CN105259353B - Kit and method for detecting sST2 (soluble ST2) in blood of abdominal aortic aneurysm and/or aortic dissection patient - Google Patents
Kit and method for detecting sST2 (soluble ST2) in blood of abdominal aortic aneurysm and/or aortic dissection patient Download PDFInfo
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Abstract
The invention discloses a kit and a method for detecting sST2 (soluble ST2) in blood of an abdominal aortic aneurysm and/or aortic dissection patient. According to the kit and the method, a pair of antibodies for identifying different epitopes of sST2 is assembled to obtain a double-antibody sandwich ELISA and immune colloidal gold test strip, so as to realize quantitive and qualitative detection of sST2. Tests prove that the ELISA kit and the immune colloidal gold test strip have relatively high specificity and sensitivity in an abdominal aortic aneurysm marker-sST2 protein of human, are simple to operate, can be utilized for detecting abdominal aortic aneurysm, aortic dissection and other diseases in scientific research and clinical application, and have the functions of auxiliary diagnosis, guide treatment and prognosis judgment.
Description
Technical field
The invention belongs to biological technical field, it is related to solvable in a kind of aortic aneurysm and/or dissection of aorta blood samples of patients
The detection kit and detection method of property ST2, and in particular to one kind can fast quantification or qualitative detection people's whole blood, serum and/or
The immunological detecting kit of blood plasma aortic aneurysm and/or dissection of aorta mark-solubility ST2 content.
Background technology
Soluble ST2 (sST2) is also called IL1RL1, is the member of Toll-like receptor superfamily, but with other members not
Together, sST2 can not cause infection reaction by activating NF- κ B.Used as the receptor family member of il-1, ST2 albumen has two
Plant form, soluble form (sST2, soluble ST2) and membrane-bound receptor form (membrane-bound receptor
Form, ST2receptor).The func-tional ligand of ST2 is IL-33, after cardiac muscle cell and fibroblast are subject to mechanically stretching,
ST2 and IL-33 contents increase.Research in recent years finds that sST2 contents levels in blood can reflect the load condition of heart.It is logical
Cross measure Peripheral Blood in Patients with Acute Myocardial Infarction ST2 and find that peripheral blood in patients ST2 levels dead and that heart failure occurs are aobvious in 30 days
Write and raise.The research to 593 acute dyspnea patients such as Januzzi finds that the median of patients with heart failure ST2 is higher than the non-heart
Decline patient, and patient ST2 medians dead in 1 year, also above the patient of survival, the rising of ST2 levels is 1 annual death rate
Independence and strength prediction index.Research to chronic heart failure finds that the rising of ST2 levels is related to the heart failure order of severity
And predict the generation of cardiac sudden death, and serious patients with heart failure raise changes of the ST2 at 2 weeks be also it is dead with need heart to move
The independent predictor of plant.
Aortic aneurysm (abdominal aortic aneurysm, AAA) is the result of the pathologic expansion of sustainer, can
With 50% more than normal blood vessels diameter.Dissection of aorta (acuteaorticdissection, AAD) is referred to due to inner membrance office
Portion tears, and is impacted by blood, and inner membrance phased separation, extension form true, false two chamber in intra-arterial.So as to cause some row bags
The performance of tear sample pain is included, if not carrying out appropriate and timely treating, the chance of rupture is very big, and the death rate is also very
It is high.Diagnosis to aortic aneurysm and dissection of aorta mainly passes through Imaging Method, and such as chest, abdominal X-ray takes pictures, CT angiograms
(CTA) check and the method such as angiography with magnetic resonance angiography (MRA).In terms of molecular marker, c reactive protein,
A series of biomarkers such as DDi, smooth muscle myosin heavy chain, calmodulin may be with Aortic Dissection
Generation is relevant, for cell factor such as Interleukin -1β, IL-6, IL-8 that the diagnosis of aortic aneurysm, some immune responses are raised
And tumor necrosis factor-alpha (tumour necrosis factor- α) and CC classes chemotactic factor (CF) all cause the note of researcher
Meaning, for matrix metalloproteinase, particularly MMP-9 and other albumen tPA, Fibrinogen and D-dimmer and sustainer
Association between the generation of knurl also has been reported that, but the limitation due to lacking specificity, half-life short or by false chamber thrombosis etc.
Factor fails to be used widely in clinical diagnosis and treatment.
The content of the invention
First purpose of the present invention is to provide the new application of the material of detection solubility ST2 protein contents.
The invention provides the material of detection solubility ST2 protein contents in preparation diagnosis or auxiliary diagnosis patient to be measured is
Application in the no product with aortic aneurysm and/or dissection of aorta.
Present invention also offers the material of detection solubility ST2 protein contents detects the material of solubility ST2 protein contents
Application in the product for preparing detection or auxiliary detection aortic aneurysm and/or dissection of aorta.
Present invention also offers the material of detection solubility ST2 protein contents is preparing prediction or is aiding in prediction aortic aneurysm
And/or the application in the product of the mortality risk state after dissection of aorta operation in patients.
In the amino acid sequence such as sequence table of the soluble ST2 albumen shown in sequence 2.
In above-mentioned application, the aortic aneurysm and/or dissection of aorta are chronic dissecting aneurysm of aorta, acute active
Arteries and veins dissecting aneurysm or Acute Aortic knurl are without interlayer.
