CN109596839A - People and peptide element fast quantitative measurement method for detecting and kit - Google Patents
People and peptide element fast quantitative measurement method for detecting and kit Download PDFInfo
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Abstract
The present invention provides the method for Full-automatic chemiluminescence method detection CPP based on MPs a kind of, by the way that common be immunized is combined with immune modulatory molecules, assisted delivery cell factor Flt3l, mGM-CSF and CCL20 while conventional common immune, obtain the antibody with high-affinity, and by being combined with Magneto separate, magnetic bead have biggish surface area and area ratio, can effectively enriching low-concentration CPP, enzyme rapidly and sensitively measured to realize CPP in human serum.The minute of CPP, sensitivity and specificity significantly improve.
Description
Technical field
The invention belongs to field of biomedicine, in particular to a kind of people and peptide element fast quantitative measurement method for detecting and kit.
Background technique
It can more quickly be diagnosed to cope with various diseases, the demand of more accurate prognosis evaluation and Treatment decsion,
The research to neoformation marker is expedited the emergence of.Arginine vasopressin (AVP) is the major hormone of hypothalamus-pituitary-adrenal axis
One of.AVP system sensitive can experience the stimulation that body is exposed to endogenous pressure, level can sensitive reaction body in disease shape
Degree is forced under state.However, because AVP is unstable, and it is mainly attached to platelet surface, it is again rapid by body after secretion
It removes, there is query accordingly, with respect to the reliability of AVP detection method.It is a kind of and smart ammonia with peptide plain (copeptin, CPP)
The homologous polypeptide comprising 39 amino acid residues of sour pitressin (AVP) is the C-terminal portion of arginine vasopressin former (pro-VAP)
Divide peptide fragment.After the activation of AVP system, stimulation of CP P is secreted into circulation with the AVP of equimolar amounts from hypophysis after brain.Therefore, CPP is straight
It is reversed to reflect AVP concentration, it can be used as the substitution biomarker of AVP secretion.One studies have shown that CPP ratio circulation cortisol more can
Delicately reflect body stress level.This new biomarker will help to make initial stage policy decision in clinical practice,
The degree of correlation with various acute disease states is shown, such as cerebrovascular events, myocardial infarction, pneumonia, kidney trouble and high blood
It presses, infiltration sexually revises, obesity, even anxiety disorder, major depressive disorder and childhood abuse etc..In the classification of chest pain patients,
Other than troponin, the measurement of CPP is assisted, improves diagnosis performance well.Early stage especially after episode,
The simultaneous determination of troponin and CPP provide significant negative predictive value, almost unrelated with the episode time, therefore help
In safely eliminate myocardial infarction in early days.In the case where acute myocardial infarction AMI (AMI), CPP is discharged from hypophysis rapidly, and several
Normal level is begun return in hour, and troponin T concentration is still normal.Cut-off is diagnosed in the CPP based on 5,000 people
In the research of value, the cut-off value of 99% percentile and 18.9pmol/L as the biomarker of generic definition, and the
97.5 percentiles are 13pmol/L, and the 95th percentile is only 9.8pmol/L.Obviously, the timeliness and sensibility of CPP are monitored
It is extremely important.Only shorten detection time as far as possible while ensuring detection sensitivity, can just make the clinical value of CPP maximum
Change.However, up to the present, being only used for determining CPP concentration, micro-pipe chemiluminescence, radiommunoassay there are four types of technology
(RIA), ELISA (ELISA) and electrochemical analysis.Although their sensitivity can satisfy requirement, when detecting
Between it is too long, therefore it is only used for scientific research, is not used in clinic.Up to now, the real-time test (POCT) of CPP is still not yet
Report, therefore it greatly limits the clinical value of CPP.
Summary of the invention
Based on this, the purpose of the present invention is to provide a kind of people quickly detected that can be used for clinical analysis and peptide element are fast
The kit of fast quantitative detection, and the detection sensitivity of kit and accuracy are high.
To achieve the above object, the present invention provides the following technical scheme that
A kind of kit of people and peptide element rapid quantitative detection includes: people and peptide element monoclonal antibody, it is described it is anti-human and
Peptide element monoclonal antibody is prepared by following methods:
With people and peptide element mice immunized with antigen；To the plasmid of the mouse delivering expression cell factor after being immunized；It collects through matter
The splenocyte or lymph node cells of the mouse of grain delivering, and the splenocyte or lymph node cells are obtained with mouse tumor cell fusion
To hybridoma, culture is secreted anti-human and peptide element monoclonal antibody.
In wherein some embodiments, the people and peptide element antigen are to contain amino acid sequence SEQ ID NO:1, SEQ ID
The polypeptide of NO:2 or SEQ ID NO:3；And/or
The people and peptide element antigen are people and the peptide element antigen that coupling has high molecular weight protein, it is preferable that the macromolecular egg
White is at least one of KLH, OVA, BSA；And/or
The cell factor includes mFlt3l, mGM-CSF and mCCL20.
In wherein some embodiments, the anti-human and peptide element monoclonal antibody and magnetic particle of the high-affinity are coupled, and are made
To capture antibody.
In wherein some embodiments, the kit also includes detection antibody, the detection antibody and the capture
The different epitopes of antibody identification people and peptide element.
In wherein some embodiments, the people of alkali phosphatase enzyme mark and peptide element monoclonal antibody, horseradish peroxidase mark
Any one of the people of note and peptide element monoclonal antibody.
In wherein some embodiments, include in the kit: capture antibody, detection antibody, people and peptide element standard
Product and luminous substrate；
The capture antibody are as follows: the anti-human and peptide element monoclonal antibody of the coated high-affinity of magnetic particle；
The detection antibody are as follows: enzyme mark people and peptide element monoclonal antibody；The luminous substrate is through enzyme mark people and peptide element Dan Ke
It shines after enzymatic on grand antibody；
The different epitopes of the detection antibody and capture antibody the identification people and peptide element.
In wherein some embodiments, the kit use when it is described capture antibody dilution ratio be 1:(50~
100), the dilution ratio of the detection antibody is 1:(50~200).Preferably, enzyme mark people described in the kit and peptide element are single
Clonal antibody is people and the peptide element monoclonal antibody of alkali phosphatase enzyme mark, and the luminous substrate is AMPPD.
