CN106996971A - Detect the kit and method of human peripheral EVs TRPC5 contents - Google Patents

Abstract

The invention provides a kind of time-resolved fluoroimmunoassay detection kit and detection method for detecting TRPC5 contents in human peripheral：The porous coating plate for being coated with TRPC5 polypeptides adds test sample into respective micropore, and the TRPC5 polypeptides added on TRPC5 and microwell plate in anti-TRPC5 antibody, oscillating reactions, the sample that dissociates compete anti-TRPC5 antibody, cleaning solution washing；Eu3+ goat anti-rabbit antibodies are added, immune response are marked, then washed with cleaning solution；Plus after enhancing liquid vibration, very strong fluorescence is launched under the exciting of uviol lamp, its fluorescence intensity cps is determined with time-resolved fluorescence instrument, fluorescence intensity is inversely proportional with the concentration in sample, reference standard curve is the amount that can determine that TRPC5 in sample.The present invention can show the situation of drug resistance of tumor cell transformation, have the advantages that detection method is simple, speed is fast, sensitivity is high, cost is low according to the height of TRPC5 expression in blood EVs.

Description

Detect the kit and method of human peripheral EVs-TRPC5 contents

Technical field

The present invention relates to the kit of detection human peripheral EVs-TRPC5 contents and method.

Background technology

Cancer (Cancer), also known as malignant tumour (Malignant neoplasm), are by control growth and proliferation of cell machine Disease caused by system is not normal.Cancer cell is out of control outer except growing, can also locally invade arround normal structure or even via internal The circulatory system or lymphatic system are transferred to body other parts.In various diseases, cancer is " number one killer ".Defend in the world Raw Organisation Report shows that the whole world in 2008 there are about 12,700,000 cancers and increase patient newly, and 7,600,000 die from cancer, especially in development Country, cancer increases number of cases newly up to 56%, and by inference to before the year two thousand twenty, the whole world every year will newly-increased 15,000,000 cancer patients.

Breast cancer is the common cancer of women, serious to threaten women physically and mentally healthy.The newest system that Chinese Anti-Cancer Association announces Numerical monitor is counted, since 1990s, breast cancer incidence and the death rate continually and steadily rise, and the incidence of disease is with annual 4.476% speed increases, and the death rate is increased with annual 3% speed.Chemotherapy is the primary treatment hand of breast cancer One of section, current clinical commonly used drug has：(i) anthracycline, Japanese yew class, anti-metabolism, anti-micro-pipe class, alkylating agent, miscellany etc.； (ii) antiestrogenic, arimedex etc.；(iii) Herceptin (anti-Her-2), bevacizumab (anti-vegf).

But, according to American Cancer Society, more than 90% tumor patient dies from different degrees of chemotherapy resistance.To chemotherapy Medicine produces a great problem that resistance has become oncotherapy.By the research of recent decades, the resistance mechanism having now been found that There is the expression enhancing of (1) cell membrane abc drug transport protein, reduce medicine intake and increase medicine discharge, P- glycoprotein (P- Glycoprotein, P-gp) it is that current most study is also mostly important abc drug transport protein, by MDRG Mdr1 is encoded；(2) pharmaceutical activity is reduced by making medicine occur alienation, common enzyme has GST, CYP450 etc.；(3) medicine is acted on Target spot is mutated or expression changes etc.；(4) Tumor Heterogeneity；(5) tumor stem cell resistance etc..

At present, newest, the most efficient method of early diagnosis tumour are to find tumor marker by blood count.Tumor-marker Thing referred to during tumorigenesis, can reacting cells canceration some intracellular matters.Clinically by each The detection of tumor markers is planted, corresponding tumour can be early diagnosed, detect curative effect and tumor recurrence situation.Therefore, in depth On the basis of entering to study resistance mechanism, exploitation tumor drug resistance molecule diagnosis index, method realize reagent, reach resistance early stage, Dynamic monitoring, it is most important in the diagnosis and treatment at this stage for breast carcinoma resistance, it is also inverse for the design of optimization medicines structure, research and development resistance Turn medicine and helpful reference is provided, thus with great and real social value.

