A simple and robust method for the complete dissociation of HIV-1 p24 and other antigens from immune complexes in serum and plasma samples.
Accuracy of antigen determination in human plasma samples is often adversely affected by immune complex formation between antigens (e.g., HIV-1 p24 protein) and specific antibodies. In this study we describe an optimized method for complete immune complex dissociation (ICD) in plasma. This method is based on heat denaturation of antibodies and utilizes a defined solution of sodium dodecyl sulfate (SDS) and diethylenetriaminepentaacetic acid (DTPA) as diluent. The efficiency of this procedure for ICD was compared with those of published methods, employing heat denaturation alone and acidification. Plasma samples from patients participating in anti-retroviral treatments and samples reconstituted in vitro were treated and analyzed in parallel. HIV-1 p24 antigen was determined by quantitative enzyme-linked immunosorbent assay (ELISA). In 312 samples from 97 patients, antigenemia was found in 44.9% when measured directly and in 87.2% after this treatment. In a subset of 56 samples, 21.4% tested positive prior to treatment, while after either novel treatment, heat denaturation or acidification, these samples tested positive in 80.4%, 62.5% and 60.7%, respectively. In 94% of cases viral RNA was detected. This improved procedure for ICD provides a reliable and convenient method for complete and accurate p24 antigen detection in human plasma and is applicable to commercially available test kits.
Institute of Applied Microbiology, University of Agricultural Sciences, Vienna, Austria. f.steindl@iam.boku.ac.at
F
F
Steindl
C
C
Armbruster
K
K
Pierer
M
M
Purtscher
H W
HW
Katinger
D000936:Antigen-Antibody Complex / Q000097:blood / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D015483:HIV Antibodies / Q000276:immunology; D016655:HIV Core Protein p24 / Q000097:blood / Q000276:immunology / Q000302:isolation & purification*; D015658:HIV Infections / Q000097:blood / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006358:Hot Temperature; D006801:Humans; D004369:Pentetic Acid; D010949:Plasma; D011489:Protein Denaturation; D012367:RNA, Viral / Q000097:blood; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate; D019562:Viral Load; D014766:Viremia / Q000821:virology*
D000936:Antigen-Antibody Complex; D015483:HIV Antibodies; D016655:HIV Core Protein p24; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D012967:Sodium Dodecyl Sulfate; D004369:Pentetic Acid