In above-mentioned application, the material is following 1) -3) shown in：
1) solubility ST2 protein antibodies；
2) contain enzyme linked immunological kit 1)；
3) contain colloidal gold strip 1).
In above-mentioned application, the soluble ST2 protein antibodies are solubility ST2 protein monoclonal antibodies or solubility ST2
Protein polyclone antibody；The soluble ST2 protein antibodies are specially solubility ST2 protein monoclonal antibodies；
The monoclonal antibody of the soluble sST2 albumen, its be it is following 1) or 2)：
1) in the amino acid sequence of the weight chain variable district of the monoclonal antibody of the soluble sST2 albumen shown in such as sequence table
Shown in sequence 5；Sequence in the amino acid sequence of the light chain variable district of the monoclonal antibody of the soluble sST2 albumen such as sequence table
Shown in row 6；
2) in the amino acid sequence of the weight chain variable district of the monoclonal antibody of the soluble sST2 albumen shown in such as sequence table
Shown in sequence 9；Sequence in the amino acid sequence of the light chain variable district of the monoclonal antibody of the soluble sST2 albumen such as sequence table
Shown in row 10.
In above-mentioned application,
1) sequence in the nucleotide sequence of the weight chain variable district of solubility ST2 protein monoclonal antibodies described in such as sequence table
Shown in 3；
1) sequence in the nucleotide sequence of the light chain variable district of solubility ST2 protein monoclonal antibodies described in such as sequence table
Shown in 4；
2) sequence in the nucleotide sequence of the weight chain variable district of solubility ST2 protein monoclonal antibodies described in such as sequence table
Shown in 7；
2) sequence in the nucleotide sequence of the light chain variable district of solubility ST2 protein monoclonal antibodies described in such as sequence table
Shown in 8；
The enzyme linked immunological kit also includes the soluble ST2 protein antibodies of Jing enzymes or compound label.
In above-mentioned application,
The enzyme is horseradish peroxidase or alkaline phosphatase；The compound is biotin molecule or fluorescence molecule；
The fluorescence molecule is Cy3, Cy5 or isothiocyanate.
In above-mentioned application,
Soluble ST2 egg of the soluble ST2 protein monoclonal antibodies in the colloidal gold strip for colloid gold label
Bai Kangti.
Second object of the present invention is to provide the new application of soluble ST2 albumen.
The invention provides solubility ST2 albumen is as detection or auxiliary detection aortic aneurysm and/or dissection of aorta
Mark in application；
In the amino acid sequence such as sequence table of the soluble ST2 albumen shown in sequence 2.
Third object of the present invention is to provide a kind of enzyme linked immunological of solubility ST2 protein contents in detection testing sample
Kit.
The present invention provide detection testing sample in solubility ST2 protein contents enzyme linked immunological kit include it is above-mentioned can
Dissolubility ST2 protein antibodies.
Whether fourth object of the present invention contains the glue of solubility sST2 albumen in being to provide a kind of detection testing sample
Body gold test paper strip.
Colloidal gold strip containing solubility sST2 albumen includes in the detection testing sample that the present invention is provided
Above-mentioned soluble ST2 protein antibodies.
5th purpose of the present invention is to provide the monoclonal antibody of soluble ST2 albumen.
The monoclonal antibody of the soluble ST2 albumen that the present invention is provided, its be it is following 1) or 2)：
1) in the amino acid sequence of the weight chain variable district of the monoclonal antibody of the soluble sST2 albumen shown in such as sequence table
Shown in sequence 5；Sequence in the amino acid sequence of the light chain variable district of the monoclonal antibody of the soluble sST2 albumen such as sequence table
Shown in row 6；
2) in the amino acid sequence of the weight chain variable district of the monoclonal antibody of the soluble sST2 albumen shown in such as sequence table
Shown in sequence 9；Sequence in the amino acid sequence of the light chain variable district of the monoclonal antibody of the soluble sST2 albumen such as sequence table
Shown in row 10.
In said monoclonal antibody,
1) sequence in the nucleotide sequence of the weight chain variable district of solubility ST2 protein monoclonal antibodies described in such as sequence table
Shown in 3；
1) sequence in the nucleotide sequence of the light chain variable district of solubility ST2 protein monoclonal antibodies described in such as sequence table
Shown in 4；
2) sequence in the nucleotide sequence of the weight chain variable district of solubility ST2 protein monoclonal antibodies described in such as sequence table
Shown in 7；
2) sequence in the nucleotide sequence of the light chain variable district of solubility ST2 protein monoclonal antibodies described in such as sequence table
Shown in 8；
In above-mentioned application or mentioned reagent box or above-mentioned test strips or said monoclonal antibody, the soluble ST2 albumen
Amino acid sequence such as sequence table in shown in sequence 2.
The invention provides biomarker solubility ST2 (sST2) albumen of a kind of human aorta knurl and dissection of aorta
Molecule and its method for quick in people's whole blood, serum or blood plasma, and using the antibodyome of a pair of identification sST2 different epitopes
Dress obtains double-antibody sandwich elisa and immunity colloidal gold test paper strip, realizes the qualitatively and quantitatively detection to soluble sST2.Pass through
Test is proved：The ELISA kit of the present invention and colloidal gold strip are to human aorta tumor markers-human soluble ST2 albumen
It is with higher specificity and sensitivity, simple to operate, can be used for the disease such as aortic aneurysm and dissection of aorta in scientific research and clinic
The detection of disease, plays a part of auxiliary diagnosis, guiding treatment, Index for diagnosis.