MUC1 is a kind of mucoprotein, is a kind of transmembrane glycoprotein when normal cell is expressed, under normal circumstances, mammary gland, The epithelial cell top expression of intestines and stomach and urogenital tract, glycosylation is complete.MUC1 degree normal epithelial plays lubrication and protection is made With, mediated signal transduction and cell adhesion.

By phosphorylation in breast cancer cell line mcf-7, MUC1 can combine Rrb/SOS, participate in receptor tyrosine kinase The signal transduction of mediation, and tyrosine phosphorylation is the committed step that membrane receptor participates in signal transduction.Breast cancer MUC1's Expression characteristic includes：Height expression, unconventionality expression are low；Glycosylation, high sialylation；Top is set to unclear, polarity confusion；Cytoplasm There is overexpression with cell surface, and these molecules can enter serum from breast cancer cell.There is researcher to detect substrate sample Patient with breast cancer, has found high expression MUC1, and about 92% patient can detect the MUC1 being overexpressed.

Immunomagnetic beads, also referred to as immune magnetic microsphere, are a kind of uniform, spherical granules with superparamagnetism and protectiveness shell Son, is substantially combined into by carrier microballoons and immunoligand.Its core is paramagnetic particles, and core outer layer wraps up polymer, Outermost layer is immunoligand.Immunomagnetic bead technique, be a kind of immunology detection based on special antigen-antibody reaction and Isolation technics.It is, using the coated magnetic bead of antibody as carrier, to be combined by antibody with specific antigen in reaction medium, forms anti- Displacement occurs in the presence of externally-applied magnetic field for original-antibody complex, this compound, so as to reach the purpose of separation antigen.

TRPC5 is that (transient receptor potential channels, TRP are logical for transient receptor potential channel Road) family a hypotype, it be on cell membrane can penetrating calcium ion non-selective passage, be distributed mainly on brain, lung, testis And placenta, and it is primarily involved in the formation of growth cone and the development of brain.The research discovery of this research department, transient receptor potential channel TRPC5 is closely related with tumor multi-medicine drug-resistant, and TRPC5 occurs obvious in the breast cancer cell (MCF-7/ADM) of adriamycin-resistant Up-regulation, and the P-gp albumen of multidrug resistance can be produced in multidrug resistance tumor cells (such as MCF-7/ADM) by mediate tumor indirectly Expression.The applicant is directed to property application Patents, and patent name is《TRPC5 is as drug target inverse Turn the application in tumor multi-medicine drug-resistant》(the patent No.：201210318389.5)《EVs-TRPC5 is in detection breast carcinoma resistance degree In application》(Authorization Notice No.：CN103954770B).

Time resolved fluoro-immunoassay (time-resolved fluoroimmunoassay, TRFIA) is special due to its The strong, sensitivity of property is high, simple to operate, cheap, and the advantages of be particularly suitable for the detection of batch samples and increasingly by people institute Pay attention to and use.There is presently no the patent and document report for the TRPC5 time fluoroimmunoassays detected.

The content of the invention

In view of the above-mentioned problems existing in the prior art, the applicant provides a kind of based on time resolved fluoro-immunoassay The kit and method of TRPC5 contents in human peripheral are detected, technical scheme is as follows：

A kind of time resolved fluoro-immunoassay kit of detection TRPC5 contents, including the coated NHS magnetic beads of MUC1, Porous coating plate, buffer solution, many poly saccharide peptide standard products of TRPC5, TRPC5 antibody, goat anti-rabbit antibody, cleaning solution and the enhancing of europium mark Liquid.

A kind of utilization mentioned reagent box detects the time-resolved fluorescence immunoassay method of TRPC5 contents, including following step Suddenly：

(1) TRPC5 polypeptides are synthesized；

(2) the TRPC5 polypeptides synthesized with step (1) are as coating primordial covering solid phase carrier；

(3) the TRPC5 polypeptides synthesized with step (1) obtain immunogene by coupling agent and KLH couplings；

(4) immunogene obtained with step (3) prepares anti-TRPC5 polyclonal antibody；

(5) with blood coagulation pigging collection new blood is promoted, 1000-2000r/min collects supernatant after centrifuging 5-20min, used After the coated immunomagnetic beads of MUC1 antibody is mixed with the supernatant, 0.5-2h is incubated or through 2-8 DEG C of reaction through 25-37 DEG C of concussion Overnight, magnetic frame absorption magnetic bead, with wash buffer and is resuspended, obtains testing sample；

(6) step (5) described testing sample is at war with and suppresses detection and analysis, measure fluorescence intensity, reference standard is bent TRPC5 contents in line computation sample.

