Specific elisa kit

The VEGF protein levels in SH-SY5Y and HCASMC cells were estimated in the respective medium with a specific ELISA kit (R&D Systems Inc.), according to the manufacturer’s instructions.
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for VEGF is already pre-coated onto a microplate. Standards and samples were pipetted into the wells and any VEGF present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for VEGF was added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added and the colour developed in proportion to the amount of VEGF bound in the initial step. The colour development was stopped and the intensity of the colour measured (570/450 nm).

The level of PGE₂ was measured using the specific ELISA kit from R&D Systems, Inc. (Minneapolis, MN, USA), according to a previously described method [29 (link)]. Briefly, BV2 cells were cultured in 48-well plates (1 × 10⁵ cell/mL) and pre-incubated with different concentrations of S. horneri extract and its fractions for 3 h. Subsequently, BV2 cells were induced by LPS (1 μg/mL for 24 h). To remove particulate matter, the supernatants were collected and then centrifuged at 13,000× g for 2 min. After that, the levels of PGE₂ were measured using the ELISA kit according to the protocol in the instructions provided by the manufacturer.

Total proteins extracted from duodenal biopsy samples as described above were analysed for the content of active TGF-β1 using a specific ELISA kit (R&D Systems) in accordance with the manufacturer’s instructions. TGF-β1 was assessed in Pakistan samples but not in those from Zambia, as the mucosal material received from the latter group was too small, and the amount of proteins extracted from such samples was not sufficient to perform an ELISA.

The macroscopic presence of gastric erosions/ulcers was examined by a gastroenterologist (N.A.-F.) immediately after euthanizing the rats. Thereafter, stomachs were frozen at −80 °C for further analysis. Upon defrosting, 100 mg of the gastric tissue (from the same zone) were homogenized as described above for the kidney (2.3.4) and mucosal PGE2 levels were determined using a specific ELISA kit (R&D Systems, Minneapolis, Minnesota). Possible blood loss (bleeding) was assessed by measuring blood hemoglobin levels and red blood cell count in the Hematology lab in SUMC, and plasma thromboxane A2 (TXA2) levels were determined using a dedicated ELISA kit (MyBioSource, San Diego, CA, USA).

Peripheral blood was obtained by venipuncture from patients and healthy adult controls using an Institutional Review Board approved protocol and informed consent. Serum was separated to be used for measurements of TNF-α, BAFF, BCMA, APRIL, and hsCRP.
Serum BAFF levels were measured using a specific ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. This assay employs the quantitative sandwich enzyme immunoassay technique. Serum BCMA (TNFRSF17) was quantitated in serum by ELISA using 96 well plates (MyBioSource, Rochester, NY). This assay also employs the quantitative sandwich enzyme immunoassay technique. Antibody specific for BCMA (TNFRSF17) has been precoated onto a microplate. APRIL was determined in the serum, using a kit from Bender MedSystems (Vienna, Austria). An anti-APRIL polyclonal coating antibody is adsorbed onto microwells.
TNF-α was done by using TNF-α ELISA kits provided by AviBon, Helsinki, Finland. The TNF-α ELISA is an enzyme-linked immunosorbent assay for the quantitative detection of human TNF-α in cell culture supernatants and plasma. High sensitive C-reactive protein (hs-CRP) in serum was performed using ELISA technique and interpreted according to the American Heart Association (CRP values < 1.0 mg/L = low risk, CRP values: 1.0–3 mg/L = intermediate risk, and CRP values > 3.0 mg/L = high risk) [12 (link)].