CN105949312A - Anti-human pct antibody and application thereof - Google Patents
Anti-human pct antibody and application thereof Download PDFInfo
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- CN105949312A CN105949312A CN201610397039.0A CN201610397039A CN105949312A CN 105949312 A CN105949312 A CN 105949312A CN 201610397039 A CN201610397039 A CN 201610397039A CN 105949312 A CN105949312 A CN 105949312A
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- 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
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/585—Calcitonins
Abstract
The invention relates to an anti-human procalcitonin (PCT) antibody and application thereof. A variety of antibodies are prepared, matched and screened, and an antibody combination with the sensitivity and specificity meeting requirements is obtained; meanwhile, mass production is convenient, and the requirement for large-scale clinic application in the future can be met. Debugging and optimization work of a detection system is carried out on the antibody combination, and a human PCT time-resolved immunofluorescence chromatography quantitative test card which is easy and convenient to operate is obtained, wherein the sensitivity, specificity and relevant detection performance can meet human clinic sample detection.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of anti-human Procalcitonin. (PCT) antibody and preparation method thereof and
The application in the former detection of human calcitonin of the above-mentioned antibody.
Background technology
Procalcitonin. (Procalcitonin, PCT) is a kind of glycoprotein without hormonal activity, by katacalein, calcitonin, N end
Residue segment composition, be the precursor of calcitonin (Calcitonin, CT), relative molecular weight 13KDa, 25-30 hour half-life.
Under physiological condition, PCT is mainly produced by the other C cell of thyroid follicle；Under pathological state, it is many that PCT can derive from liver, lung etc.
Plant organ and tissue.
PCT has extensive and important using value clinically.Human normal plasma PCT content is extremely low, in normal human blood
Less than 0.5ng/ml.When there is serious bacterial, fungus, parasitic infection and sepsis and MOFE, it
Level in blood plasma raises；And there is autoimmune, allergy and local infection, virus infection, chronic nonspecific inflammation, cancer
During the diseases such as disease heating, graft host rejection reaction, PCT level will not raise；This just determines the high degree of specificity of PCT,
Therefore can be used for the Differential Diagnosis of various clinical setting.Additionally, PCT concentration becomes positive correlation with severity of inflammation, and along with inflammation
The control of disease and the alleviation of the state of an illness and be reduced to normal level, thus PCT again can be as the reliability index judging the state of an illness and prognosis.
PCT can detect after 2 hours after infection, significant to clinical early diagnosis, within 12～24 hours, reaches after infection
To peak.
Laboratory method conventional for detection PCT at present has radioimmunology analytic process (RIA), chemiluminescence immunoassay
(CLIA), gold colloidal colorimetry (GICA) and euzymelinked immunosorbent assay (ELISA) (ELISA).Radioimmunology analytic process is to utilize artificial conjunction
The polyclonal antibody become identifies specifically and connects Procalcitonin..The method can detect blood-serum P CT of normal person, but detection is
Free PCT, conjunction type PCT and the mixture of calcitonin-gene-related peptide precursor, and cannot distinguish between above-mentioned three kinds of materials；And deposit
In detection time-consuming long (19～22h), have the pollution of radioelement, label poor stability, garbage be difficult to the shortcomings such as process and
Make it apply to be restricted.Chemiluminescence immunoassay (CLIA) is it is generally required to the fully automatic electric chemiluminescence detection of supporting costliness
Instrument, Routine Test Lab cannot be carried out, and bring unnecessary expense to patient, increase patient burden.And utilize biotin-
The electrochemiluminescence immune reagent kit of Avidin system detection PCT, it is also desirable to the fully automatic electric chemiluminescence detector of supporting costliness,
Routine Test Lab cannot be carried out, and brings unnecessary expense to patient, increases patient burden.The time resolution of the present invention is exempted from
Epidemic disease fluorescent chromatographic quantitative test card can meet quickly detection and the demand of bedside detection, and without professional and technical personnel.
Summary of the invention
The technical problem to be solved in the present invention is to provide the antibody that energy human calcitonin effective, specific binding is former.More specifically:
The first object of the present invention is to provide a kind of anti-human Procalcitonin. antibody,
Following complementary determining region is contained in its variable region of heavy chain: aminoacid sequence HCDR1 as shown in SEQ ID NO:1, as
HCDR2 shown in SEQ ID NO:2 and/or the HCDR3 as shown in SEQ ID NO:3；
And its light-chain variable sequence contains following complementary determining region: aminoacid sequence is as shown in SEQ ID NO:4
LCDR1, the LCDR2 as shown in SEQ ID NO:5 and/or the LCDR3 as shown in SEQ ID NO:6.
