Enzymes, detection production

End Point vs Kinetic Methods. Samples may be assayed for enzymes, ie, biocatalysts, and for other substances, all of which are referred to as substrates. The assay reactions for substrates and enzymes differ in that substrates themselves are converted into some detectable product, whereas enzymes are detected indirectly through their conversion of a starting reagent A into a product B. The corresponding reaction curves, or plots of detector response vs time, differ for these two reaction systems, as shown in Eigure 2. Eigure 2a illustrates a typical substrate reaction curve Eigure 2b shows a typical enzyme reaction curve (see Enzyme applications). 

Multienzyme electrodes can increase sensitivity from micromolar to nanomolar detection levels (53,57). In this case the substrate is converted to a detectable product by one enzyme, then that product is recycled into the initial substrate by another enzyme resulting in an amplification of the response signal. For example, using lactate oxidase and lactate dehydrogenase immobilized in poly(vinyl chloride), an amplification of 250 was obtained for the detection oflactate (61). 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Although this class of enzymes is involved in most electrochemical approaches, other enzymes may be investigated electrochemically indirectly. For example, the system can be arranged such that the product of the targeted nonredox enzyme serves as substrate for an appropriately selected redox enzyme. Detection then involves the redox cosubstrate of the redox enzyme. 

The reaction mixture for a coupled assay includes the substrates for the initial or test enzyme and also the additional enzymes and reagents necessary to convert the product of the first reaction into a detectable product of the final reaction. The enzyme aspartate aminotransferase (EC 2.6.1.1), for instance, results in the formation of oxaloacetate, which can be converted to malic acid by the enzyme malate dehydrogenase (EC 1.1.1.37) with the simultaneous conversion of NADH to NAD+, a reaction which can be followed spectropho-tometrically at 340 nm ... 

As the enzyme itself is usually the focus of interest, information on the behavior of that enzyme can be obtained by incubating the enzyme with a suitable substrate under appropriate conditions. A suitable substrate in this context is one which can be quantified by an available detection system (often absorbance or fluorescence spectroscopy, radiometry or electrochemistry), or one which yields a product that is similarly detectable. In addition, if separation of substrate from product is necessary before quantification (for example, in radioisotopic assays), this should be readily achievable. It is preferable, although not always possible, to measure the appearance of product, rather than the disappearance of substrate, because a zero baseline is theoretically possible in the former case, improving sensitivity and resolution. Even if a product (or substrate) is not directly amenable to an available detection method, it maybe possible to derivatize the product with a chemical species to form a detectable adduct, or to subject a product to a second enzymatic step (known as a coupled assay, discussed further later) to yield a detectable product. But, regardless of whether substrate, product, or an adduct of either is measured, the parameter we are interested in determining is the initial rate of change of concentration, which is determined from the initial slope of a concentration versus time plot. 

A modification of these coloring systems has recently been developed that leads to more sensitive detection. Chemiluminescent substrates have been designed that are converted by the enzymes to products that generate a light signal that can be captured on photographic film. This increases the level of sensitivity about 1000-fold over standard color detection methods. 

Fig. 1. Comparison of enzyme-linked immuno sorbent assay (ELISA, left) and immuno-polymerase chain reaction (IPCR, right). During ELISA, an antibody-enzyme conjugate is bound to the target antigen. The enzyme converts a substrate in solution to a detectable product. In IPCR, the antibody-enzyme conjugate is replaced by an antibody-DNA conjugate. The subsequent addition of a DNA polymerase enzyme (e.g., Taq), nucleotides and a specific primer pair uses the antibody-linked DNA marker sequence as a template for amplification of the DNA. The PCR product is finally detected as an indicator of the initial amount of antigen.

In enzyme electrochemical immunoassays the antigen or a second antibody is labeled with an enzyme that catalyzes the production of an electrochemically detectable product and the rate of the product formation is taken to quantify the antigen. 

Alternatively, in the absence of the formation of any product, continue incubation and withdraw samples every hour for analysis. The incubation can be continued for several hours with the expectation that detectable product may yet emerge. In the continued absence of any detectable product, prepare a new reaction mixture that contains more enzyme than the first. If this does not result in the formation of detectable product, the possibility should be considered that the fraction being assayed contains no activity. 

The experiments described in Section 4.2.1 will yield values for two parameters the amount of the enzyme required to form sufficient detectable product and the incubation time required to form this amount of product. Additional experiments now are required to refine the values of both parameters. Keep in mind that to generate the straight line needed to obtain the initial rate,... 

Fluorescence. The use of molecular fluorescence spectroscopy for the quantitation of enzyme reaction products has resulted in detection limits that are several orders of magnitude lower than those achieved by standard absorbance methods. At low analyte concentrations, fluorescence emission intensity is directly proportional to concentration, and its value depends on both the molar absorptivity of the analyte at the excitation wavelength, and the fluorescence quantum yield of the analyte, under the assay conditions. 

For packed-bed enzyme reactors, these results show that low flow rates ensure quantitative conversion of substrate into detectable products. The variation of K m with flow rate indicates that lower flow rates produce higher K m values, so that the linear region of the saturation kinetic curve extends to higher substrate concentrations at lower flow rates. This effect becomes significant when complete conversion does not occur during the residence time of the analyte. 

The analytical detection limits of competitive and noncompetitive immunoassays are determined principally by the affinity of the antibody and the detection limit of the label used, respectively. Calculations have indicated that a lower limit of detection of lOfmol/L (Le., 600,000 molecules of analyte in a typical sample volume of 100 jiL) is possible in a competitive assay using an antibody with an affinity of iO L/mol. Table 9-2 illustrates the detection limits for isotopic and nonisotopic labels. A radioactive label, such as l, has low specific activity (7.5 million labels necessary for detection of 1 disintegration/s) compared with enzyme labels and chemiluminescent and fluorescent labels. Enzyme labels provide an amplification (each enzyme label produces many detectable product molecules), and the detection limit for an enzyme can be improved by replacing the conventional photometric detection reaction by a chemiluminescent or bioluminescent reaction. The combination of amplification and an ultrasensitive detection reaction makes noncompetitive chemiluminescent EIAs among the most sensitive types of immunoassay. Fluorescent labels also have... 

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