Activated GTPase-based assays and kits for the diagnosis of sepsis and other infections
In one embodiment, the invention provides a method of diagnosing sepsis or a virus-related infection (often a viral hemorrhagic fever infection) in a subject by detecting and measuring the level of a set of sepsis and virus infection-associated-GTPase biomarkers in a sample obtained from the subject using multiplexed flow cytometry. Related kits are also provided. In a preferred embodiment, the invention provides point of care diagnostic methods for determining an early stage sepsis or the severity of a virus infection, especially in a hospital or other setting.
1. A kit comprising:
(a) a population of beads which have two or more sizes, which are labeled with a first fluorophore having a single wavelength and a plurality of intensity levels and which are covalently coupled to a plurality of effector proteins which bind to cognate, infection-associated-guanosine triphosphate hydrolases (GTPases) to form effector protein-infection-associated-GTPase conjugates, wherein said GTPases are ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras-related protein 1 (Rap 1) and said effector proteins which bind to Rac is 1 are selected from the group consisting of p21 activated kinase 1 rho binding domain (PAK-1 RBD), steroid receptor RNA activator 1 (Sra1), insulin receptor tyrosine kinase substrate p58/53 (IRSp58/53), p21 activated kinase 1 (PAK1), p21 activated kinase 2 (PAK2), p21 activated kinase 3 (PAK3) and mixtures thereof and said effector protein which binds to Rap1 is ra1 guanine nucleotide dissociation stimulator rho binding domain (Ra1GDS-RBD);
(b) GTPase-specific antibodies which bind to said effector protein-infection-associated-GTPase conjugates formed on the beads;
(c) detector antibodies which are specific to the GTPase-specific antibodies and which are labeled with a second fluorophore having a wavelength (color) which is different from that of the first fluorophore; and
(d) optionally, instructions with respect to the use of the beads, GTPase-specific antibodies and detector antibodies in a flow cytometry assay for detecting the existence or absence of sepsis in a patient or subject in need.
2. The kit of claim 1 , wherein:
(a) the effector proteins are PAK-1 RBD and RalGDS-RBD (a; and
(b) the first fluorophore is a red fluorophore and the second fluorophore is a green fluorophore.
3. The kit of claim 2 , wherein said red fluorophore is a red fluorescent dye and the green fluorophore is a green fluorescent dye.
4. The kit of claim 1 , wherein the first fluorophore is a fluorescent dye and the second fluorophore is a fluorescent dye.
5. The kit of claim 1 , wherein:
(a) the population of beads is defined by distinct sub-sizes, each of which conducts a unique assay; and
(b) the beads are internally covalently coupled to red and infrared fluorophores with different intensities.
6. The kit of claim 1 , wherein the beads are microbeads.
7. The kit of claim 1 , wherein the kit contains at least 10,000 beads conjugated to said effector protein.
8. The kit of claim 1 , wherein the beads are defined by at least two distinct sub-sizes.
9. The kit of claim 1 , wherein the kit can be used in conjunction with a multiplex flow cytometry assay which processes 96 or 384 well-plates.
10. The kit of claim 1 , wherein the kit comprises a positive or negative control.
11. The kit of claim 1 , wherein said flow cytometry assay is a multiplex flow cytometry assay.
12. A kit comprising:
(a) a population of beads which have two or more sizes, which are labeled with a first fluorophore having a single wavelength and a plurality of intensity levels and which are covalently coupled to a plurality of effector proteins which bind to cognate, infection-associated-guanosine triphosphate hydrolases (GTPases) to form effector protein-infection-associated-GTPase conjugates, wherein said GTPases are ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras-related protein 1 (Rap 1) and said effector protein which binds to Rac 1 is p21 activated kinase 1 rho binding domain (PAK-1 RBD) and said effector protein which binds to Rap1 is ra1 guanine nucleotide dissociation stimulator rho binding domain (Ra1GDS-RBD);
(b) GTPase-specific antibodies which bind to said effector protein-infection-associated-GTPase conjugates formed on the beads;
(c) detector antibodies which are specific to the GTPase-specific antibodies and which are labeled with a second fluorophore having a wavelength (color) which is different from that of the first fluorophore; and
(d) instructions with respect to the use of the beads, GTPase-specific antibodies and detector antibodies in a multiplexed flow cytometry assay for detecting the existence or absence of sepsis in a patient or subject in need.