CN108265034A - Hybridoma cell strain and its anti-CK18-M30 monoclonal antibodies of generation and application - Google Patents
Hybridoma cell strain and its anti-CK18-M30 monoclonal antibodies of generation and application Download PDFInfo
- Publication number
- CN108265034A CN108265034A CN201611271474.5A CN201611271474A CN108265034A CN 108265034 A CN108265034 A CN 108265034A CN 201611271474 A CN201611271474 A CN 201611271474A CN 108265034 A CN108265034 A CN 108265034A
- Authority
- CN
- China
- Prior art keywords
- cell strain
- hybridoma cell
- monoclonal antibody
- hybridoma
- application
- 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.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4741—Keratin; Cytokeratin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
-
- 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
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Abstract
The invention discloses a kind of hybridoma cell strain, deposit number is CCTCC NO：Anti-cell Keratin 18 segment M30 (CK18 M30) monoclonal antibody that C201646 and thus hybridoma cell strain generate.The invention further relates to application of the anti-CK18 M30 monoclonal antibodies in the immune detection tool for preparing CK18 M30, detection reagent, kit or test strips containing anti-CK18 M30 monoclonal antibodies can realize that highly sensitive, high specific quickly detects to CK18 M30 in sample.
Description
Technical field
The present invention relates to immunological technique field, more particularly to prepare a kind of prepare and secrete the monoclonal of anti-CK18-M30 and resist
Body hybridoma cell strain；The monoclonal antibody generated by the hybridoma and its application in the detection of CK18 related liver diseases.
Background technology
Cytokeratin 18 (CK18) is one kind of cytoskeletal protein, in addition to play the supporting role of cytoskeleton with
Outside, intracellular various reactions are also participated in through a variety of ways.Indexs of the CK18 as hepatocellular apoptosis, including non-alcohol
There is raising in many liver diseases of property fatty liver disease (NAFLD), and steatosis with NAFLD Patients ' Hepatocytes,
The degree and fibrosis that lobular inflammation, balloon sample become are related, while have 18 level of serum CK of middle severe fibrosis patients
It is apparently higher than the slight or level without fibrosis patients.
Hepatopathy is to threaten the morbidities such as the significant problem of human health, fatty liver, drug induced hepatic injury, autoimmune hepatopathy
Rate rises year by year, and a considerable amount of above-mentioned hepatopaths finally develop into liver fibrosis.Liver fibrosis is the chronic disease of all livers
The co-channel of disease, early metaphase can be reversed, and late period, that is, hepatic sclerosis is irreversible, finally develops into liver failure threat to life,
Therefore liver fibrosis early diagnosis is significant.
When cell is damaged sexual stimulus generation apoptosis, a plurality of approach is activated, while has activated Caspase systems,
Caspase is cut in two sites, and the cracking of this form is that height apoptosis is special, and apoptosis segment is to protease
The degradation of body is stablized, and can discharge into blood plasma.M30 antigens are located in CK18 crack fragments, and only in crack fragment
Can expose, therefore, M30 antibody for epithelial cell apoptosis with specificity.
Detection means non-invasive as one cytokeratin 18 segment M30 (CK18-M30), diagnosis in NAFLD,
There is important clinical value in the discriminating of NAFL and HASH and the judgement of fibrosis.
Therefore, a kind of monoclonal antibody of anti-CK18-M30 is developed to have great importance.
Invention content
The technical problem to be solved in the present invention is to provide a kind of monoclonal antibody for secreting anti-CK18-M30, is applied to
The detection of CK18-M30.
In order to solve the above technical problems, the present invention provides a kind of hybridoma cell strain, it is preserved in Chinese Typical Representative culture
Collection (referred to as CCTCC), preservation date are on March 6th, 2016, and deposit number is CCTCC NO：C201646.
The present invention also provides a kind of monoclonal antibody HR024-7-6 for specifically binding CK18-M30, by above-mentioned hybridization
Tumor cell strain generates.
The preparation method of monoclonal antibody of the present invention is as follows：
(1) synthesis of artificial antigen：The epitope peptide of artificial synthesized the 389th to the 396th amino acid containing CK18
SEQ ID NO：1.
