Enzyme-linked immunosorbent assays competitive ELISA

Enzyme-linked immunosorbent assay (ELISA). Li and Li developed an ELISA procedure for imidacloprid to determine its residues in coffee cherry and bean extracts. A 25-g amount of sample extracted with 300 mL of methanol and 1% sulfuric acid (3 1, v/v) for 3 min. An aliquot of the sample extract (0.5 mL) is mixed with 1 mL of water and a gentle stream of nitrogen is used to evaporate methanol. The solution is then extracted with 1 mL of ethyl acetate, the extract is reconstituted in 1 mL of PBST (phosphate-buffered saline containing 0.05% Tween 20) and competitive ELISA is performed to quantify imidacloprid in the extract. Eor methanol extracts of coffee cherries and beans fortified with imidacloprid at 0.5 mgL recoveries of imidacloprid by the ELISA method were 108 and 94, respectively. 

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. 

Figure 2 Schematic picture of a competitive enzyme-linked immunosorbent assay (ELISA).

Enzyme labels are usually associated with solid-phase antibodies in the technique known as enzyme-linked immunosorbent assay (ELISA). There are several variants of this technique employing both competitive and non-competitive systems. However it is best used in combination with two monoclonal antibodies in the two-site format in which an excess of antibody is bound to a solid phase such as a test-tube or microtitre plate the test antigen is then added and is largely sequestered by the antibody (Figure 7.12). After washing... 

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.)... 

Enzyme-Linked Immunosorbent Assays (ELISA). Three methods are commonly used direct competition, double antibody sandwish and antibody inhibition. 

Bober, M. A., Kurth, M. J., Milco, L. A., Roseman, D. M., Miller, R. B. and Segal, H. J. 1991. A pyrrolizidine alkaloid-enzyme-linked immunosorbent-assay detection strategy. ACS Symposium Series, 451 176-183 and, Bober, M. A., Milco, L. A., Miller, R. B., Mount, M., Wicks, B. and Kurth, M. J. 1989. A competitive enzyme-linked immunosorbent-assay (ELISA) to detect retronecine and monocrotaline in vitro. Toxicon, 27(9) 1059-1064. 

During in vivo studies under biologically relevant conditions, the cis-Pt loading of the DNA is much lower than for the above-mentioned in vitro studies. It has been calculated that mortality of HeLa cells occurs at an value of 10 5 (i.e., one bound cis-Pt molecule per 105 nucleotides) (64a). This excludes atomic absorption spectroscopy for identification of the in vivo adducts. Immunochemical techniques, however, have shown to be very promising, and high sensitivity and selectivity levels have been reached. At the moment, only a few studies in which antibodies are raised against cis-Pt-treated DNA (64) or against synthetic cis-Pt adducts with mono- or dinucleotides are available (64a). With the latter method, quantitation of the different platinum-DNA adducts formed under in vivo conditions is possible. At the moment, femtomole (10-15 mol) amounts of the adducts can be detected with competitive enzyme-linked immunosorbent assay (ELISA) techniques. It has been demonstrated in this manner that the GG-Pt adduct is also the predominant adduct under in vivo conditions. 

At their most elaborate, epitope mapping techniques can provide detailed information on the amino acid residues in a protein antigen, which are in direct contact with the antibody binding site. X-ray crystallography of antibody-antigen complexes can identify contact residues directly and unequivocally, though not surprisingly in view of the effort required, this method is not in routine use. At the other extreme, demonstration by competition enzyme-linked immunosorbent assay (ELISA) methods that two antibodies bind to different sites on... 

Pinacho, D.G., F. Sanchez-Baeza, and M.P. Marco. 2009. Development of a class selective indirect competitive enzyme-linked immunosorbent assay (ELISA) for detection of fluoroquinolone antibiotics. J. Agric. Food Chem. submitted. 

Competitive binding immunoassay (e.g., RIA) Enzyme-linked immunosorbent assay (ELISA) Immunoradiometric assay (IRMA) — dual monoclonal antibody assay Receptor binding assay Cell binding assay... 