Further, step (3) described coupling agent is Sulfo-SMCC.

Further, the immunogen immune NZw described in step (3), antibody is prepared by subcutaneous multi-point injection, Purified using antigen affinity column, obtain the polyclonal antibody of the anti-TRPC5 described in step (4).

Further, step (2) described solid phase carrier is 96 hole microwell plates.

Further, step (5) described buffer solution is phosphate buffer.

Further, step (5) described immunomagnetic beads is NHS magnetic beads.

Further, the NHS magnetic beads are purchased from Suzhou Beaver companies.

Further, step (6) described standard curve be step (1) synthesize the TRPC5 polypeptides as standard items, it is dilute It is interpreted into as 0ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 50ng/mL, 80ng/mL, 100ng/mL, 200ng/ ML, 400ng/mL, 500ng/mL, 800ng/mL, 1000ng/mL series concentration, dilution are 0.1mol/L pH7.5 phosphate Buffer solution, be at war with suppression detection and analysis, and testing result fluorescent value and standard concentration are taken the logarithm i.e. log10, Linear Quasi Close, be used as standard curve.

Further, the coated immunomagnetic beads preparation method of the MUC1 antibody is：

A. protein solution is prepared：50-100 μ g MUC1 albumen is taken thoroughly to be removed by the method for dialysis or desalination original Material containing primary amine groups in buffer, is then made into Coupling Buffer the albumen that concentration is 3.0-5.0mg/mL molten again Liquid, the protein solution prepared is saved backup in 2-8 DEG C；

B. 200-500 μ L 20% bead suspension is taken in EP pipes；

C. EP pipes are placed in magnetic separation rack, are enriched with magnetic bead, remove supernatant；

D. plus 1mL 2-8 DEG C Washing Buffer A in centrifuge tube, vortex 15-30s, make magnetic bead be well mixed；

E. EP pipes are placed in magnetic separation rack, are enriched with magnetic bead, remove supernatant；

F. plus 200-500 μ L protein solutions are in EP pipes, vortex 15-30s, it is well mixed it；

G. by EP pipe vortex 15-30s, it is placed on vertical mixed instrument, 2-8 DEG C of reaction is stayed overnight；

H. magnetic bead is enriched with using magnetic separation rack, preservation flows through liquid；

I. plus 0.5-1mL Blocking Buffer are in EP pipes, vortex 15-30s, EP pipes are placed in magnetic separation rack It is interior, magnetic bead is enriched with, supernatant is abandoned；

J. plus 0.5-1mL Blocking Buffer are in EP pipes, vortex 15-30s, EP pipes are placed in vertical mixed instrument React at room temperature 2-4h；

K. EP pipes are placed in magnetic separation rack, are enriched with magnetic bead, abandon supernatant；

L. plus 0.5-1mL ultra-pure waters are in EP pipes, it is sufficiently mixed, is enriched with magnetic bead with magnetic frame, abandons supernatant；

M. plus 0.5-1mL Storage Buffer are in EP pipes, it is sufficiently mixed, is enriched with magnetic bead with magnetic frame, abandons supernatant Liquid；Repeat the operation 2 times；

N. 0.5-1mL Storage Buffer are added in EP pipes, are sufficiently mixed, 2-8 DEG C saves backup.

The advantageous effects of the present invention are：

1st, the instrument that the present invention need not be expensive, testing cost is low, and sample pre-treatments are simple, and scene is operable detection, Meanwhile, this method sensitivity is high, and the degree of accuracy is high, easy to operate, high specificity, and detection speed is fast, is widely used, is particularly suitable for use in The quick detection of gross sample.

