Immunomagnetic quantitative immuno-PCR for detection of less than one HIV-1 virion.
Methods that allow the accurate and reliable detection of ultra-low molecular levels of proteins using techniques such as quantitative immuno-PCR (qIPCR) have demonstrated numerous technical difficulties. Protein detection methods lose specificity when the protein target is immersed within a matrix of thousands of molecules having wide ranges of concentrations. In addition, sensitivities are limited because of high background signals. To validate the performance of an immunomagnetic bead qIPCR method designed to remove the 'matrix' effect for HIV-1 p24 antigen detection, regression analyses were performed using samples from patients infected with HIV-1 diluted to approximately 100-1000, 10-100, 1-10, and 0.1-1.0 HIV-1 p24 Ag molecules/reaction. The number of HIV-1 p24 Ag molecules was derived from quantified HIV-1 RNA determinations. The modified immunomagnetic qIPCR bead assay demonstrated a limit of quantification of 10-100 HIV-1 p24 molecules per reaction, with an average correlation coefficient of 0.948+/-0.028 over a 4-log dynamic range. This method detects less than one HIV-1 virion (a limit of detection unreported previously for HIV-1), and thus, has the potential to identify HIV-1 infection and monitor the dynamics of the disease course earlier than nucleic acid methods. The immunomagnetic qIPCR bead assay is a simple and inexpensive method for ultra-low protein detection of infectious agents, toxins, and cancer markers at a level unrecognized previously using any enzymatic or molecular method.
Barletta
Janet
J
University of Maryland, Baltimore, 21201, United States. jbarletta@cdc.gov
Bartolome
Amelita
A
Constantine
Niel T
NT
D016655:HIV Core Protein p24 / Q000276:immunology / Q000302:isolation & purification; D015497:HIV-1 / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014771:Virion / Q000276:immunology / Q000302:isolation & purification*
D016655:HIV Core Protein p24; C514799:p24 protein, Human Immunodeficiency Virus Type 1