Labelling with an enzyme

Several enzymes have been used to label antibodies however allcaline phosphatase fix)m bovine intestinal mucosa and horseradish peroxidase are the most widely exploited. They both can react with a range of substrates which permits detection and measurement by hght absorbance or reflectance (via products that form insoluble complexes for example on blotting membranes) and ly fluorescence and luminescence. An important attribute of enzyme labels is that their coloured products are detectable by eye. 

Add 5% glutaraldehyde in water to the antibo slowly and with stirring to give a final concentration of 0.2%. 

Dilute the mixture to 1 ml with PBS and dialyse it against the same buffer (2 litres) at 4 °C overnight. 

Add 50 pi of freshly prepared 0.1 M sodium periodate and stir the solution at room temperature in the dark for 20 min. 

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As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

The presence of biotin labels on an antibody molecule provides multiple sites for the binding of avidin or streptavidin. If the biotin binding protein is in turn labeled with an enzyme, fluoro-phore, etc., then a very sensitive detection system is created. The potential for more than one labeled (strept)avidin to become attached to each antibody through multiple biotinylation sites provides an increase in detectability over antibodies directly labeled with a detectable tag. 

The most common of these systems is the enzyme-multiplied immunoassay technique or EMIT, which is particularly suited to the measurement of small molecules (haptens) such as drugs. EMIT is a trade mark of the Syva Corporation of Palo Alto, California. Although it does not involve the separation of bound fraction from free it is nevertheless a competitive assay system. The antigen is labelled with an enzyme in such a way that the enzyme retains its catalytic activity. When the antigen binds to the antibody the enzyme becomes inhibited, probably by an induced conformational change or by steric hindrance of the enzyme active site (Figure 7.15). 

Enzyme-linked immunosorbent assay (ELISA) is based on the specific reaction between an antibody and an antigen. One of the reagents in the reaction is labeled with an enzyme that generates a colorimetric product that can be measured with a spectrophotometric device. The color intensity correlates with the concentration of specific antibody and the respective antigen. The reaction can be formatted in various ways in a multiwell plate (microtiter plate) with the common formats being the sandwich assay, the competitive assay, and the direct assay. (See Figure 11.1.)... 

The reaction of an affinity label with an enzyme involves the initial formation of a reversibly bound enzyme-inhibitor complex followed by covalent modification and hence irreversible inhibition ... 

Immunohistochemical stains use antibodies to identify specific constituents in tissue sections. In order to detect the site of reaction, the antibody is labeled with an enzyme that can be reacted with a suitable substrate to give a colored product. The alternative is to use a fluorescent label. The advantage of an enzyme label is that the nuclei can be counter stained thereby revealing the tissue architecture, and that the stain fades slowly, if at all, allowing the slides to be stored. 

Most electrochemical immunosensors use antibodies or antigens labelled with an enzyme that generates an electroactive product which can be detected at the electrochemical transducer surface. The combination of high enzyme activity and selectivity with the sensitive methods of electrochemical detection provides a basis for the development of immunosensors. Horse radish peroxidase (HRP) and alkaline phosphatase (AP) are popular enzyme labels and can be used with a variety of substrates. 

Similar to many conventional immunoassays, the detection of the analyte of interest is performed indirectly by formation of a complex using a secondary affinity partner. In order to increase the sensitivity of the detection, this secondary affinity partner can be labelled with an enzyme. This enzyme thus serves as catalyst for the conversion of a... 

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. 

Enzyme immunoassay (ElA) These assays exploit the catalytic properties of enzymes. Typically, antibodies labelled with an enzyme are used, for example horseradish peroxidase. The enzyme, which is bound and remains after washing, is able to convert added substrate to generate a coloured product that can be measured. The major enzyme immunoassay (ElA) is ELISA, which is covered in more detail later. 

There is also another form of avidin developed by Belovo Chemical called Neutralite Avidin (available from Accurate Chemical and Scientific Corp., Westbury, NY). This avidin molecule has been engineered without sugar residues and modified to have a neutral isoelectric point. When properly labeled with an enzyme marker, this molecule should have less tendency to bind nonspecifically to charged sites on the specimen. 

In the enzyme-multiplied immunoassay techniques (EMIT), the antigen or antibody is labeled with an enzyme (e.g., iysozyme, alkaline phosphatase, horseradish peroxidase, or glucose-6-phosphate dehydrogenase) instead of a radioisotope. For example, an alkaline phosphatase-labeled drug can be made to compete with an unlabeled drug for binding sites on... 

By itself, biotin is not a detectable label. It is used as a label because a subsequent irreversible reaction with the proteins avidin or streptavidin (K > 1012 M 1) can be used for ultrasensitive detection, if these proteins have been labeled with an enzyme and an activity stain is used. Conjugates of these proteins with alkaline... 

Figure 3. (a) Schematic representation of the reaction between an antibody (Ab) and its specific antigen (Ag) to form an AbAg immunocomplex. (b) Schematic representation of sandwich immunocomplex formation. A surface-linked capture antibody (c-Ab) is reacting with its target antigen (t-Ag). Then, a labeled detection antibody (d-Ab) is reacting with the t-Ag. (c) Schematic representation of an immunocomplex formed with a d-Ab labeled with an enzyme [12]. 

For sensing low concentrated analytes without an enzymatic activity, these can be labeled with an enzyme via an immunologic reaction using an enzyme labeled antibody speciHc for the respective analyte. This results in an enzyme immunoassay with electrochemical indication. 

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