Sandwich format

Direct sandwich assays are generally used when testing for higher-molecular-weight analytes, which possess multiple epitope regions, such as macromolecules. The sample first encounters colored particles, conjugated to antibodies 

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An evanescent wave fiber optic immunosensor has been used for the detection of ricin in river water.(131) A tapered fiber optic waveguide with covalently bound anti-ricin IgG is used in a sandwich format, with tetramethylrhodamine-labeled antibody as tracer. In a two-step format, ricin in the sample is bound to the fiber first, and then the fiber is exposed to the tracer antibody. Sensitivity is 1 ng/ml for the two-step assay. The one-step assay, in which the fiber optic probe contacts the sample and labeled antibody simultaneously is less sensitive, but more convenient. 

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... 

Many strategies based on DNA hybridisation assays using AuNPs have been developed. Most of them rely on capturing the NP to the hybridised target in a three-component sandwich format. 

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...

For example, in an FRET assay for the research of a receptor ligand inhibitor, the presence of a library componnd which flnoresces at the acceptor wavelength will lead to a false negative resnlt (compensation of the decrease of the FRET signal by the fluorescent componnd) on the contrary, the same compound would give a false positive result in a cytokine assay nsing a sandwich format with two monoclonals (compound fluorescence will be interpreted as an FRET increase). In both cases, the use of HTRF would have corrected for the fluorescence of the compound in the acceptor channel. 

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique in which antibody is immobilized on the walls of a small volume plastic vessel. The ELISA technique can follow either a competitive equilibrium or a sandwich format. Both formats have been used with electrochemical detection. The general protocol for these two formats is shown in Fig. 9. 

Fig. 9 General schematic of ELISA in competitive and sandwich formats.

For example, several strategies have been used for immunoassay techniques with fiber-optic biosensors. In the sandwich format, the receptor is immobilized on the stu"face of the fiber waveguide and a secondary or tracer antibody (which is labelled with a fluorescent dye) is added to the solution. In the absence of the analyte, the tracer remains in solution and little fluorescence is observed. However, after addition of the analyte, a molecular sandwich is formed on the sensor smface within the evanescent excitation volume. The sandwich assay is usually more sensitive than a competitive-binding assay because the fluorescence intensity increases with analyte concentration. 

In heterogeneous assays, the detection is usually performed in a competitive format, in which labelled antigen is added to the sample solution, or in a sandwich format, in which after washing the siuface, secondary labelled antibodies are specifically adsorbed onto the immobilized antigens. For the latter format, the antigen must have two different epitopes—one for adsorption onto the capture antibody and one for the secondary antibody adsorption ... 

In the competitive format, the signal is inversely proportional to the antigen concentration, whereas in the sandwich format, the signal is directly proportional to the antigen concentration. 

Methods for the Determination of Elastase-1 in Feces An ELISA method in a microplate sandwich format is commercially available to measure El mass concentrations in stool samples. 

The measurement of serum hCG improved greatly in the 1970s. The assay specificity improved by using an antibody to the p-subunit of hCG that had httle cross-reactivity with other glycoprotein hormones, LH, FSH, and TSH. Currently, most hCG assays use an immunometric ( sandwich ) format. The hCG assay measures the intact (whole) molecule when an antibody for the a-subunit and an antibody for the p-subunit are used in the immunometric format. This type of assay does not measure free a- or p-subunits because free subunits cannot form a sandwich with both antibodies. The total p-hCG assay measures both the intact hCG and free p-subunits. As a tumor marker, a total P-hCG assay may be preferred, because cancer patients produce notable amounts of free P-subunit. None of the commercially available hCG assays have been approved by the Food and Drug Administration (FDA) for use as a tumor marker assay. 

Sandwich Formation between Different Particles and between Particle and Electrode... 

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