(2) by epitope peptide SEQ ID NO：1 is coupled respectively with KLH and BSA, and the polypeptide with KLH couplings is as immune
Antigen, the polypeptide with BSA couplings is as screening antigen.
(3) the polypeptide immune mouse that will be coupled with KLH
(4) screening and preparation of monoclonal antibody：The BALB/c mouse spleen cell and SP2/0 cells for taking mouse immune are melted
It closes, limiting dilution assay obtains monoclonal, and ELISA method screening positive hybridoma cell, the envelope antigen of screening is to be coupled with BSA
Polypeptide, it is final to obtain the hybridoma cell strain that can secrete anti-CK18 specific antibodies, be named as HR024-7-6, hypotype is identified
For IgG2a；Mouse ascites are prepared, pass through the anti-CK18-M30 monoclonal antibodies of Protein G affinity chromatography column purifications.ELISA is surveyed
Potency is 1: 320000.
It is another object of the present invention to provide a kind of monoclonal antibody HR024-7-6 to prepare for detecting CK18-
Application in the immune detection tool of M30.
Specifically, the immune detection tool is ELISA reagents, kit or test strips.
Monoclonal antibody specific of the present invention for CK18-M30 protein fragments, improve experiment specificity and
Reliability, and the immunoassay technology based on anti-CK18-M30 monoclonal antibodies is established, it is mainly used for Early hepatic fibrosis, liver
Fatty inflammation, drug induced hepatic injury and autoimmune hepatopathy.
Preservation information
Scientific description for the hybridoma cell strain of preservation is：Anti-human CK18-M30 monoclonal antibody hybridoma cells strain
HR024-7-6；
Depositary institution's full name：China typical culture collection center；
Depositary institution is referred to as：CCTCC；
Depositary institution address：Wuhan City, Hubei Province Wuchang District Luo Jia Shan road Wuhan University；
Preservation date：On March 6th, 2016；
Deposit number：CCTCC NO：C201646.
Description of the drawings
Fig. 1 is the segment M30 epitope peptide amino acid sequences of CK18；
Fig. 2 is the standard curve of CK18-M30 ELISA kits；
Fig. 3 is the standard curve of CK18-M30 fluorescence immune chromatographies.
Specific embodiment
Embodiment 1：The preparation of anti-CK18-M30 hybridomas
1.CK18-M30 the preparation of antigen
1) CK18-M30 epitope peptides is artificial synthesized
The peptide fragment of one section of 10 amino acid of CK18 albumen n ends the 387th to the 396th is chosen, it is artificial synthesized to contain the peptide
The M30 epitope peptides (being synthesized by Jin Wei intelligence bio tech ltd) of section：
SEQ ID NO：1：LEDGEDFNLGDALDS
The CK18 epitope peptides of the present invention have following function：1. it is moved after being connect with carrier protein as immune primary stimuli
Object generates the antibody of specificity；2. it can specifically be combined with antibody prepared by epitope peptide with the M30 segments of CK18 apoptosis.
2) preparation of M30 immunizing antigens
With BDB (Bis-diazotizedbenzidine dichloride) methods by CK18-M30 epitope peptides and carrier
Albumen KLH (keyhole limpet hemocyanin) connections are prepared into M30 antigens.
CK18-M30 epitope peptide 10.0mg are taken, are dissolved with 1ml 0.1M PBS buffer solution (pH7.4)；KLH 10mg are taken, are used
0.2M borate buffer solutions (pH9.0) 20ml dissolves；Then the two is mixed, is cooled to 0 DEG C, takes BDBCl2110 μ L, room temperature
1.5h is reacted, is dispensed after dialysed overnight, -20 DEG C of preservations.
2. animal immune
The CK18-M30 antigens of purifying mix in equal volume with complete Freund's adjuvant, are emulsified, used with the mutual pushing manipulation of double syringe
6-8 week old BALB/c mouses are immunized in subcutaneous or intraperitoneal injection method, and for 50 μ g/ only, interval carries out second to immunizing dose after two weeks
It is secondary immune, it is emulsified with incomplete Freund's adjuvant, immunizing dose is 50 μ g/.It is immune to take tail blood dilute with ELISA method gradient afterwards twice
Release measure serum titer；Booster immunization is determined whether according to result.Carry out within 3 days before fusion booster immunization, every mouse peritoneal note
The 100 μ g antigens for being not added with adjuvant are penetrated, cell fusion is carried out after 3 days.