Description of the drawings
Compositions of the Fig. 1 for sST2 immunity colloidal gold test paper strips.
Calibration curves of the Fig. 2 for ELISA detection kit.
Distributions of the sST2 of aneurysmal aortic interlayer patient in blood plasma based on Fig. 3.
Fig. 4 is the level monitoring of sST2 in 4-108 hour blood plasma after 9 patients with aortic dissection aneurysm operative treatments.
Fig. 5 is the expression and purification of restructuring sST2.Marker is molecular weight protein marker；1：The full bacterium of Bl21 not induced
The super biological result of albumen；2：The Bl21 whole bacterial proteins excusing from death result of abduction delivering；3：The wash-out result of 60mM imidazoles；4：300mM
The wash-out result of imidazoles.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
In following embodiments, material used, reagent etc., if no special instructions, commercially obtain.
The preparation of embodiment 1, sST2 monoclonal antibodies
First, immunogenic preparation
1st, the design of primer
The sequence of human soluble ST2 albumen is analyzed using protein sequence and secondary structure analysis software, according to
The surface accessibility of its domain/sequence, hydrophilic and hydrophobic, secondary structure and Amino acid profile, select suitable recombinant expressed area
Domain, devises upstream and downstream primer as follows, the synthesis of Jing Sangon Biotech (Shanghai) Co., Ltd.：ST2-F：5’-
CCAAGTTTAAACGGATCTCTAGCGAATTCGCCGCCACCATGGACTTTGGGCT CAGCTTGG-3’；ST2-R：5’-
CAAGGATCCTTGCTTCTGGGCAGCCAAGGG-3’。
2nd, the acquisition of st2 genes
People's liver cell system HepG2 (purchased from ATCC, HB-8065) is taken, after Trizol methods extract RNA, with Poly-T primers
Reverse transcription is carried out, cDNA is obtained；As template, ST2-F the and ST2-R primers designed using step 1 enter performing PCR to cDNA with acquisition
Amplification, obtains PCR primer.
PCR reaction systems (30 μ L)：The each 3 μ L of upstream primer ST2-F and downstream primer ST2-R (5 μm of ol/L), 10 × PCR
The 3 μ L of dNTP of buffer 3 μ L, 2mM, cDNA 1 μ L (about 1 μ g), 0.2 μ L of 1U thermal starting archaeal dna polymerases EZ-Taq are (from Beijing
Co., Ltd of Hua Da protein research and development centre), distilled water is supplemented to 30 μ L；
PCR reacts amplification condition：94 DEG C of denaturations 5 minutes；Then 94 DEG C of denaturation 40 seconds, 58 DEG C are annealed 30 seconds, and 72 DEG C are prolonged
Stretch 40 seconds, carry out 25 circulations altogether；72 DEG C extend 2 minutes；
PCR primer is separated in the agarose gel electrophoresis that concentration is 1%, and the centrifugal DNA glue reclaims kit of post (give birth to by Tiangeng
Change Science and Technology Ltd.) purpose fragment is reclaimed, obtain the st2 genetic fragments that size is 1003bp, its nucleotide sequence such as sequence
In table shown in sequence 1.
3rd, the acquisition of recombinant vector
St2 genes step 2 obtained with restriction enzyme NcoI and XhoI (purchased from Dalian Bao Bio-Engineering Company)
Fragment carries out double digestion, obtains the st2 genetic fragments that size is about 995bp；Will with restriction enzyme NcoI and XhoI
PET30a carriers (Invitrogen) carry out double digestion, reclaim skeleton carrier of the size for 5329bp, connection st2 genetic fragments and
Skeleton carrier, obtains recombinant vector pET30a-ST2.
Recombinant vector pET30a-ST2 is carried out into sequence verification, is as a result shown：Recombinant vector pET30a-ST2 be by
DNA sequence dna between NcoI the and XhoI restriction enzyme sites of pET30a carriers replaces with the st2 genes in sequence table shown in sequence 1, and
The constant carrier for obtaining of other sequences of pET30a carriers is kept, ST2 albumen is expressed, the amino acid sequence of ST2 albumen is sequence
2。
4th, the acquisition of recombinant bacterium
To be sequenced in correct recombinant vector pET30a-ST2 conversions Escherichia coli Bl21 (DE3), obtain recombinant bacterium Bl21-
pET30a-ST2。
5th, the abduction delivering of restructuring ST2 albumen
By 1:The Bl21-pET30a-ST2 bacterium of incubated overnight are forwarded to 100ml LB culture mediums by 100 ratio, are added eventually
Concentration is the kanamycins of 50 μ g/ml, 37 DEG C of shaken cultivations to OD600For 0.6～0.8；The IPTG of addition 0.1mol/L, 25 DEG C
Concussion and cultivate 8h, receives ultrasonication after bacterium；Supernatant (containing restructuring sST2 albumen) is collected, and the parent of protein is carried out using Ni posts
And purifying；After being eluted with the imidazole solution of variable concentrations again, by each component, loading carries out SDS-PAGE separation detections respectively.
