title: Rapid, Fully Automated Digital Immunoassay for p24 Protein with the Sensitivity of Nucleic Acid Amplification for Detecting Acute HIV Infection.
abstract: BACKGROUND
  Nucleic acid testing (NAT) has become the standard for high sensitivity in detecting low levels of virus. However, adoption of NAT can be cost prohibitive in low-resource settings where access to extreme sensitivity could be clinically advantageous for early detection of infection. We report development and preliminary validation of a simple, low-cost, fully automated digital p24 antigen immunoassay with the sensitivity of quantitative NAT viral load (NAT-VL) methods for detection of acute HIV infection.
  
  
  METHODS
  We developed an investigational 69-min immunoassay for p24 capsid protein for use on a novel digital analyzer on the basis of single-molecule-array technology. We evaluated the assay for sensitivity by dilution of standardized preparations of p24, cultured HIV, and preseroconversion samples. We characterized analytical performance and concordance with 2 NAT-VL methods and 2 contemporary p24 Ag/Ab combination immunoassays with dilutions of viral isolates and samples from the earliest stages of HIV infection.
  
  
  RESULTS
  Analytical sensitivity was 0.0025 ng/L p24, equivalent to 60 HIV RNA copies/mL. The limit of quantification was 0.0076 ng/L, and imprecision across 10 runs was <10% for samples as low as 0.09 ng/L. Clinical specificity was 95.1%. Sensitivity concordance vs NAT-VL on dilutions of preseroconversion samples and Group M viral isolates was 100%.
  
  
  CONCLUSIONS
  The digital immunoassay exhibited >4000-fold greater sensitivity than contemporary immunoassays for p24 and sensitivity equivalent to that of NAT methods for early detection of HIV. The data indicate that NAT-level sensitivity for acute HIV infection is possible with a simple, low-cost digital immunoassay.
authors.affiliation: Quanterix Corporation, Lexington MA;
authors.forename: Carlos
authors.initials: C
authors.lastname: Cabrera
authors.affiliation: Quanterix Corporation, Lexington MA;
authors.forename: Lei
authors.initials: L
authors.lastname: Chang
authors.affiliation: Blood Systems Research Institute, San Francisco, CA;
authors.forename: Mars
authors.initials: M
authors.lastname: Stone
authors.affiliation: Blood Systems Research Institute, San Francisco, CA; University of California, San Francisco, San Francisco, CA.
authors.forename: Michael
authors.initials: M
authors.lastname: Busch
authors.affiliation: Quanterix Corporation, Lexington MA; dwilson@quanterix.com.
authors.forename: David H
authors.initials: DH
authors.lastname: Wilson
mesh_terms: D002460:Cell Line; D006678:HIV / Q000302:isolation & purification*; D016655:HIV Core Protein p24 / Q000032:analysis* / Q000097:blood; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D007118:Immunoassay / Q000191:economics / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000191:economics / Q000379:methods
chemical_list: D016655:HIV Core Protein p24; D012367:RNA, Viral