Methods for determining concentration of low and high concentration proteins in a single sample
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Publication Number
US-12313630-B2
Publication Date
2025-05-27
Expiration Date
Abstract
Disclosed herein are methods for determining a concentration of at least one low concentration protein and at least one high concentration protein in a biological sample.
Core Innovation
The method determines a concentration of at least one low concentration protein and at least one high concentration protein in a biological sample using an assay plate with a plurality of sample wells. In each sample well, first capture reagents are incubated with the biological sample to produce a first binding mixture, and the first capture reagents comprise first antibodies bound to a surface that selectively bind to the at least one low concentration protein.
The first binding mixture is mixed with second capture reagents to produce a second binding mixture. The second capture reagents comprise second antibodies bound to a surface that selectively bind to the at least one high concentration protein, and the mixing comprises increasing volume in each sample well to produce a second sample well volume at least ten-fold higher than the first sample well volume.
The at least one high concentration protein is expected to be present at a concentration at least ten-fold greater than an expected concentration of the at least one low concentration protein. The method further determines a concentration of the at least one low concentration protein and the at least one high concentration protein in the biological sample based on the second binding mixtures.
Claims Coverage
Independent claim clm-00001 provides the core method framework through two-stage selective antibody capture in assay plate wells followed by determining concentrations. Dependent claims refine the workflow with specific volume constraints, flow cytometry, bead-based capture surfaces, selected cytokine pairings, and control wells with known protein standards and normalization.
Two-stage selective antibody capture in assay plate wells for low and high concentration proteins
Incubating first capture reagents with the biological sample in sample wells to form a first binding mixture using first antibodies selectively binding the at least one low concentration protein, mixing the first binding mixture with second capture reagents to form a second binding mixture using second antibodies selectively binding the at least one high concentration protein, and determining concentrations of the at least one low concentration protein and the at least one high concentration protein.
Volume increase to enable high concentration binding after low concentration binding
Increasing the volume in each sample well during mixing to produce a second sample well volume at least ten-fold higher than the first sample well volume, where the high concentration protein is expected to be present at least ten-fold greater than the expected concentration of the low concentration protein.
Second sample well volume increased between 10-fold and 100-fold
Increasing the volume in each sample well to produce a second sample well volume that is between 10-fold and 100-fold higher than the first sample well volume.
Concentration determination by flow cytometry
Determining the concentration of the at least one low concentration protein and the at least one high concentration protein in the second binding mixtures in each sample well by flow cytometry.
Bead-based surfaces for both capture stages
Defining the first capture reagents as having a surface comprising beads and defining the second capture reagents as having a surface comprising beads.
Selected cytokine pairing combinations
Selecting combinations where a low concentration protein is IL-4, IL-6, or IL-17A and a high concentration protein is INF-gamma, TNF-alpha, or IL-2.
Control wells using a protein standard and normalization
Using two or more control wells on the assay plate incubated with a protein standard of known low and high concentrations to form control binding mixtures, determining the concentrations of the at least one low concentration protein and the at least one high concentration protein in the control binding mixtures, and normalizing the determined sample concentrations based on the control well results.
Across the independent claim framework and its refinements, the central inventive concept is a two-stage binding approach in assay plate wells using selectively binding first and second antibodies, coupled to a substantial sample well volume increase for enabling detection of both low and high concentration proteins, with optional tightening of the volume range, bead-based capture surfaces, flow cytometry readout, selected protein pairings, and control wells with protein-standard normalization.
Stated Advantages
Improved linear detection ranges for simultaneously detecting low detection and high-end ranges in the same sample, including a higher-end linear range shift for IFN-gamma and TNF-alpha and low detection for IL-4 in the same sample.
Documented Applications
Simultaneous protein quantification in a biological sample for at least one low concentration protein and at least one high concentration protein using a sandwich immunoassay workflow in assay plate wells.
Measurement of low/high cytokine pairs using the described two-stage selective capture, with example pairings including IL-17A with IFN-gamma and/or TNF-alpha, IL-6 with IFN-gamma and/or TNF-alpha, IL-4 with IL-2+TNF-alpha, IL-12 with TNF-alpha, sFas with Granzyme B, and GM-CSF with IL-6.
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