Nanoparticle-based immunoassays for sensitive and early detection of HIV-1 capsid (p24) antigen

Abstract

We evaluated the feasibility of using nanoparticle (NP)-based assays for improving detection sensitivity of human immunodeficiency virus type 1 (HIV-1) p24 antigen. One assay that was evaluated is a gold NP-based biobarcode amplification (BCA) assay, which can detect HIV-1 p24 antigen at levels as low as 0.1 pg/mL. The lower limit of detection for an enzyme-linked immunosorbent assay (ELISA) is 10-15 pg/mL. These results demonstrate that the HIV-1 p24 BCA assay offers 100-150-fold enhancement in the detection limit over the traditional colorimetric ELISA. Furthermore, the BCA assay detected HIV-1 infection 3 days earlier than did ELISA in samples from patients who had experienced seroconversion. The other assay that we tested is the europium NP-based immunoassay, which uses europium NPs to replace gold NPs in the BCA assay to further simplify the detection method and decrease the incubation time. For detection of HIV-1 p24, the lower limit of detection for the europium NP-based immunoassay was 0.5 pg/mL. These results indicate that the universal labeling technology based on NPs and its application may provide a rapid and sensitive testing platform for clinical diagnosis and laboratory research.

Figure 1

Scheme of Biobarcode amplification (BCA) assay for detection of HIV-1 p24 antigen. HIV-1 p24 is captured by anti-p24 coated microtiter wells and sandwiched with biotinylated secondary anti-p24 antibody. The immune complex is then coupled with 15nm gold NPs modified with SA that recognizes and binds biotinylated biobarcode DNA. After extensive washing and heating at 80°C for 5 min, biobarcode DNAs are eluted and detected by microarray method in which the released biobarcode DNAs are first hybridized with the capture oligos immobilized on the surface of glass slides. Gold NPs coated with T20 probe complementary to the other half of the biobarcode DNA are then hybridized to the captured biobarcode DNA. Finally, silver ions in the staining solution are reduced and grow around NPs to facilitate visualization of the hybridization event. The results are recorded and quantified with the Verigene ID system Nanosphere Inc., Northbrook, IL).

Figure 2

Increased sensitivity of BCA assay compared to in-house ELISA in detecting HIV-1 p24 antigen. HIV-1 p24 positive control antigen, ranging from 0.1 to 500 pg/ml in serial dilution in PBS, served as targets. The normalized relative signal intensities are represented as the ratios of samples over the cut-off value of the negative control (S/CO). BCA assay is represented by open square; ELISA by open circle. The error bar represents the standard deviation of at least three independent repeated experiments for each assay. The correlation between the HIV-1 p24 BCA assay and the concentrations of HIV-1 p24 was r = 0.9357 (R² = 0.8756; p < 0.0001).

Figure 3

Scheme of Eu⁺ NP-based immunoassay (ENIA) for detection of HIV-1 p24 antigen. ENIA uses monoclonal anti-PA antibody coated microtiter wells to capture HIV-1 p24 that is then sandwiched with secondary biotinylated anti-p24 antibody. The Eu⁺ NPs modified with SA recognize and bind the above biotinylated antigen-antibody complex. The addition of biotinylated anti-SA antibody and SA-coated Eu⁺ chelates (PerkinElmer) facilitate the signal intensity of the binding event. After extensive washing, the fluorescence signal released from the sandwiched complex are then recorded and quantified with the Vector Mutlilabel Counter (PerkinElmer).

Figure 4

Increased detection sensitivity of ENIA in detecting HIV-1 p24 antigen. Purified HIV-1 p24 antigen, ranging from 0.5 pg/ml to 500 pg/ml in serial dilution in PBS, served as targets. The normalized relative signal intensities are represented as the ratios of samples over the cut-off value of the negative control (S/CO). ENIA assay is represented by closed cycle. The error bar represents the standard deviation of at least three independent repeated experiments for each assay. The correlation between the S/CO ratio by p24 ENIA assay and the concentrations of HIV-1 p24 was r = 0.9979; R² = 0.9959; p < 0.0001.