WO1999043287A2 - Dendrimeres a base de phosphate pour essais biologiques - Google Patents

Dendrimeres a base de phosphate pour essais biologiques Download PDF

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Publication number
WO1999043287A2
WO1999043287A2 PCT/US1999/004068 US9904068W WO9943287A2 WO 1999043287 A2 WO1999043287 A2 WO 1999043287A2 US 9904068 W US9904068 W US 9904068W WO 9943287 A2 WO9943287 A2 WO 9943287A2
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bioreagent
switch
bioreagent according
linker
dna
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PCT/US1999/004068
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WO1999043287A3 (fr
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Andrzej Wilk
Andrzej K. Drukier
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Biotraces, Inc.
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Priority to AU27880/99A priority Critical patent/AU2788099A/en
Publication of WO1999043287A2 publication Critical patent/WO1999043287A2/fr
Publication of WO1999043287A3 publication Critical patent/WO1999043287A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6832—Enhancement of hybridisation 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/534—Production of labelled immunochemicals with radioactive label
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures

Definitions

  • This invention relates to a dendrimer, a method for its radiolabeling and applications thereof.
  • Biomedical applications require ever increasing sensitivity of detection and quantitation.
  • the detection of a single organism (virus, bacteria) or even a single molecule in a few milliliters of physiological fluid is an important goal.
  • the quantitation of the number of organisms is important, for example in the case of HIV-1 viral load for AIDS.
  • each target should be characterized, e.g., the case of metastatic cancer cells which have to be distinguished from millions of virtually identical healthy cells.
  • biomedical applications not only sensitivity but also specificity is of utmost importance.
  • non-specific biological background is a limiting factor in a majority of current high sensitivity assays.
  • New methods for signal amplification and NSBB suppression are desired which may permit a major improvement (by a factor of a hundred and more) of immunoassays and nucleic acids quantitation assays.
  • I25 I-dCTP is incorporated with the help of appropriate enzymatic reactions.
  • cytosine sites For a 100 bp duplex, about fifty cytosine sites are available. However, we have been able to radiolabel only a fraction of cytosine sites. Currently, the maximum number of that can be incorporated is about 10 for 100 bp duplex. Thus, currently we can detect about 100 and 10 DNA fragments with a length of a 100 bp and 1,000 bp, respectively.
  • the labeling is not limited to dCTP and up to
  • biotins can be placed on a 100 bp duplex.
  • steric hindrance leads to less than 100% efficiency in binding of biotin with 125 I streptavidin, i.e., currently similar limits of detection were achieved for direct radiolabeling and labeling using biotin as a linker.
  • streptavidin-polyHRP wherein polyHRP is multiply radioiodinated.
  • proteins we have been able to increase the efficiency of iodination about fifty-fold.
  • Chiron Inc. uses branched DNA, i.e., a linear DNA probe linked to a large and "bushy" DNA-tree.
  • branched DNA i.e., a linear DNA probe linked to a large and "bushy" DNA-tree.
  • One can easily radioiodinate such "branched DNA” but the available literature suggests that in the case of tree-like dendrimers, the detection limits are not only due to sensitivity but also due to non-specific hybridization of such large DNA constructs.
  • the prior art limit of detection is typically on the order of pg/ml or 0.1 fmole/ml.
  • LOD limit of detection
  • NSBB elimination for microorganisms (viruses, bacteria), single cells, e.g., cancer cells, and biological molecules with molecular weight larger than about 1,000 Daltons.
  • the main focus of the invention is on nucleic acids and proteins including glycoproteins, and large lipids.
  • Immunoassays with sub-attomole/ml sensitivity may provide a much desired capacity to detect rare antigenic targets within large populations of cells. Important examples are circulating cancer cells and the initially rare HIV infected cells in individuals who will later manifest AIDS. Super-sensitive immunoassays have important implications in early diagnosis and prevention, and the control of epidemics.
  • NSBB To overcome the NSBB we disclose modifications in the use of highly specific conjugation methods, e.g., antibody-antigen or biotin-streptavidin binding.
  • SuperTracers which increase the signal as well as diminish NSBB. Techniques using appropriate solid state support(s), washing and blocking are of prior art but will be used in totally new, counter-intuitive and innovative ways in combination with other innovative steps. Some of these methods are enabled by the use of the supersensitive MPD in the step of detection.
  • Immunoassays are a classical, reliable, specific and reasonably sensitive method for quantitation of biomolecules.
  • Abs antibodies
  • targets molecules or cell surface structures.
  • NSBB non-specific biological background
  • immunoassays are two site IA's, often called “sandwich” assays. In this case, two different Ab's, each specific to a different antigen/epitope are used. Typically, the first Ab is used to immobilize the target organism/molecule and the second Ab is used to label the target.
  • immunoassays are defined as radioimmunoassays (RIAs), ELISA or fluoroimmunoassays (FIA), wherein radioisotopes, color labels or fluor labels are used, respectively.
  • immunoassays are based on the concept of the titration curve.
  • a relative measurement is performed instead of absolute quantitation.
  • This permits the use of methods such as washing, blocking etc. which improve signal/background ratio but lead to unknown loss of the signal which can be calibrated using titration curves.
  • classical RIA, ELISA and FIA have achieved essentially the same level of performance of about 1 pg/ml for proteins, larger than say 1,000 Daltons.
  • different immunoassays achieved a similar sensitivity of 0.5-5 femtomole/ml. IA's are a mature art and progress is rather slow.
  • LOD limits of detection
  • LOQ limits of quantitation
  • the IRMA is an antibody sandwich capture assay in which an immobilized antibody captures the ligand and a second radiolabeled antibody which binds to a different epitope on the ligand is used to quantitate the ligand.
  • the amount of bound tracer antibody is directly proportional to the amount of bound ligand.
  • IRMA sensitivity depends more on the sensitivity of the detection device and less on the binding affinity of the antibody.
