Automated enzyme analyzers

Other analyzers such as the Gilford Automated Enzyme Analyzer and the LKB-8600 Reaction Rate Analyzer analyze discrete samples one at a time. These instruments provide kinetic analyses, digital data reduction at the time each sample is analyzed, and excellent electronic and optical characteristics. Recently, Atwood has developed kinetic enzyme analyzers which require only 9 seconds for measuring an enzyme activity, using highly stable and sensitive electronic circuits (12). This short read out time allows a large number of samples to be processed by one instrument in an automated mode. 

The accuracy needed in the measurement of absorbances is related inversely to the measuring interval. The DuPont acauses a 17-sec measuring time for kinetic enzyme assays. Thus for a specimen with an AST activity of 20 U/L and measured using NADH in the indicator reaction, then the absorbance change in 17 sec is only 0.035 A units. A trivial error of 0.004 A introduces an 11% error. Clearly, highly accurate spectrophotometry is needed here and in other automated enzyme analyzers that use very short measuring intervals. 

EIAs can be used per se or with a spectrophotometer. Traditionally, EIAs have been developed in 96-weU microtiter plates which provide the immobilization support for the assay, the reaction vessel, and, when linked to a spectrophotometer-based reader, a rapid means to detect and quantify the color resulting from interaction of a substrate with the antibody—antigen—enzyme complex. Automated immunoassay analyzers targeted primarily for use in the clinical laboratory have taken automation one step further, utilizing robotics to carry out all reagent additions, washings, and final quantification including report preparation. 

Metabolism Bench-top or automated chemistry analyzer assays of cell lysates for key enzymes of intermediary metabolism, antioxidant system, ion transport... 

The histamine content of the supernatants can be determined using the automated fluorometric analyzer (12). Other methods used include a radioisotopic-enzymatic assay (13) or an enzyme immunoassay (14). 

E125 Smith, C.H., Landt, M., Steelman, M, and Ladenson, J.H. (1983). The Kodak Ektachem 400 analyzer evaluated for automated enzymic determination of plasma creatinine. Clin. Chem. 29, 1422-1425. 

Dorn AR, Mountain LD, Phillips M, et al. Roche Diagnostics automated cHnical analyzer enzyme/ receptor assay for mycophenolic acid measurement in transplant patient samples [Abstract]. Ther Drug Monit2003 25 510. 

Ford K, Baker H, Br J. Automated enzyme immunoassay for lutropin with the Abbott Innff " Analyzer. Clin Chem 1989 35 2333-5. 

The future of ISEs in the clinical chemistry instrumentation is quite exciting. As described in subsequent sections of this article, the coupling of enzyme and immunological reagents to ISE detectors to form bioelectrode systems appears to offer manufacturers a new approach toward the detection of metabolites such as creatinine and urea directly in blood and urine samples. Ultimately, such biosensors will be placed into complete electrode-based automated clinical analyzers. In addition, continued research on new membrane formulations, particularly liquid membrane ionophore systems, will result in the development of addition electrodes which can be incorporated into current analyzer systems to expand the electrolyte menu. Indeed, recent efforts have indicated that membranes selective fi)r bicarbonate (F5) and lithium (Z2) are likely additions in the near future. 

The development and application of centrifugal analyzers, a major advance in automated enzyme assays, is described by Tiffany et al. (5). This device was the first to use computerized process control and data reduction. There are many reports in the literature that evaluate or compare specific instruments. Some... 

The ideal analyzer described here does not exist, although various manufacturers have some of the features on their devices, the qualities described here are those currently needed in contemporary automated enzyme assays in clinical laboratories. Our aim is to set some goals for future development in this area. Owing to patients needs and physicians demands, clinical laboratories must always be ready to accept and analyze specimens making them somewhat different from most other analytical services, moreover, redundancy and ruggedness are needed at all times. The current needs are broadly grouped into three areas general human requirements, mechanical/computer needs, and analytical specifications. 

Blanks of the third type are the most difficult to handle. Examples are found in the usual Jaffe reaction for creatinine, and in certain enzyme reactions. In the creatinine [.57-00-1/, C4HgN302, assay, if potential interferences are not removed by dialysis or some similar procedure, they exhibit both slower and faster reactions compared to those of creatinine. A two-point analysis can be utilized here with the measurement times selected to be later than the time required for fast-reacting interferences and before the time for significant reaction of slow-reacting interferences. This procedure is utilized in the creatinine assay on many automated chemistry analyzers. 

The use of some additional enzyme tests has been limited by the lack of available commercially prepared reagents or methodologies unsuitable for the current generation of automated biochemical analyzers. Older manual methods, which were less precise, have largely been discarded, and they are not commonly used in human medicine. There are four broad effects on plasma enzymes in hepatotoxicity ... 

The CK isozymes may be separated by electrophoresis and detected by stains for their enzyme activity. CK-MB immunoassays have also been developed for use in humans that occasionally cross-react with some animal species, although these must first be shown to have appropriate cross-reactivity for the species tested. The LD isozymes have been commonly and readily separated by electrophoresis and stained by their enzymatic activity. For both CK and LD isozymes, their absolute activity is inferred from their relative staining on electrophoretograms and determination of total plasma enzyme activity, typically using an automated chemistry analyzer. 

Edman, Pehr Victor, 1916-1977 (pp. 118,240, Plate 15) born in Stockholm in 1916, matriculation examination in 1935, studied medicine at the Karolinska Institute Medical School in Stockholm from 1935, Bachelor of Medicine in 1938, graduation as a physician in 1946. Concurrently with his studies in medicine he started his training in biochemistry with Erik Jorpes, for a short time also with Hugo Theorell, and soon started a project on angiotensin that led to a MD thesis. Then he widened his experience in protein chemistry during one year at the Rockefeller Institute in Princeton with Northrop and Kunitz (crystallization of proteolytic enzymes). On his return to Sweden, Edman was awarded an associate professorship in Lund in 1947 where he conducted his stepwise peptide degradation work (p. 118) between 1950 and 1956. In 1957 Pehr Edman accepted an offer to be Director of Research at St. Vincent s School of Medical Research in Melbourne, Australia, where he remained for 15 years, during which the work on an automated sequence analyzer was finished in 1967. From 1972 until his death from a brain tumor in 1977 he was Director of the Department of Protein Chemistry of the Max-Planck-Institute for Biochemistry in Martinsried near Munich. 

These techniques have been used successfully in the micro-Zdman degradation of the enzyme mouse sarcoma dihydrofolate reductase to obtain the amino acid sequence of the first 25 amino acids 455). Similarly, RPC has been used in coqjunction with the automated Edman technique for sequencing 32 residues of myoglobin 456). Methionine and its oxidation products, methionine sulfoxide and methionine sulfone, in methionine fortified foods have been analyzed as their dansyl derivatives 457). Lysine has been determined as its dansyl derivative in a study in which the stability of lysine in fortified wheat flour was evaluated (458). 

In addition to analyzing compounds, enzyme sensor has been used to determine the freshness of meats. Xanthine oxidase has been used to determine the levels of xanthine and hypoxanthine that are accumulated from purine degradation during muscle aging so as to monitor fish freshness for a long time. Traditional methods including the automated colorimetric method (54) were time consuming. Jahn et al (55) developed a dipstick test by... 

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