CN105004867A - Method utilizing separate biological reagent lattices to detect biomolecules - Google Patents
Method utilizing separate biological reagent lattices to detect biomolecules Download PDFInfo
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
Abstract
The invention discloses a method utilizing separate biological reagent lattices to detect biomolecules, belongs to the field of biological detection, and solves the problems that cross-talk, poor method specificity, and the like exist in the prior art. According to the invention, capture type antibody lattices and separate antibody lattices are together used for the detection of proteins, so that various proteins, especially proteins expressed by histocyte can be detected simultaneously; as the detection antibodies are fixed on the separate antibody lattice slices, rather than the manner that free detection antibodies in the solution are used in the common double antibody sandwich method, so that the use quantity of antibodies is reduced, cross-talk is avoided; besides, the two antibodies are used for identifying one antigen, so that the detection specificity is higher.
Description
Technical field
The invention belongs to field of biological detection, more particularly, relate to a kind of method utilizing separate type biological reagent dot matrix to detect biomolecule.
Background technology
The appearance of large number of biological reagent, such as thousands of bar DNA clone sequence, numerous antibody and recombinant protein and millions of, by the molecule of chemosynthesis, facilitate the application of these reagent in biological study, clinical diagnosis and drug development etc.Biological reagent dot matrix techniques recently develops one of application technology faster.In biological reagent dot matrix, each reagent to be placed on preassigned position in case afterwards by this position to identify it.Such as, in a DNA dot matrix, (be also referred to as genetic chip), multiple cDNA or oligonucleotides are fixed on carrier, and each is placed in a preassigned position.DNA dot matrix is used in cross experiment, monitors expression (Schena et al., Science, 1995, the 270:467-470 of lots of genes; DeRisi et al., Nature Genetics, 1996,14:457-460).
In a Protein microdot arrays (or protein chip), multiple protein is fixed on a support, and each is placed on a preassigned position.There is the Protein microdot arrays of two types the most conventional: antibody dot matrix and recombinant protein dot matrix, they comprise antibody different in a large number and recombinant protein respectively.Because antibody dot matrix can with the protein combination of cells, they detect albumen activity in vivo in extremely useful.Antibody dot matrix has been used in study and has modified and protein expression (U.S. Patent Application No. is US19980126483, and publication number is US 6197599B1) after protein-protein interaction, protein translation in vivo.In addition, the lattice energy of cell, tissue, lipid molecule, polypeptide, medicine or other chemical substances composition is enough carries out (Kononen in large-scale selective mechanisms test in medical diagnosis, drug development, molecular biology, immunity and toxicology etc., et al., Nature medicine, 1998,4:844-847).
Protein is the important composition composition of cell, plays a role in cellular activity process.Protein expression kind in cell and quantity determine its shape and function, and abnormal protein expression can cause disease.A main task of biomedical research is exactly detect the expression pattern of albumen in biological sample.Due to posttranslational modification, there is the protein of specific amino acids primary sequence may occur in different forms in cell, in many cases, only have the albumen of some special shape to participate in certain specific activities of cell directly, valuable information can be provided to the activity and function understanding cell so detect at the albumen of these special shapes of cells.Albumen has various posttranslational modification, as phosphorylation, glycosylation and general elder brotherization.The phosphorylation of serine, methionine or histidine residues is an important mechanism on intracellular signaling, and abnormal protein phosphorylation result in many human diseasess.In the method detecting protein phosphorylation, the method often used detects with radioactive isotope metabolic marker cell with antibody mediated immunity, anti-phosphorylated residue specific antibody, such as PY20 and 4G10, also through being commonly used to detect phosphorylated protein.
Detect protein expression and have application in multiple field, comprise biomedical research, medical diagnosis on disease, searching treatment index and drug target and reflect in analysis at toxicity and drug effect.In basic biomedical research, often wish to know which albumen is expressed under special cell or specific condition, by the protein expression of more dissimilar cell, just may differentiate the expression of which albumen and the active character determining a special cells.In many intracellular signaling passages, some albumen are activated specifically, detect these by shock protein, such as phosphorylated protein, can provide crucial information for the principle understanding intracellular signaling passage.Numerous disease can change the expression pattern of albumen, and protein expression abnormal in many cases can be diseases induced.Therefore, the expression pattern of albumen is determined and to contrast normal and paracytic protein expression pattern for understanding the mechanism of disease be useful.The expression detecting some albumen is also widely used in clinical diagnosis.A lot of clinical testing procedure all based on the protein markers detected in sample, as the antibody produced in the antigen of virus and health.In addition, detect protein expression at course of drug development, the such as selection of medicine target application point, the index of toxicology and searching drug response is very useful.
Detection method of protein is a lot, comprises the immunization method based on Ag-Ab specific binding and other nonimmune method.In numerous nonimmune method, mass spectrum is a relatively more conventional method.This technology is by protein molecule is converted into gaseous ion, then utilizes electric field, magnetic field is separated having the protein Ion of extra fine quality with charge ratios (M/Z), collects, determines the M/Z value of ion, carry out Analysis and Identification protein.Mass-spectrometric technique can be used for identifying albumen but be unsuitable for quantitatively detecting expression and some other characteristics of comparison protein, is particularly unsuitable for studying cell inner expression high complexity and the albumen of height mobilism.The immunological method of protein detection also has a lot, has respective principle of work, feature and relative merits.The method that research field is commonly used has enzyme linked immunoassay (ELISA), protein immunoblot technology (Western blotting), immunochemistry dyeing etc.Percolation (flow through), sidestream immune chromatography (lateral flow immunoassay) etc. is also often used in disease detection.
The extensive protein that detects that appears as of protein array technology provides an effective method.According to the using forestland of Protein microdot arrays, two classes can be divided into: capture type Protein microdot arrays and separate type Protein microdot arrays.Briefly, capture type Protein microdot arrays is used for the biomolecule in biological sample to capture on dot matrix support.And separate type Protein microdot arrays, the position of albumen on dot matrix support can be maintained on the one hand; On the other hand, when after being fixed on the biomolecule on another support and being combined, albumen can leave dot matrix support and transfer on biomolecule support, and keep original relative position (Wang of each albumen, Immunostaining with dissoiable antibody microarrays.Proteomics, 2004,4,20-26).
