Enzyme-linked immunosorbant assay competitive

Muldoon et al. developed a monoclonal-based competitive inhibition enzyme-linked immunosorbent assay (cELISA) for sulfadimethoxine. The group compared... 

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. 

Schnitzius, J. M., Hill, N. S., Thompson, C. S. and Craig, A. M. 2001. Semi-quantitative determination of ergot alkaloids in seed, straw, and digested samples using a competitive enzyme-linked immunosorbent assay. Journal of Veterinary Diagnostic Investigation, 13 230-237. 

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. 

Figure 8.3. Schematic representation of a non-competitive enzyme-linked immunosorbent assay using the sandwich technique.

Indirect competitive enzyme-linked immunosorbent assay... 

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... 

Competitive electrochemical enzyme-linked immunosorbent assays based on disposable SPEs have been developed for quantitative determination of OTA. 

Competitive enzyme-linked immunosorbent assay provides an alternative to dual-antibody sandwich enzyme-linked immunosorbent assay and is widely used for the measurement of substances in biological liquids. 

Jeon, M., J. Kim, K.-J. Paeng, et al. 2008. Biotin-avidin mediated competitive enzyme-linked immunosorbent assay to detect residues of tetracyclines in milk. Microchem. J. 88 26-31. 

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. 

Ishikawa, M., Nagashima, Y., Shiomi, K. 1999. Immunological comparison of shellfish allergens by competitive enzyme-linked immunosorbent assay. Fisheries Science 65(4) 592-595. 

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