CN104880556A - An aptamer-antibody method for detecting a trace of prostate-specific antigen (PSA) in blood and a kit - Google Patents
An aptamer-antibody method for detecting a trace of prostate-specific antigen (PSA) in blood and a kit Download PDFInfo
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- CN104880556A CN104880556A CN201410066859.2A CN201410066859A CN104880556A CN 104880556 A CN104880556 A CN 104880556A CN 201410066859 A CN201410066859 A CN 201410066859A CN 104880556 A CN104880556 A CN 104880556A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
Abstract
An aptamer-antibody method for detecting a trace of prostate-specific antigen (PSA) in blood is provided. A specific PSA aptamer coupled to magnetic particles and a specific PSA antibody coupled to nanogold particles are respectively mixed with a sample to be detected so as to detect, identify and/or quantify a target molecule (the PSA). The method is simple and convenient to use, sensitive, high in specificity, good in repeatability, rapid in result production, and free of needs for complex instruments and special techniques, and can be popularized to actual industrial application.
Description
Technical field
The present invention relates to method of protein detection technical field, specifically, the present invention relates to the method for micro-PSA in detection, qualification and/or quantitative human blood.In addition, the invention still further relates to the kit and application etc. thereof for the method.
Background technology
Prostate cancer is one of modal lethal cancer of elderly men, excision is the first-selection in prostate cancer comprehensive treatment, but the recurrence rate after prostate cancer excision is high, the rate of transform is high: cancer cell is easy to enter peripheral blood generation metastases from primary tumor, and metastases is the main cause of prostate cancer therapy failure.Great many of experiments has confirmed that circulating tumor cells (Circulating Tumor Cells, CTC) detection will contribute to prostatic cancer early diagnosis, relapse and metastasis monitoring, and judge patient's prognosis, instruct postoperative adjuvant therapy etc.
Prostate specific antigen (Prostate specific antigen, PSA) is by prostate epithelial cell synthesis secretion.Within 1979, extract from prostata tissue and this protein of purifying.Because this albumen only exists in prostata tissue, be named as prostate specific antigen.PSA is one of most important mark of prostate cancer.Be mainly limited under normal circumstances in prostata tissue, during tissue canceration, a large amount of PSA enters blood circulation makes PSA level in blood raise.At present, the value of prostate cancer PSA examination has become academia and has argued one of maximum hot issue.Have research prompting, though the examination based on PSA significantly improves prostate cancer discovery rate, mostly be make slow progress, aggressive is low, without the invisible prostate cancer of clinical symptoms, only have on a small quantity because having high aggressive, occurring shift and cause death.
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Utilize the detection of anti-psa antibody on human PSA patented in the world, as US5939533A; US5599677; EP0540573 A1.The domestic detection patent also had for human prostate cancer specific antigen at present: as prostatic cancer specific detection kit 200710041914.2; Anti-prostate-specific-antigen PSA monoclone antibody 200610026505.0; A kind of kit 200610148733.5 of diagnosing prostate cancer.These methods all utilize antibody to carry out PSA detection, therefore the accuracy detected is very high to the specific requirements of antibody wherein, otherwise there will be a large amount of false-positive result, in addition because the production cost of antibody reagent own is high, antibody must be preserved, transports and use to prevent antibody titer step-down under appropriate conditions, this considerably increases the uncertainty of testing cost and testing result.
The beginning of the nineties in last century, Tuerk and Gold has founded index concentration formula aglucon systematization screening (Systematic Evolution of Ligands by Exponential Enrichment, referred to as SELEX) technology, by the external selection taken turns and amplification more, specificity aptamers (aptamer) can be filtered out from random oligonucleotide library, see (Tuerk and Gold, 1990).At present, it has been found that and permitted polymolecular specificity aptamers, as the aptamers of microorganism, biomacromolecule (as, vascular endothelial growth factor, the growth factor in blood platelet source, basic fibroblast growth factor, L-selects element, keratinocyte growth factor, IFN-γ, the cell factors such as TNF) aptamers, micromolecular aptamers, can specific bond corresponding microorganism, biomacromolecule, the target molecules such as Small molecular, see CN1490054A, CN1550501A, CN1563401A, CN101148666A, CN101835904A, bioengineering journal, 20 (4): 627-632, Deng.In the world based on the prostate cancer diagnosis of PSA aptamers and targeting therapeutic agents patented: see WIPO Patent Application W0/2012/001820.The advantage of aptamers is to have very high affinity to the identification of target molecule, and its dissociation constant even can reach 50pmol/L ~ 10nmol/L.In addition, chemical constitution due to aptamers itself is single-chain nucleic acid, the combination of aptamers and target molecule can by the interference of impurity molecule, especially when target molecule and impurity molecule be all single-chain nucleic acid (as, scDNA, mRNA) when, because nucleic acid chains itself exists the character of pairing dimerization, the situation increasing interference is occurred.These unfavorable factors have impact on accuracy and the precision of aptamers detection, make it be difficult to popularization and carry out commercialization use, and therefore the current detection of the ELISA based on antibody or PCR consuming time detection etc. are still come into operation in practice.