Antibody dot matrix is method with fastest developing speed in Protein microdot arrays technology, technically increasingly mature.Antibody dot matrix can be used for analyzing the difference of albumen between sample, the character determining respective egg white matter, analysis of cells extract or serum proteins potpourri.The conventional method using capture type antibody dot matrix is hatched by the protein sample in antibody dot matrix and solution, makes the antibody on dot matrix and the albumen in sample (antigen) react, combine, thus they are captured on dot matrix; Afterwards, captured albumen is detected further.The method detecting captured albumen in capture type Protein microdot arrays technology has: labelling method (tag label-basedapproach) and label-free (label-free approach).In labelling method, first protein sample uses label (biotin, digoxin an etc.) mark, and then hatch combination with protein chip, the albumen be combined on dot matrix shows by marking.In the most frequently used Bicolor-code method (two-color detection method), two protein samples use the label (as Cy3 and Cy5) of different colours to mark respectively, are combined after two sample mix with an antibody dot matrix.Label-free is similar to euzymelinked immunosorbent assay (ELISA) (ELISA), in this so-called ' sandwich ' method (Multiplex sandwich assay), after not having markd protein sample to be combined with Protein microdot arrays, detect with the potpourri containing Multiple Antibodies the albumen be combined on dot matrix.
Separate type antibody dot matrix is a kind of novel, special anti-dot matrix, and it can maintain the position of antibody on dot matrix support on the one hand; On the other hand, when antibody is after being fixed on the antigen molecule on another support and being combined, dot matrix support can be left and be combined in (Wang on antigen support, Immunostaining with dissociable antibody microarrays.Proteomics, 2004,4,20-26).An important application of separate type antibody dot matrix is used to the albumen detecting biological sample.Concrete grammar is, when being fixed on the antibody dot matrix on first support and contacting with the protein sample be fixed on second support, antibody combines with the corresponding antigen in protein sample, after first support is separated with second support, under proper condition, the antibody combined with antigen will be separated from first support and transfer on second support, and the quantity being transferred to antigen in the quantity of the antibody on second support and protein sample is proportional.Therefore the quantity of antigen in protein sample can be learnt by the quantity of antibody on detection second support.Separate type antibody dot matrix has been used for carrying out immunostaining to cell sample, detect the expression in cell of multiple proteins molecule and Subcellular Localization figure, analyze the difference of these protein molecules in different cell sample, but also do not have about the research report of separate type antibody dot matrix and biomolecule dot matrix or biological reagent dot matrix conbined usage at present.
Summary of the invention
1. the problem that will solve
There is the problems such as cross reaction (cross-talk), method specificity be low for existing capture type antibody dot matrix, the invention provides a kind of method utilizing separate type biological reagent dot matrix to detect biomolecule, application capture type antibody dot matrix and separate type antibody dot matrix detect multiple protein simultaneously, multiple protein can be detected simultaneously, the particularly albumen of histocyte expression, avoid cross reaction, thus improve the specificity of detection.
2. technical scheme
In order to solve the problem, the technical solution adopted in the present invention is as follows:
Utilize separate type biological reagent dot matrix to detect a method for biomolecule, its step comprises:
A. biomolecule is fixed on two or more position on first support, forms a biomolecule dot matrix;
B. separate type biological reagent is fixed on second two or more position of support, forms a separate type biological reagent dot matrix;
C. contacted with the separate type biological reagent dot matrix on second support by the biomolecule dot matrix on first support, in contact process, biological reagent is combined with biomolecule;
D. be separated with first support by second support, the separate type biological reagent combined with biomolecule is separated with second support, transfers on first support;
E. by detecting the separate type biological reagent transferred on first support, thus biomolecule is detected.
Utilize separate type biological reagent dot matrix to detect a method for biomolecule, its step comprises:
A. one or more capture type biological reagents are separately fixed on first support, form a capture type biological reagent dot matrix;
B. one or more separate type biological reagents are separately fixed on second support, form a separate type biological reagent dot matrix;
C. capture type biological reagent dot matrix one or more biomolecule in biological sample on first support are combined, thus biomolecule is captured on first support;
D. then first support is contacted with second support, the separate type biological reagent dot matrix be fixed on second support is contacted with the biomolecule on first support, and separate type biological reagent combines with the biomolecule on first support; Be separated with second support by first support subsequently, separate type biological reagent leaves second support and transfers on first support;
E. detect the separate type biological reagent on first support, thus obtain the content of the biomolecule in biological sample.
Preferably, described steps d is revised as: first captured biomolecule is transferred to the 3rd support also fixing from first support, then the 3rd support contacts with second support, the separate type biological reagent dot matrix be fixed on second support is contacted with the biomolecule on the 3rd support, separate type biological reagent combines with the biomolecule on first support, be separated with second support by 3rd support subsequently, separate type biological reagent leaves second support and transfers on the 3rd support; Described step e is revised as: detect the separate type biological reagent on the 3rd support, thus obtain the content of the biomolecule in biological sample.
Preferably, described step e is revised as: first the separate type biological reagent on first support is transferred on the 3rd support also fixing, then detect the separate type biological reagent on the 3rd support, thus obtain the content of the biomolecule in biological sample.
Preferably, described separate type biological reagent is antibody or described capture type biological reagent is antibody.
Preferably, described separate type biological reagent or described capture type biological reagent are the antibody of anti-phosphorylated protein.
Preferably, first described support or the making material of second support are nylon, or glass, or plastics, or cellulose nitrate, or polyacrylamide, or their derivant.
Preferably, the material of second described support is nylon, or its derivant.
Preferably, the capture type biological reagent in described step a or the separate type biological reagent in described step b be 5 kinds and more than, each capture type biological reagent to be fixed on first support at least one predetermined position.
Preferably, in described step c, biomolecule is captured after on first support, with crosslinking chemical, the capture type biological reagent on first support is connected with biomolecule covalency.
Preferably, with crosslinking chemical, the separate type biological reagent after transfer is connected with biomolecule covalency in described steps d.
Preferably, described crosslinking chemical is aldehydes, comprises formaldehyde and glutaraldehyde.
