Chromogenic limulus amebocyte lysate assay

EDTA plasma obtained concurrently with the isolation of PBMC was separated and snap-frozen at −70°C. LPS was later measured en bloc in thawed plasma with the Limulus Amebocyte Lysate chromogenic assay (Lonza, MD, USA) according to the manufacturer’s instructions with the following modifications: Samples were diluted 10-fold to avoid interference with background colour and preheated to 70°C for 12 minutes prior to analysis to dissolve immune complexes, as previously described [10] (link).

LPS was analyzed by Limulus Amebocyte Lysate chromogenic assay (Lonza, Walkersville, MD) according to the manufacturer's instructions, with the following modifications: In the 104 CVID patients and 30 controls, samples were diluted 10-fold to avoid interference with background color, and preheated to 68°C for 10 min prior to analyses to dissolve immune complexes as previously described (8 (link), 17 (link)).

Serum levels of sCD14, neopterin, sCD163 and sCD25 were quantified in duplicate by enzyme immunoassays obtained from R&D Systems (Minneapolis, MN). LPS was analysed by Limulus Amebocyte Lysate chromogenic assay (Lonza, Walkersville, MD) according to the manufacturer’s instructions, with the following modifications: Samples were diluted 5-fold to avoid interference with background colour, and preheated to 67 °C for 12 minutes prior to analysis to dissolve immune complexes. Serum levels of CRP were sampled together with safety blood samples via the routine hospital laboratory. B- and T-cell subpopulations were analysed by flow cytometry (Supplementary Methods).

Plasma levels of soluble (s) CD14 and sCD25 were quantified in duplicates by enzyme immunoassays obtained from R&D Systems (Minneapolis, MN). Tumor necrosis factor (TNF) and interleukin (IL)-6, IL-8, and IL-12 were analyzed using a multiplex cytokine assay (Bio-Plex Human Cytokine Plex Panel; Bio-Rad Laboratories Inc., Hercules, CA), analyzed on a Multiplex Analyzer (Bio-Rad Laboratories) according to instructions from the manufacturer. High sensitivity C reactive protein (CRP) was analyzed on a MODULAR platform (Roche Diagnostics, Basel, Switzerland). LPS was analyzed by Limulus Amebocyte Lysate chromogenic assay (Lonza, Walkersville, MD) according to the manufacturer’s instructions, with the following modifications: samples were diluted tenfold to avoid interference with background colour and preheated to 68 °C for 10 min prior to analysis to dissolve immune complexes.

Total cholesterol, HDL cholesterol, Apo A-1 and low density lipoprotein (LDL) cholesterol were measured enzymatically on a Hitachi 917 system (Roche Diagnosis GmbH, Mannheim, Germany) using the cholesterol (cholesterol CHOD-PAP), HDL-cholesterol plus (catalog no. 04713257190) and LDL-cholesterol plus (catalog no. 04714423190) kits from Roche Diagnostics. CRP levels were analysed via the routine hospital laboratory on the day of sampling using a high-sensitivity method. Plasma levels of sCD14 and sCD25 were quantified in duplicate by enzyme immunoassays obtained from R&D Systems (Minneapolis, MN). Endotoxin was analysed by Limulus Amebocyte Lysate chromogenic assay (Lonza, Walkersville, MD) according to the manufacturer’s instructions, with the following modifications: samples were diluted 10-fold to avoid interference with background colour and preheated to 68 °C for 10 minutes prior to analysis to dissolve immune complexes. Supernatant TNF and IL-6 levels were analyzed with a V-plex Proinflammatory Panel 1 kit from Meso Scale Discovery (Meso Scale Diagnostics, LLC, 1601 Research Blvd. Rockville, MD) using QuickPlex SQ120.

Recombinant HMGB1 was expressed and purified as described previously [4 (link), 15 (link)]. The cytokine-stimulating disulfide HMGB1 was characterized by LC-MS/MS [16 (link)]. HMGB1 was extracted with Triton X-114 to remove any contaminating LPS. The LPS content in HMGB1 was measured by the Chromogenic Limulus Amebocyte Lysate Assay (Lonza). The LPS content in HMGB1 protein was < 0.1 EU/mg protein as measured by the limulus assay.