Summary of the invention
The detection of specific antibody that first problem to be solved by this invention is to overcome existing human prostate cancer specific antigen requires that height, antagonist specific requirements are very high and the problem that false positive rate is higher to environment degree of purity, the suitable antibody detection method of PSA in a kind of blood is provided, the method is easy to use, sensitive, high specificity, reproducible, go out result quick, without the need to complex instrument and special skill, there will not be the situation that aptamers and target molecule (PSA) and other non-specific protein interfere with each other.
Another technical matters to be solved by this invention is to provide a kind of suitable antibody kit detecting micro-PSA in blood.
Another technical matters to be solved by this invention is to provide a kind of specific reagent box detecting micro-prostate gland cancer cell in blood, for the application in the goods of the present invention's first aspect method.
In order to solve these problems of the prior art, in first, technical scheme provided by the invention is: the invention provides a kind of method detecting PSA in blood, comprise the following steps,
(1) from prostate gland cancer cell, PSA is extracted;
(2) prepare and the specificity aptamers of magnetic-particle coupling and the PSA antibody with nanogold particle coupling, wherein specificity PSA aptamers and PSA antibody be respectively with the different parts specific bond of PSA, and PSA specificity aptamers and PSA antibody are without the Potential feasibility be combined with each other;
(3) by the aptamers of the coupling in step (2) and to be detected, identify and/or quantitative sample mix and carry out magnetic resolution,
(4) magnetic resolution product is detected.
Preferably, in the method for the present invention first aspect, wherein step (3) is:
2. by the PSA aptamers of magnetic-particle coupling and to be detected, identify and/or quantitative sample mix;
3. carry out magnetic resolution, collect the product of magnetic-adsorption gained; With
4. mix the product of magnetic-adsorption gained and the PSA antibody with nanogold particle coupling, then carry out magnetic resolution.
Preferably, in the method for the present invention first aspect, wherein PSA (PSA) is human prostate cancer specific antigen, and its albumen is preferably as shown in SEQ ID No.:1.
Preferably, in the method for the present invention first aspect, wherein PSA aptamers and human prostate cancer specific antigen entirety combine, and another binding site of PSA antibody (C-19, Santa Cruz Biotech) is positioned at the C stub area of human prostate cancer specific antigen.More preferably, wherein PSA aptamers, PSA antibody and human prostate cancer specific antigen do not have homogeneity, and most preferably PSA aptamers is as shown in SEQ ID No.2, be 5 '-dTTTTTAATTA AAGCTCGCCA TCAAATAGCT TT-3 ', PSA antibody is immunoglobulin (Ig).
Preferably, in the method for the present invention first aspect, wherein PSA antibody certification mark substance markers.Wherein, certification mark thing is one or more combinations in biotin Biotin, radioactive isotope, metal marker thing, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or acceptor, is most preferably biotin Biot in.
Preferably, in the method for the present invention first aspect, wherein PSA antibody is by the nanogold particle coupling of Avidin and streptococcus Avidin-horseradish peroxidase bag quilt.
Preferably, in the method for the present invention first aspect, wherein magnetic resolution product is the product of magnetic-adsorption gained.
Preferably, in the method for the present invention first aspect, wherein magnetic resolution product is through washing.
Preferably, in the method for the present invention first aspect, the temperature of wherein carrying out step (3) is 20-42 DEG C, is preferably 37 DEG C.
Preferably, in the method for the present invention first aspect, wherein the damping fluid of step (3) is Tris-HCl, PBS or the PBS containing Mg ion, is preferably the PBS containing Mg ion.
In second, the invention provides a kind of specific antigen kit detecting micro-prostate gland cancer cell in blood, it comprises and the PSA aptamers of magnetic-particle coupling and the PSA antibody with nanogold particle coupling, wherein PSA aptamers and PSA antibody be respectively with the aptamers of the different parts specific bond of PSA, and the potential that PSA aptamers and PSA antibody are combined without phase mutual cross.