3. beneficial effect
Compared to prior art, beneficial effect of the present invention is:
(1) a kind of method simultaneously detecting various biomolecules provided by the invention has high flux, needs less reagent and protein example amount etc., compared with use single trapping formula dot matrix or single separate type dot matrix techniques, has larger advantage; Compared with capture type dot matrix techniques, as the antibody dot matrix method of mark, the inventive method has higher specificity, because technology of the present invention employing is two kinds of same antigens of antibody recognition, and the antibody dot matrix techniques employing of mark is a kind of antigen of a kind of antibody recognition;
(2) method provided by the invention effectively can avoid the problem of cross influence, there is cross-cutting issue in existing capture type dot matrix techniques double antibody sandwich method always, and the present invention can avoid cross-cutting issue, because the antibody for detecting is fixed on the anti-dot matrix sheet of separate type, and unlike usual double antibody sandwich method, detect antibody free in the solution;
(3) method of the present invention has very high sensitivity, and relatively and the capture type dot matrix techniques of usage flag method, the present invention uses the detection antibody of separate type, can amplify detection signal, and sensitivity is strengthened further;
(4) the detection flux of the inventive method is high, due to cross-cutting issue in double antibody sandwich method, antibody type quantity is made to be limited in about tens kinds, and the antibody type quantity in the inventive method is by unrestricted, and there will not be cross-cutting issue, therefore the present invention can carry out effective quantitatively detection to the multiple antigens in protein example;
(5) in the inventive method, also biological reagent-bimolecular complexes is carried out covalently bound, make it stable further, thus improve the sensitivity detected.
Accompanying drawing explanation
Fig. 1 is the schematic diagram detecting multiple method of protein in the present invention, and wherein, 1 represents capture type antibody dot matrix; 2 represent protein example; 3 represent separate type antibody dot matrix;
Fig. 2 is the result figure that the inventive method detects the protein cleavage liquid of 293T cell line;
Fig. 3 is the result figure detecting mouse-anti body in sample in the embodiment of the present invention 2 with capture type antibody dot matrix and separate type antibody dot matrix;
Fig. 4 is the expression of results figure with two kinds of albumen in capture type antibody dot matrix and separate type antibody dot matrix bacterial detection lysate in the embodiment of the present invention 3;
Fig. 5 is the result figure being transferred to the method on the 3rd support after applying separate type antibody and protein combination in the embodiment of the present invention 4.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further.
" biological sample " refers to any material relevant with biology, as solution, and potpourri etc." protein sample " this term refers to the potpourri containing protein here.Such as, it can be the lysate from clone or tissue.The cell that it also can be complete cell or be fixed on support.Protein sample can have different sources: can from by cultured cells system, human or animal tissues, or comes from blood sample.In one case, protein sample is prepared by cell lysis or tissue.A kind of typical lysis buffer comprises detergent (detergent) as lauryl sodium sulfate (SDS), Triton X-100, etc.
" biomolecule " this term, with here for the needs being convenient to description and patent right, refers to any molecule relevant with biology, including, but not limited to protein.Biomolecule can be detected material, can with biological reagent effect, combination.
" biological reagent " this term, with here for the needs being convenient to description and patent right, refers to any material relevant with biology.Biological reagent and biomolecule action, can be used for detecting biomolecule.Biological reagent is including, but not limited to protein, antibody, antigen, polypeptide, DNA, RNA and Small molecular.
" support " this term, with here for the needs being convenient to description and patent right, refers to for placing the carrier structure with fixing biological molecules.Support has a variety of, and in the present invention, support can be cellulose nitrate (nitrocellulose), nylon (Nylon) or polyacrylamide (PVDF) film, or the film that their derivants are formed.The support that other material is made, as various glass etc., also in method used in the present invention.
" biological reagent dot matrix " or " biological reagent chip " this term refers to that a device comprises the device of a solid support and multiple biological reagent here.Biological reagent is fixed on preassigned position on support so that each biological reagent identifies by this specific position.When biological reagent is protein, be " protein array " or " protein-chip " or " Protein microdot arrays " or " protein chip ".When biological reagent is antibody, be " antibody dot matrix " or " antibody chip ".In the description of invention, when using " antibody " or " antibody dot matrix ", other biological reagent or biological reagent dot matrix also may be applicable to same method." dot matrix " and " chip " has same implication, and " biomolecule dot matrix " or " biological molecular chip " have identical implication.
" capture type biological reagent dot matrix " this term refers to that one for capturing the biological reagent dot matrix on dot matrix support by biomolecule here." capture type antibody dot matrix " this term here refer to one for by antigen capture to the antibody dot matrix on dot matrix support.Dot matrix and protein sample are hatched, and make the antibody on chip and the albumen in sample (antigen) react, combine, thus they are captured on chip support.Afterwards, captured albumen is detected further.Protein sample can in the solution, also can be fixed on solid support.
" capture type biological reagent " or " catching biological reagent " are used for refering in particular to the biological reagent be fixed on capture type biological reagent dot matrix." capture type antibody " or " capture type antibody " is used for refering in particular to the antibody be fixed on capture type antibody dot matrix.
" separate type biological reagent dot matrix " this term refers to a kind of special biological reagent dot matrix here, and it can maintain the position of biological reagent on dot matrix support on the one hand; On the other hand, when biological reagent is after being fixed on the biomolecule on another support and being combined, part or all of biological reagent can leave dot matrix support and be combined on biomolecule support.When biological reagent is antibody, " separate type biological reagent dot matrix " is " separate type antibody dot matrix "
The reagent be fixed in some applications on separate type biological reagent dot matrix is referred to as " separate type biological reagent " or " separating bio reagent " or " detection biological reagent " or " detection reagent ", as " detection antibody ".
" fix " this term with here for the needs being convenient to description and patent right, mean the motion that restriction biological reagent is supported at.Such as, when an antibody is fixed on a support, this antibody is bonded on support, under certain condition it can not from support be separated and the activity of antibody on support be also limited.But under certain conditions, as separate type antibody dot matrix described in the present invention, the antibody be fixed can be separated with support.The biological and chemical character of fixed form determines whether the reagent be fixed can depart from and second cosmic velocity, efficiency from support.
In order to make the constant intensity of the biological reagent be fixed on solid support moderate in be suitable for special application, solid support will be anticipated usually, a kind of process approach is on solid support, put one deck polymer compounds, and these polymer compounds interact by non-specific non-covalent bond and biological reagent.Such as, the condensate comprising polylysin or poly-second phthalein imido can be used to process microslide or cover glass to be used for fixing biomolecule.
Multiple Antibodies placement and the technology be fixed on solid support are had multiple, (the Hybridizationfingerprinting in genome mapping and sequencing such as picture Lehrach, genome analysis, Voll, Davies and Tilgham, Eds, Co1d Spring Harbor Press, 1990, pp.39-81) and Brown (U.S. Patent Application No. is US 19950477809, and publication number is US 5807522A) etc. described.In the article mentioned above each section any method of describing all may be relevant with the present invention, be therefore all used as the material of invention.Such as, machine point model machine is used to be placed in a glass slide by millilambda antibody-solutions.Form biological reagent dot matrix by depositing one or more a large amount of reagent on a flat solid support simultaneously, each reagent is placed on a predetermined position.
