CN1034617A - Identify the method and the device of biochemical species - Google Patents

Identify the method and the device of biochemical species Download PDF

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CN1034617A
CN1034617A CN88107039A CN88107039A CN1034617A CN 1034617 A CN1034617 A CN 1034617A CN 88107039 A CN88107039 A CN 88107039A CN 88107039 A CN88107039 A CN 88107039A CN 1034617 A CN1034617 A CN 1034617A
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dentate
cell
probe
patch
mark
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丹尼斯D·派翠欧尼格罗
约翰L·斯坦尼克
罗伯特J·路姆
玛丽露马特斯·庞德
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Nygene Corp
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

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Abstract

A kind of for the homogenizing method that detects the first anti-dentate-dentate product whether have the mark detectable label, this product is to generate on the matrix device in the liquid constituent in being contained in cell; This method comprises following each step
(a) provide be coated with the second anti-dentate carrier particle as this mark, with the matrix device of first anti-dentate-dentate product of detectable label;
(b) collect a part the liquid in this cell at least, the mark that is present in the carrier particle top with the anti-dentate-dentate product of detectable label in patch; And
(c) measure in this patch existing mark with the product quantity of detectable label.

Description

Identify the method and the device of biochemical species
The invention relates to mirror (inspection) fixed with the biological affine chemical substance of quantitative measurement for example via material that dentate-anti-dentate reaction is generated and work as in specific antibodies, cell antigen, cell fragment, protein and enzyme still be present in biological nutrition liquid or other fluids, indicate whether to have used modification method of this class material and device.Biological nutrient liquid is generally the nutrient solution that dentate or anti-dentate generate therein.
Detect particular organisms compatibility chemicals for example in monoclonal antibody, medicine, the acceptor-fields such as carrying enzyme base to carry out extensive work; Most of work relies on uses the radio chemistry product as indicator, and this kind test is called isotope.The present invention can be used in the non isotopic program especially, but also can adopt the isotope program.
The detection method in this kind immunochemistry field or identification method are classified as heterogeneous body checking method and homogeneous method.The heterogeneous body method is defined as must separate the mensuration chemical agent that optionally combined with analyte and the chemical agent of combination not from the constituent of being checked before detecting or measuring.United States Patent (USP) 4,652,533 have disclosed this kind program.Well-known ELISA(enzyme-connection in the prior art, immunoadsorption identification method) and the RIA(radioimmunoassay) program is the representative instance of heterogeneous body identification method.
The heterogeneous body identification method need be separated binding entity with binding entity not, often can be by washing/separating step repeatedly and quicken it; It is dull and stereotyped as the supplementary means of carrying out before this kind evaluation program separating and removing in conjunction with species and any constituent that contains not in conjunction with species to use magnetic bead to replace filtrator or little power valency once in a while.
The entity measuring combination of homogeneous identification method combination never, and need not separated from one anotherly, can be retained in the same cell and identify simultaneously or detection method.Existing homogeneous test does not all meet fast and responsive requirement.Wherein have minority to be suitable as outside the program of original place, the biological entities that promptly occupies major part and acceptance supervision in the method is in the program of growing or secreting.Even all these class methods more do not take manpower but still a large amount of machineries of needs and/or manually-operated than the heterogeneous body test usually.Believe in the previous existing homogeneous checking method not have a kind of method sensitive, and believe and seldom can effectively measure big molecule (for example being higher than 30,000 daltonian molecules) as the known heterogeneous body checking method of this technical field.For instance, radioimmunoassay is identified infiltration (RID) identification method and " turbidimetric analysis turbidimetry " though technology can be measured big molecule, and its susceptibility does not reach required requirement.
People's such as Wei Gena United States Patent (USP) 4,680,275 has disclosed a kind of time delay program of avoiding having in the homogeneous test inspection method coexistence of fluorescent background.
The another kind of homogeneous heterotope immunity authenticate technology (United States Patent (USP) 4 of this and Ni Kelin of the dust tinkling of pieces of jade for example, 537,861 related invention) be that scanning is via a plurality of spaced-apart electrodes or the magnet space pattern that (or it is contiguous) produced in the biological chemistry constituent of identifying.The mode of carrying out of scanning makes people can distinguish substantially background fluorescent output at random and the relevant nonrandom output of wishing to detect of cardinal principle of association reaction through mark fast.
The technology that excites fluorescent and its operation is well-known on the analytical chemistry, at the United States Patent (USP) 4,675,529 that the storehouse rising sun draws, has all done detailed description in other lists of references of above-mentioned Wei Gena patent and above citation.
The United States Patent (USP) 4,683,120 of promulgation recently shows a kind of material " patch " of centrifugal assembling, and its geometry property measured is as the standard of its assembling source constituent character.
Certainly above-mentioned description to background of the present invention has been done after knowledge of the present invention disclosed herein is understood, and the various references of being quoted from are only for reference.
The novel apparatus that fundamental purpose of the present invention provides a kind of novel homogeneous pattern method and can be used for this kind method is for detecting specific analyte-back analysis thing reaction product, for example dentate-anti-dentate reaction product or similarly reaction product, for example material that for example reacts and generated between RNA fragment (having specificity person) at not tagged dna fragmentation (for example gene) and complementary nucleic acid for dna fragmentation.Really will reach the purpose of this case, DNA/ RNA affinity normally is used as the species of analyte-back analysis thing reaction product and is looked it.
A specific purpose of the present invention provides the original place device, therefore above-mentioned these class methods can need not the device that provides loaded down with trivial details substantially, in order to avoid be subjected to from the parasitic constituent of biology, just the background energy launched of the nutrient solution that dentate-anti-dentate reaction product generates therein disturbs.
Another object of the present invention provides can carry out the device of this kind method and wherein used device, and this kind device is fit to be used for checking atomic small volume even is the biotic component thing of a time microlitre volume.
Another object of the present invention is a used device in the method that can provide sensitive and this method, thereby allow to detect very apace even indivisible specific anti-dentate-dentate reaction product, also be like this even the fluorescent coexistence that may disturb is in a large number arranged in the constituent that detects or examine and determine.
Another purpose of the present invention provides and a kind ofly can detect big dentate for example greater than 5,000 dalton except little dentate, actually greater than 30,000 dalton even be the determination method of hundreds thousand of daltonian sensitive homogeneous and general-purpose.
Another purpose of the present invention is to small amounts of analyte, that is the material of acceptance detection carries out express-analysis.
Other purposes of the present invention after it studies this disclosure carefully, will be conspicuous concerning these those skilled in the art.
