CN102590529A - Kit for detecting PCDH10 (Protocadherin10) in serum by TRFIA (Time Resolved Fluorescence Immunoassay method) - Google Patents

Kit for detecting PCDH10 (Protocadherin10) in serum by TRFIA (Time Resolved Fluorescence Immunoassay method) Download PDF

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CN102590529A
CN102590529A CN2012100347338A CN201210034733A CN102590529A CN 102590529 A CN102590529 A CN 102590529A CN 2012100347338 A CN2012100347338 A CN 2012100347338A CN 201210034733 A CN201210034733 A CN 201210034733A CN 102590529 A CN102590529 A CN 102590529A
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pcdh10n
fusion
pcdh10c
antibody
pcdh10
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邵伟
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Abstract

The invention discloses a kit for detecting PCDH10 (Protocadherin10) in a serum by a TRFIA (Time Resolved Fluorescence Immunoassay method). The kit comprises a PCDH10-terminal fusion protein, a first antibody capable of being combined with the PCDH10-terminal fusion protein, and a second antibody capable of being combined with the PCDH10-terminal fusion protein and the PCDH10-terminal fusion protein and a lanthanide series metal ion complex, and the kit is applied to the time resolved fluorescence immunoassay for detecting the PCDH10 protein in a serum sample. In comparison with the prior art, the kit has longer fluorescence service life in application, the interference of non-specificity fluorescence in the sample can be eliminated, and obtained signals are complete specific fluorescence emitted by the lanthanide series metal ion complex. In addition, the wavelength conversion of exciting light and the fluorescence reaches 270 nm, the interference of the exciting light is effectively eliminated, and the measured fluorescence is a specificity fluorescence signal emitted by the lanthanide series metal ion complex. The intensity of the measured fluorescence signal is proportional to the content of the sample to be measured.

Description

The TRFIA method of PCDH10 detects and uses kit in the serum
Technical field
The TRFIA method that the present invention relates to PCDH10 in a kind of serum detects uses kit; Relate to the preparation of two species specific antibodies of the specific polypeptide fragment of PCDH10 PROTEIN C-end and N-end; And utilize prepared double antibody, adopt time-resolved fluoroimmunoassay method (TRFIA) to detect PCDH10 method in the blood serum sample.The Antibody Preparation and time-resolved fluoroimmunoassay (TRFIA) analysis field that belong to PCDH10 albumen.
Background technology
PCDH10 is a new tumor suppressor gene, and the remarkable reduction of PCDH10 albumen in serum has suggesting effect to some tumours (like colorectal cancer etc.).PCDH10 belongs to Ca 2+A member of dependence cadherin family, the intracellular region with 6 extracellular regions that repeat to connect and 1 uniqueness.The Zhu Yong of Zhejiang University very waits the people that former generation is being found pcdh10 during the tumor stem cell gene expression profile during with viral library technology screening -/-Cell is prone to swim out of and pass the semi-permeable diaphragm of " Transwell ", and pcdh10 + /+Cell migration property a little less than.Pass through bioinformatic analysis; Further clone the full length cDNA sequence of pcdh10 and made up the eukaryotic expression vector of expressing pcdh10; Find that through liposome transient transfection k562 cell, SW480 colorectal cancer cells ectopic expression PCDH10 has suppressed these cells in external propagation, and increased the apoptosis of cell.Further find to these carefully carry out non-serum starved with add processing back PCDH10 expressions such as chemotherapeutics and increase with the increase of concentration in time, point out PCDH10 to participate in Apoptosis, possibly be a tumor suppressor gene.So it being participated in The mechanism of apoptosis does not still understand.
The Zhu Yong of Zhejiang University very waits in people's the patent of invention " to the preparation method of the specific antibody of PCDH10C-end protein sheet segment molecule " (patent No. is ZL200810060201); Having utilized round pcr from cultured cell in vitro, to clone the C-end dna sequence dna of PCDH10 albumen and be transformed in the prokaryotic expresses; Obtained the PCDH10C-end polypeptide of purifying; And immune White Rabbit, collect the also antiserum of purifying White Rabbit, obtained the antibody of PCDH10 PROTEIN C-end polypeptide behind the purifying.But do not find specific antibody as yet to PCDH10 n-end of albumen polypeptide.The preparation method's of said specific antibody to PCDH10C end protein sheet segment molecule concrete steps are: " 1) adopt polymerase chain reaction technology directly from the cultured cell in vitro strain, PCDH10C 3 ' end base sequence to be cloned into prokaryotic, make up the prokaryotic PCDH10C/pETl5b expression vector of PCDH10C end fusion; 2) the PCDH10C/pETl5b expression vector is transformed BL21 DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10C end fusion; With His metal chelating and purifying resin PCDH10C end fusion; His-tag label with Thrombin enzyme excision PCDH10C end fusion N end obtains PCDH10C end fusion; 3) PCDH10C with oneself excision label holds fusion as the antigen immune White Rabbit, produces anti-PCDH10C end antibody, collects antiserum, with ammonium sulfate precipitation, dialysis, the anti-PCDH10C end of Protein A-Sepharose affinity column purified rabbit antibody.”
Do not have commercially available reagent box to be used to detect this albumen at present on the market as yet, measure that the PCDH10 protein content is to estimate cancered probability in the serum so invent a kind of simple and highly sensitive method, meaning is very great.
Time resolved fluoro-immunoassay is from eighties of last century beginning of the eighties at the end of the seventies, the immune analysis method that new development is got up, and it has overcome, and background interference seriously influences when utilizing luciferin to carry out fluoroimmunoassay.The TRFIA principle is to utilize the sequestrant with bifunctional group structure, generally adopts BHHCT (4,4 '-two (1 ", 1 "; 1 ", 2 ", 2 "; 3 ", 3 " seven fluoro-4 ", 6 " acetyl butyryl-6 "-acyl groups)-the chlorosulfonation ortho-terphenyl); The one of which end combines with lanthanide series, and the other end combines with free amino group in antibody (protein) molecule, and that processes the lanthanide series mark (generally uses Eu 3+Mark) antibody.It and antigen undetermined are combined into immune complex.Under the ideal situation, measure Eu in the compound 3+Fluorescence intensity just can confirm the amount of antigen in the sample; But the fluorescence intensity of in fact this compound species lanthanide series very a little less than; Have only to add a kind of enhancing liquid, lanthanide series is disintegrated down from compound, and (β-NAT) forms microcapsules again with strengthening β-naphthoyltrifluoroacetone contained in the liquid; The very strong fluorescence of emission under the exciting of light such as ultraviolet, up to a million times of reinforced effects.Differentiate luminoscope with the time and measure fluorescence intensity cps, can confirm the amount in the sample antigen.
Be the time-resolved fluoroimmunoassay detection method of representative to utilize europium complex in recent years,, environmental protection easy to operate because of it and highly sensitive advantage substitute traditional ELISA assay method just gradually, are used for clinically.Europium ion (the Eu that dissociates, formed complex compound 3+) radioactive wave be 615nm, (influence of 350nm～600nm) is so be well suited for can not receive the short life background fluorescence that excitation wavelength (340nm) or certain protein causes.BHHCT-Eu 3+The complex compound compound that serves as a mark can directly combine with protein, can carry out high-sensitivity analysis through the fluorometric assay of time resolution type.BHHCT has the beta-diketon structure, with Eu 3+In conjunction with stability factor up to 10 10M -1Bibliographical information, BHHCT-Eu 3+Complex compound has been used for detecting the content of human plasma tumor markers alpha-fetoprotein and immunoglobulin E (IgE).