Antibody can be fixed by suction-operated (Trevan, 1980, Immobilized Enzymes:an introduction and theirapplication in biotechnology.Wiley, Chichester).This absorbability can be non-specific, hydrophobic or interionic interacts.The typical adsorption material used comprises clay, charcoal, hydroxylapatite and many ion exchange materials such as DEAE-cross-linked glucose.
Embedding (entrapment) is the method (Trevan of another kind of sessile antibody, 1980, Immobilized Enzemes:anintroduction and their application in biotechnology.Wiley, Chichester).In theory, the antibody be embedded does not have polymer medium and combines, and is only that their free diffusing is restricted.The embedding medium of frequent use is multi-polyamide gel.An object lesson is with kapillary fixed biologically reagent and makes dot matrix." kapillary " this term refers to the elongated structure sealed, and can be used for supporting and fixed biologically reagent.Kapillary is made up of the material such as plastics or glass, and these materials do not affect the character of biological reagent.The height of kapillary can change, in theory can from nine microns to nine meters.Biological reagent is filled in kapillary with liquid condition usually, and after filling, liquid solidifies thus biological reagent is fixed, and fixing intensity can change with different application.
Biological reagent can be fixed on solid support directly or indirectly, reagent can be secured directly to high-density and may diminish on the support of microslide, be used for preparing technology similarly (the Shalon et al. of highdensity DNA chip, Genome Reserch, 1996,6 (7): 639-645).Reagent also can be fixed on support indirectly.Such as, albumin A or G and their mutation-ure can be fixed on support, and antibody is fixed on support by the interaction with albumin A or G.The advantage of this method is that antibody acts on albumin A or G phase by means of only constant region, and the variable region (antigen binding domain) of antibody then can be used for and antigenic action completely; Another one advantage is because the binding site of albumin A or Protein G and antibody can change, and when reformed albumin A or G are used, antibody capable is fixed on support with suitable intensity.Such antibody can be fixed on support thus to keep respective site in one end, can leave support and be combined with the biomolecule with more high-affinity on the other hand.Recombinant protein combines by the acceptor that distinguished sequence and the distinguished sequence therewith on fixing support act on mutually and is fixed.Such as, part (as glutathione or nickel) can first by covalent attachment on support, the recombination fusion protein then with distinguished sequence (as GST or 6xHis) is fixed on identical support by the interaction with part.Distinguished sequence and part can be modified to change their affinity thus be fixed by recombinant protein with suitable intensity.
Although antibody is often fixed on support with the shape of round dot, antibody also can be placed on support with other shapes.Such as, antibody capable is fixed on support with rectangular shape (0.1-10 centimetre wide, 1-50 centimeter length).
The antibody of a specific antigen generally will to be fixed on support on a specific position.In general, ensure that each antibody can combine with its antigen when antibody dot matrix contacts with protein sample.Therefore perhaps each antibody be fixed on special position, and this position is decided by the position of antigen.Similar, the position that the position that antigen is fixed also can be fixed according to antibody is decided.
In a concrete optimal enforcement scheme of the present invention, two or more biomolecule is fixed on the multiple positions on first support, forms a biomolecule dot matrix.Biomolecule dot matrix contacts with the separate type biological reagent dot matrix on second support; In contact process, biological reagent is combined with biomolecule, and when second support is separated with first support, biological reagent is separated with second support, transfers on first support.By detecting the separate type biological reagent on first support, thus learn the content of biomolecule in sample.
In another optimization method, a kind of capture type biological reagent is fixed on first solid support, with the biomolecule sample incubation in biological sample, reaction, after capture type biological reagent combines with biomolecule specificity, biomolecule is captured on first solid support, then contacts with a kind of separate type biological reagent be fixed on second solid support, react.When first support and second support separate, the separate type biological reagent combined with biomolecule can be separated with second solid support and be attached on first support.Detect the separate type biological reagent on first support afterwards, thus learn the content of biomolecule in sample.
In another optimization method, the capture type biological reagent dot matrix be fixed on first solid support contacts with the biomolecule sample in biological sample, after capture type biological reagent combines with biomolecule specificity, biomolecule is captured on first solid support, then contacts with the separate type biological reagent dot matrix be fixed on second solid support, react.When first support and second support separate, can be separated with second solid support with the separate type biological reagent that biomolecule combines and being attached on first support on separate type biological reagent dot matrix.Detect the separate type biological reagent on first support afterwards, thus learn the content of biomolecule in sample.
In another concrete optimal enforcement scheme of the present invention, as shown in Figure 1, antibody is as catching biological reagent and separating bio reagent, and biological sample is protein example.In this scheme, protein example contacts with the capture type antibody dot matrix be fixed on first support, hatches.In hatching, the antigentic specificity of capture type antibody in sample is combined, thus antigen capture on first support; Then, the capture type dot matrix being combined with antigen contacts with the separate type antibody dot matrix be fixed on second support, in contact, and the separate type antibody specific binding on antigen and separate type antibody dot matrix; When separate type antibody dot matrix support (second support) is separated with capture type antibody dot matrix support (first support), separate type antibody can be separated from second support and transfer on first support, then, the separate type antibody on first support is detected.The quantity and the antigen levels captured on first support that are transferred to antigen in the quantity of the antibody on first support and protein sample are proportional.Therefore the quantity of antigen in protein sample will be disclosed by the quantity of separate type antibody on detection first support.
Capture type antibody on capture type antibody dot matrix is preferably securely fixed on first support, and this realizes by covalent bond connection.
Many known technology can be used to detect the separate type antibody transferred to and capture type antibody dot matrix is combined with antigen.Normally used method uses enzyme di-to resist exactly, the goat-anti rabbit of such as horseradish peroxidase (horseradish peroxidase, HRP) coupling and the antibody of sheep anti mouse, fluorescently-labeled two anti-etc.Other feasible technology comprise immuno-PCR (Sano et al
.,science, 1992,258:120-122), Circulating DNA amplification technique (Schweitzer et a1
.,2000, Proc.Natl.Acad., Sci.USA, 97:10113-10119), through immune detection (the Zhang et a1 of t7 rna polymerase amplification
.,proc.Natl.Acad., Sci.USA, 2001,98:5497-5502).
In another method, separate type antibody dot matrix adopts the antibody of directly mark.Mark can be enzyme (as HRP), fluorescent marker, nano particle and colloid gold particle etc.Relevant labeling method has much in the literature.
