Enzyme linked immunosorbent assay kit

To measure basal corticosterone levels, submandibular blood samples were obtained directly from the home cage condition, using animal lancets, from 7:00 to 9:00 a.m. Blood samples were collected into EDTA-coated tubes, and plasma was isolated after centrifugation of the blood samples at 2000g for 20 min at 4°C. Corticosterone in 10 μl of plasma was measured using an enzyme-linked immunosorbent assay kit (Enzo Life Sciences, NY) according to the manufacturer’s instructions. Three replicates were measured for each sample.

Plasma MPO levels were measured using a commercially available enzyme-linked immunosorbent assay kit (Enzo Life Sciences AG, Industriestrasse 17, Switzerland) according to the manufacturer’s directions. The coefficients of intra- and inter-assay variation were 4.3% (n = 10) and 5.7% (n = 10), respectively.
The serum CRP levels were measured using a nephelometric method (Immage 800 Beckman Coulter). The other biochemical parameters were calculated by routine methods with commercial kits.

A peripheral venous blood sample was taken by all study women, in the midluteal phase of the cycle, at the time of the ultrasound examination. It was collected in BD Vacutainer tubes and serum obtained after clotting was stored at -20℃ until the assays.
ANA detection was carried out by an indirect immunofluorescence method, as previously described [16 (link)]. This method is based on the use of Hep -2 cells, a human epithelial cell line, as a substrate (INOVA Diagnostics, San Diego, CA, USA). This provides cells in different stages of development, and mitotic figures aid in differential pattern recognition. Specimens were diluted with phosphate-buffered saline with a cutoff dilution of 1:80: this cut-off was chosen to define ANA positivity. Slides were examined with a fluorescence microscope at ×40 magnification. Quality was checked by using negative and positive controls with known antibody titers. Serum progesterone was assayed by a commercially available enzyme-linked immunosorbent assay kit (Enzo Life Sciences Inc., Farmingdale, NY, USA). The sensitivity of the assay is 8.5 pg/mL. The intra- and interassay coefficients of variation are <9%. All the assays were performed at the Policlinico Tor Vergata University Hospital.