Automatic elisa reader

As previously stated [9 (link)], we first used the enzymatically amplified two-site AMH-Gen-II ELISA (Beckman Coulter, Immunotech, Webster, Texas, USA) which was available until July 2013. Undiluted serum samples and controls were dispensed into the wells which were coated with anti-AMH antibody, followed by the addition of the anti-AMH detection antibody labelled with biotin. 100 μl of the streptavidin-horseradish peroxidase (HRP) was added after washing, followed by the addition of 100 μl of substrate solution containing TMB for 8–12 min. Using an automatic ELISA reader (Bio-Rad, Hercules, CA) the degree of enzymatic turnover of the substrate was determined by dual wavelength absorbance measurement at 450 nm and between 600 and 630 nm. The absorbance measured was directly proportional to the AMH concentration in the samples which was calculated from the calibration curve. The results were expressed in ng/ml. Concentrations below 0.08 ng/ml were considered undetectable.

The mAb screened by indirect ELISA was performed as follows: 96-well polystyrene microtiter plates (Corning, New York, USA) were coated with purified rIFN-γ protein (100 μL/well, 10 mg/ml) and incubated overnight at 4 °C. The plates were washed three times by PBST (PBS containing 0.05% tween-20) and blocked with 0.1 M carbonate buffer containing 5% skimmed milk powder. After washed as above, cultured supernatant from the hybridomas was added to each well and incubated for 1 h at 37 °C. Followed by previous step, 100 μL of HRP-conjugated goat-anti-mouse IgG (Bioss, Beijing, China) diluted in 1:5000 with PBST was added as secondary antibody and incubated for 1 h at 37 °C. After washing, 100 μL of 3,3′,5,5′-tetramethylbenzidine (Tiangen) was added and incubated for 18 min at 37 °C. After chromogenic termination by 0.2 M H₂SO₄, absorbencies were measured with an automatic ELISA reader (Bio-Rad, California, USA) at 450 nm.

The serum IL-35 and IL-10 levels were measured using specific ELISA kits (Elabscience, Wuhan, China) following the manufacturer’s instructions. Each sample was tested in triplicate. The optical density was measured at 450 nm using an automatic ELISA reader (Bio-Rad Laboratories, Inc.). A standard curve was generated for each plate, and the absolute concentrations of IL-35 and IL-10 were calculated. The results were expressed as picograms per milliliter.