  • the sensitivity of IRMA has been limited by the backgrounds of current gamma counters (about 1 cps), and its use has been limited by the need to use hazardous quantities of radioisotope.
  • We stress that IRMA is a much more reliable assay method than ELISA because the enzymatic signal amplification in ELISA often leads to increased inter-assay variability.
  • the antibody conjugation conditions are modifiable but always selected so that at least 80% conjugation probability is achieved. All previous methods of read-out limited the sensitivity of immunoassays, and the optimal conditions were selected to improve S/B under the constraint that the signal itself is not smaller than say 20-25% of the maximum signal available. Thus, in prior art immunoassays, the S/B was not maximized but only optimized in the presence of limitations of the read-out system. MPD permits removal of these prior art limitations.
  • MPD permits a significant new assay protocol, the super immuno-radiometric assay, or SuperlRMATM, which provides quantitative measurement of biological substances at levels as low as a femtogram/ml.
  • SuperlRMA is an MPD-based sandwich assay which uses biological procedures similar to the immunoradiometric assay (IRMA).
  • IRMA immunoradiometric assay
  • the p24 antigen ELISA is a solid phase sandwich assay which tests for the HIV-1 virus. It is the most sensitive immunoassay currently available for HIV, and permits the measurement of viral load at early stages of infection. HIV-1 p24 antigen standard curves for the Retro-TekTM ELISA and a SuperlRMA were compared. In the ELISA, absorbance values ranged from 0.795 down to 0.052, corresponding to p24 concentrations of 125 pg/ml to 7.8 pg/ml, respectively.
  • a single label is attached to a secondary Ab, i.e., a single label is attached to either a molecule or virus/bacteria/cell.
  • NSBB Another source of limitation on IA's is NSBB due to secondary Ab's spurious binding to a solid state substrate.
  • an attomole/ml LOD is roughly equivalent to the selective detection of about 15,000 viruses/ml.
  • large biological targets e.g., viruses and bacteria thousands of labeled antibodies can be conjugated to a single target.
  • the limits of detection of the disclosed immunoassay method are a few tens of bacteria or cancer cells per ml.
  • Prior art immunoassays are limited to about attomole/ml sensitivity by a combination of too low a signal and limited specificity due to use of only two different antibodies.
  • the MPD is used to permit quantitation of the labels.
  • the signal/background ratio should be considerably improved. However, the signal amplitude is diminished considerably and, for triplet assays, becomes the limiting factor.
  • the disclosed immunoassay includes five novel features:
  • the main challenge is to implement immuno reagents which permit both increased signal and reduced non-specific biological background.
  • the disclosed implementation is quite complicated and very counter-intuitive. It involves a plurality of innovative steps never before used in immunoassays.
  • the label (either enzymatic, fluorescent or radioactive) is attached through the reporter group to one small molecule, and typically one or a few labels are conjugated per target molecule.
  • the target molecule nucleic acid or protein
  • polymers are used.
  • a good example are branched DNA constructs labeled with fluorophores used in DNA quantitation.
  • Another important example is the use of polyHRP in ELISA.
  • multiple labeling usually entails use of derivatizing agents such as Bolton-Hunter reagent (N-succinimidyl-3-(4-hydroxyphenyl)propionate) to create a number of active sites for radioiodination.
  • derivatizing agents such as Bolton-Hunter reagent (N-succinimidyl-3-(4-hydroxyphenyl)propionate) to create a number of active sites for radioiodination.
  • a goal is to produce a radioactive tracer that has at least fifty times higher specific activity than 125 I-streptavidin which has on average less than one 125 I molecule per streptavidin.
  • SuperTracers with high specific activity have been developed by BioTraces. All SuperTracers employ large molecular complexes that provide multiple sites for attachment of radioisotope and functional handles for attachment to ligand. Additionally, due to special steps taken in modifying the properties of dendrimeric complexes, the recent generation of SuperTracers exhibit lower nonspecific sticking and are generally easier to remove by washing. Implementations of SuperTracers. The first generation of SuperTracers consisted of large, essentially linear polymers appropriately radiolabeled.
  • Radioiodinated SuperTracers using: 1. PolyHRP - PolyHRP is a large conglomerate of dextran/avidin/HRP which has a molecular weight in excess of 2,000,000 and which is commercially available. The multiple avidin and HRP proteins on the complex provide multiple sites for radioiodination using a standard lactoperoxidase procedure. 2. Aminodextrans - Aminodextrans are large molecular complexes of amino acids and dextranwhich range in molecular weight from 10,000 to 2,000,000 and which are commercially available. Free amine groups exposed on the surface of these molecular complexes can be used to attach either 125 I or biotin.
  • radioiodination can be performed by means of Bolton- Hunter reagent and conjugation of biotin using a succinimidyl ester biotin conjugate.
  • a succinimidyl ester biotin conjugate We used a 500,000 MW aminodextran (Molecular Probes, Eugene, OR).
  • the labeled aminodextrans were developed to permit bridge type reactions.
  • streptavidin served as the bridge between any kind of biotinylated detector antibody (or any biotinylated nucleic acid) and the biotinylated radioaminodextran.
  • polymeric derivatizing agents As an option to the use of biotin-avidin SuperTracers, larger polymeric multi-radioiodinated derivatizing agents can be synthesized to directly label antibodies, or any another type of detector molecule. Poly(4- hydroxystyrene) carboxy terminated chains can be synthesized to various lengths containing from 21 to 42 hydroxyphenyl groups for radioiodination. The radiolabeled polymer can then be chemically modified to produce either a carbonyl chloride or carbonyl hydrazide derivatizing agent for covalent attachment to free amine or carbonyl groups on target molecules, respectively. Preliminary experiments have been performed using this type of SuperTracer.
  • 2nd generation SuperTracers There are two important disadvantages of 1st generation SuperTracers.
  • the number of labels per polymer is a highly variable function of the iodination conditions. Due to steric hindrance, each polymer molecule has a different number of labels. Only when hundreds of polymers are averaged is a well defined mean value obtained.
