Enzyme immunoassays inhibitors

Enzyme catalysis Enzyme electrode Enzyme immobilization Enzyme immunoassay Enzyme inhibitors... 

Blecka, L.J. Shaffar, M. Dworschack, R. Inhibitor enzyme immunoassays for quantitation of various haptens a review. In Immunoenzymatic Techniques Avrameas, S., Dmet, P., Mosseyeff, R., Feldman, G., Eds. Elsevier Amsterdam, 1983 207. 

A collection of redox enzymes for which efficient DET with electrodes has been observed is given in Table 2.3. Most of them are metaUoenzymes containing iron or copper. Many of these enzymes are part of electron transfer chains, i.e., have macromolecular redox partners, or react on large substrates. The evidence for DET has not always been presented by direct electrochemical measurements. In many cases the DET has been proved indirectly by measurement of a substrate dependent catalytic current. Various metabolites ranging from sugars such as fructose, cellobiose and gluconate [6], amines like methylamine and histamine [123], lactate [91],p-cresol [93] and drugs such as benzphetamine [74] can be measured with enzymes in direct contact to an electrode. The bioelectrocatalytic reaction of peroxide is one of the most important reactions not only for the determination of peroxide(s) in various media but also substrates of coupled oxidase [8] and enzyme inhibitors [130, 252]. Furthermore, enzyme immunoassays have been developed based on DET of peroxidase and laccase and electrodes [7,131,132]. 

The usual technique for identification of AChE is fractionation by polyacrylamide gel electrophoresis followed by incubation of the gel with the substrate acetylcholine and copper ions. AChE migrates more rapidly than PChE and appears as a distinct white precipitate band below the PChE band. That the second band is AChE Is documented by inhibition of the enzyme activity in the band by the specific AChE inhibitor l,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide—BW284C51. No method is avail-able commercially laboratories develop their own assay systems using published methodology. Enzyme immunoassay using a monoclonal antibody specific for neurally derived AChE has also been used. ... 

H Tanaka, et al. Enzyme immunoassay discrimination of a new angiotensin converting enzyme (ACE) inhibitor, cilazapril, and its active metabolite. J Pharm Sci 76 224, 1987. 

E Ezan et al. Enzyme immunoassays for a new angiotensin converting enzyme inhibitor, zabicipril, and its active metabolite in human plasma—Application to pharmacokinetic studies. Ther Drug Monit 15 448, 1993. 

Glucagon is extensively degraded in the liver and kidney as well as in plasma and at its tissue receptor sites. Because of its rapid inactivation by plasma, chilling of the collecting tubes and addition of inhibitors of proteolytic enzymes are necessary when samples of blood are collected for immunoassay of circulating glucagon. Its half-life in plasma is between 3 and 6 minutes, which is similar to that of insulin. 

Fig. 2. Schematic representation of the principle of enzyme inhibitor immunoassay (tetrazyme). [Cited and modified from Fig. IV-5, Miyai, K.. in Ishikawa el al., eds. (15).]...

The EMMIA system was developed by Ngo and Lenhoff (N3, N4). In this assay, enzyme activity is modulated by an enzyme modulator which is coupled to antigen (free form) but not by the complex of enzyme modulator-antigen and antibody (bound form). As shown in Fig. 2 and Table 6, in an enzyme inhibitor immunoassay, an enzyme inhibitor is used as a negative modulator. For example, the reaction mixture for measuring thyroxine consists of acetylcholine inhibitor-thyroxine conjugate [I-Ag], acetylcholinesterase [E], unlabeled thyroxine [Ag], and antithyroxine antibody [Ab]. When the amount of unlabeled thyroxine, which binds to antibody [Ab Ag], is increased, the free form of acetylcholine inhibitor-thyroxine conjugate [I-Ag] increases, and the enzyme activity decreases. Therefore, the enzyme activity is inversely proportional to the concentration of unlabeled thyroxine. A tetrazyme kit (Abbott) is now available for measuring thyroxine. 

The TRH radio-immunoassay was developed as a double antibody assay, with modifications mainly depending on the antiserum used, and on the conditions of incubation. Addition of an enzyme inhibitor to the assay tubes is essential to avoid degradation of TRH in samples and of radioiodinated TRH. 

Finley, P.R. WiUiams, R.J. Lichti, D.A. Evaluation of a new homogeneous enzyme-inhibitor immunoassay of serum thyroxine with use of a bichromatic analyzer. Clin. Chem. 1980, 26, 1723-1726. 

PSA exists in two major forms in blood circulation. The majority of PSA is complexed with protease inhibitor ai-antichyraotrypsin (ACT) (MW 100,000) or with a2-macroglobulin (AMG) and a minor component of free PSA (MW 28,430). Most immunoassays measure both free and ACT-compIexed PSA but not AMG-PSA. In human seminal fluid, PSA could be fractionated into five isoforms. PSA-A and PSA-B are active, intact enzymes capable of forming complexation with ACT. PSA-C, PSA-D, and PSA-E are nicked forms with disulfide bonds cleaved they possess low or no enzymatic activities. The inactive forms of free PSA are composed of three distinct molecular forms— bPSA, pPSA, and iPSA. bPSA in tissue is relatively localized in the transition zone of the prostate and contributes to fPSA in BPH serum. pPSA is localized in the peripheral zone of the prostate and contributes to fPSA in cancer serum. 

A minor metabolite of theophylline in adults, caffeine has been shown to accumulate to significant concentrations in neonates. Caffeine itself is an effective inhibitor of apnea, which may explain the lower therapeutic concentration required for control of neonatal apnea. Therapy with caffeine alone has also been demonstrated as effective in the treatment of neonatal apnea it is gaining popularity because of caffeine s long half-life in neonates (>30 hours). Caffeine is metabolized by CyP 1A2 this enzyme is not active in neonates. The optimal therapeutic concentration of caffeine in this situation ranges from 8 to 14pg/mL. Caffeine is measured by HPLC or immunoassay. 

BM Michniewicz, JC Hodges, BG England, T Chang, CJ Blankley, BD Nordblom. A radioimmunoassay for the diacid metabolite of CI-906, a potent angiotensin-I converting enzyme inhibitor. J Clin Immunoassay 10 111, 1987. 

Between 12 and IS billion US per year are spent for analytical purposes worldwide. In this sum the analytical usage of enzymes in clinical chemistry, food and cosmetic industry, and biotechnology for the routine measurement of about 80 substances, mainly low-molecular mass metabolites but also effectors, inhibitors, and the activity of enzymes themselves, is included. A wide range of immunoassays for low-molecular mass haptens, macro-molecules, and microorganisms have been made available in recent years through the enormous progress in immunological research, especially by the preparation of monoclonal antibodies. About 1 billion immunoassays are sold per year. 

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