Sequential assay

While completing sequential assays, some residual activity might be observed. There may be some adsorbed LOX on the glass, quartz, or Teflon-coated stirrer, especially with high activities. In these cases, the sample chamber or cuvette can be washed with 0.1 % Triton X-100 or 75% methanol followed by water washes. 

Employment of glutamate oxidase enables the sequential assay of both transaminases without the need to coimmobilize a second enzyme. Using a glutamate oxidase sensor, Yamauchi et al. (1984) found a preincubation of the sample of 30 min to be necessary. The preincubation time could be lowered to 10 min by using an optimized sensor configuration (Wollenberger et al., 1989). 

The drawbacks of discrete analyzers are their mechanical complexity and high cost of operation. Sample cups, disposable cuvettes, rotors, and prepacked reagents increase the cost of individual assays above the acceptable limit for the strained budgets of most clinical laboratories. In addition, these machines are seldom used outside the clinical laboratory, because they are designed to handle three dozen of the most frequently required clinical tests. The advantages of the discrete approach are the ability of some of these instruments to perform assays via random access—which allows sequential assay of diverse analytes at will—and the capability of stat operation, which yields the analytical readout within 5-10 min after the machine has been switched on and a sample has been inserted by a technician. 

Ultrasensitive assays for PSA contribute to the earlier detection of prostate cancer relapse and (or) residual disease in prostatectomized patients as well as the more timely evaluation of response to current therapies. PSA determinations can be useful in detecting metastatic or persistent disease in patients following surgical or medical treatment of prostate cancer. Persistent elevation of PSA following treatment, or an increase in the pretreatment PSA concentrations, is indicative of recurrent or residual disease. Hence, PSA is widely accepted as an aid in the management of prostate cancer patients, and serum levels are most useful when sequential values are obtained and monitored over time. After complete removal of the prostate gland (radical prostatectomy), PSA levels should become very low or undetectable. A rise of the serum PSA level in prostatectomy patients indicates residual prostate tissue, recurrence, or metastasis of the disease (13, 16, 24, 36). 

Fakhr Eldin et al. [22] described a simple sequential spectrophotometric method for the assay of penicillamine. The method is based on the complex formed when the drug is reacted with Fe(III) solution in hydrochloric acid media. The deep blue colored drug Fe(III) complex is monitored at a maximum wavelength of 600 nm. 

Is there any other approach or concept that can directly measure protein amount in the tissue section Ten years ago, Roth et al.38 documented a novel method, named the Midwestern assay. This method is based on using two chromogens, soluble and insoluble, for the IHC staining process, to produce sequential production of soluble and insoluble reaction products. The soluble IHC product is used to measure the amount of antigen (protein) by spectrophotometry, while insoluble product indicates the localization of protein in the tissue section. Their experimental results demonstrated that soluble reac-... 

Polasek M, Skala P, Opletal L and Jahodar, L. 2004. Rapid automated assay of anti-oxidation/radical-scavenging activity of natural substances by sequential injection technique (SIA) using spectrophotomet-ric detection. Anal Bioanal Chem 379(5—6) 754—758. 

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]. 

Proceed for the assay of AP in the serum sample sequentially as follows ... 

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