But, also do not use above-mentioned Eu 3+Complex compound and PCDH10 double antibody detect PCDH10 protein process in the serum.
As stated, develop the method for PCDH10 albumen in a kind of correctly quantitative, simple to operate, stable and sensitive detection blood serum sample, will play very important effect clinical diagnosis and scientific research research.
Summary of the invention
The TRFIA method that the purpose of this invention is to provide PCDH10 in a kind of serum detects uses kit; The preparation of PCDH10 n-end of albumen peptide chain and the preparation method of specific antibody thereof are proposed simultaneously; And the method that combines the PCDH10 of Zhejiang University PROTEIN C-end peptide chain specific antibody to prepare; Antibody to utilize these two kinds of methods to prepare is material, measures the content of PCDH10 albumen in the serum with time-resolved fluorescence immunoassay method.The TRFIA method of PCDH10 detects simple and feasible, highly sensitive with kit in this serum.
The TRFIA method of PCDH10 detects and to use kit in a kind of serum of the present invention, has can hold fusion to combine with PCDH10C-and by Eu 3+The SA dried frozen aquatic products of complex compound mark; This SA dried frozen aquatic products is through 1) with the PCR PCDH10C 3 ' end base sequence is cloned into prokaryotic, make up the procaryotic cell expression carrier of PCDH10C end fusion; 2) expression vector is transformed the BL21DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10C end fusion; With His metal chelating and purifying resin PCDH10C end fusion; His-tag label with Thrombin enzyme excision PCDH10C end fusion N end obtains PCDH10C end fusion; 3) PCDH10C with oneself excision label holds fusion as the antigen immune White Rabbit, produces anti-PCDH10C end antibody, collects antiserum, with ammonium sulfate precipitation, dialysis, Protein A-Sepharose affinity column purifying gained; Be coated with the porous type trace titre plate that has the solid phase bound fraction and can hold the first antibody that fusion combines in addition with PCDH10N-.
The concrete method for making of PCDH10 protein detection kit comprises the steps:
(1) preparation of PCDH10N-end fragment protein and the preparation that can hold the first antibody that fusion combines with PCDH10N-.
A) adopt polymerase chain reaction technology directly from the cultured cell in vitro strain, PCDH10N 3 ' end base sequence to be cloned into prokaryotic, make up the prokaryotic PCDH10N/PET32a expression vector of PCDH10N-end fusion.
Described employing polymerase chain reaction technology directly is cloned into prokaryotic with PCDH10N3 ' end base sequence from the cultured cell in vitro strain, the method that makes up PCDH10N-end fusion is: will express 1 * 10 of PCDH10 5～10 7Human leukemia K562 cell centrifugation deposition adds the abundant mixing of 0.5～1.5ml Trizol, extracts total RNA, uses the reverse transcriptase transcribe rna to cDNA again; Design then that upstream primer comprises BamH I restriction enzyme site GGATCC and downstream primer comprises Hind III restriction enzyme site AAGCTT; Method through PCR directly holds dna sequence dna to be cloned into pET32aBamH I/Hind III site PCDH10N-; Behind BamH I and Hind III endonuclease digestion accreditation; Further with dna sequencing checking, the qualified PCDH10N/PET32a expression vector that is.
B) the PCDH10N/PET32a expression vector is converted into the BL21DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10N-end fusion; With His metal-chelating purifying resin PCDH10N-end fusion; His-tag label with Thrombin enzyme excision PCDH10N-end fusion N-end obtains PCDH10N-end fusion.
Described the PCDH10N/PET32a expression vector is converted into the BL21DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10N-end fusion; With His metal-chelating purifying resin PCDH10N-end fusion, the His-tag label of holding with Thrombin enzyme excision PCDH10N-end fusion N-comprises the steps:
1) the PCDH10N/PET32a recombinant plasmid of getting 1～3 μ l accreditation adds to the competent BL21 DE3 of 10～90 μ l pLysS Escherichia coli, ice bath 15～45min, 42 ℃ of 60s shocks; Add 30～44 ℃ of 30～90min of 50～350 μ lLB liquid, coating contains the LB flat board of 50～150 μ g/ml, 37 ℃ of incubator overnight incubation; Next day, manage in the new LB liquid of 6～8ml, in one to 37 ℃ of incubator hold over night with the single bacterium colony of sterilization toothpick picking; Get OD and be 0.4～0.6 spend the night bacterium liquid 300～1000 μ l in the new LB liquid of a pipe 6～8ml; 30～44 ℃ of enlarged culture 2～8h, adding final concentration are that the IPTG of 1～7mM induces 0.5～6h, centrifugal collection bacterial precipitation.
2) bacterial precipitation is added 1～5ml pH6～8 with every gram weight in wet base bacterium, contain 0.5～1.5%Trition, the phosphate buffer of 0.02～0.08M, resuspended bacterium; Ultrasonic Pulverization bacterium under the ice bath, high speed refrigerated centrifuge district supernatant, deposition continues Ultrasonic Pulverization, high speed refrigerated centrifuge; Merge supernatant, last His metal-chelating resin column is fully washed post with the phosphate buffer of pH6～8, the 0.02～0.08M that contains the 5mM imidazoles; Detect till do not have albumen and flow out with 5% Sodium Mercurothiolate, use instead and contain the 500mM imidazoles, pH6～8; 0.02 the phosphate buffer wash-out of～0.08M is collected the protein liquid that flows out, the bag filter of packing into; 2～8 ℃ of phosphate buffer dialysis desalinations to pH6～8,0.005～0.015M whenever change liquid 1 time at a distance from 3～12h, totally 3～9 times.After dialysis equilibrium finishes; Polyglycol with MW10000～30000 is concentrated into 0.5～1.5mg/ml; Get the His-tag label of 5～10mg recombinant protein, remove Thrombin albumen through the method that adds Avidin-agarose simultaneously, obtain the PCDH10N-end recombinant protein of purifying with Thrombin enzyme excision N-end; This albumen is during with the SDS-PAGE electrophoresis, and 5～20 μ g/lane should not have foreign protein district band and find.
C) hold fusion as antigen-immunized animal (adopting White Rabbit here) with the PCDH10N-that excises label; Produce anti-PCDH10N-end antibody; Collect antiserum, with ammonium sulfate precipitation, dialysis, the anti-PCDH10N-end of ProteinA-Sepharose affinity column purified rabbit antibody.
Described usefulness has been excised the PCDH10N-end fusion of label as antigen-immunized animal (adopting White Rabbit here), produces comprising the steps: of anti-PCDH10N-end antibody
1) get the PCDH10N-end recombinant protein of 1mg and freund adjuvant mixing fully respectively, fully emulsified, point surplus the inoculation White Rabbit subcutaneous 3～20; Every treated for the 3rd～4 weekend, carry out with 0.5～1.5mg PCDH10N-end recombinant protein that direct rabbit ear vein is interior to be injected at a distance from 5～12 days reinforced immunologicals 1 time; Separate the White Rabbit arteria carotis communis after 5～7 days; Cut short bloodletting to Erlenmeyer flask, tilt to place for 2～8 ℃ and spend the night, next day centrifugal collection serum.