  • An even more important disadvantage of the commonly used large linear polymers with multiple labels is their non-specific affinity to biological fluid components and surfaces of the containers used in biological assays. This problem can be easily overlooked in experiments at the femtomole/ml level which are marginally improved by increased signal, e.g., in ELISAs using polyHRP.
  • the present invention discloses a new class of SuperTracers based on "closed surface" dendrimers which are spherical polymers where polymerization is controlled in order to achieve step-wise propagation, and thus a preselected number of reporter groups (preferably amines) on the surface. Products of this step-growth are called generations, and the number of amine groups is doubled with each subsequent generation.
  • the possible range of dendrimer generations is 3-20, and preferred range is 8-12.
  • the highly branched structure of dendrimers in the latter range causes steric hindrance and forces an almost ideally spherical structure.
  • the selected dendrimer is a Starburst Polyethyleneimine (PEI) dendrimer.
  • the target molecule is labeled with dendrimers bearing multiple labels.
  • dendrimers Due to their spherical structure dendrimers have the smallest possible surface and non-specific binding is minimal in comparison with linear polymers. For example, an 8 generation dendrimer has diameter of only 97 Angstroms, and 1 ,024 amino groups on its surface.
  • dendrimers permit better elimination of non-specific binding by promoting more effective removal of weakly bound counterparts.
  • antigen- antibody complexes survive even very stringent washing.
  • 2nd generation SuperTracer Four distinctly different types of 2nd generation SuperTracer are made from the same radiolabeled dendrimer, followed by conjugation with one of four different functional groups.
  • Type 1 is conjugated with biotin using Pierce' s EZ-Link NHS-LC-LC-Biotin (Succinimidyl-6'-(biotinamido)-6-hexanamido hexanoate). This type can be used as is to directly bind to streptavidin or avidin conjugates. It also binds to biotin-DNA, biotin- antibody or other biotin-protein conjugates, provided that streptavidin is used as a bridge. The binding can be enhanced by first coating the biotin-SuperTracer with streptavidin before binding to biotin-conjugates.
  • Type 2 is conjugated with protected SH groups using SATA (N-Succinimidyl S-
  • This SuperTracer binds to maleimide-protein conjugates.
  • maleimide-streptavidin can be used to form an alternative type of Avidin-SuperTracer complex.
  • the problem with SH groups is that if oxidized they can form disulfide bonds with other SH groups. This kind of cross-linking should be avoided, and we demonstrated that it can be diminished by using protected SH groups which can be easily unprotected before binding to biotin.
  • Type 3 is conjugated with maleimide groups using Sulfo-SMCC (Sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohexane-1-caboxylate).
  • Sulfo-SMCC Sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohexane-1-caboxylate.
  • This SuperTracer will bind to reduced SH groups on proteins. It permits conjugation of antibodies to a SuperTracer. The different antibody subunits are held in place by disulfide bonds. Using gentle reducing conditions, such as mercaptolethylamine in the presence of EDTA, an antibody is split into mirror halves with exposed SH groups for conjugation with maleimide-SuperTracer.
  • the antibody- SuperTracer construct is essential for ultrasensitive immunochromatography.
  • Type 4 is conjugated with a DNA probe consisting of a double stranded DNA ending with a single strand of DNA.
  • a PNA linker to increase the specificity of the binding to single stranded target DNA.
  • the present invention comprises fully engineered, phosphate-based dendrimers, that contain only one linker group, and a strictly controlled number of reporter groups.
  • the dendrimer is composed of several modules that can be engineered and modified according to specific needs of the final application.
  • the Linker arm is a chain that provides appropriate separation of the dendrimer from the biopolymer such as nucleic acid, protein, peptide, lipid and the like, to avoid steric hindrance during bindging.
  • the linker arm contains carbon atoms with the most preferred embodiment being composed of repeating units of tetraethylene glycol phosphate, that provides appropriate hydrophilicity.
  • tetraethylene glycol phosphate can be used as a building unit such as w-O-(4,4'- dimethoxytriphenylmethyl)- 1 -O-(2-cyanoethyl)-N,N-diisopropyl tetraethylene glycol phosphoramidite (Fig. 1) in phosphoramidite method providing well defined and strictly controlled length of the linker.
  • Dendrimer.T e dendrimer is composed of units that provide an increase of the number of the functionalities available for the chemistry in the next step.
  • dendrimer is built from glycerol-2-phosphate functionalities, where step-wise synthesis occurs on the linker arm and each coupling step results in double amount of functional groups available in the next chemical transformation.
  • the dendrimer is synthesized using phosphoramidite method and (1 ,3-O,O-bis(4,4'-dimethoxytriphenylmethyl)-2-O-(2-cyanoethyl)-N,N- diisopropylglycerolphosphoramidite (Fig.) as a building block.
  • functional groups are of hydroxyl type. Throughout the text the expression "generation" will be used when referring to the number of functional groups.
  • dendrimer building steps can be alternated with additional linker building steps resulting in formation of many dendrimers on an original dendrimer core (mega-dendrimer) as exemplified in Fig. 4 where four dendrimers are built on original 2nd generation core.
  • This higher order structure will be referred to as "dimension”. That is, there are 2 X branches in the first dimension and 2 y branches in the second dimension or 2 x+y total branches.
  • a two-dimensional dendrimer as in Figure 9 is called dendrimer
  • Reporter group is a chemical moiety that is required for further derivatization and can be one of but not limited to amine, sulfhydryl, maleimide, active ester such as succinimidyl, carboxyl, allyl, alkyl, aryl, acyl, halogenoalkyl, halogenoaryl, phosphoryl, phosphorothioyl, alkyl or aryl disulfide, nucleosidyl, oligonucleotidyl, peptidyl, and the like.
  • Choice of the reporter group is based on specific implementation of the dendrimer. It is understood that should the hydroxyl be required, the functional group becomes reporter group.
