Sandwich immunoassays antibody components

Another useful heterogeneous EIA is the double antibody or "sandwich" immunoassay. In a sandwich immunoassay, an untagged antibody or capture antibody is the first reagent bound to the solid phase. The test sample and then the enzyme labeled antibody are added sequentially. The unbound components are removed by washing after each incubation. Enzyme-labeled antibody is detected by addition of a substrate as above. The capture antibody is particularly useful when the test sample consists of a complex mixture of materials. The capture antibody "fishes-out" the target antigen and other components of the test sample (which could interfere with subsequent steps in the immunoassay) are more effectively washed away. 

Antibodies are also used in combination with optical biosensors as another alternative method within direct or sandwich immunoassays. The sensors consist of three components a biological receptor with appropriate specificity for the analyte, a transducer to convert the recognition event into a suitable signal, and the detection, analysis, processing, and display system, which is usually electric. Signals can be from acoustic, electrical, mechanical, or optical sources. Technologies currently in use for... 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

Noncompetitive immunoassays This class comprises the following subtypes excess reagent, two-site and sandwich assays. A typical noncompetitive assay has the following major components (i) primary or capture antibody, (ii) sample... 

In the sandwich assay format, two biorecognition elements directed to either the same epitope present multiple times on the analyte surface or the different epitopes on the target analyte are utilized. For immunoassays, this format is useful for large analytes with multiple antigenic sites. Here, one antibody is immobilized onto a porous membrane and serves as the capture antibody, while the other is conjugated to a detectable species. The response is directly proportional to the concentration of analyte in the sample. A band consisting of a secondary antibody may also be present to serve as a control that the assay components worked and the assay was run correctly. This assay format is depicted in Fig. 4. 

Methods very similar to classical immunoassays in the sandwich format are easily implemented in flow systems (Fig. 2d). In this type of noncompetitive assays, again, antigen is captured and concentrated from an appropriate volume of sample on an immunosorbent (-Abi) column while nonantigenic components are eluted. Subsequent to the capture step, labeled second antibody (Abj-label) is introduced into the mobile phase and swept into the column, where it binds to the -Ab]-Ag complex to form -Ab -Ag-Ab2-label. Unbound Ab2-label is swept from the column, and when the label is an enzyme, antigen is quantitated indirectly by conducting an enzyme assay in the column. After substrate incubation, the reaction product is transported to a detector at the column terminus. Ag and Ab2-label can be introduced in the column sequentially or simultaneously. In some instances both modes led to similar sensitivity [55], and in other cases simultaneous injection produced a greater response than sequential injection [56]. The term sandwich has also been applied to the procedure carried out to quantitate Ab by capturing a complex Ab-Ag-label onto a protein G capillary column [57]. In this case detection is performed after elution. 

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