The inventive method uses two Protein microdot arrays, capture type antibody dot matrix and separate type antibody dot matrix.Antigen first captured formula dot matrix is caught, then the antibody on separate type antibody dot matrix is combined.The advantage that this technology has except common protein array, as high flux, needs beyond less reagent and protein example amount etc., compared with use single trapping formula dot matrix or single separate type dot matrix techniques, has larger advantage.First, compared with capture type dot matrix techniques, as the antibody dot matrix method of mark, the inventive method has higher specificity, because technology employing is two kinds of same antigens of antibody recognition, and the antibody dot matrix techniques employing of mark is a kind of antigen of a kind of antibody recognition.There is cross-cutting issue in addition in capture type dot matrix techniques double antibody sandwich method always.And this technology can avoid cross-cutting issue, because the antibody for detecting is fixed on the anti-dot matrix sheet of separate type, and unlike usual double antibody sandwich method, detect antibody free in the solution.The second, the inventive method has very high susceptibility, carries out signal amplification susceptibility is strengthened further to detection antibody.3rd, the inventive method has higher detection flux.Due to cross-cutting issue in double antibody sandwich method, antibody type quantity is made to be limited in about tens kinds, and the antibody type quantity in the inventive method is by unrestricted, and there will not be cross-cutting issue, therefore the present invention can carry out effective quantitatively detection to the antigen in protein example.
In an optimization method, comprise and antigen-antibody complex is carried out covalently bound (cross-linking) this step, concrete grammar is, the capture type antibody dot matrix be fixed on first support contacts with protein sample, in contact, the antigen of capture type antibody in sample is combined, antigen capture on first support; After the albumen of non-specific binding is removed, the compound that capture type antibody and antigen are formed makes it stable further by covalent bond.Then, the capture type dot matrix being combined with antigen contacts with the separate type antibody dot matrix be fixed on second support, and in contact, the separate type antibody of antigen on separate type antibody dot matrix is combined; When separate type antibody dot matrix is separated with capture type antibody dot matrix, separate type antibody can be separated from second support and transfer on first support, then, detects the separate type antibody on first support.In this method, after antigen is combined with separate type antibody, also can be connected between antigen with separate type antibody by the method for covalent cross-linking.Much known method and chemical reagent can be used to the compound of covalently bound two or more albumen, such as conventional homotype bi-functional cross-linking agent glutaraldehyde (glutaraldehyde) or formaldehyde.
Another method of the present invention is transferred on the 3rd solid support by the antigen of being caught by capture type antibody dot matrix.Concrete method is, first by protein sample with the capture type antibody dot matrix mixing on first support, thus capture type antibody capture can be made and antigen in protein isolate sample.After washing uncombined albumen, the albumen captured in each site can be separated with capture type antibody and is transferred to the 3rd support and is fixed in the above.Because the interaction between the comparable capture type antibody of the interaction between capture type antibody and solid support and antigen is much better than, so some conditions can be found to remove to interrupt capture type antibody and AI and not affect the effect of capture type antibody and first support.Under these conditions, antigen instead of capture type antibody capable to be transferred on other support and to be fixed in the above.In order to avoid capture type antibody is separated with first solid support, capture type antibody covalent bond can be fixed on support.In a typical transfer process, first support comprising capture type antibody and interactional albumen is with it contacted with second support, then they are placed in buffer solution to destroy the interaction of capture type antibody and antigen, meanwhile, electric current is applied so that the albumen be separated can transfer to the two support from first support.After completing, these two supports are separated, and protein delivery can occur simultaneously or separately carry out with fixing.Namely first protein be transferred, and then fixes.Protein is transferred simultaneously and fixes also is possible.If be not fixedly that picture is desirably strong, protein can be fixed further, such as, pass through covalent bond.Protein can be fixed on solid support with the form of covalent bond by many known methods.
Be transferred to antigen on the 3rd support and remain on the relative position on first support.Antigen is transferred to after on the 3rd support, contacts with the separate type antibody dot matrix be fixed on second support, and in contact, the separate type antibody of antigen on separate type antibody dot matrix is combined; When second support is separated with the 3rd support, separate type antibody can be separated from second support and transfer on the 3rd support; Then, by detecting the separate type antibody on the 3rd support, the content of antigen is learnt.
Another method of the present invention is transferred on the 3rd support by the separate type antibody transferred on capture type antibody dot matrix.Concrete method is, protein example contacts with the capture type antibody dot matrix be fixed on first support, hatches.In hatching, the antigentic specificity of capture type antibody in sample is combined, thus antigen capture on first support; Then, the capture type dot matrix being combined with antigen contacts with the separate type antibody dot matrix be fixed on second support, in contact, and the separate type antibody specific binding on antigen and separate type antibody dot matrix; When being separated with capture type antibody dot matrix support (first support) by separate type antibody dot matrix support (second support), separate type antibody can be separated from second support and transfer on first support.Then, separate type antibody can be separated from first support and transfer on the 3rd support.Be transferred to separate type antibody on the 3rd support and remain on the relative position on first support.By detecting the separate type antibody on the 3rd support, learn the amount of each antigen.
In the present invention, the method that antigen or separate type antibody are transferred on the 3rd support is had a clear superiority in.Such as, even adopt capture type and the separate type antibody of same animal origin, in method can also usage flag two resist to detect separate type antibody.
Method of the present invention can be used for detecting the interaction between albumen and albumen.A concrete grammar is, the antibody of the first protein is fixed on first support, and the antibody of the second protein is fixed on second support; First support being fixed with the first protein reacts with the protein example containing the first protein and the second protein complex, in course of reaction, compound by the antibody capture of the first protein on first support.Then, first support contacts with second support being fixed with the second protein antibody, in contact process, and the second protein bound in the antibody of the second protein and compound; Then second support is separated with first support, to be separated to transfer to first support with the antibody of the second protein bound from second support; Detect second antibody transferring on first support, thus learn the first protein whether with the second protein bound.
In another method, a capture type antibody dot matrix is used for detecting multiple protein-protein interaction in protein sample together with a separate type antibody dot matrix.In method, first group of antibody is fixed on first support and forms capture type antibody dot matrix; Second group of antibody is fixed on second support and forms a separate type antibody dot matrix; A kind of antibody of a kind of antibody of first group and second group all can respectively with interactional two protein combination; These two kinds of antibody are fixed on the relevant position of capture type antibody dot matrix and separate type antibody dot matrix, and when two dot matrix contacts, two kinds of antibody, in same position, can be combined with corresponding protein complexes.First capture type antibody dot matrix contacts with the protein example containing protein complex, and in contact, the antigentic specificity of capture type antibody in sample is combined, thus corresponding protein complex is captured on first support; Then, the capture type dot matrix being combined with compound contacts according to corresponding relative position with the separate type antibody dot matrix be fixed on second support, and in contact, the antigentic specificity of separate type antibody in compound on separate type antibody dot matrix is combined; When separate type antibody dot matrix support (second support) is separated with capture type antibody dot matrix support (first support), in conjunction with separate type antibody can be separated from second support and transfer on first support; Then, the separate type antibody on first support is detected.