Preferably the aminoacid sequence of the variable region of heavy chain of the antibody in the present invention is as shown in SEQ ID NO:7, variable region of light chain
Aminoacid sequence as shown in SEQ ID NO:8.
Second purpose of the present invention is to provide a kind of single-chain antibody, the aminoacid sequence of described single-chain antibody such as SEQ ID NO:11
Shown in.
The 3rd purpose of the present invention is to provide a kind of nucleotide sequence encoding above-mentioned single-chain antibody, the nucleotide of coding single-chain antibody
Sequence is as shown in SEQ ID NO:10.
The 4th purpose of the present invention is to provide a kind of expression vector containing above-mentioned nucleotide sequence.
The 5th purpose of the present invention is to provide a kind of recombinant host cell containing above-mentioned expression vector.Affiliated host cell can be
Escherichia coli, yeast or mammalian cell, preferably Pichia sp..
The 6th purpose of the present invention is to provide a kind of method producing above-mentioned single-chain antibody, including:
1) cultivate above-mentioned recombinant host bacterium under suitable conditions and express antibody；
2) then from Host Strains purification, collect antibody.
7th purpose of the present invention is the application providing above-mentioned anti-human Procalcitonin. antibody in the detection former content of human calcitonin.
8th purpose of the present invention is to provide a kind of pairing to detect the antibody pair that human calcitonin is former: commodity purchasedization anti-fall calcium
Element original antibody (being purchased from the PCT antibody of Hytest company entitled 42, Cat.#4PC47, the named H42 of the application) and this
Bright anti-human Procalcitonin. antibody；This antibody is high to the detection sensitivity of combination, and specificity is good.
9th purpose of the present invention is to provide a kind of time utilizing described anti-human Procalcitonin. antibody test human calcitonin former to divide
Distinguish that immunofluorescence chromatographs quantitative test card, including sample pad, fluorescence pad, reaction film and adsorptive pads；Described fluorescence pad
It is coated with the anti-human Procalcitonin. antibody of the present invention of fluorescent microsphere labelling, described reaction film has detection band and quality control band, detection
Band position is coated with antibody H42, and quality control band position is coated anti-His tag antibody or Protein L.The preferred nitric acid of described reaction film
Cellulose membrane.Described anti-His tag antibody preferred mouse-anti His antibody.
The present invention is prepared for Multiple Antibodies, and carries out pairing screening with commercial antibodies, it is thus achieved that sensitivity and specificity all can meet
The Antibody Combination (the anti-human Procalcitonin. antibody of the present invention and H42) of demand；Its convenient a large amount of productions, can meet the biggest simultaneously
The demand of scale clinical practice.Above-mentioned Antibody Combination is carried out the debugging Optimization Work of detection system, it is thus achieved that easy and simple to handle, sensitive
Degree, specificity and coherent detection performance all can meet people's clinical sample detection human calcitonin former time resolution immunofluorescence chromatography
Quantitative test card.
Accompanying drawing explanation
Fig. 1. heavy chain of antibody and chain variable region gene electrophoretogram.Lane 1 is 200bp DNA Ladder；Lane 2 is the present invention's
Anti-human Procalcitonin. antibody heavy chain variable region DNA；Lane 3 is the anti-human Procalcitonin. antibody chain variable region DNA of the present invention.
Fig. 2. single-chain antibody structural representation.VHRepresent weight chain variabl area sequence, VLRepresenting light-chain variable sequence, His label is six
Individual histidine.
Fig. 3. the agarose gel electrophoresis figure of single-chain antibody PCR primer.
Fig. 4. single-chain antibody recombinant pichia yeast strain abduction delivering supernatant culture fluid SDS-PAGE electroresis appraisal figure；Arrow institute in figure
Refer to be single-chain antibody.
Fig. 5 .SDS-PAGE gel electrophoresis purification Identification single-chain antibody.
Fig. 6. time resolution immunofluorescence of the present invention chromatography quantitative test card structural representation.1 be sample pad, 2 for reaction film, 3 for inhale
Receive pad, 4 be nature controlling line (C line), 5 for detection line (T line), 6 for fluorescence pad, 7 be PVC sheet.
Fig. 7. time resolution immunofluorescence of the present invention chromatography quantitative test card detection range matched curve.Wherein abscissa is protein concentration
(ng/mL)；Vertical coordinate is detected value；r2It is 0.996.
