Lifespan

Serum Golig glypican -73 (GP73) was investigated by using a commercially available ELISA kit provided by LifeSpan (LifeSpan BioSciences, USA). A monoclonal antibody specific for GP73 is pre-coted onto a microplate. Five standard sample wells and one blank control well were assigned to each sample. The sample dilution of 100 μl and test specimen of 15 μl were pipetted into each well, supplemented with standard samples of 0 ng, 20 ng, 50 ng, 120 ng and 250 ng (15 μl each). The microplates were sealed with sealing tapes and incubated at 37°C for 60 min. The plates were sealed with sealing tapes again and incubated at 37°C for 30 min. The plates were thoroughly rinsed for five times again and each well was supplemented with color reagents A and B (50 μl each) for the color reaction at 37°C for 20 min. Termination solution of 50 μl was added into each well and the wavelength of enzyme labeling instrument was set at 450 nm . The sample GP73 concentration was retrieved from the curve. The normal range of serum GP73 was 0-55 ng/mL and the malignancy was indicated in the case of GP73 >100 ng/mL (Shi et al. 2011).
AFP was detected by using the Cobas601 electrochemiluminescence immunoassay analyzer. It indicated positive in the case of serum AFP > 7 μg/L.

Neutralizing antibodies specific to IL-17A (100 ng-1µg) and IL-17F (1 µg) (R&D Systems or LifeSpan BioSciences) or IgG isotype controls (0.1–20µg) were added at the same time as stimuli.