Hiv 1 gag p24 elisa kit

We previously reported on the generation of Transmitted/Founder (TF) HIV-1 infectious molecular clones (IMCs) CH040.c and CH058.c [4 (link)]. TF IMC CH470 was generously provided by Beatrice Hahn (U. Pennsylvania) [69 (link)]. pCH040.c-GFP.T2A/K3223 (referred to herein as CH040.c-eGFP) was constructed from pCH040.c (GenBank #JN944939.1) by inserting eGFP using the nef ATG, followed by a self-cleaving T2A peptide cassette and nef in frame, analogous to a previous approach [70 (link)]. pCH470, pCH040.c and pCH058.c plasmids were prepared using Stbl3 cells (Invitrogen, Carlsbad, CA) and used to produce viral stocks in 293T cells as previously described [71 (link)]. Virus stocks were titered using an HIV-1 Gag p24 ELISA kit (Perkin Elmer, Waltham, Massachutsetts). Prevotella stercorea (DSM #18206, DSMZ, Braunschweig, Germany) stocks were prepared as previously detailed [35 (link)], frozen at -80°C in DPBS in single use aliquots and enumerated using CountBright counting beads and a LSRII flow cytometer (BD Bioscience, San Jose, CA).

HIV-1BaL stocks (AIDS Research Reagent Program/ARRP Catalogue #4984) were prepared by passage in MOLT4-CCR5 (ARRP #510) cells for 9 days. Virus containing supernatants were ultracentrifuged at 76,800g. T/F HIV-1 infectious molecular clones CH470, CH40, and CH58, as well as AD17, CH106, CH607, REJO, RHPA, THRO, STCOr1, STCOr2, WARO, MCST, RHGA, TRJO and WITO were generously provided by Beatrice Hahn [57 (link),58 (link)]. NL4-3 and NL4-3ΔVif were obtained from ARRP. CH470, CH40 and CH58 plasmids were re-transformed and amplified in Stbl3 cells (Invitrogen) and purified using Qiagen maxi kit. T/F maxi-preps were sequence-verified using 13 HIV-specific primers to cover the entire genome. 40 μg of T/F plasmids were used to transfect 293T cells in a T175 flask. Four flasks were transfected by CaCl2 transfection method for each virus [78 (link)]. Virus-containing supernatants were collected at 48 hrs, concentrated by ultracentrifugation at 76,800g over a 20% sucrose cushion. Virus stocks were titered using an HIV-1 Gag p24 ELISA kit (Perkin Elmer).