Rapid and automated fluorescence-linked immunosorbent assay for high-throughput screening of HIV-1 fusion inhibitors targeting gp41.
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 plays an important role in the virus entry. During the process of fusion between the viral and target cell membranes, the N- and C-terminal heptad repeat (HR) regions of the gp41 extracellular domain associate to form a 6-helical bundle, corresponding to the fusion-active gp41 core. Any compound that blocks the gp41 6-helix bundle formation between the N- and C-peptides, which are derived from the N- and C-terminal HR regions, respectively, may inhibit HIV-1 mediated membrane fusion. Based on this principle, we previously established a sandwich enzyme-linked immunosorbent assay (ELISA) for drug screening by using the N-peptide N36 and the C-peptide C34 and a monoclonal antibody (NC-1) which specifically recognizes the gp41 6-helix bundle. In the present study, a fluorescence-linked immunosorbent assay (FLISA) was developed by using fluorescein isothiocyanate (FITC)-conjugated C34 to replace C34 and by directly detecting fluorescence intensity instead of more complicated enzymatic reaction. Compared with the sandwich ELISA, this FLISA has similar sensitivity and specificity, but it is much more rapid, economic and convenient. Using an Integrated Robotic Sample Processing System, this assay has been applied for high-throughput screening of organic compounds on a large scale for HIV-1 fusion inhibitors targeting gp41.
Liu
Shuwen
S
Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10021, USA.
Boyer-Chatenet
Louise
L
Lu
Hong
H
Jiang
Shibo
S
D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000378:metabolism*; D023581:HIV Fusion Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D007163:Immunosorbent Techniques; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000378:metabolism; D012371:Robotics; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D015700:HIV Envelope Protein gp41; D023581:HIV Fusion Inhibitors; D010446:Peptide Fragments; C407149:peptide C34; C407148:peptide N36; D016650:Fluorescein-5-isothiocyanate