COVID-19 Cytokine Storm Multiplex Assays

Cytokine Release Syndrome (CRS) is a systemic inflammatory response characterized by the mass release of pro-inflammatory cytokines from immune cells. It is alternatively referred to as a cytokine storm.

Cytokines are small protein messengers that primarily function in facilitating communication between cells. They are secreted by immune cells and act on nearby cells (that possess a receptor specific to the cytokine), leading to a change of behaviour of such cells. Cytokines are an essential part of the immune response that maintain regular homeostasis by fighting infection and controlling inflammation. However, if cytokine production is dysregulated this can lead to a multitude of problems.

Cytokine release syndrome image with symptoms, and early and late onset cytokines.

Figure: CRS Cytokines and Symptoms

Symptoms of Cytokine Release Syndrome

CRS is a heterogeneous condition which can present with different symptoms that vary in severity. It most commonly begins with symptoms such as fever, chills, fatigue, nausea, vomiting, and headache. In serious cases it may lead to seizures, arrythmia, and possible organ failure.

Cytokine Storm and COVID-19

Some of the most well-known causes of CRS include allogeneic transplants, therapeutic interventions, and viral infections. Recently however, focus has been on the involvement of CRS in COVID-19. With the collation and publication of more and more clinical data, a large number of data suggest that there are mild or severe cytokine storms in severe patients, which is an important cause of death.

Pathogenesis of Cytokine Release Syndrome

The pathogenesis of CRS is not fully understood. However, certain key mediators of cytokine release syndrome have been identified. Chief among these is interleukin 6 (IL-6), an activator of the JAK/STAT signalling pathway which can bind to two forms of its receptor (soluble and transmembrane).

IL-6, along with IL-10 and IFNγ are the cytokines most consistently implicated in CRS. These three cytokines are usually elevated in the serum of patients with cytokine storm. TNF, IL-1, IL-8 and MCP-1 are more prominent early in CRS onset, with IL-6 produced later in the progression of the syndrome in higher quantities. IL-6 production is increased by TNF and IL-1, thus linking the signal of early-response cytokines to late onset profiles.

Diagnosis and Treatment of Cytokine Release Syndrome

At present, multiple therapeutics target the pathways which are affected by the pro-inflammatory cytokines of CRS. In particular, blocking IL-6 with therapies such as Tocilizumab and Siltuximab has gained much support globally.

However, to proactively tackle the issue there is need for an accurate diagnostic assay that can rapidly establish if a patient is suffering from Cytokine Release Syndrome. This may be the difference between the development of mild CRS to severe CRS, even the difference between life and death.

This is where our Genie Plex Cytokine Storm Multiplex Immunoassays come in.

GeniePlex COVID-19 Multiplex Cytokine Immunoassays

GeniePlex COVID-19 Multiplex Cytokine Immunoassays are a form of bead-based Multiplex Immunoassay kit that enables the simultaneous and quantitative detection of a range of cytokines. The selection of cytokines targeted is dependent on the species in which the kit is created for.

Not only can Genie Plex Cytokine Storm Multiplex Immunoassays detect and quantify a wide variety of pro-inflammatory cytokines, but they can do this with as little 15ul of sample, in as little as 2 hours!

Our kits can also be run on almost any Flow Cytometer using a range of samples such as Serum, Plasma, Cell culture supernatant, Cell lysates, Tissue lysates and other samples types.

Cytokine Storm Multiplex Immunoassay Kits Available

SKU Product Name Reactivity Analytes

HUAMCOV01

Human

IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNF alpha

MOAMCOV01

Mouse

IL-2, IL-7, IL-10/CSIF, GCSF/CSF-3, IP-10/CXCL10, MCP-1/JE/CCL2, MIP-1 alpha/CCL3, and TNF-alpha.

