Binding to antibodies

An epitope is an antigenic determinant of the pathogen. It consists of certain chemical groups that are antigenic, which means that it will elicit a specihc immune response by binding to antibodies. 

Figure 333 — (A) Analyte binding to antibodies immobilized onto a sensor surface (a) and electric model used to represent it (b). (B) Illustration of the concept of electrolytic capacitor (a) schematic and (b) electric description. (C) C acitance-based immunosensor (a) vertical section (b) horizontal section 1 tantalum foil 2 tantalum oxide 3 Teflon spacer 4 Teflon plates 5 metal box. (Reproduced from [234] with permission of the American Chemical Society).

Seo et al. (1999) used a planar optic biosensor that measures the phase shift variation in refractive index due to antigen binding to antibody. In this method, they were able to detect S. enterica serovar T) himurium with a detection limit of 1 x 10 cfu/ml. When chicken carcass fluid was inoculated with 20 cfu/ml, the sensor was able to detect this pathogen after 12 h of nonselective enrichment. A compact fiber optic sensor was also used for detection of S. T) himurium at a detection limit of 1 X 10" cfu/ml (Zhou et al., 1997, 1998) however, its efficacy with food samples is unproven. Later, Kramer and Lim (2004) used the fiber optic sensor, RAPTOR , to detect this pathogen from spent irrigation water for alfalfa sprouts. They showed that the system can be used to detect Salmonella spiked at 50 cfu/g seeds. An evanescent wave-based multianalyte array biosensor (MAAB) was also employed for successful testing of chicken excreta and various food samples (sausage, cantaloupe, egg, sprout, and chicken carcass) for S. T) himurium (Taitt et ah, 2004). While some samples exhibited interference with the assay, overall, the detection limit for this system was reported to be 8 x 10 cfu/g. 

Antigen A substance that stimulates the production of antibodies and binds to antibodies. 

If the on rates for small hapten binding to antibody are close to diffusion limited, say, 108 //mole-s, then with k - 3 x 106 //mole we see that the lifetime of the state in which antibody is bound to lipid hapten is approximately 10 2 to 10 3 s. Thus for nitroxide hapten concentrations of the order of 0.3 mole %, each monovalent binding is expected to be followed by divalent binding, in the absence of steric restraints. This conclusion depends strongly on the assumptions, and the kinetics of divalent binding must decrease markedly for lower values of the hapten concentration and diffusion constant. 

Not all proteins bind to antibodies in the usual binding cavity. Protein G, a cell surface protein from Streptococcus bonds to IgG molecules from many different species. Its binding site is on the outer surface of the heavy chain CH1 domain.96... 

An enzyme-linked drug derivative is added to the specimen to be tested. This competes with the drug in question for antibody. The more drug that binds to antibody, the less is bound to enzyme-linked drug. Enzyme activity is proportional to the amount of the drug that was already in the specimen. 

The EMMIA system was developed by Ngo and Lenhoff (N3, N4). In this assay, enzyme activity is modulated by an enzyme modulator which is coupled to antigen (free form) but not by the complex of enzyme modulator-antigen and antibody (bound form). As shown in Fig. 2 and Table 6, in an enzyme inhibitor immunoassay, an enzyme inhibitor is used as a negative modulator. For example, the reaction mixture for measuring thyroxine consists of acetylcholine inhibitor-thyroxine conjugate [I-Ag], acetylcholinesterase [E], unlabeled thyroxine [Ag], and antithyroxine antibody [Ab]. When the amount of unlabeled thyroxine, which binds to antibody [Ab Ag], is increased, the free form of acetylcholine inhibitor-thyroxine conjugate [I-Ag] increases, and the enzyme activity decreases. Therefore, the enzyme activity is inversely proportional to the concentration of unlabeled thyroxine. A tetrazyme kit (Abbott) is now available for measuring thyroxine. 

Haga et al. developed another type of immunosensor by combining an enzyme membrane immunoassay and an enzyme sensor using oxygen electrodes (HI). In this assay antigen molecules (theophylline) are attached on the surface of the liposomes and an enzyme (horseradish peroxidase) is encapsulated in the sensitized liposome. When antibody (antitheophylline antibody) and complement are added, the enzyme is released by the liposome lysis. The enzyme activity with the NADH-NAD reaction can be determined by the oxygen electrode. When antigen is added, it competitively binds to antibodies, then liposome lysis and enzyme activity are decreased. The sensitivity of this method for theophylline determination was reported as 0.7 ng/ml. 

Conjugation (covalent binding to antibody or avidin, for example) or noncovalent binding should not abolish enzyme activity, although it may diminish it. 

Type I Cytophilic antibody (IgE) binds to mast cells antigen binds to antibody and crosslinks receptors, causing mediator (histamine) release immediate hypersensitivity... 

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