CN101358969A - Novel method for quantitatively determining analyte by scavenger with single specificity - Google Patents
Novel method for quantitatively determining analyte by scavenger with single specificity Download PDFInfo
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Abstract
The present invention discloses a method which adopts a single specific capture agent for quantitative detection of the analyte in the biological sample, which is named as the analyte labeling and inversion recapture method, ALIRA for short, which is an improved detection method which is based on the principles of the SALRA but adopts different capture means. The method comprises: capturing the analyte on the capture device, and forming capture agent-analyte complex; labeling the complex with a labeling agent; eluting the labeled analyte from the complex; recapturing the analyte through the combination of the second recapture agent on the detection device and the labeling molecules; and adding specific detection agent, compounding the specific detection agent and the analyte, and determining the concentration of the analyte according through detecting the signals which are generated by the report modules which are carried by the specific detection agent. The invention has the advantage that the adoption of a specific detection agent can sensitively and simply complete the quantitative detection of the analyte, and the invention has excellent application prospects in the application in the disease diagnosis, medical evaluation, new drug development, protein micro-array and the like, and in the field of basic research.
Description
Technical field
The invention belongs to technical field of bioengineering, relate in particular to the new method that adopts a kind of analyte of a species specificity trapping agent detection by quantitative, be applicable to various detection platform, comprise microwell plate, filter membrane, little magnetic ball based on solid phase surface, or the like.
Background technology
The content of specific protein prime factor in detection of biological (the comprising the mankind) tissue samples has very important significance for the fundamental research and the application in fields such as medical science, biology, agricultural, food industry, environmental protection.For example, the specific protein component of detection pathogenic microorganism is the main means of current diagnosis infectious disease; Equally, the variation of the early stage biomarker rho factor content of mensuration cancer and angiocardiopathy is very important for finding early treatment and observation of curative effect the morning of these diseases.Along with the continuous progress of medical science and field of biology, the demand of the range protein factor being carried out detection by quantitative is also increasing.
For satisfying this ever-increasing requirement, range protein and proteomics detection method are arisen at the historic moment in recent years, and for example various immunoassay technologies, two dimensional gel electrophore-sis, mass spectrophotometry and peptide mapping are analyzed or the like.Wherein most convenient, range of application are the widest also is that using maximum at present is that enzyme linked immunological absorption in the immunoassay technology detects that (Enzyme-Linked ImmunoSorbent Assay is called for short ELISA; Referring to Engvall and Perlman, 1971, Immunochemistry 8:871-4.).The concrete operations mode of ELISA has some kinds, and wherein the most frequently used is that the absorption of sandwich method enzyme linked immunological detects (sandwich ELISA), is used for the concentration of certain protein of detection by quantitative biological specimen.Sandwich method ELISA adopt can with a kind of two kinds of differences of different antigenic determinant combinations of antigen protein but the antibody of working in coordination finish, the combination of another kind of antibody and same antigen molecule is not disturbed in the combination of a kind of antibody and antigen.The technical essential of this method is: (1) is coated on (the most frequently used is the micropore base plane of 96-hole micro plate) on a certain solid phase surface with capture antibody (capture antibody); (2) add biological specimen to be measured, allow the antigen protein molecule (analyte) that wherein contains combine, then the unconjugated non-specific material of eccysis with capture antibody specificity on being combined in solid phase surface; (3) add the detection antibody (detection antibody) that indicates certain reporter molecule (enzyme, biotin, haptens, fluorophor, oligonucleotide or other types molecule), make it to combine, then the unconjugated detection antibody of eccysis with captive antigen molecule specificity on the solid phase surface; (4) add the related reagent (for example enzyme mark Avidin, zymolyte etc.) that makes reporter molecule generation signal, read to survey signal intensity with 96-hole microplate reader then, perhaps directly measure the amount of reporter molecule (for example fluorescence intensity etc.), or with detecting behind the PCR in real time amplification oligonucleotide.According to the intensity of signal, with reference to the concentration known standard calibration curve of analyte, thus the concentration of analyte in definite sample.Though the specificity of sandwich method ELISA and sensitivity are all than higher, but the application of this method has a significant limitations: if set up the sandwich method ELISA detection method that detects a kind of analyte, must have two kinds of different antibodies (capture antibody and detection antibody) at this analyte, and require these two kinds of antibody that this analyte is all had high degree of specificity and height affinity, must work in coordination between them simultaneously, i.e. the combination of capture antibody and analyte does not influence and detects the combination of antibody to same analyte molecule.These require to have limited to a great extent the broader applications of sandwich method ELISA, and this is because often will pass through a lot of effort in real work, could obtain the above-mentioned two kinds of antibody at same analyte that can work in coordination for a long time.Very important protein or protein domain on the biologically many or clinical detection are although repetition test still can't obtain to be used for the pairing antibody of sandwich method ELISA for many years.And for those molecular weight the less and protein that is close together of less or antigen decision a small bundle of straw, etc. for silkworms to spin cocoons on, just two kinds of antibody of working in coordination of more difficult acquisition.Sometimes can obtain a kind of antibody that has the high specific high-affinity, but will find another kind to have similar advantage and but difficulty very of the another kind of antibody that can match mutually.This also is the antibody that why has approximately in the market at surpassing 5000 kinds of different proteins, but the 400-500 kind protein of only having an appointment can carry out detection by quantitative with sandwich method ELISA.
In order to overcome the above-mentioned limitation of sandwich method ELISA, people propose and have tested some kinds and improved one's methods, one of them be with all antigen proteins in the biological specimen in advance with reporter molecules such as fluorescent chemicals carry out direct mark (Miller etc., 2003, Proteomics.3:56-63).Then with solid phase carrier on antibodies, behind the non-specific material of eccysis, directly detect and be combined in the analyte that is labeled on the insolubilized antibody.The principle of this method has been used for their protein-chip product at present as the detection technique platform by a number of manufacturers.Though the method is simple, only need a kind of antibody (capture antibody) just can detect, but there is following shortcoming: first, usually contain thousands of kinds of sized molecules in the biological specimen, wherein much can disturb the labeling effciency to specific analyte directly or indirectly, the protein that concentration is low is often more difficult to be labeled effectively.The second, labeling process itself is modified the antigenic determinant feature that changes on the analyte molecule probably, and capture antibody combines on reduction even inhibition and the solid phase.The 3rd, the non-specific material that accounts for overwhelming majority in the sample before not being eliminated just by significant notation, because analyte does not pass through specific enrichment (concentrating), these non-specific materials that carry reporter molecule equally will produce very strong detection background noise, cause specificity and sensitivity all to reduce greatly, many with microwell plate or the little battle array of protein as the experimental result of detection platform verified this point.
