Protein quality assays

Law 480 blends corn-soy and corn-soy-milk, with respect to protein quality by animal assay, organoleptic quality and storage... 

Three animal procedures (protein efficiency ratio, net protein ratio, and protein digestibility) were used to evaluate protein quality. The AOAC (13) animal assay for protein efficiency ratio... 

A number of procedures used to determine protein quality involve bioassays. Bioassays require feeding live animals protein ingredients for a specified period of time, and then estimating the nutritive value of the protein. Two such assays are the rat-based protein efficiency ratio (PER) bioassay and the human nitrogen balance assay (Dimes et al., 1994). Animal feeding experiments require chemical analyses of both the dietary inputs and then the metabolic output of the animal (e.g., body composition analysis, fecal sample analysis, collection, and assay for urine) from which the efficiency of protein metabolism can be predicted as well as how the protein supports animal growth and cell maintenance. 

PER is a method to metabolize or determine the quality of protein in foods. Quality is measured by the amount of usable protein and the growth resulting from it through an animal assay. Formerly, this method was used as the standard method for all protein quality analysis. However, there is some question as to whether or not it is a valid measurement. This is because PER does not account for the differences in amino acid requirements between humans and rats (Seligson and Mackey, 1984), nor does PER account for the protein needed for cell maintenance. Therefore, PER results often overestimate the requirements for some amino acids and underestimate others. Specifically, PER tends to underestimate the protein quality of lysine-deficient proteins such as wheat gluten (Hackler, 1977). 

However, since the PER is an in vivo test, protein digestibility and amino acid bioavailability are encompassed to some extent within the assay. Despite these advantages, it is difficult to determine the individual contribution of digestibility and bioavailability of individual amino acids, or of individual proteins in a complex mixture, on overall protein quality. There are indications that the assay can be shortened from 4 weeks to 2 weeks with little loss in accuracy (Hackler, 1977). 

Values for a test protein will be lower than for the reference protein. The reliability of results for chemical scoring methods depends upon the accuracy of the amino acid determinations that form the basis for these assays. Chemical scoring and animal bioassays tend to provide similar relative rankings of protein quality however, the actual values may be different. Chemical scoring methods should not replace a bioassay for testing the quality of a food protein for which there is very little nutritional information. 

Bicinchoninic acid (BCA), total protein determination, 77-104 Bilirubin, in BCA protein assay, 96 Binding, water, see Water retention Biological value (BV), protein quality, 128-129, 133 Biuret assay... 

Animal assays. When assessing the nature of a protein quality reduction, the conventional methods of protein quality measurement have certain limitations. For example, a reduced TD in an NPU assay is not necessarily the result of a reduced protein digestion. Obviously the same result will be obtained if the increase of fecal nitrogen is caused by an enhanced excretion of endogenous protein or if there is a fixation of metabolic nitrogen by the colonic micro-flora. 

In order to assess the quality of the p53 protein microarrays printed on SAM2 membranes, two different assays are carried out on replica arrays. The antibodybinding assay comprises binding a Cy3-labeled anti-His antibody to the arrayed p53 proteins. This assay is independent of protein activity but provides a measurement of the relative amounts of protein immobilized in each spot. 

Laboratory tests such as urease activity, protein dispersibility index (PDI), nitrogen solubility index (NSI), thiamine, and water absorption have been found valuable in monitoring daily production for protein quality. But biological chick and/or rat assays are the only reliable means currently available for predetermining the nutritional value of whole soybean protein they must be conducted periodically to verify results of chemical tests (31). If whole soybeans are to be used in a mixture containing 20% or more soybean meal, 5% or more urea, and 20% or more molasses, or an equivalent mixture, and exposed to hot, humid storage conditions, it is advisable that the urease activity of the whole soybeans not exceed 0.12 increase in pH (31). Extruded or roasted soybeans properly treated for cattle to increase bypass protein should have urease values of less than 0.05 pH rise. A urease rise of 0.05-0.20 is an indication of proper treatment for swine and poultry. 

Nielsen, H. K., Finot, P. A., and Hurrell, R. F., Reactions of proteins with oxidizing lipids. 1. Influence of protein quality on the bioavailability of lysine, methionine, cysteine, and tryptophan as measured in rat assays, Br. ]. Nutr., 53, 75, 1985. 

In this chapter, the preparation of synaptosomal plasma membranes using centrifugation techniques will be described in detail. The method here is based on that described previously by Kristjansson et al. (14) with only slight modifications. Section 3.1. outlines protocols for dissection and homogenization of the brain, indicating the parameters most important to obtain synaptosomal plasma membrane preparations of reproducibly high quality. Section 3.2. describes the subcellular fractionation procedure itself, and Section 3.3. outlines a protocol for assessment of yield of the synaptosomal plasma membranes employing a protein determination assay described by Bradford (15). 

Protein and ash content of chitosan are very important parameters and those should be investigated early in the quality assay protocol. A high quality grade product should have less than 1 % of protein as well as ash content. 

This chapter will detail protocols used by the authors to study biochemical aspects of the plant polyadenylation complex subunits [8-11]. Three sections are presented for this. The first deals with approaches for overexpression and purification of tagged proteins produced in E. coli. The second describes the suite of electrophoretic and associated methods used to assess protein quality and the outcomes of particular biochemical reactions or assays. The third section describes a set of assays that can be performed using the same lysates, purified proteins, and electrophoretic methods to assess protein-protein interactions, RNA binding capability, and nuclease activity. 

Proteins have different biological availabilities in the human body and a number of methods have been introduced to evaluate and measure protein utilisation and retention. Two types of measurements are used to estimate protein quality biological assays and chemical analysis. The most common measures of protein quality included Biological Value (BV), Protein Efficiency Ratio (PER), Net Protein Utilisation (NPU), Amino Acid Score (AAS also known as chemical score, CS), Essential Amino Acid Index (EAAl) and Protein Digestibility Corrected Amino Acids Score (PDCAAS), amongst other procedures and modifications. 

Cytotoxicity assays based on the Fc-mediated killing can be used as bioactiv-ity/potency assays to evaluate product quality when the desired MoA of the therapeutic protein is to induce death of certain types of cells (e.g., tumor cells). It is well known in the industry and in the literature that some therapeutic proteins (mAbs or molecules of similar structure) with an intact Fc region can exhibit the nontargeted Fc-mediated functions (ADCC, CDC, phagocytosis), which can cause undesired effects, thereby compromising patient safety. The same types of assays can be used to evaluate product s safety profile by testing the ability of the therapeutic protein to exhibit the undesired, nontargeted Fc- mediated functions. In those cases when the Fc function is not a part of the MoA of the therapeutic protein, cytotoxicity assays should be used only as a characterization tool. 

Satterlee, L.D., J.G. Kendrick, D.K. Jewell and W.D. Brown, 1981. Estimating apparent protein digestibility from in vitro assays. In C.E. Bodwell, J.S. Adkins and D.T Hopkins (eds.). Protein quality in humans assessment and in vitro estimation. AVI Publishing Co., Westport, CT 316-339. 

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