Enzyme-linked immunosorbent assay substrates

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

A classical approach is the enzyme linked immunosorbent assay (ELISA), where the antigen (e.g., the protein to be quantified) is immobilized on the surface of a well. A first antigen-specific antibody is applied to occupy all antigens, before a second antibody binds all primary antibodies on the well. The second antibody carries an enzyme, which now catalyzes a color reaction. If the substrate of the enzyme is given in high excess, the enzyme is saturated and the production of product is linear with time and concentration of second antibody and antigen (Fig. 8). 

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

Enzyme immunoassay (El A) is one of such methods that label antigen or antibody with enzyme. The most representative form of El A is the enzyme-linked immunosorbent assay (ELISA) in which bound antigen or antibody is detected by a linked enzyme that converts a colorless substrate into a colored product (Figure 6.11). 

An ELISA (enzyme-linked immunosorbent assay) allows for rapid screening and quantification of the presence of an antigen in a sample (Fig. 5-28b). Proteins in a sample are adsorbed to an inert surface, usually a 96-well polystyrene plate. The surface is washed with a solution of an inexpensive nonspecific protein (often casein from nonfat dry milk powder) to block proteins introduced in subsequent steps from also adsorbing to these surfaces. The surface is then treated with a solution containing the primary antibody—an antibody against the protein of interest. Unbound antibody is washed away and the surface is treated with a solution containing antibodies against the primary antibody. These secondary antibodies have been linked to an enzyme that catalyzes a reaction that forms a colored product. After unbound secondary antibody is washed away, the substrate of the antibody-linked enzyme is added. Product formation (monitored as color intensity) is proportional to the concentration of the protein of interest in the sample. 

Enzyme-Linked Immunosorbent Assays (ELISAs) These assays are performed in the wells of a plastic microtiter dish. The antigen (protein) is bound to the plastic of the dish. The probe used consists of an antibody specific for the particular protein to be measured. The antibody is covalently bound to an ezyme, which will produce a colored product when exposed to its substrate. The amount of color produced can be used to determine the amount of protein (or antibody) in the sample to be tested. 

With regard to immunosensors, a number of different reporter groups are used, including enzymes which convert a substrate into a highly coloured product (enzyme-linked immunosorbent assay, ELISA ) or which digest a substrate to give a photon of light to expose a film (chemiluminescence). 

Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa has proven its worth as an enzyme label for many years. It is stable, has a moderate size (140 kDa), a high turnover number, and can be assayed using a variety of different substrates. Its activity is easily detected by eye in, for example, immunoblots, and it can be quantified by changes in absorbance, fluorescence, or luminescence for use in enzyme-linked immunosorbent assays. 

The enzyme-labeled antibody can be evaluated by enzyme-linked immunosorbent assay. Immobilize the appropriate antigen on the wells of a microtiter plate or strip (at a concentration of 2-10 ]Xg/ mL), incubate various dilutions of the conjugate for a few hours, wash the wells, add substrate, and measure the amount of product formed (see Chapter 15). This approach may also be used for monitoring conjugate purification in chromatography fractions. 

For all of these targets the versatility of enzyme-linked immunosorbent assays (ELISAs) qualifies this method as an almost universal detection platform, using the highly specific recognition potential of antibodies in conjugation with a detection enzyme and various signal-generating substrates [8, 9]. In contrast to the enormous amplification power of the PCR, these enzymatic methods normally have a detection limit of several millions of molecules. 

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... 

Henares et al. [210] developed a multiple enzyme linked immunosorbent assay (ELISA) chip by using capillary-assembled microchip (CAs-CHIP), which involved simple embedding of a 2 to 3 mm length of square capillaries possessing valving and immunoreaction functions into the microchannels fabricated on a PDMS substrate. The authors immobilized different anti-IgGs... 

B28. Burlingame, R. W., and Rubin, R. L., Subnucleosome structures as substrates in enzyme-linked immunosorbent assays. J. Immunol. Meth. 134, 187-199 (1990). 

Enzyme-linked Immunosorbent Assay. A promising alternative to the RIA procedure is an enzyme-linked immunosorbent assay (ELISA) which depends upon the conjugation of a functional enzyme to either an antigen or antibody. The amount of enzyme present in a competitive binding assay is quantitated instead of the amount of radiolabeled compound. The concentration of the enzyme can be determined through its subsequent reaction with a substrate which results in a measurable spectroscopic change. 

Hu, Y. Y, He, S. S., Wang, X., Duan, Q. H Grundke-Iqbal, I., Iqbal, K., Wang,). (2002). Levels of nonphosphorylated and phosphorylated tau in cerebrospinal fluid of Alzheimer s disease patients an ultrasensitive bienzyme-substrate-recycle enzyme-linked immunosorbent assay. Am J. Pathol. 160,1269-1278. 

Enzyme-linked immunosorbent assays (ELISA) are by far the most common immunological assay used in biochemical and clinico-chemical laboratories (Crowther 1995). The method is just as specific as RIA and it can achieve comparable sensitivities under optimal conditions. It can be carried out in various ways, but the common feature is that detection relies on turnover of a chromogenic substrate by an... 

Radioactivity, however, is still a very sensitive means of measuring the presence or absence of a given material. Assay methodology has now come full circle, to the development of an ultrasensitive enzyme RIA. In this technique, an antigen is bound to a solid phase. Antibody will bind to the antigen, which could be a drug-protein conjugate, and the presence of bound antibody is detected by means of a second antibody coupled to alkaline phosphatase. So far this is the standard enzyme-linked immunosorbent assay (ELISA). However, if the substrate is tritium-labeled adenosine monophosphate, it is converted by the enzyme to tritium-labeled adenosine, which may be readily separated and measured. The detection limit for this assay for cholera toxin is approximately 600 molecules of the toxin (22). 

Enzyme immunoassays (EIA) play an important role in clinical diagnostics, veterinary medicine, environmental control, and bioprocess analysis. Antibodies are coupled to enzymes like peroxidase or phosphatase, whose products can be measured after the degradation of a substrate. Because of its high selectivity and sensitivity, EIA enables the detection of a broad spectrum of analytes in complex samples. A solid phase EIA performed in a plastic microtitre plate is called an enzyme-linked immunosorbent assay (ELISA). The coloured products produced in the ELISA can be measured spectro-photometrically rather than in a scintillation counter as for the RIA. 

ELISA Enzyme-linked immunosorbent assay— heterogeneous immimoassay using enzyme-labelled antigens or antibodies Enzyme Protein capable of catalysing specific reactions of substrates into products... 

Signal antibodies have most often been radiolabeled (for immunoradiometric assays [IRMA]) with ( 25j) 7,62,i%,344,4i4 labeled with a chemiluminescent (for immunochemilummo-metric assays [ICMA]) compound, such as acridinium ester, or an enzyme (enzyme-linked immunosorbent assay [ELISA] or enzyme immunoassay [EIA]), such as ALP, converting a substrate (1,2-dioxetane phosphate) to a chemiluminescent product. 

On the basis of an enzyme thermistor, Mattiasson et al. (1977) developed one of the first immunosensors. Immobilized antibodies against albumin are placed in a column and set into an ET. After injection of an albumin-sample and a known amount of enzyme-labeled albumin, both are separated from the sample matrix by antibody-antigen-interaction. After injection of a substrate, the change in heat is a measure of analyte concentration. The less heat produced means that more albumin has been bound. An elution step regenerates the ELISA. Due to its thermal detection principle, the procedure is called TELISA (thermometric enzyme-linked immunosorbent assay). Figure 3 shows the principle of the TELISA procedure in its sandwich configuration. 

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