ELISA-like assays

Fluorescent microvolume assay technology (FMAT ) is a bead-based or cell-based fluorescent technology for homogeneous ELISA-like assays. In FMAT, a laser beam is focused on the bottom of the assay well and the localised fluorescence intensity bound to beads (or cells) is detected as an area of intense fluorescence over the unbound and background fluorescence in solution. Different analytes can be detected with appropriate fluorophores and, by using different sized beads, the assay can be multiplexed to monitor multiple analytes.13... 

A number of assays for the hydrolase-type of Hyals were developed in the last decade that facilitated their characterization. These include microtiter-based ELISA-like assays, in which a highly specific HA-binding protein substitutes for the antibody component [186]. Biotinylated HA bound to microtiter plates is subjected to Hyal activity, and the remaining HA quantified by an avidin-enzyme color reaction [107]. HA substrate gel zymography procedures were also formulated that facilitated additional studies of these enzymes [187]. 

Kow YW and Dare A (2000) Detection of abasic sites and oxidative DNA base damage using an ELISA-like assay. Methods 22(2) 164-169. 

An ELISA-like sandwich assay is illustrated for a hypothetical array format in Fig. 1.1. Spots in the array are shown on an underlying nanoparticle bed, and may contain capture antibodies or aptamers on the spots to capture analyte proteins from the sample. After washing with detergent-protein solutions designed to block non-specific binding (NSB), a labeled secondary antibody is added to bind to captured analyte proteins. Enzyme labels catalyze conversion of an added chemical substrate to produce a colored product that is usually measured with an optical... 

Like antibodies, aptamers are characterized by very impressive, unsurpassed affinity, and selectivity properties. Such remarkable feature has determined immense potentialities in the diagnostic field and various analytical systems have been developed, notably in the field of biosensors, ELISA-type assays, flow cytometry, or separation techniques [6, 7]. Specifically, aptamers constitute, with antibodies, the most popular affinity reagent employed for the development of bioaffinity-based LC and CE methodologies. However, the aptamers present many advantages over antibodies. Aptamers can be regenerated within minutes via a denaturation-renaturation... 

Studies also suggested that IL-6 causes endothelial cell dysfunction and decrease of prostacyclin production. Soluble IL-6 receptors seem to play a modulating and enhancing role in IL-6 activity (FI 8). The incidence of detection appears to be less influenced by the method of assay, as either ELISA or bioassay techniques yield consistent results, with a high correlation between these techniques. IL-6 values may be more constant and endocrine-like than those values of TNF and IL-1. 

Over the years, in addition to developments with ELISA reagents such as labels, there have been improvements in automation. This has enabled ELISA to be utilized as a high-throughput tool. Typically, ELISAs can be performed in several hours to days. The most common practice is to precoat the microtiter plate for an overnight incubation period, with the remainder of the steps performed the following day. While ELISAs are fast when compared to other assays such as bioassays, which can take days to weeks, they might be considered slow when compared to methods like HPLC, in which the time from sample injection to chromatogram is a matter of minutes. 

Enzyme-linked immunosorbent assay is a heterogenous immunoassay. Reactions involve a solid phase to which components are sequentially presented and successively bound. This method is very effective in the determination of the total alkaloid content. The positive characteristics of this method are the use of non-toxic reagents and basic equipment with low costs, a small sample volume and the ability to measure alkaloids in crude sample extracts. According to the literature, compared with results obtained from GLC, the precision of ELISA for quinolizidine alkaloids is not as high as that of the gas chromatography procedure, but is adequate for plant breeding purposes. The use of enzymes in developing the methods of quinolizidine alkaloids analysis looks likely to increase in the future. 

The other approach is based largely on informatics. In such an approach, tumors would be profiled in contrast to normal tissue. Tumor-specific markers would be identified via microarrays, as has been done in many publications already. From here, the list of tumor-specific markers would be analyzed to determine if any of these markers represented proteins which were likely to be secreted out of the cell and which may be detected in the peripheral blood stream. Preferably multiple markers would be identified that could be tested using multiplex ELISA assays (antibody arrays). Such work will take time, however, because once the potential markers are identified, antibodies must be generated, validated, and tested for effectiveness as an early diagnostic tool. Such work is being done, but little has been published so far. 

LCAT acts preferentially on lipids transported by HDL (so-called a-LCAT activity), but also on lipids transported by apoB-containing lipoproteins (so-called jS-LCAT activity) [58, 85]. In practice, LCAT activity is measured either as the activity required to esterify radioactive cholesterol that has been exogenously incorporated into native HDL or into artificial HDL-like particles (a-LCAT activity) or which has been equilibrated with endogenous lipoproteins of the plasma sample (cholesterol esterification rate, CER) [21, 58, 85]. Several variations of these assays have been reported, some of which are available as commercial test kits (e.g., Roar Biomedical, New York, USA). In addition, LCAT concentration can be determined by either laboratory-made tests or by a commercial ELISA kits [57]. However, the decrease in LCAT concentration is difficult to judge since it also decreases secondary to HDL deficiency due to causes other than LCAT deficiency. Plasma from patients with LCAT deficiency fails to esterify radioactive cholesterol provided by any substrate. By contrast, plasmas of patients with fish-eye disease show a near-normal cholesterol ester-fication rate but have a selective inability to esterify radioactive cholesterol provided to plasma with native HDL or reconstituted HDL (a-LCAT activity) [58, 85]. 

Enzyme-linked immunoabsorbent assays (ELISA) or radioimmunoassays (RIA) have been utilized to measure amounts of PBAN-like-ir in various tissues of female moths (Iglesias et al., 1998 Ma et al., 1996 Marco et al., 1995, 1996 Rafaeli et al., 1991). Consistent with the in situ immunocytochemical evidence, the highest amounts of activity were found in the SEG and CC. Lower amounts of activity were found in the thoracic and abdominal ganglia including the TAG. Although some variations were observed in the amount of PBAN-like-ir through the photoperiod, in general PBAN levels were found not to vary much with age. Males were also shown to contain PBAN-like-ir that did not vary with the photoperiod. Several studies also demonstrated that PBAN-like-ir was present in... 

ADA BEVS BHK CHO ELISA mAb MDCK MMR SCID tPA VLP adenosine deaminase deficiency baculovirus expression vector system baby hamster kidney cell line Chinese hamster ovary cell line enzyme linked immuno sorbent assay monoclonal antibodies Madin-Darby canine kidney epithelial cells measles, mumps, rubella severe combined immunodeficiency plasminogen activator virus-like particle... 

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