# US 9,005,910 B2 · Complex of labeled probes and water-soluble carrier > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,005,910 B2 - Document ID: US9005910B2 - Status: Granted - Kind: B2 - Application number: 13/640,821 - Filed: 2011-04-13 - Published: 2013-09-05 - Granted: 2015-04-14 - Earliest claimed: 2010-04-14 - Assignees: Eiken Kagaku Kabushiki Kaisha; Advanced Life Science Institute, Inc. - Applicants: Yoshiyuki Ohiro; Susumu Takayasu - Inventors: Yoshiyuki Ohiro; Susumu Takayasu - Web page: https://patentgrep.com/patent/US9005910B2 ## CPC - G01N 33/581: {with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)} - G01N 33/532: Production of labelled immunochemicals - G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)} - G01N 33/582: {with fluorescent label} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The purpose is to produce, with high reproducibility, a complex of labeled probes and a carrier, said complex being to be used for detecting and measuring a target substance to be measured with high sensitivity and high stability. The means for accomplishing the purpose is that a label is bound to a probe-water soluble carrier conjugate using specific binding of an avidin compound such as avidin, streptavidin, etc. to biotin, and the binding of the avidin compound to the probe is performed before the binding to the carrier. Namely, after conjugating the avidin compound to a substance which is capable of binding to the target substance, the conjugate is bound to a high-molecule water-soluble carrier to produce a complex of the avidinized probes and the water-soluble carrier. Then the complex of the avidinized probes and the water-soluble carrier is mixed with a biotinylated label. Thus, a stable complex of the labeled probes and the water-soluble carrier, which enables the highly sensitive detection and measurement of the target substance, can be obtained with high reproducibility via the specific binding of the avidin compound to biotin. ## Claims ### Claim 1 A method for producing a complex of labeled probes and a water-soluble carrier, comprising the following steps: (a) reacting a probe having one or more thiol groups with an avidin compound having one or more maleimide groups to obtain a probe conjugate, wherein the probe is selected from the group consisting of protein G, protein A, protein L, lectin, and at least one antibody or antigen binding fragment thereof that is selected to bind to a target substance; (b) introducing one or more thiol groups into the probe conjugate and reacting said probe conjugate having one or more thiol groups with a high-molecular water-soluble carrier having one or more maleimide groups to obtain a complex of the probe conjugate and the water-soluble carrier; (c) mixing the complex of the probe conjugate and the water-soluble carrier with a biotinylated label so that the avidin compound in the complex of the probe conjugate and the water-soluble carrier reacts with biotin in the biotinylated label to form the complex of labeled probes and the water-soluble carrier; and (d) isolating the complex of labeled probes and a water-soluble carrier. ### Claim 2 (depends on 1) The method according to claim 1, wherein the avidin compound is avidin or streptavidin. ### Claim 3 (depends on 1) The method according to claim 1, wherein the water-soluble carrier has a molecular weight of 500,000 or more. ### Claim 4 (depends on 1) The method according to claim 1, wherein the water-soluble carrier is selected from the group consisting of dextran, aminodextran, dextrin, cluster dextrin, ficoll, and pullulan. ### Claim 5 (depends on 1) The method according to claim 1, wherein the probe being capable of binding to a target substance is the at least one antibody or antigen binding fragment thereof. ### Claim 6 (depends on 1) The method according to claim 1, wherein the probe being capable of binding to a target substance is two or more kinds of antibodies or antigen binding fragments thereof. ### Claim 7 (depends on 5) The method according to claim 5, wherein the antibody or the antigen binding fragment thereof is selected from the group consisting of Fab′, F(ab′)2, Fab, IgG, and a combination thereof. ### Claim 8 (depends on 1) The method according to claim 1, wherein the biotinylated label is selected from the group consisting of biotinylated luciferase, biotinylated alkaline phosphatase, biotinylated peroxidase (POD), biotinylated glucose oxidase (GOD), biotinylated fluorescein isothiocyanate (FITC), biotinylated acridinium, biotinylated acridinium derivative, and biotinylated tris(2,2′bipyridyl)ruthenium(II). ### Claim 9 (depends on 8) The method according to claim 8, wherein the biotinylated luciferase is produced by genetic recombination. ### Claim 10 A complex of labeled probes and a water-soluble carrier produced by a method comprising the following steps: (a) reacting a probe having one or more thiol groups with an avidin compound having one or more maleimide groups to obtain a probe conjugate, wherein the probe is selected from the group consisting of protein G, protein A, protein L, lectin, and at least one antibody or antigen binding fragment thereof that is selected to bind to a target substance; (b) introducing one or more thiol groups into the probe conjugate and reacting said probe conjugate having one or more thiol groups with a high-molecular water-soluble carrier having one or more maleimide groups to obtain a complex of the probe conjugate and the water-soluble carrier; (c) mixing the complex of the probe conjugate and the water-soluble carrier with a biotinylated label so that the avidin compound in the complex of the probe conjugate and the water-soluble carrier reacts with biotin in the biotinylated label to form the complex of labeled probes and the water-soluble carrier; and (d) isolating the complex of labeled probes and a water-soluble carrier. ### Claim 11 (depends on 10) The complex of labeled probes and a water-soluble carrier of claim 10, wherein the avidin compound is avidin or streptavidin. ### Claim 12 (depends on 10) The complex of labeled probes and a water-soluble carrier of claim 10, wherein the water-soluble carrier has a molecular weight of 500,000 or more. ### Claim 13 (depends on 10) The complex of labeled probes and a water-soluble carrier of claim 10, wherein the water-soluble carrier is selected from the group consisting of dextran, aminodextran, dextrin, cluster dextrin, ficoll, and pullulan. ### Claim 14 (depends on 10) The complex of labeled probes and a water-soluble carrier of claim 10, wherein the probe being capable of binding to a target substance is the at least one antibody or antigen binding fragment thereof. ### Claim 15 (depends on 10) The complex of labeled probes and a water-soluble carrier of claim 10, wherein the probe being capable of binding to a target substance is two or more kinds of antibodies or antigen binding fragments thereof. ### Claim 16 (depends on 14) The complex of labeled probes and a water-soluble carrier of claim 14, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of Fab′, F(ab′)2, Fab, IgG, and a combination thereof. ### Claim 17 (depends on 10) The complex of labeled probes and a water-soluble carrier of claim 10, wherein the biotinylated label is selected from the group consisting of biotinylated luciferase, biotinylated alkaline phosphatase, biotinylated peroxidase (POD), biotinylated glucose oxidase (GOD), biotinylated fluorescein isothiocyanate (FITC), biotinylated acridinium, biotinylated acridinium derivative, and biotinylated tris(2,2′bipyridyl)ruthenium(II). ### Claim 18 (depends on 17) The complex of labeled probes and a water-soluble carrier of claim 17, wherein the biotinylated luciferase is produced by genetic recombination. ### Claim 19 (depends on 6) The method according to claim 6, wherein the antibodies or the antibody fragments are selected from the group consisting of Fab′, F(ab′)2, Fab, IgG, and a combination thereof.