Testing result is as shown in Figure 5：The molecular size range of restructuring sST2 albumen is 35.24kDa；The purity of the albumen of sST2
More than 90%, concentration is about 1mg/mL, can meet the requirement of immune animal and antibody screening and identification.
2nd, animal immune
Using the Freund immunogene that non-fully step one is obtained by adjuvant (Sigma companies) (restructuring ST2 albumen) emulsification, agent
Only measure as 30 μ g/, the immunogene after being emulsified；(it is purchased from the immunogen immune 4-6 week old female Balb/c mouse after emulsification
Beijing Vital River Experimental Animals Technology Co., Ltd.), 6 points of every mouse of abdominal part hypodermic, dosage are 60 μ g/；Per 14
Its booster immunization is once.
3rd, hybridoma fusion and screening
1st, hybridoma fusion
Resist so that in indirect ELISA (wavelength 450nm) detection mice serum, anti-immunity is former within 7 days after 3rd booster immunization more
Potency.Selection potency highest mouse is mixed with physiological saline with tail vein injection impact immunity, antigen, and dosage is 50 μ g/,
And the splenocyte suspension of potency highest mouse and murine myeloma cell sp2/0 (ATCC) is taken with 5:1 ratio mixes, centrifugation
1500rpm, 5min.After abandoning supernatant, centrifuge tube is put in 37 DEG C of water-baths, is slowly added to the PEG1500 (Roche of 1mL in 1 minute
Company), and stir cell.The IMDM (Sigma companies) of 10mL serum-frees after 1min is stood in warm water, is added, is mixed, centrifugation
1000rpm, 5min.After abandoning supernatant, addition 10mL serum (PAA companies) is careful to get up cell piping and druming, and adds 5mL to mix
The thymocyte of 10xHAT (Sigma companies), mixes.Add that 25mL contains 2.1% NC Nitroncellulose (Sigma companies) half
Solid medium is fully mixed, and is then uniformly poured in 20 Tissue Culture Dish.Tissue Culture Dish is put in wet box, is put
Enter 37 DEG C, 5%CO2Cultivate in incubator.After fusion, 7 days clone cells roll into a ball size medium density, under anatomical lens, absorption circle,
Real, big cloning cluster is squeezed into and is ready in 96 well culture plates of culture medium in advance, is put into 37 DEG C, 5%CO2Cultivate in incubator.
In above-mentioned indirect ELISA (wavelength 450nm) detection mice serum, how anti-the method for the former potency of anti-immunity be as follows：With
Restructuring sST2 albumen, 2 μ g/mL, 4 DEG C of coatings are overnight；With 2% skimmed milk power, 37 DEG C of closing 2h；Take mouse tail blood, by serum from
200 times start 2 times of gradient dilutions, and blank (blank) is PBS, and negative control (negative) is that 200 times of negative serum is dilute
Release.It is 100 μ L/ holes, 37 DEG C of incubators, 1h；Afterwards with wash liquid 3 times.The HRP enzyme mark sheep anti mouses for adding PBS to dilute 20000 times
IgG is anti-as two, 100 μ L/ holes, 37 DEG C of incubators, 1h；With wash liquid 3 times after taking-up.100 μ L of nitrite ion, colour developing is added per hole
Time is 5min or so.50 μ L terminate liquids (2M H are added per hole2SO4) terminate.Dual wavelength (450, light absorption value 630) is surveyed, record is protected
Deposit data, OD values are that the extension rate of maximum two/for the moment is the potency of the antibody.
2nd, the screening of hybridoma cell strain
After culture 3 days, cell concentration constitutes about floor space 2/3, and taking 100 μ l supernatants immunogenes and synthesis polypeptide is carried out respectively
ELISA is screened.Positive colony changes liquid completely, the complete medium for adding 200 μ l to contain feeder cells and 1%HT (Sigma companies).
Second ELISA screening is carried out two days later, and positive colony proceeds to 24 holes for getting out culture medium (containing feeder cells and HT) in advance
Plate culture.Taking 100 μ l supernatants after five days carries out the screening of third time ELISA, and positive colony gradually proceeds to 6 orifice plates and Tissue Culture Flask
Amplification Culture is simultaneously frozen standby, and through screening numbering be 67186-6 and the two strain of hybridoma strains of 67186-15 have well
Combination vigor and high specificity.
3rd, the variable region sequences of antibody are determined
1st, the extraction of RNA
Trizol methods are adopted to extract the hybridoma (1 × 10 numbered as 67186-6 that above-mentioned steps 2 are obtained6It is individual) and
The hybridoma (1 × 10 of 67186-156It is individual) total serum IgE.
2nd, the acquisition of cDNA
The total serum IgE of 9 μ L steps 1 acquisition is taken, 2.5 μ L oligo (dT) 12-18primer (10mM), and 5 μ L are added
DNTPs, after being well mixed, 70 DEG C are incubated 5 minutes, then put 5 minutes on ice.It is subsequently added 5 μ L RT buffer (5X), 2.5 μ L
DTT (0.1M) and 1 μ L reverse transcriptases, 42 DEG C are reacted 1 hour.70 DEG C are incubated 15 minutes with terminating reaction, obtain cDNA.
