4  List of Markers

4.1 Markers included in the exemplar assay

4.1.1 Angiopoietin-1 (Ang-1)

Constitutively expressed by pericytes and smooth muscle cells, also stored in platelets (Brouwers et al. 2013). Ang-1 is an agonist binding the TIE2 receptor. It promotes endothelial cell survival and maintains vascular quiescence. Activates or inhibits angiogenesis, depending on the context. Ang-1 mediates blood vessel maturation/stability (Leligdowicz et al. 2018).

4.1.2 Angiopoietin-2 (Ang-2)

Expressed by endothelial cells. Ang-2 binds to TIE2 in competition with Ang-1 but is an antagonist. Increased Ang-2 levels lead to increased vascular leakage, resulting from destabilised vascular endothelium, remodelling, and leakiness (Kim et al. 2016). Ang/Tie2 dysregulation is thought to be “pathogen agnostic” (Leligdowicz et al. 2018).

4.1.3 Azurocidin 1 (Azu)

Released early in infection from granules in polymorphonuclear leukocytes. Azu acts as a chemo-attractant to several leukocyte types, provokes cytokine secretion, and induces capillary leakage. Azu is directly cytotoxic to certain Gram-negative bacteria (Fisher and Linder 2017).

4.1.4 Chitinase 3-like 1 (CHI3L1)

Produced by many cell types, including immune, epithelial and others. Overexpressed in infectious and non-infectious inflammatory conditions, including sepsis and cancer. Crucial role in protecting against pathogens, antigen- and oxidant-induced injury responses, inflammation, and tissue repair and remodelling. Regulates a range of essential biological processes (Zhao et al. 2020).

4.1.5 Interleukin 6 (IL-6)

Produced by immune and other cell types during chronic and acute inflammation. Triggers acute phase proteins in the liver. Can be pro- and anti-inflammatory. Stimulates B cells to produce antibodies. Links innate to acquired immune response by promoting differentiation of CD4+ T cells. Pyrogenic and pro-coagulative (Tanaka, Narazaki, and Kishimoto 2014), (Kang and Kishimoto 2021).

4.1.6 Interleukin 8 (IL-8)

Produced by many cell types including monocytes, lymphocytes, granulocytes, fibroblasts, endothelial and epithelial cells, hepatocytes, mesangial cells and chondrocytes and is released only under inflammatory conditions. Platelets store IL-8 in their granules and rapidly release IL-8 in inflammation (Matsushima, Yang, and Oppenheim 2022).

4.1.7 Interleukin 10 (IL-10)

A key anti-inflammatory mediator. Regulator of cellular immune response. Produced by various cell types and detected by macrophages and a range of other cell types. Limits antigen presentation and anti-microbial mechanisms such as nitric oxide production (Saraiva, Vieira, and O’Garra 2019).

4.1.8 Interferon gamma inducible protein 10 (IP-10)

A chemokine released from various immune cells in response to IFNg and other cytokines. Activates and recruits lymphocytes. Elevated in viral and HIV infection, particularly acute HIV (Lei et al. 2019),(Hayney et al. 2017). Associated with disease progression.

4.1.9 Myxovirus resistance protein A (MxA)

Cytoplasmic GTPase with activity against Influenza A and a range of other viruses. Inhibits viral gene transcription by binding to the viral nucleoprotein. MxA is activated by type I and type III interferons (Haller and Kochs 2019).

4.1.10 Soluble Tumor necrosis factor receptor 1 (sTNFR1)

The soluble form of TNFR1 is a competitive inhibitor for circulating TNF, thereby reducing signalling through TNFR1 (an endogenous pyrogen), that can promote a wide range of functions from cell death, tissue damage and subsequent inflammation, to cell proliferation and immune modulation (Ruiz et al. 2021), (Bemelmans, Tits, and Buurman 2017), (Al-Lamki and Mayadas 2015)

4.1.11 Soluble Triggering receptor expressed on myeloid cells 1 (sTREM-1)

Soluble form of TREM‐1 shed from the membrane of activated phagocytes in response to bacterial and fungal infections. sTREM-1 contains an immunoglobulin-like domain that recognises ligands including those connected to bacterial-derived molecules. sTREM-1 possibly originates from the activation of TREM-1 by an infection, being at very low levels in healthy controls. sTREM-1 may also competitively bind TREM-1 ligands and be anti-inflammatory (Theobald et al. 2024)

4.2 Markers which were screened, but which failed assay quality tests

4.2.1 Procalcitonin (PCT)

Produced in the thyroid, but also in all parenchymal tissues during bacterial infection. PCT is secreted in response to bacterial endotoxins, lipopolysaccharides, and inflammatory cytokines. It is inhibited by IFN-gamma during the response to viral infections (Davies 2015)

The PCT assay was screened out of our multiplex due to difficulty in finding a working pair of antibodies that did not cross-react with other antigens in the multiplex, particularly azurocidin.

4.2.2 C-Reactive Protein (CRP)

An acute phase component of the innate immune response, decreasing within hours once the stimulus is removed. CRP is produced by hepatocytes and several other cell types in response to IL-6 in combination with other stimuli. Serum levels can rise by up to 1000-fold. CRP acts by triggering complement, and activates the cellular response by binding to the Fc portion of IgG (Sproston and Ashworth 2018).

The CRP assay was dropped from the multiplex because a reliable standard curve could not be obtained and, crucially, because of the need for much higher sample dilution than the other markers.

4.2.3 Soluble FEMS-like tyrosine kinase 1 (sFlt-1)

Flt-1, also known as VEGF Receptor 1, is primarily active in the vascular endothelium. The soluble form, sFlt-1, binds to circulating VEGF, reducing the effect of VEGF which normally induces neovascularisation. sFlt-1 is elevated in endothelial dysfunction, such as sepsis (Ugalde et al. 2024), (Wazan, Widhibrata, and Liu 2024), (Greco et al. 2018).

The sFlt-1 assay was removed from the multiplex due to difficulty in finding a working antibody pair and respective recombinant protein. All screened options gave little or no signal.