Fig. 8. time resolution immunofluorescence of the present invention chromatography quantitative test card range of linearity matched curve.Wherein abscissa is theoretical concentration
(ng/mL)；Vertical coordinate is detected value；r2It is 0.998.
Fig. 9. time resolution immunofluorescence chromatography detection by quantitative and enzyme linked immunosorbent detection results relevance.
Detailed description of the invention
Definition
" antibody " also known as immunoglobulin, is the large-scale Y shape protein secreted by bone-marrow-derived lymphocyte of a class, it is possible to by Y shape
The immunoglobulin molecules of complementary site (antigen knot bound site) the specific binding target antigen on two of which bifurcated top, described
Target antigen such as protein, sugar, polynucleotide, fat, polypeptide, micromolecular compound etc..
" single-chain antibody " (scFv) refers to the variable region of heavy chain (V of antibodyH) and variable region of light chain (VL) by 15～20
Individual amino acid short peptide (linker) connects the single chain fusion protein formed, and is generally rich in glycine and silk for the linker connected
Propylhomoserin, is beneficial to stability and the pliability of single-chain antibody.Connected mode can be by VLN end be connected to VHC-terminal, or
Person is contrary.Although eliminating constant region and introducing linker, single-chain antibody still remains the antibody specificity to antigen, and its tool
Have that molecular weight is little, penetration power is strong and the feature such as antigenicity is weak.
Complementary determining region (complementarity-determining region, CDR), also referred to as hypervariable region.Take shape in anti-
The end of body single amino acid, is the most critical zone of target antigen and antibodies, in Artificial Immune Network Theory, each antibody
Complementary determining region is otherwise known as idiotype or genotype.
The preparation of embodiment 1. anti-human Procalcitonin. hybridoma cell strain
1. animal immune
With people recombinate Procalcitonin. according to general immune programme for children immunity BALB/c female mice (purchased from this laboratory animal of Cavan, Changzhou
Company limited).Concrete Immunity sees " antibody preparation and use experiment guide ".Indirect elisa method is used to follow the tracks of immunity little
Mus serum titer, chooses the immune mouse that serum titer is the highest, carries out mouse boosting cell and murine myeloma cell carries out merging in fact
Test.
2. cell merges
(1). the preparation of spleen cell
By immune mouse, pluck eyeball and take blood, put to death to be placed in the ethanol of 75% (v/v) through disconnected cervical vertebra and soak 10 minutes, in aseptic
Operating board takes out its spleen, is placed in cell screen cloth, be fully ground cell, cross screen cloth, (be purchased from by aseptic 1640 culture medium
Gibco company) centrifuge washing for several times after, re-suspended cell is to make single cell suspension, and counts, standby.
(2). the preparation of feeder cells
Take the female BAl BIc/c mice one of 8～10 week old, pluck eyeball and obtain negative serum, put to death rearmounted 75% (v/v) through disconnected cervical vertebra
Ethanol soaks 10 minutes；Aseptic skin of abdomen of opening, exposes peritoneum, about 10mL 1640HT culture medium (is purchased with syringe
From SIGMA company) inject mouse peritoneal, abdomen massage piping and druming are for several times gently.Draw the culture medium containing macrophage to inject
In 20%1640HAT culture medium standby；
Take the female BAl BIc/c mice one of 2～3 week old, put to death through disconnected cervical vertebra and be placed in 75% (v/v) ethanol immersion 10 points
Clock；The aseptic thymus that takes, in cell screen cloth, grinds, cross screen cloth, it is thus achieved that thymocyte cell is placed in above-mentioned containing macrophage
In 20%1640HAT culture medium, standby.
(3). cell merges
Select to be in the murine myeloma cell strain SP2/0 of exponential phase, collect and count.Take about 108Individual above-mentioned splenocyte with
2×107Individual above-mentioned SP2/0 cell strain adds in fusion pipe and mixes, and 1000rpm abandons supernatant (as far as possible abandoning clean) after being centrifuged 10 minutes,
Fusion pipe is put and rubs so that precipitating loose the most gently on palm.1mL preheating is added soon after elder generation is slow in 60 seconds
PEG1450 (Polyethylene Glycol 1450, purchased from SIGMA company), add 1640HT culture medium 30mL terminate, 1000rpm from
The heart 10 minutes, removes supernatant, gently friction make precipitation loose, add step 2 obtained 20% 1640HAT culture medium in.