Another form of ELISA is so-called " antigen is at following (antigen-down) ELISA ", promptly uses the micropore of antigen coated microwell plate, adds corresponding antibody then.This form mainly is the existence that is used for detecting a certain antibody in the serum sample, the antigen protein that is used to wrap quilt be through purification or half purify, and serum to be measured is through suitably adding in it after the dilution, and then add enzyme mark secondary antibody, just can detect the antibody that whether exists in this serum sample at this antigen.This method also can be used for detecting the existence of antigen in a certain sample through after suitably modifying.Specific practice is, by micropore, adds the specific antibody that is marked with signaling molecule with the sample to be tested bag then, remove unconjugated antibody after, promptly can pass through the reporter molecule shows signal.Obviously, this method only needs a kind of antibody just can detect.Ideally, the concentration of antigen is the linear positive relation in the intensity of the signal that produces and the sample., owing to all contain thousands of middle different proteins in the biological specimen usually, antigen protein to be measured only accounts for a wherein very little part, and by micropore, bag all is non-specific protein or other molecules by last material major part with such sample packages.Therefore, the sensitivity of this method and specificity are very low, can not be used for the lower antigen of concentration ratio in the detection of complex sample.
Another improvement of sandwich method ELISA method is the immunity-PCR method that grows up recently, wherein come marker detection antibody with the DNA oligonucleotide, after with capture antibody antigen protein being captured solid phase, the detection antibody that adds the oligonucleotide mark is used the amplification of real-time polymerase chain reaction (Real-time PCR) method then and is read signal.The sensitivity of immunity-PCR method is high especially, the concentration range span of detection very big (1000-10000 times of scope), and can carry out multiplicity and detect multiple antigen simultaneously.But need the limitation of two mutual pairing antibody still to exist in the above-mentioned sandwich method ELISA method.Other shortcoming of this method also comprises, needs very careful operation steps to avoid the pollution of PCR reaction, long detection time, and higher equipment and reagent cost.
Disclosed application number is the patent documentation of 20051040295 (China), has proposed a kind of new detection method, only need to adopt a kind of trapping agent just can be delicately, a kind of analyte of detection by quantitative easily.This method is called " the specificity analyses substance markers reaches prize law again ", and English name is " Specific Analyte Labelingand Recapture Assay ", is called for short SALRA.Its step is as follows: at first go up at " acquisition equipment " and catch analyte with specificity trapping agent (being generally antibody); After removing non-specific material and enrichment of analyte by cleaning, with reporter molecule mark capturing agent-analyte complex; Then will be through the analyte of mark from the compound disassociation and elute, through after the suitable neutralization, go up bag with " pick-up unit " and combined again by same trapping agent in solid phase, determine analyte content by the marking signal that detects reporter molecule.The SALRA method only needs a kind of antibody just can detect a kind of antigen, is applicable to all kinds of detection platform based on solid phase surface, for example microwell plate, filter membrane, protein (antibody) chip, microballon (beads), or the like.The SALRA method both can be used for detecting a kind of analyte, also can be used for multiplicity and detect multiple different analyte simultaneously.Though SALRA mainly will be used to detect combining of antibody and antigen protein, also can detect combining of the protein-protein bound of non-immunity relation or protein and other types molecule.
Though the SALRA method successfully has been used for the detection by quantitative of protein, detect some kinds of protein simultaneously in particular for multiplicity, observe in the experiment, higher or contain easily and during the non-material-specific of pick-up unit solid phase surface combination, detection specificity and sensitivity meeting are subjected to bigger negative effect when non-material-specific content in some samples to be tested.This is because in above-mentioned SALRA method, the labeled molecule itself that is used for labelled analyte also is to participate in the reporter molecule that produces detection signal directly.In the labelled analyte process, any protein that is combined on the acquisition equipment solid phase surface, comprise that specific analyte and non-specific material (non-special component, the capture antibody in the sample and the protein that is used to blockade) all can be labeled simultaneously, quite a few in these non-specific materials can be entered liquid phase and enter pick-up unit by wash-out.In case and pick-up unit surface combination, the labeled molecule (reporter molecule) of carrying on these non-material-specifics will produce background noise, and this moment detection system not had to distinguish reporter molecule be from specific analyte or from the step (mechanism) of non-specific material.
Summary of the invention
In order to overcome the deficiency of sandwich method ELISA and above-mentioned SALRA method, the invention provides a kind of new immunologic detection method, adopt a species specificity trapping agent just can be delicately, the corresponding analyte of detection by quantitative easily.This method is called " analyte mark and be inverted prize law again ", and English name is " Analyte Labeling andInverse Recapture Assay ", is called for short ALIRA.This method is mainly used to detect a kind of analyte in the sample, but also can be used for multiplicity detects some kinds of analytes simultaneously; Both can be used to detect combining of antibody and antigen protein, also can detect combining of the protein-protein bound of non-immunity relation or protein and other types molecule.
Concrete technical scheme is:
(1) catch analyte: with specificity trapping agent bag by or be covalently bound to solid phase surface, become acquisition equipment; Add biological specimen to be measured, the specificity trapping agent on the acquisition equipment is combined with analyte in the sample, form trapping agent-analyte complex;
(2) labeled complex: with labelled reagent mark capturing agent-analyte complex;
(3) elution analysis thing: will elute from compound through the analyte of mark;
(4) catch again: eluent is added pick-up unit, by second trapping agent on the pick-up unit by catch analyte once more with combining of labeled molecule; Said pick-up unit is meant that bag is by the solid phase surface of second trapping agent;
(5) detect: add special detection agent and analyte combination, determine analyte concentration by the signal intensity that detects the reporter molecule generation of carrying on the special detection agent.
Specificity trapping agent in the step (1) also is called first trapping agent, in most cases is antibody, wraps quilt or covalently bind in a certain suitable solid phase surface, such as 96-hole micro plate, becomes acquisition equipment.Add biological specimen to be measured, the special trapping agent on the acquisition equipment is combined with " the specific bond site " of specific analyte in the sample, form " first trapping agent-analyte " compound; Pass through the non-material-specific of flush away and the enrichment specific analyte then.Said specific bond site is meant the domain that combines with first trapping agent on the analyte molecule.
Step (2) labeled complex adds the labelled reagent labelled analyte.The labeled molecule group that labelled reagent provides next step can be caught by " second trapping agent " on pick-up unit for analyte molecule.For example, can be with the biotin molecule that serves as a mark, corresponding second trapping agent then is an affinity prime.
Step (3) elution analysis thing, will dissociate and elute from compound through the analyte of mark enters liquid phase.The selecting for use of eluant, eluent will depend between the analyte and first trapping agent and decide in conjunction with characteristics, such as improve or reduce pH value, change ionic strength, change organic solvent concentration, change detergent concentration, the change experimental temperature, or the like; Simultaneously, when guaranteeing the elution analysis thing, will avoid non-specific protein (comprise first trapping agent, the non-specific protein of solid phase combination in the protein of blockading, sample) to enter liquid phase as far as possible.