2nd, the present invention only need to extract blood sample and just can detect, and dramatically alleviate the pain of tumour patient；

3rd, the height expressed by TRPC5 in blood EVs, can go out resistance situation of the patient in the stage with Preliminary detection, New reference project is provided, while other existing tumor drug resistance diagnostic techniques can be combined, is conducive to doctor to propose more accurate Therapeutic scheme, to obtain optimum curative effect.

Brief description of the drawings

The present invention is described in further detail with specific embodiment below in conjunction with the accompanying drawings.

Fig. 1 is that sample is pre-processed by MUC1 magnetic beads, is illustrated afterwards using indirect TRPC5-TRFIA methods clinical detection result Figure；

Fig. 2 is that sample is pre-processed without MUC1 magnetic beads, is shown using only indirect TRPC5-TRFIA methods clinical detection result It is intended to.

Embodiment

Material source or preparation method used in embodiment is as follows：

1st, main agents and consumptive material

MUC1antibody is purchased from Abcam companies of the U.S.；Sulfo-SMCC, KLH, Sulfolink Resin are purchased from the U.S. Pierce companies, Freund's complete adjuvant, freund 's incomplete adjuvant are purchased from Sigma Co., USA.Bag filter is purchased from U.S. Viskase Company, molecular cut off 14kD, P-10 post is purchased from BioRad companies of the U.S., and NHS magnetic beads are purchased from Suzhou Beaver companies.

2nd, laboratory apparatus

Magnetic stirring apparatus (Shanghai Mei Ying Pus), impeller (is transported by force in Shanghai), ultraviolet/visible light spectrophotometer (on Hai Aopule), supercentrifuge (Hitachi).

Embodiment：

1st, prepared by immunogene and coating antigen

TRPC5 polypeptides are synthesized as coating antigen, TRPC5 polypeptides are used as with KLH couplings (coupling agent is Sulfo-SMCC) exempts from Epidemic disease antigen, TRPC5 polypeptides are used as detection antigen with BSA couplings (coupling agent is glutaraldehyde), immunizing antigen is distinguished dilute with PBS 1mg/ml is interpreted as, packing freezes spends refrigerator in -20.

2nd, prepared by polyclonal antibody

2.1 animal immune

1st day, every kind of antigen took 1ml antigens to add 1ml Freund's complete adjuvants, and emulsification (examines emulsification degree：By a drop breast Change antigen liquid to instill in physiological saline, if not scattering, show to have reached requirement), the subcutaneous multiple spot of nape part (at least 8 points) injection, Every kind of 2 NZws of antigen immune.15th day, every kind of antigen took 1ml antigens to add 1ml freund 's incomplete adjuvants, emulsified, The subcutaneous multiple spot of nape part (at least 8 points) is injected, every kind of 2 NZws of antigen immune.29th day, every kind of antigen took 1ml to resist Original adds 1ml freund 's incomplete adjuvants, emulsification, the subcutaneous multiple spot of nape part (at least 8 points) injection, every kind of new west of antigen immune 2 Blue white rabbit.43rd day, every kind of antigen took 1ml antigens to add 1ml freund 's incomplete adjuvants, emulsified, the subcutaneous multiple spot of nape part is (at least 8 points) injection, every kind of 2 NZws of antigen immune.53rd day, arteria carotis took blood, and rabbit is put to death, and rabbit blood is in 4 degree of placements Overnight, centrifugation (4 degree, 10000rpm) 30 minutes, collect supernatant.

The purifying of 2.2 polyclonal antibodies

TRPC5 polypeptides are connected on the Sulfolink Resin of activation, prepare antigen affinity column, 1ml Sulfolink Resin is coupled 1mgTRPC5 polypeptides, and affinity column is balanced with 10 times of column volume PBS, flows to end solution；Rabbit anteserum is through 0.45um filter membrane mistakes Filter, crosses antigen affinity column, flows to end solution, collection is flowed through, 10 times of column volume PBS balances, flows to end solution.Add 5ml antibody elutions Liquid, is in charge of collection eluent, often pipe 1ml.Absorbance at the eluent detection 280nm of collection, absorbance is more than 1.0 component Merge, to PBS, the Identification of the antibodies after dialysis (ultraviolet absorption method detects protein concentration, and Elisa determines antibody titer).

