Sandwich assays detection

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... 

Direct detection is usually preferred in applications, where direct binding of analyte of concentrations of interest produces a sufficient response. If necessary, the lowest detection limits of the direct biosensors can be improved by using a sandwich assay. Smaller analytes (molecular weight < 10,000) are usually measured using inhibition assay, Figure 14. 

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.

However, luminescence-based detection techniques often require a high number of steps. Consider ELISA as an example. As a first step, the sample is introduced into a 96-well plate an antibody targeting the antigen of interest has been immobilized to the wells of the plate. After a rinse, the wells contain the antibody and any bound antigen. However, although the antigen has been isolated, the protocol is nowhere near completion. The remaining steps include another antibody (different from the first) to form a sandwich assay, a secondary antibody with an enzymatic label, and a substrate that is luminescent when activated by the enzyme. Finally, the sample is analyzed by relatively expensive detection optics to determine the amount of analyte that was captured in the assay. The steps are illustrated in Fig. 14.1a. 

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. 

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.

Molecular weight of the main bacterial toxins ranges from 28,000 to 150,000, which makes it possible for most sensitive SPR biosensors to measure their concentrations directly or using a sandwich assay. Examples of food safety-related toxins detected by SPR biosensors include Botulinum toxin (detection limit 2.5 pg/ml " ), . coli enterotoxin (detection limit 6 pg/ml " ) and Staphylococcal enterotoxin B (detection limit 5 ng/ml and 0.5 ng/ml for direct detection and sandwich assay, respectively" ). 

Bacterial pathogens are relatively large targets (> 1pm) and therefore, their presence can be detected directly with an optional amplification by secondary antibodies (sandwich assay). Examples of foodbome bacterial pathogens detected by SPR biosensors include Escherichia coli (detection limit 5x10 cfii/ml " " ), Listeria monocytogenes (detection limit 1 O cfii/ml " ) and Salmonella enteritidis (detection limit lO cfii/ml" ). 

Pierce introduced an array-of-arrays microplate product called Search-Light in which antibodies are directly printed into the wells of the microplate. Also, we have reviewed MSD s Multi-Spot plate products having antibodies immobilized onto multiple working electrodes. These products (albeit with some novel approaches to create microarrays and means for detection) utilize the classic immunosorbent sandwich assay but have the advantage of parallel processing using microarrays. 

Fig. 4. Classification of reported noncompetitive immunoassays for haptens based on the assay principle. (A) Assays that include a chemical modification of hapten to allow sandwich-type detection. (B1) Improved single-antibody immunometric assays that separate immune complex and excess labeled antibody, either by using a hapten-immobilized affinity column or based on differences in their physical properties. (B2) A variation of single-antibody immunometric assays based on masking of unoccupied antibody by an immunoreactive macromolecule followed by selective capture and detection of the hapten-occupied antibody. (C) Assays employing a probe molecule specific to a hapten-antibody complex.

Besides this strategy in which the DNA target can be easily attached and detected by its complementary DNA signalling probe, a sandwich assay in which the DNA target is in solution can be easily performed by a double hybridization with a capture and a signalling probe [58]. This strategy was demonstrated to be useful for the detection of a novel... 

Electrochemical methods of detection affinity interactions at the surfaces are rather effective due to their relative simplicity and low cost. Amperometric aptasensor based on sandwich assay was proposed by Ikebukuro et al. [42]. They used two aptamers selective to thrombin. 

Experiments for the detection of a single, three-base mismatch and non-complementary DNA were carried out in both assays. The results demonstrated an efficient discrimination. Figure 53.4 displays these results in sandwich assay, where the difference in current intensities is observed higher for CF-T (Fig. 53.4A), which represents the efficient hybridization electrochemical response on the M-GECE lower responses for CF-MX1 (Fig. 53.4B) and significantly lower for CF-MX3 (Fig. 53.4C) and CF-NC (Fig. 53.4D) [3],... 

Fig. 20 Mechanism of a sandwich ELISA for the detection of bovine IgG using peroxidase (POx)-labeled anti-IgG and the europium(III) tetracycline system as fluorescent reagent (top). Schematic of the microwell surface showing the assembly of the sandwich assay (bottom)...

The influence of antibody selection was also described in the work of Niemeyer et al. [37], in which an indirect sandwich assay (Fig. 3D) with a nonfunctionalized primary detection antibody was compared to a direct sandwich assay (Fig. 3C). The removal of one incubation step resulting from the functionalized primary antibody increased coupling efficiency, thereby improving the signal-to-background ratio and, ultimately, the sensitivity of the assay. 

Numata Y, Matsumoto Y. Rapid detection of alpha-human atrial natriuretic peptide in plasma by a sensitive immuno-PCR sandwich assay. Clin Chim Acta 1997 259(1-2) 169-176. 

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.)... 

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