2) with the antiserum of collecting; Method with ammonium sulfate precipitation, dialysis, the anti-PCDH10N-end of ProteinA-Sepharose affinity column purified rabbit antibody is: with the 0.005～0.03M of the rabbit anteserum of collecting with 2～6 times of precoolings; PH 6～8PBS dilution, the slow saturated (NH that adds 1～2 times of volume when constantly stirring 4) 2SO 4, place 20～30min for 2～8 ℃, centrifugal, remove supernatant, after the PBS redissolution of deposition with 0.5～2 times of original volume, use 50% and 33.3% saturated (NH more respectively 4) 2SO 4Repeat deposition each 1 time, after the final protein deposition is redissolved with 2～3ml PBS, the bag filter of packing into, 2～8 ℃ to 0.005～0.03M; PH 6～8PBS desalination of dialysing whenever changes liquid 1 time at a distance from 3～12h, adds in advance through 0.01M the ProteinA-Sepharose post of pH 6～8PBS balance; Flow velocity 0.5ml/min, after flowing to end, with 0.02～0.08M, the phosphate buffer of pH 6～8 fully is washed till till the no albumen outflow; Use 3M instead, the IgG of pH 6～8 sodium thiocyanate elution of bound merges eluent; 2～8 ℃ of phosphate buffer dialysis desalinations to pH 6～8,0.005～0.015M whenever change liquid 1 time at a distance from 3～12h, totally 3～9 times; Polyglycol with MW10000～30000 is concentrated into 0.5～1.5mg/ml, packing ,-20 ℃ of preservations.
(2) preparation of PCDH10C-end fragment protein and can hold fusion and Eu with PCDH10C- 3+The preparation of the SA that complex compound combines:
A) adopt polymerase chain reaction technology directly from the cultured cell in vitro strain, PCDH10C 3 ' end base sequence to be cloned into prokaryotic, make up the prokaryotic PCDH10C/PET32a expression vector of PCDH10C-end fusion;
Described employing polymerase chain reaction technology directly is cloned into prokaryotic with PCDH10C3 ' end base sequence from the cultured cell in vitro strain, the method that makes up PCDH10C-end fusion is: will express 1 10 of PCDH10 5～10 7Human leukemia K562 cell centrifugation deposition adds the abundant mixing of 0.5～1.5mlTrizol, extracts total RNA, uses the reverse transcriptase transcribe rna to cDNA again; Design then that upstream primer comprises NdeI restriction enzyme site CATATG and downstream primer comprises BamH I restriction enzyme site GGATCC; Method through PCR directly holds dna sequence dna to be cloned into pET32a NdeI/BamH I site PCDH10C-; Behind Nde I and BamH I endonuclease digestion accreditation; Further with dna sequencing checking, the qualified PCDH10C/PET32a expression vector that is.
B) the PCDH10C/PET32a expression vector is converted into the BL21DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10C-end fusion; With His metal-chelating purifying resin PCDH10C-end fusion; His-tag label with Thrombin enzyme excision PCDH10C-end fusion N-end obtains PCDH10C-end fusion.
Described the PCDH10C/PET32a expression vector is converted into the BL21DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10C-end fusion; With His metal-chelating purifying resin PCDH10C-end fusion, the His-tag label of holding with Thrombin enzyme excision PCDH10C-end fusion N-comprises the steps:
1) the PCDH10C/PET32a recombinant plasmid of getting 1～3 μ l accreditation adds to the competent BL21DE3pLysS Escherichia coli of 10～90 μ l, ice bath 15～45min, 42 ℃ of 60s shocks; Add 30～44 ℃ of 30～90min of 50～350 μ lLB liquid, coating contains the LB flat board of 50～150 μ g/ml, 37 ℃ of incubator overnight incubation; Next day, manage in the new LB liquid of 6～8ml, in one to 37 ℃ of incubator hold over night with the single bacterium colony of sterilization toothpick picking; Get OD and be 0.4～0.6 spend the night bacterium liquid 300～1000 μ l in the new LB liquid of a pipe 6～8ml; 30～44 ℃ of enlarged culture 2～8h, adding final concentration are that the IPTG of 1～7mM induces 0.5～6h, centrifugal collection bacterial precipitation.
2) bacterial precipitation is added 1～5ml pH6～8 with every gram weight in wet base bacterium, contain 0.5～1.5%Trition, the phosphate buffer of 0.02～0.08M, resuspended bacterium; Ultrasonic Pulverization bacterium under the ice bath, high speed refrigerated centrifuge district supernatant, deposition continues Ultrasonic Pulverization, high speed refrigerated centrifuge; Merge supernatant, last His metal-chelating resin column is fully washed post with the phosphate buffer of pH6～8, the 0.02～0.08M that contains the 5mM imidazoles; Detect till do not have albumen and flow out with 5% Sodium Mercurothiolate, use instead and contain the 500mM imidazoles, pH6～8; 0.02 the phosphate buffer wash-out of～0.08M is collected the protein liquid that flows out, the bag filter of packing into; 2～8 ℃ of phosphate buffer dialysis desalinations to pH6～8,0.005～0.015M whenever change liquid 1 time at a distance from 3～12h, totally 3～9 times.After dialysis equilibrium finishes; Polyglycol with MW10000～30000 is concentrated into 0.5～1.5mg/ml; Get the His-tag label of 5～10mg recombinant protein, remove Thrombin albumen through the method that adds Avidin-agarose simultaneously, obtain the PCDH10C-end recombinant protein of purifying with Thrombin enzyme excision N-end; This albumen is during with the SDS-PAGE electrophoresis, and 5～20 μ g/lane should not have foreign protein district band and find.
C) hold fusion as antigen-immunized animal (adopting White Rabbit here) with the PCDH10C-that excises label; Produce anti-PCDH10C-end antibody; Collect antiserum, with ammonium sulfate precipitation, dialysis, the anti-PCDH10C-end of ProteinA-Sepharose affinity column purified rabbit antibody.
Described usefulness has been excised the PCDH10C-end fusion of label as antigen-immunized animal (adopting White Rabbit here), produces comprising the steps: of the anti-PCDH10C-end antibody of rabbit
1) get the PCDH10C-end recombinant protein of 1mg and freund adjuvant mixing fully respectively, fully emulsified, point surplus the inoculation White Rabbit subcutaneous 3～20; Every treated for the 3rd～4 weekend, carry out with 0.5～1.5mg PCDH10C-end recombinant protein that direct rabbit ear vein is interior to be injected at a distance from 5～12 days reinforced immunologicals 1 time; Separate the White Rabbit arteria carotis communis after 5～7 days; Cut short bloodletting to Erlenmeyer flask, tilt to place for 2～8 ℃ and spend the night, next day centrifugal collection serum.
2) with the antiserum of collecting; Method with ammonium sulfate precipitation, dialysis, the anti-PCDH10C-end of ProteinA-Sepharose affinity column purified rabbit antibody is: with the 0.005～0.03M of the rabbit anteserum of collecting with 2～6 times of precoolings; PH 6～8PBS dilution, the slow saturated (NH that adds 1～2 times of volume when constantly stirring 4) 2SO 4, place 20～30min for 2～8 ℃, centrifugal, remove supernatant, after the PBS redissolution of deposition with 0.5～2 times of original volume, use 50% and 33.3% saturated (NH more respectively 4) 2SO 4Repeat deposition each 1 time, after the final protein deposition is redissolved with 2～3ml PBS, the bag filter of packing into, 2～8 ℃ to 0.005～0.03M; PH6～8PBS desalination of dialysing whenever changes liquid 1 time at a distance from 3～12h, adds in advance through 0.01M the ProteinA-Sepharose post of pH 6～8PBS balance; Flow velocity 0.5ml/min, after flowing to end, with 0.02～0.08M, the phosphate buffer of pH 6～8 fully is washed till till the no albumen outflow; Use 3M instead, the IgG of pH 6～8 sodium thiocyanate elution of bound merges eluent; 2～8 ℃ of phosphate buffer dialysis desalinations to pH 6～8,0.005～0.015M whenever change liquid 1 time at a distance from 3～12h, totally 3～9 times; Polyglycol with MW10000～30000 is concentrated into 0.5～1.5mg/ml, packing ,-20 ℃ of preservations.