Preferably, in the kit of the present invention second aspect, wherein PSA aptamers and human prostate cancer specific antigen entirety combine, and another binding site of PSA antibody (C-19, Santa Cruz Biotech) is positioned at the C stub area of human prostate cancer specific antigen.
Preferably, in the kit of the present invention second aspect, wherein PSA aptamers, PSA antibody and human prostate cancer specific antigen do not have homogeneity, most preferably PSA aptamers is as shown in SEQ ID No.2, be 5 '-dTTTTTAATTA AAGCTCGCCA TCAAATAGCT TT-3 ', PSA antibody is immunoglobulin (Ig).
Preferably, in the kit of the present invention second aspect, wherein PSA antibody certification mark substance markers.Wherein, certification mark thing is one or more combinations in biotin, radioactive isotope, metal marker thing, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or acceptor, is most preferably biotin.
Preferably, in the kit of the present invention second aspect, wherein PSA antibody is by the nanogold particle coupling of biotin and streptococcus Avidin-horseradish peroxidase bag quilt.
Preferably, in the kit of the present invention second aspect, the reagent that can detect certification mark thing is also comprised, as horseradish peroxidase chromogenic substrate.
Preferably, in the kit of the present invention second aspect, it also comprises damping fluid, and preferred buffer is Tris-HCl, PBS or the PBS containing Mg ion, is more preferably the PBS containing Mg ion.
Preferably, in the kit of the present invention second aspect, it also comprises magnetic separation device.
In the 3rd, the invention provides the application of kit in the goods for the preparation of the method in the present invention first of the present invention second aspect.
In the 4th, the invention provides a kind of bond, by with the PSA aptamers of magnetic-particle coupling, be combined into the PSA antibody of certification mark thing coupling and PSA, wherein PSA aptamers and PSA antibody be respectively with the aptamers of the different parts specific bond of PSA, and the potential that PSA aptamers and PSA antibody are combined without phase mutual cross.Preferably wherein, PSA is human prostate cancer specific antigen, and more preferably its albumen is as shown in SEQ ID NO:1.
Preferably, in the bond of the present invention the 4th aspect, wherein PSA aptamers and human prostate cancer specific antigen entirety combine, and another binding site of PSA antibody (C-19, Santa Cruz Biotech) is positioned at the C stub area of human prostate cancer specific antigen.
In the bond of preferred the present invention the 4th aspect, wherein PSA aptamers, PSA antibody and human prostate cancer specific antigen do not have homogeneity, most preferably PSA aptamers is as shown in SEQ ID No.2, be 5 '-dTTTTTAATTA AAGCTCGCCA TCAAATAGCT TT-3 ', PSA antibody is immunoglobulin (Ig).
Preferably, in the bond of the present invention the 4th aspect, wherein PSA antibody certification mark substance markers.
Preferably, in the bond of the present invention the 4th aspect, wherein certification mark thing is one or more combinations in biotin, radioactive isotope, metal marker thing, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or acceptor, is most preferably biotin.
Preferably, in the bond of the present invention the 4th aspect, wherein PSA antibody is by the nanogold particle coupling of biotin and streptococcus Avidin-horseradish peroxidase bag quilt.
Preferably, in the bond of the present invention the 4th aspect, it at 20-42 DEG C, can combine under being preferably the temperature of 37 DEG C.
In this article, term " detection " has implication well-known to those skilled in the art, comprises the qualification existence of target product and the quantity of quantitative objective product.The net result that detection method of the present invention obtains is existence or the quantity of PSA.(JAMA2005 is seen owing to also there is a small amount of PSA in the prostate of healthy individuals, 294 (1): 66), therefore identify that PSA directly cannot draw the result whether suffered from tumor of prostate or have prostate cancer occurence tendency; Due to implementer of the present invention not necessarily clinician, therefore and do not know that patient to be got before " sample " degree whether through treatment and treatment, the quantity of the PSA therefore quantitatively obtained directly cannot draw the result whether suffered from tumor of prostate or have tumor of prostate Preventive occurence tendency.The method of first aspect of the present invention only can regard an intermediate steps in diagnostic procedure as, what obtain is PSA result in " sample ", itself directly can not draw and whether suffer from tumor of prostate or have the diagnostic results such as tumor of prostate Preventive occurence tendency.In addition, due to the hereditary difference of individuality, the PSA level of each individual patient is caused to have different, the age of patient in addition, physiological status also can cause the difference of PSA level in blood, if directly will judge whether to suffer from tumor of prostate or have tumor of prostate to recur, the diagnostic results such as transfer occurence tendency, the result that the method for first aspect of the present invention must be obtained and corresponding normal healthy controls or individuality are before the treatment, after blood in PSA level compare, and judge whether corresponding individuality is enough to deduct individual inheritance by experienced doctor, age, the factors such as physiological status are to the systematic effects of PSA, could attempt obtaining diagnostic result.Therefore, the method for first aspect of the present invention do not comprise with corresponding normal healthy controls and individual treatment before and after the process that compares.Even if according to the specific embodiment of the present invention, the PSA level of the different prostate cancer cell lines of different individual patient also also exists greatly difference, some is even similar to background values, as negative control, directly can cannot draw diagnostic result thus.
