Enzyme automated activity assay

CK-MB can be measured in numerous ways. Immunoassays developed in recent years have improved on the analytical and clinical sensitivity and specificity of the earlier immunoinhibition and immunoprecipitation assays. These assays now (1) measure CK-MB directly and provide mass measurements, (2) are easily automated, and (3) provide rapid results (<30 minutes). Mass assays reliably measure low CK-MB concentrations in both samples with low total enzyme activity (<100 U/L) and with high total enzyme activity (>10,000 U/L). Furthermore, no interferences from other proteins have been documented. The majority of commercially available immunoassays that use monoclonal anti-CK-MB antibodies are the same as those listed in Table 5-2 for cardiac troponin assays. Excellent concordance has been shown between mass concentration and activity assays. A primary reference material is commercially available to assist in harmonization. If used for assay standardization, then this material allows... 

Soluble enzymes are employed in a wide variety of substrate and enzyme activity assays, and specialized instrumentation has been developed to automate reagent addition and quantitation. However, several disadvantages exist in the analytical use of soluble enzymes for substrate assays. Soluble enzymes are not reused or recycled, unless the cost of the enzyme justifies the lengthy repurification procedure. Furthermore, the activities of soluble enzymes decreases significantly with time, so that fresh assay solutions are frequently required. For these reasons, many assays that employ soluble enzymes have been adapted for use with immobilized enzymes. These immobilized enzymes are usually incorporated onto or into a stationary phase in a flow system substrate is introduced via a mobile, or buffer phase, and conversion into products occurs as the mobile phase flows through a column containing immobilized enzyme. A postcolumn detector allows product quantitation. 

Automated enzyme activity assays have been a valuable aid to clinical diagnosis (Coodley, 1970) but have been used only to a limited extent in... 

The use of enzyme activity assays in the presence and absence of inhibitors has some merit and can be automated. Cholinesterase variants have been detected by Kalow and Genest (1957) and Harris and Whittaker (1961) using dibucaine and fluoride inhibition, respectively they have provided a means of obtaining both qualitative information (type of variant) and quantitative information (activity of cholinesterase in the sample) about the enzyme in serum. 

Other automated systems may be purchased for a specific purpose and are called dedicated instruments, e.g. glucose analyser. Others have fairly restricted applications, an example being the reaction rate analysers which are specifically designed for the kinetic measurements of enzyme activity. Some of the more recently developed instruments employ individual pre-prepared disposable test packs or strip devices which contain all the reagents for each particular assay in a dry form. 

The ideal assay system for HTS would include automated manipulation of single wells and each should be tested for a variety of activities simultaneously. One of the main challenges is to develop a simple assay sensitive enough to pick up enzyme activities that might be below their optimal level under the assay conditions. Nowadays novel colorimetric, luminescence, and fluorescence methods have been established in HTS with automated multiplex compound testing (typically 10-20 compounds per well) (Winson, 1997). 

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