In the method that another detects albumen and Protein-protein interaction, the antibody of the first protein is fixed on first support, and the antibody of the second protein is fixed on second support; Be fixed with first support and the first proteins react of the first protein, in course of reaction, the first protein by the antibody capture of the first protein on first support.Then, and the second proteins react, the second protein and the first protein bound in course of reaction, and be captured on first support.Then, be combined with the first and contact with second support being fixed with the second protein antibody with first support of the second protein complex, in contact process, the second protein bound in the antibody of the second protein and compound; Then second support is separated with first support, to be separated to transfer to first support with the antibody of the second protein bound from second support; Detect the antibody transferring to the second protein on first support, thus learn the first protein whether with the second protein bound.
In another method, a capture type antibody dot matrix is used for detecting multiple protein-protein interaction in protein sample together with a separate type antibody dot matrix.In method, first group of antibody is fixed on first support and forms capture type antibody dot matrix; Second group of antibody is fixed on second support and forms a separate type antibody dot matrix; A kind of antibody of a kind of antibody of first group and second group all can respectively with interactional two protein combination; These two kinds of antibody are fixed on the relevant position of capture type antibody dot matrix and separate type antibody dot matrix, and when two dot matrix contacts, two kinds of antibody, in same position, can be combined with corresponding protein complexes.Capture type antibody dot matrix first with containing the sample contacts of the first histone, in contact, the antigentic specificity of capture type antibody in sample is combined, thus corresponding protein trapper is received on first support; Then, be combined with the capture type dot matrix of the first histone matter and the sample contacts containing the second histone, in contact, the corresponding protein of the albumen in first group in second group is combined, and first support forms compound.Then, first support containing protein complex contacts according to corresponding relative position with the separate type antibody dot matrix be fixed on second support, in contact, the antigentic specificity of separate type antibody in compound on separate type antibody dot matrix is combined; When separate type antibody dot matrix support (second support) is separated with capture type antibody dot matrix support (first support), in conjunction with separate type antibody can be separated from second support and transfer on first support, then, the separate type antibody on first support is detected.
Method of the present invention can be used for detecting the posttranslational modification (as protein phosphorylation) of albumen.A concrete grammar is: a kind of antibody of protein is fixed on first support, and the antibody of protein post-translational modification is fixed on second support; The antibody of protein post-translational modification can be the specific posttranslational modification albumen of identification one, as phosphorylation p53, also can be the specific posttranslational modification albumen of a class, as identified the 4G10 antibody of tyrosine phosphorylated proteins.First support being fixed with protein antibody and the example reaction containing protein, in course of reaction, antigen protein by antibody capture on first support; Then, first support contacts with second support of the antibody being fixed with protein post-translational modification, in contact process, and the antibody of the posttranslational modification of protein and protein bound; Then second support is separated with first support, to be separated to transfer to first support with the antibody of the posttranslational modification of protein bound from second support; Detect the antibody of the posttranslational modification transferred on first support, thus learn the posttranslational modification of protein.
Detect in the method for modifying after protein translation at another, capture type antibody dot matrix is used for detecting the posttranslational modification of two or more protein in protein sample together with a separate type antibody dot matrix.In method, first group of antibody is fixed on first support and forms capture type antibody dot matrix; Second group of antibody is fixed on second support and forms a separate type antibody dot matrix; A kind of antibody of a kind of antibody of first group and second group respectively with a kind of protein combination, wherein at least one antibody and posttranslational modification protein combination.Antibody is fixed on the relevant position of capture type antibody dot matrix and separate type antibody dot matrix, and when two dot matrix contacts, two kinds of antibody, can with corresponding protein combination in same position.First capture type antibody dot matrix contacts with protein sample, and in contact, the antigentic specificity of capture type antibody in sample is combined, thus corresponding protein trapper is received on first support; Then, the capture type dot matrix being combined with protein contacts according to corresponding relative position with the separate type antibody dot matrix be fixed on second support, and in contact, the separate type antibody on separate type antibody dot matrix is combined with antigentic specificity; When separate type antibody dot matrix support (second support) is separated with capture type antibody dot matrix support (second support), in conjunction with separate type antibody can be separated from second support and transfer on first support; Then, the separate type antibody on first support is detected.
Covalently boundly to can be used in the method detected in protein-protein interaction and modify after protein translation and covalently boundly can to use in one or more steps.Such as, when protein complex and capture type antibody are attached to after on capture type antibody dot matrix, covalently bound being used for reinforces albumen and albumen, the combination between albumen and antibody.After separate type antibody is combined with albumen composition, the covalently bound combination that also can be used for reinforcing them.
The capture type dot matrix used in the inventive method and separate type dot matrix can be the dot matrix of any biological reagent.Method can be used for detecting any biomolecule.Such as DNA probe instead of antibody are used as biological reagent and use in the method for the invention, then capture type and separate type DNA probe dot matrix can detect special DNA or RNA expressed in biological sample.The method for making of the method for making of capture type and separate type DNA probe dot matrix and capture type and separate type antibody dot matrix is similar.Such as, DNA probe can be fixed on by special mode on a support, makes each probe keep ad-hoc location on support.But the fixing effect cand be compared between probe and target sequence most of probe is weak, like this after probe is combined with target sequence, can depart from from support, the separate type DNA probe dot matrix made like this can be used to detect DNA or RNA sequences different in a large number simultaneously.DNA or rna probe point to support and the method be fixed thereon has a lot, as method (IanA.Darby (Editor) described in the following documents, In Situ Hybridization Protocols (Methods in Molecular Biology, 123), byHumana Press; ISBN:0896036863; 2nd edition, 2000).
Specific embodiment:
The present invention has a lot of different purposes, and because of different use, technical scheme changes to some extent, and example is below some application and some concrete case study on implementation of invention technology, and these examples can not be used for limiting the present invention.Although such as the present invention mainly describes the example that antibody dot matrix uses, similar use biological reagent dot matrix instead of antibody for people be significantly have the personage of relevant professional knowledge to know the present invention also comprises similar other biological molecular lattice.These biological reagents include, but are not limited to recombinant protein, recombinant antibodies, single-chain recombinant antibody, nucleotide, oligonucleotides, cDNA probe, carbohydrate, lipid, micromolecular compound etc.Such as, capture type and separate type cDNA probe lattice energy are used in method of the present invention and detect multiple biomolecule, as the expression of protein, mRNA.