After above-mentioned HAT culture medium is fully mixed, in 200 μ L/ hole subpackages to 96 porocyte culture plates, to put 37 DEG C, 5%CO2
Cell culture incubator in cultivate.Replace 20%1640HAT culture medium by 10%1640HT culture medium after one week, take after 3 days
Detect clearly.
The most anti-human Procalcitonin. specific hybrid tumor strain is screened
(1). the preparation of detection plate: be coated liquid dilution restructuring PCT to 1 μ g/mL with CB, be coated 96 hole ELISA ELISA Plate,
100 μ L/ holes, 2～8 DEG C are coated overnight, washed once and pat dry；Containing 2% bovine serum albumin (Bovine Serum Albumin,
BSA, buys in Sai Bao bio tech ltd, Yancheng) PBST buffer blind (200ul/ hole), close 2 little for 37 DEG C
Time；Pat dry, standby.
(2). the screening of positive colony: cells and supernatant 100 μ L/ hole to be checked is added in above-mentioned detection plate, act on 30 in 37 DEG C
Wash after minute and pat dry, adding the sheep anti-mouse igg of the HRP labelling in 100 μ L/ holes, washing also after acting on 30 minutes in 37 DEG C
Patting dry, add the TMB nitrite ion in 100 μ L/ holes, develop the color 15 minutes in 37 DEG C of lucifuges, every hole adds the 2M H of 50 μ L2SO4
Terminate reaction, and reading numerical values at OD450.Positive hole determines principle: OD450 value/negative control value >=2.1.Choose sun
Sex clone strain carries out Cell-cloned screening.After the cloning of three to four-wheel is screened, monoclonal cell strain positive rate 100%
I.e. it is defined as stable cell line, carries out determining strain to cell strain.Select to have the hybridoma cell strain of higher titer, follow-up enter
One step carries out antibody variable sequences sequencing analysis to above-mentioned hybridoma cell strain.
The mensuration of embodiment 2. hybridoma cell strain antibody variable sequences
Above-mentioned hybridoma cell strain antibody variable sequences is measured.
The extraction of a.RNA: with reference to cell total rna extraction agent box (purchased from Roche company) description to above-mentioned hybridoma
Cell strain carries out Total RNAs extraction and carries out reverse transcription immediately；
B.RNA reverse transcription becomes DNA: with reference to Thermo Scientific Reverted First strand cDNA Synthesis
Kit (purchased from Thermo company) carries out reverse transcription to the total serum IgE extracted in previous step, prepares cDNA, frozen in-20 DEG C
Standby；
C. the PCR of variable region sequences expands and reclaims: in previous step, gained cDNA is as template, with Mus IgG hypotype Dan Ke
Grand antibody variable sequences universal primer is primer, and the variable region sequences of heavy chain and light chain is carried out PCR amplification, is produced by PCR
Thing reclaims test kit (purchased from TIANGEN company) through DNA glue and reclaims, and sees accompanying drawing 1；
D. the clone of variable region sequences and sequencing: according to cloning vehicle pMD18-T kit (purchased from Takara company) explanation
Book, is attached with pMD18-T carrier respectively by heavy chain and chain variable region gene, converts bacillus coli DH 5 alpha, picking sun
Sex clone, transfers to Nanjing Genscript Biotechnology Co., Ltd. to check order.
Order-checking obtains the antibody heavy chain variable region aminoacid sequence of hybridoma cell strain as shown in SEQ ID NO:7, variable region of light chain
Aminoacid sequence is as shown in SEQ ID NO:8.The above-mentioned sequence of Vbase2 database analysis, each complementation of its variable region of heavy chain is certainly
Determine the aminoacid sequence in district respectively: the HCDR1 as shown in SEQ ID NO:1, the HCDR2 as shown in SEQ ID NO:2
And/or the HCDR3 as shown in SEQ ID NO:3；The aminoacid sequence of each complementary determining region of its variable region of light chain is: as
LCDR1 shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5 and/or such as SEQ ID NO:6 institute
The LCDR3 shown.
Recombinant expressed and the purification of embodiment 3. single-chain antibody
According to sequencing result in embodiment 2, connection peptides will be added between hybridoma cell strain heavy chain of antibody and variable region of light chain
(GGGGS)3, introduce six and histidine-tagged see SEQ ID NO:9, the histidine-tagged rear single-chain antibody aminoacid sequence of this fusion
As shown in SEQ ID NO:11, and its full genome is carried out codon optimized side according to the preferences of pichia yeast expression system
Method, codon optimized after the nucleotide sequence of single-chain antibody as shown in SEQ ID NO:10, carry out single-chain antibody recombinant expressed.
