Assay strategies studies

Because of the lack of adequately designed studies and standardized assays for many products the consequences of their immunogenicity is unclear. So assuming a product to be non-immunogenic based on literature is impossible. In other words, every new protein should be assumed to be immunogenic unless proved otherwise with a well-designed and validated assay strategy. 

Chambers et al. [80] describe a systematic and comprehensive strategy for reducing the potential for matrix effects in HPLC-MS/MS assays. They studied various... 

Jakubik, J., Wess, J., 1999. Use of a sandwich enzyme-linked immunosorbent assay strategy to study mechanism of G protein-coupled receptor assembly. J. Biol. Chem. 274, 1349-1358. 

Chemical approaches also have fueled the development of new assays to study protein-carbohydrate interactions. The features of these interactions mandate such novel strategies. When extracellular carbohydrate-binding events are investigated in solution, the resulting complexes are often of low... 

A full understanding of the role of pectin in plant development requires elucidation of the mechanisms that regulate p>ectin biosynthesis (6). Our strategy for studying the biosynthesis of HGA was to 1) establish a PGA-GalAT assay that would allow detection of synthesized HGA, 2) characterize the enzyme in microsomal membranes, 3) characterize the product synthesized by the enzyme in microsomal membranes, and 4) solubilize the enzyme and characterize the solubilized enzyme and its product. 

The potential clinical utility of the bDNA assay in AIDS patients was demonstrated by monitoring CMV DNA levels in the blood of a patient undergoing gan-cilovir therapy (Chemoff et al 1997), in semen specimens from patients being treated with the antiviral nucleotide analogue cidofovir (Lalezari et al 1995), and in cerebrospinal fluid from patients with CMV neurologic infections (Flood et ai, 1997). These studies demonstrate that bDNA can be used to measure CMV DNA in various clinical specimens. To date, no comparisons of bDNA with other strategies for detection of CMV DNA in clinical specimens have been published. 

A more successful strategy for developing sensitive and facile assays to monitor PLCBc activity involves converting the phosphorylated headgroup into a colorimetric agent via a series of enzyme coupled reactions. For example, phosphatidylcholine hydrolysis can be easily monitored in a rapid and sensitive manner by enzymatically converting the phosphorylcholine product into a red dye through the sequential action of alkaline phosphatase, choline oxidase, and peroxidase [33]. This assay, in which 10 nmol of phosphorylcholine can be readily detected, may be executed in a 96-well format and has been utilized in deuterium isotope and solvent viscosity studies [34] and to evaluate inhibitors of PLCBc [33] and site-directed mutants of PLCBc [35,36]. 

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