Elisa kit

Input virus samples for entry assays were prepared from transfected 293T cells as above, and quantified by the HIV-1 p24 antigen enzyme-linked immunosorbent assay (ELISA) kit (ZeptoMetrix Corporation). Entry assays were performed as previously described (von Schwedler et al., 1993 (link); Adachi et al., 1996 ). Briefly, HSC-F cells (10⁶) were incubated with viruses (100 ng of p24) for 2 h at 4°C, extensively washed, and collected as viral binding fractions. To determine virus entry level, HSC-F cells treated with viruses at 4°C as above were trypsinized for 5 min at 37°C, extensively washed, and incubated for 2 h at 37°C for preparation of viral entry fractions. The binding and entry fractions were lysed, and the p24 level of samples was determined by the ELISA kit as above. Entry efficiency of each sample was calculated as p24 level of entry fraction/p24 level of binding fraction. NL4-3 and NL-Kp (NL4-3ΔEnv) were used as positive and negative controls, respectively.

For infection-based assays, cells were infected with VSV-G-pseudotyped HIV-1 at an moi (multiplicity of infection) of 0.01 or 0.2 for eight hours and cultured for two days. In experiments using kinase inhibitors, cells were treated with each inhibitor at 12 h before virus infection. Virus-containing supernatants were harvested and filtered to remove cell debris, and viral p24 antigens were measured using an ELISA kit (Zepto Metrix). The cell lysates were prepared using HBST buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% Triton X-100) containing a protease inhibitor cocktail (Roche). Immunoblotting assays and the antibodies used have been described previously [61 (link)]. The culture supernatants and cell lysates were subjected to p24 ELISA or immunoblotting assays, as described above.

IL-1β ELISA assay was performed according to the manufacturer's protocol using cell culture supernatants (Chondrex, Inc., Redmond WA). Culture supernatants from cells incubated with HIV-1 alone or HIV-1 and Meth/IFNα/IL-1RA were harvested on days 0, 1, 3, and 7. P24 ELISA was performed using the Zeptometrix ELISA kit according to the manufacturer's protocol (Zeptometrix Corporation, Buffalo NY). Supernatants were stored at −80°C.

For HIV-1 infection assay, sorted TZM-bl cells (1 × 10⁵) were seeded in a 24-well plate one day before HIV-1 infection. These cells were then challenged with HIV-1_NL4-3, HIV-1_BH10, HIV-1_HXB2 or HIV-1_YU2 for 8 hours in a total volume of 500 µl serum-free medium. After removing the culture medium and washing cells with PBS 3 times, the cells were cultured in fresh complete media for indicated days. Then, the cells were harvested for luciferase activity analysis with a Bright-GloTM Luciferase Assay System Kit (Promega, WI, USA) by a luminometer (Promega Glomax, USA). At the same time, the supernatant was collected for HIV-1 p24 antigen detection using an ELISA kit (ZeptoMetrix).