The present invention also provides the method for a kind of people and peptide element rapid quantitative detection, specific technical solution is as follows:
A kind of method of people and peptide element rapid quantitative detection, comprising the following steps:
Capture antibody is separately added into sample to be tested, is incubated for；
Detection antibody is added, forms sandwich immunoassay compound, detection；
The people and peptide element standard items for detecting gradient concentration prepare standard curve；
According to standard curve calculate sample to be tested concentration to get；
The capture antibody is that the anti-human and peptide element monoclonal antibody of high-affinity described in claim 1 and magnetic particle are coupled
It obtains；The detection antibody is people and the peptide element antibody of enzyme label, knows the epitope of others and peptide element not with the capture antibody
Together.
In wherein some embodiments, the dilution ratio of the capture antibody is 1:(50~100), the detection antibody
Dilution ratio is 1:(50~200)；Preferably, when a length of 30~50min of the incubation, pH value are 8~9.
It is described to be detected as chemoluminescence method in wherein some embodiments, comprising: by the sandwich immunoassay compound magnetic point
From washing removes extra detection antibody, and luminous substrate AMPPD is added, and mixes, and incubates, and measures luminous value.
Based on the above-mentioned technical proposal, the invention has the following advantages:
The present inventor stumbles on, relative to common immune, will it is common it is immune combined with DNA immunization for
Immune response has significant adjustment effect, while the present invention expresses on the basis of conventional common immune in conjunction with assisted delivery
The plasmid of cell factor improves effective submission of body endoantigen molecule to increase the immune response of immune animal, promotes anti-
Body is effectively mature, and then obtains the antibody with high-affinity, what obtained affinity of antibody was prepared compared with conventional method
Antibody is 10~100 times high；Above-mentioned Antibody preparation is obtained into the kit of people and peptide element rapid quantitative detection, is applied to and exempts from peptide element
It in epidemic disease detection, realizes and substantially reduces detection time on the basis of guaranteeing detection sensitivity, so as to effectively realize and peptide
The POCT of element is applied, and increases the clinical value with peptide element, is provided more effectively reference for Intensive Care Therapy and prognosis treatment and is referred to
Mark.
Detailed description of the invention
Fig. 1 is the anti-human technology of preparing route with peptide element monoclonal antibody of high-affinity；
Fig. 2 is the corresponding monoclonal antibody gel electrophoresis figure being prepared of 8 plants of stable hybridoma positive cells；
Fig. 3 is the titration result figure for the high-affinity antibody that embodiment 1 is prepared；
Fig. 4~7 are the subtype identification result for the high-affinity antibody that embodiment 1 is prepared；
Fig. 8 is the result of the high-affinity antibody that embodiment 1 is prepared and the measurement of common affinity of antibody；
Fig. 9 is the result that high-affinity antibody and common antibody are used for the test of cellular immunity groupization；
Figure 10 is that AP-mAbs and mAb-MPs uses concentration optimization test result；
Figure 11 is the Optimum Experiment result of substrate pH and incubation time；
Figure 12 is the chemiluminescence standard for detecting CPP based on the Full-automatic chemiluminescence method of MPs using high-affinity antibody
Curve graph；
Figure 13 is application dilution rate of recovery appraisal procedure accuracy test result；
Figure 14 is that detection method of the invention and commercially available ELISA kit detect the result being compared.
Specific embodiment
To facilitate the understanding of the present invention, it below with reference to embodiment to invention is more fully described, is given below
Presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to described herein
Embodiment.Purpose of providing these embodiments is makes the disclosure of the present invention more thorough and comprehensive.It should be understood that
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, or according to proposed by manufacturer
Condition.Used various common agents, are commercial product in embodiment.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Used term is intended merely to describe specific reality in the description of the invention
Apply the purpose of example, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more relevant institutes
Any and all combinations of list of items.
The present invention provides the kits of a kind of people and peptide element rapid quantitative detection, which is characterized in that includes: people and peptide
Plain monoclonal antibody, described anti-human and peptide element monoclonal antibody are prepared by following methods:
With people and peptide element mice immunized with antigen；To the plasmid of the mouse delivering expression cell factor after being immunized；It collects through matter
The splenocyte or lymph node cells of the mouse of grain delivering, and the splenocyte or lymph node cells are melted with murine myeloma cell
Conjunction obtains hybridoma, and anti-human and peptide element monoclonal antibody is secreted to obtain in culture.
Optionally, the people and peptide element antigen are to contain amino acid sequence SEQ ID NO:1, SEQ ID NO:2 or SEQ
The polypeptide of ID NO:3.Preferably, people and peptide element antigen are people and the peptide element antigen that coupling has high molecular weight protein, the macromolecular
Albumen is preferably at least one of KLH, OVA, BSA.
In wherein some embodiments, the cell factor includes mFlt3l, mGM-CSF and mCCL20.Preferably, table
Plasmid up to cell factor is pCAGGS.
Preferably, the plasmid to the mouse delivering expression cell factor after being immunized are as follows: building can be thin in eukaryon lactation
The plasmid of cellular expression cell factor passes through the plasmid of the mouse tail vein injection expression cell factor.Specifically, it designs and constructs
To the plasmid of mFlt3l, mGM-CSF and mCCL20, including pCAGGS-mFlt3L, pCAGGS- can be expressed in eukaryon mammalian cell
MGM-CSF and pCAGGS-mCCL20, three kinds of plasmids, being capable of the expression cell factors after by tail vein injection to Mice Body
MFlt3l, mGM-CSF and mCCL20 play immunoregulation effect.Preferably, the expression cell factor is delivered to the mouse after being immunized
Plasmid are as follows: every mouse of pCAGGS-mFlt3L, pCAGGS-mGM-CSF and pCAGGS-mCCL20 each 9~11 μ g, it is more excellent
The mass ratio of selection of land, pCAGGS-mFlt3L, pCAGGS-mGM-CSF and pCAGGS-CCL20 is (0.5~1.5): (0.5~
1.5):1.In some other embodiment, to the plasmid that the later mouse delivering expression cell factor is immunized, it can also pass through
The modes such as particle gun, electroporation, subcutaneous injection, intracutaneous injection, intraperitoneal injection are realized, and best Delivery time, dosage and delivering
Mode is related.