3. cell fusion
PEG1500 is placed in pre-temperature in 37 DEG C of incubators before fusion, draws 1 × 107A SP2/0 myeloma cell's suspension and
5×107It is a to derive from SPF grades of Balb/c mouse spleen bone-marrow-derived lymphocytes suspensions (cell number 1: 5) to a 50ml centrifuge tube,
30mlIMDM incomplete culture mediums, abundant mixing are added, 1500rpm centrifugation 5min abandon supernatant, flick tube bottom, make cell mass loose
Paste is dissipated into, by 37 DEG C of water-baths of centrifuge tube, the 50%PEG1500 solution of 0.8ml pre-temperatures is drawn with dropper, from tube bottom about 2cm
Place is slowly added into along tube wall in cell, and edged rotation centrifuge tube in side adds in 1min or so, then stands 90s, is added dropwise 37
The incomplete culture medium IMDM8ml of DEG C pre-temperature terminates fusion, is added within 2min, and fast after speed is first slow, action is soft, will centrifuge
Pipe stands 5min in 37 DEG C of incubators, takes out centrifuge tube, and 1000rpm centrifugation 5min discard supernatant, add in 10ml HAT
(SIGMA) cell is resuspended in culture medium, gently blows and beats, mixing, fused cell is seeded to the 96 hole cells for being covered with trophocyte
Culture plate, by 100 μ l/ holes, every piece of culture plate stays 6 holes to be inoculated with SP2/0 cells, as the negative control of HAT selections, puts 37 DEG C,
It is cultivated in 5%CO2 incubators.
4. screening and clone
The growing state of cell can be observed under inverted microscope, and add 100 μ l of HAT culture mediums within the 5th day after fusion,
Hybridoma potency can be surveyed with indirect elisa method within 10th day, change within the 14th day HT (SIGMA) culture medium (containing feeder cells and 1%HT liquid
Body) 96 orifice plates in.It is put into 5%CO2Cell incubator.
One sieve：It chooses after cloning 3 days or so, when observation cell concentration accounts about floor space 2/3,100 μ L supernatants ELISA is taken to sieve
Choosing.Positive colony carries out changing liquid, adds 200 μ L complete mediums (liquid containing 1%HT)；Two sieves：Step above-mentioned steps are repeated after 2 days
Carry out postsearch screening.Positive strain is transferred to 24 orifice plates of the culture medium that is prepared in advance (containing feeder cells and 1%HT liquid).Three sieves：5
It after it, screens again, satisfactory clone is transferred to Tissue Culture Flask and expands culture.
5. hybridoma cell strain preserves
Satisfactory hybridoma cell strain is preserved in China typical culture collection center, address by inventor：Wuhan
City Wuchang Luo Jia Shan Wuhan University, preserving number are CCTCC NO：C201646.
Embodiment 2：It is CCTCC NO by preserving number：The hybridoma of C201646 prepares anti-CK18-M30 monoclonals and resists
Body
1. prepared by ascites
0.5ml atoleines are injected intraperitoneally in the male BALB/c mouse of 10-12 week old, and every mouse is noted with 1ml after a week
The monoclonal cell suspension that emitter intraperitoneal injection is resuspended through PBS washings, cell dosage are 5 × 106/.Treat that mouse ascites gather
After collect ascites.
It is 2. monoclonal antibody-purified
By ascites under the conditions of 4 DEG C, 10000 revs/min centrifuge 10 minutes, remove lipid material.Supernatant is drawn after centrifugation,
And with 0.45 μm of membrane filtration.Protein G are purified.Monoclonal antibody concentration mensuration after purification is dispensed, is frozen at -20 DEG C.