In this article, term " sample " refers to the potential in vitro circulating sample that may contain PSA, preferably from the sample of people.Although also can use in the present invention with the purification of samples of the hemorrhage total protein of extracting, sample of the present invention preferably without purify, cracking fluid sample containing blood total protein.Those skilled in the art know the nucleated blood cell cracking of solid or extracting, purifying keep the cell biological molecular techniques that wherein protein ingredient is not degraded.Sample of the present invention can be through the sample of process, as dilution process, haemocyte cracking process etc., also can be undressed sample.Treated sample can be further purified, with rich protein.Because doctor human body sampling being needed to qualification determines according to individual concrete condition, so the method for first aspect of the present invention does not comprise the process of sampling and optional sample preparation, owing to not knowing that whether sample is through the degree processed and process, and therefore directly cannot draw and whether suffer from tumor of prostate or have the diagnostic results such as tumor of prostate Preventive occurence tendency.The method of first aspect of the present invention does not comprise the process of sampling and optional sample preparation.
In the present patent application, PSA (referred to as PSA) is human prostate cancer specific antigen protein preferably, is more preferably the human prostate cancer specific antigen protein sequence as shown in SEQ ID NO:1.PSA is only expressed in prostate and prostate gland cancer cell, therefore detects PSA in circulating and may be used for judging whether the sample detected exists prostate and prostate gland cancer cell.
In this article, DNA symbol well-known to those skilled in the art represents its sequence, and if no special instructions, what sequence represented is from 5 ' end to the sequence order of 3 ' extreme direction.Be preferably 0.5 ~ 5.1 with the PSA aptamers of magnetic-particle coupling with the mol ratio of the PSA antibody detecting nanogold particle coupling, most preferably be 1:1.Owing to being employed herein aptamers/antibody, these two molecules must be attached on same target molecule, and just can obtain testing result, thus testing result is very special; If the PSA aptamers of capture probe or magnetic-particle coupling and non-specific target molecule combine, but simultaneously very little in conjunction with this non-specific target molecule possibility with the PSA antibody detecting nanogold particle coupling, the impact of non-specific binding thus still can be eliminated when detecting.Such aptamers/antibody dual fail-safe, make method of the present invention before detection row gland cancer specific antigen time greatly reduce false-positive result, improve accuracy.Due to the PSA antibody be employed herein with detect nanogold particle coupling, wherein nanogold particle is coated with multiple streptococcus Avidin-horseradish peroxidase molecule, warp and horseradish peroxidase substrate chromogenic reaction, can strengthen the detection sensitivity to PSA.
Term " aptamers " in present patent application is well-known to those skilled in the art, refers to the single-chain nucleic acid that can combine with specific target molecule (PSA), comprises DNA or RNA.The aptamers of certain target molecules can be obtained by SELEX.Owing to employing PSA aptamers in the method for first aspect present invention, the present inventor studies discovery, and PSA aptamers and PSA antibody are without the potential be combined without phase mutual cross mutually.Because the chemical classification of target molecule (PSA) belongs to memebrane protein, avoid the interference of target molecule itself.In the specific embodiment of the present invention, PSA aptamers is 5 '-dTTTTTAATTA AAGCTCGCCA TCAAATAGCT TT-3 '.
In the present patent application, " without the Potential feasibility that combines " in method step refers to can not to hybridize after PSA aptamers nucleotide sequence mixes with PSA antibody and forms stable combination, can not affect the combination of aptamers and target molecule.
In the present patent application, magnetic-particle is preferably magnetic nanoparticle (referred to as MNP), i.e. the particle of Nano Particle, and its band is magnetic, and can be separated by equipment for magnetic separation.Magnetic-particle comprises nano particle, the quantum dot or nano dot etc. of magnetic metal.Described particle is preferably superparamagnetism, comprises the particle that US6057107B, US6268222B, US6524793B etc. record.Preferably select and can obtain product by commercial channel.In the present invention, preferably the first aptamers and magnetic-particle pass through chemical reaction coupling.
