Trapping Assays

TRAP Assay (Total Radical-Trapping Antioxidant Parameter)... 

An analogous assay using a radiolabeled soft nucleophile would also be required to complement the hard nucleophile radiolabeled cyanide trapping assay. Investigations into radiolabeled glutathione have proved unsuccessful since the material is unstable due to cross-reactions induced by beta radiation. Alternate soft nucleophiles such as cysteine, N-acetyl cysteine and P-mercaptoethanol all have promise as radiolabeled substances for quantitative trapping experiments since they are more stable than GSH and equally nucleophilic, although clearly these would not be substrates for GST. 

Meneses-Lorente, G., Sakatis, M.Z., Schulz-Utermoehl, T., De Nardi, C. and Watt, A.P. (2006) A quantitative high-throughput trapping assay as a measurement of potential for bioactivation. Analytical Biochemistry, 351 (2), 266-272. 

Total radical trapping parameter (TRAP) assay is widely used in investigations and has various modifications [45-48]. This method presumes antioxidants capability to react with peroxyl radical 2.2-azobis (2-amidinopropane) dihydrochloride (AAPH). TRAP modifications differ in methods of registering analytical signal. Most often the final stage of analysis include peroxyl radical AAPH reaction with luminescent (luminol), fluorescent (dichlorofluorescein-diace-tate, DCFH-DA) or other optically active substrate. Trolox is often used as a standard. 

Fast TRAP assay of human seminal plasma was a good predictor of pregnancy after in vitro fertilization semen with a fast TRAP <1.14 mmol/L did not result in any pregnancies (R13). 

Note Albumin was determined by the trapping assay with biotin-conjugated HSA-1. aOD, optical density. 

Albumin in Tissues and Body Fluids. The presence of albumin in various tissues and body fluids was examined. The results are presented in Table V as the amount of protein per milliliter of fluid required to give 50% maximal reaction in the trapping assay. Albumin was easily detected in various body fluids and tissue extracts, including seminal fluid, milk, urine, saliva, vaginal secretions, and extracts of liver, kidney, and pancreas. The concentrations of albumin in these fluids, relative to serum, were such that assay conditions could be adjusted easily to readily distinguish between blood samples and other body fluids. 

Figure 5. Sensitivity of antigen-trapping assay with monoclonal antibodies HSA-1 and HSA-2. HSA-2 (type B antibody) was coated on the plate, and then human albumin (A) or serum (B) was diluted as indicated and added to the wells. Biotin-conjugated HSA-1 served as the specific probe. The optical density at 410 nm (OD410) was measured after 15 min.

Gray, D.A. Clarke, M.J. Baux, C. Bunting, J.P. Salter, A.M. 2002. Antioxidant aetivity of oat extraets added to human LDL particles and in free radical trapping assays. J. Cereal Sei. 36 209-218. 

Fig. 1.9. Kinesin mutant design and single-molecule motility results based on an optical trapping assay, (a) WT full CS. (b) 2G CS with mutated residues (light area in CS). (c) DEL CS is absent. The structure is based on PDB 2K1N, modified to incorporate the Drosophila CS (SwissProt ID P17210). (d) Stall force histogram. Sohd hnes Gaussian fits for WT and 2G a DEL histogram was not fitted because of the unknown number of stalls below the minimum detection force threshold. See [27] for details...

The 9-anilino proflavine derivative was designed to optimize the interaction with the intramolecular G-quadruplex from human telomere and minimize that with duplex DNA. These compounds have 60 to 100 nM potency in a modified TRAP assay and corresponding low cytotoxicity (93). The triazines have been demonstrated to produce telomere shortening, which is associated with delayed growth arrest and cell senescence (80). The fluoroquinophenoxazines are redesigned topoisomerase II poisons that now interact more specifically with G-quadruplex structures, and this activity is correlated with production of anaphase bridges (78), a property also shared by the cationic porphyrin TMPyP4 (96) and... 

In the first case study, a lead candidate 12 (Fig. 6) was assessed for its potential to form reactive metabolites in LC-MS-based trapping assays using a variety of trapping agents. There were no indications of the formation of any thiol or cyanide adducts when GSH, N-acetylcysteine, or cyanide were included in incubations of 12 with human liver microsomes. However, when a tritium-labeled derivative of 12 was incubated with human liver microsomes under standard conditions... 

Telomerase activity is typically measured using the Telomeric Repeat Amplification Protocol (TRAP) assay. In the TRAP assay, products of the telomerase reaction are quantified following their PCR amplification [20, 21], The assay is exquisitely sensitive and incorporates an internal standard (ITAS) with which to normalize signals for differences in PCR efficiency. Telomerase activity is calculated as the ratio of the intensity of the telomeric products to that of the ITAS. With this assay, telomerase activity can be measured in a wide range of specimens, from tissue biopsies to cell pellets [22]. High throughput assays have been developed to adapt the telomerase assay to the clinical environment. Many of these new assays take advantage of fluorophores that alleviate the use of radioisotopes and facilitate the quantification of PCR products. 

Much higher activity in the absence of an internal inactivator (Figure 10b and c) than in its presence (Figure 10a) suggests that the transporter has been irreversibly trapped in the vesicle without release of the probe. This indirectly demonstrates that transport occurs across the membrane of intact vesicles, because vesicle destruction would lead to inactivator dilution below the concentration at which the inactivator becomes inefficient. Whereas internal trapping assays demonstrate that vesicles remain intact during transport, they do not exclude that lipids are actively involved. 

Figure 10 Internal trapping assays can be used to prove that transport really occurs across the membrane of intact vesicles. Transporters are added in small portions well below EC50 to labeled vesicles (a) with or (b) without internal inactivators. Without inactivators, activity will gradually increase and reach the value obtained when all transporters are added at once (b vs c). With inactivators, transporters are continuously trapped intravesicularly, and no activity is observed even at high total concentrations (a vs c).

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