Embodiment 1:
In the present embodiment (Fig. 2), the protein cleavage liquid (being dissolved in the SDS physiological saline of 1% (massfraction)) of 293T cell line is fixed on a NC film (nitrocellulose filter) with the form of dot matrix by adsorbing, form the biomolecule dot matrix that has 10 points, each point fixes 2 micrograms of protein.10 kinds of different rabbits many anti-(0.2 microgram) are fixed on a nylon membrane, (in Fig. 2,1 represents beta-Catenin to form a separate type biological reagent antibody dot matrix, 2 represent Cyclin E, and 3 represent E2F1, and 4 represent Ets-1/2,5 represent GRK2,6 represent JNK1, and 7 represent MEK1, and 8 represent NF-kappa B50,9 represent Rb p107, and 10 represent Stat1).Biomolecule dot matrix contacts with the separate type antibody dot matrix on nylon membrane; In the contact process of two hours, antibody and various antigen (biomolecule) combine.When nylon membrane and NC UF membrane, the antibody combined with antigen is separated with nylon membrane, transfers on NC film.Then anti-being used for of goat-anti rabbit two of HRP mark detects the antibody shifted.In the present embodiment, tetramethyl benzidine develops the color as HRP substrate.On 10 diverse locations, the power of signal represents the binding capacity of antigen in different antibodies and cell pyrolysis liquid, is directly proportional to the content of antigen.
Embodiment 2:
In the present embodiment, with not having markd two to resist the Immunoglobulin IgG of catching in serum, and two of HRP-mark anti-are used as detection (separation) antibody.Sheep anti mouse and goat anti-rabbit igg (F
c) antibody by the special microslide (process of covalent modification is: microslide to be placed in the glutaraldehyde normal saline solution of 1% (massfraction) 15 minutes) o'clock to cross to a glutaraldehyde covalent modification and covalency be fixed in the above, make capture type antibody dot matrix.The sheep anti mouse of HRP-mark or goat anti-rabbit igg [F (ab ')
2] put to preparative separation formula antibody dot matrix on nylon membrane.Capture type antibody and separate type antibody identify the F of antibody respectively
cwith F (ab ') part.Prepare the antibody-solutions point of about 80nL during dot matrix to each point, 2 millimeters of spacing.Often kind of antibody repeats a little once.Capture type antibody dot matrix and separate type antibody dot matrix all have 6 points.6 points of capture type antibody dot matrix are respectively: on 1-4 point, point has sheep anti-mouse igg (F
c) antibody; On 5-6 point, point has goat anti-rabbit igg (F
c) antibody.6 points of separate type antibody dot matrix are respectively: on 1 and No. 2 point, point has sheep anti-mouse igg [F (ab ')
2] antibody; On 3 and No. 4 points, point has goat anti-rabbit igg [F (ab ')
2] antibody; On 5-6 point, point has sheep anti-mouse igg [F (ab ')
2] antibody.Mouse serum (1:1000 dilution) is as target protein.HRP substrate tetramethyl benzidine (Tetramethylbenzidine Dihydro-chloride) be used for observe HRP mark detection antibody.Visible at Fig. 3 A, in position (1 and 2, sheep anti mouse is at capture type dot matrix and the separate type dot matrix) visible signal of pairing antibody, and (3 and 4, sheep anti mouse is catching dot matrix, and goat-anti rabbit is at separate type dot matrix in unpaired position; 5-6, goat-anti rabbit is catching dot matrix, and sheep anti mouse is at separate type dot matrix) then there is no signal.
In control test, (Fig. 3 B) same capture type and separate type antibody dot matrix are used for detecting rabbit anteserum (1:1000 doubly dilutes) instead of mouse serum.Because the goat-anti rabbit point not having to match (namely on capture type and separate type antibody dot matrix be all goat anti-rabbit antibody without any a corresponding lattice point), so result signal do not detected.
Embodiment 3:
In the present embodiment, capture type and separate type antibody dot matrix is prepared according to method of the present invention.Linomat Ⅲ is used for a rabbit antibody (about 0.2 microgram/microlitre) point to NC film, prepares capture type antibody dot matrix, and (A arranges the consumption of antibody, 40ng from more to less; B arranges, 20ng; C arranges, 10ng; D arranges, 5ng).Mouse-anti body (40 nanogram) is put to preparative separation formula antibody dot matrix on nylon membrane.The antibody of the 1st row and the 3rd row and Connexin43 protein combination; The antibody of the 2nd row and the 4th row and GST protein combination.The mouse-anti body of identical amount is fixed on each aspect of every a line of separate type dot matrix.Antibody is fixed on NC and nylon membrane by non-covalent bond.Dot matrix is stored in 4 degree of refrigerators after having prepared, and uses in 48 hours.
Capture type antibody dot matrix first in 1% bovine serum albumin solution (1 gram of bovine serum albumin(BSA) is dissolved in 100mL physiological saline) close 1 hour, then with the bacterial lysate (Fig. 4-1) containing GST albumen, or hatch with the bacterial lysate (Fig. 4-2) containing GST and Connexin43 albumen.In the process of hatching, antibody combines with corresponding antigen.Then, fall not have the albumen combined with normal saline flushing.Capture type antibody dot matrix to be placed in the glutaraldehyde normal saline solution of 1% (massfraction) 15 minutes, makes antibody-antigene covalently bound.Afterwards, a separate type antibody dot matrix is placed on capture type antibody dot matrix, and two dot matrix alignment are so that the antibody on separate type dot matrix can with the antigen-reactive be attached on capture type antibody dot matrix.In the reaction of 1 hour, separate type antibody is combined with respective antigen.Then, separate type antibody dot matrix is separated from capture type antibody dot matrix, and the separate type antibody combined with antigen can be separated from separate type antibody dot matrix and transfer on capture type dot matrix.After physiological saline cleaning several times, resist with two of alkali phosphatase enzyme mark and hatch half an hour.After cleaning, add substrate 5-bromo-4-chloro-indolyl-phosphatase (BCIP) and nitroblue tetrazolium (NBT) produces signal, physiological saline cleaning terminates colour developing, obtains image with scanner.In the experiment only having GST albumen, can see has signal (the 2nd row in Fig. 4-1 and the 4th row) on GST antibody point, and on the antibody point of Connexin43, do not have signal (the 1st row in Fig. 4-1 and the 3rd row).In the experiment having GST and Connexin43 albumen, can see has signal in the position of GST (the 2nd row in Fig. 4-2 and the 4th row) and Connexin43 (the 1st row in Fig. 4-2 and the 3rd row) antibody point.This example demonstrates multiple protein that the inventive method can be used for detecting in protein lysate simultaneously, antibody amount (40 nanogram) used on separate type antibody dot matrix is also far less than using the consumption detecting antibody-solutions method, because often kind of detection antibody is fixed on separate type antibody dot matrix, avoid the cross reaction between antibody.