3rd, it is sequenced
The cDNA that above-mentioned steps 2 are obtained is template, enters performing PCR amplification using following primer, in 50 μ L reaction systems
The sequence of each 25pmol of primer, weight chain variable district and light chain variable district amplification primers is added to put forth energy what is edited again according to Shen《Restructuring
Antibody》The design of mouse monoclonal antibody primer sequence and synthesis in (Science Press publishes for 2005) book.
Primer for expanding weight chain variable district is as follows, and wherein MHV.B1 is until 11 primers of MHV.B12 draw for upstream
Thing, can be combined for expanding heavy chain variable region gene with heavy chain downstream primer MHC.F respectively.
MHV.B1：5’-GATGTGAAGCTTCAGGAGTC-3’；
MHV.B2：5’-CAGGTGCAGCTGAAGGAGTC-3’；
MHV.B3：5’-CAGGTGCAGCTGAAGCAGTC-3’；
MHV.B4：5’-AGGTTACTCTGAAAGAGTC-3’；
MHV.B5：5’-GAGGTCCAGCTGCAACAATCT-3’；
MHV.B6：5’-GAGGTCCAGCTGCAGCAGTC-3’；
MHV.B7：5’-CAGGTCCAACTGCAGCAGCCT-3’；
MHV.B8：5’-GAGGTGAAGCTGGTGGAGTC-3’；
MHV.B9：5’-GAGGTGAAGCTGGTGGAATC-3’；
MHV.B10：5’-GATGTGAACTTGGAAGTGTC-3’；
MHV.B12：5’-GAGGTGCAGCTGGAGGAGTC-3’；
MHC.F：5’-GGCCAGTGGATAGTCAGATGGGGGTGTCGTTTTGGC-3’.
Primer for expanding light chain variable district is as follows, and wherein MKV.B1 is until 10 primers of MKV.B10 draw for upstream
Thing, can combine the variable region gene for expanding Kappa light chains respectively with light chain downstream primer MKC.F.
MKV.B1：5’-GATGTTTTGATGACCCAAACT-3’；
MKV.B2：5’-GATATTGTGATGACGCAGGCT-3’；
MKV.B3：5’-GATATTGTGATAACCCAG-3’；
MKV.B4：5’-GACATTGTGCTGACCCAATCT-3’；
MKV.B5：5’-GACATTGTGATGACCCAGTCT-3’；
MKV.B6：5’-GATATTGTGCTAACTCAGTCT-3’；
MKV.B7：5’-GATATCCAGATGACACAGACT-3’；
MKV.B8：5’-GACATCCAGCTGACTCAGTCT-3’；
MKV.B9：5’-CAAATTGTTCTCACCCAGTCT-3’；
MKV.B10：5’-GACATTCTGATGACCCAGTCT-3’；
MKC.F：5’-GGATACAGTTGGTGCAGCATC-3’.
Remaining dNTPs and buffer solution are eventually adding 1 μ L and 1U thermal starting Taq DNA of cDNA templates according to being routinely added to
Polymerase.Arrange PCR amplification programs be 94 DEG C 40 seconds, 52 DEG C 40 seconds, 72 DEG C 40 seconds, carry out 20 to 25 circulations, last 72 DEG C
Extend 3 minutes, product is placed in 4 DEG C of standby or direct electrophoresis.Taking 20 μ L PCR primers carries out electrophoretic analysis, in 1.5% agar
Separate on sugared gel and cut glue reclaim, derived heavy chain variable region and light chain variable district are cloned into into pMD18T plasmid vectors respectively
(TaKaRa) it is sequenced.
Sequencing result shows, the heavy chain and light chain of the monoclonal antibody of the hybridoma cell strain 67186-6 secretions of the present invention
Respectively as shown in sequence 3 in sequence table and sequence 4, the amino acid sequence of its corresponding variable region divides the nucleotide sequence of variable region
Not as shown in sequence 5 in sequence table and sequence 6；The heavy chain of the monoclonal antibody of hybridoma cell strain 67186-15 secretions and light
The nucleotide sequence of chain variable region respectively as shown in sequence 7 in sequence table and sequence 8, divide by its corresponding variable region amino acid sequence
Not as shown in sequence 9 in sequence table and sequence 10.
4th, the preparation and purification of sST2 antibody
It is the miscellaneous of 67186-15 with numbering to screen the hybridoma cell strain that the numbering for obtaining is 67186-6 respectively with step 3
Hand over tumor cell strain to prepare antibody, respectively obtain sST2 capture antibody and sST2 detection antibodies.Comprise the following steps that：
1st, prepared by ascites
Take healthy BALB/c female mices, every lumbar injection saxol 0.5mL；Every lumbar injection after 1～2 week
1ml concentration is 5 × 105The hybridoma cell strain cell suspension of individual/ml；Start to collect ascites after 7 days.
2nd, the purifying of sST2 antibody
The ascites collected is centrifuged into 4000rpm, 10min in 4 DEG C, the ascites in the middle of careful sucking-off is collected in centrifuge tube, 4
DEG C or -20 DEG C preservation.SST2 is purified from ascites with HiTraprProtein A FF (GE companies) affinity chromatography by specification
Antibody.And sST2 antibody purities are identified with SDS-PAGE glue, Bradford methods determine sST2 ACs.The sST2 antibody of purifying
- 20 DEG C are stored in, for the assembling of following kits.
