Abstract
BackgroundHIV-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.MethodA 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.ResultsThe 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.ConclusionsWhen 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.
Highlights
The early stage of HIV infection is characterized by the absence of detectable antibodies and transient high viral load
Detection of human immunodeficiency virus type 1 (HIV-1) p24 antigen by enzyme-linked immunosorbent assay (ELISA) First, we examined the quantification of p24 antigen using an in-house ELISA assay with 1 G12 and 1D4 monoclonal antibodies (mAbs) pair (Figure 1A)
Serial dilutions of p24 antigens from 100,000 to 100 pg/ml were added to the microplate wells precoated with 1 G12 mAb and incubated with horseradish peroxidase labeled 1D4 mAb for colorimetric reaction
Summary
The early stage of HIV infection is characterized by the absence of detectable antibodies and transient high viral load. Patients in this window period have a high probability of HIV transmission [1,2]. The direct detection of viral nucleic acids using PCR-based approaches is of high technical complexity and requires skilled personnel, equipment and laboratory infrastructure. 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
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