CN1353313A - Paired proteins with fluorescent resonance energy transfer characteristics and usage thereof - Google Patents
Paired proteins with fluorescent resonance energy transfer characteristics and usage thereof Download PDFInfo
- Publication number
- CN1353313A CN1353313A CN 00130292 CN00130292A CN1353313A CN 1353313 A CN1353313 A CN 1353313A CN 00130292 CN00130292 CN 00130292 CN 00130292 A CN00130292 A CN 00130292A CN 1353313 A CN1353313 A CN 1353313A
- Authority
- CN
- China
- Prior art keywords
- antibody
- protein
- antigen
- albumen
- paired
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
A paried proteins with fluorescent resonance energy transfer (FRET) characteristics is composed of protein A and B. The protein A with the antigen of fluorescent substance or chemically luminescent substance is used as the energy provider. The protein B with the antigen of fluorescent substance is used as energy receptor. They have a common antigen determinant specifically bound with the same antibody molecular cluster. The homogeneous immunodetection kit prepared from them and proper buffer liquid can be used to detect the antibodies of AIDS virus, hepatitis B and hepatiti C.
Description
The paired albumen of forming by albumin A and protein B of (FRET) feature that the present invention relates to have FRET (fluorescence resonance energy transfer), albumin A is as energy donor and has fluorescent material or the antigen of chemiluminescent substance, protein B is as energy acceptor and has the antigen of fluorescent material, and albumin A and protein B have can with the common antigenic determinant of same antibody molecule specific bond, by albumin A, the homogeneous phase immune detection medicine box that B and suitable damping fluid are formed, reach them and be used for detecting the purposes that sample antibody exists, especially at AIDS virus antibody, hepatitis B virus antibody, the purposes of the context of detection of antibody of HCV.
Immunoassays utilize the micro substance in the antigen-antibody reaction detection sample, specificity and susceptibility based on antigen-antibody reaction, the range of application of immunoassays spreads all over the every field of medical test, because its status in medical test becomes more and more important, new method, new technology constantly occur in recent years, say that on the whole its progress is mainly aspect two of robotization and easy.At present recombinant antigen, monoclonal antibody and the zymotechnic of genetic engineering, cell engineering and enzyme engineering technology exploitation progressively replace original immune diagnostic reagent, for the diagnosis of various diseases has increased novel potent agent box.In existing immunologic detection method, the solid-phase immunity method is most widely used with radio-immunity detection method (RIA) and enzyme immunodetection (EIA's), generally include bag by (antigen or antibody), sealing, repeatedly hatch and wash and process such as detection, the fastest also needs more than 2 hours.And the immune response of a liquid phase method and signal measuring step in solution finishes, there is not the participation of solid phase, therefore reaction velocity is more faster than solid phase method, running program is much also simple, wherein homogeneous fluorescent immunoassays (Homogeneous Fluoroimmunoassay, hFIA) be most widely used, but the present mensuration that is only limited to small-molecule drug, this mainly is because natural specimen exists more extensive, complicated fluorescence background and the immature of chemical labeling technology to cause usually. morePeople such as Ulman at first propose the hFIA based on fluorescence resonance energy transmission (FRET), i.e. (Homogeneous Fluorescence resonanceimmunoassay hFRIA), has realized the homogeneous determination of antigen-antibody reaction in homogeneous fluorescent resonance immunoassays.
Exist a kind of FRET (fluorescence resonance energy transfer) (FRET) process between two fluorescence molecules of spectra overlapping, i.e. fluorescent material (energy donor that is stimulated, energy donor) its excited energy is transferred to the process of a light absorption molecule (energy acceptor, energy acceptor).If the excitation wavelength of acceptor and the former emission wavelength are overlapping, use the excitation of donor so, detected will be the emission light of acceptor, the emitted luminescence intensity of donor self will weaken greatly simultaneously.This process and the distance between them are closely related, have only when distance is enough near to take place.
The immunoassays of homogeneous fluorescent resonance at present have been applied to detect the antigen in the serum.[Ueda H such as Ueda, Kubota K, WangY, et al.Biotechniques, 27 (4): 738-742 (1999)] use succinimide fat and fluorescein-X heavy chain and the light chain of labelled antibody respectively, both add antigen after mixing again in cuvette, the specific bond of antigen-antibody, make succinimide fat and fluorescein-X close, excite succinimide fat with the 490nm wavelength, detect 520nm emission light and weaken, the emission light of 605nm strengthens, along with the increase of antigen amount, the fluorescence of 605nm also strengthens.[Stenroos K such as Stenroos, Hurskainen P, Eriksson S, etal.Cytokine, 10 (7): 495-499 (1998)] with the chelate mark recombination human interleukin 2 (IL-2) of europium, use the Cy5 labeled monoclonal antibody, carry out time-resolved fluorescent immunoassay.Blomberg etc. use terbium and rhodamine labelled antibody respectively, detect the β CG of human chorionic gonadotropin in the serum, and the energy transfer signal is directly proportional with β CG concentration in the sample as a result.
Existing homogeneous fluorescent resonance immunoassays all must be carried out mark to detectable with organic synthesis fluorescent dye or rare earth element, to be coupled on the different parts of antibody respectively for two kinds of right fluorescent materials of acceptor or on the different antibody molecules as energy, more be that they are coupled at respectively on antigen and the antibody, in solution, specificity by antigen-antibody is in conjunction with the distance that furthers between two fluorescence molecules, thereby the FRET phenomenon takes place.Though these methods have satisfied the needs of fast detecting, but all require to obtain highly purified antibody and antigen, make the detection previous work numerous and diverse, owing to can't reach hundred-percent purity, the non-specific mark of unavoidable generation during fluorescence labeling, very easily occurring false positive in detection, is extremely difficult and all extract antibody at every kind of immune response.And it is diagnostic reagent up to the present is all very high to the purity requirement of antigen, thereby also corresponding very high to the requirement of production technology.These are not enough has brought many limitations for their actual applying.For a change above not enough, the present invention has set up novel homogeneous fluorescent resonance immunoassay reagent and the method for a class, so that production technology is simple, false-positive appearance reduces in the antibody test, and detection can be carried out quickly and accurately.This novel homogeneous fluorescent resonance method of immunity has utilized the optical characteristics of fluorescin and luminescent protein.
The fluorescin of report mainly contains green fluorescent protein (GFP), yellow fluorescence protein (YFP), blue fluorescent protein (BFP), burst of ultraviolel green fluorescent protein (GFPuv) etc. at present, and they are all derived from the green fluorescent protein that derives from jellyfish.Green fluorescent protein (greenfluorescent protein, GFP) be a highly stable protein, total length 238 peptides, molecular weight is about 27kDa, three amino acid of 65-67 position can form a kind of serine-dehydrogenation tyrosine-glycocoll (Ser65-dehydroTyr66-Gly67) cyclized structure of uniqueness, and this structure makes GFP have stronger fluorescence.The formation of GFP fluorescence structure is the process of a self-catalysis, only by its primary structure decision, and need not special enzyme or accessory factor participation, therefore can carry out multiple heterogenous expression.N end and the C end of GFP can carry out chimeric expression and keep its fluorescence activity with other albumen, with the activity basically also not influence of GFP amalgamation and expression to many albumen.The fluorescence of GFP is very stable, is difficult for by photobleaching.These advantages make GFP become the excellent marker of gene expression in the monitoring living cells, albumen location, cell differentiation growth.Heim etc. have gone out a few strain fluorescence intensities GFP anomaly stronger and/or that fluorescence spectrum changes by screen mutation, and these anomalies and wild type GFP have only the difference of several amino acid.In other species, also separate and clonal expression has gone out some fluorescins, as from anthozoic red fluorescent protein DrFP583 (DsRed) [Matz MV, Fradkov AF, Labas YA, et al, NatBiotechnol, 17 (12): 969-973 (1999)].Mitro in 1996 etc. successfully observe the FRET phenomenon [Mitra RD, Silva CM, Youvan DC, Gene 173:13-17 (1996)] between two GFP anomaly BFP5 and the RSGFP4.FRET between BFP and the GFP has been applied to detecting that the cell programming is dead, [Mahajan NP in the researchs such as interaction in the mitochondria between Bcl-2 and the Bax, Linder K, Berry G, et al, Nat Biotechnol, 16 (6): 547-552 (1998)].