In the present patent application, be the material that can detect with the label detected on the PSA antibody of nanogold particle coupling, such as, can be excited by laser, radioactivity, chemiluminescence (comprising fluorescence), electroluminescence, electrochemiluminescence, enzymatic colour developing or any method well known by persons skilled in the art realize.Exemplary survey label includes but not limited to one or more combinations in biotin, radioactive isotope, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or acceptor.Radioisotopic suitable example has
32p,
125i,
35s,
3h etc.By conventional methods such as chemosynthesis, PCR, nick translations, radiolabeled second single-chain nucleic acid can be prepared.Chemiluminescent groups has luminol etc.; Chemiluminescence group comprises rhodamine, FITC, TRITC etc.Marked by biotin (Biotin) is undertaken by the chemical synthesis process of routine to the second single-chain nucleic acid, the above-mentioned single-chain nucleic acid with biotinylated derivative and preparation and determination methods method thereof are known, can summarize see (Tabor and Boyle, 2001).By chemical synthesis process, can be coated on nanogold particle by streptococcus Avidin-horseradish peroxidase molecule, its method can see documents such as (Long and Keating, 2006, Mayer eta1., 2000, Tang et a1., 2008).The nanogold particle of the above-mentioned single-chain nucleic acid with biotinylated derivative and streptococcus Avidin-horseradish peroxidase bag quilt can special, combine high-affinity, and form the PSA antibody complex with measuring ability.There is a large amount of commercial horseradish peroxidase detection kit available at present, as Supersignal West Pico Horseradish Peroxidase (HRP) the Detection kit etc. of the Streptavidin-Peroxidase of Sigma-Aldrich sold, Pierces (USA) sold.In the specific embodiment of the present invention, utilize nanogold particle can in conjunction with the character compared with multienzyme molecule, certification mark thing adopts the coupled product of nanogold particle and horseradish peroxidase, greatly strengthen the detection sensitivity to PSA.
In the present patent application, magnetic resolution product is the product of magnetic-adsorption gained, also can be the product be not attracted by the magnetic force, but is preferably the product of magnetic-adsorption gained.Can be attracted by the magnetic force and be detected the product that the product obtained necessarily combines PSA aptamers and PSA antibody.By equipment for magnetic separation, can the material that adsorbs by magnetic force can be attached on test tube wall, not the material that adsorbs by magnetic force then still reside in solution, when after draw solution, these two kinds of materials just can be separated, thus reach the object of separation.In theory, product and the sum total of certification mark thing in the product be not attracted by the magnetic force of magnetic-adsorption gained equal to mix the amount of the capture probe that initially adds or the PSA aptamers with magnetic-particle coupling, and both can convert according to the amount mixing the certification mark thing that initially add.Preferably in the step of magnetic resolution, finally wash magnetic resolution product, namely magnetic resolution product is through washing.
Bond of the present invention can be hatched and combined and be not separated at the temperature of 20-42 DEG C, preferably hatches at the temperature of 37 DEG C, the hybridization temperature that this temperature normally adopts in order to avoid aptamers and non-specific target hybridization; For convenience of the carrying out of operation, allow that the temperature of experimental enviroment has the deviation of 1-2 DEG C, but must carry out being less than at the temperature of 45 DEG C, preferably carry out at the temperature of 37 DEG C.As exceeded this temperature, as 50 DEG C, aptamers may be caused to be combined with specific target molecules, thus reduce the sensitivity detected.In this article, as do not added explanation, mixing refers to action and the rear process of placing (hatching) of mixing of mixing.
The method running time of the present invention is short.Wherein accounting for most the time of placing in the blend step of time is less than 60 minutes, preferred 30-45 minute, as 30,35,40 minutes.
Compared to solution of the prior art, the invention has the beneficial effects as follows:
1, simple to operate, detection time is short.In fact only need reagent due to method of the present invention and adsorb with magnetic separator after being with the sample detected simply to mix placement and detect, removing detecting step, other steps are simple, convenient, consuming time short, especially PSA is detected relative to conventional ELI SA, time much shorter.Without the need to complex instrument and special skill.
2, high specificity, reproducible, accuracy rate is high, effectively reduces false positive results.Because the present invention is by two non-interfering aptamers, adopt dual fail-safe, thus can keep the correct of result, greatly reduce the probability that false positive results occurs, make the suitable actual popularization of the present invention.
3, relatively high detection sensitivity, detects PSA than the ELISA of routine sensitive.
