HIV p24 antigen and its monoclonal antibodies
HIV-1 p24 recombinant antigen was purchased from GenWay Biotech (San Diego, CA). Mouse anti-p24 monoclonal antibody, 1 G12 and 1D4, and their corresponding horseradish peroxidase-labeled mAbs were purchased from Jing Tiancheng biology company (Beijing, China). These two mAbs recognize different p24 epitopes. The 1 G12 mAb was used to capture p24 and 1D4 mAb was used as the secondary antibody for signal detection.
Oligonucleotides and reagents
Bio-barcode DNA was 47-base in length and its sequence, 5’-CAGCTGGTCAGCAGAATGGTGTGACCCTCATGGCCGTCTTATCGGGT-3’, was derived from AT4G27630.1 (accessed December 25, 2011 at
http://www.arabidopsis.org/index.jsp). Capture DNA sequence is complementary to the Bio-barcode DNA with an addition of an SH group and 15 A residues at its 5’ end. The forward primer (5’-CAGCTGGTCAGCAGAATGGTG-3’) and reverse primer (5’-ACCCGATAAGACGGCCATGAG-3’) were used for the PCR amplification of bio-barcode DNA. All oligonucleotides were purchased from Sangon Co. Ltd. (Shanghai, China). PCR reagents were purchased from TaKaRa Biotechnology Co., Ltd (Dalian, China). Gold nanoparticles of 30 nm in diameter and tosyl-activated magnetic microparticles (MyOne™ Dynabeads®) were purchased from Ted Pella Inc (Redding, CA) and Invitrogen (Carlsbad, CA), respectively.
Preparation of functionalized MMPs
MyOne™ Dynabeads® were conjugated with 1 G12 mAb according to manufacturer’s protocol. Briefly, 40 μl 1 G12 mAb (1 mg/ml), 10 μl magnetic microparticles, 42 μl 3 M (NH4) 2SO4 and 66 μl borate buffer (0.1 M, pH 9.5) were combined in a 1.5 ml tube and incubated at 37°C at 1400 oscillations per minute for 24 h. The MMPs were separated from unbound components magnetically. Three hundred μl of blocking buffer consisting of phosphate-buffered saline (PBS), pH 7.4, 10% BSA and 0.05% Tween 20 was added and the MMPs mixture was incubated at 37°C at 1400 oscillations per minute for another 24 h. At the completion of incubation, the MMPs were separated magnetically and were washed twice with 1 ml of magnetic-probe solution containing PBS pH 7.4, 0.1% BSA and 0.05% Tween 20, and resuspended in 200 μl of the same solution and stored at 4°C until use. Functionalized MMPs retained their antigen binding activity for 4–6 months
Preparation of functionalized GNPs
GNPs functionalized with 1D4 mAb and the capture DNA oligomers were prepared as described previously
. Briefly, the pH of GNPs in aqueous solution (330 pM) was first adjusted to pH 9.2 with 1 N NaOH. One ml of the solution was incubated with 6 μg 1D4 mAb at 22°C with gentle shaking for 30 min, followed by the addition of 57.7 μg capture DNA oligomer at 10°C for 16 h. The salt concentration was then adjusted to 0.1 M using 2 M NaCl followed with the addition of 0.3 ml of 10% BSA. The mixture was incubated at 22°C for 30 min and then centrifuged at 10,000 rpm at 4°C for 30 min. The supernatant was removed and the GNPs were resuspended and washed once with 0.01 M PBS. The particles were resuspended with 400 μl PBS containing 65.5 μg of the signal DNA oligomers and allowed to hybridize at 37°C for 1 h. Finally, the GNPs were centrifuged at 10,000 rpm (Eppendorf centrifuge 5415 R, Germany) at 4°C for 30 min and then resuspended in PBS containing 0.01% (v/v) Tween 20 and 0.1% (w/v) BSA. The functionalized GNPs solution was stored at 4°C until use.
