Solid Phase Enzyme Linked Immunoassay EIA

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Probe-based methods for the detection and identification of PCR products have been developed for several different solid phases, including nylon membranes, microwell plates, microparticles, and oligonucleotide microarrays (Inouye and Hondo, 1990; Bobo et al., 1991; White et al., 1992; Schachter et al., 1994; Chee et al., 1996; DiDomenico et al., 1996; Cheung et al., 1999; Loeffelholz et al., 1999; Tang et al., 1999). Microtiter plate assays have a number of advantages, including the convenience of nonisotopic chemistry, rapid turnaround time, ease of use, and high throughput. This format is amenable to automation, with comparable sensitivity to that of Southern blot hybridization. It has many similarities with enzyme-linked immunosorbent assays (ELISAs) and is sometimes referred to as ELOSA (enzyme-linked oligosorbent assay) (Mallet et al., 1993).

Microtiter plate formats for detection of amplicon were originally described in the late 1980s and early 1990s (Keller et al., 1989,1990,1991; Kawai et al., 1993; Rapier et al., 1993) and had their foundations with hybridization-based identification of synthetic oligonucleotides (Nagata et al., 1985; Cook et al., 1988). Early procedures used sandwich hybridization, in which at least two probes (a capture probe and secondary, labeled probes) were used for amplicon detection (Kelleretal., 1989,1990; Rapier et al., 1993) (Fig. 15.2A). Typically, the secondary probe consisted of a single biotin-labeled oligonucleotide complementary to the captured amplicon (Keller et al., 1990). In a variation of this procedure, two secondary probes are used to detect hybridized amplicon; one complementary probe containing amplicon-specific sequence at the 5' end and a 3' poly-T tail, and a second probe (not sequence-specific) having a 5' poly-A tail and biotin (Rapier et al., 1993). Some have simplified the sandwich, creating a direct hybridization procedure, in which amplicon is biotin-labeled during PCR extension phase, obviating the need for a secondary probe (Keller et al., 1991; Kawai et al., 1993) (Fig. 15.2B).

biotin biotin

Figure 15.2. Schematic diagrams of microwell plate detection using sandwich (A) and direct (B) hybridization formats. Reprinted with permission from: Loeffelholz, MJ. Microwell plate detection systems for amplicon detection and characterization. In Molecular Microbiology: Diagnostic Principles and Practice, D. Persing, F. Tenover, J. Versalovic, Yi Tang, E. Unger, D. Relman, and T. White (Editors), ASM Press, Washington, DC, 2004.

Figure 15.2. Schematic diagrams of microwell plate detection using sandwich (A) and direct (B) hybridization formats. Reprinted with permission from: Loeffelholz, MJ. Microwell plate detection systems for amplicon detection and characterization. In Molecular Microbiology: Diagnostic Principles and Practice, D. Persing, F. Tenover, J. Versalovic, Yi Tang, E. Unger, D. Relman, and T. White (Editors), ASM Press, Washington, DC, 2004.

The latter direct hybridization method is simpler to perform than the sandwich method, with lower resulting background signal levels (Keller et al., 1991).

Microwell plate detection systems can be classified in two ways based on the capture molecule used (Lazar, 1994): oligonucleotide probe (sequence-specific capture) or avidin (non-sequence specific capture). Formats relying on immobilized probe for target capture (Keller et al., 1991) rely on sequence complementary hybridization of amplicon to probe. Formats using avidin (or streptavidin) for target capture agent depend on its strong affinity for biotin (Diamandis et al., 1991), incorporated into target during PCR (Cook et al., 1988; Boyle et al., 1992).

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