ELISA choice

AP is the second most popular choice for antibody-enzyme conjugation, being used in almost 20 percent of all commercial enzyme-linked assays. Although P-gal and GO are used frequendy in research and cited numerous times in the literature, their utilization for commercial ELISA applications represents less than 1 percent of the total assays available. 

Hence, this assay is an extremely useful and selective assay to measure O2 secretion. Because of this selectivity and because it measures the initial product of O2 reduction, it is often used as the method of choice to detect NADPH oxidase activity. It is suitable for semi-automation because assays can be performed in 96-well microtitre plates (using ELISA plate readers with a suitable filter), or cytochrome c reduction can be detected using simple spectrophotometers. The assay, however, is not suitable for measuring O2 that may be generated intracellularly within activated neutrophils. 

For higher throughput applications, injection-molded plastic microtiter plates have served as the formats of choice for automated assay development. Thermoplastics such as polystyrene, polycarbonate, and polypropylene are used for a variety of purposes including storage and assay plates, lids, pipette tips, and Eppendorf PCR tubes. Polystyrene plates are used for cell culture and ELISAs. Polycarbonate reagent bottles are popular, while polypropylene storage plates and PCR tubes are standards. 

The issue of which antibody to select for an assay is not a new problem. Certainly anyone involved in the development of an immunoassay has been faced with this choice. Consider attempting to create a multianalyte, microarray-based micro-ELISA of modest density (10 to 100 analytes) and determining which capture antibodies to use based upon their affinities, stabilities, and cross-reactivities. For a sandwich assay, add in the 10 to 100 analyte-specific secondary (reporter) antibodies and determine their levels of cross-reactivity with each other and with the specified antigens and capture antibodies. In other words, achieving high performance for all analytes with a microarray immunoassay is indeed a formidable challenge. 

Ironically, AP is the enzyme of choice for some applications due to its stability. Since it can withstand the moderately high temperatures associated with hybridization assays, AP is the enzyme of choice for labeling oligonucleotide probes. Alkaline phosphatase also is capable of maintaining enzymatic activity for extended periods of substrate development. Increased sensitivity can be realized in ELISA procedures by extending the substrate incubation time to hours and sometimes even days. These properties make AP the second most popular choice for antibody—enzyme conjugates (behind HRP), being used in almost 20% of all commercial enzyme-linked assays. 

Substrate one step TMB (Zymed) is a common choice see Note 4). Although many other substrates are available for HRP, TMB has high sensitivity with a quick development time. OD can be monitored at 650 nm as the color develops, then at 450 nm, when the reaction is stopped with H2S04. TMB may also be obtained in a lyophilized state and made up fresh with hydrogen peroxidase or as a preprepared one-step solution. TMB can vary considerably between different manufacturers, and this can affect the sensitivity and specificity of the ELISA. With one-step TMB, there is often large batch-to-batch variation as well. Each batch therefore needs to be tested before use. 

ELISA. The focus of this chapter, the competitive ELISA, usually is the preferred choice when a DAS ELISA is not available because it provides greater specificity than the direct ELISA, making it more reliable for the diagnostic procedures it is being used for (7,2). 

PCR-ELISA [37]) were described. The immobilized hapten-labeled amplification product was subsequently detected by an antibody-enzyme conjugate similar to conventional ELISA. By choice of a chemiluminescence- or fluorescence-inducing substrate, the sensitivity of the IPCR essay is further enhanced [37]. A comparison of different detection methods is given schematically in Fig. 6 typical results are compared in Fig. 5. 

As the standardized 96-well microplate format (and multitudes thereof) has found universal prevalence as the platform of choice in routine laboratory applications (ELISA and PCR), the full compatibility of the IPCR... 

HbsAg was also the antigen of choice for the first evaluation of the properties of an in situ IPCR Cao et al. [81, 82] introduced this method in direct similarity to the Universal-IPCR (2.1.2) and PCR-ELISA (2.2.2) protocols, respectively, for the detection of HbsAg in tissue samples. Comparing samples from 17 explanted livers of hepatitis B (HBV)-infected... 

Antibodies have been raised against representative compounds from the major classes of pesticides. Although the ELISA will be useful for individual analysis of a wide variety of compounds, if one needed to analyze several different compounds simultaneously in one matrix immunoassay may not be the method of choice, due to the large amount of controls and standards needed. However, it could be successfully used for the rapid screening of a large number of samples for the presence of specific types of pesticides and for confirmatory tests (Ji). The work reported here with paraquat,... 

RIA) using 2 I-labeled antibodies and enzyme-linked immunosorbent assay (ELISA) using alkaline phosphatase conjugates, wll be described. It is assumed that in most cases, the second antibodies will be bought either as purified material for radiolabeling or already conjugated to fluorescein, biotin, or the enzyme of choice (seeref. 2 for additional methods). 

Choice of Enzyme for EMIT. Since EMIT does not include any separation of immune reactants from sample before enzyme assay, the choice of enzyme becomes restricted to a much less variable group than those that can be used for ELISA. 

However, in the long term, ELISA is an ephemeral format. Even when streamlined and automated, it has too many steps. Certainly we should realize that it will be replaced by other systems, the most exciting of which will be biosensors. Also, other formats offer a proprietary edge in the market place which will be very important in the maturation of immunoassay systems in the environmental field. Finally, different formats will lend themselves to different environmental problems. We should continually emphasize that the same reagents can be used in many formats. Possibly in small letters we also should caution that certain antibody characteristics may be more important in one format than another, that some formats are more resistant to matrix effects, and that relative cross reactivities of compounds can change as one changes the subtle principles upon which an immunoassay works. For this reason a clear choice of formats should be made before initiating validation studies. 

Currently, immunoassay is the practical method of choice for measuring cytokines and their receptors. As cytolanes are proteins, specific antibodies can be raised against recombinant cytokines and have also been measured as an indicator of cytokine presence, The general characteristics of cytokine immunoassays are comparable with the classical immunoassays, with monoclonal, oligoclonal, or polyclonal antibodies all being used (see Chapter 9). The most popular formats are immunoradiometric assay (IRMA) and ELISA, which use a first monoclonal antibody for the capture and a second antibody labeled with a radioisotope or an enzyme. 

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