Protein quantitative tests

Riccardi D, Schaefer P, Cui Q (2005) pKa Calculations in Solution and Proteins with QM/MM Free Energy Perturbation Simulations A Quantitative Test of QM/MM Protocols. J Phys Chem B 109 17715-17733. 

Patients with type 2 DM should have a routine urinalysis at diagnosis as the initial screening test for albuminuria. If positive, a 24-hour urine for quantitative assessment will assist in developing a treatment plan. If the urinalysis is negative for protein, a test to evaluate the presence of microalbuminuria is recommended. 

First, laboratory testing is conducted to ascertain the stability of the wine. Like tests for protein stability, tests for determining stability and method for correcting instability vary from winery to winery. Berg (34) suggested that a wine stored at — 4° C for four days, without a bitartrate crystalline deposit, may be considered stable. The wines usually are allowed to warm to room temperature before test results are read. Absence of crystals indicates stability. A quantitative method, the concentration product (36), also can be used to evaluate tartrate stability. 

Standardization and Testing. Potency is determined by quantitating the Hepatitis B antigen by an antibody-binding assay combined with a determination of the amount of protein. Safety testing typical for cell culture-derived products is also performed, and includes assuring the absence of live vims. 

Fluorescein retention measurements are converted to relative amount of fluorescence normalized against protein concentration. Test chemical exposure causing disruption of tight junctions will allow fluorescein molecules to penetrate the comeal tissue and become trapped within its epithelial layer. Thus, fluorescein molecules are used as a permeability tracer and applied in defining comeal epithelial and endothelial permeability (Watsky et al. 1989). FITC-retention provides a quantitative assessment for paracellular leakage... 

The function of clinical chemistry in toxicology (as well as in human and veterinary medicine) is to provide, via laboratory analysis, evaluations of the qualitative and quantitative characteristics of specific endogenous chemical components present in samples of blood, urine, feces, spinal fluid, and tissues. The purpose is to help identify abnormal or pathological changes in organ system functions. The most common specimens used in clinical chemistry are blood and urine, and many different tests exist to test for almost any type of chemical component in blood or urine for example, blood glucose, electrolytes, enzymes, hormones, lipids (fats), other metabolic substances, and proteins. The tests used were all initially applied to human clinical medicine, and may not possess the same utility when performed as part of nonclinical toxicity studies in a wide variety of other species. 

Riccardi, D., Schaefer, R, Cui, Q. pK(a) calculations in solution and proteins with QM/MM free energy perturbation simulations A quantitative test of QM/MM protocols. J. Phys. Chem. A 2005, 109,17715-33. 

Turbidimetry can be used for the measurement of plasma proteins by their reaction with antisera to form light scattering immune precipitates. Many qualitative and semi quantitative tests are also used in clinical chemistry, e.g. the flocculation tests. 

Colorimetric Methods.— Many of the protein colour tests can be made quantitative by comparing the intensity of the colour with a standard test containing a known amount of the same protein. 

Standardization and Testing". RequHemeats are geaerally specified within Hceases Hi the United States, and include a variety of Hi-process tests to assess purity, safety, and potency of the iadividual components and potency and safety of the final product. Potency is standardized by determining the size of the conjugate and the quantitative amount of saccharide that is bound to the carrier protein. General safety and immunogenicity is assessed Hi animals. 

Standardization and Testing". The final vaccine is tested for safety, potency, and residual chemicals. Safety includes testing for endotoxin and stetihty. Potency is evaluated by quantitative determination of the amount of hemagglutinin in the vaccine. Antibody to this glycoprotein is associated with protection. The single radial immunodiffusion (SKID) technique is used to standardi2e the mass of this protein in comparison to a reference preparation. 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

The discrete protonation states methods have been tested in pKa calculations for several small molecules and peptides, including succinic acid [4, 25], acetic acid [93], a heptapeptide derived from ovomucoid third domain [27], and decalysine [61], However, these methods have sofar been tested on only one protein, the hen egg lysozyme [16, 61, 71], While the method using explicit solvent for both MD and MC sampling did not give quantitative agreement with experiment due to convergence difficulty [16], the results using a GB model [71] and the mixed PB/explicit... 

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