Enzymatic incubation procedures

Some authors have used carrier-free enzymatic incubation mixtures at pH 8.0-8.3 (J5, P3, W12). In general, the final concentrations used (incubation at 37°C) were 5-10-fold higher than the solubility of bilirubin at 25°C (B25). Although solubility data at 37°C are not available, it is likely that in most instances the solubility was exceeded. It is not known whether, and to what extent, bilirubin is solubilized in an aspecific way, e.g., by dissolution in lipid membrane regions. Formation of colloidal bilirubin is possible (B25). Aging of the initial, supersaturated (B25) bilirubinate solution is expected to depend (B26) on the procedure of initial solubilization, the time elapsed between lowering the alkaline... 

Most diazo methods (Table 2), with the exception of a 1-minute diazo procedure, are acceptable. For a number of reasons (Section 3) firm recommendations regarding the conditions of enzymatic incubation could not be made. Therefore, it is gratifying that, to a large extent, the choice of the analytical assay system is independent of previous enzymatic incubation. 

Besides the enzymatic incubation in the reaction mixture, all procedures are carried out at 4°C. GTPCH activity is assayed by measuring the neopterin produced upon enzymatic incubation at 37°C for 60 min in a final volume of 0.1 ml in the dark (due to light sensitivity of pterins), followed by chemical oxidation and dephosphorylation. Two separate blanks are prepared, a blank reaction with cell lysate that is immediately oxidized to detect the neopterin that was present in the lysate, and a blank reaction without cell lysate to detect the neopterin that is generated from the incubation (substrate) buffer. The sum of both blanks is later subtracted from the value of the incubation reaction to determine the enzymatically produced neopterin. 

Ironically, AP is the enzyme of choice for some applications due to its stability. Since it can withstand the moderately high temperatures associated with hybridization assays better than HRP, AP often is the enzyme of choice for labeling oligonucleotide probes. AP also is capable of maintaining enzymatic activity for extended periods of substrate development. Increased sensitivity can be realized in ELISA procedures by extending the substrate incubation time to hours and sometimes even days. These properties make AP the second most popular choice for antibody-enzyme conjugates (behind HRP), being used in almost 20 percent of all commercial enzyme-linked assays. 

In the procedure of Wong (W12) bilirubin is incubated enzymatically with [U- C]UDP-glucuronic acid of known specific activity. The derived azo pigments are transferred quantitatively to a thin-layer plate and are separated. The spot of conjugated azo pigment is eluted and counted. With other radioactive UDP-sugars, extension of the procedure to the corresponding transfer processes is obvious. 

The ATP test is a bioluminescence procedure based on the reaction between adenosine-5-triphosphate (ATP) and a luciferin-luciferase enzymatic system (42, 43). The principle of the test relies on the fact that after a certain incubation period the intracellular ATP level, which gives a reliable indication of the state of development of a suitable bacterial culture (44), will remain low relative to a control, when antimicrobial residues are present. In its first version the ATP test employed Bacillus subtilis ATCC 6633 as the test organism, but a current version is based on the use of Streptococcus thermophilus T.J. culture. 

Calibration prior to HPLC requires 1 or 2 days to perform all necessary steps (i.e., prepare stock solutions, filter, measure absorbance, dilute, and HPLC injection of the working solutions). Once the stock solution and working solutions have been prepared, a calibration check can be performed on a UV spectrophotometer within 1 hr. The sample clean-up procedure for HPLC analysis may require afew hours. Enzymatic hydrolysis requires overnight incubation. Each HPLC analysis requires 50 min. When an autosampler is available,... 

Offidani et al. compared chemical hydrolysis with enzymatic hydrolysis utilizing hair samples obtained from heroin users. Hair samples were subjected to incubation in a solution of 1 M NaOH, then neutralized with hydrochloric acid and pH 7, 1 M phosphate buffer. Samples were also treated with a 1-mg/mL pronase solution in 0.05 M Tris buffer for 24 h at 39°C, followed by neutralization with pH 7, 1 M phosphate buffer. Both procedures yielded comparable results for morphine by RIA. 

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