Specifically, immune mouse is repeatedly immune, and the 6th~8 day after being immunized every time carries out a cell factor delivering, often
The secondary immunization interval time is 13~15 days.It is when being immunized, antigen is isometric with Freund's complete adjuvant in some of embodiments
Mixing, after fully emulsified, initial immunity carries out back of mice subcutaneously more site injections according to the amount of every 90~110 μ g, is immunized
Carrying out within the 6th~8 day afterwards can be in the tail vein injection of the plasmid of the expression of eukaryon mammalian cell mFlt3l, mGM-CSF and mCCL20
(pCAGGS-mCCL20, pCAGGS-mFlt3L, pCAGGS-mGM-CSF: each 9~11 μ g/ are only)；It is carried out after another week second
Immune, antigen dosage is 45~55 μ g, cannots be used up full adjuvant substitution Freund's complete adjuvant.Hereafter, the latter Zhou Jinhang of each subcutaneous inoculation
Cell factor delivering.Preferably, it is immunized and the process of cell factor delivering carries out altogether three times.Optionally, exempt from for the second time
After epidemic disease, third time are immune antibody titer measurement can be carried out to mice serum.Preferably, go mouse liver or lymphocyte with it is small
When rat bone marrow tumour cell fusion is prepared before hybridoma 3 days, abdominal cavity booster immunization can be carried out.
In some other embodiment, to the plasmid of the mouse delivering expression cell factor after being immunized, it can also use
3 ligand of mouse tyrosine kinase receptor (the Mouse fms-like tyrosinekinase of direct tail vein injection recombination
3ligand, Flt3l), mouse granulocyte-macrophage colony stimutaing factor (Mouse granulocyte-macrophage
Colony-stimulating factor, mGM-CSF) and 20 (Mouse CC subtype of mouse CC hypotype chemotactic factor (CF)
Chemokine ligand 20, mCCL20).
On the basis of mouse is immunized in above-mentioned immunization strategy, immune mouse spleen and/or lymph node are taken, with mouse
Myeloma cell SP2/0 fusion, obtains hybridoma.Specifically, fused cell is trained in the Selective agar medium containing HAT
It supports, cell culture supernatant antibody titer is detected after 7~10d, screen positive hybridoma cell.Further, using limited dilute
Interpretation of the law is subcloned through 3~4 times, until the cloned culture supernatant antibody positive that individual cells are formed, measures supernatant potency, obtain
Obtain the monoclonal cell strain of stably excreting antibody.The monoclonal cell strain of the stably excreting antibody have can stably excreting it is anti-human
With peptide element monoclonal antibody, and antibody affinity with higher has good antigen rich when for detecting people and peptide element
Collection ability can effectively improve detection sensitivity.
Further, the monoclonal cell strain of hybridoma of aforementioned stable secretory antibody is expanded into culture, induced in vivo
Method prepares ascites.Preferably, by the ascites being prepared centrifugation, the Dan Ke for taking supernatant, purifying plain to get the anti-human and peptide of purifying
Grand antibody.Specifically, described to induce method preparation ascites in vivo are as follows: with every 4.5 × 105A~5.5 × 105A cell abdominal cavity note
Mouse is penetrated, collects ascites after 7~10d；The centrifugation are as follows: 9000r/min~11000r/min is centrifuged 8~12min.Further
Ground, monoclonal antibody purification are the continuous double dilution mouse ascites supernatant of PBS (pH 7.4) using 0.01mol/L, use grape
Pneumococcal proteins A affinity chromatography (is operated) monoclonal antibody purification by reagent specification.After the monoclonal antibody purified, also
Spectrophotometry instrument method can be selected and measure protein concentration after purification, be convenient for subsequent storage and application.
Preferably, in the kit of people and peptide element rapid quantitative detection, the anti-human and peptide element Dan Ke of the high-affinity
Grand antibody and magnetic particle are coupled, as capture antibody.Preferably, the anti-human and peptide element monoclonal antibody of high-affinity passes through the end N
Amino End Group and magnetic particle (MPs) covalent coupling.
Specifically, the anti-human and peptide element monoclonal antibody of high-affinity includes: firstly, with being combined with magnetic particle coupling step
MPs is placed on magnetic force inspissator in washing process and removes supernatant by buffer washing MPs；Then, MPs is resuspended in
In combination buffer, oscillation incubation is overnight, is added the anti-human and peptide element monoclonal antibody solution of high-affinity；After incubation, set
The separation removal supernatant in magnetic force inspissator；Close the residual binding sites on MPs, washing to get the anti-human of high-affinity and
The coated immunomagnetic beads of peptide element monoclonal antibody (mAb-MPs).Preferably, the closing are as follows: sealed with bovine serum albumin(BSA) (BSA)
It closes, it is highly preferred that BSA concentration is 2%~3%, is incubated for 2~3h.Specific surface area of the magnetic-particle (MPs) due to its sessile antibody
It is bigger, have the characteristics that capture rate improves, incubation time shortens, is easily separated, is for the various biology marks of highly sensitive detection
The suitable tools for remembering object, are conducive to the quick detection for further realizing people and peptide element.Moreover, being effectively enriched in for MPs is extremely low dense
The quantity of the catch of people and peptide element are increased under degree, provide guarantee for its highly sensitive, accuracy and specificity.
Further, the kit also includes detection antibody, and the detection antibody and the capture antibody identify people
With the different epitopes of peptide element.Preferably, the detection antibody are as follows: the people of alkali phosphatase enzyme mark and peptide element monoclonal antibody, peppery
Root peroxidase label people and peptide element monoclonal antibody it is any.It is highly preferred that the detection antibody alcaline phosphatase mark
The people of note and peptide element monoclonal antibody.
Preferably, kit of the present invention includes: capture antibody detects antibody, people and peptide element standard items and shines
Substrate；The capture antibody are as follows: the anti-human and peptide element monoclonal antibody of the coated high-affinity of magnetic particle；The detection antibody
Are as follows: enzyme mark people and peptide element monoclonal antibody；The luminous substrate is sent out after the enzymatic on enzyme mark people and peptide element monoclonal antibody
Light；The different epitopes of the detection antibody and capture antibody the identification people and peptide element.When using the kit, sent out using chemistry
Light method detects people and peptide element, and calculates to obtain people and peptide element concentration by calibration curve method.Preferably, capture antibody when use
Dilution ratio be 1:(50~100), it is described detection antibody dilution ratio be 1:(50~200).Preferably, the enzyme mark people
With people and peptide element monoclonal antibody that peptide element monoclonal antibody is alkali phosphatase enzyme mark, the luminous substrate is AMPPD.