4, commercialization is easy, and testing cost is low.Because reagent itself can be prepared by business-like method, and in detecting, required instrument also can be bought and obtains, therefore can fast in large, medium and small type hospital, even clinic popularization and application; It is cheap that wherein used instrument comprises equipment for magnetic separation, and thus easily commercialization is promoted.
5, constant product quality, condition of storage is less demanding.Aptamers is more stable than protein antibodies, not changeableness, easily long-time preservation, and this causes the term of validity of product greatly to extend.
6, cost is low, because aptamers can chemosynthesis in a large number, thus production cost comparatively antibody detection method can significantly reduce.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described:
1, Fig. 1 illustrates:
The left figure of Fig. 1 shows in the present invention, fit link with magnetic-particle formed link compound, antibody links with nanogold particle the method being formed and link compound; The right figure of Fig. 1 shows in the present invention, detects load procedure and the principle of PSA in blood sample.
2, Fig. 2 illustrates:
Fig. 2 shows method of the present invention and detects PSA and comparing by ELISA method detection sensitivity.Fig. 2 .1 shows the sensitivity that method of the present invention detects PSA; Fig. 2 .2. shows the sensitivity detecting PSA by ELISA method.
Embodiment
For the ease of understanding, below will be described in detail the present invention by specific embodiment.It is important to note that these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this instructions, many changes of the present invention, to change concerning one of ordinary skill in the art be all apparent.
Below will be described by way of example, if any not detailed part, can see conventional laboratory manual, as the manufacturers instruction of " Molecular Cloning: A Laboratory handbook " and agents useful for same and instrument.Wherein, all chemical reagent all adopt AG, and experimental water filters through Mi l l i-xQ, and each reagent all can obtain by commercial channel with material, specifically: there is the magnetic corpusculum (1.5M, 20mg/mL) of carboxyl terminal purchased from Polysciences company (U.S.); 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (referred to as EDC) is purchased from Alfa Aesar company (U.S.); (particle diameter is 5nm to nanogold particle, 10nm, 30nm and 50nm), streptococcus Avidin (streptavidin) wraps the gold nano grain (particle diameter is 10nm and 40nm) of quilt all purchased from BB Internat i onal (U.S.); Bovine serum albumin, streptococcus Avidin-horseradish peroxidase (S2438) are purchased from Sigma-Aldrich (U.S.); Horseradish peroxidase substrate reagent box is purchased from Millipore Corporation (U.S.); PSA aptamers entrusts Sigma-Aldrich company (USA) synthesis, and its sequence is as follows: 5 '-5 '-dTTTTTAATTA AAGCTCGCCA TCAAATAGCTTT-3 '-3 '; Biotinylation PSA antibody; LNCaP/PC3 cell all can purchased from American Type Culture Collection (ATCC), and the nutrient culture media cultivating it is nutrient culture media (the DMEM nutrient culture media that interpolation 10% hyclone cow's serum (Gibco company), Pidolidone (Gibco company), the penicillin of 50U/ml and the Dulbecco of 5 mcg/ml streptomysins (Gibco company) improve Eagle; Sigma-Aldrich company); Total protein of cell extracts reagent related reagent purchased from (Gibco company, the U.S.).
Embodiment 1 detects the preparation with prostate gland cancer cell protein sample and contrast thereof
In vitro culture LNCaP (PSA+) cell (The Prostate2011, Vol71, Issue15, pg1668-79.), attached cell makes cell suspend through trypsinization, centrifugally removes nutrient solution, then adds physiological saline, prepare single cell suspension, get a certain amount of cell (10 through cell count
7) test.Illustrate (liking rich abio) according to manufacturer, extract reagent with total protein of cell and extract LNCaP total protein of cell, measure the amount of quantitative total protein.Then total protein PBS damping fluid (pH7.2) being carried out gradient dilution, comprising respectively from being equivalent to 10 in each dilution solution equal-volume
5(A group), 10
4(B group), 10
3(C group), 10
2(D group) and 10
1the total protein of (E group) individual LNCaP prostate gland cancer cell.
According to said method, extracting the total protein of PC3 (PSA-) (The Prostate2011, Vol71, Issue15, pg1668-79.) cell, comprising respectively from being equivalent to 10 in each dilution solution equal-volume after dilution
5(A
0group), 10
4(B
0group), 10
3(C
0group), 10
2(D
0group) and 10
1(E
0group) total protein concentration of individual PC3 (PSA-) prostate gland cancer cell.