It is right for acceptors that the various anomalies of GFP can be formed many group energy: the S65G/V68L/S72A/T203Y anomaly (excitation peak 513nm, the yellow fluorescence protein of emission peak 527nm, YFP) and the F64L/S65T anomaly (excitation peak 488nm, emission peak 507nm, EGFP); Y66H anomaly (excitation peak 382nm, the blue fluorescent protein of emission peak 448nm, BFP) and EGFP, T203I/S72A/Y145F anomaly (excitation peak 395nm, emission peak 509nm, GFPuv) and S65G/S72A/K79R/T203Y anomaly (excitation peak 513nm, emission peak 527nm, [Heim R such as EYFP), Prasher DC and Tsien RY, Proc.Natl.Acad.Sci.U.S.A., 91:12501-12504 (1994), Heim R, Cubitt AB, Tsien RY, Nature, 373:663-664 (1995)].With they respectively with a kind of antigen chimeric expression, the existing immunogenicity of its product possesses fluorescent characteristic again, is special existence in system, just Interference Detection can not occur, cause false-positive factor through preliminary purification, can be applied as detectable.
Naturally exist a kind of FRET phenomenon in Victoria medusoma, promptly the energy between aequorin (apoaequorin) and the green fluorescent protein (wtGFP) shifts.Aequorin is a kind of bioluminescent protein, works as Ca
2+After reaching enough concentration and with it combination taking place, oxidation reaction will take place and launch blue light in it, its maximum emission wavelength is 466nm, close on the maximum emission wavelength 470nm of wtGFP, in medusoma, because distance is minimum between two kinds of albumen, and the transfer of energy must take place, the form with wtGFP transmitting green fluorescence (maximum emission wavelength 504nm) embodies at last.Present these two kinds of albumen have all obtained separating and at multiple allos biological species activity expression, when GFP was by renovation and utilization, multiple anomaly had also appearred in luminescent protein, comprise type [the Casadei J that biologically active improves, Powell MJ, KantenJH, Proc.Natl.Acad.Sci.U.S.A., 87:2047-2051 (1990), Zenno S, Inouye S, Biochem.Biophys.Res.Commun., 171:169-174 (1990), Nomura M, Inouye S, Ohmiya Y, et al.FEBS Lett., 295:63-66 (1991), Shimomura O, Inouye S, Musicki B, et al.Biochem., J.270:309-312 (1990), Kurose K, Inouye S, Sakaki Y, et al.Proc.Natl.Acad.Sci.U.S.A., 86:80-84 (1989)], with the two respectively with a kind of antigen chimeric expression, add Ca
2+As inducing agent, the existing immunogenicity of its product possesses fluorescent characteristic again, it in system special existence, just Interference Detection can not occur, cause false-positive factor through preliminary purification, could detect FRET but also need to add derivant, make detection reaction more accurate, also can constitute the sensitive detectable of a cover.
Biomacromolecules such as protein, enzyme, nucleic acid, sugar, can pass through bi-functional cross-linking agent, between biomacromolecule or carry out covalently boundly, can prepare enzyme labelled antibody, artificial antibody, enzyme-labelled antigen, medication of guiding antibody, immunotoxin, nucleic acid probe, immobilised enzymes, biological affinity chromatography material, modification property enzyme molecule etc. with biological micromolecules such as medicine, haptens.Various homotypes and special-shaped bi-functional cross-linking agent all develop successively.The fluorescent material of chemosynthesis can be marked on amino, carboxyl or the sulfydryl of purifying antigen after crosslinking chemical is modified; With bifunctional group sequestrant and rare earth ion Eu
3+Deng forming chelate, can combine with purifying antigen and obtain fluorescent antigen [Evangelista RA, et al, Clin.Biochem, 21:173 (1988), DiamandisEP, Clin.Biochem., 21:139 (1988), Diamandis EP, et al, J.Immunol.Methods, 112:43 (1988), Diamandis EP, et al, Anal.Chem., 61:48 (1989)].It is right for acceptor that labelled protein cooperates the albumen of amalgamation and expression fluorescin to constitute energy.Though this operation is comparatively complicated in step, it is non-specific to exist, but the existence of fluorescin/luminescent protein fusion will make non-specific reduction greatly, the reagent production technology is oversimplified, and still keeps the idiosyncrasy of antigen, antibody and FRET apart from dependence, and testing result is sensitive and special.
The objective of the invention is to seek and provide detectable with FRET feature.
The inventor finds to have the paired albumen of being made up of albumin A and protein B of this detection of FRET (fluorescence resonance energy transfer) (FRET) feature after deliberation, it can be used for detecting the existence of antibody in the sample, and this detection has simply, fast, the characteristics that specificity is high, therefore, this paired albumen can be used in the detection of disease.
First aspect present invention relates to the paired albumen of being made up of albumin A and protein B with fluorescent resonance energy transfer characteristics, wherein, albumin A is as energy donor and has fluorescent material or the antigen of chemiluminescent substance, protein B is as energy acceptor and has the antigen of fluorescent material, and albumin A and protein B have can with the common antigenic determinant of same antibody molecule specific bond.
First aspect present invention relates to the paired albumen be made up of albumin A and the protein B purposes as detectable.
The invention still further relates to the kit that is used for disease detection, it comprises the paired albumen that (1) is made up of albumin A and protein B, and (2) damping fluid reaches ion and/or surfactant if necessary.
The present invention relates on the other hand and is used for detecting the method that sample antibody exists, and this method comprises: (a) antibody in arbitrary paired albumen provided by the present invention and the sample to be measured is in contact with one another, forms antigen antibody complex; (b) detect the existence of this antigen antibody complex, the existence with the antibody of this paired albumen common antigen specific bond is indicated in this sample in the existence of this antigen antibody complex.
According to the present invention, the albumin A in the paired albumen of the present invention can be selected from virus, bacterium, fungi, mycoplasma, Chlamydia, tumor associated antigen or other has the material of antigenic determinant.
According to the present invention, the protein B in the paired albumen of the present invention can be selected from virus, bacterium, fungi, mycoplasma, Chlamydia, tumor associated antigen or other has the material of antigenic determinant.
According to the present invention, the optional autofluorescence albumen of fluorescent material of the present invention, fluorescein, rare earth element, cancellation material etc.
According to the present invention, optional autoluminescence albumen of chemiluminescent substance of the present invention or chemosynthesis luminescent substances such as luminol, peroxide oxalates.
