Heterogeneous Assay Format

Fig. 13. General protocol for heterogeneous enzyme immunoassay preparation of reagent cuvettes by coating Ab, competitive assay format, and sandwich assay format. (Reprinted with permission from W. R. Heineman and H. B. Halsall, Anal. Chem, 1985, 57, 1321A. Copyright 1985, American Chemical Society)...

PERFORMANCE AS A REAGENT ON STRATUS SYSTEMS 4.2.1 Heterogeneous Assay Format... 

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. 

Immunoassays can be classified according to different criteria. The particular type selected has a strong influence on the assay performance with regard to precision and sensitivity. The main criteria include [3, 22, 23] (1) labeled or unlabeled assay formats, with different type of labels (2) competitive or non-competitive immunoassays, and (3) homogeneous or heterogeneous immunoassays. These classifications can also be extended to MIP-ILAs (Fig. 2) ... 

Nelson, N. C., Hammond, P. W., Weise, W. A., and Arnold, L. J., Jr., Homogeneous and heterogeneous chemiluminescent DNA probe-based assay formats for the rapid and sensitive detection of target nucleic acids. In Luminescence Immunoassay and Molecular Applications (K. Van Dyke and R. Van Dyke, eds.), pp. 293-309. CRC Press, Boca Raton, FL, 1990. Nelson, R., Jiang, N.-S., and Shellum, C., A simple and sensitive immunochemiluminescent assay for tumor necrosis factor-alpha. Ctin. Chem. (Winston-Satem, N.C.) 38, 1079-1080 (abstr) (1992). 

Enzyme immunoassays are generally performed using a heterogeneous assay format on a microtitre plate containing typically 96 wells. These heterogeneous assays are referred to as enyzme-linked immunosorbent assays (ELISA). ELISAs are widely used in clinical testing. Eor example, one type of ELIS A is currently used for the clinical screening of blood supplies for HIV, the Human Immunodeficiency Virus that causes acquired immunodeficiency syndrome (AIDS). This assay is discussed in more detail below as a typical example of an ELISA. 

Fig. 1 (a-f) Several heterogeneous protein assay formats (Reprinted from [2]). A variety of protein binding events can be studied, ranging from antibody/antigen, to protein/protein, to enzyme/ substrate interactions. 

There are two basic formats in which assays are incorporated in microfluidic devices homogeneous, or solution-based, assays, and heterogeneous, or surface-based, assays. Both approaches significantly reduce sample and reagent consumption relative to their macroscale counterparts, but heterogeneous assays are emerging as the dominant method for proteomics, as they can be adapted to many different types of interactions (Fig. la-f) [2],... 

In this chapter, we first present the different formats used in ECI according to the type of label used. Since enzymes are by far the most common label type, we organize this in two parts according to whether an enzyme label is used or not. Each of these is further divided as heterogeneous, which refers to assays that require the separation of the Ab-bound Ag from the free Ag, and homogeneous, which refers to assays with no separation. Because of their importance to ECI, the different enzyme-substrate-product (E-S-P) systems and their properties are tabulated in Table 1. The sections on assay formats are followed by a discussion on the implementation of ECI in the form of miniaturized... 

Heterogeneous enzyme immunoassay is by far the most common assay format used in ECI. Most of the heterogeneous assays are based on the enzyme-linked immunosorbent assay (ELISA) technique where Ah is immobilized on the walls of small (<500 gL) microtiter wells or cuvettes [105]. The general procedure of heterogeneous immunoassay is outlined... 

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