Related Experiment Video
Updated: Aug 8, 2026
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Published on: August 31, 2014
Detection and molecular mass determination of an HIV replication-enhancing female genital tract factor using a blot
F B Hashemi1, G T Spear, L Madsen
1Department of Immunology/Microbiology, Rush University, Chicago, IL 60612, USA. fhashem2@rush.edu
Insights
Cervicovaginal lavages contain a HIV-inducing factor (HIF) that enhances HIV replication. A novel BioBlot assay determined this factor has a molecular mass of approximately 14 kDa.
Area of Science:
- Virology
- Immunology
- Biochemistry
Background:
- Cervicovaginal lavages (CVL) were previously found to contain a factor enhancing human immunodeficiency virus (HIV) replication.
- This factor, termed HIV-inducing factor (HIF), increases HIV transcription in T cells and monocytoid cells.
Purpose of the Study:
- To determine the molecular mass of the HIV-inducing factor (HIF).
- To establish a novel method for detecting and characterizing bioactive secretory substances.
Main Methods:
- Adaptation of a blot technique involving nonreducing SDS-PAGE of CVL samples.
- Electrophoretic transfer of separated proteins onto nitrocellulose paper.
- Incubation of nitrocellulose paper slices with HIV-infected U1 cells and detection of enhanced HIV production via p24 ELISA, termed the BioBlot assay.
Main Results:
- The BioBlot assay successfully detected HIF bioactivity anchored to nitrocellulose.
- The molecular mass of HIF was determined to be approximately 14 kDa.
- SDS-PAGE and nitrocellulose binding did not alter the bioactive domain(s) of HIF.
Conclusions:
- The BioBlot assay is a viable method for determining the molecular mass and detecting the bioactivity of HIF.
- This technique provides a rapid alternative for studying receptor-ligand interactions, testing bioactivity, and determining molecular masses of secretory substances.
Abstract:
We previously reported that cervicovaginal lavages (CVL) contain a factor that enhances the replication of human immunodeficiency virus (HIV) by increasing virus transcription in T cells and monocytoid cells. This factor was named the HIV-inducing factor (HIF). To determine the molecular mass of HIF, we adapted a blot technique that involves nonreducing SDS-PAGE of CVL samples and electrophoretic transfer onto nitrocellulose paper followed by incubation of paper slices with HIV-infected monocytoid U1 cells. The slices with HIF bioactivity were detected by increased HIV production and measured by an HIV core protein (p24) ELISA. We refer to this technique as the "BioBlot" assay. The BioBlot assay successfully detected bioactivity of HIF anchored onto nitrocellulose and determined that HIF has a molecular mass of about 14 kDa. Paper slices with HIF-negative CVL samples as well as nitrocellulose paper samples without CVL did not enhance HIV production. This finding suggested that SDS-PAGE and nitrocellulose binding do not functionally alter the bioactive domain(s) of HIF structure. In addition to the detection of HIF bioactivity anchored to nitrocellulose and HIF molecular mass determination, the BioBlot technique offers an alternative, rapid method for other applications. These include the study of receptor-ligand interactions of mucosal proteins, direct bioactivity testing and molecular mass determination of secretory substances.