  • Terminus is a label to be used in further implementation. Introduction of the terminus into the dendrimer can be achieved with or without the reporter group.
  • radiolabels such as Bolton-Hunter reagent and its [ l 5 I]iodinated derivative; P o phosphate; S phosphorothioate; * chelated metals such as Eu +3 ;
  • antigens such as digoxigenine or bromouracyl
  • fluorophores such as fluorescein, acridine
  • assymetric reagent can be used in order to provide assymetric forking where one of the branches is transformed into a reporter group, and the other is used for further dendrimer generations.
  • the resulting dendrimer has reporter groups, and alternatively the labels, buried under subsequent layers of the dendrimer. This feature can be important when non-specific biological binding is major concern and the surface of the dendrimer can be used for "passivation" with reagents known to provide least non-specific binding.
  • Linker arm terminus is the moiety that is generated after appropriate cleavage from the solid support and deprotection of the dendrimer on the end of the linker opposite to the dendrimer.
  • the linker arm terminus is covalently attached to the oligonucleotide.
  • oligonucleotide is assembled first on the solid support and the appropriate modules are chemically attached to the 5' end of the oligonucleotide.
  • the linker arm terminus can be derivatized according to known chemical methods such as use of amine generating supports. Appropriate chemistry can be used to obtain similar variety of chemical moieties as described above for reporter groups.
  • Figure 1 is a schematic structural representation of a bioreagent according to the invention and which is an example of the 4 th generation having 16 reporter groups R 2 and linker arm terminal group Rj , for example, an affinity group or reactive group
  • Figure 2 is a schematic structural representation of tetraethylene glycol repeating units of a linker arm moiety;
  • Figure 3 is a schematic structural representation of a forking moiety or unit in a polymer
  • FIG. 4 is a schematic structural representation of a bioreagent which is a SuperTracer according to the invention and which is an example of the dendrimer [2,2], i.e., two dimensions and two generations each, with 16 reporter groups R 2 ;
  • Figure 5 is a schematic flow diagram of a method for preparing dendrimers according to the invention using phosphoramidite as the forking material;
  • Figure 6 is a schematic structural representation of a phosphoramidite - tetraethylene glycol monomer for producing a linker arm polymer
  • Figure 7 is a schematic structural representation of an embodiment where Ri is an oligonucleotide
  • Figure 8 is a schematic structural representation of a branching monomer reagent to provide the branching unit of Figure 3;
  • Figure 9 is a schematic structural representation of a bioreagent which is a
  • Figure 10 is a schematic structural representation of a substrate for making amino end groups for functionalizing
  • Figure 11 schematically illustrates a reaction between a Bolton-Hunter reagent and the amino terminal groups of the forking unit chains to provide an iodinated terminal reporting group for use in the method of the invention
  • Figure 12 is a schematic structural representation of a substrate for asymmetric forking (DMT-levulinyl glycol phosphoramidite. DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • Starburst dendrimers rely on cationic alkylamine chemistry. As described in Serial No. 60/065,063 filed November 10, 1997, incorporated herein by reference, with a starburst dendrimer, there are about 1000 active amino groups. One can iodinate up to 100-200 of them. One can passivate by attaching a "shell" PEG with chain length of 2000-3000 having low specific binding. The ratio of affinity probe to starburst dendrimer is statistical, not one- to-one or stoichiometric.
  • the inventive dendrimers relies instead on anionic phosphate chemistry.
  • Prior use of phosporamidite chemistry has been limited to single forked moieties bearing two labels, or with linear chains with one arm of each fork being labeled, the other forming a chain.
  • a highly branched three dimensional dendrimer is formed.
  • One can iodinate the dendrimer inside and outside the branched spherical structure, and can passivate the surface.
  • the ratio of dendrimers to affinity group probes is stoichiometric, typically one to one.
  • the dendrimers are useful in any type of bioassay, including DNA, immunoassay, protein-ligand interactions, and so on.
  • the dendrimers provide extraordinary signal amplification.
  • a library of oligonucleotide sequences or other terminal active groups may be prepared and used to select the appropriate analyte of interest.
  • the method of preparing the dendrimers is advantageous. It can be anhydrous, and handling of the solid supports is easy.
  • the dendrimers may be constructed, dried, stored as a powder, and used when needed. The appropriate number may be counted or estimated microscopically by counting beads for precise quantitative chemistry.
  • the inventive dendrimer has several advantages. It increases signal where a signal is weak, for example, a single copy or a few copies of DNA. Signal amplification allows fast measurement.
  • the inventive dendrimer also diminishes non-specific background because it permits exponential washing as discussed in U.S.S.N 60/065,065, filed November 10, 1997, incorporated herein by reference.
  • “Exponential” or stringent as it refers to washing conditions is a term of art understood by those of ordinary skill to refer to those conditions of dilution, turbulence, forcefulness, temperature, chaotrophic acidity, buffer and ionic strength which permit association of a particular molecule with a conjugate molecule for which it has an intended binding affinity, while substantially inhibiting the association of molecules which have no intended binding affinity. Washing may be done under aggressive physical conditions such as ultrasonic, shade wave, vortex, agitation or by using streams of medium.
  • a stringent wash step is directed at reducing background at least about twice as effectively as reducing signal.
  • Signal may be reduced below half of the original amount if background is reduced to less than a quarter and so on.
  • signal is no more than about 25% of the preceding level and background is less than about 10%.
  • the signal may be reduced to about 20% or less by washing, while background may be reduced to less than about 1%.
  • Appropriate conditions may be determined for each specific interaction using a standard control and a level of experimentation that is not considered to be undue by those of skill in the art. However, it is not easy to implement such washing because it requires high enough forces to break non-specific binding without breaking specific binding.
  • a linker is long enough to reduce steric inference between the probe and its target at one end, and the spherical forking unit with reporter groups at the other.
  • the inventive structure is built from the probe, from scratch.