The present invention provides the method for a kind of people and peptide element rapid quantitative detection on the basis of mentioned reagent box, including
Following steps: capture antibody is separately added into sample to be tested, is incubated for；Detection antibody is added, forms sandwich immunoassay compound, inspection
It surveys；The people and peptide element standard items for detecting gradient concentration prepare standard curve；The concentration of sample to be tested is calculated according to standard curve, i.e.,
?.Wherein, the capture antibody is that the anti-human and peptide element monoclonal antibody of high-affinity described in claim 1 and magnetic particle are coupled
It obtains；The detection antibody is people and the peptide element antibody of enzyme label, knows the epitope of others and peptide element not with the capture antibody
Together.
Preferably, the dilution ratio of the capture antibody is 1:(50~100), the dilution ratio of the detection antibody is 1:
(50~200).Preferably, when a length of 30~50min of the incubation, pH value are 8~9.
It is further preferred that prepare design parameter when standard curve include: select 1:100 dilution ratio detection antibody and
The capture antibody of the dilution ratio of 1:50, incubation time are the condition of 30~40min, and a series of standard CPP for measuring concentration is molten
Liquid (S0~S9,0~1250pmol/L) constructs the calibration curve of CPP standard items.Wherein, the detection range of the standard curve
For 1.2~1250pmol/L, detection limit (LOD) is 6.25pmol/L, related coefficient 0.9993.
It is described to be detected as chemoluminescence method in wherein some embodiments, comprising: by the sandwich immunoassay compound magnetic
Separation, washing remove extra detection antibody, and luminous substrate AMPPD is added, and mix, and incubate, and measure relative light unit value.Chemistry
Luminescence immunoassay further ensures the high sensitivity of detection method, wide detection range, methodology reliability and relatively stable
Property, convenient for being widely used in routine clinical analysis.
Present invention application magnetic bead is coated with the high-affinity antibody that can recognize CPP c-terminus site, alkalinity as solid phase carrier
The monoclonal antibody in the phosphatase enzyme mark identification another site CPP.It is enterprising in Full-automatic chemiluminescence apparatus using sandwich reaction pattern
Row detection serum and peptide element concentration.The Full-automatic chemiluminescence method established after optimization shows good in human serum sample tests
Good specificity, stability and reproducibility, and substantially reduce detection time (30min).With common commercial ELISA kit
It compares, the result of the CLIA does not show significant difference, it is meant that method constructed by the present invention has good practical value.
The anti-human preparation method with peptide element monoclonal antibody of 1 high-affinity of embodiment
1.1 Peptide systhesis
Synthesis and peptide element antigen polypeptide (cppF, N-cpp, C-cpp) are simultaneously coupled KLH and prepare comlete antigen, obtain KLH-cppF,
KLH-N-cpp,KLH-C-cpp.Amino acid sequence therein are as follows:
CppF:ATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY (SEQ ID NO:1),
N-cpp:CATQLDGPAGALLLRLV (SEQ ID NO:2),
C-cpp:CLAGAPEPFEPAQPDAY (SEQ ID NO:3)
That is the comlete antigen that coupling KLH is obtained are as follows:
KLH-cppF(KLH-C-ATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY)；
KLH-N-cpp(KLH-C-ATQLDGPAGALLLRLV)；
KLH-C-cpp(KLH-C-LAGAPEPFEPAQPDAY)；
It Peptide systhesis and couples using conventional method, gill biochemistry Shanghai Co., Ltd is entrusted to complete.High performance liquid chromatography
(HPLC) method measures purity, equal > 95%.
1.2 plasmid construction
Using pCAGGS carrier, building can be in the matter of eukaryon mammalian cell expression people and peptide element and immunity regulatin remedy molecule respectively
Grain.Immune modulatory molecules are respectively as follows: 3 ligand of mouse tyrosine kinase receptor (mouse Fms-like tyrosinekinase 3
Ligand, mFlt3l), mouse granulocyte-macrophage colony stimutaing factor (mouse granulocyte-macrophage
Colony-stimulating factor, mGM-CSF), 20 (mouse CC subtype of mouse CC hypotype chemotactic factor (CF)
Chemokine ligand 20, mCCL20).It can divide in the plasmid that eukaryon mammalian cell expresses Flt3l, mGM-CSF and CCL20
Not are as follows: 1. pCAGGS-mFlt3L；②pCAGGS-mGM-CSF；③pCAGGS-CCL20.Plasmid commission general strong biological (Wuhan) is raw
The preparation of object Science and Technology Ltd..
1.3 experimental animals and cell line
8~12 week old female BAl BIcs/c mouse is purchased from Nanjing Medical University's Experimental Animal Center.SP2/0 cell line is Guangzhou
Ten thousand, which inspire confidence in biotechnology joint-stock company, freezes, and Human cervical cancer cell lines are purchased from China typical culture collection center.
1.4 reagent
Fu Shi is helped completely, freund 's incomplete adjuvant, (H- hypoxanthine, A- aminopterin-induced syndrome, T- thymus gland are phonetic by selection system HAT
Pyridine), source of people and peptide element, horseradish peroxidase (HRP) label sheep anti-mouse igg, tetramethyl benzidine (TMB) and IgG subclass
It identifies Isotype specific kit (Sigma company)；Staphylococcal protein A (Amersham company)；Immunohistochemistry is used
SABC-AP immunohistochemical kit (Bioengineering Research Institute is built up in Nanjing)；Positive control people and peptide element monoclonal antibody 4081/
4082/4084/4086 (Medix company).
1.5 experimental method
1.5.1 immunization strategy
Antigen KLH-cppF is mixed into (in other embodiments, antigen with Freund's complete adjuvant in equal volume in the present embodiment
It includes the people of KLH-C-cpp sequence and the sequence of peptide element C-terminal that KLH-C-cpp or other, which can also be selected), after fully emulsified,
Back of mice subcutaneously more site injections are carried out according to the amount of every 100 μ g, are initial immunity；Cell factor is carried out after a week to pass
It send: by plasmid (pCAGGS-mCCL20, pCAGGS-mFlt3L, pCAGGS-mGM- of the tail vein injection expression cell factor
CSF: each 10 μ g/ only)；It carries out second after another week to be immunized, antigen dosage is 50 μ g, cannots be used up full adjuvant substitution assistant completely
Agent.Hereafter, cell factor delivering of the latter Zhou Jinhang of each subcutaneous inoculation.It carries out 3 times altogether.It is imitated after two exempt from, three exempt from
Valence measurement.Merge first 3 days progress abdominal cavity booster immunizations.Control group is delivered without cell factor, and other conditions are consistent.