3rd, MUC1 is coated with magnetic bead method

A. protein solution is prepared：50 μ g MUC1 albumen are taken thoroughly to be removed by the method for dialysis or desalination original Material containing primary amine groups in buffer, is then made into Coupling Buffer protein solution of the concentration for >=3.0mg/mL again, The protein solution prepared is saved backup in 4 DEG C.

B. 500 μ L 20% bead suspension is taken in 1.5mL EP pipes.

C. EP pipes are placed in magnetic separation rack, are enriched with magnetic bead, remove supernatant.

D. plus 2~8 DEG C of 1mL Washing Buffer A in centrifuge tube, vortex 15s, make magnetic bead be well mixed.

E. EP pipes are placed in magnetic separation rack, are enriched with magnetic bead, remove supernatant.

F. plus 200 μ L protein solutions are in EP pipes, vortex 30s, make its mixing equal.

G. by EP pipe vortex 15s, it is placed on vertical mixed instrument, 4 DEG C of reactions are stayed overnight.

H. magnetic bead is enriched with using magnetic separation rack, preservation flows through liquid.

I. plus 1mL Blocking Buffer are in EP pipes, vortex 30s, EP pipes are placed in magnetic separation rack, are enriched with magnetic Pearl, abandons supernatant.

J. plus 1mL Blocking Buffer are in EP pipes, vortex 30s, it is anti-that EP pipes are placed in into room temperature in vertical mixed instrument Answer 2h.

K. EP pipes are placed in magnetic separation rack, are enriched with magnetic bead, abandon supernatant.

L. plus 1mL ultra-pure waters are in EP pipes, it is sufficiently mixed, is enriched with magnetic bead with magnetic frame, abandons supernatant.

M. plus 1mL Storage Buffer are in EP pipes, it is sufficiently mixed, is enriched with magnetic bead with magnetic frame, abandons supernatant.Weight Operation 2 times again.

N. 1mL Storage Buffer are added in EP pipes, are sufficiently mixed, 4 DEG C save backup.

* 4, sample pretreating method (steps necessary)

A. with blood coagulation pigging collection human peripheral is promoted, 1000r/min collects supernatant after centrifuging 5min；

B. with the NHS magnetic beads and supernatant concussion incubation 1h for being surrounded by MUC1 antibody；

C. magnetic frame adsorbs magnetic bead, and PBS is washed once；

D. it is resuspended with PBS and is used as testing sample.

5th, reagent preparation box and detection sample

It is prepared by 5.1Eu3+- goat anti-rabbit antibodies

5g/L 1~the 2mL of goat anti-rabbit antibody for being dissolved in 50mmol/L PBS pH7.0 are taken, through the conversion buffered bar of PD-10 posts Part, eluent is the 50mmol/L Na2CO3-NaHCO3pH8.5 buffer solutions of the NaCl containing 0.155mmol/L.Collect protein peak, It is quantitative (1.46A280-0.74A260) through UV absorption analysis, rabbit anti-mouse antibody is diluted to 2g/L with above-mentioned eluent.Take 500 Goat anti-rabbit antibody after~1000 μ L dilutions adds Eu3+-N2- [p- isocyanic acids-benzyl]-divinyl three containing 0.2~0.4mg In the bottle of amine tetraacethyl (Eu3+-DTTA), 30 DEG C of magnetic agitations are reacted 20 hours.Reaction solution is through with 80mmol/LTris-HCl Protein peak is collected in SepharoseCL-6B posts (1 × 40cm) chromatography of pH7.8 buffer solutions balance, A280 monitorings, and dilution packing is standby With.

It is prepared by 5.2 coating plate solid phase antigens

TRPC5 polypeptides are diluted to 0.19 μ g/L coating buffer with 50mmol/L Na2CO3-NaHCO3pH9.6 buffer solutions, 96 holes each hole of coating plate adds 100 μ L, and 4 DEG C stand overnight.Coating buffer is discarded, flushing 2 times, plus 150 μ L BSA's containing 3g/L are above-mentioned Buffer blind, room temperature places 2h.Confining liquid is discarded, vacuum is drained, lath seals rearmounted -20 DEG C of freezen protectives.

