Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

Background The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR. Results A liposome detection reagent was prepared, which consisted of a population of liposomes ~120 nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA) in human serum. This ILPCR assay exhibited a linear dose–response curve from 10-10 M to 10-16 M CEA. Within this range the assay coefficient of variance was <6 % for repeatability and <2 % for reproducibility. The assay detection limit was 13 fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed. Conclusions The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the liposomes allows nonspecific DNA in the assay medium to be degraded with DNase I prior to quantification of the encapsulated reporter by PCR, which reduces false-positive results and improves quantitative accuracy. The ability to encapsulate multiple reporters per liposome also helps overcome the effect of polymerase inhibitors present in biological specimens. Finally, the biotin-labeled liposome detection reagent can be coupled through a NeutrAvidin bridge to a multitude of biotin-labeled probes, making ILPCR a highly generic assay system.


Preparation of the β 2 -myoglobin reporter
Materials PCR primers were purchased from Integrated DNA Technologies (Coralville, IW) and AmpliTaq Gold DNA polymerase, Taqman universal PCR mastermix, and the PCR Taqman probes were purchased from Applied Biosystems (Carlsbad, CA). The TOPO TA cloning kit with pCR2.1-TOPO T/A plasmid vector and One-Shot E. coli was purchased from Invitrogen (Carlsbad, CA). The TRizol Plus RNA purification kit, SuperScript First-Strand Synthesis System for RT-PCR and 10x PCR buffer were also purchased from Invitrogen. The Plasmid DNA Mini-Prep kit and QIAquick PCR purification kit were purchased from Qiagen (Valencia, CA). Sodium acetate solution (3 M), glycogen, and absolute ethanol were purchased from Sigma-Aldrich (St. Louis, MO). HeLa cells were obtained from the ATCC (Manassas, VA).

Preparation of the β 2 -microglobin amplicon
HeLa cells (~1 x 10 7 cells) in PBS, pH 7.4, were pelleted by centrifugation at 300 x g for 10 min. The total RNA was extracted from the cell pellet using the TRizol Plus RNA Purification kit. The isolated RNA was then converted to cDNA with the SuperScript RT-PCR First-Strand Synthesis System using random primers.
The β 2 -microglobin amplicon was amplified from the cDNA derived from the HeLa cells by PCR using the β 2 M-246F and β 2 M-330R primers whose sequences are given in the following section. All primer and probe design was performed using Taqman Probe & Primer Design software (Applied Biosystems). The presence of the amplicon was confirmed by agarose gel electrophoresis. If more than one band was present the correct amplicon was isolated by extraction from the appropriate gel band.
The amplicon was then cloned into the pCR2.1-TOPO T/A plasmid vector, which was used to transform One-Shot chemically-competent E.coli using the TOPO TA cloning kit. The E. coli were then incubated overnight at 37°C on appropriate LB plates (Ampicillian and X-gal). Ten white or light blue colonies were isolated and cultured overnight in LB medium containing 50 μg/mL Ampicillin.

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The positive transformants were analyzed by PCR using forward or reverse M13 primer plus the β 2 M-246F or β 2 M-330R primer for the β 2 -microglobin insert. If desired, a restriction analysis can be performed in parallel. Once the correct clone was identified, a stock solution was prepared by culturing the colony in 1-2 mL of LB containing 50 μg/mL of Ampicillin. The culture was allowed to reach stationary phase, after which 1mL of the culture was combined with 0.15 mL of sterile glycerol, transferred to a cryovial, and stored at -80°C.
A stock solution of the amplicon was prepared by extracting the plasmid DNA using the Plasmid Mini-Prep kit. A 328 bp DNA fragment containing the β 2 -microglobin transcript was amplified from the recombinant plasmid by using M13 forward and reverse primers. This was done to ensure that only the β 2 -microglobin reporter was amplified in the final PCR step. PCR amplification was carried out using AmpliTaq Gold DNA polymerase, Taqman universal PCR mastermix, and the standard protocol of 29 cycles as shown below.
Following PCR, the presence of the 328 bp fragment was confirmed by agarose gel electrophoresis.

72°C for 3 min
The 84-bp DNA reporter was generated by amplifying the 328-bp amplicon with the β 2 M-246F and β 2 M-330R primer set (15 μM each) using the same PCR protocol described above. Following PCR, the presence of the 84-bp reporter was confirmed by agarose gel electrophoresis. The reporter was purified using a QIAquick PCR purification kit and then precipitated at -20°C overnight by adding 1/10 (v/v) of 3M sodium acetate, pH 5.2, and three volumes of absolute ethanol containing glycogen (1 ng/mL) as a carrier. The DNA solution was centrifuged at 16,000 x g for 25 min at 23°C. The pellet was washed with 70% ethanol and dried under a stream of nitrogen. 4 The reporter was then dissolved in 500 μL of 10 mM Tris-HCl, pH 7.4, and the reporter concentration was determined by measuring the absorbance at 260 nm. For the β 2 -microglobin reporter an absorbance of 1.0 at 260 nm corresponds to a concentration of 50 μg/mL. The reporter was diluted to 667 μg/mL and stored at -80°C. The reporter is stable for ~ 2years [1]. Other reporters can be prepared as described above by using appropriate primers. Alternately, reporters can be ordered from commercial sources including Invitrogen, Integrated DNA Technologies, and GenScript (Piscataway, NJ).