Above-mentioned purpose can reach via adopting following method substantially, matrix device used in this method should use semitransparent materials, for example be loaded with the particle of magnet, will pluck through the dentate-anti-dentate product of mark and bring into through separating and being preferably in opaque " patch " that material to be determined arranged.In the preferred embodiment of the present invention, the patch maskable has zone to be determined not to be subjected to background interference and no matter this interference source is unconjugated mark material or certain volume of liquid biological material or nutrient solution, and patch separates this kind background interference." background interference " refractive power line that reflects from residual labeling dye that comprises as described herein just can be used on this look assessment patch, because intrinsic property and can not produce the width of cloth and penetrate the mark that the inventive method is useful.Yet, comprise isotope labeling suitably using other marks under the situation.Can use the matrix thing such as glass, but people must device focus on so that receive from wherein and the light that comes, so the background fluorescent can be blocked in beyond the optical measurement of for example measuring fluorescent labelling.The representative of " opaque " matrix material one speech can generate the material of opaque patch substantially.Thin patch meets the requirements, and therefore, with the limited magnetic particle of translucence, for example ferriferous oxide is the magnetic particle of base, generates thin opaque patch for best.Obtain to have about 4% transmission or following patch easily.
Must notice that dentate one speech is used for the chemical substance that the anti-dentate of chemical substance to be broad sense representative be called as to(for) another kind has special affinity herein.This is defined in can accept usually in the biology techniques field and replace that to require dentate be that one group of atom is around a central metal ion old definition.
" liquid " speech is used for describing any material, though this material of thickness is in the example that dentate and anti-dentate are arranged can move into the test patch; Therefore multiple gel is all true executes suitable " liquid " of the present invention.
Some personnel of present technique field are called " analyte " with determined material, and its material with biological affinity then is called the back analysis thing.Dentate and anti-dentate are used for herein, but must understand that these terms are not that broad sense is considered as containing analyte-back analysis produce things such as speech.
Among the embodiment of one of the present invention excellence, the method will utilize the used this magnetic bead of technological field of biochemistry to attract the bioaffinity material as the carrier particle device, for generating opaque patch.Yet other these particles are for example based on carbon black, and barium metal or other materials can be used as quite opaque carrier.The optimal selection of this kind carrier is the material with evening chemical characteristic and size dimension distribution character, certainly also tackle in bioprocess and have material to be assessed to have minimum interference: non magnetic or magnetic particle can be separated via the centrifugal force of sedimentation or even showy power, for example its buoyancy of some plastic cement or hollow-particle can utilize the effect of floating to be separated.Yet one of magnetic classification particle excellent characteristics be for moving in the opaque patch via applying limit, suitable magnetic field, easily but optionally transportable on contiguous precalculated position, the biological cell wall district that remains to be tested.In this kind precalculated position, use pick-up unit (for example impinge upon the excitation source in the patch and detect) from the used device of this district fluorescent easily mensuration from the fluorescent of minimum special separation material patch.Arbitrary characteristics as the used magnetic material for making clothes of the microgranular matrix material of activity are quite easy in the use, and this particle can be scattered in the biological substance of identifying or detecting once again.
Generally speaking, when the check point superordinate becomes opaque patch near the carrier that is loaded with tagged interested anti-dentate-dentate is being loaded with the hold-up vessel of liquid to be determined or little locular wall, discovery can provide a kind of antagonism background or the unusual effective sieve of noise, the expection of these backgrounds or noise may be disturbed and penetrate and produce homogeneous determination method that the material fluid carries out and produce and disturb having the remaining width of cloth, for example unconjugated fluorescent probe and/or from the fluorescent interference in biological or other sources.Carrier can be single-matrix structure or a plurality of particle (for example pearl).
Like this, the culture medium natural background in not only growing, and the fluorescent background can all be sifted out; Have powerful connections from the institute of incorporation of markings or label not simultaneously, the mark that also promptly can induce fluorescent also all sifts out via opaque matrix of microparticles.Sift out without what utilize, just unconjugated mark has meaning especially, because so sift out the correlationship that can significantly reduce detection method value and use up major part or whole fluorescent labellings before measurement." label " used herein or " mark " but can combine with anti-dentate and generate detector probe for arbitrary group, for example radioactivity reflection fluorescent labelling or chemiluminescent labeling.
When the pearl that is separated into patch is when separating by gravity techniques, preferably the shape of cell will make pearl be deposited in the cell bottom, and cell is generally the reactor that analyte generates therein and concentrates in the patch.In order to ensure reaching this purpose requirement, cell is not only more microscler along the tubular axis limit, simultaneously approximately slightly spherical perpendicular to tube axial direction or even slightly microscler; When the polymer pipe with size described herein and classification is generated this kind cell, the biological culture cell that remains to be tested can be with used perfluorocarbon fluid, better be about 25 lis of history holders, for example the name of stepping on D-25 with height is reduced to less than 1 microlitre from the volume of A Ximo group company (Meng Tefule subsidiary company) gained, and is for example little of about 0.5 microlitre or littler.
Have now found that and to use the perfluorocarbon liquids coat to come " stopping " to contain the pipe face of reaction small chamber.1%BSA solution also can be for the usefulness that stops.
" probe " typically is can for example fluorescent labelling combines or the anti-dentate of coupling with detection agent; Probe is suspended in the liquid at first, and probe will be bonded on the matrix after dentate to be detected is bonded on the matrix.It is because anti-dentate coexistence (this anti-dentate can be identical or different with the existing anti-dentate that has in the probe) is arranged that dentate combines with matrix.Certainly must understand anti-dentate and dentate can change mutually, the easness of antibody attraction antigen is as synantigen attraction antibody; Therefore in this case, use all these class terms all for comparatively speaking, summary, " anti-dentate " represented usually and is fixed on the matrix and is present in attractant in the probe; And dentate is to be present in the alternative material that is attracted on the anti-dentate in the liquid constituent.
Certainly also can generate and have known dentate and attach to probe on the mark, this kind probe is particularly useful in the competition assay.This routine middle probe with sample in the irrelevant quantity of competitive coordination base directly attach on the coordination matrix.Simultaneously also can in probe, be incorporated into a kind of matrix, this kind matrix is to generate via the matrix of using anti-dentate and handle through the dentate of mark, the sample that will remain to be tested then adds in the dentate of mark, and through the dentate of mark be with sample in the proportional quantity of dentate quantity and replaced.
Other surprising advantages of the present invention are conspicuous, for example, when carrier particle is assembled in the opaque patch, a kind of unusual effective baseline can be provided, even also can be detected from the light of the tagged back analysis produce of the thing by analysis thing of seeking especially on a small quantity, that is the compound of matrix, anti-dentate, dentate and probe with respect to this patch.
