Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

Abstract

BackgroundThe 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.ResultsA 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.ConclusionsThe 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.

The principles and applications of avidin-based nanoparticles in drug delivery and diagnosis

“…Upon binding to the target antigen, the liposome bursts to release the reporter DNA, which permits the target protein to be quantified using real-time PCR. [129]…”

Recent progress in prostate-specific antigen and HIV proteases detection

“…HIV-1 DNA ILPCR 0.013 pg/ml 2012 [83] HIV-1 gp41 Biomimetic sensor (epitope imprinted hydrophilic polymer-coated quartz crystal microbalance) 2 Â 10 3 pg/ml 2012 [84] HIV-1 p24 BCA-MMP 0.1 pg/ml 2012 [85] HIV-1 p24 ELISA 1 Â 10 3 pg/ml 2012 [85] HIV-1 p24 BCA-based microplate method assays 1 pg/ml 2012 [85] HIV-1 p24 Amperometric immunosensor (using GNP, MWCNTs and an AEP) 6.4 pg/ml 2011…”

Protein detection through different platforms of immuno-loop-mediated isothermal amplification

“…The application of DNA-encapsulated liposome has been reported in a study, where a modified version of iPCR, called as immunoliposome-PCR, has been utilized to measure the concentration of carcinoembryonic antigen (CEA) in human serum. This study showed that this novel method is 1,500 times more sensitive than common methods of CEA detection [ 56 ]. Similarly, immunoliposome-LAMP method can be designed to considerably enhance the detection limit of iLAMP.…”