Antigen plastics, adsorption

The adsorption process, unlike antigen-antibody interactions, is nonspecific. Thus, during the incubation of the immobilized antigen or antibody with enzyme-labeled antigen or antibody, the latter binds specifically to the immobilized immune reactant, but may also be adsorbed directly onto the solid phase. This nonspecific adsorption of enzyme activity can be minimized by inclusion of a nonionic detergent such as Triton X-lOO or Tween 20. These do not interfere with the antigen-antibody reaction but prevent formation of new hydrophobic interactions between added proteins and the solid phase without disrupting to any appreciable extent the hydrophobic bonds already formed between the previously adsorbed protein and the plastic surface. 

The simplest way of fraction separation, however, is when one of the immunoreactants (i.e., either the antigen or the antibody) is immobilized on a solid phase, i.e., immunoreagent modified solid-phase separations, such as plastic tubes, microtitre plate, latex-, glass- or magnetic beads, dipsticks or nitrocellulose membranes [121]. The efl ciency of separation is determined by the nature of the solid surface. Unspecific binding of the label to the solid phase can occur, especially when the latter has hydrophobic properties [96]. The adsorption of the antibody to the solid surface causes its partial inactivation, which can explain some unexpected effects when comparing the same antibody used in liquid and solid-phase systems (see also Section 9.3.4.4). 

Adsorption of antigen or antibody to the plastic solid phase... 

A key feature of the solid-phase ELISA is that antigens or antibodies can be attached to smfaces easily by passive adsorption. This proeess is commonly called coating. Most proteins adsorb to plastic smfaces, probably as a result of hydrophobie interactions between nonpolar protein substructures and the plastie matrix. The interactions are independent of the net charge of the protein, and thus each protein has a different binding constant. The hydrophobieity of the plastic/protein interaction can be e qjloited to increase binding since most of proteins hydrophilic residues are at the outside and most of the hydrophobic residues orientated towards the inside (1). 

It is important to realize that plastic surfaces have a finite capacity for adsorption. The capacity for proteins to attach to microplate wells is influenced by the exact nature of the protein adsorbed to the specific plate used. Saturation levels of between 50 and 500 ng per well have been found valid for a variety of proteins when added as 50-pL volumes. The effective weight of protein per well can be increased if the volume of the attaching protein is increased, effectively increasing the surface area of the plastic in contact with the coating antigen. In cases in which there is an obvious discrepancy between the actual concentration of protein added (where known) and the values just given, then the titration of the ELISA should be re-examined. Thus, if concentrations of, e.g., L CIO mg/mL (or greater) of sample are needed to coat wells, this is not have an ideal situation. 

ELISAs can be used in two modes, qualitatively to determine the presence or absence, or quantitatively to determine the amount of antigen present. ELISA kits often depend on the adsorption of either the antibody or antigen to a solid phase, e.g., wells of a microtiter plate, surface of plastic beads, or plastic stick. The choice of antibody (or antibodies) used determines the specificity of the ELISA assay, which can range from genus-specific to strain-specific. The principle on which ELISA methods are based usually prevents them from being used for the determination of total microbial counts. However, they can be used to detect pathogens such as Salmonella spp.. Listeria spp. 

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