Another advantage of this kind trace routine need not for this material detects by the precision optics eyeglass or even optical material, for example glass or quartz: find really light with any suitable angle focus on select for use on the storage wall that detects very effective; But most convenient and the most satisfactory mode are fluorescents around measuring in many cases; Usually storage is a kind of unmeasured liquid " bubble ", because bubble will be along plastic cement pipe a series of storages and moving in the fluorocarbons pipe for example.
A speech is to be used for measuring using the tagged immunochemistry component of dyestuff " to be added with optical markings " among the present invention, because the result that labels is so can use suitable light source and detector means to detect.Fluorescent one emission dyestuff is the mark that very is fit to, and its operation technique is that technical field is well-known, but also can use other dyestuffs under some situation.The sensitivity of fluorescent detection technique and the preferred technique that is the most embodiment of invention with the common ability that exists of living cells.When using other marks for example labelled with radioisotope generates probe, can use the suitable detection width of cloth to penetrate method.Must understand and add that mark can utilize any reasonable manner and reaches, be included in pearl coated with before the anti-dentate with fluorescent substance blending (that is finally can attracted to a carrier pearl probe pearl) in second group of beaded glass or plastic cement pearl, generate complete probe thus.
Note also that fluorescent labelling can be attached on second pearl and generate the probe pearl that available then anti-dentate is handled and generated probe.
Can use in addition beaded glass together with this pearl jointly as stroma ground substance.Measure various different parameters and can use various pearls, for example on various pearls, use different anti-dentates and reach.
One of the present invention major advantage is to have biochemical to be detected to measure in the original place, that is in the production medium of material to be detected is arranged, measure; Therefore this material volume of using for test objective need not in the storage that carries biological entities to be detected (that is cell).Minimum as volume to be analyzed, that is this factor becomes very important in 0.25 to 10 microlitre scope the time.
Can be to carrying out in the small enclosed cell that contains a little volume biotic component thing with reference to the visible detection of the present invention of the 7th figure and determination techniques, though the cell in secreting is being grown and various antibody or other materials for example glycoprotein or glycolipid matter or carbohydrates belong to also like this by emiocytosis.In typical case, storage or reactor will be enclosed in that (being by No. 18 FEP(fluorine one ethene polymers) generates in the poly-fluorocarbons pipe, that is 0.042 inch of internal diameter, 0.012 inch of wall thickness).This kind pipe is by the New Jersey, and this industrial products company of the road of blue Te Shi is with this sale of trade name road, and its internal diameter is about about 0.01 to 0.02 inch of 0.03-0.04 inch and wall thickness.This piping is aseptic, and is nontoxic and can be not moistening by water institute, but is gas penetration potential.
A specific question is the special monoclonal antibody of early detection.Having suitable secretion blendes together oncocyte and also can distinct antibodies occur in the medium to be detected and differentiate out via having.Material described here is a kind ofly can detect antibody by means of homogeneous via blending together under the coexistence in the growth of secretory antibody and in the middle of the division but (that is in original place) and detect the homogeneous calibrating technology that whether has specific monoclonal antibody.
Very need to point out fast whether to have to secrete for example cell existence of monoclonal antibody.This kind mensuration can be undertaken by cell, and the volume of cell is little more favourable to measuring, and its reason is that the secretion sexual cell tends to secrete the antibody as at the larger volume moderate; Small-sized in addition or more spherical small-sized or more spherical cell (or shape, vertical elongated shape) has bigger relative surface area with respect to each cell volume and goes in the cell for gas exchange.
This kind size I must be adjusted the biological culture liquor capacity and be pumped into the perfluorocarbon fluid ratio in managing and reach in ground via little; Therefore find that the little and shape of cell size suitably not only helps to concentrate patch to be assessed is arranged, can promote that also the possible concentration of monoclonal antibody to be detected is arranged simultaneously in this cell (indicating whether to have the secretion sexual cell to exist) thereby promote testing process.Blend together the antibody that oncocyte produces and describe the present invention though below put up with the secretion of the performance monoclonal antibody of selecting especially to have the ability, need to understand the present invention and also can be used to detect and be used for the specific antibodies that the oncocyte process that blendes together of individual plant production generated in secretion and whether exist.
As this paper special this method of describing have remarkable ability and can detect in the described the type cell of embodiments disclosed herein existence less than 10 secretion sexual cell/10 microlitre culture mediums.Even the concentration of antibody is for 40ng/ml or lowlyer also can detect.
It not is unique mode of utilizing of the present invention that the person skilled in the art will understand from the particle patch that the width of cloth of measuring certain kind penetrates.The mark fluorescent or other mark quantity that remain in the liquid phase of particle source also can be measured pull conversely or be deposited in the interior material quantity in " patch " zone.In the method the width of cloth of mark penetrate characteristic also can be substantially different with background.
Also need understand simultaneously has first analyte to be detected can utilize magnetic force or gravity means and related with separated matrix phase; Another kind of have analyte to be detected then to be separated by different programs; So a kind of have the reliable magnetic force technical point of material to be detected to separate another kind then to separate by gravity; Perhaps there is material to be determined to be and is dispersed in the medium of being measured, for example utilize optical measurement to measure in conjunction with pattern; Another kind of material then hand separates with the gravity or the magnetic force industrial crops rational faculty; When mark be not fluorescent or when background (biogenic) fluorescent extremely low in the last kind technology particularly useful.
In a single day the personnel that are familiar with the known biological running program of this technology study this disclosure carefully and can carry out all these programs easily afterwards.
For simplicity, with reference to using single anti-dentate, that is detect specific monoclonal antibody dentate useful and use from the light of single fluorescence light source radiation and describe the present invention, can be applicable to the different anti-dentates of use and detect two or more antibody simultaneously yet the person skilled in the art will understand the present invention.Therefore each anti-dentate can be and carries the not probe of isolabeling, and for example different fluorescently-labeled probes utilize optical instrument one via different marks radiation or reflect different wave length and be distinguished from each other easily.
The substantial advantage that also need understand simultaneously the inventive method can have particle to be analyzed and reaches via removing from cell before analyzing; For example removing patch can carry out optical evaluation afterwards and need not washing step.
The promotion that concentrates
The valuable especially method of changing of the inventive method is greatly to help early detection to go out interested secretory cell, is to use " the concentrated effect " of cell itself and Bao Gu at any interested secretory product that encloses inside, phase matrix porous networking.Matrix can be by the opaque particle that is loaded with anti-dentate or pearl and is generated.The fundamental purpose of this kind matrix is the activity of restriction analysis thing and obtains Cmax, for example the analyte that analyte and cell produced be concentrated in lucky encircling cell around, herein with the analyte mark with multiple probe; So help to identify very apace specific secretion sexual cell or do not have this kind cell to exist.