  • the chemistry is well known and has high coupling efficiencies in the range of 99.7%. It works well with solid supports and provides modules that can be arranged in many ways.
  • a nucleic acid probe is attached to the dendrimer, it can be made in the same equipment as the linker and forking unit, as in Figure 5.
  • Each dendrimer is made one by one, and when the synthesis is finished, it is cleaved from the CPG glass balls.
  • phosphoramidite chemistry and equipment may be adapted from automatic DNA synthesis to make the inventive dendrimers, using phosphoramidite reagents instead of nucleosides. See Figure 5.
  • a nucleotide is constructed or attached to a solid support and then it is reacted with the phosporamidite linker monomer of Figure 6.
  • Detritylation removes the protecting groups on the reactive group, as in Figure 5.
  • DMT is a protecting group (dimethoxytriphenymethyl or dimethoxytrityl).
  • a hydroxyl group of the nucleotide in the general method reacts with phosphoramidite attacking the phosphorus with release of amine and then the chain is prolonged or elongated by one building unit, as shown in Figure 5 and 7. The whole cycle is repeated with oxidation and other technical transformations, each cycle adding new building blocks.
  • the linker is a tail that provides some space between the oligonucleotide and forking unit dendrimer.
  • the spacer may be a polymer made of tetraethylene glycol phosphoramidite monomers, as shown in Figures 2 and 7. The monomers are shown in 6. Tetraeethylene glycol is protected at one end with a compatible group such as dimethoxytrityl. This is reacted with DNA on a solid support and gives the same reaction as with normal coupling.
  • a forking monomer is shown, and it is shown as a unit of a polymer in Figure 3.
  • This reagent after coupling, oxidation, and deprotection, provides a doubling of active hydroxyl groups, as shown in Figure 3. Instead of extending chain length, each coupling using the forking monomer doubles the terminal ends to provide forking.
  • Figure 9 shows a resultant dendrimer after six levels of doubling of the dendrimer, having 2 6 or 64 branches. After building required generations with preset numbers of hydroxyl groups, hydroxyl groups are converted into amino groups which are the most compatible with all conjugation reactions on macromolecules. 90% of derivatising agents are directed to amino group and again this can be easily done as with Figure 10.
  • Figure 10 shows a protected substrate for providing amino groups. It is an amino spacer, a derivative of hexamethylene amino hexanol protected with monomethoxytrityl (MMT). MMT is more stable with amino groups than dimethoxytrityl.
  • This reagent is reacted with as many as all hydroxyl groups on the forking unit dendrimer to convert them into amino groups. Then the amino groups can be efficiently and easily iodinated e.g. with Bolton Hunter reagent or otherwise. As shown in Figure 11, the Bolton Hunter reagent may be mono- or di-iodinated (as shown with square brackets).
  • the reagent optionally contains a sulfate group to make the reagent soluble in water. Without the sulfate group, the reagent is more soluble in organic solvents. This is a simplest example of building a multi-labeled dendrimer.
  • An advantage of the phosphoramidite chemistry with DMT protecting groups is that for each coupling cycle, when this group is removed, it provides a colorimetric assay that shows the efficiency of the cycle.
  • the residue of DMT is red in the solution and can be measured by conventional assays to confirm that, up to iodination, the dendrimer is properly constructed. After each step, if saturation was not achieved, further treatment such as passivating is possible.
  • Figure 4 shows a skeletal model of the beginning of a four ball dendrimer. According to computer modeling of the forking unit, the diameter of each ball is about 40 Angstroms, as shown in Figure 9.
  • selective deprotection permits use of one arm for iodination, and the other arm for the next generation.
  • Figure 12 shows an asymmetric reagent that can be used with one of the two arms selectively protected. Then one of the arms can be used for conversion into amino group and iodination and the other arm can be used for growing next generations.
  • Figure 9 at the point marked "1" one can stop growth and after synthesis, deprotect and iodinate.
  • iodination will be buried under the subsequent layers of the forking reagent and this can be done at any preselected generation.
  • This provides more reporter groups, avoiding the following problem.
  • Non-specific background is typically surface proportional, but surface-bound signal is also limited by surface area.
  • the surface can be passivated, i.e., coated with polyethylene glycol or something else that provides very little interaction with biological systems like proteins or DNA or cells.
  • the dendrimer is a type of hydrogel or ionic polymer, similar in some respects to DNA.
  • a forking unit On the outside of a forking unit one can put various labels other than or in addition to iodine. Other halogens may be appropriate, or other reporter groups.
  • the linker arm terminal group may be any affinity group or reactive group, such as those described in U.S.S.N. 08/679,671, filed July 12, 1996, incorporated herein by reference.
  • the reporter group may be any radiolabel for MPD or other radiodetection, or it may be another reporter group for e.g. enzymatic, immunoassay, fluorescence, or so on.
  • the common elements remain the linker arm and spherical forking unit or units.
  • Preferred implementation of the proposed phosphate-based dendrimer is schematically shown on Figure 1 , where elements of the proposed structure are shown after deprotection and cleavage from the solid support. In this example the terminus groups are not attached yet.
  • the synthesis is preferably performed on the solid support and using phosphoramidite method analogous to the one used in the oligonucleotide chemistry. After deprotection of the support the linker reagent is added (w-O-(4,4'- dimethoxytriphenylmethyl)- 1 -O-(2-cyanoethyl)-N,N-diisopropyl tetraethylene glycol phosphoramidite, Figures 2 and 6) in the presence of tetrazole.
  • This reagent is protected with a monomethoxytrityl group (4- monomethoxytriphenylmethyl, MMT), which is more appropriate for the N-protection, but provides similar quantitative assay of the coupling efficiency as the DMT group.
  • MMT monomethoxytrityl group
  • the last coupling of aminomodifier can be followed by deprotection of the MMT group, and then the reaction column can be stored at low temperature and used as a stock for further reaction with Bolton-Hunter reagent ( Figure 11).
