Heterogeneous immunoassay fluorescence immunoassays

Figure 14.3. Distinction between homogeneous and heterogeneous immunoassay formats. Haptenic analytes are indicated as triangles, and conjugated fluorescent probes are indicated by the letter F. In this hypothetical depiction, the homogeneous immunoassay is quantitated in the original reaction mixture. The heterogeneous immunoassay requires removal of unreacted tracer, further addition of reagents such as an enzyme to release a fluorescent molecule F, followed by quantitation.

Heterogeneous fluorescence immunoassays have many different assay formats, but all possess one unifying feature the free fluorophore remaining in solution after binding of some of the fluorophore to antibody must be removed before quantification can be achieved. Again, both negative and positive reading assay formats have been employed. 

In the past ten years, numerous applications of fluorescence methods for monitoring homogeneous and heterogeneous immunoassays have been reported. Advances in the design of fluorescent labels have prompted the development of various fluorescent immunoassay schemes such as the substrate-labeled fluorescent immunoassay and the fluorescence excitation transfer immunoassay. As sophisticated fluorescence instrumentation for lifetime measurement became available, the phase-resolved and time-resolved fluorescent immunoassays have also developed. With the current emphasis on satellite and physician s office testing, future innovations in fluorescence immunoassay development will be expected to center on the simplification of assay protocol and the development of solid-state miniaturized fluorescence readers for on-site testing. 

In another report, pFNs were also formed from PDMS chips to create patterns of immobilized chicken IgG. Heterogeneous immunoassay with antispecies IgG (fluorescently labeled) was then conducted [247]. 

Ml. Maggio, E. T., Recent advances in heterogeneous fluorescence immunoassays. "Immunoassays Clinical Laboratory Techniques for the 1980s, pp. 1-12. Liss, New York, 1980. 

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. 

The front surface approach shows similar sensitivity to the right angle detectors hut is more susceptible to background light scatter. Front surface fluorometry has been widely applied to heterogeneous solid-phase fluorescence immunoassay systems. 

Heterogeneous immunoassay in which a nanometer-sized (less than lOnm) semiconductor quantum dot is used as a label. A quantum dot is a highly fluorescent nanocrystal composed of CdSe, CdS, ZnSe, InP, or InAs or a layer of ZnS or CdS on, for example, a CdSe core. Multiplexing is possible with these labels because the emission properties can be modulated by changing the size and composition of the nanocrystal (e.g., CdS emits blue light, InP emits red light). 

Heterogeneous fluorescent immunoassays for T4 based on lanthanide rare earth ions and time-resolved fluorescence were also developed. The use of europium chelates as fluorescent probes is particularly attractive because of their extraordinarily long Stokes shifts and long fluorescence decay times. Thus the sharp emission peak of europium (613 nm) can be easily separated fr om scattering caused by excitation light (340 nm) or by interfering substances in... 

Heterogenous fluorescence immunoassays can be carried out with the aid of the same separation procedures used in radioimmunoassay. The more expedient homogenous fluorescence immunoassays require quenching, enhancement, polarization, or shifting of the fluorescence of the label upon binding of the labeled analyte to its antibody. Occasionally, a second antibody, directed at the anti analyte antibody, will be used in a double-antibody method to precipitate the bound labeled and unlabeled analyte or to alter the optical properties of the label in such a way as to make the analysis more sensitive. 

The fluorescence of a tagged antigen is often quenched upon immunochemical reaction, and the decrease in fluorescence can be measured without going through a physical separation. This serves as the basis for homogeneous immunoassays, in contrast to heterogeneous immunoassays, which require a separation. 

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. 

In heterogeneous immunoassays in batch format the problem of background fluorescence is reduced by separating the analyte from other fluorescent species present in the sample matrix. The use of enzymes with fluorogenic substrates in fluorescence enzyme-linked immunosorbent assays (F-ELISA) and Eu(in)-chelates in time-resolved fluoroimmunoassay (TRFIA) permits to achieve very low LODs for pesticides (e.g., between 0.023-0.3 and 0.05-0.4pgl for F-ELISA and TRFIA, respectively). 

There are different formats of heterogeneous fluorescence immunoassays. 

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