Matrix can be void volume in the middle of implementing, and promptly the space in the matrix can be filled by liquid.Volume is healed little then better, and this is that the little pericellular secretory product concentration that is looped around just that is then obtained heals high because volume is healed.The voidage of patch must enough allow the infiltration of dentate and probe to pass through; But the most suitable level of factor of porosity will with the cell size, the analyte size, factors such as quantity and probe character are relevant.
Must stressed cell need not integral body and be connected on matrix inside, for example the thickness of the comparable matrix of its diameter is bigger, and best in such cases most of cell volume system position is in matrix inside.
Have now found that cell itself can with analyte response before be contained in matrix inside (for example the patch matrix that generates in advance, for example patch through applying wherein also can contain at pearl that anti-dentate is arranged and probe) via japanning or any method easily.When required antibody is when being come out by emiocytosis, antibody must attach on the matrix that applies through anti-dentate just, makes the fluorescence probe material thereon attached subsequently thus, therefore makes to be present near the cell the easiest the detecting of cell.Quite be confined to cell peripheral at the antibody that probe is arranged: often generate a kind of " ring of light " effect, this is that (really typically be the individual plant for dentate analyte one person) is attached near the cell the probe not via diluting in a large amount of culture sample to fall to slow down or hinder this method because the analyte secretory product; This shows solid-based know from experience restriction remain in the surveyed area amount of fluid and promote analyte (or simple cell) and product increases with respect to liquid in the system and the concentration that is generally culture medium.But this kind program also can be slowed down the activity of cell and secretory product thereof and be improved its degree of detection simultaneously.Can use this kind program to detect to produce the single cell of for example required individual plant secretory product; Note also that simultaneously the secretion sexual cell can constitute the part of patch but not add the part of the culture medium in the patch after the patch generation; Probe can still be included in the patch or probe can constitute and places a part that adds liquid constituent wherein after the patch in such cases.
Have now found that can reach one of the concentrated effect of this kind good way be guarantee cell group and through anti-dentate handle pearl for example magnetic bead contact closely.Comprise the pearl that is loaded with the back analysis thing and remain to be monitored a kind of substrate mixture of cell of its analyte production situation can be in all the other culture feed cells or the bottom of the little power valency of 96 1 grooves flat board etc. before be coated on anti-born of the same parents' device surface i.e. tube-type reactor inside: the pearl that is loaded with the back analysis thing in addition can be coated on the reactor surface before cell adds Zhu Ding.
Also cell can be layered on simultaneously on the groove surfaces of 96 1 grooving slabs certainly or directly be layered on above or below the porous bead patch, for example on clear and bright optical surface.
Another kind generates and utilizes the mode of cell/pearl patch is to place it on the film tablet.If fixed with required use gel coat; After having the cell compartment or the evaluation of cell process that to secrete required antibody, cut and remove this zone then as the part in the cell one results process.This tablet is to install especially easily, because tablet can make the zone that is loaded with required secretion sexual cell utilize punching to go out and be easy to reclaim.
All these programs all provide the patch that is loaded with analyte to be positioned at just near the transparent surface, utilize easily herein the strongest person of light for peripheral reflection light or on every side fluorescence monitored.Magnetic screen
Except using opaque pearl as the matrix, when and uncoated anti-dentate in also can use the agent of sieving as a setting of opaque pearl.
Be that anti-dentate is coated on residuite that is dull and stereotyped book film of little power valency or the film in such cases.
Decide according to the optical density (OD) of special test system, may need to move opaque patch to cellular layer or other non-opacity matrix patch rears after opaque patch reacts with probe, so the maskable detection system does not contact background radiation.The opaque patch of this kind can be used on every side in fluorescence or the peripheral reflection system especially, and is then utterly useless in the transmittance system; Need not in this kind reaction to make that the example that can generate opaque patch is coated with biochemical.
The opaque mask effect that must understanding can utilize magnetic bead to carry out easily can effectively be used " placing patch in advance " and reach, even also like this after analyte response.Primary culture adds that the used basic substance of the present invention is placed in the measuring vessel in such cases; Second group of strain can add in the culture that is covered in the patch top for the pearl magnetic bead in addition that is used in placement in advance or " japanning " patch easily.
Then with these second group of opaque pearl (for example need not to use magnetic force then to utilize gravity) pull via magnetic means or in suitable excessive power to paint patch or fixedly the matrix rear further shield the background light of being launched in its optical parameter mensuration process medium used in fluorescence or phosphorescence or any detection method.Thus, the program of finding above-mentioned research patient tumour cell is a kind of typical case, wherein can borrow magnetic bead to be shielded in the reaction rear backdrop; Certainly when all reactants all in the placement positioning, the opaque mask patch can be moved the reactant rear to before reaction, voidage through shielding patch thus and permeability can make and mark present whole patch and will appear at tightly on the light stimulation face of patch once again.
Can generate above matrix out of a clear sky and contain antigen (back analysis thing) layer, this matrix is for example for using opaque pearl to cover 96 1 grooving slab matrix of back analysis thing layer; And have only and on pearl, add at this moment and wherein contain the probe that adds anti-dentate mark by the culture that is loaded with cell.Anyly all can arrive the back analysis thing by the opaque mask pearl via the secreted analyte of cell.Probe can be before seeing through screen layer, among or attach on the analyte afterwards; Attention ought only use opaque pearl as preferably using its surface of resistance magnetic such as BSA in the shielding in order to avoid biochemical attaching on the pearl on demand not.
Except analyzing or using the cell surface analysis, also can use the present invention to come the interior function of analysis of cells; Therefore nobody can utilize for example existing fluorescence labeling of technos of cell inner mark.The used mark substance of mark if not enter in the cellular environment then can't be detected like this, and the mark-probe that is generated can quantitative and qualitative indication specific cells function then; Existing this class mark comprises enzyme labeling, to mark and pH-class mark.Multiple mark can obtain from heavily good diagnostic companies of Kao Baikai immune chemical company, hundred and branch office of Hoechst enterprise.
Equally, cell can generate the dyestuff of dna marker thing at the cell interior mark to react with DNA, for example Yi Siliu bromide (Kao Baikai immune chemical company) or derive from Binzhou, the DAPI of Washington city Li Saisi company.
Among one of the present invention embodiment, little power valency groove can be coated with a series of materials, comprise just be coated with under the culture medium-culture medium of analysis of experiments thing that the layer that is loaded with anti-dentate is adjacent to the cell lower wall and this lower wall can pearl or some other matrix be substrate.Jie is at these two layers of intermediate fixed pole glue-line that is one deck printing opacity and utmost point book agaropectin for example.
Certainly the surface need not to be little power valency groove.One of the present invention important feature adopts the susceptibility of fluorescence on every side for the present invention.