Embodiment 2: with the comparing of method detecting PSA with ELISA
According to the condition determined, ELISA (Human PSA ELISA Kit is carried out respectively to the known LNCaP total protein of cell (A, B, C, D, E group dilution) containing target molecule (PSA albumen) prepared by embodiment 1, GenWay Biotech Inc) detect, the existence of protein target molecule is judged according to the power of chromogenic reaction.Target molecule (PSA) expresses negative PC3 total protein of cell (A
0, B
0, C
0, D
0, E
0group dilution) be corresponding control group. result is as shown in Figure 1, the OD value that target molecule expresses negative PC3 (PSA-) total protein of cell is very low, use can be contrasted as a setting, using its detection line as the standard baseline detected (be that testing result positive higher than this line); And in three duplicate detection to the known LNCaP cell series diluted protein sample containing target molecule PSA albumen, even if lower dilution D sample (10
2cells/ml), OD value is also significantly greater than minimum dilutability (10
5cells/ml) the result of contrast (PC3), its sensitivity at least can reach other dilutability of 10,000/level (Fig. 2 .1).Adopt this kit, three duplicate detection have been carried out, except lower dilution D sample (10 to the known LNCaP cell series diluted protein sample containing target molecule PSA albumen
2cells/ml) be positive outside result, minimum dilutability (10
5cells/ml) LNCaP cell dilution protein sample is also positive result, and its sensitivity at least can reach other dilutability of 100,000/level (Fig. 2 .2).
This shows, the ELISA detection detection sensitivity that method of the present invention can substitute complicated operation is effectively higher.
Above-mentioned example, only for technical conceive of the present invention and feature are described, its object is to person skilled in the art can be understood content of the present invention and implement according to this, can not limit the scope of the invention with this.All equivalent transformations of doing according to Spirit Essence of the present invention or modification, all should be encompassed within protection scope of the present invention.
Claims (28)
1. detect the suitable antibody method of micro-PSA (PSA) in blood, its step comprises,
(1) from prostate gland cancer cell, PSA is extracted;
(2) prepare and the specificity PSA aptamers (PSA aptamers) of magnetic-particle coupling and the specificity PSA antibody (PSA antibody) with nanogold particle coupling, wherein PSA aptamers and PSA antibody be respectively with the different parts specific bond of PSA, and PSA aptamers and PSA antibody are without the Potential feasibility be combined with each other;
(3) by the aptamers of the coupling in step (2) and to be detected, identify and/or quantitative sample mix and carry out magnetic resolution,
(4) magnetic resolution product is detected.
2. method according to claim 1, wherein step (3) is:
1. by the PSA aptamers of magnetic-particle coupling and to be detected, identify and/or quantitative sample mix;
2. carry out magnetic resolution, collect the product of magnetic-adsorption gained; With
3. mix the product of magnetic-adsorption gained and the PSA antibody with nanogold particle coupling, then carry out magnetic resolution.
3. the method according to any one of claim 1-2, wherein PSA (PSA) is human prostate cancer specific antigen, and its protein sequence is preferably as shown in SEQ ID No.:1.
4. method according to claim 3, wherein PSA aptamers and human prostate cancer specific antigen entirety combine, and another binding site of PSA antibody (C-19, Santa Cruz Biotech) is positioned at the C stub area of human prostate cancer specific antigen.
5. method according to claim 4, wherein PSA aptamers, PSA antibody and human prostate cancer specific antigen do not have homogeneity, most preferably PSA aptamers is as shown in SEQ ID No.2,-5 '-dTTTTTAATTA AAGCTCGCCA TCAAATAGCT TT-3 ', PSA antibody is immunoglobulin (Ig).
6. the method according to any one of claim 1,2,3 or 4, wherein PSA antibody certification mark substance markers, as one or more combinations in biotin, radioactive isotope, metal marker thing, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or acceptor.
7. method according to claim 6, wherein certification mark thing is biotin.
8. method according to claim 7, wherein PSA antibody is the nanogold particle coupling by biotin and streptococcus Avidin-horseradish peroxidase bag quilt.
9. according to claim 1-3,5,6, method described in 8 any one, wherein magnetic resolution product is the product of magnetic-adsorption gained.
10. according to claim 1-3,5,6,8, method described in 9 any one, wherein magnetic resolution product is through washing.
11. according to claim 1-3,5,6,8,9, method described in 10 any one, the temperature of wherein carrying out step (2) is 20-42 DEG C.
12. methods according to claim 11, the temperature of wherein carrying out step (2) is preferably 37 DEG C.
13. according to claim 1-3,5,6,8,9, method described in 10 any one, wherein the damping fluid of step (2) is Tris-HCl, PBS or the PBS containing Mg ion.
