# IL40: A Newly Described Cytokine With Conflicting Measurements and Detection Variability—Are There Different Forms?

## TL;DR

This paper investigates IL40, a newly described cytokine, and finds inconsistencies in its measurement using commercial assays, suggesting possible different forms of the protein.

## Contribution

The study reveals discrepancies in IL40 detection and suggests structural or modified forms of the cytokine.

## Key findings

- Two commercial ELISAs showed large discrepancies in IL40 plasma levels.
- Abbexa IL40 levels correlated with BAFF and APRIL but were not elevated in rheumatic diseases.
- Neither ELISA detected recombinant IL40, while a Human Protein Atlas antibody did.

## Abstract

Interleukin‐40 (IL40) is a recently described 27 kDa cytokine encoded by C17orf99, originally suggested to play a role in B‐cell biology, but its function is largely unknown. However, elevated serum levels have been reported in rheumatic diseases. Most published studies focus on IL40 measurements in serum/plasma using commercial sandwich ELISAs. Here we found large discrepancies between two IL40 ELISAs (Mybiosource and Abbexa), with Abbexa reporting significantly higher plasma levels. In our investigation, IL40 (Abbexa) was not elevated in patients with ANCA‐associated vasculitis, early or established rheumatoid arthritis (RA), or RA patients who had developed B cell lymphoma (RA‐L), compared to healthy donors. Yet, we found significant correlation of Abbexa IL40 levels with BAFF and APRIL. We next compared the binding of IL40 between the two commercial assays. Pre‐adsorption experiments showed that the Mybiosource capture antibody bound the same target as the Abbexa capture antibody but did not detect the same IL40. Moreover, neither assay detected the reciprocal IL40 kit reference nor mammalian expressed recombinant IL40. In contrast, a Human Protein Atlas (HPA) antibody towards the unstructured C‐terminal of IL40, despite being only partly validated by HPA, detected recombinant IL40 in Western blot and ELISA. We speculate that there may be different structural or modified forms of IL40. The discrepancy between the IL40 Abbexa results and the literature also highlights the difficulties in interpreting results from commercial antibodies and assays.

IL40 has previously been reported to be elevated in rheumatic disease. Here we did not observe higher IL40 plasma levels in rheumatoid arthritis (RA) patients or ANCA‐associated vasculitis (AAV) patients compared to healthy donors and further found large discrepancies in IL40 measurements by two commercial ELISAs.