The relevant cell of placing in advance in the patch among some embodiment of the present invention, a group cell can be made the usefulness of matrix, for example the usefulness of analyte matrix.Can for example on tossing about, the distance fluorescence light source generate opaque sieve at the patch rear in this kind purposes; For example need in this kind purposes can use the patch tumour cell to mark it is elaborated the matrix that is only second to the transparent vessel surface, and can detect through surface thus with being attached to one of tumor cell surface group monoclonal antibody when patch sieves out.In fact the kinds of tumor cells sample all can add in the different cells, for example in the groove of the little power valency of 96 1 grooves flat board.
Add different monoclonal antibodies, known antigenic action of each antibody then in each groove corresponding to different cell surfaces.If monoclonal antibody combines with patient's tumour cell and then can make probe (for example fluorescence anti-antibody) combine with the cell face so again.Can measure the fluorescence of planting cell association therewith then, fluorescence is measured material as described later around preferably utilizing, and light can be only limited to the cellular matrix patch via limiting the depth of focus or via shielding particle one these particle pulls being generated opaque sieve to the cell rear.
With regard to this purposes, describe a kind of preferred embodiment of the present invention, and propose its various variation examples and modification, be not to enumerate and can make within the scope of the present invention other to change example and modification but need to understand these.Select these suggestion contents and be included in and only supply to illustrate purpose in this paper, the personnel that are familiar with this technical field can be easier to understand the present invention and principle thereof thereby can be in a variety of forms with its modification and concrete manifestation it, make indivedual forms condition of suitable various special cases.
Fig. 1 is a synoptic diagram, shows that containing the liquid bio nutrient solution that blendes together the oncocyte constituent is isolated in the plastic cement pipe cell, as shown in Figure 8 the best.
Fig. 2 example is the situation of explanation identical culture shown in Figure 1 after showing antibody via this kind cell.
Fig. 3 represent same culture antibody and fluorescence probe the two all substantially with situation after magnetic bead combines.
Fig. 4,5 and 6 show that magnetic material is drawn in the opaque patch of cell sidepiece.
Fig. 7 is the synoptic diagram of light source collection with measuring system.
Fig. 8 illustrates in a schematic way can be for the typical tubular operating means of handling a series of biological chemistry constituents.
Fig. 9 is for can the most advantageously carrying out the synoptic diagram of the preferable little chamber shape of the inventive method.
The following example illustrates together with Fig. 1 to 6 can be for detecting in forming culture medium whether have the original place homogeneous determination method that blendes together the secreted antibody of oncocyte.The tubular configuration of reaction small chamber and letter nurse this people such as grade be at United States Patent (USP) 3,491, and 141 is used identical.Can suitably sterilize to pipe and perfluocarbon carrier liquid.
Embodiment 1
But this assay validation detected magnitude surpasses 100,000 dalton's antibody.
As shown in Figure 1, use the polystyrene magnetic bead 20(silicon Luo Jun company supply of 1.75 microns of item diameters) via absorb goat anti--mouse (GAM) Ig G antibody (deriving from the heavy chain and the light chain type of Sai Mide company) and coating in addition.Use bovine serum albumin(BSA) (BSA) hinders any further response location above the magnetic bead.Magnetic bead 20 can be used as probe matrix.This matrix will combine with pearl and finally be drawn in the opaque patch.This pearl is scattered in the reactor that is limited to by pipe, that is cell 22 wherein contains tissue culturing medium 24: also be dispersed with so-called mark chemicals or probe 26 in the cell 22, this be the tagged goat of FITC anti--mouse multi-strain antibody (also being to obtain from Sai Mide company) and some oncocyte 28(55.2 that blendes together that selects in advance blend together oncocyte and can produce Ig G 2a).Cell uses hanks' balanced salt solution to wash 4 times approximately and removes any free antibodies before using.Other cells contain non-secretory 653 murine myeloma cells as negative control or contain Ig G 1a, mouse myeloma protein (ICN company product) is as the positive antibody contrast.
Fig. 2 point out the emerging widow of a kind of new nose pavilion an ancient drinking vessel  wash with watercolours climb to the tired in the anti-Pu of Lai solid 0 at least from some cells 28 that is from the secretion sexual cell secretion come out.
Magnetic bead 20 utilize its goat anti--mouse antibodies can be described as the MB/GAM coating and the antibody 30(that is attached to secretion recently can be described as Ab) on, as the tagged goat of " probe " FITC anti--mouse multi-strain antibody 26(can be described as GAM): the gained compound is as shown in Figure 3, for have as shown in the formula chemical composition thing 32, use above-mentioned each term in the formula:
MB/GAM-Ab-FITC/GAM
Mouse antibodies is dies probe-antibody 26 because its top has, thereby only links with strain.
By Fig. 4 to 6 as can be seen, the 7 kilogauss magnet of about 1 * 1.4 * 7cm of magnet 40(size) the material pull that is used for being loaded with magnetic particle on the small size, therefore obtains opaque this kind group of hill substantially around the cell 22 in the being seen patch 42 of Fig. 6.
Known as technos, in response to matching through suitable, thorn laser rays institute emitted fluorescence quantity must have direct relation with the secreted antibody quantity of cell 28, and this antibody attaches to MB/GAM with above the FITC/GAM and generation MB/GAM-Ab-FITC/GAM material; Certainly have the required magnetisable material that does not attach to antibody as yet and also can be contained in the magnetic patch 42, but can't facilitate any fluorescence.
In the typical mark, being used for the fluorescence of measurement markers product quantity is to produce and measure by comprising, so that the fluorescent microscope of the fluorescence light source device of quantitative measurment fluorescence, this device that is photomultiplier (PMT) and suitable readout device.Microscope is typically provided with the aperture so that hold receptacle, and for example PMT etc. is as described below.Must understand this example and be the relevant FITC of use, and other light sources also can optimum strange land be used in other fluorescence labelings with different the most suitable stimulating frequencies.
Other optical systems also can be used to check whether have probe in the path, for instance, use Ar Laser 46 to produce a branch of smooth 66(wavelength and be 488nm), this kind light is removed insignificant light by filter 68, that is the irrelevant light of being launched by this class laser usually; The light beam expansion that generated this moment and the combination 70 of collimating optics lens, and set up particular beam in patch 72, for example about 10 microns of its diameter is typically by about 100 microns of the collected mean diameter of magnet 84.The specific big I of light beam in the patch has the intensity (watt/cm that can avoid the excessive reversibility of fluorescence probe " bleaching " (forfeiture fluorescent emission ability) through selecting 2).
