HIV Lateral flow methods
Test Strip Configuration
A noncontact jetting system (XYZ series 3050; Biodot, Ervine, CA) was used to generate 3 lines on to 2.5 Ã 30-cm strip of backed nitrocellulose membrane (HF120; Millipore Corp, Billerica, MA). Neutravidin protein (Pierce, Rockford, IL), whole mouse IgG, and antimouse IgG (Biospacific, Emeryville, CA) was diluted to 2 mg/mL in 100 mM ammonium acetate and jetted at 0.6 ÎŒL/cm with 100 micron ceramic tips yielding 0.5-mm-thick lines. The mouse IgG line was positioned 15 mm from the bottom of the nitrocellulose membrane and the neutravidin test and antimouse IgG control lines were positioned at 19 mm and 23 mm, respectively. The nitrocellulose was transferred to a desicating chamber and dried at 37°C for 24 hours. Test strips were assembled using an adhesive polystyrene backing card 6 Ã 30 cm (#GL-49901; G&L Precision Die Cutting, San Jose, CA) over which was applied 2.5 cm of nitrocellulose membrane, 2 cm of sample pad (grade 8301; Ahlstrom, Holly Springs, PA), and 1.5 cm of blotter pad (grade 205; Ahlstrom). Strips were lightly pressed with a hand roller and cut into 3-mm strips.
Biotinylated Antibody and Latex Bead Conjugate Formation
One of the monoclonal antibodies targeting an epitope of the HIV p24 protein (115B151-423; Abbott Laboratories) was biotinylated. The antibody was diluted to 5 mg/mL in phosphate-buffered saline (PBS) and reacted with 85 ÎŒL of 10 mM Sulfo-NHS-LC-Biotin (Pierce) for 1 ml of antibody solution. Reactions were left at room temperature (RT) for 2 hours before being transferred to 4°C overnight. The following day purification of the antibody was performed over a PD10 column (PD 10; Sigma Chemical Co, St. Louis, MO), resulting in desalting into PBS. The final concentration of the antibody stock was 2.5 mg/mL with approximately 9-11 biotins per antibody as determined by the HABA dye (4'-hydroxyazobenzene-2-carboxylic acid) reaction (Pierce).
Another monoclonal antibody targeting a second epitope of the HIV p24 protein (108394-470; Abbott Laboratories) was used for coating latex particles as a detection conjugate. Two hundred fifty microliter of 500 nm beads (1% w/v) containing Nile Red dye (CFP-0556-2; Spherotech, Lake Forest, IL) was placed in a microfuge tube. The sample was sonicated with microtip probe sonicator for 3-5 seconds at 0-1 setting (Sonic Dismembrator Model 100; Fisher Scientific, Pittsburgh, PA). The beads were spun down at 1 K rpm for recollection. Fifty microliter of 108 antibody (5mg/ml) was added and the mixture incubated for 5 minutes after vortexing. Then 250 ÎŒL of 150 mM MES pH = 5.8 (Sigma Chemical Co) was added followed by vortexing and incubation for 10 minutes. Fifty microliter of 10 mM EDAC (Sigma Chemical Co) was added to the beads and vortexed. Reaction was carried out at RT for 2 hours, then transferred to 4°C overnight. For purification, beads are spun down at 8 k for 5 minutes. The first buffer change is with PBS/0.05% Tween-20. Three subsequent washes are performed in PBS/0.05% Tween-20/0.1% bovine serum albumin (BSA). Finally, the beads in are resuspended in 240 ÎŒL of PBS and stored at 4C.
Mix and Run Assay
Twenty-five microliter of plasma with or without p24 antigen is added to 10 ÎŒL of acid-shock buffer (200 mM citric acid/0.5% Triton X-100) in a 12 Ã 75-mm polypropylene tube and mixed. After 1 minute, the reaction is neutralized with 35 ÎŒL neutralization buffer (1M Tris HCl pH = 7.8 + 0.16% Triton X-100 + 0.03% sodium dodecyl sulfate) containing 5% BSA. After 1 minute of neutralization, 3 ÎŒL of biotinylated antibody is added (1:500 dilution of 2.5 mg/mL stock). Next, 3 ÎŒL of conjugates is added (1:10 dilution of 1% solids stock). The reaction is mixed for 1 minute and finally applied to a test strip.