RTAMCOV01

Rat

IL2, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα

MKAMCOV01

Non-Human Primate

IL-2, IL-10, IP-10/CXCL10, MCP-1/JE/CCL2, and TNFalpha

HUAMCOV02

Human

IL-6, IL-10, IFN-gamma

MOAMCOV02

Mouse

IL-6, IL-10, and IFN-gamma

RTAMCOV02

Rat

IL-6, IL-10, and IFN-gamma

MKAMCOV02

Non-Human Primate

IL-6, IL-10, and IFN-gamma

HUAMCOV04

Human

IL-6, IL-10, IFN-gamma, CCL2/SYCA2/MCP-1, and GM-CSF/CSF-2

MOAMCOV04

Mouse

IL-6, IL-10, IFN-gamma, MCP-1/JE/CCL2, and GM-CSF/CSF-2

RTAMCOV04

Rat

IL-6, IL-10, IFN-gamma, MCP-1/JE/CCL2, and GM-CSF/CSF-3

MKAMCOV04

Non-Human Primate

IL-6, IL-10/CSIF, IFNgamma, and MCP-1/JE/CCL2

HUAMCOV03

Human

IL-6, IL-10, IFN-gamma, TNF-alpha, IL-1beta, IL-8/CXCL8, and CCL2/SYCA2/MCP-1

MOAMCOV03

Mouse

IL-6, IL-10, IFN-gamma, TNF-alpha, IL-1beta, CXCL1/KC/CINC1/KC/Gro alpha (there is no IL-8 in Mouse. KC is a homologous), and MCP-1/JE/CCL2

RTAMCOV03

Rat

IL-6, IL-10, IFN-gamma, TNF-alpha, IL-1beta/IL-1F2, GRO alpha/KC/CINC1 (there is no IL-8 in Rat. KC is a homologous), and MCP-1/JE/CCL2

MKAMCOV03

Non-Human Primate

IL-6, IL-10/CSIF, IFN-gamma, TNF-alpha, IL-1beta, IL-8/CXCL8, and MCP-1/JE/CCL2

HUAMCOV05

Human

IL-6, IL-10, IFN-gamma, CCL2/SYCA2/MCP-1, GM-CSF/CSF-2, TNF-alpha, IL-1beta, IL-2, and IL-8/CXCL8

MOAMCOV05

Mouse

IL-6, IL-10, IFN-gamma, MCP-1/JE/CCL2, GM-CSF/CSF-2, TNF-alpha, IL-1beta, IL-2, and CXCL1/KC/CINC1/KC/Gro alpha

RTAMCOV05

Rat

IL-6, IL-10, IFN-gamma, MCP-1/JE/CCL2, GM-CSF/CSF-2, TNF-alpha, IL-1beta, IL-2, and GRO alpha/KC/CINC1.

MKAMCOV05

Non-Human Primate

IL-6, IL-10/CSIF, IFN-gamma, MCP-1/JE/CCL2, TNF-alpha, IL-1beta, IL-2, and IL-8/CXCL8

HUAMCOV06

Human

IFNγ, IL-1RA, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, MCP-1, MIP-1α, RANTES and TNFα

Multiplex Assay Video Protocol

Multiplex ELISA Protocol Overview

Figure 1. Protocol Overview
1.) Add you Genieplex antibody bead conjugates to your well. 2.) Add your samples and standards to the wells. Incubate your samples at room temperature for 60 mins. Followed by washing the plate X3 using a filter plate vacuum system. 3) Add your biotinylated antibodies and incubate for 30 mins. Followed by washing the plate X3 using a filter plate vacuum system. 4) Add your streptavidin-PE solution and incubate for 20 mins. Followed by washing the plate X3 using a filter plate vacuum system. 5) Add Reading Buffer and read your samples on a flow cytometer. 7) Analyze your data.

Protocol Steps

Step Procedure

1.

Prepare the filter plate template. Mark the standard, sample and blank wells. Standards and samples should be run in duplicates or triplicates. If the whole plate will not be used, seal the unused well with a plate seal.

IMPORTANT: Place the filter plate on top of the filter plate lid during the entire assay process to prevent touching the plate bottom on any surface.

2.

Vortex working bead suspension for 15 seconds.

3.

Add 45 µL of capture bead working suspension to each well. NOTE: Save the remaining capture bead working suspension and store at 2-8°C with light protection. It can be used for setting up acquisition parameters on the flow cytometer.

4.

Remove buffer in the wells by using the “flow-through“ Filter Plate Washer connected to a vacuum source that has been adjusted according to the Filter Plate Washer Instructions.

5.

Gently tap the plate bottom onto several layers of paper towels to remove residual buffer after the “flow-through” removal of the buffer.

6.

Add 30 µL of CCS, SPB or TL Assay Buffer to each sample well. NOTE: Cell culture supernatant samples can be run without diluting in Assay Buffer if very low levels (less than 20 pg/mL) of cytokines are expected. If it is the case, skip this step and add 45 µL of cell culture supernatant samples to each sample well in Step 7.

7.

Add 15 µL of samples to each sample well.

8.

Add 45 µL of standards to each standard well.

9.

Cover the plate with a plate seal.

10.

Incubate on the shaker (set at 700 rpm) for 60 min at room temperature. Protect from light by wrapping the filter plate in aluminum foil.

11.

Remove the plate seal.