Embodiment 3：ELISA method identifies monoclonal antibody subclass
1. experimental implementation
Coating sheep anti-mouse igg (Zhong Shan Golden Bridge) is diluted with 100mM PBS (pH7.4) and is diluted to 0.5 μ g/mL, adds 100 per hole
μ L, are stayed overnight by 4 DEG C.Liquid is emptied, is washed 3 times with the PBS (PBST) containing 0.05%Tween, the 200 μ L confining liquids of addition per hole, 37 DEG C
It is incubated 1 hour.Liquid is emptied, is cleaned 3 times with PBST.0.1mL hybridoma supematants are added in per hole, 37 DEG C are incubated 1 hour.It is emptied liquid
Body is cleaned 3 times with PBST.Sheep anti mouse (κ, the λ) antibody or 1: 2000 dilution HRP labels of HRP labels are diluted with confining liquid 1: 1000
Sheep anti mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody 0.1mL per hole, be separately added into appropriate hole, 37 DEG C
With incubation 1 hour.Liquid is emptied, is cleaned 3 times with PBS-T.Add 50 μ L substrate solutions per hole, 450nm wavelength is surveyed in 10-20 minutes
Under OD values.
2. experimental result
Experimental result shows that monoclonal antibody of the present invention is IgG2a type mouse resource monoclonal antibodies, is named as HR024-7-6.
Embodiment 4：CK18-M30 ELISA kits
In the present embodiment, using the CK18-M30 monoclonal antibodies obtained in embodiment 1 as the enzyme mark in this kit
Antibody is remembered, using homemade CK18 polyclonal antibodies as coated antibody.
The preparation and operation of CK18-M30 external diagnosis reagent cases are as follows：
1. the preparation of various buffer solutions and reagent：
A, it is coated with buffer solution：The CB (carbonate buffer solution) of 0.050M, pH9.6
B, sample/washing buffer：10 × PBS-Tween20 of pH7.2
C, enzyme marker dilution：10 × PBS-Tween20 of pH7.2, containing 2%FCS, 0.1% enzyme stabilizers, 0.02%
Procl in300
D, color developing agent A：
Citric acid：35.5 grams
Urea peroxide：10 grams
It distills water-soluble to 1000ml
Tween20：10ml
E, color developing agent B：
Citric acid：120 grams
EDTA-2Na：1 gram
TMB·2HCl：2 grams
It distills water-soluble to 1000ml
F, terminate liquid：2M H2SO4
2. the preparation of pre-coated plate：
CK18 polyclonal antibodies are dissolved in the carbonate buffer solution of the 0.05M of pH=9.6, pre-coated liquid are made, in enzyme
100 μ l are added in by 0.1 μ g/ holes per hole on target, 4 DEG C is put and places 20 hours, take out, get rid of coating buffer, wash, closed through BSA
16 hours, be dried overnight after be fitted into aluminide-coating bag and vacuumize sealing, be placed in 4 DEG C of preservations.
3.CK18-M30 monoclonal antibody is coupled with HRP.
5mg HRP and 10mg CK18-M30 monoclonal antibodies are weighed, are coupled using sodium periodate improved method.
4. the composition of kit：
Pre-coated plate：48/96 hole
CK18-M30 standard items：Standard items 7 are (respectively：0mU/ml, 75mU/ml, 150mU/ml, 250mU/ml,
500mU/ml, 750mU/ml, 1000mU/ml).
Enzyme labelled antibody：1 × 10ml (through 1: 5000 dilution)
Concentrated cleaning solution (25 × PBS-Tween20)：1×20ml
Color developing agent A：1×6.0ml
Color developing agent B：1×6.0ml
Terminate liquid：1×6.0ml
5. the operating procedure of kit：
100 μ l/ holes of blood sample and standard items to be checked are separately added into each hole of pre-coated plate, are diplopore, 37 DEG C incubate
It educates 60 minutes, is washed 5 times, patted dry with washing buffer.The 100 μ l/ holes of HR024-7-6 monoclonal antibodies of HRP labels are added in each hole,
37 DEG C are incubated 30 minutes, are washed 5 times, patted dry with 1 × washing buffer.Add in enzyme-linked 100 μ l/ holes of object in each hole again, 37 DEG C
It is incubated 30 minutes, is washed 5 times, patted dry with 1 × washing buffer.Color developing agent A, B liquid is added in, per each 50 μ l in hole, mixing, 37 DEG C incubate
It educates 15 minutes.50 μ l/ holes of terminate liquid is added to terminate reaction, absorbance is detected with enzyme detector dual wavelength (450nm, 620nm).
