ELISA competitive inhibition

The most common types of assays employed to quantitate protein concentrations in biological matrices are listed in Table 32.4. Enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunoradiometric assays (IRMAs) require protein-specific antibodies, labeled proteins, or labeled antibodies as reagents, and are generally competitive inhibition assays. Radioimmunoassays measure concentrations by displacing ligands from cell-bound receptors. The most common assay, the... 

Very recently we raised three monoclonal antibodies against human liver Mn-SOD. The epitope of one of these antibodies was found to be a COOH-terminal peptide, as judged by competitive inhibition assay using synthetic peptides (K4). Using this antibody we developed an ELISA method and found that the enzyme is also present in human serum. Measurement of the serum immunoreactive Mn-SOD protein levels in various diseases revealed that the enzyme levels are... 

ELISAs are the most prevalent methods used for detecting antibodies. These assays can be conducted using a variety of approaches, which include direct, indirect, bridging, and competitive inhibition formats (Fig. 8.1). These assay formats offer high throughput, are easily automated, use simple technology, require low capital investment, and enjoy comparatively short method development times. A major... 

R.O. Harrison, M.H. Goodrow, B.D. Hammock, Competitive Inhibition ELISA for the s-Triazine Herbicides Assay Optimization and Antibody Characterization , /. Agric. Food Chem., 39,122-128 (1991). 

The systems can all be used in competition/inhibition ELISA for examination of antibodies. The target antigen can be captured first and then competition performed for the detecting antibody. 

The procedures for production of specific antibodies and their application in a competitive inhibition ELISA (Enzyme-Linked Immunosorbent Assay) are discussed in detail in the preceding chapter (Vanderlaan et al., this volume). In addition, other comprehensive overviews of the immunoassay development process in the pesticide field are available in general, a... 

The evaluation of a number of immunoassay diagnostic kits was undertaken to determine their usefulness in a regulatory analytical laboratory environment in the food, feed and pesticide areas. Four rapid enzyme immunoassay tests for the detection of aflatoxin residues at the 20 ppb level in animal feeds were compared to the official HPLC procedure. In the pesticide area, a commercial pentachlorophenol competitive inhibition assay for residues in water was investigated as to its applicability to poultry and pork liver matrices. In addition, an ELISA screening procedure for the herbicide fusilade was developed. Modifications were incorporated into the rapid immunoband 1-2 Test procedure for the detection of motile Salmonella in various food and animal feed products resulting in quicker analysis than the standard culture method. Also, a comparative evaluation of a Quik-Card Test for sulphamethazine drug residues in pork urine, liver and muscle tissue, is described. 

Fig. 1 Competitive inhibition of antibody binding to DMAB-DNA by ELISA. Each well was coated with 10 ng of denaturated DMAB-DNA (5 pmol adducts/ug DNA) and antibody was 25 ng of IgG per well. The inhibiter contained fmol adducts in ug of DNA ( ) 26, (O) 710, (A) 5,140 and ( ) 13,600, respectively.

Figure 1. Sugars and sugar derivatives used as competitors for the competitive inhibition ELISA.

Soluble cytldylyltransferase protein levels were measured using a competitive Inhibition enzyme-linked Immunosorbent (ELISA) method . A polyclonal antibody was raised in rabbit to the purified cytldylyltransferase and this was used in the ELISA with a goat anti-rabbit IgG-horseradish peroxidase conjugate. The colorimetric reaction was obtained with a substrate containing hydrogen peroxide and 0-phenylenediamlne. ... 

Enzyme-Linked Immunosorbent Assays (ELISA). Three methods are commonly used direct competition, double antibody sandwish and antibody inhibition. 

RIAs are highly sensitive and quantitative, capable of detecting small amounts of Ag or Ab. As a result, they are often used to measure the quantities of hormones or drugs present in a patient s serum. In this case, RIAs are performed in a manner similar to the competitive ELISA. The presence of the hormone in the serum sample inhibits binding of the radiolabeled hormone. Thus, the amount of radioactivity present in the test is inversely proportional to the amount of hormone in the serum sample. A standard curve using increasing amounts of known concentrations of the hormone is used to determine the quantity in the sample. 

The ELISA tests are performed using different incubation steps, depending on the assay format, namely sandwich, competitive or inhibition immunoassays that correspond to the way the analyte is captured and further revealed, which is generally dictated by the nature of the analyte. 

The competition assay was designed which followed the standard indirect ELISA format (17-18 . The methoprene conjugate was bound to a solid support in the form of a microtiter plate. Free methoprene in methanol (5 fib) was added to the pre-coated wells followed by methoprene-specific antiserum. The antibodies were allowed to compete for both immunogen-bound and free methoprene. Enzyme-conjugated, goat-antirabbit antibody was added, followed by substrate, and the color was allowed to develop. The absorbance of substrate over a range of methoprene concentrations can be drawn as a standard curve, which is presented as percent inhibition of the assay (Figure 6). The 50% inhibition (I5g) of methoprene was at a concentration of approximately 50 ng/mL. 

Many types of assay are available to be used in HTS protocols to identify inhibitors of PPIs, but a competition assay, in which inhibition of complex formation is measured, is most common. Fluorescence polarization (FPA), fluorescence resonance energy transfer (FRET), enzyme-linked immunosorbent assays (ELISA), and other assay formats have been used. The interacting proteins can be used in their full-length forms though, more frequently only the interacting domains are employed, and if possible the excised interacting peptide is usually preferred. 

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