  • SuperTracers with low NSBB may permit further diminishment of nonspecific biological background (NSBB).
  • Second and third generation SuperTracers are essentially spherical in shape and very large. Thus, there is a mismatch between its size and the size of topological defects on the surface of the plastic. Furthermore, the forces imparted on the dendrimer by streaming washing liquid are very large, i.e., stronger than the chemical forces of non-specific binding, and washing is therefore very efficient. However, these forces are lower than the strength of covalent bounding, biotin-avidin or antibody-epitope binding. The NSB kinetics suggest a saturation phenomenon.
  • Direct quantitation of DNA using the SuperTracers Direct DNA quantitation will become a necessary tool in assays both for genome incorporation tests as well as expression modulation tests using mRNA screening.
  • the tool of choice is polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • methylated DNA PCR fails and direct hybridization based methods should be developed.
  • First method to be used with the phospate based SuperTracer are hybridization tests on the membranes: dot blot, and Southern blot. The first involves immobilization of the long DNA target on the membrane (positively charged) and hybridization of the labeled probe - usually labeled oligonucleotide.
  • Southern blot involves separation step using electrophoresis, and subsequent transfer of the DNA onto the membrane (blotting). Again detection is achieved using labeled hybridization probe.
  • Super tracer by introduction of multiple label attached through the linker arm to the 5'-end of the oligonucleotide provides strong signal without duplex destabilization.
  • membranes introduce significant background due to non specific binding of the probe. This background becomes very pronounced when the quantitation is at the attomole level.
  • An alternative method that provides diminished background involves use of primary hybridization probe for the target DNA.
  • This primary probe is immobilized on an appropriate support, preferably polystyrene plates or magnetic beads. Immobilization can be achieved either by using chemical methods or biotin-streptavidin binding.
  • Hybridization of the target DNA in this case serves also as a purification step and allows using a mixture of DNA fragments e.g., resulting from restriction enzyme digestion. Subsequently, labeled probe is used followed by detection/quantitation.
  • This pseudo-sandwich assay uses additional coincidence i.e., the signal is obtained only when both probes are hybridized, which makes it equivalent to logic operator AND.
  • a third method that provides increased specificity is based on the format described above (capture of the target strand on an immobilized probe). The difference is in the use of a third probe, where target DNA, detection probe and auxiliary probe form a cruciform-type structure of three "branches".
  • the branch formed by the detection and auxiliary probes is designed to contain cleavable ⁇ Switch ⁇ structure, preferably restriction enzyme cleavage site.
  • appropriate cleavage reagent preferably restriction enzyme is used, and quantitation is done only on the material released from the solid support. This method introduces an additional proof-reading step that increases specificity of the whole assay.
  • the preferred method for dqDNA involves a place on the Double stranded DNA that can be cleaved in a controlled manner (environment).
  • the preferred implementation uses double stranded DNA and a sequence recognized by an appropriate restriction enzyme as a ⁇ Switch ⁇ . This allows the implementation of a large family of switches, because there are a few hundred restriction enzymes. For example, one can use the family of restriction enzymes with the highest specificity, i.e., those restriction enzymes which recognize the sixmer pattern of oligonucleotides.
  • a particular implementation uses high specificity restriction enzymes selected from the following list:
  • restriction enzyme site consisting of a six nucleotide pattern is quoted within in the bracket.
  • a particularly advantageous implementation of the invention uses restriction enzyme sites which contain only G and C bases, i.e., nucleotide patterns which after complementation to double stranded DNA fragments have a high melting temperature.
  • the said restriction enzymes are indicated in the above list by the underlined base sequences.
  • the recognition site is a restriction enzyme site of the two stranded DNA used as ⁇ Switch/linker ⁇ .
  • ⁇ DNA ⁇ i is a two stranded DNA
  • ⁇ RES ⁇ s is a restriction enzyme site
  • ⁇ DNA ⁇ ⁇ two stranded DNA linker ⁇ + ⁇ single stranded DNA ⁇
  • ⁇ ST/DNA c2 ⁇ is a SuperTracer conjugated to a single stranded DNA the end of which is complementary to a single stranded DNA which is part of ⁇ DNA ⁇ .
  • this construction permits us to operate with reagents which are not radioactive, and then perform the step of conjugating the radiolabeled conjugate the ⁇ ST/DNA c2 ⁇ only after stringent wash.
  • the "three-probe" direct DNA quantitation using SuperTracers.
  • the disclosed implementation of the "three-probe" DNA quantitation consists of a series of steps:
  • each of the washes in steps 2, 4 and 6 should be performed in optimal conditions, which may involve a change of temperature and/or pH. Furthermore, step 7, i.e., adjustment of the buffer is necessary if sub-attomole sensitivity is required.
  • SuperTracer can be attached to antibodies by using the innovative procedures described in the following.
  • Amino groups can be attached to DNA molecule and can be utilized for derivatization with a number of available agents. Preferably the amino group is reacted with active carboxyl esters such as 4-nitrophenyl, succinimidyl, sulfosuccinimidyl, or pentachlorophenyl. If anhydrous conditions can be appropriate it is possible to use a reaction with acyl or sulfonyl chlorides.
  • oligonucleotides can be modified at the 5' end to incorporate reporter groups on an appropriate linker.
  • the oligonucleotide contains an amino or mercapto group at the 5' end. It is possible to introduce other groups, but most preferable are those that are stable in deprotection conditions (i.e., aqueous ammonia at elevated temperature).
  • oligonucleotide is modified with a mercapto group at the 5' end and reacted with a maleimide group generated on the dendrimer surface by means of a reagent such as N-(4- maleimidobutyryloxy) succinimide ester. It is possible to achieve conjugation of an oligonucleotide to the dendrimer by using the following chemical reactions involving the amino group and the DNA:
  • photoactivated reagents such as 4-azidosalicylic, 2-nitro-4- azidophenyl, 4- azidophenyl, and the like; * active esters as described above;
  • phosphorothioate group on the DNA can be alkylated with a variety of alkylating agents such as iodoacetic acid.