Embodiment 2, a kind of double crush syndrome kit of detection sST2 contents and its detection method
First, the composition of kit
The double crush syndrome kit of the present invention includes that sST2 captures prepared by the embodiment 1 for being coated in ELISA Plate are anti-
SST2 detection antibodies that body, Jing horseradish peroxidases (HRP) are marked and sST2 protein standard substances (R＆D system,
DST200), Sample dilution, confining liquid, nitrite ion and terminate liquid are constituted.
The preparation method of the sST2 detection antibodies of Jing horseradish peroxidase-labeleds：
1st, weigh 6mg HRP (horseradish peroxidase, purchased from Sigma) to be dissolved in 1mL tri-distilled waters, per mL solution dropwise
It is slowly added to the 0.1M NaIO that 0.30mL newly matches somebody with somebody4Solution, 4 DEG C of lucifuges stir 35min, activate HRP, and color is changed into green from brown
Color.Above-mentioned solution loads in bag filter, and with 0.01M, pH is 4.4 sodium-acetate buffer, and 4 DEG C of dialysed overnights, color are changed into palm fibre
Green.Precipitation is seen whether, and has analyzed precipitation proterties, 10000r/min, 4 DEG C, 10min, centrifugation remove precipitation.Dialysed
HRP afterwards.
2nd, 1 sST2 detection antibodies 0.05M for filtering out will be implemented, pH is 9.5 4 DEG C of dialysed overnights of carbonic acid buffer.
Precipitation is seen whether, and has analyzed precipitation proterties, 10000r/min, 4 DEG C, 10min, centrifugation remove precipitation, after being dialysed
Antibody.
3rd, 0.16M ethylene glycol (adding 0.1mL per mg enzymes), 4 DEG C of lucifuges is added to stir 1h, be subsequently adding the HRP after dialysis
Antibody after dialysis；Both use 0.05M after mixing, and pH is 9.5 4 DEG C of dialysed overnights of carbonic acid buffer, obtain HRP- antibody and mix
Close liquid.
4th, the 5mg/mL NaBH of 0.1mL are added in HRP- antibody mixed liquors4Solution (adds 0.1-0.2mg's per mg enzymes
NaBH4), 4 DEG C of lucifuges stir 3h.Equal-volume saturation sulfate of ammoniac is added dropwise over, 4 DEG C of lucifuges stir 2h.4 DEG C, 10000rpm centrifugations
10min, abandons supernatant.PBS dissolution precipitations, obtain the sST2 detection antibodies of Jing horseradish peroxidase-labeleds.
2nd, the detection method of sST2 contents
1st, with Na that pH is 9.62CO3,、NaHCO3Coating sST2 captures antibody (2 μ g/mL), as capture antibody, 96
100 μ L, 4 DEG C of coating 12h so as to combine closely with ELISA Plate are added in hole elisa Plates per hole.
2nd, with cleaning solution (PBST) board-washing 6 times after being coated with, per 2% bovine serum albumin of 300 μ L of Kong Zaijia as confining liquid,
37 DEG C of incubation 3h.Closing terminates to add testing protein extract solution in ELISA Plate hole (with testing sample and PBS
Volume ratio is extension rate) and sST2 protein standard substances (per 5 μ g/mL of hole, with 1:5 gradient dilutions to 10pg/mL, each sample
Do three repetitions), 100 μ L/ holes, 37 DEG C of incubation 1h.
3rd, the anti-sST2 detection antibodies of horseradish peroxidase-labeled, 100 μ L/ holes, 37 DEG C of incubation 1h are subsequently added into.
4th, it is last to add developer TMB solution in the compound for being formed, per 100 μ L of hole, 3min is incubated at room temperature, in horseradish
Under peroxide enzyme effect, there is color change in developer, add 2M, H2SO4, 50 μ L/ holes terminating reactions, according to OD450Value is big
The presence of sST2 albumen and concentration in little judgement sample.
Result judgement：Taken the logarithm with the concentration of sST2 protein standard substances and do abscissa, OD450Be worth standard is made for ordinate
Curve (Fig. 2), obtains computing formula, calculates the sST2 contents in testing sample according to the OD values of testing sample.
The preparation of embodiment 3, sST2 immunity colloidal gold test paper strips
1st, the preparation of collaurum
3 milliliter of 1% tetra chlorauric acid is taken in 500 milliliters of round-bottomed flask with 5 milliliters of micropipettors, 297 milliliters are measured
Ultra-pure water also add flask, be configured to 0.01% tetra chlorauric acid reactant liquor, be sufficiently stirred for mixing, be placed in magnetic force heating and stir
Mix on device, heating is boiled.3 milliliters of ammonium citrate solutions are rapidly joined after being sufficiently stirred for, and aqueous solution of chloraurate is changed into ash from gold
Color, became aubergine after about 2 minutes, stopped heating after continuing to boil 5 minutes, after collaurum cooling, was sub-packed in glass reagent
In bottle.
