Immunolon 4hbx

Isolated bone marrow neutrophils were applied to pRGD (20 µg/ml)-, ICAM-1 (1 µg/ml)–, or fibrinogen (150 µg/ml)-precoated 96-well plates (Immunolon-4 HBX; Thermo Fisher Scientific) with 1 mM CaCl₂, 1 mM MgCl₂, 0.1 mM cytochrome c (Sigma-Aldrich), and 50 ng/ml rmTNF-α, where indicated. For oxidative burst in suspension, wells were blocked with 10% FCS for 1 h at 37°C, and cells were stimulated with 100 nM PMA (Sigma-Aldrich) or 1 µg/ml rmTNF-α. 45 units of superoxide dismutase (Sigma-Aldrich) were added to each control sample to confirm that reduction of cytochrome c was mediated by superoxide production. Absorbance at 550 and 490 nm was recorded every 10 min for 90 min at 37°C in a plate reader. For calculation, each wavelength value was corrected by its superoxide dismutase control value.

A 90 amino acid fragment of human-derived KEX1 was cloned into the pET28b(+) expression vector (GenScript) in Escherichia coli BL21(DE3) pLysS (Thermo Fisher Scientific) and used to produce an approximately 11 kDa recombinant protein. Microtitre plates (Immunolon 4HBX; Thermo Fisher Scientific) were coated with 5 µg/mL of purified KEX1 in phosphate-buffered saline (PBS). Heat-inactivated plasma was diluted 1:100 in blocking buffer (PBS with 5% non-fat milk); 50 µL of plasma were plated into KEX1-coated wells, and serial dilutions were made to determine end point titres. Goat antihuman immunoglobulin-conjugated horseradish peroxidase (1:10 000, IgG; Sigma-Aldrich) was used for detection, and plates were developed by standard methods. Plasma samples from this cohort that had an undetectable Pc antibody titre were used as negative controls. The reciprocal end point titre (RET) was calculated as the highest dilution at which the optical density was the same or less than that of the negative control. To determine whether patients with low anti-KEX1 antibody titres had a generalised defect in humoral immunity, tetanus toxoid antibody levels were determined using commercial ELISA kit (MyBioSource).