Sandwich elisa kit

The circulating oxLDL concentration was determined in serum (100 µl) with a rat-specific commercial Sandwich ELISA kit (USCN Life Sciences, Texas) according to the manufacturer’s instructions. This assay uses rat polyclonal antibodies against oxLDL and has a measurement range between 31.2 and 2000 pg/ml. In this range, intra- and inter-assay imprecision is below < 12%. NO was estimated in serum (100 µl) by measuring the degradation products nitrite (NO₂⁻) and nitrate (NO₃⁻) using a commercial photometric method (NO quantification kit, Active Motif, California) on a FlexStation3 (Molecular devices, California). In addition, plasma concentrations of homo-arginine (h-arg), ADMA and symmetrical dimethyl-arginine (SDMA) were quantified by a reverse-phase high-pressure liquid chromatography (HPLC) method as described previously [30 (link), 31 (link)].

The EM2 concentration in CSF was measured using a sandwich ELISA kit (Uscn Life Science Inc, Wuhan, China) according to the manufacturer’s protocol. 96-well micro-titers were used for incubation and measurement. The optical density of each sample was read at 450 nm by a microplate reader (Bio-Rad Laboratories, Shanghai, China). Endomorphin-2 concentration was determined according to the standard curve as described previously [22 ].

BALF was centrifuged at 1000 × g for 5 min at 4 °C. After centrifugation, IL-4, IL-17, and IFN-γ protein expression levels in the BALF supernatant were measured using a sandwich ELISA Kit (USCN, Life Science Inc., China), according to the manufacturer’s instructions. Samples were read at 450 nm using a SpectraMax Plus 384 microplate reader (Molecular Devices) and SoftMax Pro software.

A venous blood sample was collected from each subject in the morning of the surgical procedure and another sample 5 days after the intervention. Both samples were collected in sodium citrate tubes, and the plasma obtained after centrifugation was preserved at −80 °C until analysis.
For the assessment of tMGP plasma levels a sandwich ELISA kit (USCN Life Science Inc., Wuhan, China) was used, and results were read with an Organon Reader 230S (Organon Teknika, Oss, The Netherlands). The detection range for tMGP in plasma was 39–2500 pg/mL. The sensitivity of the assay was 20 ng/L, and our intraday CV was 6.1%.

Measurements of serum IL-33 were carried out both in women with osteoporosis and in healthy controls. Blood samples, taken by venipuncture, were centrifuged at 3000 rpm for 5 min and the sera obtained were frozen and stored at −20 °C until processing.
The levels of IL-33 protein were assessed by using a sandwich ELISA kit, according to the standard procedure recommended by the manufacturer (USCN LIFE SCIENCE Houston, TX, USA).
Using a microspectrophotometer mod. 340 ATTC (SLT Lab. Instruments Salzburg, Austria), the absorbance was measured at 450 nm and the values obtained were expressed as pg/ml.
The minimum detectable level of IL-33, as reported by the manufacturer of the assay kit, was lower than 5.3 pg/ml. We have also established the lower limit of detection (LLD), according to the following procedure, as suggested by the manufacturer: (mean negative control optical density) + 2 * (Stde v of negative control optical density). Our LLD was 1.9 pg/ml. We also performed a standard curve starting from 7.8 pg/ml to more accurately detect the IL-33 concentrations in the tested serum samples.