Detection of HIV-1 p24 antigen by ELISA
Microplate strips (Corning Costar Inc., Lowell, MA) were coated with 100 μl per well of 1 G12 mAb (5 μg/ml in 0.5 M carbonate buffer) at 4°C for 24 h. After incubation, the strips were washed twice with PBS and then treated with 10% BSA at 37°C for 24 h to block the surface. The strips were washed again and then coated with 100 μl per well of serially diluted p24 antigen (ranged from 0.1 to 100,000 pg/ml) by incubating at 37°C for 1.5 h. Unbound antigens were washed 3 times with PBS. One hundred μl of 1,000 fold diluted HRP-labeled 1D4 was added to each well and the plate was incubated at 37°C for 1 h followed with 6 washes with PBS. One hundred μl of tetramethylbenzidine was then added to each well and the plate was incubated at 22°C for 15 min. The enzymatic reaction was terminated with the addition of 50 μl of 2 M H2SO4, and the color was quantified in an ELx808 plate reader (BioTek, Seattle, WA) at the wavelength of 450 nm.
Detection of HIV-1 p24 antigen by BCA based on microplate
The assay scheme is shown in Figure
1B. The coating of BCA and blocking processes of microplates were the same as in ELISA. One hundred μl of 10 fold serially diluted HIV-1 p24 (0.1 to 10,000 pg/ml) in PBS containing 3% BSA was added into 1 G12 mAb coated wells. After incubation at 37°C for 60 min, the wells were washed 5 times with PBS containing 5 mM EDTA and 0.05% (v/v) and Tween 20 (PBSET). Then 100 μl GNPs (with a final concentration of 50 pM in 10% BSA) were added into each well and incubated at 37°C for 20 min. The wells were washed 7 times with PBSET and 3 times with PBS and once with 50 μl of Milli-Q distilled water. Finally, the wells were membrane sealed and heated in a water bath at 80°C for 5 min to release the captured signal DNA oligomers.
PCR amplification was performed by adding 1 μl of the free bio-barcode DNA solutions as templates. Total volume of the PCR reaction mixture was 25 μl containing 2.5 μL 10x Ex Taq Buffer (Mg2+), 2 μl dNTPs (2.5 mM each), 0.5 μl each of forward and reverse primers (with final concentration of 20 μM each), 1 μl Bio-barcode DNA, and 0.8 unit of TaKaRa Ex Taq polymerase. PCR reaction was run on Veriti 96-well thermal Cycler (Applied Biosystems, Foster City, CA) with the following temperature profile: initial denaturation, 95°C, 1 min; 25 cycles of denaturation (95°C, 30 s), annealing (54°C, 30 s), and extension (72°C, 45 s); and final extension (72°C, 5 min). Five μl of the PCR product was electrophoresed in a 4% agarose gel and the signal of the amplified 47-bp bio-barcode DNA was measured using GeneGenius Gel Imaging System (Syngene, Cambridge, UK) and BandScan version 5.0
[29, 30]. The 100 bp DNA band of the 20 bp DNA Ladder (Takara, Dalian, China) standard with known input amount prior to gel electrophoresis was used as the DNA reference. The gray scale of electrophoresed 47 bp product was directly compared with the reference and its DNA amounts (in pmoles) were automatically determined by BandScan.
Detection of HIV-1 p24 antigen by BCA based on MMPs
The assay scheme is shown in Figure
1C. One hundred μl of ten-fold serial dilutions of HIV-1 p24 antigen (10,000 ~ 0.01 pg/ml) in 3% BSA, 0.2% Tween 20, PBS (pH 7.4) and 40 μl diluted functionalized MMPs (1:100 diluted in PBS containing 1% BSA and 0.2% Tween 20) were mixed in a tube of 1.5 ml for 60 min (1,400 oscillations/min) at 37°C, followed by 5 washes with PBS containing 0.1% BSA, 0.05% Tween 20. After magnetic separation, 0.5 μl functionalized GNPs and 3.3 μg tRNA (66 mg/ml, Sigma-Aldrich, Inc., St. Louis, MO) in PBS containing 10% BSA and 0.05% Tween 20 in a total volume of 200 μl were added in this reaction, followed by mixing for 20 min (1400 oscillations/min). The mixture was washed 8 times with PBS containing 0.1% BSA and 0.05% Tween 20 and 4 times with PBS. After magnetic separation, 50 μl of Milli-Q distilled water was added and the tubes were incubated at 80°C for 5 min to release bio-barcode DNA. One μl of the DNA solution was used for PCR amplification. PCR products were quantified as described in the previous section.