Step (4) is caught analyte again, and eluent is added pick-up unit.Pick-up unit is to be coated on solid phase surface (for example 96-hole micro plate) with said second trapping agent (for example Avidin or streptavidin) to be made.The labeled molecule that analyte carries by the surface (normally biotin) combines with second trapping agent on the pick-up unit and is trapped in solid phase once more.If eluent is former because of pH, ionic strength, organic solvent concentration, detergent concentration etc. thereby the combination of the impact analysis thing and second trapping agent, before adding pick-up unit, need to handle eluent, such as taking to regulate temperature, regulate pH, regulate ionic strength, regulating measures such as organic solvent concentration or detergent concentration, to promote the combination of the analyte and second trapping agent.Remove not bound substances then.This catching of analyte is called " (Inverse Recapturing) caught in inversion ", this be because with acquisition equipment on catching of analyte compared, catching on the pick-up unit is the situation of a kind of " upside down ", thereby outside original " the specific bond site of describing in the step (1) " that combines with first trapping agent on acquisition equipment be exposed to.
Step (5) adds special detection thing: adding can with " specific bond site " the special detection thing that combines on the analyte.Special detection thing is generally the first trapping agent or derivatives thereof (or analog) that is marked with reporter molecule, have specific binding capacity and the binding mechanism to analyte identical with first trapping agent, promptly can discern and bound analyte on same specific bond site.The difference of the two is that special detection thing indicates reporter molecule and first trapping agent does not have.If first trapping agent is an antibody, special detection thing then is the same a kind of antibody that indicates a certain reporter molecule, is similar to the detection antibody in the sandwich method ELISA on function.After the specific bond site combination on special detection thing and the analyte, flush away is bound substances not, adds suitable reagent then and makes the reporter molecule shows signal.For example, if reporter molecule is that (for example horseradish peroxidase HRP), then adds the substrate of this enzyme to enzyme, makes it to produce signals such as color, reads to survey color reaction then.Determine analyte content by the signal intensity of reporter molecule at last.
Said first trapping agent can be an antibody in the inventive method, but also can be Fab fragment, Fab ' fragment, F (ab) 2 ' fragment, Fv fragment or the scFv fragment of antibody, or non-antibody proteinoid or antibody dummy (antibody mimetics) or have peptide, oligonucleotide or the micromolecular compound of specificity capturing function.When first trapping agent was antibody, said antibody is monoclonal antibody preferably, but also can be polyclonal antibody.
Mainly refer to can be by the non-antibody proteinoid of the said first trapping agent specificity combination for said analyte in the inventive method, but also can be antibody, peptide, oligonucleotide is fit (aptamers), the big molecule of other biological and compound thereof, non-peptide micromolecular compound, subcellular structure, or the like.
Be meant can be with the compound of particular molecule group (labeled molecule) mark to the analyte for said labelled reagent in the inventive method, and this labeled molecule can be by the said second trapping agent specificity combination.The biotin of activation (promptly can the compound of biotin labeling to the analyte) is the labelled reagent of using always, but said labeled molecule also can be a peptide, for example FLAG peptide, streptavidin binding peptide, MYC peptide, histidine-tagged peptide (HIS-tag peptide), or haptens, for example digoxin (digoxin Digoxigenin), fluorescent dye (for example fluorescein) etc., or oligonucleotide, for example DNA (deoxyribonucleic acid) (DNA), RNA (ribonucleic acid) (RNA) or peptide nucleic acid (PNA) etc.Table 1 is listed these labeled molecule commonly used and corresponding specific binding molecules (being said second trapping agent) thereof:
Table 1, labeled molecule commonly used and think the second corresponding trapping agent
| Labeled molecule commonly used | The second corresponding trapping agent |
| Biotin | Avidin, streptavidin, anti-biotin antibodies |
| The streptavidin binding peptide | Streptavidin |
| The FLAG peptide | Anti-FLAG antibody |
| The MYC peptide | Anti-MYC antibody |
| Histidine (HIS-6) peptide | Anti-histidine (HIS-6) antibody |
| Digoxin (Digoxigenin) | Anti digoxin antibody |
| Oligonucleotide | Complementary oligonucleotide |
| Various fluorescent dyes | Anti-corresponding fluorescent dye antibody |
| Other hapten molecules | The antibody of anti-corresponding hapten molecule |
The said labeled molecule of the inventive method generally realizes by the side-chain radical formation covalent bond with analyte the mark of analyte.If analyte is a protein, can form covalent bond by activated group on the labelled reagent and the reaction of the special groups on the protein side chain.Table 2 is listed the activated group protein side chain special groups corresponding with it commonly used on the labelled reagent or the relation of other analytes.
The relation of table 2, activated group commonly used and corresponding protein side chain group
| Activated group commonly used | Corresponding side-chain radical |
| NHS (succinimido) | The amino of the primary amine of protein (protein N-terminal and lysine residue) |
| TFP(Tetrafluorophenyl Ester) | (N-holds amino for the primary amine of protein and secondary amine; Amino on lysine, histidine and the tryptophane) |
| TFPA(tetraflurophenyl azide) | The C-H of protein and N-H key |
| (Biotin) Hydrazide (biotin) hydrazides | Glycosyl on the protein |
| (Biotin)-Amine (biotin) amine | The carboxyl of protein (aspartic acid or glutamic acid) |
| BMCC | Protein-the SH group, form the mercapto ester bond |
| Iodoacetyl (indoles acetyl) | Protein-the SH group, form the mercapto ester bond |
| HPDP | Protein-the SH group, form disulfide bond |
| Maleimide (maleimide) | Protein-the SH group, form the mercapto ester bond |
| (Biotin-LC)-ASA | Under illumination activates, with biomacromolecule covalent bond such as nucleic acid |
| Photoactivatable (Biotin) optical activation (biotin) | Under ultraviolet light activates, activate with biomacromolecule covalent bond such as nucleic acid |
Said second trapping agent of the inventive method is the material of combination of energy specificity and capture of labels molecule (and the analyte that is attached thereto).When labeled molecule was the biotin or derivatives thereof, said second trapping agent was Avidin or streptavidin or derivatives thereof; When labeled molecule is the haptens of a kind of antigen of representative decision a small bundle of straw, etc. for silkworms to spin cocoons on or peptide, said second trapping agent is the antibody that can determine a small bundle of straw, etc. for silkworms to spin cocoons on specific bond with this antigen, when labeled molecule is oligonucleotide, antibody or other protein of the oligonucleotide that said second trapping agent is a base complementrity with it or energy this oligonucleotide of specific recognition (labeled molecule).When second trapping agent was antibody, said antibody is monoclonal antibody preferably, but also can be polyclonal antibody.