1.5.2 cell fusion, screening and clonal cell line
It is sterile to take immune mouse spleen, lymph node, progress is merged with murine myeloma cell SP2/0 using cell fusion apparatus
Fusion.Fused cell is cultivated in the Selective agar medium containing HAT, with indirect elisa method detection cell training after 7~10d
Object supernatant antibody titer is supported, positive hybridoma cell is screened.It is subcloned using limiting dilution assay through 3~4 times, until individual cells
The cloned culture supernatant antibody positive of formation measures supernatant potency with indirect elisa method, obtains the list of stably excreting antibody
Clonal cell line.Expand culture, dispenses postposition liquid nitrogen cryopreservation.
1.5.3 it merges positive rate and stablizes strain number and calculate
Hybridoma hole count × 100% after positive rate=positive hole count/selection culture
Wherein, stablizing strain number is the cell strain number that finally subclone culture obtains in Single cell fusion.
1.5.4 the preparation of anti-human and peptide element monoclonal antibody
The hybridoma cell strain of acquisition is expanded into culture, induces method preparation ascites in vivo, i.e., with every 5 × 105A cell
Intraperitoneal injection of mice, ascites is collected after 7~10d, and 12000r/min is centrifuged 10min, stays supernatant.Utilize the PBS of 0.01mol/L
(pH 7.4) continuous double dilution mouse ascites supernatant (is operated) with staphylococcal protein A affinity chromatography by reagent specification
Monoclonal antibody purification, Nanodrop2000 spectrophotometry instrument method measure protein concentration after purification, dispense, -70
DEG C save.The technical solution of above-mentioned preparation method and subsequent characterizations is as shown in Figure 1.
2 immune effect of embodiment and antibody titer measurement
Cell factor group: immunization strategy described in embodiment 1: i.e. cell of the latter Zhou Jinhang of each subcutaneous inoculation because
Sub- tail vein injection.
Control group 1: compared with Example 1, without cell factor tail vein injection, remaining step side after being immunized every time
Method is same as Example 1.
Control group 2: compared with Example 1, after the initial immunity of subcutaneous loci injection, pass through tail vein injection after a week
The plasmid of the expression cell factor: pCAGGS-mFlt3L and pCAGGS-mGM-CSF does not inject pCAGGS- that is, compared with experimental group
mCCL20。
Above-mentioned two groups of immune effect comparing result is as shown in table 1.
The comparison of 1 immune effect of table
As shown in Table 1, cell factor group, that is, method described in embodiment 1 is immunized, and immune effect is obviously preferable.Its
In, in cell factor group, 8 plants of stable hybridoma positive cells have been prepared, after it is expanded culture respectively, have induced in vivo
Method prepares ascites, i.e., with every 5 × 105A cell intraperitoneal injection of mice collects ascites, 12000r/min centrifugation after 7~10d
10min stays supernatant.Monoclonal antibody purification (is operated) by reagent specification using staphylococcal protein A affinity chromatography,
Nanodrop2000 spectrophotometry instrument method measures protein concentration after purification.Gel electrophoresis therapy determining antibody purification
Purity.As a result as shown in Figure 2.Wherein, Lane1 Marker, Lane2~Lane9 (9#~16#) are respectively cell factor group institute
Obtain the corresponding monoclonal antibody being prepared of 8 plants of stable hybridoma positive cells.As it can be seen that method preparation described in embodiment 1
The purity of obtained antibody is 90% or more.And the positive hybrid rate of control group 1 is very low, only obtains 1 plant of stable cell strain, and
Control group 2 does not screen the cell strain for stablizing expression.
Coating is added in (1:1000,1:10000,1:100000,1:1000000) after 10 times of gradient dilutions of antibody after purification
In the ELISA reaction plate micropore of people and peptide element, after 37 DEG C of water-bath 30min, secondary antibody (1: 20000 dilution), 37 DEG C of water-baths are added
After 30min, 50 μ L 2mol/L H are added in the colour developing of TMB developing solution2SO4Terminate reaction, microplate reader (Thermo fisher company)
Measure absorbance value at 450nm.As a result as shown in Figure 3.Wherein, OD 450 >=0.3 is considered as the positive.As it can be seen that 9#~16# antibody is pressed
After 1:1000,1:10000,1:100000,1:1000000 dilution, OD 450 be all larger than 0.3,2# antibody (control group 1 gained),
The potency of 9#~16# (obtained by cell factor group) antibody is up to 10-6, with positive control people and peptide element monoclonal antibody
The potency of Medix4084 and Medix4086 is suitable.
3 monoclonal antibody subgroup identification of embodiment
Packet is added in (1:1000,1:10000,1:100000,1:1000000) after 10 times of gradient dilutions of antibody after purification
By in the ELISA reaction plate micropore of people and peptide element, after 37 DEG C of water-bath 30min, it is separately added into IgG subgroup identification Isotype
Subgroup identification secondary antibody (including IgA, IgG1, IgG2a, IgG2b, IgG3, IgM) in specific kit (Sigma company),
After 1: 1000 dilution, after 37 DEG C of water-bath 30min, 50 μ L 2mol/L H are added in the colour developing of TMB developing solution2SO4Terminate reaction, enzyme
Absorbance value at instrument (Thermo fisher company) measurement 450nm is marked, and Subclass of antibody is judged according to result.As a result as Fig. 4~
Shown in 7, the subtype identification result that Fig. 4 is the subtype identification result of 9# antibody, Fig. 5 is 13# antibody, the hypotype that Fig. 6 is 14# antibody
Qualification result, the subtype identification result that Fig. 7 is 15# antibody.Complex chart 4~7 can determine whether the anti-human and peptide that the present invention is prepared
The hypotype of plain monoclonal antibody is IgG1 type.
The measurement of 4 affinity of antibody of embodiment
It is measured using biomembrane interference technique.Avidin sensor solidifies antigen molecule, and antibody gradient dilution passes through
Association and dissociation rate characterizes affinity of antibody size.As a result as shown in Figure 8.Wherein Fig. 8 A is positive control monoclonal antibody
The affinity measurement result of MEDIX 4084, obtains affinity 1.693nM, and Fig. 8 B is the height that the embodiment of the present invention 1 is prepared
The anti-human affinity measurement result with peptide element antibody 9# of affinity, affinity 0.14nM, Fig. 8 C are common in control group 1
The affinity determination of antibody (2#) (being delivered in immunization method without cell factor, remaining step method is same as Example 1)
As a result, its affinity is 19.3nM.As it can be seen that the anti-human and peptide element affinity of antibody that the embodiment of the present invention 1 is prepared is compared with market
Upper common 4084 affinity of control antibodies MEDIX improves 10 times or more, relative to not carrying out the immune of cell factor delivering
The common affinity of antibody that mouse is prepared has more obvious raising.
