Bovine serum albumin (bsa)

Immunoreactivity of pin-bound synthesized epitopes was tested at RT by ELISA [51 (link),53 (link)] against three groups of pooled sera: initial CDI episode, umbilical cord blood, or healthy volunteers. The assay consisted of four main steps: pins incubation with: (1) blocking solution containing 1% BSA (SeraCare, Milford, MA, USA) in TBS-T; (2) primary antibodies in a 1:1000 dilution in TBS-T with 0.1% BSA in TBS-T; (3) secondary antibodies – anti-human IgG conjugated with AP (cat. A1543-1ml, Sigma-Aldrich) in a 1:10000 dilution in TBS-T; and (4) Alkaline Phosphatase Yellow Liquid Substrate System for ELISA (AP Yellow, Sigma-Aldrich). Plates were read at 405 nm (PowerWave HT, BioTek Instruments, Winooski, VT, USA). After assay, bound antibodies were stripped off by sonication in disruption buffer (1% sodium dodecyl sulfate, 0.1% 2-mercaptoethanol and 0.1 M Na₃PO₄) preheated to 60 °C, washed in water and methanol, and left to dry.
Based on the results obtained in ELISA assays, the baseline was calculated, and peptides were selected for further analysis. In his work, Carter suggested that the background should be calculated as the mean of the 10–25% of the lowest results [51 (link)]. We, however, considered the baseline as a mean of the results obtained for all peptides, which is a more restrictive approach.

PGG-loaded nanoparticles were obtained by dissolving 250 mg of bovine serum albumin, BSA (Seracare, MA) in 4 mL of deionized (DI) water as stated previously. PGG (125 mg) was dissolved in 400 μl of dimethyl sulfoxide (DMSO) then the solution was added to BSA solution. Glutaraldehyde solution at a concentration of 12μg/mg of protein (BSA) was added while stirring. After an hour of continuous stirring, the mixture was added dropwise to 24 mL of ethanol under continuous probe sonication. The sonication was continued for an additional 30 mins. The nanoparticles were separated by centrifugation and washed.

A human NRGN ELISA that our group developed was used as previously described [24 (link)], an electro-chemiluminescent sandwich immunoassay for NRGN based on the MesoScale Discovery platform (MesoScale Discovery, Gaithersburg, MD). A purified, mouse monoclonal anti-NRGN was used as the capture antibody, and an unlabeled polyclonal rabbit anti-NRGN was used for detection, and identified by a MesoScale Discovery Sulfo-TAG-labelled goat anti-rabbit antibody (R32AB). The standard curve (from 40–0.055 ng/mL) was constructed by serial dilutions of purified recombinant hNRGN in 1 X PBS containing 1% bovine serum albumin (SeraCare Life Sciences, Milford, MA).