Said special detection thing in the inventive method is meant on to the identification specificity of analyte identically with first trapping agent, but indicates the reagent of reporter molecule.The same with special trapping agent, special detection thing can be an antibody, but also can be Fab fragment, Fab ' fragment, F (ab) 2 ' fragment, Fv fragment or the scFv fragment of antibody, or non-antibody proteinoid or antibody dummy (antibody mimetics) or have peptide, oligonucleotide or the micromolecular compound of specificity capturing function.When first trapping agent was antibody, said antibody is monoclonal antibody preferably, but also can be polyclonal antibody.
Said reporter molecule includes but not limited in the inventive method: enzyme (HRP, AP[alkaline phosphatase] or other enzymes), a kind of antigen decision a small bundle of straw, etc. for silkworms to spin cocoons on of representative thereby the haptens that can be discerned by secondary antibodies or peptide, fluorescent chemicals, oligonucleotide (showing detection signal), biotin or derivatives thereof (when the labeled molecule on the analyte is not biotin), collaurum as the PCR substrate, or the like.When thereby said reporter molecule is the haptens of a kind of antigen of representative decision a small bundle of straw, etc. for silkworms to spin cocoons on or peptide can be discerned by secondary antibodies the time, said secondary antibodies is connected with secondary reporter molecule for example enzyme or video screen material, and monoclonal antibody preferably.
The ALIRA method is applicable to various detection platform based on solid phase surface, comprises microwell plate, filter membrane, little magnetic ball, or the like.The ALIRA method is mainly used to detect a kind of analyte in the sample, but also can be used for multiplicity detects some kinds of analytes simultaneously; Both can be used to detect combining of antibody and antigen protein, also can detect combining of the protein-protein bound of non-immunity relation or protein and other types molecule.
Below with a kind of antibody as first trapping agent, its corresponding antigen protein is analyte, the NHS-biotin is a labelled reagent, and streptavidin is second trapping agent, further specifies the method by " analyte mark and be inverted again prize law (ALIRA) " detection by quantitative specific antigen:
(1) make acquisition equipment: can specificity wrap quilt to solid phase surface in conjunction with the monoclonal antibody (first trapping agent) of determined antigen, the most frequently used is the microwell plate surface, but also can be nylon (or other medium) filter membrane surface, bead surface, or the like; The effect of this solid phase surface is the specific antigen of catching in the sample, so be called " acquisition equipment ".According to detection system, detect the different of target and sample characteristics, the antibody of bag quilt or a kind of on the acquisition equipment, or multiple mixing (being used for detecting simultaneously multiple antigen).After bag is done, with excessive nonspecific proteins matter (for example skimmed milk power or bovin serum albumin) or nonprotein blockade unconjugated plane space on liquid (for example Protein Free Blocking Reagent of the U.S. PIRECE company) solid phase surface of blockading and then the unconjugated liquid of blockading of flush away.
(2) capture antigen: add biological specimen to be measured, place certain hour, for example 1-2 hour, allow antibodies on the acquisition equipment also " catch " specific antigen (analyte) in the sample, form the antibody-antigenic complex of combining closely; Then flush away not with the nonspecific proteins matter of antibodies and other constituents.The effect of this process is enrichment antigen and removes most non-material-specifics in the sample.
(3) labelled antigen: use the labelled reagent labelled antigen-antibody camplex.(or its analog for example streptavidin [Streptavidin]) combines because biotin and Avidin high degree of specificity and high affinity, adopt the biotin molecule that serves as a mark in most cases, and with Avidin as second trapping agent on the pick-up unit (section as follows).For example, N-hydroxy-succinamide (N-hydroxysuccinimide is called for short NHS)-biotin (NHS-Biotin) can covalently bind in biotin on the primary amine group (primaryamine) of protein lysine.Because lysine almost is prevalent in all proteins, so the NHS-biotin can the nearly all protein of mark.Simultaneously, combining closely of antigen and antibody is protected specific bond site (specific antigen determinant) on the antigen molecule and not by the NHS-biotin modification, still keeps the binding ability to this antibody in the compound.Therefore, after antibody-antigenic compound dissociated separately, this antigen molecule can form new compound with the detection antibodies.Except NHS, also can with bioactive molecule that can other group of covalently bound protein (as sulfydryl, carboxyl or hydroxyl) with biotin labeling to antigen.If the biotin molecule that can not serve as a mark for a certain reason also can be with molecules that serves as a mark such as certain haptens or little peptide molecules, correspondingly use antibody at this labeled molecule simultaneously as second trapping agent.
(4) wash-out antigen: with solution (for example Tris-hydrochloride buffer or Tris-glycine buffer) cancellation that contains excessive primary amine group and the free NHS-biotin of flush away.If labelled reagent adopts other activated group outside the NHS, then will adopt corresponding solution cancellation and the unreacted labelled reagent of flush away as the case may be, for example, if labelled reagent is indoles acetyl-LC-biotin, then add excessive halfcystine cancellation it.Add the antigen eluent then, the antigen protein that allows mark cross comes out from antibody-antigenic compound disassociation.The antigen eluent contains the composition of the failure analysis thing and the first trapping agent combination, for antibody-antigenic compound, can adopt low pH solution, 0.1M citric acid (pH2.8) for example, the antigen eluent (for example ImmunoPure eluent of U.S. PIERCE company) that perhaps uses existing market to sell; Also can adopt high pH solution, 0.1M triethylamine (Triethylamine) for example elutes the protein on the acquisition equipment and enters liquid phase.The eluent that will contain antigen protein shifts out then, adds suitable neutralization buffer (containing nonspecific proteins matter such as skimmed milk power etc.) neutralization.Perhaps before shifting out antigen eluent general, directly neutralization buffer is joined on the acquisition equipment.The purpose that adds neutralization buffer is that the pH value is returned near neutral, and reduces ionic strength to a certain extent, makes the combination that directly no longer influences next step labeled molecule on second trapping agent and antigen protein on the pick-up unit.
(5) capture antigen once more: the eluent after will neutralizing joins " pick-up unit " immediately.Pick-up unit still also can be by filter membrane, glass or plastic surface thin slice, the carrying of microballon solid surface such as (magnetic balls) usually by the microwell plate preparation.The pick-up unit solid phase surface is coated with second trapping agent, and passes through blockading of nonspecific proteins matter.Adopt streptavidin (or Avidin) as second trapping agent in this example.High affinity between Avidin and the biotin has guaranteed to catch quickly and effectively antigen and the non-specific protein that carries the labeled molecule biotin.The bag quilt of Avidin and blockade and carry out in advance on the pick-up unit is finished with above-mentioned " 4 " step of box lunch, after the antigen that just is labeled elutes from acquisition equipment, can be transferred to pick-up unit immediately.
