Enzymes conjugates with antibodies

EMIT homogenous enzyme assay - Reaction of ligand-enzyme conjugate with antibody to ligand. Free ligand increases enzyme activity (NAD - NADH). 

Secondary antibodies incubate sections for 60 min at room temperature with the corresponding enzyme-conjugated secondary antibody. Wash sections in a buffer recommended for the corresponding enzyme substrate development. 

It should be noted that an alternative approach that avoids some of the disadvantages of immunofluorescence is the use of enzyme-conjugated secondary antibodies (see Chapter 23). Although this approach sacrifices the resolution of a light-emitting source, low-power objectives compatible with thicker whole-mounts give optimal images (13). 

They are incubated in the primary antibody (PC10, diluted 1 200 in PBS containing 0.2% BSA for PCNA) for 1-2 hr at room temperature. The sections are washed five times for 5 min each with PBS and then incubated in the enzyme-conjugated secondary antibody in PBS containing 1% heat-inactivated normal rat serum for 1 hr. Horseradish peroxidase... 

Yazynina et al. (1999) introduced novel reagents into the inhibition step with the goal of decreasing the long analysis times typically required for ELISA measurements. A polyanion-protein A conjugate was introduced into the atrazine antibody-enzyme conjugate solution. Antibody bound by protein A was in turn bound to the walls of a microplate coated with a polycation. This innovation reduced overall time of analysis to 40 min and achieved a detection limit of 0.03 ppb of atrazine. 

In Chapter 1, the structure, synthesis and toxicity of ricin, a protein present in the endosperm cells of the seeds of the castor oil plant, are described and possible uses of this toxic agent in immunology as conjugates with antibodies are discussed. The biochemical and pharmacological evidence for the presence of functional histamine receptors in the mammalian central nervous system is set out in Chapter 2. Chapter 3 covers the chemical properties of molybdenum-containing enzymes such as aldehyde oxidase and xanthine oxidase, although their physiological roles are by no means clear. 

ELISA - Enzyme linked immunosorbent assay - Reaction of antibody - enzyme conjugate with ligand. Free ligand or antibody affects enzyme activity (NAD NADH). 

Muzykantov et al. (1987) postulated that an antioxidant enzyme conjugated with a specific endothelial ligand will bind to endothelial cells and protect them against oxidative injury. Sakharov et al. (1987) documented that indirect targeting of catalase mediated by polyclonal anti-endothelial antibody protects cultured endothelial cells against the cytotoxic effects of H2O2. Atochina et al. (1998)... 

Another class of immunosensors utilizes enzyme conjugates with antigens at the surface of antibody coated capillaries or thick film patterned glass plates (15). This report describes a PMT and CCD camera based immunosensor for the herbicide (2,4-D) and fertihzers (Atrazine and Simazine). The detection principle improves the sensitivity of the sensor by atleast three orders of magnitude compared to a microtitre plate assay. 

An antibody-enzyme conjugate with specificity against the first antibody is added. The second antibody is species specific. This means that it binds to any immunoglobulin of the species from which the first antibody is derived, e.g., goat anti-mouse antibody. Conjugated to the second antibody is an enzyme such as alkaline phosphatase which can be detected by a colorimetric assay. These conjugated anti-Ig antibodies are available commercially. 

There are many commercially available labeled secondary antibody products. The most common conjugated enzymes are alkaline phosphatase and horseradish peroxidase. Alkaline phosphatase, discussed earlier in the section on histochemistry, dephosphorylates the substrate 5-bromo-4-chloro-3-indolyl phosphate which can be oxidized by nitroblue tetrazolium. The resulting signal would appear as a blue/ purple dye. Horseradish peroxidase can catalyze the oxidation of 4-(chloro-l-naphthol), in the presence of hydrogen peroxide, into an insoluble blue dye. Apart from using antibody-enzyme conjugates, secondary antibodies can be labeled with to permit detection by autoradiography. 

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