As a specific tumor marker, prostate-specific antigen (PSA) is widely used for the early diagnosis of prostate cancer. Sensitive and specific methods are required to improve the diagnostic accuracy of PSA detection. In the current study, we compared the immuno-polymerase chain reaction (immuno-PCR) method with the solid-phase proximity ligation assay (SP-PLA) with respect to the detection of PSA. Using oligonucleotide-labeled antibody probes, we used both immuno-PCR and SP-PLA to detect trace levels of PSA. The nucleic acid sequences can be monitored using real-time PCR. SP-PLA, however, was found to be superior in terms of both the detection limit and the dynamic range. To detect even lower levels of PSA, we used the loop-mediated isothermal amplification (LAMP) method to measure the levels of reporter DNA molecules in SP-PLA. The sensitivity of the LAMP method is 0.001 pM, which is approximately 100-fold higher than the sensitivities of the other assays. The results suggest that an SP-PLA- and LAMP-based protocol with oligonucleotide-labeled antibody probes may have great application in detecting PSA or other proteins present at trace levels.
"Fig. 3E shows results obtained using a standard qPCR thermal cycler (CFX Connect, Biorad, Hercules, CA, USA) or a digital PCR (Constellation, Formulatrix, Bedford, MA, USA) setup. In addition, flow cytometry , loop-mediated isothermal amplification  or DNA sequencing   have also been employed. The indirect form of PLA follows the same principle, except that unmodified primary antibodies are detected with secondary antibodies that are conjugated to the DNA strands. "
[Show abstract][Hide abstract] ABSTRACT: Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein–protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression.
[Show abstract][Hide abstract] ABSTRACT: A DNA-labeled immunosensor for melamine (MEL) detection is presented which combined the exponential amplification and quantitative effect of Real-Time quantitative PCR (RT-qPCR) with the simplicity and specificity of competitive antigen-antibody reaction. An excellent linear relationship between cycle threshold (Ct) and MEL concentration in the range of 0.001-10pgg(-1) was obtained with a limit of detection of 0.3fgg(-1). Compared with other methods, the sensitivity of this DNA-labeled immunosensor showed a 1000-fold improvement, and was below the strictest safety limit of 1ppm. Furthermore, the specificity was excellent and the recovery in liquid milk samples spiked with MEL was satisfactory. With the advantages of high sensitivity and a low limit of detection (LOD), this sensor is a powerful and promising tool for the detection of other small molecules besides MEL.
[Show abstract][Hide abstract] ABSTRACT: The development of an indirect competitive immunomagnetic-proximity ligation assay (ICIPLA), which is a novel method for detecting small molecules, is described in this report. Free small molecules in samples can be detected using a proximity ligation assay (PLA); the detection is based on the proximity effect caused by a high concentration of small molecule-BSA conjugates bound to streptavidin magnetic beads. As an indirect format competitive immunoassay, the ICIPLA method has the advantage in that the quantity of monoclonal antibody (mAb) used for small-molecule detection is 8-fold lower than that required for the competitive immunomagnetic-proximity ligation assay (CIPLA) described in our previous work. Small molecules can be detected using a single monoclonal antibody, and the PLA method can be used to amplify high-performance signals. In this work, the small molecular compound ractopamine (RAC) was selected as a target for ICIPLA. The limit of detection (LOD) was 0.01 ng ml(-1), and the method exhibited a broad dynamic range of up to six orders of magnitude. We also employed the ICIPLA method to detect RAC in serum, urine, and muscle extracts; the results indicated that the LOD and dynamic range were not altered. The cross-reactivity studies showed that the cross-reactivity values for all RAC analogs were below 0.01%. These results suggest that ICIPLA is a sensitive, specific and practical method for small-molecule detection. This is the first report of the improved PLA technology for small-molecule detection by indirect competitive formats in the biological samples.
The Analyst 11/2012; 138(2). DOI:10.1039/c2an36447f · 4.11 Impact Factor
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