According to the present invention, fluorescin of the present invention can be selected from green fluorescent protein, yellow fluorescence protein, blue fluorescent protein, red fluorescent protein and other fluorescin.
According to the present invention, luminescent protein of the present invention can be selected from aequorin and its various anomalies.
According to the present invention, fusion fluorescin or luminescent protein had both kept the antigenic determinant that combines with the original antibody molecular specific with the fusion of antigenic determinant fragment in the paired albumen of the present invention, the optical characteristics that has fluorescin or luminescent protein again, purifying requires simple, to cause the significant change of fluorescence spectrum in pairs after the reaction of albumen and specific antibody, and easily detect and find.The damping fluid that adds special homogeneous fluorescent immune response adjuvant also helps to enlarge the degree of spectrum change, therefore can be used as detectable, be used for detecting the specific antibody of sample, as AIDS virus antibody, hepatitis B virus antibody, antibody of HCV.
The gene of fluorescin or luminescent protein gene and certain antigen of coding can be entrenched togather with engineered the whole bag of tricks, place suitable carriers to express.This antigen can be that virus, bacterium, fungi, mycoplasma, Chlamydia, tumor associated antigen or other have the material of antigenic determinant.In genetic engineering, utilize the technology of Escherichia coli production foreign protein very ripe, chimeric protein can be by adjusting expression condition, occur with the soluble protein form, and be unique existing immunogenicity in the expression system, again can emitting fluorescence or carry out chemiluminescent antigen protein, purifying just can be used as detectable slightly.In even liquid-phase system, optical characteristics just constitute a pair of energy for right its identical antigenic determinant of paired albumen of acceptor respectively in conjunction with two arms of specific antibody, utilize unique structure phase of antibody itself, distance between them is furthered, reach in the required distance that FRET takes place, be in the 1-10nm, FRET (fluorescence resonance energy transfer) takes place, the significant change of fluorescence spectrum appears, this obvious variation just can take place in 20 minutes, judge by corresponding fluoroscopic examination instrument such as the full spectrometer of fluorescence or naked eyes, confirm the existence of specific antibody in the testing sample, disease is made diagnosis as detecting index.Because the specificity of albumen existence has only in the system that has specific antibody FRET to occur in pairs, in the sample solution of the non-positive, albumen is free in the solution in pairs, and the distance between them is not enough to take place FRET.Has a fluorescin or a luminescent protein that relates to amalgamation and expression in this a pair of albumen of the present invention at least, therefore the false positive of having avoided using chemical method to carry out marking operation numerous and diverse and be difficult to avoid in the immune detection has in the past guaranteed the simplification that detectable is produced and simple, the quick and accuracy of application.
The homogeneous fluorescent immune response adjuvant that the present invention adopts has polarity based on antibody itself, the slight polarity that changes solution behind adding ion or the surfactant in solution, the structure phase of corresponding change antibody, make the distance between antibody two arms more approaching, the distance that is combined between the fluorescent antigen on antibody two arms is also nearer, thereby improves the sensitivity of energy transfer efficiency and detection.
The present invention selects the fluorescin of maximum emission wavelength more than 500nm to be built into albumen according to the autofluorescence characteristic of detected object-serum.For example, existence for the antibody of the green fluorescent protein GFPuv of checking in the serum, with green fluorescent protein GFPuv and tailor-made respectively albumin A of yellow fluorescence protein EYFP and protein B, 95.8% homology has been guaranteed the immunogenicity that they are similar between them, and the former maximum emission wavelength (509nm) is very approaching with the latter's maximum excitation wavelength (513nm), it is right for acceptor to can be used as a pair of energy, after the specific antibody reaction in they and the serum, form immune complex, wherein FRET will take place in the form of GFPuv-antiGFPuv-EYFP, exciting light (395nm) with GFPuv excites, in 20 minutes, the fluorescence intensity at its maximum emission wavelength 509nm place will than add before the serum reduce greatly and the fluorescence intensity at EYFP maximum emission wavelength 527nm place will strengthen.
According to a specific embodiments of the present invention, for detecting in the serum whether have the HIV antiviral antibody, select Recombinant HIV-1 envelope protein ENV2 and fluorescin GFPuv, the chimeric protein ENV2-GFPuv of EYFP, ENV2-EYFP is respectively as albumin A and protein B, constitute detectable by them, when there is the test serum of HIV-1 antiviral antibody in adding, to detect the FRET phenomenon in 20 minutes, promptly the maximum excitation light 395nm with GFPuv excites, and the 509nm place emission maximum light intensity of GFPuv adds and reduces before the serum and the emission maximum light intensity at EYFP527nm place adds rising before the serum.Do not observe this variation and add HIV-1 negative antibody serum.During the acquired immune deficiency syndrome (AIDS) that therefore can be applied in the paired albumen that above-mentioned albumin A and protein B constitute detects.
According to another preferred embodiment of the present invention, the present invention also makes up another set of HIV-1 antibody test reagent, its with chimeric protein ENV2-GFPuv as albumin A, the albumen of the plain BODIPY507/545 of mark fluorescent is as protein B, they all have common antigenic determinant---Recombinant HIV-1 envelope protein ENV2, the maximum emission wavelength of GFPuv (509nm) is very approaching with the maximum excitation wavelength (507nm) of BODIPY507/545, be that a pair of energy is right for acceptor, when there is the serum of HIV-1 antiviral antibody in adding, to detect the FRET phenomenon in 20 minutes, extremely significant spectrum change appears, promptly the maximum excitation light 395nm with GFPuv excites, the liquid phase systems maximum emission wavelength moves to about 530nm about by the 507nm that adds before the serum, and the system maximum emission wavelength that adds HIV-1 negative antibody serum moves hardly.This system has higher sensitivity and accuracy in acquired immune deficiency syndrome (AIDS) detects.
The paired albumen of using among the present invention all is genetic engineered product, utilize Escherichia coli structure and expression plasmid pGFPuv, pEYFP, pRSETB-env2, pRSETB-env2-gfpuv, pRSETB-env2-eyfp, obtain Protein G FPuv, EYFP, ENV2, ENV2-GFPuv, ENV2-EYFP respectively.With purification technique purifying expression product commonly used or that know in the genetic engineering, ENV2 just can match respectively by BODIPY507/545 on the sulfydryl reaction marking, forms albumin A and protein B in the paired albumen.
Medicine box provided by the invention is selected polarity damping fluid (10% cow's serum, 0.5% casein, 2%BSA, 1 ‰ TritonX-100, the 1ppm aminopyrin), can slightly change the conformation of antibody and the quantum yield of organic fluorescent substance, help the reaction of albumin A, B and specific antibody, form the detection of FRET behind the immune complex.According to the present invention,, promptly can be used as the existence of specific antibody in the kit test sample preferably with mole albumin A and protein B such as damping fluid dilutions.
The object of immune detection generally all is a serum, to many parts of serum carry out fluorescence compose full scanning discovery with ultraviolet excitation usually can occur maximum emission wavelength about 450nm emission peak and very long end peak is arranged.For avoiding the interference of the background in the serum, the emission maximum spectrum of selecting detectable is all more than 500nm in the present invention.In the fluorescent reagent of various saltants of existing GFP and chemosynthesis, selected GFPuv and EYFP, GFPuv and BODIPY507/545 are right for acceptor as energy.