Its structure forms as shown in Figure 2.The recombinant expressed step of above-mentioned single-chain antibody is specific as follows:
A) expression plasmid of single-chain antibody gene builds
Single-chain antibody full genome upstream after optimizing introduces XhoI restriction enzyme site in pPICZ α A carrier, and downstream introduces XbaI
Restriction enzyme site, is building up in pUC57 carrier, obtains one and preserves plasmid for a long time, and plasmid is designated as pUC57-T01 (Nanjing gold
Si Rui Science and Technology Ltd. provides).Carrying out PCR amplification, wherein forward primer P1 is: 5'-TGT AAA ACG ACG GCC
AGT-3'；Downstream primer P2 is: 5'-CAG GAA ACA GCT ATG AC-3'.Purpose is expanded according to Standard PCR program
Gene, carries out agarose gel electrophoresis analysis (accompanying drawing 3), and display product size is consistent with expection size (750bp).By PCR
Obtain gene outcome to reclaim after purification, with XhoI (#R0146S, purchased from New England Biolabs company) and
XbaI (#R0145V, purchased from New England Biolabs company) double digestion, and with T4 ligase, recovery product is connected to
In pPICZ α A (V19520, purchased from Invitrogen) plasmid, it is transformed in DH5 α competent cell, containing Zeocin
37 DEG C of overnight incubation in the LB flat board of (R250-01, purchased from Invitrogen company).Screening positive clone bacterium order-checking in second day,
Comparison, completely the same with expected sequence, i.e. obtain the expression plasmid of single-chain antibody, be designated as pPICZ α-T01.
B) single-chain antibody gene Pichia sp. host's engineered strain structure, screen and express
YPDS solid medium is prepared: with reference to Invitrogen company EasySelect Pichia Expression Kit description；Finish
Prepared by red competent yeast cells: with reference to EasySelect Pichia Expression Kit description；BMGY culture medium is prepared:
With reference to Invitrogen company Multi-Copy Pichia Expression Kit description；BMMY culture medium is prepared: reference
Invitrogen company Multi-Copy Pichia Expression Kit description.
By pPICZ α-T01 plasmid, with SacI digestion with restriction enzyme linearisation.By linearized vector after ethanol precipitation, point
Electricity converts and enters into X-33 competence yeast cells, is respectively coated the YPDS solid medium containing Zeocin, 30 DEG C
Cultivate 3-5 days, just have positive colony to produce.
The monoclonal of the above-mentioned acquisition of picking, in 5mL BMGY culture medium, is cultivated to OD for 30 DEG C600When=2.0～6.0, take 1mL
Preserve strain, and by remain bacterium solution resuspended after transfer to BMMY Small Amount abduction delivering, add methanol to final concentration every 24h
It is 1% (v/v).After one week, centrifugal collection bacterium solution supernatant, by PAGE gel electrophoresis, object observing protein expression feelings
Condition (accompanying drawing 4).
The present invention anti-human Procalcitonin. antibody recombination engineered strain of above-mentioned acquisition is inoculated in 500mL BMGY culture medium
In, 30 DEG C, 220rpm cultivation to cell density to OD600=2.0～6.0, added methanol every 24 hours to final concentration of 1.0%
(v/v).After one week, collect fermentation culture.
C) single-chain antibody purification
Histidine-tagged affinity column antibody purification fusion protein, prepackage pillar is used to be chosen as HisTrap HP, specifically comprise the following steps that
Step one, fermentation liquid except cell pretreatment: above-mentioned expression is obtained antibody fusion protein fermented liquid supernatant, centrifugal collects
Supernatant, and add combine buffer so that supernatant final concentration of 300mM NaCl, 20mM NaH2PO4,10mM Imidazole,
Adjust pH7.5,0.45 μm membrane filtration.
Step 2, HisTrap HP affinity column purification: (AKTA avant150, purchases to use fully-automatic intelligent protein purification system
From GE healcare company) the antibody fusion protein fermentation liquid that obtains pretreatment carries out affinity purification, and pillar is HisTrap
HP (17-5248-02, purchased from GE healcare company).It is 300mM NaCl in conjunction with buffer, 20mM NaH2PO4, 10mM
Imidazole, pH7.5, elution buffer is 300mM NaCl, 20mM NaH2PO4, 500mM Imidazole, pH7.5.
Carry out linear elution during eluting, and collect each eluting peak.By SDS-PAGE electroresis appraisal purity, from accompanying drawing 5,
Purity of protein after purification reaches more than 95%, changes buffer and is PBS solution and (1mg/ml), filtration sterilization are concentrated by ultrafiltration
Save backup in-20 DEG C.
