Streptavidin horseradish peroxidase

Bone tissue sections were rehydrated through xylene and graded ethanol, incubated in the proteinase K (20 μg/ml) for 10 min and then sections were blocked for endogenous peroxidase with 3% hydrogen peroxide (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min in room temperature. Sections were permeabilized and blocked in 10% goat serum for 1 hr. The primary antibodies were anti-collagen I (1:600, ab138492 and ab21286, Abcam, Cambridge, MA, USA) diluted in the 5% goat serum and incubated at 4°C overnight. Sections were then incubated with a biotinylated goat anti-rabbit antibody (BD Pharmingen, San Diego, CA, USA). For detection, Streptavidin-Horseradish Peroxidase and DAB Substrate Kit (BD Pharmingen, San Diego, CA, USA) were used and the counterstain was done with hematoxylin. For osteocalcin (AbD Serotec 7060–1815, 1:1200 dilution), androgen receptor (Santa Cruz, sc-816, 1: 200) and ERβ (Santa Cruz, sc-8974) antigen retrieval was done with 0.5% trypsin at 37°C for 30 min and for estrogen receptor alpha (Santa Cruz 8002, 1:150), antigen retrieval was done in 10 mM sodium citrate, pH 6, at 95°C for 15 min.

IL-26 was quantified in serum samples collected between 2007 and 2009 for the PLUS study. Serums were stored in -80°C. IL-26 was quantified by ELISA using home-made anti-IL-26 monoclonal antibodies (mAbs), as previously described (15 (link), 16 (link)). Sensitivity and specificity of this assay are detailed in Supplementary Figure 1. The detection limit is 200 pg/ml. Briefly, 96-well plates (Maxisorp; Nunc) were coated with 5 µg/mL anti-IL-26 mAb (clone 13C9). After saturation with phosphate-buffered saline containing 5% bovine serum albumin (w/v), plates were successively incubated with the serum samples, 1 µg/mL biotinylated anti-IL-26 mAb (clone 8G3), and then with streptavidin-horseradish peroxidase (BD Biosciences). Bound Abs were detected using the TMB substrate (Sigma-Aldrich). The optical density was measured at λ = 450 nm. IL-26 was also quantified in serum samples from 47 healthy human volunteers (Blood Collection Center, Angers, France; agreement PLER ANG 2017-01). As IL-26 can bind to circulating DNA we made sure that the latter did not interfere with IL-26 detection (Supplementary Figure 1C).

The level of human IL-4 protein was measured using the Human IL-4 ELISA Set BD OptEIA™ (BD Biosciences) according to the manufacturer’s instructions. The protocol for specific IgG antibody detection has been previously described (41 (link)). Briefly, micro-wells of microtiter plates (Thermo Fisher Scientific) were coated with anti-human Igs (for total IgG detection), CH401MAP (for specific IgG detection), or BAL6MAP, a third-party antigen (N:NYYAGGFNSVRGFKDSTLGP) (1 μg/mL) diluted in carbonate buffer (pH 9.5), and the antigens were adsorbed to microtiter plates overnight at 4°C. The wells were washed with PBS-Tween (0.05% v/v) and blocked with 3% BSA-PBS at RT for 2 h. After three washes with PBS-Tween, 10-fold serial dilutions of mouse plasma were added to the wells and incubated for 2 h at RT. The plates were washed three times before adding biotin-conjugated mouse anti-human IgG mAb (BD Pharmingen, San Diego, USA) (1:3,000). After 2 h incubation at 37°C, the plates were washed 3 times, followed by the addition of streptavidin-horseradish peroxidase (1:50,000 v/v; BD Pharmingen). The plates were incubated for 1 h at RT, and unbound conjugates were removed by washing. Then, the EIA substrate kit solution (Bio-Rad Laboratories, Hercules, CA, USA) was added to each well. The reaction was stopped with 10% HCl, and the absorbance was measured at 450 nm.