(6) add detectable, for example detect antibody: on pick-up unit, antigen by its labeled molecule of carrying (biotin) and Avidin in conjunction with being trapped in solid phase, thereby with specific bond site (antigen decision a small bundle of straw, etc. for silkworms to spin cocoons on) overturn be exposed to outside.Behind the unconjugated non-specific protein of flush away, add and detect antibody (special detection thing).Detect antibody and connect reporter molecule by capture antibody (first trapping agent) and be made, can discern and conjugated antigen on the specific bond site.If the trapping agent on the acquisition equipment is not antibody but other types molecule, then special detection thing is that same molecule connects reporter molecule and forms.
(7) read to survey signal: behind the unconjugated detection antibody of flush away, add the reagent that makes reporter molecule shows signal on the detection antibody.Character according to reporter molecule, signal produces and detection method includes but not limited to following several: (a) enzyme (for example HRP, AP etc.) is directly as reporter molecule: the substrate that adds this enzyme, make it to produce color, fluorescence or signal such as luminous, can be by reading to survey the activity that signal intensity is identified enzyme.(b) haptens micromolecular compound or little peptide are as reporter molecule: the effect of this class reporter molecule has provided specific antigen decision a small bundle of straw, etc. for silkworms to spin cocoons on, can add antibody (secondary antibodies) at this haptens or little peptide, carry secondary mark signal on the secondary antibodies, for example, enzyme, luminescent dye molecule etc. add corresponding zymolyte again or directly read and survey fluorescence intensity with identified activity.(c) fluorophor is as reporter molecule, reads or scanning of fluorescent intensity is determined signal intensity by direct survey; Perhaps utilize the haptens characteristic of some fluorophors, add enzyme labelled antibody, detect the activity of enzyme according to the method described above at fluorophor.(d) oligonucleotide will carry out real-time PCR reactions to detect its content as substrate behind this oligonucleotide wash-out as reporter molecule; Carry out immunity-PCR with oligonucleotide as reporter molecule, not only can improve detection sensitivity, and can carry out multiplicity and detect some kinds of antigens in the sample simultaneously.
The concrete operations technology that relates in each step of ALIRA detection method disclosed in this invention, for example with the trapping agent bag by the technology of the technology of microwell plate, trapping agent and analyte combination, with the technology of labeled molecule labelled proteins such as NHS-biotin, reporter molecule is connected to technology, demonstration that detects antibody and the technology of measuring the reporter molecule signal intensity etc., be well-known to those skilled in the art.
Obviously, the method of " analyte mark and be inverted prize law again " disclosed by the invention quantitative detecting analysis thing, only need just a kind of analyte of energy detection by quantitative of single specificity trapping agent, can be applied in disease clinical diagnosis, disease biomarker (biomarkers) identification and analysis, molecular biology and every field such as RESEARCH ON CELL-BIOLOGY analysis, proteomics research analysis, new drug target position identification and analysis, clinical pharmacokinetics and pharmacodynamic analysis.According to the principle of this method, can develop, produce, allocate and pack various testing products, comprise the ingredient of complete kit and kit, for example acquisition equipment, pick-up unit, various related reagent, or the like.
The present invention compared with prior art its beneficial effect is:
A kind of new technological process that the single specificity trapping agent just can the detection by quantitative specific analyte that only needs is provided.This new technological process is different from traditional sandwich method ELISA and SALRA method on committed step.See Fig. 1.
1 and the comparison of traditional sandwich method ELISA:
Compare with the sandwich method ELISA method of widespread use both at home and abroad at present, the major advantage of ALIRA detection method proposed by the invention is as follows:
(1) the most important advantage of ALIRA method is, only needs to use just energy quantitative detecting analysis thing of a species specificity trapping agent, that is to say that as long as a kind of antibody just can detect a kind of antigen protein, and sandwich method ELISA needs two kinds of antibody that match mutually.Obviously, it is more much easier than the monoclonal antibody that obtains two high degree of specificity high affinities and mutual pairing to obtain a high degree of specificity high affinity monoclonal antibody.For a lot of protein, for some reason or other, obtain very difficulty of ELISA pairing antibody.Therefore, do not match antibody at present for those and carry out the antigen protein that sandwich method ELISA detects,, just can utilize the ALIRA method to set up detection method rapidly as long as there is the high antibody of a kind of high specificity affinity.This is specially adapted to detect protein molecule or the molecular structure territory that only has one or several adjacent very near antigenic determinant, such as the very little protein of molecular weight, the antigenic determinant very near protein that leans on of distance, the phosphorylation state of protein, the critical function territory and the active state thereof of protein mutually, or the like.The same with the SALRA method, but the ALIRA method provides simple and efficient detection by quantitative approach for those extremely important each proteinoid of still not having desirable detection means so far in clinical practice and fundamental research.This has not only shortened the time of setting up detection method greatly, and provide new approaches for detection range: the technician needn't spend a large amount of manpower and materials and time to go to carry out paired experiment between antibody again, can concentrate on screening high specific, high-affinity, bag to energy by the good monospecific antibody of performance.Owing to only need a kind of antibody just can detect; therefore the scope of application of ALIRA method is very extensive, will promote the work of medical diagnosis on disease, new drug development, molecular biology and RESEARCH ON CELL-BIOLOGY, proteomics research application, food and agricultural product sanitary inspection and compound residue detection, environmental protection or the like every field effectively.
(2) principle of ALIRA and method both be applicable to the existence that utilizes antibody to detect antigen in the sample, also be applicable between the protein-protein that utilizes non-antibody-antigen relation or the interaction between protein and other molecule, detect a certain rho factor.These do mutually and in conjunction with for disclose the life secret, study cell processes, the process that diagnoses the illness, the development newtype drug is all extremely important.For example, for the content of certain rho factor X in the identification of organism sample, can adopt the principle of ALIRA with being coated on the solid phase surface of acquisition equipment as first trapping agent with the another kind of protein Y of protein X combination, add sample to be tested, form protein X-protein Y compound; Use the biotin labeling compound then, behind the wash-out, catch the protein X that carries biotin, add enzyme target protein Y then, can carry out detection by quantitative protein X by the Avidin on the pick-up unit.Except protein, can also be with other materials as trapping agent, such as peptide, nucleic acid, polysaccharide, lipid, haptens micromolecule, or the like, detect all kinds of analytes that combine with its specificity, for example protein, peptide, aptamer (aptamer), or the like.