14. suitable antibody method and the kits detecting micro-PSA (PSA) in blood, comprise and the PSA aptamers of magnetic-particle coupling and the PSA antibody with nanogold particle coupling, wherein PSA specificity aptamers and PSA antibody be respectively with the different parts specific bond of PSA, and PSA specificity aptamers and PSA antibody are without the Potential feasibility be combined with each other;
Wherein PSA aptamers, PSA antibody and human prostate cancer specific antigen do not have homogeneity, and most preferably PSA aptamers is as shown in SEQ ID No.2, and be 5 '-dTTTTTAATTA AAGCTCGCCA TCAAATAGCT TT-3 ', PSA antibody is immunoglobulin (Ig).
15. kits according to claim 14, wherein PSA aptamers and human prostate cancer specific antigen entirety combine, and another binding site of PSA antibody (C-19, Santa Cruz Biotech) is positioned at the C stub area of human prostate cancer specific antigen.
Kit described in 16. any one of claim 14,15, wherein PSA antibody certification mark substance markers.
17. kits according to claim 16, wherein certification mark thing is one or more combinations in biotin, radioactive isotope, metal marker thing, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or acceptor.
18. kits according to claim 16, wherein PSA antibody is the nanogold particle coupling by biotin and streptococcus Avidin-horseradish peroxidase bag quilt.
19. kits according to any one of claim 17-18, it also comprises the reagent that can detect certification mark thing.
20. kits according to any one of claim 17-18, it also comprises damping fluid, and preferred buffer is Tris-HCl, PBS or the PBS containing Mg ion.
21. kits according to any one of claim 15,16,18,19, it also comprises magnetic separation device.
22. application of kit in the goods for the preparation of method described in claim 1 according to any one of claim 14,15,16,18.
23. 1 kinds of bonds, it is by with the PSA aptamers of magnetic-particle coupling, be combined into the PSA antibody of certification mark thing coupling and PSA antigen protein, wherein PSA aptamers and PSA antibody are respectively with the different parts specific bond of PSA albumen, and PSA aptamers and PSA antibody are without the potential be combined with each other;
Wherein PSA aptamers and human prostate cancer specific antigen entirety combine, and another binding site of PSA antibody (C-19, Santa Cruz Biotech) is positioned at the C stub area of human prostate cancer specific antigen;
Wherein PSA aptamers, PSA antibody and human prostate cancer specific antigen do not have homogeneity, and most preferably PSA aptamers is as shown in SEQ ID No.2, and be 5 '-dTTTTTAATTA AAGCTCGCCA TCAAATAGCT TT-3 ', PSA antibody is immunoglobulin (Ig).
24. bonds according to claim 23, wherein PSA antigen is people PSA albumen, and preferably its protein sequence is as shown in SEQ ID NO:1.
25. bonds according to any one of claim 23,24, wherein PSA antibody certification mark substance markers.
26. bonds according to claim 27, wherein certification mark thing is one or more combinations in biotin (B i ot i n), radioactive isotope, metal marker thing, chemiluminescent groups, chemiluminescence group, fluorescin, enzyme, antigen or antibody, part or acceptor.
27. bonds according to any one of claim 23,24, wherein PSA antibody is the nanogold particle coupling by biotin and streptococcus Avidin-horseradish peroxidase bag quilt.
28. bonds according to any one of claim 23,24, can combine at the temperature of 20-42 DEG C.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106525814A (en) * | 2016-11-07 | 2017-03-22 | 华南师范大学 | PSA detection method based on magnetic core-gold satellite assembly body |
| CN107748257A (en) * | 2017-10-25 | 2018-03-02 | 湖南科技大学 | Preparation method and application based on gold nanoclusters chitosan complexes membrane reagent box |
| CN108956991A (en) * | 2018-07-25 | 2018-12-07 | 南通大学 | A kind of fluorescence resonance energy transfer biosensor and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106525814A (en) * | 2016-11-07 | 2017-03-22 | 华南师范大学 | PSA detection method based on magnetic core-gold satellite assembly body |
| CN106525814B (en) * | 2016-11-07 | 2018-12-25 | 华南师范大学 | It is a kind of based on magnetic core-gold satellite assembly PSA detection method |
| CN107748257A (en) * | 2017-10-25 | 2018-03-02 | 湖南科技大学 | Preparation method and application based on gold nanoclusters chitosan complexes membrane reagent box |
| CN108956991A (en) * | 2018-07-25 | 2018-12-07 | 南通大学 | A kind of fluorescence resonance energy transfer biosensor and its application |
| CN108956991B (en) * | 2018-07-25 | 2021-04-27 | 南通大学 | Fluorescence resonance energy transfer biosensor and application thereof |
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