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

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.

[1]  J. Bremer,et al.  Comparison of Two Human Immunodeficiency Virus (HIV) RNA Surrogate Assays to the Standard HIV RNA Assay , 2005, Journal of Clinical Microbiology.

[2]  R. Holzman,et al.  Human Immunodeficiency Virus (HIV) Reverse Transcriptase Activity Correlates with HIV RNA Load: Implications for Resource-Limited Settings , 2005, Journal of Clinical Microbiology.

[3]  R. Hodes,et al.  Loss of Dact1 Disrupts Planar Cell Polarity Signaling by Altering Dishevelled Activity and Leads to Posterior Malformation in Mice* , 2010, The Journal of Biological Chemistry.

[4]  Y. P. Bao,et al.  Detection of protein analytes via nanoparticle-based bio bar code technology. , 2006, Analytical chemistry.

[5]  Xavier Anglaret,et al.  Transfer and Evaluation of an Automated, Low-Cost Real-Time Reverse Transcription-PCR Test for Diagnosis and Monitoring of Human Immunodeficiency Virus Type 1 Infection in a West African Resource-Limited Setting , 2005, Journal of Clinical Microbiology.

[6]  Chad A Mirkin,et al.  Nanoparticle-Based Biobarcode Amplification Assay (BCA) for Sensitive and Early Detection of Human Immunodeficiency Type 1 Capsid (p24) Antigen , 2007, Journal of acquired immune deficiency syndromes.

[7]  Y. Laurian,et al.  SEROLOGICAL MARKERS IN EARLY STAGES OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION IN HAEMOPHILIACS , 1986, The Lancet.

[8]  B. Clotet,et al.  Lack of Longitudinal Intrapatient Correlation between p24 Antigenemia and Levels of Human Immunodeficiency Virus (HIV) Type 1 RNA in Patients with Chronic HIV Infection during Structured Treatment Interruptions , 2004, Journal of Clinical Microbiology.

[9]  Longyan Chen,et al.  Gold nanoparticle enhanced immuno-PCR for ultrasensitive detection of Hantaan virus nucleocapsid protein. , 2009, Journal of immunological methods.

[10]  M. Martins-Green,et al.  The cxc chemokine cCAF stimulates differentiation of fibroblasts into myofibroblasts and accelerates wound closure , 2002, The Journal of cell biology.

[11]  J. Chu,et al.  Establishment of one-step SYBR green-based real time-PCR assay for rapid detection and quantification of chikungunya virus infection , 2010, Virology Journal.

[12]  D. Edelman,et al.  Lowering the detection limits of HIV-1 viral load using real-time immuno-PCR for HIV-1 p24 antigen. , 2004, American journal of clinical pathology.

[13]  Chad A Mirkin,et al.  Multiplexed detection of protein cancer markers with biobarcoded nanoparticle probes. , 2006, Journal of the American Chemical Society.

[14]  C. Mirkin,et al.  Nanoparticle-Based Bio-Bar Codes for the Ultrasensitive Detection of Proteins , 2003, Science.

[15]  D. Daskalakis.  HIV diagnostic testing: evolving technology and testing strategies. , 2011, Topics in antiviral medicine.

[16]  K. Fransen,et al.  Performance of a quantitative human immunodeficiency virus type 1 p24 antigen assay on various HIV-1 subtypes for the follow-up of human immunodeficiency type 1 seropositive individuals. , 2003, Journal of virological methods.

[17]  Indira Hewlett,et al.  Nanoparticle-based immunoassays for sensitive and early detection of HIV-1 capsid (p24) antigen. , 2010, The Journal of infectious diseases.

[18]  Chad A Mirkin,et al.  A bio-bar-code assay based upon dithiothreitol-induced oligonucleotide release. , 2005, Analytical chemistry.

[19]  B. Branson,et al.  Evaluation of an Ultrasensitive p24 Antigen Assay as a Potential Alternative to Human Immunodeficiency Virus Type 1 RNA Viral Load Assay in Resource-Limited Settings , 2005, Journal of Clinical Microbiology.

[20]  R. Sutthent,et al.  Antigen Detection Assay Modified with a Booster Step for Diagnosis and Monitoring of Human Immunodeficiency Virus Type 1 Infection , 2003 .

[21]  P. Wright,et al.  Potential of a Simplified p24 Assay for Early Diagnosis of Infant Human Immunodeficiency Virus Type 1 Infection in Haiti , 2007, Journal of Clinical Microbiology.

[22]  L. Wang,et al.  Virology Journal , 1966, Nature.

[23]  C. Mirkin,et al.  Nanoparticle-based detection in cerebral spinal fluid of a soluble pathogenic biomarker for Alzheimer's disease. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Michael P Busch,et al.  Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection , 2003, AIDS.

[25]  Jwa-Min Nam,et al.  Colorimetric bio-barcode amplification assay for cytokines. , 2005, Analytical chemistry.

[26]  J. Phair,et al.  Detection of HIV-1 p24 Gag in plasma by a nanoparticle-based bio-barcode-amplification method. , 2008, Nanomedicine.

[27]  H. Klocker,et al.  Nanoparticle-based bio-barcode assay redefines “undetectable” PSA and biochemical recurrence after radical prostatectomy , 2009, Proceedings of the National Academy of Sciences.

[28]  R. Lodha,et al.  Evidence behind the WHO guidelines: hospital care for children: what is the role of HIV antigen testing in infants <12-months old? , 2009, Journal of tropical pediatrics.

[29]  Ruengpung Sutthent,et al.  p24 Antigen Detection Assay Modified with a Booster Step for Diagnosis and Monitoring of Human Immunodeficiency Virus Type 1 Infection , 2003, Journal of Clinical Microbiology.

[30]  Chad A Mirkin,et al.  A fluorophore-based bio-barcode amplification assay for proteins. , 2006, Small.

[31]  J. Böni,et al.  Heat‐mediated immune complex dissociation and enzyme‐linked immunosorbent assay signal amplification render p24 antigen detection in plasma as sensitive as HIV‐1 RNA detection by polymerase chain reaction , 1996, AIDS.

[32]  M. Pruvost,et al.  Minimizing DNA contamination by using UNG-coupled quantitative real-time PCR on degraded DNA samples: application to ancient DNA studies. , 2005, BioTechniques.