Enzyme bioanalytical method

Pharmacokinetic data analysis requires determination of the analyte in various body fluids. In the case of therapeutic antibodies, serum is the most common matrix to be analyzed. For a critical interpretation of pharmacokinetic data the chosen bioanalytical methods must be considered. The most frequently used for mAbs include enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE)/polyacrylamide gel electrophoresis (PAGE), fluorescence-activated cell sorting (FACS), and surface plasmon resonance (SPR). The challenges and limitations of bioanalytical methods used for the analysis of mAb concentrations are discussed in detail in Chapter 6. 

Due to the high specificity and sensitivity of immunoassays, there are bioanalytical methods for the measurement of an analyte of interest, with little or without preconcentration or purification of the samples. The principle behind immunoassays is based on an interaction between an antibody and a corresponding antigen, and the detection of the specific interaction using radiolabels (247), enzyme, fluorescent and luminescent compounds (178, 179,181,183), electroactive markers (177,180, 228, 248), or nanomaterials (249-251). 

Enzyme Immunoassay and Related Bioanalytical Methods / 1566 Equipment Cleaning / 1580... 

To understand how enzymes are used in bioanalytical methods, and how their concentrations are represented and determined, it is first necessary to examine the kinetics of one- and two-substrate enzymatic reactions. 

The ultimate goal of an assay or an analytical procedure is to measure accurately a quantity or a concentration of an analyte, or to measure a specific activity, as in some assays for biomarkers. However, many activity assays, such as cell-based and enzyme activity assays, may not be very sensitive, may lack precision, and/or do not include the use of definitive reference standards. Assays based on measurements of physicochemical (such as chromatographic methods) or biochemical (such as ligand-binding assays) attributes of the analyte assume that these quantifiable characteristics are reflective of the quantities, concentration, or biological activity of the analyte. For the purpose of bioanalytical method validation, we will follow the recently proposed classifications for assay data by Lee et al. [4,5]. These classifications, as summarized below, provide a clear distinction with respect to analytical validation practices and requirements. 

LC-MS is a powerful method used to detect and quantify CWAs. The use of LC-MS for CWA and hydrolysis product detection has been reviewed [7,20,26]. LC-MS methods are often used to detect CWA hydrolysis/ degradation products instead of the active agents [27-28]. LC-MS serves as a bioanalytical method for CWA detection in living systems and its contributions have also been reviewed [7, 26, 29]. A LC-MS method using an on-line trypsin digestion is used to identify GB and sulfur mustard adducts with proteins and enzymes like human butyryl cholinesterase [30]. This technique, along with similar techniques, could be applied to confirm CWA exposure when illness is suspected from an unknown toxin. 

In the near future we will probably see a sharp increase in the importance of bioanalytical methods as tools for finding new crop protection agents. In this connection, it would be useful to give a comparative assessment of metabolic profiling versus enzyme assays. 

In terms of apphcations, reviews on novel bioanalytical methods based on the use of enzymes, DNAzymes, antibodies, cell slices, to mention the more typical ones, are highly welcome. Articles on subjects related to the areas including genomics, prote-omics, metabolomics, high-throughput screening, but also bioinformatics and statistics as they relate to bioanalytical methods are of course also welcome. Reviews cover both fundamental aspects and practical applications. 

Cetuximab serum concentrations were measured using validated enzyme-linked immunosorbent assay (ELISA) methods or a validated surface plasmon resonance assay. These bioanalytical assays were crossvalidated to allow pooling of PK data across studies. 

Immobilization provides great flexibility, particularly in the design of enzyme-based bioanalytical systems. Some relatively recent developments in protein immobilization methodology which can be broadly classified as reversible immobilization methods, have resulted in novel analytical approaches such as bioaffinity sensors (7) and flow injection binding reactions (8). 

Volume 36 of Methods of Biochemical Analysis focuses on the bioanalytical applications of enzymes. Because enzymes facilitate rapid and highly specific molecular transformations under mild conditions, this class of protein has become increasingly important in analysis, synthesis, manufacturing, and medical diagnosis. 

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