The preparation of 5.3 reagents

A.TRPC5 standard solutions are prepared：By TRPC5 standard items, dilution turns into 10ng/mL, 20ng/mL, 40ng/mL, 50ng/mL, 80ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 500ng/mL, 800ng/mL, 1000ng/mL series are dense Degree, dilution is 0.1mol/L pH7.5 phosphate buffers；

B. buffer solution：8mmol/L NaCl, 0.2%OVA, 50 μm of ol/L diethylene triamine pentacetic acid (DTPA)s (DTPA), 0.1mL/ LTweeen-80 and 0.1 mass %NaN3 50mmol/L Tris-HCl pH7.8；

C. cleaning solution is：14.5mmol/L NaCl, 0.2mL/L Tweeen-80 and 0.2 mass %NaN3 50mmol/ LTris-HCl pH7.8；

D. the preparation of liquid is strengthened：By 15 μm of ol β-naphthoyltrifluoroacetones, 50 μm of ol TOPOs and 1mL Qulas Lead to X-100 to add in pH3.2 Potassium Hydrogen Phthalate buffer solutions, then be settled to 1L and be formulated.

Points for attention before 5.4 measure：

A. all reagents are returned before use and be warmed to room temperature (18-30 DEG C).

B. all reagents are put back to 2-8 DEG C immediately after use.

If the c. big suggestion of sample size uses Multi-channel liquid transfer device.

D. the microwell plate and framework of quantity need to be used by taking out, by no microwell plate put into former Fresco Bag and with offer Drier is resealed together, is stored in 2-8 DEG C.

5.5 specific detecting steps are as follows：

Take the porous coating plate for being coated with coating antigen (TRPC5 polypeptides), add 50 μ L TRPC5 standard items or handle well Sample is into respective micropore, plus the anti-TRPC5 antibody that 50 μ L are diluted with buffer solution, and 25 DEG C are vibrated 1 hour, and cleaning solution is washed 4 times, It is subject to 100 μ L Eu3+- goat anti-rabbit antibodies of buffer solution dilution, 25 DEG C are vibrated 1 hour, and cleaning solution is washed 6 times, plus 200 μ L enhancing liquid Vibration measures fluorescence intensity cps after 5 minutes, and the TRPC5 contents in sample are calculated according to standard curve.

6 indirect TRPC5-TRFIA methods clinical practices

Collect clinical serum sample, mammary cancer chemotherapy resistance 21, fine 21 of chemotherapy, normal person 12, using indirect TRPC5-TRFIA methods detection breast cancer source TRPC5 contents, are as a result shown in Fig. 1.Control group is compared without substantially for fine group with chemotherapy Difference (p>0.6), and chemotherapy resistance group and chemotherapy still organize the statistically significant (p of difference<0.0001).

Comparative example：

1st, indirect TRPC5-TRFIA methods clinical practice of sample pretreatment is not carried out

1.1 operating procedure

The porous coating plate for being coated with coating antigen (TRPC5 polypeptides) is taken, 50 μ L TRPC5 standard items or patients serum is added Into respective micropore, plus the anti-TRPC5 antibody that 50 μ L are diluted with buffer solution, 25 DEG C are vibrated 1 hour, and cleaning solution is washed 4 times, is subject to 100 μ L Eu3+- goat anti-rabbit antibodies of buffer solution dilution, 25 DEG C are vibrated 1 hour, and cleaning solution is washed 6 times, plus 200 μ L enhancing liquid vibrations Fluorescence intensity cps is measured after 5 minutes, the TRPC5 contents in sample are calculated according to standard curve.

1.2 experimental result

Collect clinical serum sample, mammary cancer chemotherapy resistance 21, fine 21 of chemotherapy, normal person 12, using indirect TRPC5-TRFIA methods detection breast cancer source TRPC5 contents, are as a result shown in Fig. 2.Chemotherapy resistance group is compared nothing for fine group with chemotherapy Notable difference (p>0.1).