2nd, the preparation of Immuno gold
Protein quality to be marked according to required for the total amount of the collaurum that need to be marked is calculated.With the potassium carbonate of 0.1M
Or the pH value of 0.1M hydrochloric acid regulation collaurum is 7.8, under magnetic stirrer, the sST2 detection antibodies that embodiment 1 is filtered out
It is added dropwise in colloidal gold solution, antibody and colloidal gold reaction add final concentration of 1% under magnetic stirring apparatus after about 5 minutes
Bovine serum albumin (BSA), after 10 minutes, add final concentration of 0.3% PEG2000.Continue reaction 1 hour.To mark
Colloidal gold solution in 2000rpm, 4 DEG C be centrifuged 10 minutes, take supernatant in 10000r/min, 4 DEG C be centrifuged 30 minutes, discard
Clearly, precipitation is dissolved with the 0.01MPBS pH8.2 of original volume, repeated centrifugation three times, the careful supernatant discarded of last time, precipitation
It is dissolved in the 1/50PBS (including 1%BSA) of original volume, obtains Immuno gold.
3rd, prepared by test strips
SST2 capture antibody prepared by embodiment 1 loads the micro spray membranous system of albumen, presses 1 μ on nitrocellulose filter
The amount line of L/cm, obtains detection line；Sheep anti-mouse igg antibody (Sigma, M4280) coating buffer is loaded into the micro spray membrane system of albumen
System, rules by the amount of 1 μ l/cm on nitrocellulose filter, obtains nature controlling line, and 37 DEG C are coated with 2 hours, and 37 DEG C are closed 30 minutes,
Nitrocellulose filter after coating is put in vacuum desiccator and is dried 20 hours, closed to preserve stand-by.
When preparing pad, the OD values for adjusting Immuno gold are 30, and Immuno gold is added in the plastic containers on the machine left side, will
Immuno gold is sprayed in glass fiber conjugate pad, after having sprayed pad, is placed in 37 DEG C of baking ovens and is dried 30 minutes, glass after marking
Glass fiber is put in vacuum desiccator and is dried 20 hours, takes out closed preservation stand-by.
When preparing detection card, tear the adhesive tape being overlying on above in the central authorities of plastic bottom board off, stick the nitric acid for being coated with antibody
Cellulose membrane, tears the adhesive tape of a width of 5 millimeters below plastic plate central authorities off, sticks the pad for spraying collaurum, before pad
The nitrocellulose filter at end overlaps 2 millimeters, tears the bottom a width of 20 millimeters adhesive tape of plastic plate off, sticks the sample through pre-processing
Product pad, the front end of sample pad are Chong Die with pad about 5 millimeters, tear the adhesive tape of about 25 millimeters of the top of plastic bottom board, patch off
Upper blotting paper, blotting paper are Chong Die with nitrocellulose filter about 2 millimeters, after being completed, each accessory are compacted, big by what is assembled
Card cutting machine is cut into the test strips of 3 mm wides, loads in test card, obtains sST2 immunity colloidal gold test paper strips (Fig. 1).
Application of the embodiment 4, sST2 in detection aortic aneurysm dissection of aorta
First, with the method for above-described embodiment 2 and kit to 207 aortic aneurysms altogether and dissection of aorta patient and
In the plasma sample (plasma sample derives from Beijing An Zhen hospital, and patient knows the inside story) of 615 healthy populations, the content of sST2 is carried out
Detection.207 aortic aneurysms and dissection of aorta patient information are referring to table 1, wherein, 21 entitled chronic dissectings aneurysm of aorta
Patient；113 entitled Acute Aortic knurls are without interlayer patient；73 entitled Acute Aortic Dissecting Aneurysm patients.
Testing result is as shown in table 1 and Fig. 3：The intermediate value of healthy population is 4.99ng/mL；Chronic dissecting aneurysm of aorta
The intermediate value of patient is 7.77ng/mL；Intermediate value of the Acute Aortic knurl without interlayer patient is 11.84ng/mL；Aortic Dissection
The intermediate value of aneurysm patient is 18.87ng/mL.Compare with the intermediate value of healthy population sST2 contents (ng/mL), chronic aorta clamp
Layer aneurysm patient, sST2 content (ng/ of the Acute Aortic knurl without interlayer patient and Acute Aortic Dissecting Aneurysm patient
ML) substantially increase.Illustrate sST2 can become diagnosis (positive or negative diagnosis) for aortic aneurysm and/or dissection of aorta,
The biomarker candidate that the monitoring of the monitoring of the course of disease and progress and CHF treatments is used with control level.
The content distribution situation of table 1, detection crowd blood plasma sST2
CI=confidential intervals
2nd, 4- after 9 patients with aortic dissection aneurysm operative treatments is detected with the method described in embodiment 2 and kit
(plasma sample derives from Beijing An Zhen hospital to 108 hours blood plasma, and patient knows the inside story, and all suffers from dissection of aorta and move before corrective surgery
Arteries and veins knurl) in sST2 content, by detecting the content of the sST2 in postoperative patient blood plasma judging the mortality risk state of patient
Size.
As a result it is as shown in Figure 4：After postoperative 24 hours, the sST2 contents of 2,3,9 patients exceed threshold value (64ng/mL),
Judge which has the excessive risk state died of illness；1st, the sST2 contents of 4,5,6,7,8 patients are below threshold value (5ng/mL), judge
Its mortality risk is very low.As a result show：SST2 contents after operation in patients to be measured are postoperative with patients with aortic dissection aneurysm
Mortality risk state size it is related, the sST2 contents after detecting operation in patients to be measured can be determined that the mortality risk shape of patient
State.
