Enzyme-labeled antigen

D. Enzyme-labeled antigen is used at a constant (and excess) concentration of approximately 10 pg/tnL (such that it is not the limiting factor in the assay), allowed to react, and excess is washed away (PBST). 

B. Enzyme-labeled antigen is serially diluted across the plate, allowed to react, and excess is washed away (PBST). 

Figure 7.15 Enzyme-multiplied immunoassay (EMIT). The three reactants, test (or standard) antigen, enzyme-labelled antigen and a limited amount of antibody are allowed to react and reach an equilibrium position. The unbound labelled antigen which remains is the only source of enzyme activity, the bound enzyme being inactivated. This free enzyme can be quantitated using a direct kinetic assay method and is proportional to the amount of unlabelled antigen originally present.

ELISA assays may be competitive or noncompetitive. As the name imphes, in a competitive ELISA, enzyme-labeled antigen competes with free antigen (the analyte of interest) for a fixed and limited quantity of immobihzed antibody binding sites. After incubation, the microtiter plate (sohd support) is rinsed to remove all unbound species and the enzyme substrate is added in saturating concentration. The conversion of substrate to produce can be measured continuously (kinetic assay) or, more commonly. 

Engvall E, Jonsson K, Perlmann P. 1971. Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes. Biochim Biophys Acta 251 427-434. 

Direct competition. The solid phase (a microtiter plate) is coated with an antibody specific for the antigen being assayed. The sample and enzyme-labelled antigen (antibiotic) are added. There is a competition for the antibody between the labelled and unlabelled antigen (antibiotic). Substrate is added and the color produced by the enzymatic hydrolyse is inversely proportioned to the concentration of antigen in the sample... 

The enzyme-labelled antigen was obtained using the same procedure. 

In order to detect the analyte specifically, a complex has to be formed first. To this end, the revelation moiety (e.g. an enzyme-labelled antigen or antibody) is for instance incubated in the chip so as to bind to the analyte that has previously been captured within the microchannel. In another scheme, the analyte solution is first mixed with the revelation moiety, and the formed complex is then incubated in the chip in order to be captured on the bed of antibodies coating the walls of the micro-channel. After a washing step (to remove the excess affinity partner), the microchannel is filled with the substrate which shall thus react... 

Enzyme immunoassays using enzymes as markers have a number of advantages. Detection limits for enzymes are very low, because enzyme reactions can be amplified as catalytic reactions. The enzyme-labeled antigen and antibody are considerably stable. No expensive equipment is required to determine the enzyme activity. 

Enzyme-labeled antigen Antigen.antibody Enzyme reaction... 

Fig. 5. An assay cycle using the flow-ELISA principle. The sample supplied with a fixed amount of enzyme-labeled antigen is introduced at the arrow sample into the continuous flow stream. Buffer is fed for a shon period before substrate for the marker enzyme is given as a pulse. The system is rinsed by a pulse of glycine buffer, pH 2.2. After reconditioning, the system is ready for a new assay. (After Mattiasson et al. (135).)...

One characteristic of these assays is that the calibration curve obtained for such an immunosorbent column is valid throughout the lifespan of the preparation. One needs only to compensate for decrease in capacity. This means that a value obtained with just enzyme-labeled antigen passed through the column intermittently, and subsequent reading is then compared to that calibration value. 

Incubate with enzyme-labeled antigen in presence or absence of standard antigen or unknown sample... 

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. 

Mix enzyme-labeled antigen. Limited concentration of anti-antigen antibody, and sample containing free antigen... 

The antigen in the sample competes with the enzyme-labeled antigen for a limited number of antibody binding sites... 

Unbound enzyme-labeled antigen converts the substrate to a colored product... 

Rubenstein, Schneider, and Ullman described a HOIA method for morphine using lysozyme as the enzyme label. The covalent enzyme labeled antigen (Ag ) competes with sample antigen (Ag) for a limited concentration of antibody (Ab) to form a complex. The resultant complex exhibits very little enzyme activity because of either steric hindrance or... 

Enzyme-Labeled Antigen. In this method, the sample is first incubated with a moderate excess of solid-phase immobilized antibody (Ab ). After washing, excess enzyme-labeled antigen (Ag ) is allowed to bind to unreacted Ab . 

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