We developed fluorescence-linked antigen quantification (FLAQ) assays for HIV-1 and SIV antigen quantitation. The assays utilize polystyrene microspheres coated with monoclonal antibodies against HIV-1 Gag p24 or SIV Gag p27, which are incubated with unknown samples, flourochrome-conjugated detector antibody, and lysing agent. The fluorescence of individual microspheres is measured using flow cytometry. The speed, simplicity, and wide dynamic range of FLAQ assays makes them superior to enzyme-linked immunosorbent assays for many applications performed in research laboratories.
Flow cytometry has several advantages for quantitative assays, including rapid readout fluorescent indicators, the capacity to distinguish particle-associated and free fluorescence, and a four log dynamic range. To address the limitations of enzyme-linked immunosorbent assay (ELISA) techniques that are used for viral quantitation in research laboratories, we developed rapid, single-step and single-tube approaches called fluorescence-linked antigen quantification assays (FLAQ) for the HIV-1 p24 and SIV p27 capsid proteins (Fig. 1a).
In these assays, 100 μg of capture antibody (anti-HIV-p25/24 Gag monoclonal antibody 76C, AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH Rockville, MD, USA; or anti-SIV p27 clone M612441, Fitzgerald Industries, Concord, MA, USA)  is passively absorbed to 3.6 × 106 15 μm Polybead polystyrene microspheres according to the manufacturer's protocol (Polysciences, Inc., Warrington, PA, USA). Antibody-conjugated microspheres are counted by hemacytometer and stored at 4°C. Undiluted viral samples (160 μl) are added to a 20 μl reaction mixture containing 3000 antibody-conjugated microspheres, 1.9 μg/ml phycoerythrin-conjugated monoclonal detector antibody (Coulter Immunotech, Miami, FL, USA), 2% fetal bovine serum, and 1% Triton-X in phosphate-buffered saline. Reactions are performed at 37°C for 30 min in sealed 96-well conical bottom plates or single tubes. A 30 min incubation time was empirically determined to be sufficient for the completion of the binding reactions. Bound complexes are stabilized by pelleting at 1400g for 5 min and resuspending in 1% paraformaldehyde in phosphate-buffered saline. Samples were read immediately or stored in the dark at 4°C.
The size, opacity, and fluorescence intensity of each bead is measured by flow cytometry by forward scatter, side scatter, and emitted light intensity, respectively (Fig. 1b). Flow cytometric data were acquired using a FASCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) and analysed using either CELLQuest (Becton Dickinson), or FlowJo (Tree Star, San Carlos, CA, USA). Five hundred gated events were collected and analysed to determine the geometric mean fluorescence intensity of each unknown and standard reaction. Forward and side scatter properties revealed a microsphere population that is easily distinguished from spurious events (Fig 1b; inset). The averaged fluorescence intensities of the microsphere populations at various antigen concentrations are easily distinguished (Fig. 1b). Assay standards were quantified in quadruplicate using commercially available HIV-1 Gag p24 and SIV Gag p27 ELISA (NEN Life Science Products, Boston, MA, USA, and Coulter Corporation, Miami, FL, USA, respectively).
To assess the reproducibility and dynamic range of the assay, we performed three independent FLAQ determinations, each in triplicate, using seven concentrations of HIV-1 or SIV that spanned the linear range of the assays (Fig 1c). Individual aliquots of virus stock were thawed for each FLAQ. Both our HIV-1 p24 FLAQ and SIV p27 FLAQ have a three log dynamic range of 0.1–100 ng/ml, over which the assay has a coefficient of variation of less than 6% (Fig. 1c, and data not shown). Similar results were obtained for both p27 and p24 when quantifying purified protein and culture supernatants of multiple primary isolates from primary blood lymphocytes and macrophages (data not shown). We have successfully combined, lyophilized, and reconstituted microspheres, detector antibody, and the lysing reagent. This may allow long-term storage and decreased lot-to-lot variation. The storage of microspheres with bound capture antibody at 4°C did not result in a detectable decrease in the amount of capture antibody bound to the microspheres for at least 4 months.
The FLAQ requires less time and effort than traditional immunoassays because antigen and antibody binding and detection occur rapidly and in a single step. In traditional immunoassays, it is necessary to remove unbound reagents thoroughly because the final readout is performed on a solution. In FLAQ, however, unbound detector antibody does not need to be washed away because bead-associated and free fluorescence are distinguished using flow cytometry . Theoretically, extremely high concentrations of antigen could inhibit the bead-associated fluorescence by saturating both capture and detector antibodies with free antigen. In such situations, it may be necessary to remove excess antigen before adding detector antibody. Even with this additional step, however, a FLAQ has fewer steps than an ELISA, which contributes to the improved reproducibility that we observed.
The FLAQ is an immunoassay technique with great promise in clinical and research settings. The rapid quantification of primate virus antigens made possible with these assays has been useful for monitoring viral replication in tissue culture in real-time, and for the purpose of harvesting viral stocks with optimal titers. We have demonstrated that FLAQ can be developed and optimized easily and affordably using available reagents. Our HIV-1 Gag p24 and SIV Gag p27 FLAQ require less time, less effort, cost less, and have improved precision and dynamic range than commercially available ELISA. The larger dynamic range of the FLAQ decreases the need to test multiple dilutions of sample unknowns. A further advantage of the FLAQ technique, not presented here, is that they can be multiplexed for single-tube determinations of multiple analytes by varying several parameters, including microsphere size, microsphere fluorescence intensity, microsphere fluorescence spectra, and detector antibody fluorescence spectra . This would allow the concurrent quantitation of microbial and host antigens involved in host-dependent infections.
M.S.H. and E.H.P. contributed equally to this work. The authors would like to thank the Gladstone Institute of Virology and Immunology Flow Cytometry Core Facility for technical input, and S. Ferrel for the HIV-1 transfection product used in the p24 FLAQ experiments.
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