6. result judgement：
Table 1：ELISA standard concentrations and corresponding mean light absorbency (OD) value
ELISA kit CK18-M30 standard concentrations 0mU/ml, 75mU/ml, 150mU/ml, 250mU/ml, 500mU/
Ml, 750mU/ml, 1000mU/ml mean OD value are 0.085,0.182,0.300,0.508,1.087,1.642,2.354, with mark
Quasi- product concentration and corresponding absorbance carry out quadratic polynomial fitting, draw standard curve, R2=0.9989, see Fig. 2.According to standard
Curve calculates detected sample CK18-M30 concentration results.
Serum CK 18-M30 detections, NAFLD patient's people's blood are carried out in a manner described to 30NAFLD patient and 81 healthy persons
CK18-M30 contents in clear are apparently higher than healthy control group, and difference is statistically significant (P ＜ 0.01), is shown in Table 2.
Table 2：Two groups of sample CK18 concentration compare
Embodiment 5：CK18-M30 fluorescence immune chromatography test paper bars (card)
1. fluorescent microsphere labelled antibody
1) activation of fluorescent microsphere
Take fluorescent microsphere (purchased from Guangzhou Growth hormone secretagogue company) aqueous dispersions of 500 μ l, content 1 (w/v) %, at 10 DEG C with
12000rpm is centrifuged 10 minutes, removes supernatant, sediment is distributed to the distilled water of 500 μ l or first wash buffer (the MES water of 0.1M
Solution) in, ultrasonic wave (240W) is handled 2 minutes, repeats above procedure three times, adds in carbodiimide 50mg, is stirred 15 minutes, from
And activate the fluorescent microsphere.
2) with the fluorescent microsphere labelled antibody activated
The fluorescent microsphere activated under 12000rpm is centrifuged 10 minutes, removes supernatant, sediment is distributed to 1ml couplings
In buffer solution (citrate buffer solution of the N- hydroxy thiosuccinimides of 50mM), ultrasonic wave (240W) is handled 2 minutes, is repeated
Above procedure three times, obtains the buffer solution 1ml for being dispersed with fluorescent microsphere.It has been activated according to 3 μ l antibody (8mg/ml)/100 μ l
The ratio of fluorescent microsphere adds in CK18 monoclonal antibodies thereto, stirs 2 hours at normal temperatures, adds in 1ml Block buffers (1
(w/v) %BSA-0.05M ethanol amines), continue stirring 1 hour, later, centrifuged 10 minutes under 12000rpm, repeated centrifugation 3
It is secondary, sediment is distributed in 500 μ l ends wash buffers (0.5 (w/v) %BSA-0.11 (v/v) % Tween solutions), ultrasound
Wave (240W) is handled 2 minutes, and 500 μ l are settled to above-mentioned whole wash buffer.
2. it is coated with the preparation of bonding pad
The fluorescent microsphere that the label of above-mentioned preparation has antibody is diluted to 1mg/ml with antibody diluent, obtains work
Liquid, the amount even application for then pressing 4 μ l/cm with micropipettor is dried on bonding pad with 37 DEG C of baking ovens later, wet 45%
It is saved backup under degree.
3. the preparation of reaction film
Anti- CK18-M30 monomers HR024-7-6 and the sheep anti-mouse igg monoclonal antibody PBS buffer solution of 50mM pH7.2 are distinguished
1mg/ml is diluted to, the detection line of metal spraying machine and nature controlling line spacing parameter are set as 6mm, package amount is respectively set to 1.0 μ
L/cm draws CK18-M30 monoclonal antibodies (coating T detection lines) and sheep anti-mouse igg list with metal spraying machine on nitrocellulose filter
Clonal antibody (coating C detection lines), room temperature dries spare.
