Enzyme-linked immunosorbent assay methodology

To summarize proposed methodology Future-active Future-passive This project will involve development and applications of... (From Aga, 2002) These studies will be pursued through... (From Kinsel, 1999) Enzyme-linked immunosorbent assays will be employed for the analysis of antibiotics. (From Aga, 2002)... 

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). 

Linked with the previous basic description on antibodies, there is an other form of labelling, which employs an enzyme rather than a radioisotope. In this case the tag (the enzyme) is enormous with respect to the subject molecule. Since the concentrations are very low, recovery is undertaken using an antibody. The best known methodology of this type is the enzyme-linked immunosorbent assay... 

Current methodology frequently applies methods for the analysis of PUHs in soil involving classic LSE with organic solvents at room temperature. Henze et al. performed the extraction of linuron and its metabolites from soil samples by LSE with acetone followed by SPE cleanup. Performing LSE, Liegeois et al. proposed a quantification procedure for isoproturon in soil samples using an enzyme-linked immunosorbent assay technique. Perez et al. isolated chlorotoluron, isoproturon. 

Among the recent publications that detail IsoP quantification in urine, Yan et al. (2007) have reported the detection of three class III isomers and two class VI isomers. Concentrations determined by LC—MS were then correlated with those obtained by enzyme-linked immunosorbent assay (ELISA) measurements. Interestingly, the amount reported by LC-MS was approximately half that found by ELISA. The report by Yan et al. is not the only example of this problem, which most likely results from the cross reactivity of the antibody with other IsoPs in the urines. LC-MS/MS methods are potentially more accurate than ELISA-based methodology because they can separate the individual isomers and reduce interference from the same class of IsoP. 

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