The test of 5 cellular immunity groupization of embodiment
It is (immune using human cervical carcinoma cell (Hela) and high-affinity antibody (embodiment 1 is prepared), common antibody
Delivered in method without cell factor, remaining step method is same as Example 1) immunohistochemical assay is carried out, as a result such as Fig. 9
It is shown.Wherein, Fig. 9 A, which is the result of high affinity antibody (9#), 9B is negative control, 9C is that control group 1 is prepared common resists
Body (2#) ImmunohistochemistryResults Results, the ImmunohistochemistryResults Results that 9D is control antibodies (MEDIX 4084).As it can be seen that the present invention is prepared
Antibody can be in conjunction with natural and peptide element.For high affinity antibody due to its affinity outstanding, the more common antibody of background colour developing is obvious
It reduces.
Embodiment 6 detects CPP based on the Full-automatic chemiluminescence method of MPs
1, the antibody of CPP different loci is identified
Using with peptide element antigen polypeptide (cppF, N-cpp, C-cpp) and be coupled KLH KLH-cppF, KLH-N- be prepared
Mouse is immunized as comlete antigen in cpp, KLH-C-cpp, and the antibody of identification CPP different loci can be prepared, comprising: identification
The antibody of the antibody in the site N-cpp and the identification site C-cpp, wherein N-cpp site sequence CATQLDGPAGALLLRLV (SEQ
ID NO:2), the position 132-147 of pre-proAVP is represented plus N- terminal cystein residue, C-cpp site sequence
CLAGAPEPFEPAQPDAY (SEQ ID NO:3) represents the position 149-164 of pre-proAVP plus N- terminal cysteine
Residue.
CppF sequence ATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY (SEQ ID NO:1) then represents pre-
The position 132-164 of proAVP.
2, CPP is coated with the preparation of immunomagnetic beads
The high-affinity CPP monoclonal antibody for identifying the site C-cpp is covalent by N-terminal amino and magnetic particle (MPs)
Coupling, as capture antibody.Specific step is as follows:
Firstly, the MPs of 20mg/mL is placed in 2.0mL Ep pipe.It is washed MPs five times with combination buffer.Washed
Pipe is placed on magnetic force inspissator and removes supernatant by Cheng Zhong.Then, MPs is resuspended in 2mL combination buffer.At 37 DEG C
Lower shaken cultivation is stayed overnight, and antibody-solutions are added in above-mentioned suspension with CPP antibody and MPs.After incubation, Ep pipe is placed in magnetic
With by they and supernatant separation in power inspissator.The residual binding sites on MPs are closed with 3% bovine serum albumin(BSA) (BSA),
It is incubated at 37 DEG C simultaneously gentle agitation 2 hours.After washing 5 times, coated immunomagnetic beads (mAb-MPs) are dispersed in 2mL buffer
In and save at 4 DEG C, it is spare.
3, glutaraldehyde coupling method AP (alkaline phosphatase) marks the preparation of CPP antibody
Enzyme mark CPP antibody (AP-mAbs) is prepared in the antibody in the site alkaline phosphatase (AP) marker recognition N-cpp, makees
To detect antibody.Specific step is as follows:
Firstly, AP and anti-CPP antibody are suspended in ultrapure water and are diluted to 4 and 8mg/mL respectively.By 250 μ L aliquots
4mg/mL AP solution is transferred in 1.5mL Ep pipe and mixes with the 250 anti-CPP antibody-solutions of μ L 8mg/mL.Secondly, to solution
The middle 0.1mol/L phosphate buffer (pH7.4) that 0.5mL is added and contains 1% glutaraldehyde.By gained mixture black at 37 DEG C
Slight oscillatory incubates 4 hours in the dark.Third step 0.1mL 1mol/L monoethanolamine solution is added into mixture, then in room
Temperature is incubated under agitation 2 hours lower.Mixture is used into PBS solution dialysed overnight at 4 DEG C.After dialysis, enzyme mark CPP antibody is transferred to
In Ep pipe, and mixed with isometric glycerol and 1%BSA.Finally, enzyme mark CPP antibody (AP-mAbs) is stored in -20 DEG C, it is standby
With.
3, AP-mAbs and mAb-MPs uses concentration optimization
Using magnetic bead coated antibody (mAb-MPs) and enzyme labelled antibody (AP-mAbs) sandwich reaction pattern in full-automatic chemical
CPP detection is carried out on light-emitting appearance.
By the mAb-MP (1:20,1:50,1:100,1:200,1:500) of 50 μ L various concentrations and CPP sample or standard
Product (30 μ L) are pipetted into respectively in the pipe with Instrument Matching, and are gently shaken at 37 DEG C and be incubated for 20 minutes (capture time).Then
Pipe is washed 3 times by cleaning station with cleaning solution (the 0.01mol/L PBS containing 0.05%Twe), and nonspecific knot is removed
It closes.It is then respectively adding the AP-mAbs (1:50,1:100,1:200,1:500) of various concentration, 37 DEG C gently shake 10 points of incubation
Clock.At this point, sandwich immunoassay compound MPs-CPP-AP is formed.By the sandwich immunoassay compound Magneto separate of formation, and pass through washing
Remove excessive AP-mAbs.Then, sandwich complex is added in the solution containing luminous substrate AMPPD (200 μ l).By gained
Mixture incubates in immunoassay instruments, and measures the value of relative light unit (RLU), and the results are shown in Figure 10.As it can be seen that working as
When the thinner ratio of AP-mAbs increases to 1:50 from 1:500, RLUS3/RLUS0Increase (P < 0.05).The thinner ratio of AP-mAbs is 1:
When 50 and 1:100, RLUS3/RLUS0Difference it is not significant.For mAb-MPs, RLUS3/RLUS0 occurs when thinner ratio is 1:50
Peak value, when mAb-MPs amount is reduced to 1:500 from 1:50, RLUS3/RLUS0 decline shows not capture all CPP.When
When the mAb-MPs of 1:50 is added, antigen is sufficiently combined with mAb-MPs, highest RLU occursS3/RLUS0Ratio.When addition is more than
When the mAb-MPs of 1:50 (1:20), excessive mAb-MPs leads to sensitivity decrease, because the particle of mAb-MPs is too close, inhales
The light penetrated is received and dispatched, to stop to shine.Accordingly, it is considered to arrive sensitivity and cost of determination, the dilution of 1:100 and 1:50 is selected respectively
Ratio is used for AP-mAbs and mAb-MPs.
