Haptens enzyme-labeled

Conventional ion-selective electrodes have been used as detectors for immunoassays. Antibody binding measurements can be made with hapten-selective electrodes such as the trimethylphenylammonium ion electrode Enzyme immunoassays in which the enzyme label catalyzes the production of a product that is detected by an ion-selective or gas-sensing electrode take advantage of the amplification effect of enzyme catalysis in order to reach lower detection limits. Systems for hepatitis B surface antigen and estradiol use horseradish peroxidase as the enzyme label and... 

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

Figure 3 Immobilized antibody ELISA. Primary antibody (Y) is passively adsorbed to the surface of a polystyrene microtiter plate. Analyte (free H) and an enzyme-labeled hapten (H-E) compete for the fixed number of primary antibody binding sites. Following a wash step (dotted line), the substrate for the enzyme is added O) and a colored product formed ( ). The amount of product is inversely proportional to the amount of analyte present...

Common haptens used for labeling DNA probes for BISH assays are biotin, DIG, DNP, FITC, and Texas Red. Based on the size of your DNA targets, you may choose from a direct detection or an indirect detection for BISH assays. In general, an indirect detection system can provide better sensitivity compared to a direct detection system. For an indirect detection, you need to select a combination of two antibodies raised with two different animal species, such as a mouse anti-DIG antibody and a rabbit anti-DNP antibody, so that enzyme-labeled anti-mouse antibody and anti-rabbit antibody can be applied for signal detection. If a direct BISH detection is going to be applied, anti-hapten antibodies raised in the same animal species that are labeled with either AP or HRP enzyme molecules... 

Enzyme-linked immunosorbent assays. An indirect application of enzymes in analysis is as a marker or label in enzyme-linked immunosorbent assays (ELISA). In ELISA, the enzyme does not react with the analyte instead, an antibody is raised against the analyte (antigen or hapten) and labelled with easily assayed enzyme, usually a phosphatase or a peroxidase. The enzyme activity is proportional to the amount of antibody in the system, which in turn is proportional, directly or indirectly depending on the arrangement used, to the amount of antigen present (Morris and Clifford, 1984). 

An enzyme immunoassay using adenosine deaminase as the enzyme label has been described (318). Potentiometric rate measurements were made with an ammonia gas-sensing electrode. The immunoassay system employed is based on competition between a model haptenic group, dinitrophenyl (DNP) covalently coupled to adenosine deaminase, and free DNP hapten for the available binding sites on the anti-DNP antibody molecules. The detection limit is 50 ng antibody. 

The use of enzyme labels in place of radioisotopes for the measurement of antigens, antibodies, and haptens has stimulated the new and expanding field of enzyme immunoassay (EIA). This technique has been the focus of several recent reviews, - and its merits compared to radioimmunoassay (RIA) have been discussed. In many cases, EIA can match RIA in terms of sensitivity and selectivity, yet has advantages of speed, convenience, and reduced cost. EIA sensitivity and simplicity is, however, dependent on the choice of enzyme label. It is the purpose of this work to introduce urease as a new enzyme label and to demonstrate the... 

A separation step may not always be necessary when measuring low molecular weight haptens by EIA. In some cases, antibody binding to the enzyme-labeled hapten causes complete inhibition of enzyme activity. This may occur when antibody binding either sterically hinders substrate access to the active site of the enzyme or induces enzyme conformational changes. Whatever the cause, a more convenient homogeneous EIA system can result. - ... 

Results here give evidence that urease is a good choice of enzyme label for sensitive and simple assays. We further show that urease can be effectively applied to hapten assays by developing an EIA system for cAMP. 

Labelling with enzymes drastically alters the properties of the labelled compounds, because of the large sizes of the enzjune labels, and the effect is more pronounced for small haptens than for larger compounds (e.g., peptides and proteins). A practice often used is to optimize the length of the spacer arm between the structure containing the antigenic determinants and the enzyme label, so that hindering effects are avoided [97,98]. 

Several ELISA formats have been considered, and the one currently being tested is called the "hapten tracer" method. First introduced to this project by Dr. Freya Jung while a postdoctoral fellow in Dr. Hammock s laboratory, this method uses microtiter plates coated with a commercial preparation of goat anti-mouse antibody. Enzyme-labeled hapten competes with analyte for receptor sites on a mouse monoclonal antibody specific for the analyte. The amount of enzyme left after washing is inversely proportional to the... 

Enzyme-Multiplied Immunoassay Technique. EMIT is a homogeneous method for the quantitation of haptens, especially hormones, therapeutic drugs, and drugs of abuse. This method is a competitive assay, in which hapten and enzyme-labeled hapten compete for a fixed, insufficient quantity of antibody (Eq. 6.16). 

Free enzyme-labeled hapten exhibits high enzyme activity, but if antibody is bound to the hapten moiety, enzyme activity is drastically reduced due to steric... 

The basic tool for detecting antibodies binding to a specific antigen is the ELISA, or Enzyme Linked Immuno-Sorbent Assay [3]. In this assay, antigen-specific antibodies are detected by antigen-mediated attachment to a solid support and secondary detection with an Fc-specific enzyme-labeled secondary antibody (Fig. 2). For hapten immunization, ELISA is performed using a carrier protein, typically BSA (bovine serum albumin), different from the carrier protein used for immunization. In this manner only antibodies with binding specificity to the hapten are revealed by the assay. 

Incubation ot enzyme-labeled hapten reagent with the specimen. 

Addition ot antibody specitic lor hapten, which competes for binding with either tree hapten or enzyme-labeled hapten... 

Substrate Incubation with specific substrate, and quantitation oi product formed, it any. Enzyme-labeled hapten, when bound with the antibody specific to hapten,... 

Competitive homogeneous immunoassays using enzyme-labeled hapten or antigen... 

Crosslinking of haptenated or enzyme-labeled basic polymers... 

---