庄洹众生物科技有限公司 | Testing device for quantitatively detecting soluble growth stimulation expression protein 2 |
| CN104151416A (en) * | 2013-05-15 | 2014-11-19 | 中国科学院上海巴斯德研究所 | Anti-human sST2 monoclonal antibody and application thereof |
| CN106199002A (en) * | 2016-07-19 | 2016-12-07 | 河南华程氏生物科技股份有限公司 | A kind of for detecting the chemical luminescence immune analysis reagent box of ST2, preparation method and application |
| CN107340386A (en) * | 2017-06-09 | 2017-11-10 | 厦门大学附属中山医院 | SST2 detection kits based on microparticle chemiluminescence immunoassay |
-
2017
- 2017-12-12 CN CN201711318542.3A patent/CN107991485A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102830234A (en) * | 2012-08-10 | 2012-12-19 | 杭州华得森生物技术有限公司 | Rapid diagnostic kit for detecting novel marker ST2 of human heart failure |
| CN104151416A (en) * | 2013-05-15 | 2014-11-19 | 中国科学院上海巴斯德研究所 | Anti-human sST2 monoclonal antibody and application thereof |
| CN103487586A (en) * | 2013-09-04 | 2014-01-01 | 石家庄洹众生物科技有限公司 | Testing device for quantitatively detecting soluble growth stimulation expression protein 2 |
| CN106199002A (en) * | 2016-07-19 | 2016-12-07 | 河南华程氏生物科技股份有限公司 | A kind of for detecting the chemical luminescence immune analysis reagent box of ST2, preparation method and application |
| CN107340386A (en) * | 2017-06-09 | 2017-11-10 | 厦门大学附属中山医院 | SST2 detection kits based on microparticle chemiluminescence immunoassay |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108982871A (en) * | 2018-07-19 | 2018-12-11 | 北京市心肺血管疾病研究所 | Application of the serum sST2 in Children with Dilated Cardiomyopathy prognosis |
| CN108982871B (en) * | 2018-07-19 | 2021-09-24 | 北京市心肺血管疾病研究所 | Application of serum sST2 in children dilated cardiomyopathy prognosis |
| CN110208549A (en) * | 2019-07-01 | 2019-09-06 | 北京利德曼生化股份有限公司 | A kind of Soluble growth stimulation 2 albumen sST2 luminescence reagent box of expressing gene |
| WO2021164109A1 (en) * | 2020-02-21 | 2021-08-26 | 苏州旭光科星抗体生物科技有限公司 | Enzyme-linked immunoassay kit for detecting content of soluble st2, and use thereof |
| CN112710841A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | ST2 protein detection kit and detection method |
| CN113777324A (en) * | 2021-09-10 | 2021-12-10 | 普十生物科技(北京)有限公司 | Growth stimulation expression factor 2 instant detection kit, preparation method and application thereof |
| CN113846066A (en) * | 2021-09-15 | 2021-12-28 | 海珂分子（北京）科技有限责任公司 | Hybridoma cell strain secreting ST2 antibody and application thereof |
| CN113846066B (en) * | 2021-09-15 | 2024-06-07 | 海珂分子（北京）科技有限责任公司 | Hybridoma cell strain secreting ST2 antibody and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107991485A (en) | For detecting the kit of soluble ST2 albumen | |
| JP3108103B2 (en) | Monoclonal antibodies against human cardiac myoglobin | |
| US20040106143A1 (en) | Monoclonal antibody against estrogen stimulated leucine aminopeptidase | |
| US7196169B2 (en) | Isolated post-translationally modified mammalian proteins for monitoring and diagnosing muscle damage | |
| CN109613265A (en) | A kind of kit with latex immunoturbidimetry measurement apoC 3 | |
| CN111808198B (en) | Antibody specifically binding RANKL targeted therapeutic drug and application thereof | |
| CN107045062A (en) | Detect colloidal gold immuno-chromatography test paper strip, kit of human neutrophil genatinase associated lipocalin and preparation method thereof | |
| CN108982861A (en) | A kind of blood vessel dilatation type stimulation phosphoprotein detection kit | |
| CN113817062B (en) | Anti-human hydroxysteroid 17-beta dehydrogenase 13 (HSD 17B 13) rabbit monoclonal antibody and application thereof | |
| CN105849562B (en) | The measuring method of soluble g PC3 protein | |
| JP2001519894A (en) | Early detection method for lysosomal storage disease | |
| US11067579B2 (en) | Target marker GP73 for detecting steatohepatitis and detection application method | |
| CN107478848B (en) | The kit and preparation method thereof of quantitative detection people NT-proBNP | |
| CN110092824A (en) | Amino-terminal brain natriuretic peptide Precursor Peptide, antibody and preparation method thereof, detection kit and detection method | |
| US20050202513A1 (en) | Method of judging cardiotoxicity of anthracycline-type anticancer chemical therapeutic by detecting human h-fabp and reagent therefor | |
| CN108802367A (en) | A kind of chemical luminescence ELISA detection kit of vancomycin | |
| CN104165992A (en) | Kit for detecting apolipoprotein A5 for diagnosis of hypertriglyceridemia | |
| CN113956355B (en) | Anti-human Brain Natriuretic Peptide (BNP) rabbit monoclonal antibody and application thereof | |
| CN103797369B (en) | The assay method of anti-WT1 antibody | |
| CN106220722B (en) | The antigen polypeptide and application thereof of DKK1 autoantibody identification | |
| CN113929776B (en) | Antifungal (1, 3) -beta-D glucan monoclonal antibody, encoding gene and expression and application thereof | |
| KR20230059115A (en) | An antibodies specifically binding to NT-proBNP and uses thereof | |
| CN109765385B (en) | A method of improving emulsion reagent sensitivity and linear | |
| CN112074738B (en) | Heart failure detection method, heart failure detection device, sandwich immunoassay, and antibody combination | |
| CN107177557B (en) | Hybridoma cell capable of secreting anti-H-FABP monoclonal antibody, and preparation methods and applications thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180504 |