title: 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.
  
  
  METHODS
  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.affiliation: 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.
authors.forename: Huahuang
authors.initials: H
authors.lastname: Dong
authors.forename: Jianli
authors.initials: J
authors.lastname: Liu
authors.forename: Hong
authors.initials: H
authors.lastname: Zhu
authors.forename: Chin-Yih
authors.initials: CY
authors.lastname: Ou
authors.forename: Wenge
authors.initials: W
authors.lastname: Xing
authors.forename: Maofeng
authors.initials: M
authors.lastname: Qiu
authors.forename: Guiyun
authors.initials: G
authors.lastname: Zhang
authors.forename: Yao
authors.initials: Y
authors.lastname: Xiao
authors.forename: Jun
authors.initials: J
authors.lastname: Yao
authors.forename: Pinliang
authors.initials: P
authors.lastname: Pan
authors.forename: Yan
authors.initials: Y
authors.lastname: Jiang
mesh_terms: D000911:Antibodies, Monoclonal; D004587:Electrophoresis, Agar Gel / Q000379:methods; D015483:HIV Antibodies; D016655:HIV Core Protein p24 / Q000032:analysis*; D015658:HIV Infections / Q000175:diagnosis* / Q000821:virology; D015497:HIV-1 / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D053758:Nanoparticles; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D016655:HIV Core Protein p24