Inhibitory activity of peptides on 6-HB formation

The inhibitory activity of peptides on 6-HB formation was measured by a modified ELISA as previously described [48 (link)]. Briefly, a 96-well polystyrene plate (Costar, Corning Inc., Corning, NY, USA) was coated with 50 μL 2 μg/mL NY364 (a polyclonal antibody for HIV-1 gp41 subunit) in 0.1 M Tris buffer (pH 8.8). A mixture of 1 μM N46 and C-peptide with graded concentrations was added to the coated plate after incubation at 37 °C for 30 min. After another 1 hour incubation at 37 °C, the plate was washed with washing buffer (PBS containing 0.1% Tween 20) three times and refilled by 50 μL 1 μg/mL NC-1 (a monoclonal antibody specific for HIV-1 gp41 6-HB formed by NHR and CHR) [49 (link)]. Afterwards, 50 μL of horseradish peroxidase (HRP)-labeled rabbit anti-mouse antibody (Sigma, St. Louis, MO, USA) (1:4000 diluted) were added to the wells of plate, followed by incubation for 1 h and washing. Finally, the substrate 3,3,5,5-tetramethylbenzidine (TMB, Sigma, St. Louis, MO, USA) was added. Absorbance at 450 nm (A450) was tested by an ELISA reader (Ultra 384, Tecan, NC, USA).