  • the sandwich immunoassay enhanced by the use of SuperTracers.
  • the disclosed implementation of the 2-plex immunoassay consists of a series of steps: 1) capture the target on a solid surface using appropriate antibody Abi;
  • block using appropriate blockers e.g., the noniodinated starburst dendrimers; 6) stringent wash;
  • step 7 i.e., adjustment of pH before the conjugation of SuperTracers to biotinylated Ab 2 , is necessary if sub- attomole sensitivity is required.
  • the disclosed duplex immunoassay has considerable advantages over the classical sandwich immunoassay, because it allows considerable signal amplification.
  • a single reporter label e.g., a fluorphore or a single radiolabel
  • a large number of labels are attached to each Ab 2 . This leads to a 100 to 1,000 fold signal amplification.
  • a shorter incubation time is required which usually considerably diminishes the NSB and tends to improve the signal/background ratio.
  • the wash conditions may include higher temperature and cycling the pH from acidic to neutral to basic to neutral.
  • block using the appropriate blockers e.g., the noniodinated starburst dendrimer
  • the step of SuperTracer hybridization can be made more specific by temperature cycling around the melting temperature of the said specially designed DNA probe with appropriate washing. More specific hybridization can be achieved leading to elimination of this component of nonspecific background.
  • Our previous MPD enabled studies of the DNA hybridization background suggest that NSB of a few zeptomole is achievable.
  • the SuperTracer enhanced superfast immunoassay. Antibody binding is a very characteristic function of time. Typically, good binding probability, say above 90%, is achieved after quite a long period (longer than an hour). For short binding times, the binding probability is a linear function of time.
  • the slope depends on the antibody quality and buffer conditions, e.g., 37°C temperature increases the binding probability. It also depends on the format of the immunoassay, and usually the use of beads, e.g., magnetic beads is favored for short assay times. For a short binding time (10 minutes), the binding probability may be as low as 10%. Thus, in the case of sandwich assays the probability that both antibodies will find and bind to their respective epitopes is very small, say a few percent. Therefore, the use of both very sensitive detectors, e.g., MPD instrumentation and signal amplification, e.g., the use of SuperTracers, are especially advantageous for implementing immunoassays shorter than 10 minutes.
  • very sensitive detectors e.g., MPD instrumentation and signal amplification, e.g., the use of SuperTracers
  • the magnetic beads removal time is shorter than the duration of stringent wash and has been selected as the preferred implementation of the SuperTracer enabled "fast immunoassay”.
  • the preferred implementation of the disclosed 2-plex fast immunoassay consists of the following steps: 1 ) capture the target on magnetic beads using an appropriate antibody Ab i ;
  • the capture and conjugation steps are expected to take less than three minutes, each.
  • the conjugation of SuperTracer is expected to take less than one minute. Each wash step is expected to take about 15 seconds.
  • the read-out time may be as short as 30 seconds and all titration curves will be measured in parallel. Thus, a less than 10 minutes immunoassay seems feasible.
  • SuperTracers with an attached DNA probe are very important innovative reagents for a plurality of applications in which signal amplification is used to enable the direct detection and quantitation of nucleic acids interactions with other biological macromolecules. These methods are enabled by both the use of super-sensitive MPD technique and the signal amplification by means of radiolabeled SuperTracer. Once more, the main challenge is the rejection of non-specific biological background.
  • the application of SuperTracers to direct detection of DNA/protein interaction is disclosed in a patent application titled "New methods for DNA/protein interaction".
  • EXAMPLE 1 Synthesis of 4 th generation dendrimer on a 30-mer oligonucleotide probe.
  • Synthesis of the oligonucleotide hybridization probe was performed on the solid support using 0.2 ⁇ mol synthesis column (1000 A, Long Chain Alkylamine Controlled Pore Glass, LCA CPG, Applied Biosystems) with 5'-O-dimethoxytriphenylmethyl-(4-N-benzoyl)- 2'-deoxycytidine attached as a first building block.
  • the synthesis was performed in an automatic DNA synthesizer (Applied Biosystems, 380B).
  • Standard nucleoside phosphoramidites were purchased from Perkin Elmer (Applied Biosystems, MasterPiece, 500mg). Modified phosphoramidites were purchased from Clontech:
  • linker reagent - ( ⁇ -O-(4,4'-dimethoxytriphenylmethyl)-l-O-(2-cyanoethyl)- N,N- diisopropyl tetraethylene glycol phosphoramidite 1 (lOOmg).
  • linker reagent - (l,3-O,O-bis(4,4'-dimethoxytriphenylmethyl)-2-O-(2- cyanoethyl)- N,N-diisopropylglycerolphosphoramidite 2 (lOOmg).
  • Each nucleoside phosphoramidite was dissolved in 3.0ml of anhydrous acetonitrile.
  • Each modified phosphoramidite (1,2, and 3) was dissolved in 1.2ml of anhydrous acetonitrile.
  • DMT assay dimethoxytrityl cation assay
  • Solid support containing PDP5.4.1 can be stored at -20°C for at least several months. Prior to radioiodination reaction, the support is deprotected with trichloroacetic acid (3% w/v in methylene chloride, 3ml), and washed with acetonitrile.
  • trichloroacetic acid 3% w/v in methylene chloride, 3ml

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Abstract

L'invention concerne une nouvelle classe de bioréactifs fondée sur des dendrimères à 'surface fermée' comportant un bras de liaison, une fraction en fourche et une fraction rapporteur qui peuvent se conjuguer à une étiquette et à une biomolécule. Les bioréactifs comportent des polymères sphériques dans lesquels la polymérisation est régulée afin d'obtenir une propagation par étapes, et par conséquent un nombre présélectionné de groupes rapporteurs (de préférence des amines) sur la surface. Les produits de cette croissance par étapes sont appelés générations, et le nombre de groupes amines est doublé à chaque nouvelle génération. En ce qui concerne la présente invention, la fourchette de générations de dendrimères est d'environ 3-20, et la fourchette préférée est de 8-12.