D) anti-PCDH10C-end antibody of rabbit and one step of anhydrous DTPA is crosslinked, cross the SephadexG-75 gel filtration chromatography free europium ion (Eu 3+) remove.
(3) have solid phase bound fraction and the solid phase embedding that can hold the first antibody that fusion combines with PCDH10N-.
Can use the solid matter of arbitrary shape and material as " solid phase ", if can carry out the combination of antibody and do not hinder above-mentioned compound to form and after the fluorometric assay stated.No matter which kind of carrier, all can screen before use: antigen coated with equivalent, under same experiment condition, react, observe whether homogeneity of its chromogenic reaction, distinguish in view of the above whether its absorption property is good.The porous type trace titre plate of using among the present invention for Corning company.
" first antibody " is to exist with the state that combines with above-mentioned solid phase, and through antigen-antibody reaction and the desired protein bound antibody of PCDH10.
Encapsulate the step on solid phase carrier to first antibody: use 0.05M, pH9.6 carbonate encapsulates damping fluid, and first antibody is diluted to protein content is 1～10 μ g/ml.In the reacting hole of each XPS, add 0.1ml, 4 ℃ are spent the night.Discard solution in the hole next day, washes 3 times each 3 minutes with lavation buffer solution.-20 ℃ of preservations are before needs are measured.
(4) add testing sample and mark Eu 3+The SA of complex compound:
A) add serum sample 100 μ l, 37 ℃, hatch 1～2h; (cleansing solution is Tris-Hcl, can add an amount of non-ionic surfactant with the short molten ability of protein in case of necessity, for example Tween in washing; Concentration is representational to be about 0.005～about 0.2%, preferred 0.01～about 0.1%).(doing blank well simultaneously, negative control hole and positive control hole).
B) add Eu 3+The SA of mark: add the SA 100 μ l that suitably dilute with BSA.37 ℃, hatch 2h, fully washing.Here add concentration and be about the preferred bovine serum albumin(BSA) (BSA) about 0.5%, purpose is to eliminate to test middle nonspecific reaction, improves sensitivity.
In this step, be present in the desired PCDH10 albumen in the blood serum sample or as the PCDH10 albumen of standard control, it is through combining to be fixed on the solid phase with first antibody.PCDH10 albumen needn't contact with first antibody with free state, it can with mark Eu 3+The SA of complex compound combines with first antibody after combining again.Sample and mark Eu among the present invention like this 3+The adding order of the SA of complex compound in no particular order.
(5) adding and time-resolved fluorescence appearance (TRFIA) result that strengthen liquid measure.
A) add enhancing liquid: 100 μ l.Strengthening liquid is 1 by umbrella ketone, popop, and two (5-phenyl-2-oxazolyl) benzene of 4-, methyl alcohol are formed, and its objective is the enhancing fluorescence intensity, improve detection sensitivity.
The result measures: measure the compound device that contains the group of the lanthanides complex compound that above-mentioned steps obtains and be by Wellac company provide time resolution type fluorescence detector.Condition determination is, representational is to be about 0.2～about 0.3 millisecond (ms) time delay, and the window time is about 0.2～about 0.6ms, and flash speed is about 0.5～about 1.5ms, and excitation wavelength is 337.1nm (a nitrogen Wavelength of Laser), and the mensuration wavelength is 615nm.Measure the data of contrast combination sample sets, can obtain the content of PCDH10 albumen in the sample by typical curve.
Embodiment
Below practical implementation of the present invention is explained in more detail.
Kit of the present invention is based on the proposition of time-resolved fluorescence double antibody immune detection (TRFIA) method." time-resolved fluoroimmunoassay detection " is the fluorescent chemicals of using like lanthanide series metal network of the present invention and thing that can launch long-life fluorescence; Through immunological response marker determination object; After more short-life background fluorescence disappears, the fluorescence signal that comes the self-marker is carried out the assay method that the time resolution type is measured.
Kit of the present invention is used for surveying blood-serum P CDH10 albumen and has high sensitivity, high accuracy and advantage simple to operate.
The application of kit of the present invention for desired PCDH10 albumen in selectivity seizure, the mark blood serum sample, forms the compound that contains this albumen on solid phase.Particularly, the compound that contains this PCDH10 albumen is to be formed on suitable solid phase by following composition:
(a) have solid phase bound fraction and the first antibody that can hold fusion to combine with PCDH10N-;
(b) PCDH10 albumen;
(c) have and to hold fusion and Eu with PCDH10C- 3+The SA that complex compound combines.
In view of the above, comprising PCDH10N-in the kit of the present invention holds fusion, can hold fusion, can hold fusion and Eu with PCDH10C-with first antibody and the PCDH10C-that PCDH10N-holds fusion to combine 3+The SA that complex compound combines.
Below, each composition is described.
1, can encapsulate on solid phase with the first antibody that solid phase and PCDH10N-end fusion combine.Can use the solid matter of arbitrary shape and material as " solid phase ", if can carry out the combination of antibody and do not hinder above-mentioned compound to form and after the fluorometric assay stated.No matter which kind of carrier, all can screen before use: antigen coated with equivalent, under same experiment condition, react, observe whether homogeneity of its chromogenic reaction, distinguish in view of the above whether its absorption property is good.The porous type trace titre plate of using among the present invention for Corning company.
First antibody is to exist with the state that combines with above-mentioned solid phase, and through antigen-antibody reaction and the desired protein bound antibody of PCDH10.
The preparation process of first antibody is following:
1) adopts polymerase chain reaction technology directly from the cultured cell in vitro strain, PCDH10N 3 ' end base sequence to be cloned into prokaryotic, make up the prokaryotic PCDH10N/PET32a expression vector of PCDH10N-end fusion;
2) the PCDH10N/PET32a expression vector is converted into the BL21DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10N-end fusion; With His metal-chelating purifying resin PCDH10N-end fusion; His-tag label with Thrombin enzyme excision PCDH10N-end fusion N-end obtains PCDH10N-end fusion;
3) hold fusion as the antigen immune White Rabbit with the PCDH10N-that excises label, produce anti-PCDH10N-end antibody, collect antiserum, with ammonium sulfate precipitation, dialysis, the anti-PCDH10N-end of ProteinA-Sepharose affinity column purified rabbit antibody.
2, be present in desired PCDH10 albumen in the blood serum sample, representational is through combining to be fixed on the solid phase with first antibody.PCDH10 albumen needn't contact with first antibody with free state, it can with after the SA stated combine the back and combine with first antibody.The formation of compound is not limited to the order that each composition combines among the present invention like this.
