Enzyme with antibodies

Fig. 12. Diagram showing sequence for detecting viral antibody by the peroxidase-antiperoxidase (PAP) method of Stemberger et al. This method uses a bridging antibody (anti-primate antibody) between the human and simian antibody. Artificial (chemical) linkage of enzyme with antibody is replaced by an antigen-antibody reaction.

Homogeneous, AM assays pose different requirements. Here, the enzyme should be easily conjugated near the active site without altering its activity. The reaction of hapten- or antigen-labeled enzyme with antibody should affect strongly the enzyme activity, e.g., through steric inhibition of the substrate at the catalytic site. The requirements for optimal ionic conditions and temperature for both enzyme activity and antigen-antibody interaction should be compatible. 

Biosensors ai e widely used to the detection of hazardous contaminants in foodstuffs, soil and fresh waters. Due to high sensitivity, simple design, low cost and real-time measurement mode biosensors ai e considered as an alternative to conventional analytical techniques, e.g. GC or HPLC. Although the sensitivity and selectivity of contaminant detection is mainly determined by a biological component, i.e. enzyme or antibodies, the biosensor performance can be efficiently controlled by the optimization of its assembly and working conditions. In this report, the prospects to the improvement of pesticide detection with cholinesterase sensors based on modified screen-printed electrodes are summarized. The following opportunities for the controlled improvement of analytical characteristics of anticholinesterase pesticides ai e discussed ... 

The enzyme /i-phenylethanolamine-A-methyl transferase, which is required to convert noradrenaline (NA) to adrenaline (Ad), is present in the CNS and there is histofluoro-metric evidence (positive staining with antibodies to that enzyme and to tyrosine hydroxylase and dopamine /i-hydroxylase as well) for adrenergic cell bodies in two groups (nuclei) alongside NA neurons of the locus coeruleus (EC) but ventral and lateral (Ci) and dorsal and medial (C2) to it. Projections go to the hypothalamus and in... 

Homogeneous immunoassays rely on a change in the intensity of the label signal that occurs when labeled antigen binds with antibody. When the label is an antibody, a reduction in the rate of enzyme catalysis forms the basis for the assay. This technique... 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

Remarkably, Brassica napus pollen was reported to have a 22 kDa cutinase that cross-reacted with antibodies prepared against F. solani f. pisi cutinase [134]. Although a 22 kDa and a 42 kDa protein that catalyzed hydrolysis of p-nitrophenyl butyrate were found in this pollen, only the former catalyzed cutin hydrolysis. Immunofluorescence microscopic examination suggested that the 22 kDa protein was located in the intine. Since the nature of the catalytic mechanism of this enzyme has not been elucidated, it is not clear whether this represents a serine hydrolase indicating that plants may have serine and thiol cutinases. The role of the pollen enzyme in controlling compatibility remains to be established. 

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

One of the best ways to ensure retention of activity in protein molecules is to avoid doing chemistry at the active center. The active center is that portion of the protein where ligand, antigen, or substrate binding occurs. In simpler terms, the active center (or active site) is that part that has specific interaction with another substance (Means and Feeney, 1971). For the preparation of enzyme derivatives, it is important to protect the site of catalysis where conversion of substrate to product happens. For instance, when working with antibody molecules, it is crucial to stay away from the two antigen binding sites. 

SMCC frequently is used to prepare hapten-carrier or antibody-enzyme conjugates. In both applications, one of the molecules is activated (usually the carrier or the enzyme) with the... 

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