Frontiers in Immunology (Nov 2022)

Distinction of early complement classical and lectin pathway activation via quantification of C1s/C1-INH and MASP-1/C1-INH complexes using novel ELISAs

  • Lisa Hurler,
  • Erik J. M. Toonen,
  • Erika Kajdácsi,
  • Bregje van Bree,
  • Ricardo J. M. G. E. Brandwijk,
  • Wieke de Bruin,
  • Paul A. Lyons,
  • Paul A. Lyons,
  • Laura Bergamaschi,
  • Laura Bergamaschi,
  • Cambridge Institute of Therapeutic Immunology and Infectious Disease-National Institute of Health Research (CITIID-NIHR) COVID BioResource Collaboration,
  • György Sinkovits,
  • László Cervenak,
  • Reinhard Würzner,
  • Zoltán Prohászka,
  • Zoltán Prohászka,
  • Stephen Baker,
  • John R. Bradley,
  • Patrick F. Chinnery,
  • Daniel J. Cooper,
  • Gordon Dougan,
  • Ian G. Goodfellow,
  • Ravindra K. Gupta,
  • Nathalie Kingston,
  • Paul J. Lehner,
  • Paul A. Lyons,
  • Nicholas J. Matheson,
  • Caroline Saunders,
  • Kenneth G. C. Smith,
  • Charlotte Summers,
  • James Thaventhiran,
  • M. Estee Torok,
  • Mark R. Toshner,
  • Michael P. Weekes,
  • Gisele Alvio,
  • Sharon Baker,
  • Areti Bermperi,
  • Karen Brookes,
  • Ashlea Bucke,
  • Jo Calder,
  • Laura Canna,
  • Cherry Crucusio,
  • Isabel Cruz,
  • Rnalie de Jesus,
  • Katie Dempsey,
  • Giovanni Di Stephano,
  • Jason Domingo,
  • Anne Elmer,
  • Julie Harris,
  • Sarah Hewitt,
  • Heather Jones,
  • Sherly Jose,
  • Jane Kennet,
  • Yvonne King,
  • Jenny Kourampa,
  • Emily Li,
  • Caroline McMahon,
  • Anne Meadows,
  • Vivien Mendoza,
  • Criona O’Brien,
  • Charmain Ocaya,
  • Ciro Pascuale,
  • Marlyn Perales,
  • Jane Price,
  • Rebecca Rastall,
  • Carla Ribeiro,
  • Jane Rowlands,
  • Valentina Ruffolo,
  • Hugo Tordesillas,
  • Phoebe Vargas,
  • Bensi Vergese,
  • Laura Watson,
  • Jieniean Worsley,
  • Julie-Ann Zerrudo,
  • Laura Bergamaschi,
  • Ariana Betancourt,
  • Georgie Bower,
  • Ben Bullman,
  • Chiara Cossetti,
  • Aloka De Sa,
  • Benjamin J. Dunore,
  • Maddie Epping,
  • Stuart Fawke,
  • Stefan Gräf,
  • Richard Grenfell,
  • Andrew Hinch,
  • Josh Hodgson,
  • Christopher Huang,
  • Oisin Huhn,
  • Kelvin Hunter,
  • Isobel Jarvis,
  • Emma Jones,
  • Maša Josipović,
  • Ekaterina Legchenko,
  • Daniel Lewis,
  • Joe Marsden,
  • Jennifer Martin,
  • Federica Mescia,
  • Francesca Nice,
  • Ciara O’Donnell,
  • Ommar Omarjee,
  • Marianne Perera,
  • Linda Pointon,
  • Nicole Pond,
  • Nathan Richoz,
  • Nika Romashova,
  • Natalia Savoinykh,
  • Rahul Sharma,
  • Joy Shih,
  • Mateusz Strezlecki,
  • Rachel Sutcliffe,
  • Tobias Tilly,
  • Zhen Tong,
  • Carmen Treacy,
  • Lori Turner,
  • Jennifer Wood,
  • Marta Wylot,
  • John Allison,
  • Heather Biggs,
  • Helen Butcher,
  • Daniela Caputo,
  • Debbie Clapham-Riley,
  • Eleanor Dewhurst,
  • Christian Fernandez,
  • Anita Furlong,
  • Barbara Graves,
  • Jennifer Gray,
  • Tasmin Ivers,
  • Emma Le Gresley,
  • Rachel Linger,
  • Mary Kasanicki,
  • Sarah Meloy,
  • Francesca Muldoon,
  • Nigel Ovington,
  • Sofia Papadia,
  • Christopher J. Penkett,
  • Isabel Phelan,
  • Venkatesh Ranganath,
  • Jennifer Sambrook,
  • Katherine Schon,
  • Hannah Stark,
  • Kathleen E. Stirrups,
  • Paul Townsend,
  • Julie von Ziegenweidt,
  • Jennifer Webster,
  • Ali Asaripour,
  • Lucy Mwaura,
  • Caroline Patterson,
  • Gary Polwarth,
  • Katherine Bunclark,
  • Michael Mackay,
  • Alice Michael,
  • Sabrina Rossi,
  • Mayurun Selvan,
  • Sarah Spencer,
  • Cissy Yong,
  • Petra Polgarova

DOI
https://doi.org/10.3389/fimmu.2022.1039765
Journal volume & issue
Vol. 13

Abstract

Read online

The most commonly used markers to assess complement activation are split products that are produced through activation of all three pathways and are located downstream of C3. In contrast, C4d derives from the cleavage of C4 and indicates either classical (CP) or lectin pathway (LP) activation. Although C4d is perfectly able to distinguish between CP/LP and alternative pathway (AP) activation, no well-established markers are available to differentiate between early CP and LP activation. Active enzymes of both pathways (C1s/C1r for the CP, MASP-1/MASP-2 for the LP) are regulated by C1 esterase inhibitor (C1-INH) through the formation of covalent complexes. Aim of this study was to develop validated immunoassays detecting C1s/C1-INH and MASP-1/C1-INH complex levels. Measurement of the complexes reveals information about the involvement of the respective pathways in complement-mediated diseases. Two sandwich ELISAs detecting C1s/C1-INH and MASP-1/C1-INH complex were developed and tested thoroughly, and it was investigated whether C1s/C1-INH and MASP-1/C1-INH complexes could serve as markers for either early CP or LP activation. In addition, a reference range for these complexes in healthy adults was defined, and the assays were clinically validated utilizing samples of 414 COVID-19 patients and 96 healthy controls. The immunoassays can reliably measure C1s/C1-INH and MASP-1/C1-INH complex concentrations in EDTA plasma from healthy and diseased individuals. Both complex levels are increased in serum when activated with zymosan, making them suitable markers for early classical and early lectin pathway activation. Furthermore, measurements of C1-INH complexes in 96 healthy adults showed normally distributed C1s/C1-INH complex levels with a physiological concentration of 1846 ± 1060 ng/mL (mean ± 2SD) and right-skewed distribution of MASP-1/C1-INH complex levels with a median concentration of 36.9 (13.18 - 87.89) ng/mL (2.5-97.5 percentile range), while levels of both complexes were increased in COVID-19 patients (p<0.0001). The newly developed assays measure C1-INH complex levels in an accurate way. C1s/C1-INH and MASP-1/C1-INH complexes are suitable markers to assess early classical and lectin pathway activation. An initial reference range was set and first studies showed that these markers have added value for investigating and unraveling complement activation in human disease.

Keywords