4. the assembling and cutting of test paper
Mutually overlap joint pastes sample pad, bonding pad, reaction film and absorbent filter successively on bottom plate, obtains test paper plate, will
It cuts into the test strips that width is 5mm.
The preparation of 5.CK18-M30 fluorescence immunoassays detection card：
The test paper of above-mentioned well cutting is fixed on plastic bottom card, test paper surface is compressed with face card, and face is stuck in test strips
Well and observation window are provided on the position of sample pad and reaction film.Detection card is fitted into after assembling in aluminium foil bag, adds in drying
Agent sealing preserves, and can be preserved 1 year or more under the conditions of drying at room temperature.
6. standard curve
Fluorescence immune chromatography CK18-M30 standard concentrations are set as：0mU/ml、30mU/ml、90mU/ml、300mU/ml、
50 μ l more thans calibration objects are added drop-wise on well, are examined after 15 minutes with fluorescence detector by 600mU/ml, 1200mU/ml respectively
It surveys, fluorescence can be collected on detection line T and nature controlling line location of C.Quadratic polynomial fitting is carried out with sample concentration and T/C values,
Draw standard curve, R2It is 0.9985, sees Fig. 3.Nature controlling line is made corresponding for test paper Effective judgement and to detection line signal
Correction if nature controlling line does not occur band, then illustrates that test paper fails.
Table 3：Fluorescence immune chromatography standard concentration and corresponding average signal value
7. sample detection
The blood sample to be checked of 50 μ l is taken, is added drop-wise on well, is detected after 15 minutes with fluorescence detector, if detection line goes out
Existing band illustrates to obtain according to calibration curve containing CK18, concentration in sample.
Claims (6)
1. a kind of hybridoma cell strain, on March 6th, 2016 precious deposits in China typical culture collection center, deposit number
For CCTCC NO：C201646.
2. a kind of monoclonal antibody of anti-CK18-M30 is secreted by hybridoma cell strain described in claim 1 and generated.
3. application of the monoclonal antibody in CK18-M30 immune detection tools are prepared described in claim 2.
4. application according to claim 3, which is characterized in that the immune detection tool is reagent, kit or test paper
Item.
5. the monoclonal antibody described in claim 2 is preparing the application in detecting CK18 related liver diseases diagnostic reagents.
6. a kind of preparation method of hybridoma cell strain described in claim 1, which is characterized in that include step：
1) the epitope peptide SEQ ID NO of artificial synthesized the 389th to the 396th amino acid containing CK18：1；
2) by epitope peptide SEQ ID NO：1 is coupled respectively with KLH and BSA, with the polypeptides of KLH couplings as immunizing antigen,
Polypeptide with BSA couplings is as screening antigen；
3) the polypeptide immune mouse that will be coupled with KLH；
4) myeloma cell's suspension and the spleen bone-marrow-derived lymphocyte of immune mouse carry out cell fusion；
5) cell after culture fusion, surveys hybridoma potency, screens positive cell strain, obtain the hybridoma cell strain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611271474.5A CN108265034A (en) | 2016-12-30 | 2016-12-30 | Hybridoma cell strain and its anti-CK18-M30 monoclonal antibodies of generation and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611271474.5A CN108265034A (en) | 2016-12-30 | 2016-12-30 | Hybridoma cell strain and its anti-CK18-M30 monoclonal antibodies of generation and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108265034A true CN108265034A (en) | 2018-07-10 |
Family
ID=62771387
