Enzyme antigen-antibody reaction

The basis for this procedure is the antigen-antibody "reaction"—i e., specific binding of an antibody to the molecule being assayed. Among the many different immunoassay techniques that have been developed—e.g., radioimmunoassay (RIA), and chemoluminescence immunoassay (CIA)—a version of the enzyme-linked immunoassay (ElA) is shown here. 

Further improvement of microchemical methods for proteinaceous media was based on immunological techniques. The high specificity of the antigen-antibody reaction enables the discrimination of the same protein coming from different species, or the detection of multiple antigens in the same sample. Application to the analysis of artwork has been reported in two types of immunological techniques immunofluorescence microscopy (IFM), and enzyme-linked immunosorbent assays (ELISA) [31]. 

NG14. This mutant is capable of elaborating 15 FP units/mL with a productivity of 45 units/L/hr. The final concentration of soluble extracellular protein is 21.2 mg/mL. Samples of this enzyme preparation were sent to G. Pettersson at the University of Uppsala, Sweden, for quantification of each of the enzymes in the cellulase complex using purified antibodies to the individual enzymes. The quantitative antigen-antibody reaction showed that 600 mg/g of this enzyme preparation was one enzyme, cellobiohydrolase. This represents a yield of 13 g/L of cellobio-hydrolase, which is a 100-fold increase over the amount of cellobiohydrolase obtained with strain QM 9414 (130 mg/L) the best previously existing cellulase mutant (G. Pettersson, personal communication). 

The term homogeneous immunoassay may be defined as an immunoassay in which the extent of the antigen-antibody reaction can be determined without physical separation of the free and bound forms. This term is usually used for immunoassays such as enzyme and fluorescence immunoassays in which labeled substances (markers) are used. It does not include immunoassay systems such as nephelometry in which no labeled substances are used. Homogeneous immunoassays are widely used as routine tests because the procedures involved are simple. The principle of homogeneous immunoassay is based on changes in signals of the indicators by the antigen-antibody reaction (N5, U2). 

Many molecules including biopolymers participate in biological functions as a molecular assembly or tissue the self-assembly of the microtublin of bacterial flagella, antigen-antibody reactions, the high activity and selectivity of enzymes, etc. are skillfully and accurately achieved by intermacromolecular interactions. 

After establishing a molecular bridge between antigen and the detection enzyme by means of specific antigen-antibody reactions, the antigen-antibody-enzyme complexes are visualized typically by chromogenic reactions. A variety of enzyme-substrate systems have been defined which yield soluble or insoluble, pigmented end products of different colors based on the needs of the researcher. 

The fractionation and purification of deteriorated proteins is undoubtedly one of the least successful techniques. This is simply because all of the methods that have been developed, with very few exceptions, are directed toward purifying the undeteriorated protein. The methods available are usually based on some particular biochemical activity of the protein, usually enzyme activity, and sometimes an affinity column or affinity adsorbent could be used to separate the native protein from the deteriorated one. Quite often a good affinity adsorbent is unavailable. This procedure, however, does not always work properly even when an adsorbent is available, because the deteriorated protein may possess some activity or an affinity for the adsorbent even though it has lost its natural enzyme activity (see Figure 24). The antigen-antibody reaction can also be used by means of precipitation with antibodies against the native proteins or adsorption on the immobilized antibodies. But here again, the specific antibody must be available, and the deteriorated protein may retain so much affinity for the antibody that differential separations will be impractical in some cases. 

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.

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. 

Glycoproteins play major roles in antigen-antibody reactions, hormone function, enzyme catalysis, and cell-cell interactions. Membrane glycoproteins have domains of hydrophilic and hydrophobic sequences and are amphi-pathic molecules. The carbohydrate moieties of glycoproteins are distributed asymmetrically in cell membranes, cluster near one end of the protein molecule (Figure 10-7), and constitute a hydrophilic domain of amino acid residues (Chapter 21) as well as carbohydrates. The hydrophobic domain of the molecule interacts with the lipid bilayer. 

The basic IA measures the reaction of an analyte with its specific antiserum. A labeled antigen or antibody is used as the tracer to quantitate the extent of the reaction. The label can be radioisotopic or nonisotopic. In enzymatic IA, the antigen-antibody reaction is linked to an enzyme reaction which releases a chro-mophore detected by colorimetric, UV, or fluorimetric devices. 

Nonspecific interference can be encountered as a result of changes in temperature, ionic strength, and pH, or as a result of the presence of hemolysis or excessive quantities of bilirubin, heparin, and urea. Any of these factors can alter the composition of the incubation medium and affect the kinetics or equilibrium of the antigen-antibody reaction. Nonspecific interference contributes to assay variability and results in a decrease in sensitivity. This is particularly prevalent in early enzyme IA applications. Assay sensitivity can be greatly improved with increased assay specificity. 

Immunoassay techniques are known for their high selectivity and sensitivity. Radioimmunoassay (RIA) as well as enzyme-linked immunosorbent assay (ELISA) are often used in clinical analysis. However, to use these techniques it is necessary that more attention should be given to sample pickup due to the possibility of producing an antigen-antibody reaction that can act as an interferent for the analyte that is to be assayed. When an immunoassay is used in clinical analysis, the utilization of laser techniques makes sample collection possible without any adverse reaction from the body.317... 

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