Those skilled in the art know, and recombiant protein can not tape label, it is possible to carry other labels, it is possible to add the company of other forms
Connect peptide.Regardless of whether the label of tape label or band multi-form all can use Capto L purification.
The performance evaluation of embodiment 4. single-chain antibody
1. the Western blot of antibody identifies
A. polyacrylamide gel electrophoresis: configuration 12%SDS-polyacrylamide gel；Loading standard protein and restructuring respectively
PCT albumen, electrophoresis 1 hour under constant voltage；
B. transferring film: transferring film 1 hour under the conditions of constant current (35mA/ film), shifts respectively by the protein in polyacrylamide gel
To nitrocellulose filter.The polyacrylamide gel completing transferring film is dyeed by Coomassie brilliant blue G250, observes the residual of albumen
Show mercy condition；
C. close: the TBST buffer blind (confining liquid) containing 5% defatted milk, 4 DEG C overnight；Cleaning mixture after closing (TBST,
Refer to TaKaRa company's T BST buffer) washed once, 10 minutes；
D. antigen antibody reaction: confining liquid dilution (by 1:400 volume ratio) Horseradish Peroxidase Conjugates (1mg/mL,
Our company uses classical Over-voltage protection labelling, lower same), add in above-mentioned nitrocellulose filter, room temperature reaction 1 hour；TBST
Wash 5 times, each 10 minutes；
E. developing the color and take pictures: blotting residual liquid on nitrocellulose filter, every nitrocellulose filter is separately added into 2mL and stablizes
Type Peroxidase Solution (1mL) and the mixed liquor (buying in Thermo company) of luminol/reinforcing agent solution (1mL),
The surface of uniform wet nitrocellulose filter, room temperature lucifuge is reacted after one minute in gel imaging system (buying in GE company)
Take pictures, leave and take result.
Test result indicate that, antibody can be with PCT albumino reaction, it was demonstrated that the adhesion of single-chain antibody of the present invention and PCT albumen.
2. single-chain antibody is in the evaluation of time-resolved fluorescence detection platform
With the PBS of 0.05mol/L pH7.2, H42 antibody (buying in Hytest company) is diluted to 1mg/ml, lines nitric acid
On cellulose membrane；With the borate buffer of 0.05mol/L pH8.0, the antibody of time-resolved fluorescence microsphere labelling is diluted 20 times, spray
It is applied on pad；As shown in accompanying drawing 6 pad pasting, cutting, be installed (concrete preparation see embodiment 5).Detection card is detected respectively
Levels is 10, the PCT recombiant protein of 1ng/ml and the PBS of 0.05mol/L pH7.2, and testing result is as follows:
From the above results, H42, the double-antibody sandwich detecting system of single-chain antibody of the present invention composition can be applicable to time resolution
Fluoroscopic examination platform, it is possible to detect certain density PCT recombiant protein.
The preparation of the time-resolved fluoroimmunoassay detection card of embodiment 5. Procalcitonin.
This detection method uses the principle of double-antibody sandwich immunochromatographic method.
1, solution preparation
Prepared by the borate buffer of 0.05mol/L pH8.0: take the H of 0.1mol/L3BO370ml, with 0.025mol/L's
Na2B4O7·10H2O regulates pH to 8.0, and is settled to 100ml, be placed in 4 DEG C standby, 3 months effect duration.
Prepared by 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC, purchased from SIGMA company) solution: 1.5gEDC
Add 100ml deionized water, be made into aqueous solution be placed in 4 DEG C standby, 3 months effect duration.
The preparation of confining liquid: containing 10%BSA (described percentage ratio is mass volume ratio), the boric acid of 0.05mol/L pH8.0 delays
Rush liquid, use 0.22um membrane filtration, be placed in 4 DEG C standby, 7 days effect duration.
Prepared by fluorescent antibody diluent: containing 3% trehalose, 0.05% tween 20, the borate buffer of 0.05mol/L pH8.0, use
0.22U membrane filtration, be placed in 4 DEG C standby, 7 days effect duration.
Prepared by coated antibody diluent: containing 1% trehalose, 2% methanol, the PBS of 0.05mol/L pH7.2, by 0.22U film mistake
Filter, be placed in 4 DEG C standby, 7 days effect duration.
Sample dilution: containing 0.05% tween 20,0.05% phenazone, 0.05%proclin300,0.05mol/L pH8.0's
Borate buffer, 2-28 DEG C preserves 1 year effect duration.
