Assay formats sandwich assays

Figure 2.1. Schematic representation of aptamer-based assa configuration. [A] Direct assay [B] Sandwich assay format with two aptamers (C] Sandwich assay format with an aptamer used as the primary ligand and an antibody as the secondary ligand (D] The opposite configuration to case C [E] Competitive assay.

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

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)...

Very recently, a sandwich assay for prostatic acid phosphatase antigen was carried out using two cascaded enzyme reactions to provide amplification of the immunochemical event. In one format, an optical readout was used whereby a forma-zan dye was generated by reaction of a dye precursor and NADH generated from the second enzyme cycle. In the electrochemical format, the NADH generated in the second enzyme cycle was used to reduce Fe(CN) to FeCCN) " which was then detected amperometrically. While the use of Fe(CN) in ECIA has appeared in the... 

Figure 14. Detection formats used in affinity biosensors based on spectroscopy of guided waves a) direct detection, b) sandwich assay, and c) competitive inhibition assay.

The most common use of protein microarrays is in immunoassays. In particular, antibody-based immunoassays are the main stream of diagnostic assays due to their specificity. The assay usually runs in a multiplexed mode where the antibodies or other capture agents are immobilized and then exposed to a biological sample. There are four immunoassay formats direct binding, sandwich (ELISA), competitive, and displacement. Direct-binding and sandwich assays are the most common. There are some reports on the use of competitive assays and displacement assays, which are usually associated with high surface area/volume systems [72-76],... 

Enzyme-linked immunosorbent assay (ELISA) is based on the specific reaction between an antibody and an antigen. One of the reagents in the reaction is labeled with an enzyme that generates a colorimetric product that can be measured with a spectrophotometric device. The color intensity correlates with the concentration of specific antibody and the respective antigen. The reaction can be formatted in various ways in a multiwell plate (microtiter plate) with the common formats being the sandwich assay, the competitive assay, and the direct assay. (See Figure 11.1.)... 

The sandwich assay is the format used most often to quantitate a target antigen or analyte. In the sandwich assay, two antibodies are used that bind to different parts of the antigen. One of the antibodies is bound to, or coated on, the solid surface (mictotiter plate wells), whereas the other has a label attached to it (Figure 11.1a). Alternatively, a secondary conjugated antibody can be used to detect the bound primary antibody (Figure 11.1b). If the antigen is present in the sample solution, it links the two antibodies. Therefore, the label is retained on the plate where it can be detected by use of a colorimetric substrate. 

The most commonly used format for quantitation assays is the sandwich assay format. Typically, a monoclonal antibody (MAb) is used to capture the product. It is then detected by another antibody, usually enzyme-labeled. A reference standard is used from which to compare the response of an unknown test sample. There is a relative increase in measured response (optical density) with increasing analyte concentration. Figure 11.2 is an example of a typical ELISA standard curve. 

The binding of the antigen and antibody can be affected by several factors, including the conjugated label chosen for detection and the method used to conjugate the label, as well as the assay format itself. The selectivity of the ELISA can be affected by the assay format. In an ELISA with a two-site sandwich format, independent epitopes are bound by different antibodies.26 The specificity comes from multiple site recognition. Polyclonal antibodies can react with many epitopes on a complex antigen surface.24... 

SPR affinity biosensors have been developed to detect an analyte in a variety of formats. The choice of detection format for a particular application depends on the size of target analyte molecules, binding characteristics of available biomolecular recognition element, and range of concentrations of analyte to be measured. The main detection formats used in SPR biosensors include direct detection (Fig. 11), sandwich assay (Fig. 12) and inhibition assay (Fig. 13). 

Figure 12. Detection formats used in SPR affinity biosensors sandwich assay.

Sandwich assay — This format works well for ELISA. The success and potential shortcomings for microarrays are discussed in the next section. 

The ELISA tests are performed using different incubation steps, depending on the assay format, namely sandwich, competitive or inhibition immunoassays that correspond to the way the analyte is captured and further revealed, which is generally dictated by the nature of the analyte. 

HYBRIDIZATION OF DNA STRAND RELATED TO CYSTIC FIBROSIS GENE, USING A SANDWICH ASSAY FORMAT... 

Fig. 6.3 Scheme of an ELISA sandwich assay. Left panel A typical assay format using a 96-well microplate, pre-coated with a capture antibody (Ab). The drug analyte in the sample is bound to the immobilized capture Ab. The extraneous matrix components are washed away before addition of the detector second Ab, which is conjugated with horseradish per-... 

Figure 10.14. Scheme 1 depicts the formation of the three-component sandwich assay discussed in the text. (a) and (b) show flatbed scanner images of microarrays treated with gold nanoparticles before and after silver enhancement, respectively, (c) shows a typical Raman spectrum acquired from one of the silver spots. (d) shows a profile of the Raman intensity at 1192 cm-1 as a function of position on the chip the laser beam from the Raman instrument is moved over the chip from left to right as defined by the line in (b). (With permission from Ref. 40.)... 

Homogeneous immunoassay using FRET is an attractive assay format, since the antigen-antibody binding reaction is much accelerated compared to that of solid phase sandwich im-... 

Figure 12.3 Scheme of multianalyte immunoassay. Four analytes (A, B, C, and D) can be detected simultaneously in a sandwich assay format in a single well of a microtiter plate. Antibodies against all four analytes are adsorbed on the wells of the plate, exposed to a mix of all four analytes, and analytes are detected by anti-toxin antibodies conjugated to various QDs. (See color insert.)... 

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...

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