PCT/US1999/004068 1998-02-25 1999-02-25 Dendrimeres a base de phosphate pour essais biologiques WO1999043287A2 (fr)

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Cited By (10)

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WO2001002861A1 (fr) * 1999-06-29 2001-01-11 Dako A/S Detection a l'aide de sondes et de marqueurs a dendrimeres
EP1136569A2 (fr) * 2000-03-24 2001-09-26 Bayer Corporation Sondes d'acides nucléiques qui ont des marqueurs non-nucléosidiques à haute hydrophilicité comprenant plusieurs autres marqueurs et leur utilisation
WO2001081924A2 (fr) * 2001-04-23 2001-11-01 Biotraces, Inc. Micro-arrangements reutilisables pour quantification de proteines peu abondantes
WO2002033412A1 (fr) * 2000-10-14 2002-04-25 Macrogen Inc. Support biologique et methode de preparation dudit support
KR100377946B1 (ko) * 2000-07-15 2003-03-29 한국과학기술원 덴드리머를 이용한 단분자막의 제조방법
WO2005026191A2 (fr) * 2003-09-18 2005-03-24 Posco Macromolecule a taille regulee
WO2007080114A2 (fr) * 2006-01-11 2007-07-19 Biotech Igg Ab Conjugué de macromolecule
JP2010151828A (ja) * 2003-09-18 2010-07-08 Posco サブストレート、製造方法、診断システム及び検出方法
EP2208998A2 (fr) * 2005-05-02 2010-07-21 ANP Technologies, Inc. Essais biologiques améliorés par conjugués polymères
EP3201628B1 (fr) * 2014-10-02 2020-11-18 Ventana Medical Systems, Inc. Polymères et conjugués comprenant ces polymères

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US5001072A (en) * 1984-05-23 1991-03-19 Icn Biomedicals Inc. Compositions and methods for multiple simultaneous immunoradiometric assay (IRMA) of analytes using radioisotope chelate labels
US5435990A (en) * 1988-06-24 1995-07-25 The Dow Chemical Company Macrocyclic congugates and their use as diagnostic and therapeutic agents
US5527524A (en) * 1986-08-18 1996-06-18 The Dow Chemical Company Dense star polymer conjugates

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US5001072A (en) * 1984-05-23 1991-03-19 Icn Biomedicals Inc. Compositions and methods for multiple simultaneous immunoradiometric assay (IRMA) of analytes using radioisotope chelate labels
US5527524A (en) * 1986-08-18 1996-06-18 The Dow Chemical Company Dense star polymer conjugates
US5435990A (en) * 1988-06-24 1995-07-25 The Dow Chemical Company Macrocyclic congugates and their use as diagnostic and therapeutic agents

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001002861A1 (fr) * 1999-06-29 2001-01-11 Dako A/S Detection a l'aide de sondes et de marqueurs a dendrimeres
EP1136569A2 (fr) * 2000-03-24 2001-09-26 Bayer Corporation Sondes d'acides nucléiques qui ont des marqueurs non-nucléosidiques à haute hydrophilicité comprenant plusieurs autres marqueurs et leur utilisation
EP1136569A3 (fr) * 2000-03-24 2004-01-28 Bayer Corporation Sondes d'acides nucléiques qui ont des marqueurs non-nucléosidiques à haute hydrophilicité comprenant plusieurs autres marqueurs et leur utilisation
KR100377946B1 (ko) * 2000-07-15 2003-03-29 한국과학기술원 덴드리머를 이용한 단분자막의 제조방법
WO2002033412A1 (fr) * 2000-10-14 2002-04-25 Macrogen Inc. Support biologique et methode de preparation dudit support
WO2001081924A2 (fr) * 2001-04-23 2001-11-01 Biotraces, Inc. Micro-arrangements reutilisables pour quantification de proteines peu abondantes
WO2001081924A3 (fr) * 2001-04-23 2003-08-28 Biotraces Inc Micro-arrangements reutilisables pour quantification de proteines peu abondantes
US9201067B2 (en) 2003-03-05 2015-12-01 Posco Size-controlled macromolecule
KR101125787B1 (ko) 2003-09-18 2012-04-12 주식회사 포스코 분자 크기 제어된 거대분자
AU2004272465B2 (en) * 2003-09-18 2008-11-06 Posco Size-controlled macromolecule
AU2004272465B8 (en) * 2003-09-18 2008-12-18 Posco Size-controlled macromolecule
JP2010151828A (ja) * 2003-09-18 2010-07-08 Posco サブストレート、製造方法、診断システム及び検出方法
WO2005026191A3 (fr) * 2003-09-18 2005-12-01 Posco Macromolecule a taille regulee
WO2005026191A2 (fr) * 2003-09-18 2005-03-24 Posco Macromolecule a taille regulee
EP2208998A2 (fr) * 2005-05-02 2010-07-21 ANP Technologies, Inc. Essais biologiques améliorés par conjugués polymères
US8563329B2 (en) 2005-05-02 2013-10-22 Anp Technologies, Inc. Polymer conjugate enhanced bioassays
US9176142B2 (en) 2005-05-02 2015-11-03 Anp Technologies, Inc. Polymer conjugate enhanced bioassays
WO2007080114A2 (fr) * 2006-01-11 2007-07-19 Biotech Igg Ab Conjugué de macromolecule
WO2007080114A3 (fr) * 2006-01-11 2008-03-27 Biotech Igg Ab Conjugué de macromolecule
EP3201628B1 (fr) * 2014-10-02 2020-11-18 Ventana Medical Systems, Inc. Polymères et conjugués comprenant ces polymères
US11079372B2 (en) 2014-10-02 2021-08-03 Ventana Medical Systems, Inc. Polymers and conjugates comprising the same

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