2, prepared by Procalcitonin. fluoroscopic examination card
1) time-resolved fluorescence microsphere labelling
Single-chain antibody labeling method is following (as a example by 500ul reaction system): add 400ul borate buffer solution in 2ml centrifuge tube
In, adding the unloaded fluorescent microsphere (purchased from Thermo company) of 100ul concentration 1% particle diameter 200nm, vortex oscillation mixes.
Add 10ulEDC solution, shaken at room temperature 15min.10 DEG C of centrifugal 10min of 14000rpm, go supernatant, precipitation 0.5ml
Borate buffer dissolves, ultrasonic disperse, power 100W, time 1min (ultrasonic 3s is spaced 3s).
Microsphere after activation adds the single-chain antibody 125ul of concentration 1mg/ml, 250r/min25 DEG C of isothermal vibration reaction 2h；Add
Enter 62ul confining liquid, isothermal vibration reaction 4h.10 DEG C of centrifugal 15min of 14000rpm, wash 2 times, go supernatant, precipitation to use
0.5ml borate buffer dissolves, and last centrifuged deposit fluorescent antibody diluent dissolves and ultrasonic disperse, is placed in 4 DEG C
Constant temperature preserves.
2) spraying of fluorescence pad
Single-chain antibody fluorescence pad specking method is as follows: with fluorescent antibody diluent by the strand fluorescent-labeled antibody of above-mentioned preparation
Dilute 20 times, be sprayed on whole piece pad (long 300mm, the glass fibre of wide 12mm).
3) being coated of nitrocellulose filter
Nitrocellulose filter method for coating is as follows: take the H42 antibody of 1ml concentration 2mg/ml, is added in 5ml graduated centrifuge tube,
Add coated antibody diluent to 1ml, be coated in T line 5 position of nitrocellulose filter 2.Taking 0.5ml concentration is that 4mg/ml resists
HIS antibody, is added in centrifuge tube, adds coated antibody diluent to 1ml, is coated in the C of nitrocellulose filter (reaction film) 2
Line 4 position.
4) pad pasting, cutting, be installed
Sample pad 1, fluorescence pad 6, it is coated with the nitrocellulose filter 2, by adsorptive pads 3 the most successively of H42 antibody
Arrange, and contact the most a little, the T line 5 of described nitrocellulose filter at left, C line 4 in right (as shown in Figure 6),
And cut according to the size that gets stuck, loading is got stuck, and completes detection card preparation.
5) test kit assembles
Take aluminium foil bag and desiccant；Open heat sealing machine, preheating；Detection blocked, desiccant loads in aluminium foil bag；Seal with heat sealing machine
Good aluminium foil bag；Labelled.
3, the using method of Procalcitonin. fluoroscopic examination card
1) dilution sample to be tested: add 1ml Sample dilution at clean centrifuge tube, more accurately draw 100 μ L serum/plasma
Sample, joins in centrifuge tube, vibration fully mixing.
2) sample-adding and interpretation: the sample after drawing 50 μ L dilutions with liquid-transfering gun is slowly added in well, starts timing, 10～15
Result is quantitatively judged with fluorescence immune chromatography instrument in minute.Judging more than 15 minutes, result is invalid.
4, Procalcitonin. fluoroscopic examination card Detection results assessment
1) elaboration: by H42 (being coated)-single-chain antibody of the present invention (labelling) detection card by detection card using method detection 0.1,1,
Each 25 replications of PCT recombiant protein of 20ng/ml, calculate detection card precision after rejecting outlier.Experimental result shows
Three Concentration Testing result coefficient of variation CV < 15%.
| Concentration point (ng/ml) | 0.1 | 1 | 20 |
| CV | 14% | 6% | 11% |
2) detection range: by the PCT recombiant protein of H42 (being coated)-single-chain antibody of the present invention (labelling) detection card detection variable concentrations
0.1,0.2,0.4,0.8,1.6,3.2,6.4,12.8,25.6,51.2ng/ml, matched curve and detection range are 0.1-51.2ng/ml
(such as accompanying drawing 7).
3) range of linearity: high level sample and Sample dilution are configured to according to a certain percentage 6 series concentration samples (0.1,
0.5,2,10,20,50ng/ml), with H42 (being coated)-single-chain antibody of the present invention (labelling) detection card detection, each sample is examined
Survey 3 times, result and theoretical concentration are carried out regression calculation, it is judged that the most linear in this concentration range.The range of linearity is
0.1-50ng/ml (such as accompanying drawing 8).Sensitivity is 0.1ng/ml.