4, Matrix buffer pH and incubation time optimization
PH value and incubative time are also two important parameters, because the acidity of solution greatly influences the work of coated antibody
Property, and too long of incubative time may result in the dissociation of antigen-antibody complex.
The two parameters are assessed by detecting the maximum RLU value of CPP standard sample (S9,1250pmol/L).By AP-
MAbs solution (1:100,50 μ L), mAb-MPs solution (1:50,50 μ L) and CPP standard sample (S9,30 μ L) are incubated at 37 DEG C
0~60 minute.Every 5 minutes washing immune complex MPs-CPP-AP, the chemistry hair with different pH (200 μ L) is then added
Light substrate is to measure RLU.NaOH solution by adding 1mol/L prepares the substrate buffer solution of different pH.Figure 11 show pH and
Incubative time is on photoemissive dynamic (dynamical) influence.As the extension RLU intensity of incubation time gradually increases, until reaching stable
State.When pH is 7.0, RLU value increases from 0~20min, and is subsequently forming unstable platform, and the maximum under the pH
RLU is lower than other values.RLU obviously reaches maximum value at about 9.0 pH, therefore pH value is selected as 9.0.It is maximum for incubation time
RLU minute increase from 0 to 30 with incubation time.However, not changing between 30~50min, show antigen-antibody complex
Formation has reached balance.Therefore, 30min is most suitable incubation time.
Embodiment 8 detects CPP based on the Full-automatic chemiluminescence method of MPs
1, the foundation of standard curve
The dilution ratio of 1:100 and 1:50 is selected to be used for AP-mAbs and mAb-MPs, incubation time is the condition of 30min,
Measure a series of standard CPP solution (S0~S9,0~1250pmol/L) of concentration.The calibration curve of CPP standard items is constructed, such as
Shown in Figure 12.By adding twice of standard deviation to determine minimum detection limit (LOD) average value in 10 holes S0.The inspection of standard curve
Survey range is 1.2~1250pmol/L, and detection limit (LOD) is 6.25pmol/L, related coefficient 0.9993.
2, the Full-automatic chemiluminescence method detection CPP based on MPs is compared with existing common method
Detection method of the present invention can meet clinical diagnosis demand (lower than 18.9pmol/L).By the method and before
Report about CPP detection is compared, to assess the advantage of the method for the invention.As shown in table 2.
The comparison of 2 difference CPP detection method of table
| Method | LOD | Detection range | Assay Time | Reference |
| Tubes CLIA | 2.25pmol/L | 2.25-1215pmol/L | >2h | [1] |
| RIA | ― | 0-2500pmol/L | >24h | [2] |
| ELISA | 19.5pmol/L | 19.5-1250pmol/L | >5h | [3] |
| Electrochemical assay | 37pmol/L | 25-125pmol/L | 5min | [4] |
| Fully automated CLIA | 6.25pmol/L | 1.2-1250pmol/L | 30min | The present invention |
Reference:
[1]N.G.Morgenthaler,J.Struck,C.Alonso,A.Bergmann,Assay for the
Measurement of Copeptin,a Stable Peptide Derived from the Precursor of
Vasopressin,Clinical Chemistry,52(2006)112.
[2]Y.Xiao,Z.G.Zhang,S.B.Chai,etermination of Plasma Copeptin Levels
in Some Cardiacand Pulmonary Disorders,Journal of Radioimmunology,21(2008)97-
100.
[3]X.D.Zhu,J.S.Chen,F.Zhou,Q.C.Liu,G.Chen,J.M.Zhang,Detection of
copeptin in peripheral blood of patients with aneurysmal subarachnoid
hemorrhage,Critical Care,15,6(2011-11-29),15(2011)1-13.
[4]Y.Yang,Development of an Ultrasensitive Electrochemical Method for
Copeptin Content Determination,International Journal of Electrochemical
Science,(2017)6694-6704.
As it can be seen that detection time of the invention only needs 30min well below the method being previously reported, including TubesCLIA (>
2h), RIA (> for 24 hours) and ELISA (> 5h).Shortening detection time is also the prerequisite realized with peptide element POCT.In addition, although
Electrochemical determination method[4]Minute be only 5 minutes, but its LOD be 37pmol/L, be unable to satisfy clinical application requirement, and
LOD in the method for the invention is 6.25pmol/L.To sum up, these results indicate that based on the complete of enrichment with magnetic bead building
Robotics shines and peptide element analysis method has a clear superiority, and can effectively realize and apply with the POCT of peptide element, increases and peptide element
Clinical value, thus for Intensive Care Therapy and prognosis treatment more effective reference index is provided.
Accuracy, accuracy and the stability assessment of 9 detection method of embodiment
The present embodiment assesses the Full-automatic chemiluminescence method detection of the present invention based on MPs using the dilution rate of recovery
The method accuracy of CPP.Steps are as follows for specific experiment:
Five blood serum samples are tested after gradient dilution (at most 1:32), as a result as shown in figure 13.Measured value is multiplied
With dilution gfactor and compared with original undiluted concentration.> 15% deviation is not shown in dilution, in 5 samples.For
The accuracy of confirmation this method, using the CPP standard items of three kinds of various concentrations be measured batch in and difference between batch, wherein 6 times
It is recycled and reused for being measured in 7 days.As shown in table 3, the two CV is below 15%.
The present embodiment is investigated the stability of Full-automatic chemiluminescence method.AP-mAbs is stored at 4 DEG C, at -20 DEG C
Store CPP standard items and after AP-mAbs 20 days, RLUS3/RULS0Value without significant variation.This full-automatic chemical as the result is shown
Luminous excellent properties.
Between 3 batches, table, criticize internal difference analysis
Note.Intraassay and interassay tests were performed using three
different concentrations of CPP standards.CV,coefficient of variation；SD,
standard deviation.