(3) detection specificity is strong, highly sensitive.Specificity and reduction that the ALIRA method has two steps to guarantee to detect detect noise: the first, and acquisition equipment is only caught the specific analyte in the sample.When carrying out mark, most non-specific materials are by flush away, and analyte is by highly enriched.The second, on pick-up unit, the detection antibody that is marked with reporter molecule combines and shows signal with the specific bond site of the analyte of being caught again.Owing to only need a kind of special trapping agent just can detect, when selecting antibody as trapping agent, needn't be subject to the consideration whether two antibody match, can stress to select for use those specificity height, affinity height, bag by the good antibody of performance, thereby improve detection specificity and sensitivity.In addition,, join pick-up unit after can suitably concentrating analyte from acquisition equipment because acquisition equipment and pick-up unit separate, thus the amplification detection signal.For example, can adopt area bigger or shaggy micropore as the carrier of acquisition equipment, increase the surface area of catching solid phase, the area of plane of dwindling pick-up unit simultaneously is to improve the concentration of analyte on the pick-up unit.
2, compare with the SALRA method:
ALIRA detection method proposed by the invention is the same with previously described SALRA method, all only need the just corresponding analyte of energy detection by quantitative of a species specificity trapping agent, but as shown in Figure 1, the two is different in following committed step.
(1) major different is to the difference of catch mechanism again of the analyte that is labeled on the pick-up unit.In the SALRA method, trapping agent on the pick-up unit and the trapping agent on the acquisition equipment are with a kind of molecule (normally same antibody) or possess with a kind of catch mechanism that the two is to realize by the same site on the bound analyte (specific bond site) to catching of analyte; And in ALIRA method described in the invention, the trapping agent on the pick-up unit (i.e. second trapping agent) is different from the trapping agent (i.e. first trapping agent) on the acquisition equipment fully.Here, second trapping agent is to reach the purpose (promptly be inverted and catch) that analyte is attached to solid phase by the labeled molecule on identification and the bound analyte.This difference has caused other step of testing process that corresponding difference (as follows) is also arranged.
(2) in the SALRA method, what be used for that the labeled molecule of analyte-trapping agent compound plays on the mark capturing device is the effect of reporter molecule, this labeled molecule or itself are exactly signaling molecule (for example fluorescein), or the directly generation of mediation participation signal (biological example element-enzyme mark Avidin signal system).And in the ALIRA method, analyte is carried out mark, be for provide can detected device on the site of the second trapping agent combination, labeled molecule is no longer as reporter molecule, therefore needn't have directly or mediation produces the function of detection signal.The signal report mechanism of ALIRA method is finished by the special detection thing of next step.
(3) in the SALRA method, on analyte and the pick-up unit trapping agent in conjunction with after, the generation of the direct mediated detection signal of its reporter molecule that carries, any mark the non-specific binding thing of reporter molecule all can produce noise signal.Since mark the non-material-specific of reporter molecule may comprise the trapping agent in the acquisition equipment, some the non-special component in blockade protein or the sample to be tested, complicated component, and the history with the non-specific bond of acquisition equipment is all arranged once, therefore bigger by the possibility of non-specific bond once more on pick-up unit.In case combination, because the SALRA method does not have the subsequent step of further discriminance analysis thing, the reporter molecule that carries on these non-material-specifics will directly produce the detection noise.And in the ALIRA method, second trapping agent is at labeled molecule rather than analyte itself to catching again of analyte on the pick-up unit.If with the biotin molecule that serves as a mark, Avidin is as second trapping agent, the high affinity of the two has guaranteed to greatest extent the analyte in the sample to be caught, and then adding detects antibody, identification and in conjunction with once on acquisition equipment by the specific bond site of the first trapping agent combination, produce detection signal by detecting the reporter molecule that carries on the antibody again.Though any non-material-specific that carries labeled molecule all can be captured on the pick-up unit, owing to detect the specific bond site of an antibody discriminance analysis thing, therefore the non-material-specific of nonrecognition does not produce the detection noise basically.In other words, the ALIRA method has last " safety inspection " mechanism, thereby reduces the background noise that comes from non-specific material.Therefore the ALIRA method aspect sensitivity and specificity two all than SALRA method height.Especially be fit to detect those biological specimens that analyte content is low, nonspecific composition is complicated.
(4) in the SALRA method, the effect of specificity trapping agent is to wrap be hunted down device and pick-up unit.And in the SALIRA method, the specificity trapping agent is used for wrapping the device that is hunted down as first trapping agent on the one hand, does not carry out mark; Be as special detection thing (detection antibody) on the other hand, then need to be marked with reporter molecule.The mark that detects antibody can prepare in advance.
(5) in the SALRA method, pick-up unit for catching again of analyte be based on to the specificity of analyte itself in conjunction with and realize, therefore when carrying out the multiplicity detection, multiple capture antibody is mixed the back wrap the device that is hunted down, and capture antibody wraps quilt separately on pick-up unit, the specific geographical determining positions of different antibodies the captive once more geographic position of corresponding different analytes, therefore can in little gust of arrangements such as protein-chip, directly carry out multiplicity detection multiple analytes with fluorescence molecule as reporter molecule.And in the ALIRA method, pick-up unit is based on for catching again of analyte the combination of label is realized, because labeled molecule is identical, therefore can't on pick-up unit different analyte solid phases be combined in different geographic position.Generally speaking, the SALRA method can be used for single detection, also can be used for multiplicity and detect, and comprises adopting little gust of arrangement such as protein-chip to detect as the multiplicity of load-bearing surface; And SALIRA is more suitable in a kind of analyte is carried out single detection.But, the ALIRA method also can be used to carry out multiplicity and detect multiple analytes, and its principle with step is: adopt the different special detection thing of different reporter molecule marks in advance, will mix the back at first trapping agent of different analytes and wrap the device that is hunted down; Add sample to be tested, corresponding analyte is trapped in solid phase; With labeled molecule labelled analyte and wash-out it, join pick-up unit, the special detection thing that will indicate different reporter molecules then mixes the back and adds, and through washing, then according to the character of reporter molecule, shows and reads to survey signal.If reporter molecule is a fluorescent chemicals, can read respectively to survey according to the exciting light and the wavelength of transmitted light characteristics of different fluorescent chemicalses; If reporter molecule is different enzyme, can be with special detection thing wash-out, after the neutralization, break into portions joins respectively in the micropore that contains different zymolytes, detects the reaction signal of enzyme then; If reporter molecule is an oligonucleotide, can use the special detection thing of eluent (for example 0.1M triethylamine) wash-out, after the neutralization, use PCR lead at different oligonucleotides to (primer pairs), carry out PCR in real time, then according to the concentration of the CT value computational analysis thing of sample and normal concentration.
Description of drawings
Fig. 1 be the inventive method schematic flow sheet and with the comparison diagram of ELISA and SALRA method;
Fig. 2 is the testing result figure of embodiment 1.