The excitation wavelength (513nm) of the wavelength of transmitted light of GFPuv (509nm) and EYFP is overlaid almost, fluorescence resonance energy transmission (FRET) can take place in the two in enough near distance (10nm), promptly the exciting light (395nm) with GFPuv excites, the energy that GFPuv obtains passes to in-plant EYFP in radiationless mode, excitation energy as the latter, last this energy shows as yellow fluorescence with the optical radiation form emission of 527nm.95.8% homology is arranged between GFPuv and the EYFP, and the antiserum that obtains for the immunogen immune animal with GFPuv also has good immune response to EYFP.Two arms of antibody can further these two kinds of fluorescins in the positive antiserum, and FRET takes place.The situation of change that detects fluorescence spectrum can reflect immunoreactive power.Will with a kind of antigen respectively with these two kinds of fluorescin chimeric expressions, utilize antibody two arms with the effect that two kinds of fluorescent antigens further, make between them the FRET phenomenon take place.Detect the variation of fluorescence spectrum in the liquid phase at this moment and can measure whether combination has taken place between antigen-antibody.
The fluorescent material BODIPY 507/545 of Molecular probe company can react with the sulfydryl in the protein, covalent bond is marked on the protein, makes the latter obtain the photoluminescent property of maximum excitation wavelength 507nm and maximum emission wavelength 530nm and keeps most of biologically active.With BODIPY 507/545 mark purifying antigen.It is right for acceptor that the isoantigen of this fluorescent antigen and GFPuv chimeric expression constitutes a pair of energy, after being incorporated on two arms of same antibody jointly, exciting light (395nm) with GFPuv excites, the energy that GFPuv obtains passes to in-plant BODIPY 507/545 in radiationless mode, then FRET can take place, detect the increase of the light intensity of 530nm.Whether generation with FRET can be used as the standard that whether specific antibody exists and even whether disease takes place.
Technology with gene engineering expression fluorescin/luminescent protein fused antigen is very ripe, they generally occur with the form of supernatant, the purifying facility, active high, easily produce, utilize the special conformation of antibody itself that they are furthered during detection and FRET takes place, reaction fast and accurate, being fit to the extensive needs that detect, is a kind of very promising detection method.
PGFPuv, pEYFP are respectively the expression vectors of two kinds of fluorescin GFPuv and YFP, and with these two kinds of plasmids difference Transformed E .coli expression strain BL21, low temperature induction is expressed to guarantee the correct folding and fluorescence activity of fluorescin.The expression supernatant is a soluble component, and fluorescence is obvious.Because having, GFPuv, EYFP, EGFP, EBFP be higher than 95% homology, the GFPuv immune mouse of the molecular exclusion high performance liquid chroma-tography purifying of learning from else's experience, and can obtain all has very strong immunoreactive antibody to these fluorescins.
For example, moles such as GFPvu and EYFP are mixed, dilute the serum that the back adds immune mouse, the change in fluorescence before and after detecting can be verified the power that whether has been produced antibody and antibody generation by mice immunized.When the ratio of two kinds of fluorescins and fluorescin antibody is 1: 1: 2 just, should there be 50% antigen, antibody complex to exist with the form of GFPuv-antiGFPuv-EYFP, two arms of antibody further the distance of GFPuv and EYFP, produce FRET.With 395nm light whole liquid phase systems is excited, the variation of fluorescence spectrum reached stable in 20 minutes.Can see that add in the system of GFPvu antibody positive serum, the emission photopeak value of GFPuv obviously descends, and does not have special antibody that two kinds of fluorescins are furthered in the negative serum, and FRET does not take place.Owing to add variation that serum causes fluorescence background generally all before 500nm, can ignore.
Acquired immune deficiency syndrome (AIDS) (AIDS, aids) is increased sharply to the mankind's threat, and the development of detectable also seems and be important.EIA is present the most universal HIV detection means, is based on mainly that the immunogenicity of the capsid protein p24 of the glycoprotein gp120, the gp41 that are embedded in coating of HIV env gene code and gag gene code develops.According to the present invention, the present invention can fluorescence resonance energy transmission homogeneous phase detection method detect acquired immune deficiency syndrome (AIDS).
Specifically, constructing in the mode of molecular cloning can the amalgamation and expression fluorescin and the fusion protein expression plasmid of antigen protein.Plasmid pRSETB-env2 can go out to have reorganization AIDS virus envelope protein than the strong antigen activity at expression in escherichia coli, from the plasmid pGFPuv (available from Clonetech company) that contains the gfpuv gene, cut out the gfpuv genetic fragment of 750bp with restriction enzyme, be connected into env2 gene 3` end, obtain recombinant plasmid pRSETB-env2-gfpuv (2u).Equally, connect the 750bp eyfp gene that comes from plasmid pEYFP and hold, obtain recombinant plasmid pRSETB-env2-eyfp (2y) in the 3` of env2 gene.
The plasmid 2u, the 2y transformed into escherichia coli expression strain BL21 that make up, low temperature induction express 2U, 2Y with the fluorescin that guarantees amalgamation and expression and ENV2 structure correct with function mutually, the thalline supernatant of getting ultrasonic degradation is used for detection.Fluoroscopic examination and immune detection find that fusion has been preserved the biology and the chemical activity of fluorescin and reorganization AIDS virus envelope protein, but a little less than these two kinds of protein of single expression.
Moles such as 2Y and 2U are mixed, and the dilution back adds serum, the change in fluorescence before and after detecting.When the ratio of two kinds of fluorescent antigen 2U, 2Y and envelope protein antibody is 1: 1: 2 just, it is 2U-Ig-2Y that 50% antigen, antibodies form will be arranged in theory, all the other 50% are the 2U-Ig-2U and the 2Y-Ig-2Y of equal proportion, just will have 50% antigen, antibody complex that FRET can take place.Exciting light with GFPuv excites whole liquid phase systems, and it is stable that the variation of fluorescence spectrum reaches in 20min.Add in the system of HIV-1 antibody positive serum, the emission photopeak value of GFPuv obviously descends, and it is caused that this is that the exciting light energy of its absorption passes to EYFP.The system that adds the HIV-1 negative serum does not detect the obvious decline of GFPuv emission photopeak value, does not promptly have special antibody that two kinds of fluorescence antibodies are furthered yet, and FRET does not take place.Replace serum with dilution and join in the detection architecture, observe, thereby reduce the influence of fluorescence intensity because the change of the entire reaction volume that adding serum causes is big, protein concentration reduces.But experiment finds that this influence is very little, is lower than 1%, can ignore.
HIV-1 recombinant envelope protein ENV2 to purifying has carried out chemiluminescent labelling in addition.The fluorescence probe that Molecular probe company produces can carry out mark to protein, polypeptide, nucleic acid etc., forms stable covalent bond.BODIPY 507/545 is the sulfydryl reaction fluorescence probe that the said firm produces, and is dissolved into the storing solution of 10mmol/l with DMSO.The HIV-1 recombinant envelope protein ENV2 of purifying, earlier PB (pH=7.3) is dialysed before the mark, and be condensed into 2mg/ml with PEG10000 and be stored in solution among the PB, ratio with 1: 100 (V/V) under room temperature, lucifuge, the condition of not stopping shaking slowly adds BODIPY507/545 storing solution (now with the current), starvation, keep room temperature, lucifuge, concussion reaction 3 hours, to PB (pH=7.3) dialysis cessation reaction, obtain ENV2-BODIPY507/545 (2D) at last.Detection learns that its fluorescence spectrum is constant, and fluorescence intensity and antigen active slightly descend, and can be used as the FRET detectable.
