Serum protein binding assay

Serum protein binding is an important contributor to distribution and clearance in vivo. In addition, results from in vitro tests of compounds with high serum protein binding are expected to be sensitive to the presence of serum or other proteins in assay incubations. 

Artefactual increases of as much as 50% in total thyroxine, estimated by a competitive protein-binding assay, and of as much as 30% in triiodothyronine resin uptake are probably due to rapid and continuing lipolytic hydrolysis of triglycerides after blood has been drawn (126). Thyroid function tests should therefore always be performed on blood samples taken before (or a sufficient time after) heparin treatment (127). An increase in serum-free thyroxine concentrations has also been reported after low molecular weight heparin, by up to 171% in specimens taken 2-6 hours after injection. When specimens were obtained 10 hours after injection, the effects were smaller, but with concentrations still up to 40% above normal the results can still cause errors of interpretation (128). 

Competitive Protein-Binding Assays with Vitamin D-Binding Protein. Before the development of immunoassays, 25(OH)D was measured primarily with CPBA with DBP as the specific binder, and with tritiated 25(OH)D3 (>100 Ci/mmol) as tracer. Rat serum, diluted approximately... 

Consistent wdth a major role of the liver in maintaining normal somatomedin levels in the circulation, low levels of somatomedin-A activity have been found in unextracted plasma of patients with cirrhosis of the liver and chronic hepatitis (S14, T5). Significant correlations were seen between somatomedin-A and albumin, cholinesterase, and other indicators of liver function (T5). The decrease measured in this RRA appears to be due to low levels of both somatomedins and binding protein, since Zapf et al. (Z5) have shown an 89% decrease in immunoreactive IGF-I, a 74% decrease in total IGF by protein binding assay (which preferentially measures IGF-II), and a 57% decrease in specific binding of somatomedin tracer to stripped serum, in patients with cirrhosis. 

Schalch, D. S., Heinridt, U. E., Koch, J. C., Johnson, C. J., and Schlueter, R. J., Non-suppressible insulin-like activity of human serum. 1. Development of a new sensitive competitive protein-binding assay. /. Clin. Endocrinol. Meted). 46, 664-671 (1978). 

Z4. Zapf, J., Kaufinann, U., Eigenmann, E. J., and Froesch, E. R., Determination of nonsuppressible insulin-like activity in human serum by a sensitive protein-binding assay. Clin. Chem. (Winston-Salem, N.C.) 23, 677-682 (1977). 

A3. Archibald, E. L., Mincey, E. K., and Morrison, R. T., Estimation of serum folate levels by competitive protein binding assay. Clin. Biochem. (Ottawa) 5, 232-241 (1972). 

Shaw, W., Slade, B. A., Harrison, J. W., and Nino, V., Assay of serum folate Difference in serum folate values obtained by L. casei bioassay and competitive protein binding assay. Clin. Biochem. (CHtawa) 7, 165-178 (1973). 

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. 

C19. Cook, D. B., and Latner, A. L., Saturation in the competitive protein-binding assay of serum thyroxine. Clin. Chim. Acta 47, 183-189 (1973). 

Oers, R. J. M. van, and Gijzen, A. H. J., A systematic investigation on the competitive protein binding assay of th)rroxine in serum with ion exchange strips. Clin. Chim. Acta 38, 363-370 (1972). 

Immunoassays, specific protein-binding assays, and radioisotope tests are sometimes used for the determination of water-soluble vitamins. These are the only feasible and practical methods for the quantification of certain vitamins in physiological samples. The principle of competitive protein binding (CPB) using labeled radioactive or fluorescent tracer is still routinely applied to quantification of serum vitamin... 

Property Structural integrity Solubility (nephelometry) Stability (buffer, plasma) Serum protein binding (see Section 6.3.2.4) Assessing physicochemical properties Providing context to in vivo data interpretation Assay (protein binding) Serum from multiple species 5-10 pM compound concentration Ultrafiltration or equilibrium dialysis in 96-well format Analysis Samples in both buffer and serum matrices " PPT followed by LC-MS/MS... 

Figure 26.4 Automatic measurement of cobalamin (Cbl) in human serum samples on platforms design of the methods. All three methods employ protein binding assays using intrinsic factor (IF) but differ in the detailed assay design. See the main text for details. The figure is published with permission from Clinical Chemistry Laboratory Medicine (Lildballe et al. 2009).

We chose the method developed by Abbott as an example of automatic protein binding assays for analysis of serum cobalamin. Minor variations occur for other automatic platforms. 

Measurement of serum cobalamin includes release of cobalamin from its binding proteins transcobalamin and haptocorrin, conversion of the various forms of cobalamin into one form of the vitamin, and quantification employing microbiological or protein binding assays. 

Standardized protein binding assays have been developed for measurement of total cobalamin in serum. Further studies are required to optimize assays of cobalamin in other matrixes than serum and in order to explore the use of measurement of sub-fractions of cobalamin such as holoTC. 

The methods now used to measure 25-OH-D are competitive protein-ligand binding assays that use either serum globulin (diluted rat serum) (27)28) or a vitamin D-deficient rat kidney... 

For ionizable lipophilic molecules, the right pH gradients can drive the solute in the acceptor compartment to the charged (impermeable) form the uncharged fraction is then further diminished in concentration by binding to the serum protein or surfactant, in the double-sink assay. 

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. 

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. 

Rather than using the initial donor concentration, the final donor concentration at the termination of incubation can be used. Another approach to reduce nonspecific binding involves the addition of serum proteins [80, 120] or micelleforming excipients, such as Gelucire 44/14, Cremophor EL, or TPGS, [45] to the receiver compartment, leading to an improved assay recovery and better predictability of the model [64],... 

Additional assays used in early pharmaceutical property profiles usually include plasma protein binding, individual cytochrome P450 assays, stability in the presence of serum, production of metabolites likely to be involved in covalent binding to biomolecules, and interaction with efflux pumps ... 

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