ELISA systems

Perhaps the most common conjugates of (strept)avidin involve attaching enzyme molecules for use in ELISA systems. As in the case of antibody-enzyme conjugation schemes (Chapter 20), by far the most commonly used enzymes for this purpose are HRP and alkaline phosphatase. Other enzymes such as (3-galactosidase and glucose oxidase are used less often, especially with regard to assay tests for clinically important analytes (Chapter 26). 

To date, complex proteins with biological activity (6), for use in X-ray crystal structure analysis (7) and ELISA systems (8), and for the development of animal drugs (9) have successfully been produced by using this system. Therefore, the Kaiko-baculovirus protein production system has broad applicability across the field of reverse chemical genetics for the analysis of protein function on the basis of interactions with chemical compounds. 

A comparison of ELISA, HPLC, and HPLC-ELISA methods was published for the determination of SAL in chicken liver tissue. Samples were homogenized with MeOH and extracted with methylenechloride. Some samples were analyzed by HPLC using the isocratic solvent system and postcolumn derivatization (vanillin in MeOH containing sulphuric acid), with the eluent monitoring at 520 nm. The HPLC-ELISA system was used to characterize nonspecific effects analyzing column fractions by ELISA, since this detection is over 1000 times more sensitive than the spectrophotometric one. This alleviated the need to derivatize the drug prior to the detection (104). 

The following sections briefly describe the principal enzymes utilized for conjugation with other protein molecules, particularly in the design of ELISA systems. 

Antibody against the target protein (unlabeled or labeled, usually with an enzyme). The enzyme-linked immunosorbent assay (ELISA) system needs to be designed according to each situation. 

Sato, K., Yamanaka, M., Tokesih, M., Morishima, K., Kitamori, T., Multichannel micro ELISA system. Micro Total Analysis Systems 2003, Proceedings 7th p lAS Symposium, Squaw Valley, CA, Oct. 5-9, 2003, 781-782. 

Jensen, M Hartmann, T., Engvall, B et al. (2000) Quantification of Alzheimer amyloid beta peptides ending at residues 40 and 42 by novel ELISA systems. Mol. Med. 6, 291-302. 

Miyaguchi, H., Tokeshi, M., and Kikutani, Y., et al. J. Chromatogr. A 1129,105-110 (2006). Ohashi, T., Mawatari, K., Sato, K., et al. Automated micro-ELISA system for allergy checker a prototype and clinical test, in "Proceedings of Micro Total Analysis Systems 2006" (T. Kitamori, H. Fujita, S. Hasebe Eds.), pp. 858-860. Japan Academic Association Inc., Japan (2006). 

An example of the immobilization on polystyrene beads is the bead-bed micro-ELISA system of Kitamori et al. [403]. The beads were coated with a capture antibody and loaded into the reaction channel of the ELISA microchip (Scheme 4.92a). Next, solutions containing antigen (Scheme 4.92b), a biotinylated secondary antibody (Scheme 4.92c) and a streptavidin-peroxidase conjugate were introduced into the channel (Scheme 4.92d). Finally, the substrate for peroxidase was continuously... 

Instead of the sandwich ELISA-system, which was applied by Damle et al. (1998), Hildebrand et al. (1996) and Audebert et al. (1994), the competitive RIA-system was applied by Wring et al. (1996) and Basu et al. (1996) for toxicokinetic assays. The competitive RIA-system needs only one antibody but this may lead to a loss of selectivity. 

Cell-Based Cell-based ELISA are indirect or direct ELISA systems that use intact cells... 

Recently, LSPCF fiber-optic biosensors are used in clinical diagnosis for alpha-fetoprotein (AFP) identification in human serum. Figure 8.28 shows the relationship between the fluorescent signals and the AFP concentrations of human serum measured by ELISA system (ABBOTT, ARCHITECT i-2000). The preparation and the measurement of human serum were completed at Taipei City Hospital (Taipei, Taiwan), The correlation coefficient R is 0.9331. 

Fig. 10.10. Determination of thermogenin amount in brown adipose tissue mitochondria by the enzyme-linked immunosorbent assay (ELISA) system. The amount of thermogenin was determined as elsewhere described (Cannon et al. [13] Sundin et al. [40] Hansen et al. [56]) in an assay system based on the competition between absorbed and added thermogenin for rabbit on/r-rat-thermogenin antibodies. The interaction was followed with a sheep onri-rabbit-IgG antibody conjugated to alkaline phosphatase. The reaction was linearized as indicated (abs 0 is the absorbance developed in the absence of competing thermogenin). It is seen that this assay can detect less than 0.25 fig thermogenin, i.e., the content in less than 10 fig of mitochondria. It is also seen that the thermogenin content of rat brown fat mitochondria is approximately doubled after a 24 h cold stress. (Our unpublished observations.)...

Several enzyme-linked immunosorbent assays (ELISAS) have been developed for trinitrotoluene, trinitrobenzene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene using polyclonal antibodies raised in New Zealand white rabbits. Nitro substituted benzoic and phenyl acetic acids were used as haptens by conversion to the correspond NHS esters followed by coupling to protein carriers.The antibodies which were developed to 1,3-dinitroaromatic haptens had the greatest specificity and sensitivity when the nitroaromatic analytes contained a 1,3-dinitro functionality. In one ELISA system a lower detection limit for various 1,3-dinitroaromatics analytes of 1 ng/mL with an I50 of 5 ng/mL was observed. No cross reactivity with mononitroaromatic compounds was observed. Antibodies developed to mononitroaromatic haptens showed high affinity for a variety of coating antigens but would not compete with nitroaromatic analytes in a normal ELISA. 

Table II Cross Reactivity Comparison for Various Analytes on Different Antisera/Coating Antigen Systems (NI refers to non inhibition of ELISA system by that analyte)...

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