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611271474.5A Withdrawn CN108265034A (en) | 2016-12-30 | 2016-12-30 | Hybridoma cell strain and its anti-CK18-M30 monoclonal antibodies of generation and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108265034A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110003320A (en) * | 2019-03-22 | 2019-07-12 | 深圳市龙华区人民医院 | Immunogenic polypeptide and anti-TTC36 antibody BP1-7 and application |
| CN110988357A (en) * | 2019-12-03 | 2020-04-10 | 南方医科大学南方医院 | Marker for early hepatic fibrosis diagnosis, kit and application |
| CN111269314A (en) * | 2019-12-31 | 2020-06-12 | 苏州和锐生物科技有限公司 | anti-CK 18 monoclonal antibody and application thereof |
| EP4047017A1 (en) * | 2021-02-22 | 2022-08-24 | Sysmex Corporation | Monoclonal antibody, measurement reagent for cytokeratin 18 fragment, reagent kit, and measurement method |
| CN116987185A (en) * | 2023-05-25 | 2023-11-03 | 首都医科大学附属北京佑安医院 | Keratin18 monoclonal antibody, hybridoma cell strain and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020081310A1 (en) * | 1997-09-30 | 2002-06-27 | Peviva Ab | Apoptosis-related compounds and their use |
| CN102453093A (en) * | 2010-10-28 | 2012-05-16 | 华中农业大学 | Screening and application of single-chain antibody against fumonisin |
| CN102838676A (en) * | 2012-09-26 | 2012-12-26 | 李彬 | Carcino-embryonic antigen monoclonal antibody, chip containing same and application |
| CN103421742A (en) * | 2013-05-31 | 2013-12-04 | 华中农业大学 | Recombinant monoclonal antibodies H2 resisting fumonisins B1 |
| WO2015143277A1 (en) * | 2014-03-20 | 2015-09-24 | Wellstat Diagnostics, Llc | Antibodies and methods for the detection of cell death |
| CN105592846A (en) * | 2013-03-15 | 2016-05-18 | 持田制药株式会社 | Compositions and methods for treating non-alcoholic steatohepatitis |
| CN106866820A (en) * | 2017-02-17 | 2017-06-20 | 艾比玛特医药科技（上海）有限公司 | A kind of monoclonal antibody and its application for capturing the anti-human Keratin 18 of tumour cell |
| CN108267586A (en) * | 2016-12-30 | 2018-07-10 | 苏州和锐生物科技有限公司 | A kind of people CK18 protein assay reagents and preparation method thereof |
| CN111269314A (en) * | 2019-12-31 | 2020-06-12 | 苏州和锐生物科技有限公司 | anti-CK 18 monoclonal antibody and application thereof |
-
2016
- 2016-12-30 CN CN201611271474.5A patent/CN108265034A/en not_active Withdrawn
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020081310A1 (en) * | 1997-09-30 | 2002-06-27 | Peviva Ab | Apoptosis-related compounds and their use |
| CN102453093A (en) * | 2010-10-28 | 2012-05-16 | 华中农业大学 | Screening and application of single-chain antibody against fumonisin |
| CN102838676A (en) * | 2012-09-26 | 2012-12-26 | 李彬 | Carcino-embryonic antigen monoclonal antibody, chip containing same and application |
| CN105592846A (en) * | 2013-03-15 | 2016-05-18 | 持田制药株式会社 | Compositions and methods for treating non-alcoholic steatohepatitis |
| CN103421742A (en) * | 2013-05-31 | 2013-12-04 | 华中农业大学 | Recombinant monoclonal antibodies H2 resisting fumonisins B1 |
| WO2015143277A1 (en) * | 2014-03-20 | 2015-09-24 | Wellstat Diagnostics, Llc | Antibodies and methods for the detection of cell death |
| CN108267586A (en) * | 2016-12-30 | 2018-07-10 | 苏州和锐生物科技有限公司 | A kind of people CK18 protein assay reagents and preparation method thereof |
| CN106866820A (en) * | 2017-02-17 | 2017-06-20 | 艾比玛特医药科技（上海）有限公司 | A kind of monoclonal antibody and its application for capturing the anti-human Keratin 18 of tumour cell |
| CN111269314A (en) * | 2019-12-31 | 2020-06-12 | 苏州和锐生物科技有限公司 | anti-CK 18 monoclonal antibody and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| MATHIE P.G.LEERS等: "Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis", 《THE JOURNAL OF PATHOLOGY》 * |
