Continuous assay method

Figure 1.2 The assay of an enzymatic activity by the continuous assay method. In the illustration, the reaction mixture is transferred to a cuvette, which is shown in place in the light path of the spectrometer. The addition of the enzyme directly to the cuvette initiates the reaction. Product formation results in a change in absorbance, which is monitored continuously by the detector. This change signals a deflection on a recorder. Note that product formation requires neither termination of the reaction nor separation of the substrate from the product.

Not all assays require a separation step, and this fact may be used to develop a classification scheme for assay methods. Assays that require no separation have been grouped under the heading continuous assay methods, while discontinuous methods incorporate those that do. 

The enzyme and radioactive substrate are incubated for a known period of time together with appropriate blank samples. The reaction is stopped, often by acid precipitation of the enzyme, and the unreacted substrate and product separated from one another. An aliquot of the isolated product is then taken for radioassay. In only a few cases have continuous assay methods been used in radioisotopic enzyme procedures [310]. 

Udenfriend, S., Gerber, L., and Nelson, N., Scintillation proximity assay a sensitive and continuous isotopic method for monitoring ligand/receptor and anti gen/anti body interactions, Anal. Biochem., 161, 494, 1987. 

A titrametric assay of PLCSc, alternatively called the pH-stat method, was the workhorse in early studies [28]. This method simply involves titrating the acidic product of the PLC reaction as it is formed with a solution of standard base. An advantage of this continuous assay is that it can be used to detect the turnover of both synthetic and natural substrates, and its sensitivity has been estimated to be in the 20-100 nmol range. However, the pH-stat assay has low throughput capability, and it cannot be easily performed in a parallel fashion with multiple substrate concentrations. It is also necessary to exclude atmospheric carbon dioxide from the aqueous media containing the enzyme and substrate. 

The velocity of an enzyme-catalyzed reaction can be measured either by a continuous assay or by a stopped-time protocol. Whenever possible, the continuous measurement of a velocity (e.g., the increase or decrease in absorbance vx. time) should be utilized. In stopped-time assays, the investigator must demonstrate that the reaction is completely terminated at the specified point in time and that products are readily and quantitatively separated from substrates. In addition, one must show that the system is under initial rate conditions. Thus, at least three or four different time points should be chosen. Stopped-time assays also require an assay blank (for t = 0). In this blank, typically the quenching conditions are applied prior to the initiation step. Whenever practicable, replicate kinetic analyses should be done, even with continuous assay protocols. See Enzyme Assay Methods Basal Rate... 

The greatest advantage of the spectrophotometric method is that it is direct and rapid, requires no sample workup, and allows for continuous assays of lipase activity compared to the multiple fixed-time-point analyses incumbent within Basic Protocols 1 and 2. The spectrophotometric method can also be done using very small volumes (as small as 1 ml) and is suitable for following the course of purification (such as in chromatographic fractions) or adaptable to 96-well plates (and subject to automation, if available). Thus, it is the method of choice for screening several samples or preparations for lipase (esterase) activity. 

In practice it is often more convenient to measure the release of a phenol from an aryl phosphomonoester. Standard serum phosphatase methods employ phenyl phosphate (188), p-nitrophenyl phosphate (189), phenolphthalein monophosphate (140), or thymolphthalein monophosphate (141) where the phenol released can be determined spectrophoto-metrically [only the Bodansky method (13) uses a Pi determination]. A number of fluorogenic substrates have been used for phosphatase studies, e.g., jS-naphthyl phosphate (30, 148), 4-methylumbelliferyl phosphate (143), and 3-O-methylfluorescein phosphate (144) The main advantage here is the much greater sensitivity of fluorescence as compared with spectrophotometric assays as little as 1 pmole of 4-methyl-umbelliferone can be detected in continuous assay. 

Israel and Lesbats reported a chemiluminescence method for the determination of acetylcholine, and continuous detection of its release from torpedo electric organ synapses and synaptosomes [55], Birman described a new chemiluminescence assay method for the determination of acetylcholineesterase activity with the natural substrate [56], The method involved monitoring the increase in light emission produced by accumulation of choline, or measuring the amount of choline generated. 

Koops, J., Klomp, H., van Hemert, H. 1990. Rapid enzymatic assay of free fatty acids lipolysis in farm tank milk by a segmented continuous flow method. Comparison of the results with those obtained by the BDI procedure. Neth. Milk Dairy J. 44, 3-19. 

Figure 9.111 Adenylate kinase activity measured by the HPLC assay method. The assay mixture contained 200 fiM ATP, 20 fiM pH]AMP (approximately 120,000 cpm), 50 mM Tris-HCl (pH 7.4), and 32 fig of protein of the enzyme from Dictyostelium discoideum. Samples (20 juL) were injected and the radioactivity monitored continuously with a Berthold LB 503 detector using PICO-Fluor 30 scintillant. Absorbance was measured at 254 nm. Three representative time points were shown (A) 1 minute (B) 30 minutes, and (C) 60 minutes after initiation of the reaction. (From Rosso-mando, 1987.)... 

Enzymatic assay methods are classified as fixed-time assays, fixed-change assays, or kinetic (initial rate) assays. Kinetic assays continuously monitor concentration as a function of time pseudo-first-order conditions generally apply up to 10% completion of the reaction to allow the initial reaction rate to be determined. If the initial substrate concentration is > 10Km, then the initial rate is directly proportional to enzyme concentration. At low initial substrate concentrations (< 0.1 Km), the initial rate will be directly proportional to initial substrate concentration (cf. Chapter 2). For enzyme quantitation, a plot of initial rate against [E] provides a linear... 

All assay methods are based on the forward ALD-catalyzed reaction. Both photometric fixed-time and continuous-monitoring procedures have been developed. In the analytical approach on which all the commonly used procedures and kits are based, the ALD reaction is coupled with two other enzyme reactions. Triosephosphate isomerase (EC 5.3.1.1) is added to ensure rapid conversion of all GLAP to DAP. Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) is added to reduce the DAP to glycerol-3-phosphate, with NADH acting as hydrogen donor. The decrease in NADH concentration is then measured. 

Continuous-monitoring methods for assay of TR-ACP activity are based on the principle introduced by Hillmann in which a-naphthoi released from its phosphate ester forms a colored product with the stabilized diazonium salt of 2-amino-5-chlorotoluene-1,5-naphthalene disulfonate (Fast Red TR). The introduction of alcohols, such as 1,5-pen-tanediol, accelerates the reaction and increases sensitivity by acting as phosphate acceptors in transfer reactions. The addition of sodium tartrate inhibits the sensitive isoenzymes (i.e., prostatic and lysosomal ACPs) if they are present in the sample. 

A number of commercial kits are available for determining PRA " the basic elements of these procedures are summarized in the following sections. With continued improvements and availability of the immunometric assay methods for determining renin mass, renin activity assays will likely be replaced by the direct mass assays for the routine assessment of plasma renin. 

The current approach of using assay methods that did not require quantitative International Chemical Reference Substances (ICRS) should be continued with respect to APIs. 

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