Above result of the test illustrates that aortic aneurysm and interlayer morbidity acute and chronic and the order of severity are had with sST2 contents in blood plasma
Close correlation, this causes sST2 to become diagnosis (positive or negative diagnosis) for aortic aneurysm and interlayer, the course of disease and progress
Monitoring and CHF treatments the biomarker candidate that is used of monitoring and control level.
Claims (9)
1. detect that whether the material of solubility ST2 protein contents is preparing diagnosis or auxiliary diagnosis patient to be measured with aortic aneurysm
And/or the application in the product of dissection of aorta；
Or the material of detection solubility ST2 protein contents is preparing prediction or is aiding in prediction aortic aneurysm and/or dissection of aorta
Application in the product of the mortality risk state after operation in patients；
In the amino acid sequence such as sequence table of the soluble ST2 albumen shown in sequence 2.
2. application according to claim 1, it is characterised in that：
The aortic aneurysm and/or dissection of aorta be chronic dissecting aneurysm of aorta, Acute Aortic Dissecting Aneurysm or
Acute Aortic knurl is without interlayer.
3. the application according to claim 1 or 2, it is characterised in that：The material is following 1) -3) shown in any one：
1) solubility ST2 protein antibodies；
2) contain enzyme linked immunological kit 1)；
3) contain colloidal gold strip 1).
4. application according to claim 3, it is characterised in that：The enzyme linked immunological kit also includes Jing enzymes or compound
The soluble ST2 protein antibodies of mark；
The enzyme is horseradish peroxidase or alkaline phosphatase；The compound is biotin molecule.
5. application according to claim 3, it is characterised in that：Soluble ST2 albumen in the colloidal gold strip resists
Soluble ST2 protein antibodies of the body for colloid gold label.
6. it is a kind of detection testing sample in solubility ST2 protein contents enzyme linked immunological kit, including described in claim 3
Soluble ST2 protein antibodies；
The soluble ST2 protein antibodies are solubility ST2 protein monoclonal antibodies；
The monoclonal antibody of the soluble ST2 albumen, its be it is following 1) or 2)：
1) sequence 5 in the amino acid sequence of the weight chain variable district of the monoclonal antibody of the soluble ST2 albumen described in such as sequence table
It is shown；6 institute of sequence in the amino acid sequence such as sequence table of the light chain variable district of the monoclonal antibody of the soluble ST2 albumen
Show；
2) sequence 9 in the amino acid sequence of the weight chain variable district of the monoclonal antibody of the soluble ST2 albumen described in such as sequence table
It is shown；10 institute of sequence in the amino acid sequence such as sequence table of the light chain variable district of the monoclonal antibody of the soluble ST2 albumen
Show.
7. whether the colloidal gold strip of solubility ST2 albumen is contained in a kind of detection testing sample, including institute in claim 3
The soluble ST2 protein antibodies stated；
The soluble ST2 protein antibodies are solubility ST2 protein monoclonal antibodies；
The monoclonal antibody of the soluble ST2 albumen, its be it is following 1) or 2)：
1) sequence 5 in the amino acid sequence of the weight chain variable district of the monoclonal antibody of the soluble ST2 albumen described in such as sequence table
It is shown；6 institute of sequence in the amino acid sequence such as sequence table of the light chain variable district of the monoclonal antibody of the soluble ST2 albumen
Show；
2) sequence 9 in the amino acid sequence of the weight chain variable district of the monoclonal antibody of the soluble ST2 albumen described in such as sequence table
It is shown；10 institute of sequence in the amino acid sequence such as sequence table of the light chain variable district of the monoclonal antibody of the soluble ST2 albumen
Show.
8. a kind of monoclonal antibody of soluble ST2 albumen, its be it is following 1) or 2)：
1) sequence 5 in the amino acid sequence of the weight chain variable district of the monoclonal antibody of the soluble ST2 albumen described in such as sequence table
It is shown；6 institute of sequence in the amino acid sequence such as sequence table of the light chain variable district of the monoclonal antibody of the soluble ST2 albumen
Show；
2) sequence 9 in the amino acid sequence of the weight chain variable district of the monoclonal antibody of the soluble ST2 albumen described in such as sequence table
It is shown；10 institute of sequence in the amino acid sequence such as sequence table of the light chain variable district of the monoclonal antibody of the soluble ST2 albumen
Show.
9. enzyme linked immunological kit according to claim 6 or the colloidal gold strip described in claim 7 or right will
Seek the monoclonal antibody described in 8, it is characterised in that：2 institute of sequence in the amino acid sequence such as sequence table of the soluble ST2 albumen
Show.
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| CN105925705A (en) * | 2016-06-14 | 2016-09-07 | 北京市心肺血管疾病研究所 | Marker of aortic aneurysm |
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Effective date of registration: 20220824 Address after: No. 3-11A78, Building 3-1 Floor, No. 8 Chuangye Road, Haidian District, Beijing 100085 Patentee after: Huaxin Anping (Beijing) Biopharmaceutical Co.,Ltd. Address before: Institute of Cardiopulmonary and Vascular Diseases, Beijing Anzhen Hospital, No. 2 Anzhen Road, Chaoyang District, Beijing 100029 Patentee before: BEIJING INSTITUTE OF HEART LUNG AND BLOOD VESSEL DISEASES |