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

被引：30

作者：

Dong, Huahuang ^{[1
]}

Liu, Jianli ^{[2
]}

Zhu, Hong ^{[2
]}

Ou, Chin-Yih ^{[3
]}

Xing, Wenge ^{[1
]}

Qiu, Maofeng ^{[1
]}

Zhang, Guiyun ^{[1
]}

Xiao, Yao ^{[1
]}

Yao, Jun ^{[1
]}

Pan, Pinliang ^{[1
]}

Jiang, Yan ^{[1
]}

机构：

[1] Chinese Ctr Dis Control & Prevent, Natl HIV HCV Reference Lab, Natl Ctr AIDS STD Control & Prevent, Beijing, Peoples R China

[2] Beijing Exit & Entry Inspect & Quarantine Bur, HIV Cent Confirmatory Lab, Beijing, Peoples R China

[3] US Ctr Dis Control & Prevent, Global AIDS Program, China Off, Beijing, Peoples R China

来源：

VIROLOGY JOURNAL | 2012年 / 9卷

关键词：

Human immunodeficiency viruses; Bio-barcode amplification; p24; detection; RESOURCE-LIMITED SETTINGS; REAL-TIME-PCR; ULTRASENSITIVE DETECTION; VIRUS-INFECTION; IMMUNO-PCR; RNA; DIAGNOSIS; PROTEIN; PLASMA; LOAD;

D O I：

10.1186/1743-422X-9-180

中图分类号：

Q93 [微生物学];

学科分类号：

071005 ; 100705 ;

摘要：

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.

引用

页数：7

共 30 条

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