Light beam is opened from 76 reflections of dichroscope beam splitter with miter angle, makes light beam 74 focus on by object lens 75 then.The selection of beam splitter 76 makes it can reflect greater than the 90% Ar Laser light velocity 66 transmission less than 10% light beam; The beam splitter transmissive is greater than 90% fluorescence spectrum (centre bit is at about 518nm) in addition, and this kind spectrum is to launch from patch in response to fluorescence thorn laser beam, but reflection is lower than 10% this kind wavelength.The fluorescence 78 that this kind launched from patch is come by object lens 75 transmissions, and object lens make light become parallel then by beam splitter 76.Light beam 78 is loaded with the reflex irritation light beam 74 of substantial amount, therefore can filter by obstacle filter 80 before by another eyeglass 81, and eyeglass 81 can be with the collected light focusing that gets on detecting device 82, and detecting device is silicon photoelectricity diode easily.The function that the photoelectricity diode is output as bunchy 78 amount of light is exaggerated into output signal.
The personnel that are familiar with proper technology will select suitable hardware easily after studying above-described invention carefully, that is eyeglass, filter, mirror, amplifying circuit and signal output apparatus.
Pay special attention to may since the background fluorescence of unreacted fluorescence probe and tissue culture itself via the complete conductively-closed of opaque characteristic of magnetic patch thereby can not cause any materially affect to the light that reflects from patch 42.The device that makes light be incident in the patch is included in no counting method known in the optical technical field, comprises that only non-being limited to wherein use fibre optical device that light is bridged in the patch, and will bridge to the analyst from the light of patch.
Fig. 8 is typical case's synoptic diagram of running program successively, generally, adopts a kind of scheme, the polyfluortetraethylene pipe 52 that the cell 22 of about 10 microlitres of the volume of wherein analyzing is separated through bubbles 50 and moving.Liquid substance (the perfluorocarbon liquids constituent is sold by 3M company with the name of fluorine Rui Neite FC-77) can provide the means that help separate and help reactor to do lubricated progressive moving along reaction tube 52 inwalls simultaneously.Pipe 52 constitutes gas and the CO that allows enough nutrition 2Device is therebetween passed through in infiltration.The gas of position between cell or air bubble can constitute and transport gas by fluorine Rui Neite supporting agent 60 additional surface mutually.This supporting agent around bubble with hold bio-culture solution cell the two.Its clinical procedure is as follows:
Through applying and add 96 1 grooves that contain secretion property or non-secretory cell through the magnetic bead that hinders, in tissue training base is dull and stereotyped-or contain Ig G at experiment contrast example middle plateform, or nutrient medium (or " tissue culture " medium).
All cells all contain the magnetic bead through processing that typical concentration is about 1.12 * 10 pearls/milliliter.The concentration of pearl can reach lower.Probe adds wherein with the concentration of 375ng FITC/GAM/ml.4 to 250 blend together oncocyte or the non-secretory myeloma cell is positioned in the appropriate reaction cell at first; The Ig G that ICN company is supplied 2a, mouse myeloma protein also combines with fluorescence probe with magnetic bead and as positive control, is positioned in other reactors with the concentration of 37.5ng/ml.
Pipe internal coat a small amount of carrier fluid, that is fluorine Rui Neite FC-77, this kind constituent can be through sterilization, wettable tube wall and be gas permeable, and must have suitable transparency and carry out optical assay.Reaction mixture uses bifurcated pipe and syringe pumping system and is pumped in the not imperial pipe of iron from different grooves, thus generate reactor cell in pipe.Capacity FC-77 also is pumped in the pipe so that be looped around the biological culture cell, that is around reaction mixture the dyspoiesis layer.With known in the air inlet tube, so that separate the cell of adjacency as this technical field.To manage interior cell then and be positioned in 37 ℃ the cultivating container, and be in 5%CO 2Experience is 24 hours in-95% air ambient, and be pumped into magnetic bead in the opaque patch and measure patch fluorescence this moment.
From containing secretion sexual cell or Ig G 2a, all reactors of groove fluorescence all is positive.Cell with non-secretory cell or medium is then to the fluorescence reaction that is negative.
The foregoing description uses and the irrelevant cell of matrix, and the cell relevant with matrix for example liver cell and fibroblast also can be arranged.Must at first attach to (sale of Niu Bulangwei company) on the known suitable miniature base of this technical field.
Embodiment 2
Use the quite high particle of transparency, that is beaded glass replaces magnetic bead and repeats the step of embodiment 1.Beaded glass utilizes gravity separation to go in the patch.When the enough thin and transmissive light of patch, must avoid collected light fully from patch.Excitation beam can become certain angle with the emission light beam and introduce a fine variety the result so that link.
Embodiment 3
Further understanding can utilize different probe and detect two or more characteristics of single dentate; Illustrate thus and can wish to obtain that a kind of analyte has specificity to a kind of anti-dentate but another kind is not had a specificity.Mark even can be for example two kinds of different fluorescence labelings of same big class for instance, can use a kind of probe to detect the fluorescence spectrum of launching from the specific anti-dentate of dentate, and use another probe to measure the classification and the same type of dentate.
Thus secreting of people's protoheme (α chain but not β chain) being blended together knurl antibody (Ig G) can use following manner to select.GAMY-I is as the coat on the magnetic bead matrix.Probe 1 is for to be marked with fluorescence labeling No. 1 that is people's protoheme α chain of FITC.Probe 2 is for to be marked with fluorescence labeling No. 2 that is people's protoheme β chain of Texas redness; Then can in cell, identify and to secrete Ig G 1Blend together oncocyte (people's protoheme α chain specific antibody), but patch is No. 1, mark but unmarked No. 2 one and also may have the cell of a group secretory antibody but have the specificity of No. 1, probe but not No. 2, probe in this cell.Vice versa for No. 2 specific cells of antibody tool probe.The cell that can detect two kinds of marks in patch is then represented the 3rd example, that is indivedual two kinds of probes are all had specific cell mixture or all have specific antibody for two kinds of probes.In order to distinguish this kind possibility, in the 3rd example, cell in cell is grown arbitrary probe that (that is cell launches, thereby each cell only has a cell) use two probes again and is examined each cell again and only measure a probe is had specific cell in different cells according to the present invention in same system.
Fig. 9 is a synoptic diagram, shows that this kind has the subcircular cell of being separated by bubble 92 in the plastic cement pipe 93 of perfluorocarbon liquids 94 91 and help cell and bubble to separate, and helps moving of bubble that cell is moved along pipe 93.
Can be observed the typical scenario that concentrates effect is:
Step 1: with 2ml L45-50 2Oil adds round bottom Tai Lasa Supreme Being flat board 3Groove is interior and use asptic technique to add the following reactant solution of 3.5-4.0 μ l.
Medium-or contain 20%FCS or Pu Timo difficult to understand for RPMI1640 4Contain 20%FCS.
Goat anti-mouse coat latex magnetic bead 50.7mm diameter 1.95g/ml density or 1.7mm diameter, 1.07g/ml density, concentration are 0010%w/v to 0.003%w/v.