| PATUMA SINTHUJAROEN等: "Elavation of serum fortilin levels is specific for apoptosis and signifies cell death in vivo", 《BBA CLINICAL》 * |
| 魏飞力等: "细胞角蛋白18凋亡片段M30单克隆抗体的制备及其在慢性乙型肝炎肝损伤检测中的初步应用", 《中华临床医师杂志(电子版)》 * |
| 鲁大鹏等: "细胞角蛋白18及其基因在牙源性角化囊肿衬里上皮中表达的意义", 《华西口腔医学杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110003320A (en) * | 2019-03-22 | 2019-07-12 | 深圳市龙华区人民医院 | Immunogenic polypeptide and anti-TTC36 antibody BP1-7 and application |
| CN110988357A (en) * | 2019-12-03 | 2020-04-10 | 南方医科大学南方医院 | Marker for early hepatic fibrosis diagnosis, kit and application |
| CN110988357B (en) * | 2019-12-03 | 2023-10-03 | 南方医科大学南方医院 | Marker for early liver fibrosis diagnosis, kit and application |
| CN111269314A (en) * | 2019-12-31 | 2020-06-12 | 苏州和锐生物科技有限公司 | anti-CK 18 monoclonal antibody and application thereof |
| EP4047017A1 (en) * | 2021-02-22 | 2022-08-24 | Sysmex Corporation | Monoclonal antibody, measurement reagent for cytokeratin 18 fragment, reagent kit, and measurement method |
| CN116987185A (en) * | 2023-05-25 | 2023-11-03 | 首都医科大学附属北京佑安医院 | Keratin18 monoclonal antibody, hybridoma cell strain and application thereof |
| CN116987185B (en) * | 2023-05-25 | 2024-01-23 | 首都医科大学附属北京佑安医院 | Keratin18 monoclonal antibody, hybridoma cell strain and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108265034A (en) | Hybridoma cell strain and its anti-CK18-M30 monoclonal antibodies of generation and application | |
| CN111620945B (en) | Monoclonal antibody or derivative thereof for resisting novel coronavirus | |
| CN107438621B (en) | anti-PD-1 antibodies | |
| US9333255B2 (en) | Methods of enhancing antibody-based antiviral therapies by administering KIR2DL-binding antibodies | |
| CA2406240C (en) | Hcv anti-core monoclonal antibodies | |
| TW201920279A (en) | B7-H4 antibodies and methods of use thereof | |
| CN102143978B (en) | Anti- P2X7 peptides and epitopes | |
| CN112794899B (en) | Fully human monoclonal neutralizing antibody for resisting novel coronavirus and application thereof | |
| EP2174957A1 (en) | Monoclonal antibodies binding to avian influenza virus subtype h5 haemagglutinin and uses thereof | |
| UA128057C2 (en) | Humanized or chimeric cd3 antibodies | |
| CA2637837A1 (en) | Monoclonal antibodies binding to avian influenza virus subtype h5 haemagglutinin and uses thereof | |
| Herlyn et al. | Specific detection of anti-idiotypic immune responses in cancer patients treated with murine monoclonal antibody | |
| JP2009536641A (en) | VCAM-1-specific monoclonal antibody | |
| US11084869B2 (en) | Antibody-mediated neutralization of Marburg virus | |
| US20060240006A1 (en) | Novel antibody structures derived from human germline sequences | |
| CN111518202B (en) | Novel coronavirus antibody and ELISA detection kit for same | |
| CN109734801A (en) | A kind of Preparation method and use of GII.4 type norovirus broad-spectrum monoclonal antibody | |
| CN104220090A (en) | Enterovirus 71 specific antibodies and uses thereof | |
| Skvaril et al. | Characterization of the subclasses and light chain types of IgG antibodies to rubella. | |
| CN111269314A (en) | anti-CK 18 monoclonal antibody and application thereof | |
| WO2022267936A1 (en) | Antibody specifically bound to glycosylated ceacam5 | |
| CN106749645B (en) | A kind of neutralizing antibody of full source of people anti-hepatitis c virus | |
| CN112094346B (en) | Monoclonal antibody of mouse anti-cell single-chain transmembrane glycoprotein CD142 capable of being applied to tumor cell capture | |
| KR102021537B1 (en) | A rapid diagnosis kit for diagnosis of yellow fever virus infection and a yellow fever virus detection method using thereof | |
| CN112094345B (en) | Mouse anti-immunoglobulin associated beta CD79b monoclonal antibody applicable to tumor cell capture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20180710 |