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

Virology Journal

Yan Jiang¹  Pinliang Pan¹  Jun Yao¹  Yao Xiao¹  Guiyun Zhang¹  Maofeng Qiu¹  Wenge Xing¹  Chin-Yih Ou²  Hong Zhu³  Jianli Liu³  Huahuang Dong¹ 

[1] National HIV/HCV Reference Laboratory, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, The People’s Republic of China;Global AIDS Program-China office, US Centers for Disease Control and Prevention, Beijing, The People’s Republic of China;HIV Central Confirmatory Laboratory, Beijing Exit and Entry Inspection and Quarantine Bureau, Beijing, The People’s Republic of China

关键词: p24 detection;    Bio-barcode amplification;    Human immunodeficiency viruses;   

Others  :  1153984
DOI  :  10.1186/1743-422X-9-180

 received in 2012-01-19, accepted in 2012-08-27,  发布年份 2012

【 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.

【 授权许可】

   
2012 Dong et al.; licensee BioMed Central Ltd.

【 图 表 】

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