Elisa assays

Circulating levels of NLRP3 was estimated using enzyme-linked immunosorbent assays (ELISA), catalog number CSB-E15885h, Cusabio, Houston, TX, USA. According to the manufacturer’s protocol, the minimum detectable dose for this assay was <0.039 ng/mL of human NLRP3 with a CV% of <8% and <10% for intra- and inter-assay precision, as previously described [8 (link)]. Circulating levels of IL-1β, IL-18 and IL-37 were assessed using the Flex MAP-3D System (Luminex Corporation, Austin, TX, USA), which utilized human cytokine magnetic bead panels (IL-18 and IL-1β: HCYTA-60K human cytokine, chemokine growth factor panel A—immunology multiplex assay) (IL-37: HCYP4MAG-64K human cytokine chemokine magnetic bead panel IV). The intra- and inter-assay % CVs for IL-18, IL-1β and IL- 37 were <15 and <20 and <10 and <15, respectively. Circulating levels of IL-1α and IL-33 were estimated using ELISA assays (Bio Vendor, R & D systems, Brno, Czech Republic) Cat No. RAF045R and RAF064R, respectively. The intra-assay % CVs for both were <5.4% and 4.7%, respectively, while the inter-assay % CVs for both were <10% and 6.9%, respectively. The controls and standards implemented in all the biochemical assays were regularly reviewed by the quality assurance team of CBCD in KSU.

Women were screened between 24 and 28 weeks of gestation using the two-step criteria: first step, non-fasting 50 g oral glucose challenge test (GCT); then, if positive: second step, 100 g GCT administered in the fasting state. GDM diagnosis was confirmed if a participant exceeded threshold levels in both criteria. This screening protocol is widely used in US institutions and recommended by the American College of Obstetrics and Gynecology (ACOG) [18 (link)]. Freshly drawn blood samples were sent to Quest Diagnostics (Las Vegas, NV, USA) for analyses of blood glucose, lipids, C-reactive protein, and glycosylated hemoglobin. Serum interleukin-6 and total antioxidant capacity were measured using ELISA assays according to the manufacturer’s protocol (R&D Systems).

At the endpoint, serum levels of VEGF were determined using ELISA assays (R&D Systems, USA). Optical density of each well was determined using a microplate reader (Stat Fax-2100, Awareness Corp., USA) at 450 nm. The optical density of each sample was reported as the mean optical density at 450 nm. The obtained values were converted to VEGF levels (picograms per milliliter) through standard formulas.

The supernatant culture media were collected at 1 and 3 days post cell seeding. MMP2 (DY902, range = 0.625–20.00 ng/mL) and IL-1β/IL-F2 (DY201, range = 3.91–250 pg/mL) were quantified using an ELISA assays (R&D Systems, Minneapolis, MN, USA) according to manufacturer’s protocol as previously described [31 , 32 (link)]. Briefly, 100 μL of assay diluents and 100 μL of sample were incubated for 2 h at room temperature in antibody-precoated 96-well plates. Wells were washed 3 times with washing buffer, incubated for 2 h with peroxidase-conjugated antibody solution, washed again, followed by the addition of 100 μL of substrate solution for 20 min and 50 μL of stopping solution for 20 min. Absorbance was measured at 450 nm and 570 nm on an ELx808 Absorbance Reader and subtract at 570 nm from the readings at 450 nm.

The concentration of secreted GDF-15 was determined in macrophage culture supernatants using ELISA assays from R&D systems (Wiesbaden, Germany) according to the manufacturer’s instructions.