Enzyme-linked immunosorbent quantification

Stanker, L. H., Kamps-Holtzapple, C., Beier, R. C., Levin, C. E., Friedman, M. (1996). Detection and quantification of glycoalkaloids comparison of enzyme-linked immunosorbent assay and high-performance liquid chromatography methods. ACS Symp. Ser, 243-255. 

The assessment of DNA adducts may provide a sensitive indicatCH of previous exposure. The enzyme-linked immunosorbent assay (ELISA) has a lower limit of detection of about 0.08 femtomol per microgram of DNA (Perera et oL, 1982). This assay requires (1) the development of an antibody specific for a certain chemical metabolite bound covalently to DNA and (2) the isolation of DNA from some tissue sample, ag., skin biopsy, or lymphocytes of an exposed individual. It is anticipated that further refinement of such immunologic techniques may lower the threshold of sensitivity by one or two orders of magnitude. One such refined test is the ifitrasensitive ymatic radioimmunoassay (USE-RIA), purported to be about five times more sensitive than ELISA (Hsu et oL, 1981 Shamsuddin et oL, 1985 Harris et oL, 1985). Quantification by the development of monoclonal antibodies to aflatoxin Bj metabolites bound to DNA (Groopman et oL, 1982 Sizaret et oL, 1982) has now been reported. 

An ELISA (enzyme-linked immunosorbent assay) allows for rapid screening and quantification of the presence of an antigen in a sample (Fig. 5-28b). Proteins in a sample are adsorbed to an inert surface, usually a 96-well polystyrene plate. The surface is washed with a solution of an inexpensive nonspecific protein (often casein from nonfat dry milk powder) to block proteins introduced in subsequent steps from also adsorbing to these surfaces. The surface is then treated with a solution containing the primary antibody—an antibody against the protein of interest. Unbound antibody is washed away and the surface is treated with a solution containing antibodies against the primary antibody. These secondary antibodies have been linked to an enzyme that catalyzes a reaction that forms a colored product. After unbound secondary antibody is washed away, the substrate of the antibody-linked enzyme is added. Product formation (monitored as color intensity) is proportional to the concentration of the protein of interest in the sample. 

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... 

Niemeyer CM, Adler M, Blohm D. Fluorometric polymerase chain reaction (PCR) enzyme-linked immunosorbent assay for quantification of immuno-PCR products in microplates. Anal Biochem 1997 246(1) 140-145. 

Nording, M., K. Freeh, Y. Persson, et al. 2006. On the semi-quantification of polycychc aromatic hydrocarbons in contaminated soil by an enzyme-linked immunosorbent assay kit. Anal. Chim. Acta 555 107-113. 

Sanchez, F.G., A.N. Diaz, A.F.G. Diaz, et al. 1999. Quantification of 2,4,5-trichlorophenoxyacetic acid by fluorescence enzyme-linked immunosorbent assay with secondary antibody. Anal. Chim. Acta 378 219-224. 

L Hocine, L., Boye, J.I., Munyana, C. 2007. Detection and quantification of soy allergens in food Study of two commercial enzyme-linked immunosorbent assays. J Food Sci 72 C145-C153. 

Nygren H, Stenberg M. Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) Quantification of antigen by diflusion over an antibody-coated surface. Scand J Clin Lab Invest 1982 42 355-9,... 

Leith, A.G., Griffiths, G.D. and Green, M.A. (1988) Quantification of ricin toxin using a highly sensitive avidin/biotin enzyme-linked immunosorbent assay. J Eorensic Sci Soc, 28, 227-236. 

After more than two decades, advances in IHC have provided a feasible approach to performing immunostain-ing on routinely processed tissues, such that this method is now routine for the performance of IHC special stains in surgical pathology laboratories using EEPE tissues (see Appendix lA). However, demands for improved reproducibility and for quantification have led to a growing recognition that IHC has the potential to be more than just a special stain. If properly controlled in all aspects of its performance, IHC can provide a tissue-based immunoassay with the reproducibility and quantitative characteristics of an ELISA (enzyme-linked immunosorbent assay) test, which not only detects the presence of an analyte (protein or antigen) but also provides an accurate and reliable measure of its relative or real amount (see Quality Control and Standardization section). 