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. 

The most common types of assays employed to quantitate protein concentrations in biological matrices are listed in Table 32.4. Enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunoradiometric assays (IRMAs) require protein-specific antibodies, labeled proteins, or labeled antibodies as reagents, and are generally competitive inhibition assays. Radioimmunoassays measure concentrations by displacing ligands from cell-bound receptors. The most common assay, the... 

Fig. 10.10. Determination of thermogenin amount in brown adipose tissue mitochondria by the enzyme-linked immunosorbent assay (ELISA) system. The amount of thermogenin was determined as elsewhere described (Cannon et al. [13] Sundin et al. [40] Hansen et al. [56]) in an assay system based on the competition between absorbed and added thermogenin for rabbit on/r-rat-thermogenin antibodies. The interaction was followed with a sheep onri-rabbit-IgG antibody conjugated to alkaline phosphatase. The reaction was linearized as indicated (abs 0 is the absorbance developed in the absence of competing thermogenin). It is seen that this assay can detect less than 0.25 fig thermogenin, i.e., the content in less than 10 fig of mitochondria. It is also seen that the thermogenin content of rat brown fat mitochondria is approximately doubled after a 24 h cold stress. (Our unpublished observations.)...

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique in which antibody is immobilized on the walls of a small volume plastic vessel. The ELISA technique can follow either a competitive equilibrium or a sandwich format. Both formats have been used with electrochemical detection. The general protocol for these two formats is shown in Fig. 9. 

Alternatively, MIPs have also been used in biological receptors for competitive binding assays. The assay principle is similar to that in other known biological assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) except that instead of antibodies, MIPs are utilized. This method is often called molecularly imprinted assay (MIA). Typically, in MIA methods, a marker molecule (a labeled analyte analogue) is incubated together with the sample and the MIPs. Analyte and marker molecules compete for the binding... 

Enzyme-Linked Immunosorbent Assay Both competitive and sandwich ELlSAs are available. Although the competitive ELISA is faster because it uses only one incubation with an antibody, it is reported to be less sensitive and exhibits large imprecision. ELISA can be performed on a microplate reader, allowing semiautomation. In the sandwich assay, the primary antibody (antialbumin antiserum) is fixed on the plastic plate, which is then washed. Samples, controls, and calibrators are added, and the complexes detected and quantified by a second antibody conjugated to an enzyme label. 

In most cases, competitive immunoassays for the analysis of small molecules are carried out in microtitre plates. The majority of these assays use an enzyme as label, thus leading to the term enzyme immunoassay, and the most commonly used is the ELISA (enzyme-linked immunosorbent assay) that has a heterogeneous format (separation of bound and unbound). This format can be set up either in the enzyme-tracer format (Figure 3.3.1 A) or in the coating antigen format (Figure 3.3. IB). The result shows a... 

Antibodies can be used for a variety of applications in the molecular characterization of receptors and receptor-hgand interactions. Antibodies can be used for the detection of receptors in tissue shces. Western blot experiments [48], or ELISAs (enzyme-linked immunosorbent assays) [49]. They can also be used in competition experiments to map the binding epitope of a hgand [50]. Even though the use of antibodies is routine, fhere is no general protocol for fheir generation. 

Ricin can be detected in the blood or other bodly fluids of exposed animals using competitive radioimmunoassays or enzyme-linked immunosorbent assays (ELISA). These methods generally do not distinguish between active ricin molecules versus partially degraded or otherwise inactivated toxin. The postexposure time limit for accurate antibody-based detection of ricin in biological samples varies and depends on the route of exposure and the absorbed dose. In the laboratory, ELISA detects ricin in oro-nasal swabs of NHP exposed to ricin aerosol up to 24 h after exposure (Franz and Jaax, 1997). Likewise, ELISA detects ricin in selected tissues of laboratory rats up to 48 h after an i.m. challenge (Leith et al., 1988). 

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