Non-invasive, ultra-sensitive, high-throughput assays to quantify rare biomarkers in the blood

Abstract

Many diseases are easier to treat and control when detected at an early stage of disease progression. Often, disease-related antigens or biomarkers are shed from the primary site and present in the blood. Unfortunately, there are very few tests capable of detecting these rare biomarkers in the blood. A blood test would be very useful to diagnose the disease earlier, monitor effectiveness of treatments, predict recurrence, and monitor recurrence. There is certainly a need to develop assays that are ultrasensitive, non-invasive and high-throughput. Here we describe several highly sensitive immunological assays we have developed to detect rare serum antigens. Initially we created an assay named immunodetection amplified by T7 RNA polymerase (IDAT). To enhance the effectiveness and streamline the procedure, this assay was amended to the facile amplification system termed fluorescent amplification catalyzed by T7 polymerase technique (FACTT). These assays have been used to analyze the tumor antigen HER2 and the prion protein PrP Sc . They can also be applied to other tumor markers or antigens from a variety of diseases such as cardiovascular disease, rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, and hepatitis. These tests are not limited to testing only serum, but may also be applicable to detecting biomarkers in tissue, saliva, urine, cerebrospinal fluid, etc. Clearly, the FACTT-based technology represents an important step in the detection of rare molecules in fluids or tissues for a variety of diseases.

Sensitive Ligand-Based Protein Quantification Using Immuno-PCR: A Critical Review Of Single-Probe And Proximity Ligation Assays

“…This scheme requires other means of signaling than those using Taq polymerase 5-3 exonuclease activity, where the signal is obtained by a reporter dye, freed from its quencher situated in close proximity on the hydrolysis probe. Here, RNA-intercalating dyes can be incorporated as reporter molecules and read on a fluorometer (18, 19). This method, superseding the earlier gel-based isotope detection by Zhang et al (17), is known as fluorescent amplification catalyzed by T7 polymerase technique, or FACTT for short.…”

DNA-based immunoassays for sensitive detection of protein

“…By incorporating the 32 P-labeled cytidine triphosphate (CTP) to RNA synthesis by T7 RNA polymerase, the IDAT method exhibited a 1000-fold increase in sensitivity over an ELISA assay for the detection of prion aggregates in serum or her2/neu breast cancer tumor markers in tissue samples [32]. There are many benefits of the IDAT assay, for example, it is more sensitive, less cumbersome, less time consuming and extremely versatile [33]. IDAT offers a broad dynamic range due to the linear amplification by T7 RNA polymerase.…”

“…Recently, this method was modified by using an RNA intercalating fluorescent dye in place of radioactive isotopes for RNA detection. This method is referred to as "Fluorescent Amplification Catalyzed by T7-polymerase Technique", or "FACTT" [33]. The amplified RNA was quantified by addition of the RNA-intercalating dye RiboGreen and subsequent fluorescence detection.…”

“…The amplified RNA was quantified by addition of the RNA-intercalating dye RiboGreen and subsequent fluorescence detection. The FACTT assay has numerous advantages over the IDAT assay, as this quantitative assay is isothermal, it requires less time, the RiboGreen dye quantifies amplified RNA and thus eliminates the use of radioactivity, and there is no need for a tedious electrophoresis step [33].…”

Sensitive monitoring of RNA transcription levels using a graphene oxide fluorescence switch

“…Through the detection of transcription products, more than one thousand times signal amplification can be achieved in protein detection [8,9]. Recently, some high-sensitivity biological analysis tools were developed based on transcription mediated amplification [8][9][10][11][12][13][14][15].…”