Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen
Autor: | Maofeng Qiu, Yao Xiao, Jun Yao, Jianli Liu, Yan Jiang, Wenge Xing, Hong Zhu, Huahuang Dong, Chin-Yih Ou, Pinliang Pan, Guiyun Zhang |
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Jazyk: | angličtina |
Předmět: |
medicine.drug_class
HIV Core Protein p24 HIV Infections Biology HIV Antibodies Monoclonal antibody Sensitivity and Specificity law.invention lcsh:Infectious and parasitic diseases chemistry.chemical_compound Antigen law Human immunodeficiency viruses Virology medicine Humans lcsh:RC109-216 Polymerase chain reaction Gel electrophoresis Detection limit Electrophoresis Agar Gel p24 detection Oligonucleotide Methodology Antibodies Monoclonal virus diseases Nucleic acid amplification technique Molecular biology Bio-barcode amplification Infectious Diseases chemistry Molecular Diagnostic Techniques HIV-1 Agarose Nanoparticles Nucleic Acid Amplification Techniques |
Zdroj: | Virology Journal Virology Journal, Vol 9, Iss 1, p 180 (2012) |
ISSN: | 1743-422X |
DOI: | 10.1186/1743-422x-9-180 |
Popis: | 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. |
Databáze: | OpenAIRE |
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