2nd, detected using MUC1 magnetic beads and TRPC5 sandwich methods

2.1 operating procedure

With blood coagulation pigging collection human peripheral is promoted, 1000-2000r/min collects supernatant after centrifuging 5-20min；With being surrounded by The NHS magnetic beads of MUC1 antibody are incubated 1-1.5h, plus the anti-TRPC5 antibody that 50 μ L are diluted with buffer solution, 25-37 with supernatant concussion DEG C vibration 1-1.5 hour, Magnetic Isolation, cleaning solution is washed 4 times, be subject to buffer solution dilute 100 μ L Eu3+- goat anti-rabbit antibodies, 25-37 DEG C vibrate 1-1.5 hour, Magnetic Isolation, cleaning solution is washed 6 times, plus 200 μ L enhancing liquid vibrate 5 minutes after measure fluorescence it is strong Cps is spent, the TRPC5 contents in sample are calculated according to standard curve.

2.1 have problem

(1) there is non-specific adsorption in magnetic bead, and influence of the non-specific adsorption to result be can not ignore；

(2) magnetic bead is pure manual operations, causes batch interior, differences between batches (CV) larger, influences experimental result.

Assay method of the present invention：The basis of measure is labelled immune reaction.The porous coating plate of TRPC5 polypeptides is coated with, Test sample is added into respective micropore, the TRPC5 and micropore added in anti-TRPC5 antibody, oscillating reactions, the sample that dissociates TRPC5 polypeptides on plate compete anti-TRPC5 antibody, cleaning solution washing, the TRPC5 antibody not connected in washing step by except Go.Eu3+- goat anti-rabbit antibodies are added, immune response is marked, then are washed with cleaning solution, the Eu3+- for not having connection after reaction Goat anti-rabbit antibody is removed in washing step.Plus after enhancing liquid vibration, very strong fluorescence is launched under the exciting of uviol lamp, is used Time-resolved fluorescence instrument determines its fluorescence intensity cps, and fluorescence intensity is inversely proportional with the concentration in sample, reference standard curve Determine the amount of TRPC5 in sample.

It should be appreciated that for those of ordinary skills, can according to the above description be improved or converted, And all these modifications and variations should all belong to the protection domain of appended claims of the present invention.

Exemplary description has been carried out to patent of the present invention above in conjunction with accompanying drawing, it is clear that the realization of patent of the present invention not by The limitation of aforesaid way, if the various improvement that the method design of patent of the present invention and technical scheme are carried out are employed, or without The design of patent of the present invention and technical scheme are directly applied to other occasions by improvement, within the scope of the present invention.

Claims (10)

1. a kind of time resolved fluoro-immunoassay kit of detection TRPC5 contents, it is characterised in that：It is coated including MUC1 NHS magnetic beads, porous coating plate, buffer solution, many poly saccharide peptide standard products of TRPC5, TRPC5 antibody, the goat anti-rabbit antibody of europium mark, washing Liquid and enhancing liquid.

2. the kit described in a kind of utilization claim 1 detects the time-resolved fluorescence immunoassay method of TRPC5 contents, its It is characterised by comprising the following steps：

(1) TRPC5 polypeptides are synthesized；

(2) the TRPC5 polypeptides synthesized with step (1) are as coating primordial covering solid phase carrier；

(3) the TRPC5 polypeptides synthesized with step (1) obtain immunogene by coupling agent and KLH couplings；

(4) immunogene obtained with step (3) prepares anti-TRPC5 polyclonal antibody；

(5) with blood coagulation pigging collection new blood is promoted, 1000-2000r/min collects supernatant after centrifuging 5-20min, uses MUC1 After the coated immunomagnetic beads of antibody is mixed with the supernatant, it is incubated 0.5-2h through 25-37 DEG C of concussion or was reacted through 2-8 DEG C Night, magnetic frame absorption magnetic bead with wash buffer and is resuspended, obtains testing sample；

(6) step (5) described testing sample is at war with and suppresses detection and analysis, measure fluorescence intensity, reference standard curve meter Calculate the TRPC5 contents in sample.