The most prominent of these assays are radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). In the case of RIA, the analyte sample is either extracted or used directly, and mixed with a constant amount of antibody and radiolabeled analyte [tracer mostly fi-emit-ters (e.g., H, " C) or y-emitters (e.g., I)]. After equilibration and separation of the free and bound antigen, radioactivity is measured for quantification of the analyte. In the case of sandwich-RIA, two antibody preparations are used the first serves as the binding partner of the analyte, whilst the second - which is radiolabeled - is directed either against the analyte or the first... 

P15. Pronovost, A. D., Baumgarten, A., and Hsiung, G. D., Sensitive chemiluminescent enzyme-linked immunosorbent assay for quantification of human immunoglobulin G and detection of Herpes Simplex virus. J. Clin. Microbiol. 13, 97-101 (1981). 

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. 

Tayab Z R, Balthasar J P (2004). Development and validation of enzyme-linked immunosorbent assays for quantification of anti-methotrexate IgG and Fab in mouse and rat plasma. J. Immunoassay Immunochem. 25 335-344. 

B.D. Banerjee, Development of an Enzyme-linked Immunosorbent Assay for the Quantification of DDA (2,2-bis (p-Chlorophenyl) Acetic Acid) in Urine , Bull. Environ. Contam. Toxicol, 38, 798 (1987). 

Hefle SL, Bush RK, Yunginger JW, Chu FS (1994). A sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of selected peanut proteins in foods. J. Food. Prot., 57 (suppl.) 419 23. 

This part will mainly focus on host cell proteins (HCPs) quantification. Process-specific HCP assays are in general targeted to be in place prior to the initiation of phase III clinical trials. Immunoassays are the most specific and sensitive techniques available for detecting and quantifying protein impurities. There are two methods commonly employed to quantify protein impurities in biopharmaceuticals enzyme-linked immunosorbent assays (ELISA) and immunoligand assays (ILA). Both methods are able to detect very low ppm level of impurities. ELISA have been developed to measure host protein impurities in a number of recombinant proteins including human growth hormone (Anicetti et ah, 1986), insulin (Baker et al, 1981) and staphylokinase (Wan et ah, 2002). ILA assays have been used to detect protein impurities in recombinant bovine somatotropin (Whitmire and Eaton,... 

Zhang, Y. and Lynd, L.R. (2003) Quantification of cell and cellulase mass concentrations during anaerobic cellulose fermentation development of an enzyme-linked immunosorbent assay-based method with application to Clostridium thermocellum batch cultures. Anal. Chem., 75 (2), 219—227. 

The primary alternative to the instrumental methods summarized above is immunodetection of EA with specific antibodies. Antiserum raised in response to immunization with lysergol (6a) coupled to human serum albumin recognizes several EA (100). Used for competitive enzyme-linked immunosorbent assay (ELISA), this broad-spectrum antiserum allows rapid detection of total EA in many samples concurrently. However, it does not separately quantify the different EA that contribute to the profile, and different EA show different reactivities to the antiserum. Monoclonal antibodies specific for individual EA species, such as ergotamine, also have been raised and provide an effective means for detection and quantification of the particular EA, as well as a valuable tool for physiological studies (101). 

The quantitative aspects of changes in various intracellular pools of NADPH protochlorophyllide oxidoreductase (POR) are important for understanding the principles of operation of the multienzyme system of chlorophyll biosynthesis which proteins are encoded by nuclear DBA and synthesized in the cytoplasm. The application of immunochemical methods /I,2/ allowed to detect loss of POR in the system of intraplastid membranes of etiolated leaves under illumination. In this work the enzyme-linked immunosorbent assay (ELISA) was employed for quantification of POR in different intracellular compartments of postetio-lated and green barley seedlings. 

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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