Serum shift assays

Serum shift assays are popular ways of looking at protein binding during lead optimization... 

Applicability in biological ion assay is an important factor for biocompatible potentio-metric ion sensors. Attempts were made to determine Na" " concentrations in human blood sera by using silicone-rubber membrane Na+-ISFETs based on (5) [Fig. 17(a)] [29]. The found values for Na concentration in undiluted, 10-fold diluted, and 100-fold diluted serum samples are in good agreement with the Na" " calibration plots. Even in the undiluted serum samples, only a slight potential shift was observed from the calibration. This indicates that the calixarene-based silicone-rubber-membrane Na+-ISFETs are reliable on serum Na assay. For comparison with the silicone-rubber membrane, Na -ISFETs with corresponding plasticized-PVC membrane containing (2) or (5) were also tested for the Na assay. The found values of Na" " concentration... 

A similar strategy was employed to identify a DPP-IV inhibitor (6) with good in vivo potency in a mouse model of diabetes [44], Plasma protein binding, as assessed by shift assay (50% serum), was presented for all final compounds. The compound selected as having the best overall profile was active in vivo at 0.1 mg/kg. The activity at 1 h post-dose was consistent with the free drug principle - total plasma concentration 269 nM murine-free fraction 4% unbound plasma concentration 11 nM in vitro potency versus murine DPP-IV 6nM. 

Changes in analytical bias directly shift the distribution of the patient test values. If the elevated values on a test are associated with specific clinical actions, then the shifts in analytical bias can notably alter the number of patients having test values that exceed the action limit. For example, for a serum calcium assay with an action limit of lO.i mg/dL, an upward bias of 0.2 mg/dL changes the number of patients subjected to further investigation from 6.5% to 15.0%. Similarly, analytical shifts in other critical analytes can cause notable clinical problems, such as false elevations of prostate-specific antigen values triggering prostate ultrasound examinations and biopsies and false elevation of TSH triggering additional thyroid examinations. These small analytical shifts can have major downstream effects on healthcare costs. 

The first studies to address whether STAT proteins can be activated in neutrophils were published 5 months apart in 1995-1996. In one of them, Tweardy et al. [32] investigated the effect of several neutrophil agonists on this response. Whole-cell extracts from neutrophils disrupted by freeze-thaw cycles were analyzed in an electrophoretic mobility shift assay (EMSA) using a human serum-inducible element (hSIE/m67) oligonucleotide probe. A specific complex was induced in cells treated with G-CSF, but could not be supershifted using antibodies raised against individual STAT proteins. The authors concluded that granulocytes express a novel STAT-like protein, which they called STAT-G... 

For quantitative analysis of protein concentration the colorimetric Bradford-assay [147] is most commonly used. Here another Coomassie dye, Brilliant Blue G-250, binds in acidic solutions to basic and aromatic side chains of proteins. Binding is detected via a shift in the absorption maximum of the dye from 465 nm to 595 nm. Mostly calibration is performed with standard proteins like bovine serum albumin (BSA). Due to the varying contents of basic and aromatic side chains in proteins, systematic errors in the quantification of proteins may occur. 

The most frequently used protein assay is based on a method after Bradford (Bradford, 1976), which combines a fast and easily performed procedure with reliable results. However, the Bradford assay has sensitivity limitations and its accuracy depends on comparison of the protein to be analyzed with a standard curve using a protein of known concentration, commonly bovine serum albumin (BSA). Many commercially available protein assays such as those from Pierce or BioRad rely on the Bradford method. The assay is based on the immediate absorbance shift from 465 nm (brownish-green) to 595 nm (blue) that occurs when the dye Coomassie Brilliant Blue G-250 binds to proteins in an acidic solution. Coomassie dye-based assays are known for their non-linear response over a wide range of protein concentrations, requiring comparison with a standard. The dye is assumed to bind to protein via an electrostatic attraction of the dye s sulfonic groups, principally to arginine, histidine, and lysine residues. It also binds weakly to the aromatic amino acids, tyrosine, tryptophan, and phenylalanine via van der Waals forces and hydrophobic interactions. 

The assay sample buffer we have used is 0.14 M sodium chloride with 10 mM phosphate, pH 7.0, phenol red at 10 mg/liter, and an inert protein, usually 0.1% gelatin. The protein is included in all samples to minimize adsorptive losses. Gelatin has proved to be most useful because it is free of most potentially cross-reactive proteins that occasionally contaminate some preparations (e.g., luteinizing hormone in crystalline bovine serum albumin) it is free of most small, nonproteinaceous molecules that occasionally contaminate other preparations (e.g., steroids in ovalbumin) it effectively reduces nonspecific adsorption it is inexpensive and it does not cause foaming or create problems with valves on some automatic pipetting equipment. The concentration of phosphate is low and could be increased or supplemented. In effect we accomplish this by including 50 mM EDTA in the buffer used for the first antibody. The phenol red is included to serve as an indicator of dangerous pH shifts upon addition of sample. 

Blood bacterial culture - positive Blood bacterial stain - positive Blood creatinine - increased Blood immunoabsorbent assay - positive Blood liver enzymes - increased Blood sodium - decreased Blood serum creatinine, total - increased Blood WBC - left shift... 

Rusnak, D.W., et al. A simple method for predicting serum protein binding of compounds from IC50 shift analysis for in vitro assays. Bioorg. Med. Chem. Lett. 2004, 14, 2309-2312. 

No standard reference material and procedure is actually available for molal ion activity assays. Calibration of the electrode is required. The calibration solution should match the mean sample composition as closely as possible. Addition of albumin to the calibration standard does not guarantee high accuracy of the results due to a shift of the assay standard potential that is different for plasma, serum, and albumin. Reports on comparisons of clinical analyzers show that interinstrumental deviations may be considerable. Recommendations for calibration and standardization by the IFCC Committee on Electrolytes and Blood Gases are in preparation. 

The chromatography of bovine serum albumin on gels of hyaluronic acid cross-linked with diepoxybutane has been investigated. A very sensitive spectrophotometric assay of hyaluronidase activity is based on the binding of a carbocyanine dye to the undegraded substrate-this results in a spectral shift... 

Neonatal thyroid screening is an important additional tool for evaluating the potential consequences of iodine deficiency on brain development and for monitoring iodine prophylaxis at the population level. Indeed, neonates, whose very low thyroidal iodine stores make them hypersensitive to iodine deficiaicy, frequently have altered thyroid function tests (elevated serum TSH, possibly low T4) even at moderate degrees of iodine deficiency that do not appear to affect thyroid function in the adults. Therefore, a shift towards higher neonatal TSH values has been proposed as a useful tool to detect and monitor iodine deficient newborn populations. However, such a shift can be interpreted validly if representative sampling of newborns is carried out (at least one hundred determinations in randomly selected populations), with the use of sensitive assays for TSH measurements. 

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