Human albumin elisa quantification set

To quantify albumin (ALB) and α-fetoprotein (AFP) synthesis, the culture medium used for each culture condition was collected 24 h after the medium was refreshed at a specific time point and frozen at −20 °C until analysis. AFP levels were measured using the AFP Human ELISA Kit (Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. In parallel, albumin secretion was quantified using the human albumin ELISA Quantification Set (Bethyl Laboratories, Montgomery, TX, USA), according to the manufacturer’s description.

Urea and albumin levels were measured in the CM of HepG2 or HepG2 co-cultured with UC-MSCs or BM-MSCs by quantitative colorimetric and ELISA, respectively.The number of the cells and the condition of (co)-cultivation were similar to VEGF ELISA test. The levels of Urea and albumin level were measured on days 1 and 7. For urea measurement, urea assay kit (DIUR-100, BioAssay System, Tokyo, Japan) was used following the manufacturer’s instructions. For albumin measurement, albumin ELISA test kit (Human albumin ELISA Quantification Set, Bethyl Laboratories Inc., Tokyo, Japan) was used following the manufacturer’s instructions. Optical density was read at 430nm on BIO RAD 680 Microplate Reader. Each experiment was performed in triplicate. Three independent experiments were performed in this study.

The concentrations of the secreted products were determined from the culture supernatants using sandwich ELISA assays as previously described (Maccani et al. 2014 (link)). Briefly, 96-well microtiter plates (Nunc MaxiSorp, Thermo Fisher Scientific, Waltham, MA, USA) were coated with 0.33 μg mL⁻¹ goat anti-human IgG (γ-chain specific) antibody (I3382, Sigma-Aldrich, St. Louis, MO, USA) to detect 3D6scFv-Fc. Affinity purified 3D6scFv-Fc was used as a standard. Standard and samples were applied in serially twofold dilutions, and captured 3D6scFv-Fc was incubated with 0.5 μg mL⁻¹ horseradish peroxidase conjugated goat anti-human IgG (γ-chain specific) antibody (62–8420, Life Technologies, Carlsbad, CA, USA). Staining was conducted using o-phenylenediamine and H₂O₂. The absorption was measured at 492 nm with an infinite M1000 microplate reader (Tecan, Männedorf, Switzerland). HSA concentrations were determined using the Human Albumin ELISA Quantification Set (E80-129, Bethyl, Montgomery, TX, USA) according to the manufacturer’s instructions.
The specific product secretion rate q_P (pg cell⁻¹ d⁻¹) during steady-state cultivation was calculated according to Eq (1), where D (d⁻¹) represents the dilution rate. VCC (cells mL⁻¹) is the viable cell concentration and C_P (μg mL⁻¹) the product concentration. ${\mathrm{q}}_{\mathrm{p}}=\mathrm{D}\times \frac{{\mathrm{C}}_{\mathrm{p}}}{\mathrm{VCC}}\times {10}^6$