3, have and to hold fusion and Eu with PCDH10C- 3+The preparation process of the SA that complex compound combines is following:
1) adopts polymerase chain reaction technology directly from the cultured cell in vitro strain, PCDH10C 3 ' end base sequence to be cloned into prokaryotic, make up the prokaryotic PCDH10C/PET32a expression vector of PCDH10C-end fusion;
2) the PCDH10C/PET32a expression vector is converted into the BL21DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10C-end fusion; With His metal-chelating purifying resin PCDH10C-end fusion; His-tag label with Thrombin enzyme excision PCDH10C-end fusion N-end obtains PCDH10C-end fusion;
3) hold fusion as antigen-immunized animal (adopting White Rabbit here) with the PCDH10C-that excises label; Produce anti-PCDH10C-end antibody; Collect antiserum, with ammonium sulfate precipitation, dialysis, the anti-PCDH10C-end of ProteinA-Sepharose affinity column purified rabbit antibody.
4) anti-PCDH10C-end antibody of rabbit and one step of anhydrous DTPA is crosslinked, cross the SephadexG-75 gel filtration chromatography free europium ion (Eu 3+) remove.
Also need add preferred bovine serum albumin(BSA) (BSA) in the SA, its concentration is representational to be about 0.05～about about 0.5%, preferred about 0.1～about 0.3%.Its objective is and eliminate nonspecific reaction in the test, thereby improve its sensitivity.
The selection of SA working concentration: the titration of at first taking preliminary experiment tentatively to tire, and then fixing other conditioned disjunction is taked " square formation method " (reference the article and the europium labelled antibody of encrusting substance, sample to be checked are respectively different dilutabilitys) its working concentration of titration exactly in formal experimental system.
The step of first antibody embedding and sample detection can be summarized as follows in the inventive method:
1) encapsulate first antibody on solid phase carrier: use 0.05M, pH9.6 carbonate encapsulates damping fluid, and first antibody is diluted to protein content is 1～10 μ g/ml.In the reacting hole of each XPS, add 0.1ml, 4 ℃ are spent the night.Discard solution in the hole next day, washes 3 times each 3 minutes with lavation buffer solution.-20 ℃ of preservations are before needs are measured.
2) application of sample: add serum sample 100 μ l; 37 ℃, hatch 1～2h, (cleansing solution is Tris-Hcl in washing; Can add an amount of non-ionic surfactant in case of necessity with the short molten ability of protein; Tween for example, concentration is representational to be about 0.005～about 0.2%, preferred 0.01～about 0.1%).(doing blank well simultaneously, negative control hole and positive control hole).
3) add SA: add the SA 100 μ l that suitably dilute with BSA.37 ℃, hatch 2h, fully washing.
4) add enhancing liquid: 100 μ l.Strengthen liquid and form, its objective is the enhancing fluorescence intensity, improve detection sensitivity by umbrella ketone, popop, methyl alcohol.
5) result measures: measure the compound device that contains the group of the lanthanides complex compound that above-mentioned steps obtains and be by Well ac company provide time resolution type fluorescence detector.Condition determination is, representational is to be about 0.2～about 0.3 millisecond (ms) time delay, and the window time is about 0.2～about 0.6ms, and flash speed is about 0.5～about 1.5ms, and excitation wavelength is 337.1nm (a nitrogen Wavelength of Laser), and the mensuration wavelength is 615nm.Time-resolved fluoroimmunoassay detects (TRFIA) method, is to be the basis with the immunological response, with specific reaction and the susceptibility of fluorescent material detection and a kind of immuno analytical method that intuitive combines of antigen, antibody.Time-resolved fluorescent immunoassay is with lanthanide metal ion complex compound (like the Eu3+ complex compound) labelled antibody in this invention, corresponding PCDH10 antigen in the test sample.This complex compound has longer fluorescence lifetime, utilizes the time-resolved fluorescence analyser to prolong Measuring Time, can get rid of the interference of non-specific fluorescence in the sample, and the gained signal is the specific fluorescence of lanthanide metal ion complex compound emission fully.Another characteristics are wavelength significant differences of exciting light and fluorescence, and its Wavelength conversion reaches 270nm, can effectively get rid of the interference of exciting light, the specificity fluorescent signal that the fluorescence that records sends for the lanthanide metal ion complex compound.Measured fluorescence signal intensity is directly proportional with testing sample content.Standard of comparison curve and sample data can obtain the numerical value of PCDH10 albumen in the serum.
Embodiment 1: have the solid phase bound fraction and can prepare with the first antibody that PCDH10N-holds fusion to combine
1, adopt polymerase chain reaction technology directly from the cultured cell in vitro strain, PCDH10N 3 ' end base sequence to be cloned into prokaryotic, the method that makes up PCDH10N-end fusion is: will express 1 * 10 of PCDH10 5～10 7Human leukemia K562 cell centrifugation deposition adds the abundant mixing of 0.5～1.5mlTrizol, extracts total RNA, uses the reverse transcriptase transcribe rna to cDNA again; Design then that upstream primer comprises BamH I restriction enzyme site GGATCC and downstream primer comprises Hind III restriction enzyme site AAGCTT; Method through PCR directly holds dna sequence dna to be cloned into pET32aBamH I/Hind III site PCDH10N-; Behind BamH I and Hind III endonuclease digestion accreditation; Further with dna sequencing checking, the qualified PCDH10N/PET32a expression vector that is.
2, the PCDH10N/PET32a expression vector is converted into the BL21DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10N-end fusion; With His metal-chelating purifying resin PCDH10N-end fusion, the His-tag label of holding with Thrombin enzyme excision PCDH10N-end fusion N-comprises the steps:
1) the PCDH10N/PET32a recombinant plasmid of getting 1～3 μ l accreditation adds to the competent BL21DE3pLysS Escherichia coli of 10～90 μ l, ice bath 15～45min, 42 ℃ of 60s shocks; Add 30～44 ℃ of 30～90min of 50～350 μ lLB liquid, coating contains the LB flat board of 50～150 μ g/ml, 37 ℃ of incubator overnight incubation; Next day, manage in the new LB liquid of 6～8ml, in one to 37 ℃ of incubator hold over night with the single bacterium colony of sterilization toothpick picking; Get OD and be 0.4～0.6 spend the night bacterium liquid 300～1000 μ l in the new LB liquid of a pipe 6～8ml; 30～44 ℃ of enlarged culture 2～8h, adding final concentration are that the IPTG of 1～7mM induces 0.5～6h, centrifugal collection bacterial precipitation.
2) bacterial precipitation is added 1～5ml pH6～8 with every gram weight in wet base bacterium, contain 0.5～1.5%Trition, the phosphate buffer of 0.02～0.08M, resuspended bacterium; Ultrasonic Pulverization bacterium under the ice bath, high speed refrigerated centrifuge district supernatant, deposition continues Ultrasonic Pulverization, high speed refrigerated centrifuge; Merge supernatant, last His metal-chelating resin column is fully washed post with the phosphate buffer of pH6～8, the 0.02～0.08M that contains the 5mM imidazoles; Detect till do not have albumen and flow out with 5% Sodium Mercurothiolate, use instead and contain the 500mM imidazoles, pH6～8; 0.02 the phosphate buffer wash-out of～0.08M is collected the protein liquid that flows out, the bag filter of packing into; 2～8 ℃ of phosphate buffer dialysis desalinations to pH6～8,0.005～0.015M whenever change liquid 1 time at a distance from 3～12h, totally 3～9 times.After dialysis equilibrium finishes; Polyglycol with MW10000～30000 is concentrated into 0.5～1.5mg/ml; Get the His-tag label of 5～10mg recombinant protein, remove Thrombin albumen through the method that adds Avidin-agarose simultaneously, obtain the PCDH10N-end recombinant protein of purifying with Thrombin enzyme excision N-end; This albumen is during with the SDS-PAGE electrophoresis, and 5～20 μ g/lane should not have foreign protein district band and find.