According to the present invention, in suitable reaction buffer, add 2U and 2D composition detection architecture by the antigen active that equates, the serum that adds corresponding titre again, excitation with GFPuv, the emission light of scanning 500-550nm, contrast add the variation of serum front and back emission spectrum, find after 20 minutes, the system maximum emission wavelength that adds HIV-1 antibody positive serum moves to about 530nm about by 507nm, and the system maximum emission wavelength that adds HIV-1 negative antibody serum moves hardly.Promptly detected specific antigen, the antibody response of HIV-1ENV2, can be applicable in the acquired immune deficiency syndrome (AIDS) detection (seeing Fig. 3, Fig. 4) with FRET.
Green fluorescent protein has now developed and multiple anomaly, and their characteristic comprises moving of excitation spectrum, emission spectrum, and the variation of quantum yield is so that the variation of fluorescence intensity, be easier to [(Heim R such as expression, Prasher DC, Tsien RY, Proc.Natl.Acad.Sci.U.S.A., 91:12501-12504 (1994), Heim R, Cubitt AB, Tsien RY, Nature, 373:663-664 (1995)].Development along with biology techniques, technology such as the prediction of X-diffraction analysis, protein structure, rite-directed mutagenesis, DNA shuffling are applied in the design and transformation of protein, may produce more GFP saltant, it is right for acceptor to become the FRET energy for you to choose.From other species, also separate and cloned some fluorescins, as coral polyp fluorescin etc.The nucleotide sequence of fluorescence protein gene and antigen is linked to each other at amino terminal or carboxyl terminal with molecule clone technology, can prepare fusion, obtain fluorescent antigen.Biological fluorescent antigen of expressing, fluorescin as molecular probe high special ground mark on specific antigen, the interference in the time of can avoiding detection that the purifying process deficiency causes, its physicochemical property also is very stable.
For the existing very long history of the research of chemosynthesis fluorescent material and rare earth element, the fluorescent characteristic of many groups or rare earth element has all obtained sufficient understanding, after bi-functional cross-linking agent is modified, can carry out mark, obtain fluorescent antigen behind the purifying the groups such as amino, carboxyl or sulfydryl of virus protein or polypeptide.Their characteristics are quantum yield height of fluorescence, and the variation of reaction back fluorescence spectrum clearly is easy to detect.
According to the present invention, can fluorescent antigen be detectable, set up an even liquid-phase reaction system, adding may contain the serum of specific antibody, in case the idiosyncrasy of antigen, antibody takes place, antibody two arms further to fluorescent energy in the detectable this for acceptor, just can observe FRET.Add some special adjuvant,, thereby may slightly change the structure phase that electrically changes protein of solution, draw the distance between antibody two arms nearer as Triton, metallic ion etc.Because the generation of FRET and six powers of distance are inversely proportional to, nearer distance will make the generation of FRET more obvious, thereby improve the sensitivity that detects.According to the present invention, can chemiluminescent antigen and fluorescent antigen be detectable, set up even liquid-phase reaction system, adding may contain the serum of specific antibody, in case the idiosyncrasy of antigen, antibody takes place, antibody two arms further to fluorescent energy in the detectable this for acceptor, as long as add suitable inducing agent, just can observe FRET.Because chemiluminescence needs inductive condition, can be used to set the size of detection time, control survey value, more helps normalizing operation and mensuration.
Following with reference to form, accompanying drawing and describe example to illustrate in greater detail the present invention.
In the form:
Form 1 shows the comparison that adds positive and negative serum front and back GFPuv fluorescence intensity in detectable GFPuv, the EYFP system
Form 2 shows the comparison that adds positive and negative serum front and back GFPuv fluorescence intensity in detectable 2U, the 2Y system
Compare on the fluorescence peak that adds positive and negative serum front and back system in form 3 demonstration detectable 2U, the 2D system
Description of drawings:
Fig. 1 shows that the fluorescence intensity that adds the positive serum front and back in GFPuv, the EYFP detection architecture changes
Fig. 2 shows that the fluorescence intensity that adds the negative serum front and back in GFPuv, the EYFP detection architecture changes
Fig. 3 shows that the fluorescence intensity that adds the positive serum front and back in detectable 2U, the 2Y system changes
Fig. 4 shows that the fluorescence intensity that adds the negative serum front and back in detectable 2U, the 2Y system changes
Fig. 5 shows the variation that adds positive serum front and back fluorescence spectrum in detectable 2U, the 2D system
Fig. 6 shows the variation that adds negative serum front and back fluorescence spectrum in detectable 2U, the 2D system
The terminological interpretation fluorescence resonance energy transmits (Fluorescence Resonance Energy Transfer, FRET) refer to that a pair of suitable fluorescent material can consist of an energy donor (Donor) and energy acceptor (Acceptor) is right, when the distance between them reaches in the scope of 1-10nm, because the interaction of dipole-dipole, the photon energy h ν of excited donor molecule may be passed to acceptor molecule, and then acceptor molecule relaxes by launching photon h ν ' (h ν＞h ν '). By F rster this theory was proposed at first in 1948. Homogeneous fluorescent immunoassays (Homogeneous Fluoroimmunoassay, hFIA) refer to that in same homogeneous solution system step finishes the mensuration of immune response and signal, detection method comprises two kinds of sandwich method and competition laws, all is to be caused the change of label fluorescence signal wherein and detected by forming of antigen, antibody complex.
Embodiment 1 GFPuv and EYFP are used for detecting the whether existence of serum antibody as albumen in pairs
PGFPuv, pEYFP are respectively the expression vectors of two kinds of fluorescin GFPuv and YFP, with these two kinds of plasmids difference Transformed E .coli expression strain BL21, the picking monoclonal shakes bacterium to OD600=0.6 for 30 ℃, adds IPTG to final concentration 0.5mmol/l, and 24hr is cultivated in 15 ℃ of 200rpm concussions.Centrifugal thalline with lysate (100mMNaCl pH=8.0) blows afloat for 20mM Tris-Cl, 5mM EDTA, ultrasonication, centrifugal 10 minutes of 12000rpm, supernatant fluorescence is obvious, is used for detecting.
Detect expression supernatant, GFPuv excitation wavelength 395nm, emission wavelength 507nm with Russian Lumex company's product full spectrometer of fluorescence (FLUORAT-02 PANORAMA); EYFP excitation wavelength 513nm, emission wavelength 527nm, fluorescence intensity is suitable.