4) the accuracy response rate: the detection card of H42 (being coated)-single-chain antibody of the present invention (labelling) is detected addition and is respectively
0.2,1, the PCT recombiant protein of 10ng/ml, testing result such as following table.
| Concentration point (ng/ml) | 0.2 | 1 | 10 |
| The response rate | 113% | 107% | 89% |
5, accuracy methodology comparison:
The PCT detection card performance of the above results display H42 (being coated)-single-chain antibody of the present invention (labelling) is more excellent, selects like product
In obtain Guangzhou Wondfo Biotech. Co., Ltd.'s Procalcitonin. immue quantitative detection reagent box of good prestige in the market and (exempt from
Epidemic disease chromatography) compare product comparison checking.Select 20 parts of clinical patient specimen, by the serial number of 1 to 20, with comparison
The PCT fluoroscopic examination card of product and H42 (being coated) to be evaluated-single-chain antibody of the present invention (labelling) is tested simultaneously, according to 1,
The sample order of 2,3......18,19,20,20,19,18......3,2,1 is measured.Comparison and the inspection of product to be evaluated
Survey result coefficient R2=0.98, illustrate that two kinds of method testing results have preferable dependency (such as accompanying drawing 9).
6, recipe determination
In addition to above-mentioned optimum preparation example 1, applicant also attempts multiple preparation scheme, such as below 5 groups of detection card preparations and application
Result such as following table:
Claims (8)
- The most anti-human Procalcitonin. antibody, including:Following complementary determining region is contained in variable region of heavy chain: aminoacid sequence HCDR1 as shown in SEQ ID NO:1, as HCDR2 shown in SEQ ID NO:2 and/or the HCDR3 as shown in SEQ ID NO:3；And light-chain variable sequence contains following complementary determining region: aminoacid sequence is as shown in SEQ ID NO:4 LCDR1, the LCDR2 as shown in SEQ ID NO:5 and/or the LCDR3 as shown in SEQ ID NO:6.
- Anti-human Procalcitonin. antibody the most as claimed in claim 1, is characterized in that the aminoacid sequence of the variable region of heavy chain of described antibody Row as shown in SEQ ID NO:7, the aminoacid sequence of variable region of light chain is as shown in SEQ ID NO:8.
- Anti-human Procalcitonin. antibody the most as claimed in claim 1, is characterized in that the aminoacid sequence such as SEQ ID of described antibody Shown in NO:11.
- 4. the nucleotide sequence of antibody described in coding claim 3, described nucleotide sequence is as shown in SEQ ID NO:10.
- 5. contain the expression vector of nucleotide sequence described in claim 4.
- 6. contain the recombinant host cell of expression vector described in claim 5.
- 7. produce the method for antibody described in claim 3, including:1) cultivate recombinant host cell described in claim 6 under suitable conditions and express antibody；2) then from host cell purification, collect antibody.
- 8. any one of claim 1-3 anti-human Procalcitonin. antibody application in the detection former content of human calcitonin.
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| CN110747171A (en) * | 2019-09-17 | 2020-02-04 | 华南理工大学 | A pair of hybridoma cell strains capable of secreting anti-procalcitonin monoclonal antibody, anti-procalcitonin monoclonal antibody and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103304666A (en) * | 2013-05-21 | 2013-09-18 | 上海贝西生物科技有限公司 | Procalcitonin monoclonal antibody and application thereof |
| CN203858248U (en) * | 2014-05-26 | 2014-10-01 | 安徽惠邦生物工程股份有限公司 | Procalcitonin test strip |
| CN104745534A (en) * | 2015-03-02 | 2015-07-01 | 南方医科大学 | Procalcitonin monoclonal antibody hybrid tumor 2H4 and monoclonal antibody |
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2016
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103304666A (en) * | 2013-05-21 | 2013-09-18 | 上海贝西生物科技有限公司 | Procalcitonin monoclonal antibody and application thereof |
| CN203858248U (en) * | 2014-05-26 | 2014-10-01 | 安徽惠邦生物工程股份有限公司 | Procalcitonin test strip |
| CN104745534A (en) * | 2015-03-02 | 2015-07-01 | 南方医科大学 | Procalcitonin monoclonal antibody hybrid tumor 2H4 and monoclonal antibody |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110747171A (en) * | 2019-09-17 | 2020-02-04 | 华南理工大学 | A pair of hybridoma cell strains capable of secreting anti-procalcitonin monoclonal antibody, anti-procalcitonin monoclonal antibody and application thereof |
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