3, hold fusion as antigen-immunized animal (adopting White Rabbit here) with the PCDH10N-that excises label, produce comprising the steps: of anti-PCDH10N-end antibody
1) get the PCDH10N-end recombinant protein of 1mg and freund adjuvant mixing fully respectively, fully emulsified, point surplus the inoculation White Rabbit subcutaneous 3～20; Every treated for the 3rd～4 weekend, carry out with 0.5～1.5mg PCDH10N-end recombinant protein that direct rabbit ear vein is interior to be injected at a distance from 5～12 days reinforced immunologicals 1 time; Separate the White Rabbit arteria carotis communis after 5～7 days; Cut short bloodletting to Erlenmeyer flask, tilt to place for 2～8 ℃ and spend the night, next day centrifugal collection serum.
2) with the antiserum of collecting; Method with ammonium sulfate precipitation, dialysis, the anti-PCDH10N-end of ProteinA-Sepharose affinity column purified rabbit antibody is: with the 0.005～0.03M of the rabbit anteserum of collecting with 2～6 times of precoolings; PH 6～8PBS dilution, the slow saturated (NH that adds 1～2 times of volume when constantly stirring 4) 2SO 4, place 20～30min for 2～8 ℃, centrifugal, remove supernatant, after the PBS redissolution of deposition with 0.5～2 times of original volume, use 50% and 33.3% saturated (NH more respectively 4) 2SO 4Repeat deposition each 1 time, after the final protein deposition is redissolved with 2～3ml PBS, the bag filter of packing into, 2～8 ℃ to 0.005～0.03M; PH6～8PBS desalination of dialysing whenever changes liquid 1 time at a distance from 3～12h, adds in advance through 0.01M the ProteinA-Sepharose post of pH 6～8PBS balance; Flow velocity 0.5ml/min, after flowing to end, with 0.02～0.08M, the phosphate buffer of pH 6～8 fully is washed till till the no albumen outflow; Use 3M instead, the IgG of pH 6～8 sodium thiocyanate elution of bound merges eluent; 2～8 ℃ of phosphate buffer dialysis desalinations to pH 6～8,0.005～0.015M whenever change liquid 1 time at a distance from 3～12h, totally 3～9 times; Polyglycol with MW10000～30000 is concentrated into 0.5～1.5mg/ml, packing ,-20 ℃ of preservations.
Embodiment 2: have and can hold fusion and BHHCT-Eu with PCDH10C- 3+The SA preparation that complex compound combines
1, adopt polymerase chain reaction technology directly from the cultured cell in vitro strain, PCDH10C 3 ' end base sequence to be cloned into prokaryotic, the method that makes up PCDH10C-end fusion is: will express 1 * 10 of PCDH10 5～10 7Human leukemia K562 cell centrifugation deposition adds the abundant mixing of 0.5～1.5ml Trizol, extracts total RNA, uses the reverse transcriptase transcribe rna to cDNA again; Design then that upstream primer comprises NdeI restriction enzyme site CATATG and downstream primer comprises BamH I restriction enzyme site GGATCC; Method through PCR directly holds dna sequence dna to be cloned into pET32a Nde I/BamHI site PCDH10C-; Behind Nde I and BamH I endonuclease digestion accreditation; Further with dna sequencing checking, the qualified PCDH10C/PET32a expression vector that is.
2, the PCDH10C/PET32a expression vector is converted into the BL21DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10C-end fusion; With His metal-chelating purifying resin PCDH10C-end fusion, the His-tag label of holding with Thrombin enzyme excision PCDH10C-end fusion N-comprises the steps:
1) the PCDH10C/PET32a recombinant plasmid of getting 1～3 μ l accreditation adds to the competent BL21DE3pLysS Escherichia coli of 10～90 μ l, ice bath 15～45min, 42 ℃ of 60s shocks; Add 30～44 ℃ of 30～90min of 50～350 μ lLB liquid, coating contains the LB flat board of 50～150 μ g/ml, 37 ℃ of incubator overnight incubation; Next day, manage in the new LB liquid of 6～8ml, in one to 37 ℃ of incubator hold over night with the single bacterium colony of sterilization toothpick picking; Get OD and be 0.4～0.6 spend the night bacterium liquid 300～1000 μ l in the new LB liquid of a pipe 6～8ml; 30～44 ℃ of enlarged culture 2～8h, adding final concentration are that the IPTG of 1～7mM induces 0.5～6h, centrifugal collection bacterial precipitation.
2) bacterial precipitation is added 1～5ml pH6～8 with every gram weight in wet base bacterium, contain 0.5～1.5%Trition, the phosphate buffer of 0.02～0.08M, resuspended bacterium; Ultrasonic Pulverization bacterium under the ice bath, high speed refrigerated centrifuge district supernatant, deposition continues Ultrasonic Pulverization, high speed refrigerated centrifuge; Merge supernatant, last His metal-chelating resin column is fully washed post with the phosphate buffer of pH6～8, the 0.02～0.08M that contains the 5mM imidazoles; Detect till do not have albumen and flow out with 5% Sodium Mercurothiolate, use instead and contain the 500mM imidazoles, pH6～8; 0.02 the phosphate buffer wash-out of～0.08M is collected the protein liquid that flows out, the bag filter of packing into; 2～8 ℃ of phosphate buffer dialysis desalinations to pH6～8,0.005～0.015M whenever change liquid 1 time at a distance from 3～12h, totally 3～9 times.After dialysis equilibrium finishes; Polyglycol with MW10000～30000 is concentrated into 0.5～1.5mg/ml; Get the His-tag label of 5～10mg recombinant protein, remove Thrombin albumen through the method that adds Avidin-agarose simultaneously, obtain the PCDH10C-end recombinant protein of purifying with Thrombin enzyme excision N-end; This albumen is during with the SDS-PAGE electrophoresis, and 5～20 μ g/lane should not have foreign protein district band and find.
3, hold fusion as antigen-immunized animal (adopting White Rabbit here) with the PCDH10C-that excises label, produce comprising the steps: of anti-PCDH10C-end antibody
1) get the PCDH10C-end recombinant protein of 1mg and freund adjuvant mixing fully respectively, fully emulsified, point surplus the inoculation White Rabbit subcutaneous 3～20; Every treated for the 3rd～4 weekend, carry out with 0.5～1.5mg PCDH10C-end recombinant protein that direct rabbit ear vein is interior to be injected at a distance from 5～12 days reinforced immunologicals 1 time; Separate the White Rabbit arteria carotis communis after 5～7 days; Cut short bloodletting to Erlenmeyer flask, tilt to place for 2～8 ℃ and spend the night, next day centrifugal collection serum.
