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Assay conditions, ELISA

In summary, the simple and flexible Universal-IPCR, with or without nonfunctionalized primary antibody, is well suited for a proof of principle of the general feasibility of the IPCR for a given problem in clinical laboratory applications, as successfully shown in several examples. It allows for an easy access to ultrasensitive research studies—if the necessary time and expertise for some fine-tuning of assay conditions is available. Under these conditions, a typical 1000- to 10,000-fold improvement of ELISA sensitivity is accessible in the research laboratory (see Table 1). [Pg.253]

Figure 1. Dose-response antigen inhibition curves derived from ELISA (enzyme-linked immunosorbant assay), utilizing an antiserum raised against synthetic pigment-dispersing factor (PDF) of Romalea, Assay conditions antigen coat, 200 fmol Romalea PDF/well primary antibody dilution 1 140,000 secondary antibody (peroxidase-labelled antirabbit IgG), 1 1000 other details as described earlier (63). When no Romalea PDF is present in the assay, the obtained value is defined as 100%. Figure 1. Dose-response antigen inhibition curves derived from ELISA (enzyme-linked immunosorbant assay), utilizing an antiserum raised against synthetic pigment-dispersing factor (PDF) of Romalea, Assay conditions antigen coat, 200 fmol Romalea PDF/well primary antibody dilution 1 140,000 secondary antibody (peroxidase-labelled antirabbit IgG), 1 1000 other details as described earlier (63). When no Romalea PDF is present in the assay, the obtained value is defined as 100%.
Several assay conditions were also tested to determine if the ELISA is applicable to analysis procedures for environmental samples. The assay was performed at pH s 5-9 and also in the presence of 0.02 - 2.0% bovine serum albumin. In addition, tap water, water collected from a rice field, and effluent water from a sewage treatment plant were spiked with alkaline-dissolved toxin and then assayed with the ELISA. [Pg.356]

Kinase assays run in ELISA format. IL-2 stimulated T ceU blast proliferation assay. Kinase and cell assay conditions and results for 1 under these conditions have been previously reported. ... [Pg.288]

Note-. (1-9) Organic acid disorders, (10-14) fatty acid oxidation disorders, (15-20) amino acid disorders, (21-23) hemoglobinopathies, (24 and 25) endocrinopathy, (26) other inborn error of metabolism, (27) carbohydrate disorders, (28) miscellaneous genetic conditions, and (29) infectious diseases. MS/MS tandem mass spectrometry, HPLC high pressure hquid chromatography, lEF isoelectrofocusing, RIA radioimmuno assay, and ELISA enzyme-linked immunosorbent assay. [Pg.492]

Van Emon et al. ° developed an immunoassay for paraquat and applied this assay to beef tissue and milk samples. Milk was diluted with a Tween 20-sodium phosphate buffer (pH 7.4), fortified with paraquat, and analyzed directly. Fortified paraquat was detected in milk at less than 1 pgkg , a concentration which is considerably below the tolerance level of 10 pg kg Ground beef was extracted with 6 N HCl and sonication. Radiolabeled paraquat was extracted from ground beef with recoveries of 60-70% under these conditions. The correlation coefficient of ELISA and LSC results for the ground beef sample was excellent, with = 0.99, although the slope was 0.86, indicating a significant but reproducible difference between the assays. [Pg.698]

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. [Pg.193]

For ELISA, an enzyme is linked chemically to the antibody. The labeled antibody is allowed to bind to the unlabeled antigen, under conditions where nonspecific adsorption is blocked, and any unbound antibody and other proteins are washed away. Binding is detected by a reaction that converts a colorless substrate into a colored reaction product. The color change can be read directly in the reaction tray, making data collection very easy, and ELISA also avoids the hazards of radioactivity. This makes ELISA the preferred method for most direct-binding assays (Plested et al. 2003). [Pg.171]

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. [Pg.185]

Another format to test for newly expressed proteins is provided through different ELISA assays. Typically, one antibody is coated on a microtiter plate and serves as a capture antibody while a second antibody (added later in the process) is labeled with a reporter molecule allowing the read-out with optical devices. These ELISAs can be operated in a quantitative manner, but need to be calibrated. The measurement unit can be traced back to the amount of protein present in the calibrator, independent of whether the calibrator consists of purified proteins or other biological materials (e.g., seeds, leaves). The amount of proteins within a plant-derived matrix (leaves, seeds, grains), however, depends on several factors, including environmental conditions and can thus not directly be related to thresholds expressed in weight-%. [Pg.136]

Pentosidine is determined by HPLC with spectrofluorimetric detection (excitation and emission wavelengths of 335 and 385 nm, respectively) (S14), although immunochemical and ELISA assays for determination of various protein oxidative modification products have become increasingly popular (08). Protein-aldehyde adducts can be estimated using adduct-specific antibodies (U2, Wl). Another approach requires stabilization of adducts, producing derivatives resistant to conditions used in protein acid hydrolysis and quantification of hydrolysis products by gas chromatography-mass spectrometry (R7). [Pg.229]

Enzyme-linked immunosorbent assays (ELISA) are by far the most common immunological assay used in biochemical and clinico-chemical laboratories (Crowther 1995). The method is just as specific as RIA and it can achieve comparable sensitivities under optimal conditions. It can be carried out in various ways, but the common feature is that detection relies on turnover of a chromogenic substrate by an... [Pg.230]

ELISA), radioimmunoadsorbent assays (RIA), Western blotting, or other techniques (17,22-24). These methods will detect the presence and also to some extent the specificity of a particular antibody, but will not ensure that the antibody is also suitable for immunocytochemistry (25). For this reason, the antibody should be tested under the experimental conditions of fixing, embedding, and staining, and on the desired tissue to be used subsequently. [Pg.8]

The results have shown that we have developed a sensitive and specific ELISA assay for the analysis of clomazone residues from soil samples. The procedures have demonstrated good recovery of clomazone from soil, and excellent correlation of the ELISA test results with standard GLC methodology. In addition, the results of the ELISA tests demonstrate good correlation between the observed soil levels of clomazone, and crop injury when the bioassay is performed under controlled, greenhouse conditions. This assay could, therefore, be used as a more rapid and convenient analytical method over the standard GLC technique after further validation. [Pg.178]


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