Goat anti-mouse FITC 6-concentration is 0.375mg/ml to 1.5mg/ml.
Step 2: generation beads (3 to 24 hours) back adds the mouse that includes RPMI1640+20%FCS or Pu Timo+20%FCS difficult to understand and blendes together oncocyte solution, and blending together oncocyte concentration is 25 cells/groove.
Added behind the cell about 3 to 24 hours, the visible fluorescence light source blendes together oncocyte around mouse, cell itself then be surrounded with GLM BM.Can then can make hithermost pearl edge become fluorescence Hena at the cell of GLM BM granule inside, add behind the cell 3 to 24 hours can be in GLM BM granule inside with and see fluorescence device " focus " on every side; Adding behind the cell 24 to 48 hours " focus " in addition merges and makes whole granule all produce fluorescence.
1Mouse inner at GLM BM granule or on every side blend together the secreted mouse antibodies of oncocyte in conjunction with and free GLM FITC combine with mouse antibodies and produce fluorescence.
2L45-50 oil one is united the silicon ketone oil that calcium carbide company makes forever fully.
3Safe Lhasa Supreme Being flat board-Luo Bin chemical company makes.
4Ao Putimo-jeep button corporate system
5Lactic acid magnetic bead-Sai Luoding company makes.
6Goat and mouse FITC-Bai Midiou company make.
7Via Ou Lin Paasche IMT2 fluorescence microscope fluorescence.
Need understand also that following claim scope is intended that all summarys of the present invention described herein and specificity speciality and to all statements of the scope of the invention wherein done.

Claims (52)

1, a kind ofly whether have the homogenizing method of mark with the first anti-dentate one dentate product of detectable label for detecting, this product is the speciality that generates on the matrix device in the liquid constituent in being contained in cell; This method comprises the following steps:
(a) provide be coated with the second anti-dentate carrier particle as this mark, with the matrix device of the first anti-dentate one dentate product of detectable label;
(b) collect a part the liquid in this cell at least, be present in the mark of carrier particle top, with a certain dentate product of the anti-coordination of detectable label in patch; And
(c) measure existing mark in this patch, with the product quantity of detectable label.
2, the method for claim 1 is characterized in that when patch still is in the cell, measure this patch kind with this carrier barrier from the optics background interference of at least one part constituent in this cell in order to avoid disturb the patch of being assessed.
3, method as claimed in claim 2, it is characterized in that said assessment system utilize opaque carrier as shielding from the optics background interference of the constituent in the cell device with the obstruction free surveyed area.
4, the method for claim 1, it is characterized in that said measurement through detectable label product quantity is: after fluid composition does not contain the contained anti-dentate-dentate product of matrix substantially through collecting step, measure in the liquid constituent the inverse function of residual number of probes.
5, the method for claim 1, the anti-dentate-dentate that it is characterized in that said mark are the nucleotide one belly nucleotide products through mark.
6, the method for claim 1 is characterized in that using multiple different detectable label to generate different probe, and each probe has the anti-dentate that can represent this dentate different qualities.
7, the method for claim 1 it is characterized in that said particle is a magnetisable material, and this patch is opaque material.
8, the method for claim 1 is characterized in that said assessment is to adopt the mark that can carry out optical detection.
9, the method for claim 1 is characterized in that said patch is opaque material.
10, the method for claim 1 is characterized in that comprising the probe pearl that wherein contains fluorescent signal constituent through the anti-dentate-dentate product of detectable label.
The elaborate ɡ  of 11 ⑷ , 2,3,4,5,6 or 7 described methods is characterized in that said method is to carry out under the living cells coexistence.
12, method as claimed in claim 9 is characterized in that said magnetic particle utilized magnetic field to be collected in the opaque patch before assessment.
13, the method for claim 1 is characterized in that collecting step and utilizes gravity or centrifugal force and carry out.
14,, it is characterized in that this collection carries out along the translucent area of little locular wall as claim 2,3,4,5 or 6 described methods.
15,, it is characterized in that being marked with a kind of mark of launching fluorescent at least through the anti-dentate-dentate product of optical markings as claim 1,2,3,4,5 or 9 described methods.
16, as claim 1,2,3,4,5 or 9 described methods, it is further characterized in that it comprises the following steps: that also (a) disperses to make having of the patch anti-dentate-dentate material through the matrix place of mark to be detected again, and (b) subsequently, collect again and assess this material once again so that carry out another time optical evaluation by optical mode.
17,, it is characterized in that saidly having dentate to be detected-anti-dentate compound to comprise monoclonal antibody as the described method of claim 1 to 10.
18, method as claimed in claim 12, it is characterized in that saidly having dentate to be detected-anti-dentate compound to comprise monoclonal antibody, wherein but the cell concentration of secretory antibody is lower than 10 cells/10 microlitre culture mediums in this cell, and wherein the concentration of monoclonal antibody is lower than 40ng/ml.
19, the method for claim 1, the first anti-dentate and the second anti-dentate that it is characterized in that being stated from the matrix are monoclonal antibody.
20, the method for claim 1, the first anti-dentate and second dentate that it is characterized in that being stated from the matrix are many strains dentate.
21, the method for claim 1 is characterized in that the said first anti-dentate is many strains or individual plant, if the first anti-dentate be individual plant then the second anti-dentate be many strains, but to be transported to the position be individual plant for the second anti-dentate then if open one.
22, the method for claim 1 is characterized in that the size of said cell is about 1 microlitre or following, and almost can't prolong in the horizontal direction.
23, a kind of employing comprise known dentate and mark the control pin and quantitatively or the test specimen competitive assays of the known dentate quantity of qualitative determination, in this determination method
(1) probe is attached on the matrix that is loaded with anti-dentate via known dentate component, and its quantitative value is relevant with the dentate competition quantity in being present in test specimen at first;
(2) be collected in the probe that has been attached in the patch on the matrix that is loaded with anti-dentate; And
(3) detecting the probe that attaches on the anti-dentate matrix is present in the competitive dentate quantity in the test specimen at first and whether has competitive dentate as reverse mensuration.
24, a kind of utilization contains the determination method through label probe of known dentate and mark, in this determination method
(1) this probe constitutes the matrix of using anti-dentate and handling through the dentate of mark;
(2) will remain to be tested the sample that whether has dentate adds in this probe;
(3) will be through the mark dentate from the matrix displacement that is loaded with probe and its quantity is proportional with the dentate quantity that remains the test specimen;
(4) receiving the subtle punishment straw of   merchant's tool sodium waterside the sound of sth. throbbing or bursting Yun curtain  reaches
(5) detect whether the probe that attaches on the anti-dentate exists as dentate or the reverse mensuration of its quantity.
