Multiplex Immune Effector Molecule Assay
AssigneeSouth Dakota State University
InventorsJane Christopher-Hennings, Steven Lawson, Eric Nelson, Ying Fang, Joan K. Lunney
Life of this patent
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Abstract
Methods for detecting at least seven cytokines in a porcine biological sample are provided. Also provided are multiplex assay kits that allow for the detection and quantification of the cytokines in a single reaction mixture. Use of the methods and kits for diagnosis, prognosis, and monitoring of immunity is also contemplated.
Claims21
A method of detecting the presence or concentration of a plurality of immune effector molecules in a biological sample, comprising:
- (a) incubating a biological sample under suitable conditions with (i) at least seven capture particles, wherein each capture particle is specific for an immune effector molecule, further wherein only one capture particle is specific for each immune effector molecule, and (ii) at least seven detection particles specific for the same immune effector molecules as the at least seven capture particles, wherein each detection particle comprises a reporter, further wherein only one detection particle is specific for each immune effector molecule;
- (b) forming a complex by (a) immobilizing each immune effector molecule to a capture particle specific for the immune effector molecule and (b) immobilizing each immune effector molecule to a detection particle specific for the immune effector molecule;
- and
- (c) measuring the presence or concentration of the reporter in a multiplex assay, wherein the presence or concentration of the reporter corresponds to the presence or concentration of each immune effector molecule in the biological sample.
A method of determining immunity status of a subject, comprising:
- (a) detecting the concentration of at least seven immune effector molecules in a biological sample of interest in a multiplex assay, and
- (b) determining immunity status of a subject based on concentrations of the at least seven immune effector molecules in the biological sample of interest.
A kit comprising reagents for simultaneously detecting at least seven immune effector molecules in a porcine biological sample of interest.
A method of detecting the presence or concentration of a plurality of immune effector molecules in a biological sample, comprising:
- (a) incubating porcine serum under suitable conditions with nine monoclonal capture antibodies, wherein the monoclonal capture antibodies are immobilized on a microsphere bead, further wherein each monoclonal capture antibody is specific for an immune effector molecule consisting of IL-1β, IL-4, IL-8, IL-10, IL-12, IFN-α, IFN-γ, or TNF-α;
- (b) forming a microsphere bead/monoclonal capture antibody/immune effector molecule complex;
- (c) washing the microsphere bead/monoclonal capture antibody/immune effector molecule complex in a buffer;
- (d) incubating the micro sphere bead/monoclonal capture antibody/immune effector molecule complex under suitable conditions with nine biotinylated monoclonal detection antibodies, wherein each monoclonal detection antibody is specific for one microsphere bead/monoclonal capture antibody/immune effector molecule complex;
- (e) forming a microsphere bead/monoclonal capture antibody/immune effector molecule/monoclonal detection antibody complex;
- (f) washing the microsphere bead/monoclonal capture antibody/immune effector molecule/monoclonal detection antibody complex in a buffer;
- (g) incubating the microsphere bead/monoclonal capture antibody/immune effector molecule/monoclonal detection antibody complex with strepavidin-R-phycoerthrin,
- (h) washing the microsphere bead/monoclonal capture antibody/immune effector molecule/monoclonal detection antibody/strepavidin-R-phycoerthrin complex in a buffer;
- and
- (i) detecting the presence or concentration of a phycoerthrin fluorescence by flow cytometry, wherein the presence or concentration of phycoerthrin fluorescence corresponds to the presence or concentration of each immune effector molecule in the porcine serum.
Dependencies are inferred from the claim text.