2) with the antiserum of collecting; Method with ammonium sulfate precipitation, dialysis, the anti-PCDH10C-end of ProteinA-Sepharose affinity column purified rabbit antibody is: with the 0.005～0.03M of the rabbit anteserum of collecting with 2～6 times of precoolings; PH 6～8PBS dilution, the slow saturated (NH that adds 1～2 times of volume when constantly stirring 4) 2SO 4, place 20～30min for 2～8 ℃, centrifugal, remove supernatant, after the PBS redissolution of deposition with 0.5～2 times of original volume, use 50% and 33.3% saturated (NH more respectively 4) 2SO 4Repeat deposition each 1 time, after the final protein deposition is redissolved with 2～3ml PBS, the bag filter of packing into, 2～8 ℃ to 0.005～0.03M; PH6～8PBS desalination of dialysing whenever changes liquid 1 time at a distance from 3～12h, adds in advance through 0.01M the ProteinA-Sepharose post of pH 6～8PBS balance; Flow velocity 0.5ml/min, after flowing to end, with 0.02～0.08M, the phosphate buffer of pH 6～8 fully is washed till till the no albumen outflow; Use 3M instead, the IgG of pH 6～8 sodium thiocyanate elution of bound merges eluent; 2～8 ℃ of phosphate buffer dialysis desalinations to pH 6～8,0.005～0.015M whenever change liquid 1 time at a distance from 3～12h, totally 3～9 times; Polyglycol with MW10000～30000 is concentrated into 0.5～1.5mg/ml, packing ,-20 ℃ of preservations.
Embodiment 3: encapsulate first antibody on solid phase carrier
Use 0.05M, pH9.6 carbonate encapsulates damping fluid, and first antibody is diluted to protein content is 1～10 μ g/ml.In the reacting hole of each XPS, add 0.1ml, 4 ℃ are spent the night.Discard solution in the hole next day, washes 3 times each 3 minutes with lavation buffer solution.-20 ℃ of preservations are before needs are measured.
Embodiment 4: the TRFIA of standard items
In encapsulating the 96 holes trace titre plate of first antibody, add and contain PCDH10 protein standard solution (100 μ l), hatch 2h for 37 ℃, with cleansing solution washing 3 times; Add and use BHHCT-Eu 3+The SA 100 μ l of mark are hatched 2h, are fully washed with cleansing solution for 37 ℃; Add and strengthen liquid 100 μ l; The 1420 type multiple labeling calculating instruments of using Well ac company carry out solid phase fluorescent to this plate and measure.
Embodiment 5: the preparation of blood serum sample
Extract blood with BD company disposable vein vacuum test tube (do not contain the anti-coagulants pipe: red cap or yellow are covered), 3000～5000rpm is centrifugal 5～10 minutes then, obtains serum.Preserve blood serum sample for-80 ℃, melt before analyzing and use, not multigelation.
Embodiment 6: the TRFIA that carries out with blood serum sample
In encapsulating the 96 holes trace titre plate of first antibody, add blood serum sample and each 100 μ l of positive and negative contrast, hatch 2h for 37 ℃, with cleansing solution washing 3 times; Add and use BHHCT-Eu 3+The SA 100 μ l of mark are hatched 2h, are fully washed with cleansing solution for 37 ℃; Add and strengthen liquid 100 μ l; The 1420 type multiple labeling calculating instruments of using Well ac company carry out solid phase fluorescent to this plate and measure.

Claims (10)

1. the TRFIA method of a blood-serum P CDH10 detects and to use kit, has can hold fusion to combine with PCDH10C-and by Eu 3+The SA dried frozen aquatic products of complex compound mark; This SA dried frozen aquatic products is through 1) with the PCR PCDH10C 3 ' end base sequence is cloned into prokaryotic, make up the procaryotic cell expression carrier of PCDH10C end fusion; 2) expression vector is transformed the BL21DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10C end fusion; With His metal chelating and purifying resin PCDH10C end fusion; His-tag label with Thrombin enzyme excision PCDH10C end fusion N end obtains PCDH10C end fusion; 3) PCDH10C with oneself excision label holds fusion as the antigen immune White Rabbit, produces anti-PCDH10C end antibody, collects antiserum, with ammonium sulfate precipitation, dialysis, Protein A-Sepharose affinity column purifying gained; It is characterized in that: be coated with the porous type trace titre plate that has the solid phase bound fraction and can hold the first antibody that fusion combines in addition with PCDH10N-.
2. claims 1 described kit is characterized in that: wherein first antibody is the animal's antibody of PCDH10N-end fusion; Wherein SA is the animal's antibody of PCDH10C-end fusion.
3. claims 1 described kit is characterized in that: this detection kit also comprises PCDH10 standard protein dried frozen aquatic products, cleansing solution, preferred cow's serum BSA and strengthens liquid.
4. claims 1 described kit is characterized in that: wherein be coated with and have the solid phase bound fraction and can hold the preparation method of the first antibody that fusion combines to be with PCDH10N-:
(a) adopt polymerase chain reaction technology directly from the cultured cell in vitro strain, PCDH10N 3 ' end base sequence to be cloned into prokaryotic, make up the prokaryotic PCDH10N/PET32a expression vector of PCDH10N-end fusion.The cDNA fragment that it is characterized in that comprising the primer of BamH I restriction enzyme site and the primer that downstream comprise Hind III restriction enzyme site at the design upper reaches, cDNA two ends that obtain through the reverse transcriptase transcript mRNA through the amplification of high temperature polymerase chain reaction be contain the double-stranded DNA PCDH10N-end group in BamH I and Hind III site because of, be cloned into the BamH I/Hind III site of PET32a carrier then;
The concrete grammar that makes up PCDH10N-end fusion is: will express 1 * 10 of PCDH10 5～10 7Human leukemia K562 cell centrifugation deposition adds the abundant mixing of 0.5～1.5ml Trizol, extracts total RNA, uses the reverse transcriptase transcribe rna to cDNA again; Design then that upstream primer comprises BamH I restriction enzyme site GGATCC and downstream primer comprises Hind III restriction enzyme site AAGCTT; Method through PCR directly holds dna sequence dna to be cloned into PET32a BamH I/Hind III site PCDH10N-; Behind BamH I and Hind III endonuclease digestion accreditation; Further with dna sequencing checking, the qualified PCDH10N/PET32a expression vector that is;
(b) the PCDH10N/PET32a expression vector is converted into the BL21DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10N-end fusion; With His metal-chelating purifying resin PCDH10N-end fusion; His-tag label with Thrombin enzyme excision PCDH10N-end fusion N-end obtains PCDH10N end fusion;
Described the PCDH10N/PET32a expression vector is converted into the BL21DE3pLysS Escherichia coli; Set up the procaryotic cell expression system of PCDH10N-end fusion; With His metal-chelating purifying resin PCDH10N-end fusion, the His-tag label of holding with Thrombin enzyme excision PCDH10N-end fusion N comprises the steps:
(1) the PCDH10N/PET32a recombinant plasmid of getting 1～3 μ l accreditation adds to the competent BL21DE3pLysS Escherichia coli of 10～90 μ l, ice bath 15～45min, 42 ℃ of 60s shocks; Add 30～44 ℃ of 30～90min of 50～350 μ l LB liquid, coating contains the LB flat board of 50～150 μ g/ml, 37 ℃ of incubator overnight incubation; Next day, manage in the new LB liquid of 6～8ml, in one to 37 ℃ of incubator hold over night with the single bacterium colony of sterilization toothpick picking; Get OD and be 0.4～0.6 spend the night bacterium liquid 300～1000 μ l in the new LB liquid of a pipe 6～8ml; 30～44 ℃ of enlarged culture 2～8h, adding final concentration are that the IPTG of 1～7mM induces 0.5～6h, centrifugal collection bacterial precipitation;
Bacterial precipitation is added 1～5ml pH6～8 with every gram weight in wet base bacterium, contain 0.5～1.5%Trition, the phosphate buffer of 0.02～0.08M, resuspended bacterium; Ultrasonic Pulverization bacterium under the ice bath, high speed refrigerated centrifuge district supernatant, deposition continues Ultrasonic Pulverization, high speed refrigerated centrifuge; Merge supernatant, last His metal-chelating resin column is fully washed post with the phosphate buffer of pH6～8, the 0.02～0.08M that contains the 5mM imidazoles; Detect till do not have albumen and flow out with 5% Sodium Mercurothiolate, use instead and contain the 500mM imidazoles, pH6～8; 0.02 the phosphate buffer wash-out of～0.08M is collected the protein liquid that flows out, the bag filter of packing into; 2～8 ℃ of phosphate buffer dialysis desalinations to pH6～8,0.005～0.015M whenever change liquid 1 time at a distance from 3～12h, totally 3～9 times; After dialysis equilibrium finishes; Polyglycol with MW10000～30000 is concentrated into 0.5～1.5mg/ml; Get the His-tag label of 5～10mg recombinant protein, remove Thrombin albumen through the method that adds Avidin-agarose simultaneously, obtain the PCDH10N-end recombinant protein of purifying with Thrombin enzyme excision N-end; This albumen is during with the SDS-PAGE electrophoresis, and 5～20 μ g/lane should not have foreign protein district band and find.