25, detect the device of using through the chemicals of optical markings, comprising
(a) at least one has the cell of transparent wall part;
(b) chemicals can be concentrated in the device of using in the patch in the cell;
(c) transparent wall of cell formation can be observed the device that patch is used; And
(d) but the device that the Radiation Emission character of this zonule part of optical evaluation or light reflectance properties are used.
26, device as claimed in claim 25, the device that it is characterized in that said concentrating chemical thing comprises with magnet as the device that attracts the magnetic grain, after in a single day these particles are attracted, promptly can generate the not device used of contact optical apparatus for evaluating of shielding background radiation, and as can generating the device that this matrix that chemicals to be detected are arranged is used, and as being loaded with the device that the fluorescent emission mark is used.
27, device as claimed in claim 25, it is characterized in that said cell is not prolonging the axle prolongation of cell substantially, the cell axle is vertical with the light path of pick-up unit on impinging upon cell, and wherein uses gravity patch is concentrated near the transparent wall that is pressed with the device for the treatment of the chemistry assessment.
28, the method for claim 1 is characterized in that saidly having dentate to be detected to surpass 5,000 dalton.
29, the method for claim 1 is characterized in that saidly having dentate to be detected to surpass 10,000 dalton.
30, the method for claim 1 is characterized in that said patch is the material of non-opacity.
31, the method for the analyte in a kind of analytical biochemistry environment, this method comprises following each step:
(a) anti-dentate is fixed on the matrix;
(b) make anti-dentate contact, and contain the dentate through mark of this fixing anti-dentate in the culture medium with culture medium;
(c) generate a kind of compound substance that is associated with dentate and anti-dentate, this material comprises that dentate and anti-dentate are incorporated on the matrix; And
(d) make with light appraisal agency assessment and the character of compound substance is carried out optical evaluation from this material reflection or emission light property.
32, method as claimed in claim 31 is characterized in that said matrix is pearl, and this method comprises the step that some pearls is shaped to opaque little patch, and the step of carrying out optical evaluation on the fraction of patch; This part is culture medium and towards the light apparatus for evaluating dorsad.
33, a kind of helping, carried out the method that homogeneous detects the secretion sexual cell in culture medium, and this method comprises:
(a) make dielectric sample in the cell that is less than 10 microlitres;
(b) with one period that makes that enough cell is grown again of this sample culturing; And
(c) secretory product of detection cell indicates whether to have this secretion sexual cell in each cell.
34, method as claimed in claim 31 is characterized in that said cell content is less than 1 microlitre.
35, method as claimed in claim 32 is characterized in that said cell content is less than 1 microlitre.
36, method as claimed in claim 33 is characterized in that said cell content is less than 1 microlitre.
37, method as claimed in claim 33 is characterized in that said secretory product has size and surpasses 100,000 daltonian antibody.
38, method as claimed in claim 34 is characterized in that said secretory product has size and surpasses 100,000 daltonian antibody.
39, a kind of method that detects the back analysis thing-analyte complex in the culture medium, this compound comprises cell at least and engages the probe that is generated via the back analysis thing with detectable label, this cell carries out the tester via the secretion of using the probe mark analyte, and this method comprises following each step
(a) a part of cell is positioned in the porous matrix and reduces culture medium near the quantity of cell and the analyte secretion of cell, this matrix is made of material that can not blending, and can be permeated and be permeated by the analyte through mark that is loaded with in the medium by culture medium;
(b) similarly, reduce the activity through the labelled analyte probe via using porous matrix, this probe is accommodated in the basal body structure, and is positioned near the cell, can make thus through labelled analyte probe environment at cell peripheral; And
(c) measure near be positioned at just the cell the standard of whether secreting this analyte as cell through the detected output of detectable label probe area.
40, method as claimed in claim 39 is characterized in that said clone is in abutting connection with carrying out the superficial cell that fluorescent detects.
41, method as claimed in claim 39 is characterized in that said target area is to be limited to the zone of environment around the secretion sexual cell.
42, method as claimed in claim 39 is characterized in that the fluorescent on every side that said detection is measured from the target area carries out.
43, a kind ofly detect the method that back analysis thing-analyte complex engages with fluorescent labelling in the culture medium, wherein this probe at first with cell is positioned in the medium jointly and experience can contact with analyte time:
Make the generating along the transparent surface of container of probe one analyte, at this moment in conjunction with product
From medium, utilize the some magnetic beads of magnetic attraction and generating opaque mask in conjunction with the position between product and culture medium, so that the background fluorescent in the shielding medium, and then
Measurement as the combining of measuring probe and analyte, still is maintained at described position and shield simultaneously from fluorescent around the target area.
44, method as claimed in claim 43 is characterized in that the combination in cell of said probe and analyte.
45, method as claimed in claim 44 is characterized in that said probe and analyte are in the extracellular combination.
46, a kind of analyze launch from the target area that comprises fluorescent-mark-anti-dentate binding element around the method for fluorescent, this method via the goal in research zone launched fluorescent is for the reaction of source stimulating on every side, this method comprises following each step
(1) bottom of transparent panel applies and generates tagged dentate one anti-aglucon for example observing on the surface;
(2) at the draw opaque sieve of magnetic grain of tossing about of target area and light source; And
(3) opaque sieve the most away from this side of light source on to any background fluorescent of target area elimination.
47, a kind of method that detects tagged back analysis thing-analyte complex wherein from (a) back analysis thing of culture medium or (b) at least one in the cell, is fixed in just near the coating the transparent surface, and this method comprises following each step again
(1) analyte that makes back analysis thing and this cell be produced reacts; And
(2) by this transparent surface via emission analysis on every side and the anti-product of analyte-back analysis thing.
48, method as claimed in claim 47, it is characterized in that said analysis by around fluorescent and carrying out.
49, method as claimed in claim 47 is characterized in that said patch is opaque material.
50, method as claimed in claim 48 is characterized in that said method comprises the step of pulling out opaque magnetic patch, so that as the sieve that resists the background fluorescent in face of the analytical procedure.
51, the method for claim 1 it is characterized in that the said first anti-dentate is many strains, and this second anti-dentate is an individual plant.
52, the method for claim 1 it is characterized in that the said first anti-dentate is an individual plant, and second dentate is many strains.
CN88107039A 1987-10-09 1988-10-03 Identify the method and the device of biochemical species Pending CN1034617A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
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CN104395736A (en) * 2012-06-29 2015-03-04 皇家飞利浦有限公司 Processing of bound and unbound magnetic particles
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JPH03501884A (en) 1991-04-25
AU2790889A (en) 1989-05-02
EP0397659A4 (en) 1991-01-30
WO1989003533A1 (en) 1989-04-20
EP0397659A1 (en) 1990-11-22

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