PSA software analysis result shows that GFPuv, EYFP, EGFP, EBFP have very high homology, all in that (GFPuv:EGFP 96.7% more than 95%, GFPuv:EBFP95.8%, GFPuv:EYFP95.8%, EGFP:EBFP99.2%, EGFP:EYFP97.9%).Based on this point, get the GFPuv immune mouse, to obtain fluorescin antibody.GFPuv is expressed supernatant precipitate with saturated sulphur ammonium, the content of GFPuv was more than 60% during wherein 50% saturated sulphur ammonium precipitated.With PBS (20mMNaCl, 2.68mM KCl, 10mM Na2HPO4,1.76mM KH2PO4 pH7.4) behind the resuspension, to PBS dialysis, gets the dialysis product and carries out the molecular exclusion high performance liquid chroma-tography (TSK-GELHPLC, BECKMAN), product purity is more than 95%.Measure and adjust concentration to 2mg/ml with MBA-2000 nucleic acid, Protein Detection instrument, get 30 μ l and isopyknic freund adjuvant mixing, limb muscle equivalent injecting immune mouse, and after 2 all and 7 weeks respectively once with same dosage booster immunization.All cut the blood sampling of mouse tail before and after the immunity, separation of serum is used for immune detection.EIA measures discovery, and GFPuv, EYFP express supernatant with 1: 2000 dilution bag of carbonic acid buffer quilt, still have good immune response, contrast the P/N value all more than 10 with negative serum, are applicable to that FRET detects.
With GFPvu and EYFP mixed in equal amounts, get 2ul and become 100ul (dilution: 10% cow's serum, 0.5% casein, 2%BSA, 1 ‰ TritonX-100,1ppm aminopyrin) with 1: 50 dilution proportion, add 5ul serum, the change in fluorescence before and after detecting.With 395nm light whole liquid phase systems is excited, it is stable that the variation of fluorescence spectrum reaches in 20min.Can see that behind the adding GFPvu antibody positive serum, the emission photopeak value of GFPuv obviously descends, reach more than 17% that this is that its emission luminous energy is absorbed caused by EYFP.Because covering of its end peak, the increase of the emission photopeak value of the EYFP of 527nm place generally can not be seen.Do not have special antibody that two kinds of fluorescence antibodies are furthered in the negative serum, FRET does not take place.GFPuv emission light also has decline after adding negative serum, is lower than 5%, and this may be to be caused by some false-positive antigen-antibody reactions.But can embody the difference between the positive and negative serum.Because the change of the entire reaction volume that adding serum causes is big, protein concentration reduces, thereby the influence that fluorescence intensity reduces is very little, is lower than 1%, can ignore.(seeing Table 1, Fig. 1, Fig. 2)
Embodiment 2 ENV2-GFPuv (2u) and ENV2-EYFP (2y) are as whether having HIV-1 antibody in the Protein Detection serum in pairs
Detected most serum and all existed, avoided the EBFP of emission maximum light when making up fluorescent antigen at 448nm with the emission light about the 450nm that produces behind the ultraviolet excitation; Directly provide excitation energy for the exciting light of avoiding energy donor simultaneously for energy acceptor, also avoided the very near EGFP (EX=488nm of distance between maximum excitation optical wavelength and the emission maximum optical wavelength, EM=507nm), selected GFPuv (EX=395nm, EM=509nm) as energy donor, (constructing in the mode of molecular cloning can the amalgamation and expression fluorescin and the fusion protein expression plasmid of antigen protein for EX=513nm, EM=527nm as energy acceptor for EYFP.Plasmid pRSETB-env2 can go out to have reorganization AIDS virus envelope protein than the strong antigen activity at expression in escherichia coli, handles with restriction enzyme PstI/KpnI, cuts a bit of base before its termination codon.The plasmid pGFPuv (available from Clonetech company) that will contain the gfpuv gene simultaneously also handles with restriction enzyme PstI/KpnI, reclaims the gfpuv genetic fragment of 750bp, is connected into env2 gene 3` end, obtains recombinant plasmid pRSETB-env2-gfpuv (2u).Equally, handle plasmid pRSETB-env2 and pEYFP simultaneously with restriction enzyme BamHI/KpnI, the eyfp gene that connects 750bp obtains recombinant plasmid pRSETB-env2-eyfp (2y) in the 3` of env2 gene end.
The plasmid Transformed E coli BL21 bacterial strain that makes up, 30 ℃ are shaken bacterium to OD600=0.6, add IPTG to final concentration 0.5mmol/l, and 24hr is cultivated in 15 ℃ of 200rpm concussions.Centrifugal thalline with lysate (100mM NaCl pH=8.0) blows afloat for 20mM Tris-Cl, 5mM EDTA, ultrasonication, centrifugal 10 minutes of 12000rpm gets supernatant and is used for detecting.
Observe with 12%SDS-PAGE, 2Y, 2U are 43KD (ENV2 16KD, GFPuv, EYFP27KD), and content accounts for 20% of supernatant.Detect the fluorescence of 2Y, 2U respectively with the full spectrometer FLUORAT-02 of fluorescence (Russia produces), 2U maximum excitation optical wavelength Ex=395nm, emission maximum optical wavelength Em=509nm, 2Y maximum excitation optical wavelength Ex=513nm, emission maximum optical wavelength Em=527nm, identical with GFPuv, the EYFP of single expression, but fluorescence intensity weakens relatively.EIA detects the two all stronger immunologic competence, but is lower than independent ENV2.
With 2Y and 2U mixed in equal amounts, get 100ul and become 3ml (dilution: 10% cow's serum, 0.5% casein, 2%BSA, 1 ‰ TritonX-100,1ppm aminopyrin) with 1: 30 dilution proportion, add 50ul serum, the change in fluorescence before and after detecting.With 395nm light whole liquid phase systems is excited, it is stable that the variation of fluorescence spectrum reaches in 20min.. can see that behind the adding HIV-1 antibody positive serum, the emission photopeak value of GFPuv obviously descends, reach more than 14% that this is that its emission luminous energy is absorbed caused by EYFP.Because the covering of its end peak, the increase of the emission photopeak value of the EYFP of 527nm place generally can not see, when abundant, the phenomenon of enhancing is arranged also but the reaction ratio of strong positive serum and antigen is suitable.Do not have special antibody that two kinds of fluorescence antibodies are furthered in the negative serum, FRET does not take place.GFPuv emission light also has decline after adding negative serum, is lower than 5%, and this may be to be caused by some false-positive antigen-antibody reactions.But can embody the difference between the positive and negative serum.Because the change of the entire reaction volume that adding serum causes is big, protein concentration reduces, thereby the influence that fluorescence intensity reduces is very little, is lower than 1%, can ignore.(seeing Table 2, Fig. 3, Fig. 4)
Embodiment 3 2u and ENV2-BODIPY 507/545 (2D) are used for detecting serum as paired albumen and whether have HIV-1 antibody.
HIV-1 recombinant envelope protein ENV2 to purifying has carried out chemiluminescent labelling.Fluorescent material is purchased the company in Molecular probe.BODIPY 507/545 is the red solid powder, lucifuge, drying, is stored in-20 ℃.An amount of dry powder of picking before the mark is dissolved in 200 μ l DMSO and becomes red solution, surveys its light absorption value A under the 507nm exciting light.Calculate the actual concentrations of BODIPY 507/545 according to formula C=A/ ε (ε=69000), add DMSO again and adjust concentration to 10mM.The HIV-1 recombinant envelope protein ENV2 of purifying, purity reaches 95%, and concentration is 0.5mg/ml, is stored in the Tris-Cl damping fluid, earlier to PB (pH=7.3) dialysis, and concentrates with PEG10000 and to become 2mg/ml and be stored in solution among the PB before the mark.With antigen ENV2 five equilibrium, per 200 μ l lucifuge under the room temperature, do not stop to shake, slowly add 2 μ l and dilute good BODIPY507/545 solution (now with the current) in a 0.5ml eppendorf pipe, build the pipe lid, keep room temperature, lucifuge, concussion reaction 3 hours.After the reaction, solution obtains ENV2-BODIPY507/545 (2D) to PB (pH=7.3) dialysis cessation reaction.Detect its fluorescence spectrum and antigenicity.Spectrum is exciting light 504nm, emission light 530nm, and EIA detects antigen active and descends 10%, can be used as the FRET detectable.