(c) hold fusion as antigen-immunized animal with the PCDH10N-that excises label, produce anti-PCDH10N-end antibody; Specifically comprise the steps:
(1) PCDH10N that gets 1mg respectively holds recombinant protein and complete freund adjuvant mixing, and is fully emulsified, point surplus in the of 3～20 under the inoculation animal skins; Every treated for the 3rd～4 weekend, carry out with 0.5～1.5mg PCDH10N end recombinant protein that direct rabbit ear vein is interior to be injected at a distance from 5～12 days reinforced immunologicals 1 time; Separating animal's arteria carotis communis after 5～7 days; Cut short bloodletting to Erlenmeyer flask, tilt to place for 2～8 ℃ and spend the night, next day centrifugal collection serum;
(2) with the antiserum of collecting; Method with ammonium sulfate precipitation, dialysis, the anti-PCDH10N-end of ProteinA-Sepharose affinity column purified rabbit antibody is: with the 0.005～0.03M of the rabbit anteserum of collecting with 2～6 times of precoolings; PH 6～8PBS dilution, the slow saturated (NH that adds 1～2 times of volume when constantly stirring 4) 2SO 4, place 20～30min for 2～8 ℃, centrifugal, remove supernatant, after the PBS redissolution of deposition with 0.5～2 times of original volume, use 50% and 33.3% saturated (NH more respectively 4) 2SO 4Repeat deposition each 1 time, after the final protein deposition is redissolved with 2～3ml PBS, the bag filter of packing into, 2～8 ℃ to 0.005～0.03M; PH 6～8PBS desalination of dialysing whenever changes liquid 1 time at a distance from 3～12h, adds in advance through 0.01M the ProteinA-Sepharose post of pH 6～8PBS balance; Flow velocity 0.5ml/min, after flowing to end, with 0.02～0.08M, the phosphate buffer of pH 6～8 fully is washed till till the no albumen outflow; Use 3M instead, the IgG of pH 6～8 sodium thiocyanate elution of bound merges eluent; 2～8 ℃ of phosphate buffer dialysis desalinations to pH 6～8,0.005～0.015M whenever change liquid 1 time at a distance from 3～12h, totally 3～9 times; Polyglycol with MW10000～30000 is concentrated into 0.5～1.5mg/ml, packing ,-20 ℃ of preservations.
5. claims 1 described kit is characterized in that: having the solid phase bound fraction and can hold the porous type trace titre plate solid-phase coating method of the first antibody that fusion combines to be with PCDH10N-wherein: encapsulate first antibody on solid phase carrier thereby under same experiment condition, react as solid phase carrier and equivalent first antibody with the micro-titre plate of the porous type of Corning company; Its concrete steps are: use 0.05M, pH9.6 carbonate encapsulates damping fluid, and first antibody is diluted to protein content is 1～10 μ g/ml; In the reacting hole of each XPS, add 0.1ml, 4 ℃ are spent the night; Discard solution in the hole next day, washes 3 times each 3 minutes with lavation buffer solution.-20 ℃ of preservations after the freeze-drying are before needs are measured.
6. claims 1 described kit, it is characterized in that: said expression vector is PCDH10C/PET32a among the preparation method of SA dried frozen aquatic products wherein.
7. claims 1 described kit is characterized in that: having wherein can hold fusion to combine with PCDH10C-and by Eu 3+The Eu of the SA dried frozen aquatic products of complex compound mark 3+The complex compound labeling method is that anti-PCDH10C end antibody of rabbit and one step of anhydrous DTPA is crosslinked, crosses the SephadexG-75 gel filtration chromatography free Eu 3+Remove, freeze-drying is subsequent use.
8. claims 1 described kit is characterized in that: its method of application is Standard PC DH10 albumen and the mark Eu that on solid-phase coating has the porous type trace titre plate of first antibody, adds testing sample box and finite concentration gradient 3+The SA of complex compound, its addition sequence are not limit and are formed the double-antibody sandwich immunocomplex, with cleansing solution flush away free SA and other foreign proteins, carry out TRFIA time resolution immunofluorescence analysis after adding synergy liquid again; Measure the data of control group and sample sets, can obtain the content of PCDH10 albumen in the sample by typical curve;
Its concrete steps are following: add testing sample and mark Eu 3+The SA of complex compound:
(1) add serum sample 100 μ l, 37 ℃, hatch 1～2h, washing, cleansing solution is Tris-HCl, or also is added with 0.005～0.2% Tween, does blank well simultaneously, negative control hole and positive control hole and Standard PC DH10 contrast;
(2) SA of adding Eu3+ mark: add SA 100 μ l with the BSA dilution.37 ℃, hatch 2h, fully washing; Here add concentration and be about the preferred bovine serum albumin(BSA) about 0.5%, purpose is to eliminate to test middle nonspecific reaction, improves sensitivity;
(3) adding and the time-resolved fluorescence appearance result that strengthen liquid measure;
(a) add enhancing liquid: 100 μ l; Strengthening liquid is 1 by umbrella ketone, popop, and two (5-phenyl-2-oxazolyl) benzene of 4-, methyl alcohol are formed, and its objective is the enhancing fluorescence intensity, improve detection sensitivity;
(b) TRFIA condition determination characteristic is, is about 0.2～about 0.3 millisecond time delay, and the window time is 0.2～0.6ms, and flash speed is 0.5～1.5ms, and excitation wavelength is 337.1nm, and the mensuration wavelength is 615nm; Measure the data of contrast combination sample sets, can obtain the content of PCDH10 albumen in the sample by typical curve.
9. claims 3 described kits is characterized in that cleansing solution wherein is Tris-HCl.
10. claims 3 described kits is characterized in that wherein synergy liquid is 1 by umbrella ketone, popop, and two (5-phenyl-2-oxazolyl) benzene of 4-and methyl alcohol are formed.
CN2012100347338A 2012-02-16 2012-02-16 Kit for detecting PCDH10 (Protocadherin10) in serum by TRFIA (Time Resolved Fluorescence Immunoassay method) Pending CN102590529A (en)

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