Detection system enzyme-labeled antigen

There are variety of sandwich ELISA systems. Eor example, in indirect sandwich ELISAs, the assay uses an unlabeled primary antibody for the sandwich step in conjunction with an enzyme-labeled anti-immvmoglobulin secondary antibody. This system can also employ a biotinylated sandwich antibody and enzyme-labeled streptavidin for detection. The indirect sandwich ELISA can be used not only for detection of antigens but also for measurement of antibody concentration, a system more specifically referred to as an indirect antibody capture immunoassay. In this case, an antigen-coated solid phase is prepared and then incubated with a serum or plasma test sample. Next, a secondary enzyme-labeled antiimmunoglobulin antibody or enzyme-labeled antigen is added, unbound reagents are removed by washing, and enzyme activity is assayed. 

Conventional ion-selective electrodes have been used as detectors for immunoassays. Antibody binding measurements can be made with hapten-selective electrodes such as the trimethylphenylammonium ion electrode Enzyme immunoassays in which the enzyme label catalyzes the production of a product that is detected by an ion-selective or gas-sensing electrode take advantage of the amplification effect of enzyme catalysis in order to reach lower detection limits. Systems for hepatitis B surface antigen and estradiol use horseradish peroxidase as the enzyme label and... 

A common application for (strept)avidin-biotin chemistry is in immunoassays. The specificity of antibody molecules provides the targeting capability to recognize and bind particular antigen molecules. If there are biotin labels on the antibody, it creates multiple sites for the binding of (strept)avidin. If (strept)avidin is in turn labeled with an enzyme, fluorophore, etc., then a very sensitive antigen detection system is created. The potential for more than one labeled (strept)avidin to become attached to each antibody through its multiple biotinylation sites is the key to dramatic increases in assay sensitivity over that obtained through the use of antibodies directly labeled with a detectable tag. 

Other immunoassays are based on the same antibody-antigen binding reaction but use a different labeling system for detection. Instead of an enzyme label, there are radioactive isotopes, and fluorescent and luminescent labels. Some important immunoassays are defined below ... 

Proteins can be detected in tissue sections or cell cultures using similar immune detection systems. Use of an antibody to detect specific proteins in tissues is called immunohistochemistry, whereas detection of proteins in cell suspensions is called immunocyto-chemistry. Tissues can be prepared by fixation and embedding in paraffin wax, or by rapid freezing in a compound that inhibits ice formation in the tissue, so as to preserve cell morphology. Some antibodies do not work well with paraffin-embedded tissues, probably because the antibody cannot access the antigen properly (133). The most common labeling system used for detection of the bound antibody is an enzyme-coupled secondary antibody that produces a color reaction... 

Direct or Sandwich ELISA The direct format represents the most simplified ELISA system. In this assay, serum or plasma samples are incubated with the antigen (usually the drug), which has been previously immobilized directly onto well surfaces of microtiter plates. The bound antibody is detected using an enzyme-labeled anti-immunoglobulin reagent of appropriate specificity. 

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