In 100 μ l reaction buffers (containing 10% cow's serum, 0.5% casein, 2%BSA, 5 ‰ TritonX-100,1ppm aminopyrin), add 5 μ l 2U and 5 μ l 2D, in the micropore of the enzyme linked immunosorbent detection of packing into microwell plate, add 3 μ l serum again, the fluorescence spectrum before and after detecting changes.With the 395nm optical excitation, the emission light of scanning 500-550nm, contrast adds the variation of serum front and back emission spectrum, finds after 20 minutes, and the fluorescence intensity of system is whole to be improved, and may be the result that TritonX-100 has improved the fluorescence efficiency of fluorophore.The micropore maximum emission wavelength that adds HIV-1 antibody positive serum moves to about 530nm about by 507nm, and the micropore maximum emission wavelength that adds HIV-1 negative antibody serum moves hardly, about 510nm.Promptly detected specific antigen, the antibody response of HIV-1ENV2, can be applicable to during acquired immune deficiency syndrome (AIDS) detects, determined whether contain positive antibody in the serum (see Table 3, Fig. 5, Fig. 6) with the position of seeking maximum emission wavelength with FRET.
Table 1. adds the comparison of different serum front and back 507nm fluorescence intensity
| Add sample | The GFPuv fluorescence intensity | GFPuv emission peak decline number percent | ||
| Before the increase serum | Behind the increase serum | |||
| Damping fluid | ??0.00561 | ??0.00539 | ????3.92 | |
| The clear u v of the anti-G F blood P positive | ???G2531 | ??0.00706 | ??0.00577 | ????18.27 |
| ???G3531 | ??0.0076 | ??0.00577 | ????24.08 | |
| ???G255 | ??0.0066 | ??0.00494 | ????25.15 | |
| ???G355 | ??0.00687 | ??0.00475 | ????30.86 | |
| Anti-G F P u v negative serum | ???Co | ??0.00784 | ??0.00747 | ????4.72 |
| ???182 | ??0.00795 | ??0.00681 | ????14.34 | |
| ???183 | ??0.00736 | ??0.00708 | ????3.80 | |
| ???184 | ??0.00687 | ??0.00693 | ????-0.87 | |
| ???185 | ??0.00731 | ??0.00715 | ????2.19 | |
| ???186 | ??0.00657 | ??0.00675 | ????-2.74 | |
| ???187 | ??0.00681 | ??0.00679 | ????0.294 | |
| ???190 | ??0.00615 | ??0.00518 | ????15.77 | |
The 509nm fluorescence intensity that table 2. adds positive and negative serum compares
50ul 2Y+50u12U dilution → 3000ul exciting light Ex=395nm
| Add sample | Before the adding | After the adding | GFPuv emission peak decline number percent |
| Damping fluid | ??0.021569 | ??0.021401 | ????0.776 |
| ??HIV-1(+)C46 | ??0.028322 | ??0.02468 | ????12.9 |
| ??HIV-1(-)BD15 | ??0.025702 | ??0.024084 | ????6.29 |
| ??HIV-1(-)BD23 | ??0.0232 | ??0.02216 | ????4.48 |
| ??HIV-1(-)BD33 | ??0.02225 | ??0.021739 | ????2.30 |
| ??HIV-1(-)BD35 | ??0.023009 | ??0.02253 | ????2.08 |
Table 3. adds the variation of different serum front and back fluorescence maximum emission wavelength
Ex=395nm scanning Em 480 ~ 550nm
| Add serum | Before the increase serum | Behind the increase serum | |||
| Maximum excitation wavelength (nm) | Fluorescence intensity | Maximum emission wavelength (nm) | Fluorescence intensity | ||
| The serum-free contrast | ????509 | ??0.00315 | ????509 | ??0.00361 | |
| HIV-1 antibody positive serum | ??C46 | ????510 | ??0.00166 | ????525 | ??0.00425 |
| ??P/40 | ????508 | ??0.00298 | ????529 | ??0.00582 | |
| ??FJ4 | ????508 | ??0.00344 | ????528 | ??0.00512 | |
| HIV-1 negative antibody serum | ??BD7 | ????508 | ??0.0029 | ????513 | ??0.00421 |
| ??BD8 | ????508 | ??0.0032 | ????500 | ??0.0111 | |
| ??BD9 | ????507 | ??0.00313 | ????504 | ??0.00737 | |
| ??158 | ????510 | ??0.00164 | ????516 | ??0.00647 | |
| ??162 | ????506 | ??0.00196 | ????513 | ??0.0022 | |
| ??180 | ????510 | ??0.00167 | ????519 | ??0.00746 | |
| ??181 | ????507 | ??0.00167 | ????519 | ??0.00612 | |
| ??182 | ????510 | ??0.00164 | ????519 | ??0.0063 | |
| ??184 | ????513 | ??0.00163 | ????519 | ??0.00539 | |
| ??185 | ????507 | ??0.00163 | ????519 | ??0.00586 | |
| ??187 | ????507 | ??0.00166 | ????480 | ??0.0203 | |
| ??189 | ????510 | ??0.00164 | ????519 | ??0.00573 | |
| ??191 | ????510 | ??0.00166 | ????519 | ??0.00598 | |
Claims (10)
1, the paired albumen of forming by albumin A and protein B that has FRET (fluorescence resonance energy transfer) (FRET) feature, wherein albumin A is as energy donor and has fluorescent material or the antigen of chemiluminescent substance, protein B is as energy acceptor and has the antigen of fluorescent material, and albumin A and protein B have can with the common antigenic determinant of same antibody molecule specific bond.
2, according to the paired albumen of claim 1, wherein albumin A can be selected from virus, bacterium, fungi, mycoplasma, Chlamydia, tumor associated antigen or other has the material of antigenic determinant.
3, according to the paired albumen of claim 1, wherein protein B can be selected from virus, bacterium, fungi, mycoplasma, Chlamydia, tumor associated antigen or other has the material of antigenic determinant.
4, according to the paired albumen of the arbitrary requirement of claim 1-3, the wherein optional autofluorescence albumen of fluorescent material, fluorescein, rare earth element, cancellation material etc.
5, according to the paired albumen of the arbitrary requirement of claim 1-3, wherein optional autoluminescence albumen of chemiluminescent substance and chemosynthesis luminescent substances such as luminol, peroxide oxalates.
6, according to the paired albumen of claim 4, wherein fluorescin can be selected from green fluorescent protein, yellow fluorescence protein, blue fluorescent protein, red fluorescent protein and other fluorescin.
7, according to the paired albumen of claim 5, wherein luminescent protein can be selected from aequorin and its various anomalies.
8, a kind of being used for detected the method that sample antibody exists, and this method comprises: (a) the paired albumen of the arbitrary requirement of claim 1-7 and the antibody in the sample to be measured are in contact with one another, and form antigen, antibody complex; (b) detect the existence of this antigen antibody complex, the existence with the antibody of this paired albumen common antigen specific bond is indicated in this sample in the existence of this antigen antibody complex.
