Amplified ELISA

If the major aim of the ELISA is to obtain quantification of substances present in extremely low concentrations there are a number of adaptations to the technique that can be used. Such techniques often use alkaline phosphatase enzyme systems, which can be used, for example, to lock into a circular redox cycle producing an end product such as red formazan which, is hugely amplified in comparison to standard amplification methods (4). Chemiluminescent amplified ELISA principles have also been shown to give very high... 

The majority of the papers cited above under the individual hormones contain experimental details of how to set up and use immunoassays to quantify these hormones in extracts of plant tissue. Additional procedures can be found elsewhere for auxins [102-105], cytokinins [106-108], abscisic acid [109-115] and gibberellins [116-120]. The sensitivity of immunoassays for most of the plant hormones is generally in the pmole range but occasionally, when high affinity antibodies (K3=10 lmol ) are available, analysis is possible at the fmol level. Using amplified ELISA assays sensitivity down to 200 amol (200 x 10 mol) has been claimed for abscisic acid [113]. 

We perceived the need for sensitive assays that do not rely on the use of radioisotopes or extensive analytical methodology and that could accurately detect protein-bound acetaminophen in biological fluids in the presence of unbound acetaminophen. To this end, we recently developed sensitive avidin biotin-amplified ELISA (A-B ELISA) and particle concentration fluorescence immunoassays (PCFIA) which use antiserum specific for the major acetaminophen-protein adduct associated with toxicity (13-16). These assays are new tools to study the relation between formation of the 3-Cys-A protein adduct and acetaminophen-induced toxicity. In this report we review how these assays were developed, validated in laboratory animals, and used to quantify 3-Cys-A protein adduct formation in human acetaminophen overdose patients. 

Kendall C, lonescu-Matiu I, Dreesman GR. (1983) Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA). J Immunol Methods 56, 329-39. 

Determination of the amount offosor mycprotein in the extract by ELISA using specific antibodies, one of which is conjugated to alkaline phosphatase (AP) and is detected by the AMPAK amplifier system. 

Fig. 1. A schematic representation of the twin-site ELISA for fos and myc proteins. The signal from the alkaline phosphatase label is amplified via the AMPAK enzyme cycle to generate the red formazan dye.

In the procedure described below, the amplified DNA is detected by an ELISA. The detection limit for RT is slightly above the background of this ELISA. It must be realized that the generated signal is dependent on several variables, including incubation time or temperature for reverse transcription, the number of amplification cycles, or the source and properties of enzymes and cofactors. Therefore, to fit signals into the window provided by the ELISA, it will be necessary to adjust some of these variables. 

Surface-dependent effects. It is an underappreciated fact that 384- and 1,536-well formats have a proportionately higher contacted surface area versus contained assay volume see Table 2), so any surface-dependent phenomena is amplified with both positive (e.g., plate coating or ELISA assays) and deleterious (e.g., limiting enzyme depletion by absorption) effects. 

JE Butler, JH Peterman, TE Koertge. The amplified enzyme-linked immunosorbent assay (a-ELISA). In TT Ngo, HM Lenhoff, eds. Enzyme-Mediated Immunoassay. New York Plenum Press, 1985, p 241. 

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