Homogeneous single-label biochemical Ras activation assay using time-resolved luminescence.

Mutations of the small GTP-binding protein Ras have been commonly found in tumors, and Ras oncogenes have been established to be involved in the early steps of cancerogenesis. The detection of Ras activity is critical in the determination of the cell signaling events controlling cell growth and differentiation. Therefore, development of improved methods for primary screening of novel potential drugs that target small GTPase or their regulators and their signaling pathways is important. Several assays have been developed for small GTPases studies, but all these methods have limitations for a high-throughput screening (HTS) use. Multiple steps including separation, use of radioactive labels or time-consuming immunoblotting, and a need of large quantities of purified proteins are decreasing the user-friendliness of these methods. Here, we have developed a homogeneous H-Ras activity assay based on a single-label utilizing the homogeneous quenching resonance energy transfer technique (QRET). In the QRET method, the binding of a terbium-labeled GTP (Tb-GTP) to small GTPase protein H-Ras protects the signal of the label from quenching, whereas the signal of the nonbound fraction of Tb-GTP is quenched by a soluble quencher. This enables a rapid determination of the changes in the activity status of Ras. The assay optimization showed that only 60 nM concentration of purified H-Ras protein was needed. The functionality of the assay was proved by detecting the effect of H-Ras guanine nucleotide exchange factor, Son of Sevenless. The signal-to-background ratio up to 7.7 was achieved with an average assay coefficient of variation of 9.1%. The use of a low concentration of purified protein is desirable and the signal-to-background ratio of 3.4 was achieved in the assay at a concentration of 60 nM for H-Ras and SOS proteins. The need of only one labeled molecule and the ability to decrease the quantities of purified proteins used in the experiments are valuable qualities in HTS showing the potential of the QRET method.

High sensitivity luminescence nanoparticle assay for the detection of protein aggregation.

Nanoparticle assay utilizing time-resolved luminescence resonance energy transfer (TR-LRET) was developed for the detection of protein aggregation. This mix-and-measure nanoparticle assay is based on the competitive adsorption of the sample and the acceptor-labeled protein to donor europium(III) polystyrene particles. The protein aggregation was detected with the developed TR-LRET nanoparticle assay, UV240 absorbance and dynamic light scattering (DLS). All methods well equally detected the aggregation and aggregates, whose size ranged from single protein to more than 1000 nm aggregates. The developed method allowed the aggregation detection of the entire size range at more than 10,000 times lower concentration, 30 µg/L, compared to UV240 and DLS. The simple-to-use and sensitive nanoparticle assay with existing microtiter plate luminometric instrumentation can find use as a routine tool for protein aggregation studies in biochemical laboratories and for quality assessment of protein products in industry.

Comparison of homogeneous single-label fluorometric binding assays: fluorescence polarization and dual-parametric quenching resonance energy transfer technique.

The time-resolved fluorescence technique, quenching resonance energy transfer, QRET, relies on a single-labeled binding partner in combination with a soluble quencher. The quencher reduces efficiently the fluorescence of the unbound labeled ligand, whereas the fluorescence of the bound fraction is detectable. This approach allows the development of homogeneous screening assays in a simple and cost-effective manner. In this study, two single-label fluorometric methods, fluorescence polarization (FP) and the QRET technique, are compared in a simple biochemical model immunoassay of estradiol. Estradiol-6-amino was labeled with fluorescein and lanthanide(III) chelates for the FP and QRET assays, respectively. The labeled estradiols were allowed to compete against free estradiol, and the assay parameters were investigated. The EC(50) value of QRET assay using europium(III)-labeled estradiol was 0.1 nM, and the assay sensitivity was approximately 10 pM. These values were more than 10-fold lower than those for the FP assay with Z' values higher than 0.75 for both assays. The high sensitivity was attributed to a low concentration of antibody fragment and labeled estradiol used in the QRET assay. This reduces cost in screening studies without sacrificing the assay performance. A signal-to-background ratio (S/B) of more than 20 was reached in the QRET assay at elevated concentration of the assay components, whereas S/B of 3.6 was measured in the FP assay when both assays shared the same EC(50) value of 1 nM. Multiplexing of assays is a cost-effective means to run screening studies as multiple data can be extracted from a single experiment. Therefore, multiplexing of the QRET assay was investigated and its feasibility was successfully demonstrated in a dual-parametric assay using estradiol labeled with europium(III) and terbium(III) chelates.

Ultrasensitive protein concentration measurement based on particle adsorption and fluorescence quenching.

A new easy-to-use method for quantification of proteins in solution has been developed. It is based on adsorption competition of the sample protein and fluorescently labeled bovine serum albumin (BSA) onto gold particles. The protein concentration is determined by observing the magnitude of fluorescence altered by quenching the fluorescence on the gold particles in a homogeneous assay format. Under optimal low pH conditions, the assay allowed the determination of picogram quantities (7.0 microg/L) of proteins with an average variation of 4.5% in a 10 min assay. The assay sensitivity was more than 10-fold improved from those of the commonly used most sensitive commercial methods. In addition, the particle sensor provides a simple and rapid assay format without requirements for hazardous test compounds and elevated temperature. Eleven different proteins were tested with the constructed sensor exhibiting a protein-to-protein variability less than 15% allowing protein concentration measurements without the need for recalibration of different proteins.