9, the method for claim 8, antibody wherein to be measured can be AIDS virus antibody, hepatitis B virus antibody, antibody of HCV.
10, a kind of detection box, it comprises the paired albumen of the arbitrary requirement of (1) claim 1-7, (2) damping fluid reaches (3) ion and/or surfactant if necessary.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001302922A CN1136450C (en) | 2000-11-03 | 2000-11-03 | Paired proteins with fluorescent resonance energy transfer characteristics and usage thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001302922A CN1136450C (en) | 2000-11-03 | 2000-11-03 | Paired proteins with fluorescent resonance energy transfer characteristics and usage thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1353313A true CN1353313A (en) | 2002-06-12 |
| CN1136450C CN1136450C (en) | 2004-01-28 |
Family
ID=4594090
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB001302922A Expired - Lifetime CN1136450C (en) | 2000-11-03 | 2000-11-03 | Paired proteins with fluorescent resonance energy transfer characteristics and usage thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1136450C (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100473988C (en) * | 2004-07-08 | 2009-04-01 | 厦门大学 | Protein composition possessing fluorescent resonance energy transfer characteristics and use thereof |
| CN101806802A (en) * | 2010-04-29 | 2010-08-18 | 华中科技大学 | Fluorescence resonance energy transfer-based homogeneous immunoassay method for multi-component simultaneous detection |
| CN1711476B (en) * | 2002-11-18 | 2010-09-08 | 芬兰技术研究中心 | Non-competitive immunoassay for small analytes |
| CN103293145A (en) * | 2013-05-16 | 2013-09-11 | 赫利森（厦门）生物科技有限公司 | Chemiluminescence reagent |
| CN105784657A (en) * | 2016-03-25 | 2016-07-20 | 鲁延军 | Method for efficiently screening positive clones of monoclonal antibody by using TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) |
| CN106290988A (en) * | 2015-05-25 | 2017-01-04 | 中国科学院上海应用物理研究所 | saturated resonance energy transfer super-resolution probe and its preparation method and application |
| CN113125712A (en) * | 2019-12-31 | 2021-07-16 | 博阳生物科技（上海）有限公司 | Homogeneous phase chemiluminescence detection kit for hepatitis C virus antibody and application thereof |
| CN114252622A (en) * | 2020-09-23 | 2022-03-29 | 中国科学院大连化学物理研究所 | Method for screening novel coronavirus inhibitor in vitro |
| CN114252621A (en) * | 2020-09-23 | 2022-03-29 | 中国科学院大连化学物理研究所 | Method for detecting novel coronavirus spike protein in real time based on interaction of RBD and ACE2 |
-
2000
- 2000-11-03 CN CNB001302922A patent/CN1136450C/en not_active Expired - Lifetime
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1711476B (en) * | 2002-11-18 | 2010-09-08 | 芬兰技术研究中心 | Non-competitive immunoassay for small analytes |
| CN100473988C (en) * | 2004-07-08 | 2009-04-01 | 厦门大学 | Protein composition possessing fluorescent resonance energy transfer characteristics and use thereof |
| CN101806802A (en) * | 2010-04-29 | 2010-08-18 | 华中科技大学 | Fluorescence resonance energy transfer-based homogeneous immunoassay method for multi-component simultaneous detection |
| CN103293145A (en) * | 2013-05-16 | 2013-09-11 | 赫利森（厦门）生物科技有限公司 | Chemiluminescence reagent |
| CN103293145B (en) * | 2013-05-16 | 2016-08-10 | 赫利森（厦门）生物科技有限公司 | A kind of chemical illuminating reagent |
| CN106290988A (en) * | 2015-05-25 | 2017-01-04 | 中国科学院上海应用物理研究所 | saturated resonance energy transfer super-resolution probe and its preparation method and application |
| CN105784657A (en) * | 2016-03-25 | 2016-07-20 | 鲁延军 | Method for efficiently screening positive clones of monoclonal antibody by using TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) |
| CN113125712A (en) * | 2019-12-31 | 2021-07-16 | 博阳生物科技（上海）有限公司 | Homogeneous phase chemiluminescence detection kit for hepatitis C virus antibody and application thereof |
| CN113125712B (en) * | 2019-12-31 | 2023-06-16 | 科美博阳诊断技术(上海)有限公司 | Homogeneous chemiluminescence detection kit for hepatitis C virus antibody and application thereof |
| CN114252622A (en) * | 2020-09-23 | 2022-03-29 | 中国科学院大连化学物理研究所 | Method for screening novel coronavirus inhibitor in vitro |
| CN114252621A (en) * | 2020-09-23 | 2022-03-29 | 中国科学院大连化学物理研究所 | Method for detecting novel coronavirus spike protein in real time based on interaction of RBD and ACE2 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1136450C (en) | 2004-01-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Patterson et al. | Förster distances between green fluorescent protein pairs | |
| US20050282225A1 (en) | Quantitative binding assays using green fluorescent protein as a label | |
| Fradkov et al. | Far-red fluorescent tag for protein labelling | |
| CN1136450C (en) | Paired proteins with fluorescent resonance energy transfer characteristics and usage thereof | |
| EP2663642B1 (en) | Treponema pallidum triplet antigen | |
| EP1548107B1 (en) | Chromoprotein and fluoroproteins | |
| CN111521816A (en) | Time-resolved fluorescence immunochromatographic assay test strip for echinococcosis granulosus of cattle and preparation method thereof | |
| CN113447658B (en) | Kit for detecting anti-peroxiredoxin-1-IgG antibody | |
| CN105807064B (en) | A kind of complementary quanta point biological sensor of luciferase and its construction method and its application | |
| US20110300558A1 (en) | Nanoparticle for bioaffinity assays | |
| JP4214206B2 (en) | Fluorescent indicator using FRET | |
| CN102838663A (en) | Viromembrane protein with site-specific mutagenesis and site-specific decoration, preparation method and applications of viromembrane protein | |
| CN114324862A (en) | Homogeneous detection reaction reagent and detection method for African swine fever virus | |
| Frank et al. | Use of proZZ-obelin fusion protein in bioluminescent immunoassay | |
| CN100473988C (en) | Protein composition possessing fluorescent resonance energy transfer characteristics and use thereof | |
| CN107216381A (en) | CTnI cTnC cTnT trimer proteins and preparation method thereof and cTnI detection kits | |
| US6350574B1 (en) | Fluorescence polarization—based diagnostic assay for equine infectious anemia virus | |
| JP2007511226A (en) | Resonant energy transfer assay system for multiple component detection | |
| CA1321765C (en) | Immunodiagnostic assays using chimeric antigens | |
| JP2001124770A (en) | Method for measuring concentration of substance | |
| US10739347B2 (en) | Fluorescent fusion proteins for use as reporters in multiplexed bioassays | |
| WO2007033514A1 (en) | Protein combination characterized in fluorescence resonance energy transfer, and the use thereof | |
| US20060148016A1 (en) | Nanoparticle for bioaffinity assays | |
| JP2002325578A (en) | Photoprotein of symplectoteuthis oualaniensis and gene coding for photoprotein of symplectoteuthis oualaniensis | |
| CN112194709A (en) | Immunochromatographic test strip for detecting feline panleukopenia syndrome virus antibody, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term | ||
| CX01 | Expiry of patent term |
Granted publication date: 20040128 |