Fig. 3 is the testing result figure of embodiment 2.
Embodiment
Step below in conjunction with SALIRA method among Fig. 1 is further set forth the inventive method.
Embodiment 1: detect human P 53 protein with HRP as reporter molecule
In present embodiment, determined antigen protein is for having the tumor suppression function, promoting apoptotic human body p53 protein, and acquisition equipment and pick-up unit all are 96-hole micro plates.The micropore bag of acquisition equipment is by anti-p53 monoclonal antibody, and the pick-up unit bag is by streptavidin.Detecting antibody is the same a kind of anti-p53 monoclonal antibody that is marked with fluorescein, and reporter molecule is a fluorescein here.
(1) capture antigen protein:
A, the bag antibody that is hunted down: it is (flat to get two 96-hole micro plates, to the combination of protein height), one is used for catching the antigen for the treatment of in the sample as acquisition equipment, adding 100 μ l concentration in the micropore is that 2 μ g/ml (are diluted in bag and are cushioned liquid, 0.05M sodium bicarbonate buffer liquid, pH9.6) anti-p53 monoclonal antibody (U.S. CalBiochem company) adds two stringers totally 16 micropores.Another micro plate is used for catching the also antigen of certification mark as pick-up unit, and adding 100 μ l concentration in the micropore is the streptavidin of 10 μ g/ml (being diluted in the PBS damping fluid), adds two stringers totally 16 micropores.Place 4 to spend night micro plate.
B, the nonspecific binding site of blockading: remove the antibody-solutions in acquisition equipment and the pick-up unit micropore, clean 1 time with PBS+0.05%Tween 20 (PBST).Remove PBST, add 2% skimmed milk power (being dissolved in PBS) of 380 μ l, at room temperature blockade.The time of blockading of acquisition equipment is 1 hour; Blockading of pick-up unit is 2 hours.
C, capture antigen protein: remove the liquid of blockading, the purification reorganization human P 53 protein that adds the variable concentrations of 100 μ l process serial dilution in micropore (from the purchase of U.S. BD Biosciences Pharmingen company, is that carrier is recombinant expressed in insect cell with Baculovirus; Be diluted in the PBS+1% skimmed milk power).Following 1.5 hours of room temperature.
(2) labelled antigen protein:
Empty micropore, clean 2 times with PBST, PBS cleans 1 time.Each micropore adds 100 μ l 0.02%NHS-biotins (being dissolved in PBS), and room temperature kept 30 minutes.Remove the NHS-biotin, add the Tris-hydrochloride buffer (pH8.0) of 380 μ l 10mM, the NHS-biotin of cancellation and erase residual, room temperature kept 5 minutes.Water cleans 1 time.
(3) wash-out antigen:
Aim at the micropore middle part, add 20 μ l antigen eluents (using the ImmunoPureElution Buffer of U.S. PIERCE company), room temperature keeps 15 minutes (micro plate is covered to keep humidity).Meanwhile, remove the solution of blockading in the pick-up unit micropore, add 80 μ l PBS+1% skimmed milk powers.
(4) antigen is caught again:
To from the acquisition equipment micropore, be transferred on the pick-up unit by the antigen (about 20 μ l) of wash-out, suitable shaking mixing, room temperature kept 1 hour.Because the pick-up unit micropore has contained 80 μ l PBS+1% skimmed milk powers, the antigen eluent is neutralized and dilutes 5 times, can not influence the combination of streptavidin on biotin (labeled molecule) and the pick-up unit (second trapping agent).
(5) detect:
A, adding detect antibody: remove not bound substances, clean 2 times with PBST, PBS cleans 1 time.Add the anti-p53 monoclonal antibody (U.S. CalBiochem company of 100 μ l through fluorescein-labeled same clone; Be diluted to 0.5 μ g/ml with the PBS+1% skimmed milk power), room temperature kept 1 hour.
B, shows signal: remove not bound substances, clean 3 times with PBST.The anti-fluorescein antibody (American I nvitrogen company is diluted to 0.5 μ g/ml with the PBS+1% skimmed milk power) that adds 100 μ l horseradish peroxidase-labeled, room temperature kept 1 hour.Clean 4 times with PBST.Add 100 μ l peroxidase substrate solution (the SureBlue Reserve HRP of U.S. KPL company substrate reactions liquid, TMB is a substrate), 37 degree 20 minutes.Add 50 μ l 2M sulfuric acid, read to survey the 450nm wavelength light and absorb.
C, testing result: Fig. 2 shows the testing result of embodiment 1.For from 100ng/ml to 1ng/ml, all representing good linear relationship between p53 signal intensity of trying and the concentration, prove that the ALIRA method can detect this protein in this concentration range, sensitivity (blank mean value adds three standard deviations) reaches 0.6ng/ml.
Embodiment 2: detect human C-reactive protein with oligonucleotide as reporter molecule
In present embodiment, determined antigen protein is that (C-reactive protein, CRP), acquisition equipment and pick-up unit all are 96-hole micro plates to the human body C-reactive protein that plays an important role in immune response.The micropore bag of acquisition equipment is by anti-CRP monoclonal antibody, and the pick-up unit bag is by streptavidin.Detecting antibody is the same a kind of anti-CRP monoclonal antibody that is marked with oligonucleotide (60 bases are long), and reporter molecule is an oligonucleotide here.
(1) capture antigen protein:
A, the bag antibody that is hunted down: it is (flat to get two 96-hole micro plates, to the combination of protein height), one is used for catching the antigen for the treatment of in the sample as acquisition equipment, adding 100 μ l concentration in the micropore is that 2 μ g/ml (are diluted in bag and are cushioned liquid, 0.05M sodium bicarbonate buffer liquid, anti-CRP monoclonal antibody (U.S. R﹠amp pH9.6); D Systems company), add two stringers totally 16 micropores.Another micro plate is used for catching the also antigen of certification mark as pick-up unit, and adding 100 μ l concentration in the micropore is the streptavidin of 10 μ g/ml (being diluted in the PBS damping fluid), adds two stringers totally 16 micropores.Place 4 to spend night micro plate.
B, the nonspecific binding site of blockading: with embodiment 1.
C, capture antigen protein: remove the liquid of blockading, the purification recombined human CRP protein that adds the variable concentrations of 100 μ l process serial dilution in micropore (obtains from U.S. BioCheck is public; Be diluted in the PBS+1% skimmed milk power).Following 1.5 hours of room temperature.
(2) labelled antigen protein:
With embodiment 1.
(3) wash-out antigen:
Aim at the micropore middle part, add 20 μ l antigen eluents (0.1M triethylamine), room temperature keeps 15 minutes (micro plate is covered to keep humidity).Add 80 μ l and contain the RBST of 1% skimmed milk power and 0.1M Tris-HCl (pH8.0).Meanwhile, remove the solution of blockading in the pick-up unit micropore.