Embodiment 4:
In the present embodiment, rabbit anti-mouse antibody 32 points be fixed in a glass slide form a capture type antibody dot matrix.The sheep anti-mouse antibody of HRP mark is fixed on 32 positions on a nylon membrane and forms a separate type antibody dot matrix.The position that on two dot matrix, antibody is fixed corresponding with the contact of box lunch two dot matrix time, they can closely stick together, and can be combined with same antigen.First capture type dot matrix hatches 2 hours with the mouse-anti liquid solution of 20 nanograms, and the mouse-anti body in sample is captured formula antibody capture on microslide.After physiological saline is used for washing away other albumen not having to combine, the compound covalency glue crosslinking agent (the glutaraldehyde normal saline solution of 1% (massfraction)) that rabbit anti-mouse antibody is combined with mouse-anti body comes covalently bound.Then contact with separate type antibody dot matrix.In contact, separate type antibody (sheep anti-mouse antibody of HRP mark) and mouse-anti precursor reactant, combination.In conjunction with after, separate type antibody dot matrix is separated with capture type antibody dot matrix, and the separate type antibody combined with mouse-anti body will be separated with separable dot matrix and transfer on capture type antibody dot matrix.Then, the capture type dot matrix being combined with separate type antibody contacts with another (the 3rd) support (NC film), and separate type antibody is transferred on NC film.After adding HRP substrate, chemiluminescence signal G-Box cooled camera obtains.As shown in Figure 5, the 3rd support detects signal, separate type antibody is described and is trapped in the mouse-anti body on capture type dot matrix and is combined, transfers on capture type dot matrix, and then successfully transferred on the 3rd support.
Claims (12)
1. utilize separate type biological reagent dot matrix to detect a method for biomolecule, its step comprises:
A. biomolecule is fixed on two or more position on first support, forms a biomolecule dot matrix;
B. separate type biological reagent is fixed on two or more position on second support, forms a separate type biological reagent dot matrix;
C. contacted with the separate type biological reagent dot matrix on second support by the biomolecule dot matrix on first support, in contact process, biological reagent is combined with biomolecule;
D. be separated with first support by second support, the separate type biological reagent combined with biomolecule is separated with second support, transfers on first support;
E. by detecting the separate type biological reagent transferred on first support, thus biomolecule is detected.
2. utilize separate type biological reagent dot matrix to detect a method for biomolecule, its step comprises:
A. one or more capture type biological reagents are separately fixed on first support, form a capture type biological reagent dot matrix;
B. one or more separate type biological reagents are separately fixed on second support, form a separate type biological reagent dot matrix;
C. capture type biological reagent dot matrix one or more biomolecule in biological sample on first support are combined, thus biomolecule is captured on first support;
D. then first support is contacted with second support, the separate type biological reagent dot matrix be fixed on second support is contacted with the biomolecule on first support, and separate type biological reagent combines with the biomolecule on first support; Be separated with second support by first support subsequently, separate type biological reagent leaves second support and transfers on first support;
E. detect the separate type biological reagent on first support, thus obtain the content of the biomolecule in biological sample.
3. a kind of method utilizing separate type biological reagent dot matrix to detect biomolecule according to claim 2, it is characterized in that: described steps d is revised as: first captured biomolecule is transferred to the 3rd support also fixing from first support, then the 3rd support contacts with second support, the separate type biological reagent dot matrix be fixed on second support is contacted with the biomolecule on the 3rd support, separate type biological reagent combines with the biomolecule on first support, subsequently the 3rd support is separated with second support, separate type biological reagent leaves second support and transfers on the 3rd support, described step e is revised as: detect the separate type biological reagent on the 3rd support, thus obtain the content of the biomolecule in biological sample.
4. a kind of method utilizing separate type biological reagent dot matrix to detect biomolecule according to claim 2, it is characterized in that: described step e is revised as: first the separate type biological reagent on first support is transferred on the 3rd support also fixing, then detect the separate type biological reagent on the 3rd support, thus obtain the content of the biomolecule in biological sample.
5. a kind of method utilizing separate type biological reagent dot matrix to detect biomolecule according to claim 2-4, is characterized in that: described separate type biological reagent is antibody or described capture type biological reagent is antibody.
6. a kind of method utilizing separate type biological reagent dot matrix to detect biomolecule according to claim 5, is characterized in that: described separate type biological reagent or described capture type biological reagent are the antibody of anti-phosphorylated protein.
7. a kind of method utilizing separate type biological reagent dot matrix to detect biomolecule according to claim 2-4, it is characterized in that: first described support or the making material of second support are nylon, or glass, or plastics, or cellulose nitrate, or polyacrylamide, or their derivant.
8. a kind of method utilizing separate type biological reagent dot matrix to detect biomolecule according to claim 7, is characterized in that: the material of second described support is nylon, or its derivant.
9. a kind of method utilizing separate type biological reagent dot matrix to detect biomolecule according to claim 2-4, it is characterized in that: the capture type biological reagent in described step a or the separate type biological reagent in described step b be 5 kinds and more than, each capture type biological reagent to be fixed on first support at least one predetermined position.
10. a kind of method utilizing separate type biological reagent dot matrix to detect biomolecule according to claim 2 or 4, it is characterized in that: in described step c, biomolecule is captured after on first support, with crosslinking chemical, the capture type biological reagent on first support is connected with biomolecule covalency.
11. a kind of according to Claims 2 or 3 utilize separate type biological reagent dot matrix to detect the method for biomolecule, it is characterized in that: connected with biomolecule covalency by the separate type biological reagent after transfer with crosslinking chemical in described steps d.
12. a kind of according to claim 10 or 11 utilize separate type biological reagent dot matrix to detect the method for biomolecule, it is characterized in that: described crosslinking chemical is aldehydes, comprise formaldehyde and glutaraldehyde.
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