ELISA modifications

Enzyme-linked immunosorbent assay (ELISA) Modification of radioimmimo-assay in which the anti-antibody, instead... 

Enzyme-Linked Immunosorbent Assay (ELISA) Modification of radioimmunoassay in which the antiantibody, instead of being radioactive, is attached to an enzyme that causes a color change in its substrate. 

Among the many immunological assay methods, the enzyme-linked immunosorbent assay methods (ELISA) are the most popular methods. ELISA can detect both antigen molecules and antibody molecules with only a slight modification of the procedure. The direct-binding and sandwich methods that are used for the... 

Double antibody sandwich ELISA along with their modifications (Hefle et al., 1994) (Figure 3.1.1)... 

The ELISA method (in its various modifications), employing radionuclide markers (radioimmunoassay RIA), enzymatic markers (enzyme immunoassay EIA) (Dill et al., 2006), or fluorescent dyes (fluorophores or fluorochromes) (Hildebrand et al., 2005) (FEIA—fluoroenzyme immunoassay, Immuno-CAP-system) or nanoparticles (Dequaire et al., 2000) are commonly used as materials for protein labeling in an immunoassay. 

The results obtained with the ELISA method have shown that acetylation and succinylation were useful methods for modification of immunoreactive properties of whey allergens (Table 6.3.3). The addition of acetic or succinyl anhydride to milk proteins caused a significant reduction of a-la and P-lg immunoreactivity (Jydrychowski et al. 2005, Wroblewska and J drychowski 2002). 

The main advantage of the ELISA over Western blotting is its ability to analyze many samples at one time. In addition, modifications to the general ELISA protocol can be used to quantify the exact concentration of antigen in an unknown solution, the concentration of a specific antibody in an unknown solution, the dissociation constant for a particular antigen-antibody interaction, or the relative affinity of two different antibodies for the same antigen. An example of an indirect ELISA, and its use in the determination of the concentration of an antigen in an unknown solution, is the subject of Experiment 17. 

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). 

We have already described the design and use of 24-place and 60-place devices for use in solid phase RIA or ELISA techniques. Since that time, we have designed modifications that have increased the numbers of specimens handled simultaneously to 400 and have miniaturized the me-... 

The following gives, for the convenience of newcomers in the ELISA field, some colorimetric methods for measuring HRP activity. Some of the methods are taken from the literature, and some are our own modifications of published assays. 

ELISA is also of limited application when the antigen is extremely diluted, such that other molecules interfere with the performance of the technique. One approach in this case is the so-called substrate-capture ELISA . Here, a substrate for a protease, for example, is bound to the plastic plate in order to absorb and concentrate the specific enzyme for detection by immune reaction. The first modification of a standard capture assay technique in which a metalloprotease substrate is used to capture the enzyme of interest was described by Wacher et al. in 1990, and is summarized in generalized form below. The substrate-capture ELISA greatly simplifies the mixture in which the enzyme is detected and removes potentially interfering substances, thus avoiding some of the difficulties... 

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