3. a kind of time-resolved fluorescence immunoassay method of detection TRPC5 contents according to claim 2, its feature exists In：Step (3) described coupling agent is Sulfo-SMCC.

4. a kind of time-resolved fluorescence immunoassay method of detection TRPC5 contents according to claim 2, its feature exists In：Immunogen immune NZw described in step (3), antibody is prepared by subcutaneous multi-point injection, uses antigen affinity column Purified, obtain the polyclonal antibody of the anti-TRPC5 described in step (4).

5. a kind of time-resolved fluorescence immunoassay method of detection TRPC5 contents according to claim 2, its feature exists In：Step (2) described solid phase carrier is 96 hole microwell plates.

6. a kind of time-resolved fluorescence immunoassay method of detection TRPC5 contents according to claim 2, its feature exists In：Step (5) described buffer solution is phosphate buffer.

7. a kind of time-resolved fluorescence immunoassay method of detection TRPC5 contents according to claim 2, its feature exists In：Step (5) described immunomagnetic beads is NHS magnetic beads.

8. a kind of time-resolved fluorescence immunoassay method of detection TRPC5 contents according to claim 7, its feature exists In：The NHS magnetic beads are purchased from Suzhou Beaver companies.

9. a kind of time-resolved fluorescence immunoassay method of detection TRPC5 contents according to claim 2, its feature exists In：Step (6) described standard curve be the TRPC5 polypeptides that synthesize of step (1) as standard items, dilution turns into 0ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 50ng/mL, 80ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 500ng/mL, 800ng/mL, 1000ng/mL series concentration, dilution are 0.1mol/L pH7.5 phosphate buffers, are carried out competing Suppression detection and analysis is striven, testing result fluorescent value and standard concentration are taken the logarithm i.e. log10, linear fit is used as standard bent Line.

10. a kind of time-resolved fluorescence immunoassay method of detection TRPC5 contents according to claims 2 to 9, it is special Levy and be, the coated immunomagnetic beads preparation method of MUC1 antibody is：

A. protein solution is prepared：50-100 μ g MUC1 albumen is taken thoroughly to be removed by the method for dialysis or desalination original Material containing primary amine groups in buffer, is then made into Coupling Buffer the albumen that concentration is 3.0-5.0mg/mL molten again Liquid, the protein solution prepared is saved backup in 2-8 DEG C；

B. 200-500 μ L 20% bead suspension is taken in EP pipes；

C. EP pipes are placed in magnetic separation rack, are enriched with magnetic bead, remove supernatant；

D. plus 1mL 2-8 DEG C Washing Buffer A in centrifuge tube, vortex 15-30s, make magnetic bead be well mixed；

E. EP pipes are placed in magnetic separation rack, are enriched with magnetic bead, remove supernatant；

F. plus 200-500 μ L protein solutions are in EP pipes, vortex 15-30s, it is well mixed it；

G. by EP pipe vortex 15-30s, it is placed on vertical mixed instrument, 2-8 DEG C of reaction is stayed overnight；

H. magnetic bead is enriched with using magnetic separation rack, preservation flows through liquid；

I. plus 0.5-1mL Blocking Buffer are in EP pipes, vortex 15-30s, EP pipes are placed in magnetic separation rack, it is rich Collect magnetic bead, abandon supernatant；

J. plus 0.5-1mL Blocking Buffer are in EP pipes, vortex 15-30s, EP pipes are placed in room temperature in vertical mixed instrument React 2-4h；

K. EP pipes are placed in magnetic separation rack, are enriched with magnetic bead, abandon supernatant；

L. plus 0.5-1mL ultra-pure waters are in EP pipes, it is sufficiently mixed, is enriched with magnetic bead with magnetic frame, abandons supernatant；

M. plus 0.5-1mL Storage Buffer are in EP pipes, it is sufficiently mixed, is enriched with magnetic bead with magnetic frame, abandons supernatant；Weight Operation 2 times again；

N. 0.5-1mL Storage Buffer are added in EP pipes, are sufficiently mixed, 2-8 DEG C saves backup.