(4) antigen is caught again:
Will be from the acquisition equipment micropore antigen (about 100 μ l) of wash-out and neutralization be transferred on the pick-up unit, suitable shaking mixing, room temperature kept 1 hour.Because the antigen eluent has been neutralized and has diluted 5 times, can not influence the combination of streptavidin on biotin (labeled molecule) and the pick-up unit (second trapping agent).
(5) detect:
A, adding detect antibody: remove not bound substances, clean 2 times with PBST, PBS cleans 1 time.Add the anti-CRP monoclonal antibody (U.S. R﹠amp of 100 μ l through the same clone of oligonucleotide mark; DSystems company; Be diluted to 0.5 μ g/ml with the PBS+1% skimmed milk power), room temperature kept 30 minutes.
B, wash-out reporter molecule: remove not bound substances, clean 6 times with PBST.The 0.1M triethylamine that adds 100 μ l, room temperature kept 20 minutes.The 1M Tris-HCl (pH8.0) that adds 50 μ l after the mixing, gets 5 μ l and carries out real-time PCR reactions as substrate.
C, shows signal: with u.s.a. applied biosystem company (Applied Biosystems) ABI7000 PCR in real time detection by quantitative reporter molecule.The PCR reaction conditions is: 55 degree 2 minutes, 95 degree 10 minutes then are that spend 1 minute 95 15 seconds of degree and 60 of 40 reaction times.Reaction primer: 1mM; Fluorescent marker: FAM; Signal generating method: TaqMan method; Reaction volume: 40 μ l.
D, testing result: Fig. 3 shows the testing result of embodiment 2.For from 500ng/ml to 2ng/ml, all representing good linear relationship between CRP signal intensity of trying and the concentration, prove that the ALIRA method can detect CRP protein in this concentration range.The detection sensitivity of this experiment (blank mean value adds three standard deviations) is 0.45ng/ml.
Claims (15)
1, a kind of method with single specificity trapping agent quantitative detecting analysis thing is characterized in that:
(1) catches analyte: specificity trapping agent bag is arrived solid phase surface, become acquisition equipment; Add biological specimen to be measured, the specificity trapping agent on the acquisition equipment is combined with analyte in the sample, form trapping agent-analyte complex;
(2) labeled complex: with labelled reagent mark capturing agent-analyte complex;
(3) elution analysis thing: will elute from compound through the analyte of mark;
(4) catch again: will be in the analyte of wash-out and the back add pick-up unit, by second trapping agent on the pick-up unit by once more analyte being caught with combining of labeled molecule.Said pick-up unit is meant that bag is by the solid phase surface of second trapping agent;
(5) detect: add special detection agent and the combination of analyte specificity, determine analyte concentration by the signal intensity that detects the reporter molecule generation of carrying on the special detection agent.
2, quantitative detecting method according to claim 1 is characterized in that: wherein said special trapping agent is antibody, antibody fragment, non-antibody proteinoid, peptide, oligonucleotide or micromolecular compound.
3, quantitative detecting method according to claim 1 is characterized in that: wherein said second trapping agent is the material that can combine with said labeled molecule, includes but not limited to Avidin or streptavidin, antibody or other protein.
4, quantitative detecting method according to claim 1 is characterized in that: wherein said special detection agent is the special trapping agent that indicates reporter molecule; Special detection agent and special trapping agent combine with the same site of analyte, the two be not both the former and carry reporter molecule and the latter does not carry reporter molecule.
5, quantitative detecting method according to claim 1 is characterized in that: wherein said analyte is can be fit with antigen protein, antibody, peptide, the oligonucleotide that said trapping agent specificity combines, the big molecule of other biological and compound, micromolecule and subcellular structure.
6, quantitative detecting method according to claim 1 is characterized in that, said labelled reagent is can be with the reagent of labeled molecule mark to the analyte; Said labeled molecule is a biotin preferably; Other compounds that can be used as labeled molecule comprise: all kinds of haptens micromolecule, peptide or oligonucleotide.
7, quantitative detecting method according to claim 1 is characterized in that: wherein said reporter molecule is fluorescence molecule, biotin, haptens micromolecule, enzyme or oligonucleotide.
8, quantitative detecting method according to claim 1 is characterized in that, wherein the solid phase surface of said acquisition equipment be test tube, micro plate, filter membrane, can conjugated protein test paper or microballon; The solid phase surface of said pick-up unit is micro plate, filter membrane, test paper, microballon, glass or plastic tab carrier.
9, the described quantitative detecting method of claim 1, the application in clinical detection diagnosis, biomarker identification and analysis, molecular biology and RESEARCH ON CELL-BIOLOGY, proteomics research analysis, new drug target position identification and analysis, clinical pharmacokinetics and pharmacodynamic analysis field.
10, a kind of said kit of using the method for single specificity trapping agent quantitative detecting analysis thing of claim 1 that is used for, it is characterized in that, comprise acquisition equipment, pick-up unit, labelled reagent, analyte neutralizer and eluent, the special detection agent that is marked with reporter molecule, the reagent that makes the reporter molecule shows signal, analyte standard model, directions for use etc.
11, kit according to claim 10 is characterized in that, said acquisition equipment is that bag is by the 96-hole micro plate of monoclonal antibody specific; Said pick-up unit is that bag is by the 96-hole micro plate of streptavidin; Said labelled reagent is the reagent that biotin can be connected on the analyte, for example NHS-biotin; The said special detection agent that is marked with reporter molecule is connect to go up reporter molecule and produce by being used to wrap the be hunted down monoclonal antibody specific of device.
12, according to claim 10 and 11 described kits, it is characterized in that, the special detection agent of said reporter molecule mark is the monoclonal antibody specific with horseradish peroxidase-labeled, and the said reagent of reporter molecule shows signal that makes includes but not limited to horseradish peroxidase substrate and reacting terminating solution.
According to claim 10 and 11 described kits, it is characterized in that 13, the special detection agent of said reporter molecule mark is with a kind of haptens micromolecule or peptide-labeled monoclonal antibody specific; The enzyme di-level antibody that the said reagent that makes the reporter molecule shows signal includes but not limited to combine with said haptens micromolecule or peptide specific, the substrate of enzyme, reacting terminating solution etc.。
According to claim 10 and 11 described kits, it is characterized in that 14, the special detection agent of said reporter molecule mark is the monoclonal antibody specific with the oligonucleotide mark.
According to claim 10 and 11 described kits, it is characterized in that 15, the special detection agent of said reporter molecule mark is the monoclonal antibody specific with the fluorescent chemicals mark.
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