The methods comparison of 10 detection method of embodiment
The validity of the analysis method for further evaluation analyzes 37 serum samples and tries with commercially available ELISA
Agent box is compared.As shown in figure 14, there is good consistency between two methods.The equation of linear regression of data is as follows: Y=
0.58782X+0.94386 (R=0.9813).Data show that measuring method newly developed can be used as the measurement of CPP in blood serum sample.
In the present invention, by combining with Magneto separate, it is quick to realize CPP in human serum, delicately measures.CPP's
Minute, sensitivity and specificity obtain significant improvement.Magnetic bead has biggish surface area and area ratio, can effectively be enriched with
The CPP of low concentration.This method is 1.2~1250pmol/L for the detection range of CPP measurement, and detection is limited to 6.25pmol/L,
Far below the standard requirements of detection CPP (< 18.9pmol/L).In addition, the application of robotics luminescent immunoassay instrument is significantly
Minute (in 30 minutes) are shortened, and are required in other methods more than 180 minutes.This method is used for clinical serum
The detection of CPP in sample detects ELISA kit testing result correlation with higher with commercially available CPP.Therefore, we
Method can provide tool for the quick and accurate detection of CPP in clinical sample.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to the above reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Guangzhou Wondfo Biotech. Co., Ltd.
<120>people and peptide element fast quantitative measurement method for detecting and kit
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<170> SIPOSequenceListing 1.0
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<212> PRT
<213>artificial sequence (Artificial Sequence)
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Ala Thr Gln Leu Asp Gly Pro Ala Gly Ala Leu Leu Leu Arg Leu Val
1 5 10 15
Gln Leu Ala Gly Ala Pro Glu Pro Phe Glu Pro Ala Gln Pro Asp Ala
20 25 30
Tyr
<210> 2
<211> 17
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Cys Ala Thr Gln Leu Asp Gly Pro Ala Gly Ala Leu Leu Leu Arg Leu
1 5 10 15
Val
<210> 3
<211> 17
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Cys Leu Ala Gly Ala Pro Glu Pro Phe Glu Pro Ala Gln Pro Asp Ala
1 5 10 15
Tyr
Claims (10)
1. the kit of a kind of people and peptide element rapid quantitative detection, which is characterized in that include: people and peptide element monoclonal antibody,
Described anti-human and peptide element monoclonal antibody is prepared by following methods:
With people and peptide element mice immunized with antigen；To the plasmid of the mouse delivering expression cell factor after being immunized；Collection is passed through plasmid
The splenocyte or lymph node cells of immune mouse are sent, and the splenocyte or lymph node cells are melted with murine myeloma cell
Conjunction obtains hybridoma, and anti-human and peptide element monoclonal antibody is secreted to obtain in culture.
2. kit according to claim 1, which is characterized in that the people and peptide element antigen are to contain amino acid sequence
The polypeptide of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3；And/or
The people and peptide element antigen are that coupling has people and the peptide element antigen of high molecular weight protein, the high molecular weight protein be KLH, OVA,
At least one of BSA；And/or
The cell factor includes mFlt3l, mGM-CSF and mCCL20.
3. described in any item kits according to claim 1, which is characterized in that the anti-human and peptide element Dan Ke of the high-affinity
Grand antibody and magnetic particle are coupled, as capture antibody.
4. kit according to claim 3, which is characterized in that the kit also includes detection antibody, the inspection
Survey the different epitopes of antibody and capture antibody the identification people and peptide element.
5. kit according to claim 4, which is characterized in that the detection antibody are as follows: the people of alkali phosphatase enzyme mark
Any one of the people marked with peptide element monoclonal antibody, horseradish peroxidase and peptide element monoclonal antibody.
6. described in any item kits according to claim 1~5, which is characterized in that include: capture antibody, detection antibody,
People and peptide element standard items and luminous substrate；
The capture antibody are as follows: the anti-human and peptide element monoclonal antibody of the coated high-affinity of magnetic particle；
The detection antibody are as follows: enzyme mark people and peptide element monoclonal antibody；The luminous substrate is anti-through enzyme mark people and peptide element monoclonal
It shines after enzymatic on body；
The different epitopes of the detection antibody and capture antibody the identification people and peptide element.
7. kit according to claim 6, which is characterized in that the dilution ratio of the capture antibody is 1 when use:
The dilution ratio of (50~100), the detection antibody is 1:(50~200)；And/or
The enzyme mark people and peptide element monoclonal antibody are people and the peptide element monoclonal antibody of alkali phosphatase enzyme mark, the luminous bottom
Object is AMPPD.
8. the method for a kind of people and peptide element rapid quantitative detection, which comprises the following steps:
Capture antibody is separately added into sample to be tested, is incubated for；
Detection antibody is added, forms sandwich immunoassay compound, detection；
The people and peptide element standard items for detecting gradient concentration prepare standard curve；
According to standard curve calculate sample to be tested concentration to get；
The capture antibody is that the anti-human and peptide element monoclonal antibody of high-affinity described in claim 1 and magnetic particle are coupled
It arrives；The detection antibody is people and the peptide element antibody of enzyme label, and it is different to know the epitope of others and peptide element from the capture antibody.
9. the method for people according to claim 7 and peptide element rapid quantitative detection, which is characterized in that the capture antibody
Dilution ratio is 1:(50~100), the dilution ratio of the detection antibody is 1:(50~200)；And/or
When a length of 30~50min of the incubation, pH value are 8~9.
10. the method for people and peptide element rapid quantitative detection according to claim 8 or claim 9, which is characterized in that described to be detected as
Chemoluminescence method, comprising:
By the sandwich immunoassay compound Magneto separate, washing removes extra detection antibody, and luminous substrate AMPPD is added, and mixes,
It incubates, measures luminous value.
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| CN111007270A (en) * | 2019-11-27 | 2020-04-14 | 迪瑞医疗科技股份有限公司 | Peptide-neutralizing chemiluminescence immunoassay kit and preparation method thereof |
| CN111751533A (en) * | 2019-06-19 | 2020-10-09 | 吉林大学 | Kit for establishing chemiluminescence immunoassay detection and peptide |
| CN111896741A (en) * | 2019-07-01 | 2020-11-06 | 吉林大学 | Preparation and detection method of copeptin antibody |
| CN114184603A (en) * | 2021-11-11 | 2022-03-15 | 宁波海壹生物科技有限公司 | Kit for determining copeptin by magnetic particle chemiluminescence method |
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