Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen.

Abstract

BACKGROUND:

HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen.

METHOD:

A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified.

RESULTS:

The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml.

CONCLUSIONS:

When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3-4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.

• Authors: Huahuang Dong, Jianli Liu, Hong Zhu, Chin-Yih Ou, Wenge Xing, Maofeng Qiu, Guiyun Zhang, Yao Xiao, Jun Yao, Pinliang Pan, Yan Jiang
• Journal: Virology journal (Virol J) Vol. 9 Pg. 180 (Aug 31 2012) ISSN: 1743-422X [Electronic] England
• PMID: 22935171 (Publication Type: Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies, Monoclonal
• HIV Antibodies
• HIV Core Protein p24

Topics

• Antibodies, Monoclonal
• Electrophoresis, Agar Gel (methods)
• HIV Antibodies
• HIV Core Protein p24 (analysis)
• HIV Infections (diagnosis, virology)
• HIV-1 (immunology, isolation & purification)
• Humans
• Molecular Diagnostic Techniques (methods)
• Nanoparticles
• Nucleic Acid Amplification Techniques (methods)
• Sensitivity and Specificity
• Virology (methods)