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US 2019/0154677 A1Published application
Method of multiplex immunoassays utilizing differential affinity and methods for synthesizing aptamer-based reagents for multiplex immunoassays
OwnerChin-Yih Hong, Herng-Er Hornginventors
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Abstract
Multiplex immunoassays utilize the differential affinities among the conjugation pairs between the capture ligands and target analytes are proposed. Window magnetic-assisted rapid aptamer selection (window-MARAS) methods for selecting aptamers with desirable affinity toward the target analytes and methods for generating reagents for multiplex immunoassays or multiplex detection in one assay by utilizing the selected aptamers as capture ligands in reagents are described and used to demonstrate the feasibility of multiplex immunoassays based on the differential affinity of conjugation pairs between the capture ligands and target analytes.
Claims16
A method of aptamer-based multiplex immunoassay for detecting target analytes in a biological sample, comprising:
- a) providing one or more types of aptamers having a different binding affinity toward each type of I types of target analytes (i), wherein i is a variable integer with a value from 1 to I and each type of I types of target analytes (i) is a different type of target analytes from the others;
- b) providing a reagent to a multiplex immunoassay by supplying the one or more types of aptamers from step a) as capture ligands that can bind to all of I types of different target analytes (i), wherein the aptamers are dispersed in a first binding buffer, and heated and quenched to induce the formation of secondary structures;
- c) preparing a plurality of magnetic particles conjugated with a plurality of biomolecules in a biological sample to form sample-MPs, wherein the magnetic particles (MPs) are nanoparticles (MNPs) or microparticles (MMPs) and the biological sample constitutes of a plurality of various types of biomolecules;
- d) incubating the sample-MPs from step c) with the reagent from step b) to form an aptamer-sample bound mixture denoted a bound mixture in the multiplex immunoassay, wherein if the biological sample contains at least one type of I types of target analytes (i), the aptamers in the reagent and the at least one type of I types of target analytes (i) in the biological sample form at least one type of target analyte-MPs (PS-MPs(i));
- e) subjecting the bound mixture to an affinity differentiating means to differentiate and collect different fractions containing aptamers capable of binding to a different and corresponding type of I types of target analytes (i) in the sample-MPs, wherein the binding affinity between the aptamers and each type of I types of target analytes (i) is different from the others and each fraction (i) contains only aptamers with a specific range of binding affinity toward a corresponding type of I types of target analytes (i) in the biological sample;
- f) detecting and quantifying the quantity of the aptamers in each collected fraction (i) obtained in step e) and converting the measured quantity of the aptamers in each collected fraction (i) to the quantity of each type of I types of target analytes (i) by using a corresponding predetermined standard calibration curve for each type of I types of target analytes (i), respectively, to quantity each type of I types of target analytes (i) in the biological sample.
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A method of multiplex immunoassay for detecting target analytes in a biological sample, comprising:
- a) providing one or more types of capture ligands capable of binding to all of I types of target analytes (i), wherein i is a variable integer with a value from 1 to I, each type of I types of target analytes (i) is a different type of target analytes from the others, and the binding affinity of the capture ligands toward each corresponding type of I types of target analytes (i) is different from the others;
- b) providing a reagent constituting of the one or more types of capture ligands from step a) to a multiplex immunoassay, wherein the one or more types of capture ligands are dispersed in a first buffer;
- c) providing a biological sample constituting of a plurality of various types of biomolecules to the multiplex immunoassay, wherein the various types of biomolecules include none or more types of I types of target analytes (i);
- d) incubating the biological sample from step c) with the reagent from step b) to form a ligand-sample bound mixture in the multiplex immunoassay, wherein if the biological sample contains at least one type of target analytes (i), the capture ligands in the reagent and the at least one type of target analytes (i) in the biological sample form at least one type of ligand-analyte complex (i);
- e) subjecting the ligand-sample bound mixture to an affinity differentiating means to differentiate and collect different fractions, wherein each collected fraction (i) contains a specific type of ligand-analyte complex (i), and wherein the type of ligand-analyte complex (i) and the binding affinity of the capture ligands toward the corresponding type of target analytes (i) forming the ligand-analyte complex (i) in each collected fraction (i) are different from the others;
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- f) measuring the target analytes (i) of the ligand-analyte complex (i) in each collected fraction (i) and quantifying each type of I types of target analytes (i) in the biological sample by a detection means.
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Dependencies are inferred from the claim text.