12.

Remove solutions in the wells by using the Filter Plate Washer connected to a vacuum source.

13.

Wash the wells three times with 100 µL 1x Wash Buffer using the Filter Plate Washer.

14.

Gently tap the plate bottom onto several layers of paper towels to remove residual buffer on the plate bottom after the last wash.

15.

Add 25 µL of biotinylated antibody working solution to each well.

16.

Cover the plate with a plate seal.

17.

Incubate on the shaker (set at 700 rpm) for 30 min at room temperature. Protect from light by wrapping the filter plate in aluminum foil.

18.

Remove the plate seal.

19.

Remove solutions in the wells by using the Filter Plate Washer.

20.

Wash the wells three times with 100 µL 1x Wash Buffer using the Filter Plate Washer.

21.

Gently tap the plate bottom onto several layers of paper towels to remove residual buffer on the plate bottom after the last wash.

22.

Add 25 µL of streptavidin-PE working solution to each well.

23.

Cover the plate with a plate seal.

24.

Incubate on the shaker (set at 700 rpm) for 20 min at room temperature. Protect from light by wrapping the filter plate in aluminum foil.

25.

Remove the plate seal.

26.

Remove solutions in the wells by using the Filter Plate Washer.

27.

Wash the wells twice with 100 µL 1x Wash Buffer.

28.

Gently tap the plate bottom onto several layers of paper towels to remove residual solution. 2 Add 150 µL to 300 µL of 1x Reading Buffer to each well depending on the sample loading mechanism of a flow cytometer to re-suspend the beads.

29.

Cover the plate with a plate seal.

30.

Place the plate on the microtiter shaker and shake for 30 seconds at 700 rpm. NOTE: If the flow cytometer has no 96-well plate loader and more than 200 µL of 1x Reading Buffer is needed to re-suspend the beads, do not shake the plate. Re-suspend the beads in each well by pipetting up and down 6–8 times with a P200 pipette then transfer to a test tube for acquisition.

31.

Remove the plate seal.

32.

Read on a flow cytometer.

Browse all Cytokine Release Syndrome Multiplex Products

Product Name Reactivity Analytes

Human

IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNF alpha

Mouse

IL-2, IL-7, IL-10/CSIF, GCSF/CSF-3, IP-10/CXCL10, MCP-1/JE/CCL2, MIP-1 alpha/CCL3, and TNF-alpha

Rat

IL2, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα

Non-Human Primate

IL-2, IL-10, IP-10/CXCL10, MCP-1/JE/CCL2, and TNFalpha

Human

IL-6, IL-10, IFN-gamma

Mouse

IL-6, IL-10, and IFN-gamma

Rat

IL-6, IL-10, and IFN-gamma

Non-Human Primate

IL-6, IL-10, and IFN-gamma

Human

IL-6, IL-10, IFN-gamma, TNF-alpha, IL-1beta, IL-8/CXCL8, and CCL2/SYCA2/MCP-1

Mouse

IL-6, IL-10, IFN-gamma, TNF-alpha, IL-1beta, CXCL1/KC/CINC1/KC/Gro alpha (there is no IL-8 in Mouse. KC is a homologous), and MCP-1/JE/CCL2

Rat

IL-6, IL-10, IFN-gamma, TNF-alpha, IL-1beta/IL-1F2, GRO alpha/KC/CINC1 (there is no IL-8 in Rat. KC is a homologous), and MCP-1/JE/CCL2

Non-Human Primate

IL-6, IL-10/CSIF, IFN-gamma, TNF-alpha, IL-1beta, IL-8/CXCL8, and MCP-1/JE/CCL2

Human

IL-6, IL-10, IFN-gamma, CCL2/SYCA2/MCP-1, GM-CSF/CSF-2, TNF-alpha, IL-1beta, IL-2, and IL-8/CXCL8

Mouse

IL-6, IL-10, IFN-gamma, MCP-1/JE/CCL2, GM-CSF/CSF-2, TNF-alpha, IL-1beta, IL-2, and CXCL1/KC/CINC1/KC/Gro alpha

Rat

IL-6, IL-10, IFN-gamma, MCP-1/JE/CCL2, GM-CSF/CSF-2, TNF-alpha, IL-1beta, IL-2, and GRO alpha/KC/CINC1

Non-Human Primate

IL-6, IL-10/CSIF, IFN-gamma, MCP-1/JE/CCL2, TNF-alpha, IL-1beta, IL-2, and IL-8/CXCL8

Human

IFNγ, IL-1RA, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, MCP-1, MIP-1α, RANTES and TNFα