Antigen capture fluorescence immunoassay

Figure 5. Antigen capture fluorescence Immunoassay for human IgG.

Figure 4.12 describes two popular antibody microarrays formats that are constructed for antigen capture in small sample volumes with detection by either sandwich immunoassay or antigen labeling. In sandwich immunoassay, capture antibodies are arrayed and immobilized to select specific proteins which are then found by a second labeled detection antibody. In protein target labeling, all proteins in the sample are prelabeled (i.e., fluorescent dyes) prior to capture by immobilized antibody arrays. In direct assay systems, sample proteins are directly immobilized onto... 

Fig. 5 NRL Array Biosensor mixed format immunoassays (modified from [119]). Schematic of (a) the sandwich and (b) the competitive immunoassay fonnats used in the detection of the Campylobacter jejuni and aflatoxin Bi (AFBi), respectively, (a) Sandwich format antigen captured by the immobilized antibody then quantified by passing a second, fluorescently labeled, antibody over the surface, (b) Competitive format competition for binding sites on the fluorescently labeled antibody occurs between the unlabeled antigen in solution and the surface-bound antigen analog, (c) Final charge-coupled devices image taken with the NRL Array Biosensor of a waveguide exposed simultaneously to the C. jejuni (5 x 10" cfu/mL) sandwich assay (SAND) and the aflatoxin Bj (AFBi-1 ng/mL) competitive assay (COMP) in various combinations...

The detection of flu viruses via a fluorescent sandwich immunoassay was reported by Bucher.(10) However, the method sensitivity was too low for direct detection of the virus. A novel sandwich immunoassay was described by Ogcr((lff7 for the detection of Botulinum Toxin A. Antibodies specific for Clostridium botulinum were covalently attached to the surface of a tapered fiber. After the capture of the antigen, a sandwich was formed with a rhodamine-labeled anti-toxin IgG, and the evanescent wave was measured. The assay was highly specific with detection limits near 5 ppb. 

An NIR biosensor coupled with an NIR fluorescent sandwich immunoassay has been developed. 109 The capture antibody was immobilized on the distal end of an optical fiber sensor. The probe was incubated in the corresponding antigen with consecutive incubation in an NIR-labeled sandwich antibody. The resulting NIR-labeled antibody sandwich was excited with the NIR beam of a laser diode, and a fluorescent signal that was directly proportional to the bound antigen was emitted. The sensitivity of the technique increased with increasing amounts of immobilized receptor. There are several factors involved in the preparation of the sandwich type biosensor. A schematic preparation of the sandwich optical fiber is shown in Figure 7.14. 

In sandwich immunoassays, a pair of antibodies binds to different epitopes of an analyte and one of the antibodies (the so-called capture antibody ) is immobihzed on the substrate. The second antibody is labeled with a probe (e.g. a fluorescent dye), it allows detection of the analyte molecules that bind to the capture antibody. We use this approach when the antigen is a well-characterized protein. Multiplexing is possible by immobilizing the capture antibodies in an array or on individually coded beads for medium- to low-volume amounts of biological liquids. The submicroliter version of this approach, in which capture antibodies are immobilized in stripes and the sample is guided to the capture sites by means of microfluidic networks, is described in Section 3.1 of this chapter. 

A sandwich immunoassay is another widely used detection scheme and involves the use of two antibodies. The first antibody is immobilized on the fiber and is used to capture the antigen, and the second antibody, which is conjugated to a fluorescent dye or enzyme, is used to generate the signal (Fig. 16c). When an enzyme is used for antibody labeling, the enzymatic conversion of a nonflu-orescent substrate to a fluorescent product is measured. The enzyme-labeling method is more sensitive since the signal is amplified by the enzymatic reaction. 

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