Heterogeneous Immunoassays, detection

Heineman s group [13,14] proposed the method of heterogeneous immunoassay to detect human serum albumin (HSA) labeled with In... 

Heterogeneous immunoassay has also been conducted with the antibody immobilized on beads. For instance, mouse IgG (50-100 ng/mL) was detected by ELISA in a glass chip. First, mouse IgG (antigen) was captured by magnetic beads coated with sheep anti-mouse antibody (1.02 x 107 beads/mL). Then the secondary antibody, which was rat anti-mouse conjugated with alkaline phosphatase (0.7 pg/mL), was delivered. Thereafter the substrate, PAPP, was added. It was enzymatically converted to p-aminophenol (PAP), which was electrochem-ically detected by the on-chip interdigital microelectrodes [1016]. 

Heterogeneous immunoassay has also been conducted without the use of an enzyme label. For instance, electrochemical immunoassay of mouse IgG (antigen) was carried out in glass chip. The chip contained magnetic beads coated with the sheep anti-mouse antibody. After flowing in the secondary antibody (rat antimouse conjugated with PAPP), electrochemical oxidative detection of PAP was achieved (i.e., PAP was oxidized to p-quinoneimine) [1016]. 

After patterning antibodies as an array using the PDMS microchannel, TNT (2,4,6-trinitrotoluene) was detected using the heterogeneous immunoassay format... 

Immunoassays detect and quantify unknown levels of antibody or antigen and may be competitive or noncompetitive and heterogeneous or homogeneous. 

Most immunoassays currently employed in the biomedical field are either radioimmunoassays, enzyme immunoassays, or luminescence immunoassays (including fluorescence immunoassays [FIA] and chemiluminescence immunoassays). Although radioimmunoassay is currently the most sensitive of these (10 -10 M concentrations are often detectable), due to the problems inherent to dealing with radioactive materials, such as licensing, radiation hazard, short shelf-life of expensive radioisotopes, the expense of the counting equipment, and the tedium associated with heterogeneous immunoassay, it has fallen, in popularity, behind the non-isotopic methods of analysis. 

Enzymes are currently the most widely used and investigated labels for immunoassays, because a single enzyme label can provide multiple copies of detectable species. This catalytic amplification results in immunoassay detection limits that rival those of radioimmunoassay without the storage and disposal problems associated with radioisotopes. Enzyme immunoassays label either ligands or antibodies with enzyme, and enzyme activity in bound or free fractions is measured. Heterogeneous immunoassays employing enzymatic labels have been named enzyme-linked immunosorbent assays (ELISAs). ELISA methods usually employ antibody immobilized onto the wells of polystyrene microtiter plates, and may be... 

Heterogeneous immunoassays may be used with conventional microtiter systems in clinical laboratories, but have also been adapted for doctor s office and home diagnostic test devices. For example, a device for detecting antibodies to the human immunodeficiency virus (HIV), operates as shown in Figure 6.10.19... 

Other Detection Systems for Heterogenous Immunoassays. Tags other than enzymes which have been used successfully in heterogeneous immunoassay systems include radioisotopes, fluorescent dyes, and colloidal gold. Similar assay procedures are performed using these tags, although different detection methods are employed to determine the result of the assay. 

Microfiuidic devices for heterogeneous immunoassays consist of microchannels for transporting solutions, reaction solid phase, and detection area. There are several important factors to develop a... 

Like enzymatic assays, immunoassays are conventionally implemented in microtiter wells, but they are even more easily adapted to the microscale. Homogeneous immunoassays are typically employed for the study of small molecules, while heterogeneous immunoassays are preferred for large-molecule detection. 

By using the micro/nanoparticles and magnetic beads, the homogenous immimoassay without the separation of whole blood can be manipulated. However, the detection sensitivity is limited due to the complex whole blood enviromnent. For the heterogeneous immunoassay, the commercial... 

The next significant development was the immuno-radiometric assay (IRMA), in which the isotopic label is attached to the antibody rather than the analyte, thus resolving problems associated with labeling diverse analytes. This was the first example of a sandwich immunoassay, in which two antibodies are used firstly to capture the analyte and then to detect and quantify it. It was also the first example of a solid-phase heterogeneous immunoassay, i.e., the first (capture) antibody is immobilized on a solid substrate such as the surface of a microtiter dish or membrane. The surface is then flooded with the... 

Electrochemical detection in a heterogeneous immunoassay, whether competitive or sandwich, can he done either directly in the container in which the assay was... 

An exciting possibility of heterogeneous immunoassay with ASV detection is its extension to multianalyte detection using multiple metal labels. This concept was successfully applied by Hayes and coworkers in the simultaneous detection of HSA and human IgG in perhaps what is the first multianalyte ECI [74]. Because ASV is an extremely sensitive method that can detect up to about six metals simultaneously, it... 

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