Sandwich immunoassays assays

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)...

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],... 

Phase-separation immunoassays have been reported, in which the solid phase particles are formed after the immunoreaction is completed.(42) Phase-separation immunoassays are advantageous since the unstirred layer of solution near a solid surface alters diffusion and binding kinetics at the surface in comparison with the properties of the bulk solution. In phase-separation assays for IgG and IgM, capture antibodies are bound with monomers suitable for styrene or acrylamide polymerization.(42) Monomer-labeled capture antibodies are reacted with analyte and with fluorescein- and/or phycoerythrin-labeled antibodies in a sandwich assay, followed by polymerization of the monomers. Fluorescence of the resulting particles is quantitated in a FACS IV flow microfluorometer, and is directly proportional to analyte concentration. 

The issue of which antibody to select for an assay is not a new problem. Certainly anyone involved in the development of an immunoassay has been faced with this choice. Consider attempting to create a multianalyte, microarray-based micro-ELISA of modest density (10 to 100 analytes) and determining which capture antibodies to use based upon their affinities, stabilities, and cross-reactivities. For a sandwich assay, add in the 10 to 100 analyte-specific secondary (reporter) antibodies and determine their levels of cross-reactivity with each other and with the specified antigens and capture antibodies. In other words, achieving high performance for all analytes with a microarray immunoassay is indeed a formidable challenge. 

Fig. 3. Schematic representation of noncompetitive immunoassays. (A) Two-site immunometric assays (sandwich assays) and (B) single-antibody (single-epitope) immunometric assays.

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors require multiple steps for analyte determination, and either sandwich assays or competitive binding assays may be used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. 

Figure 12 Examples of immunoassay systems and DNA assays performed with magnetic bead technology (a) sandwich assay (b) competitive assay (c) bridge assay to determine proteins (d) DNA and RNA assays.

Arenkov et al. prepared poly(acrylamide) gel pads for use in protein microarrays [199], The gels were prepared by photopolymerization of acrylamide and crosslinkers. Capture probes were immobilized, either by use of glutaraldehyde or by converting some of the acrylamide groups into hydrazides and subsequent coupling of aldehyde-modified antibodies to the pending hydrazide groups. Then, immunoassays were performed on the pads, either assays with directly labeled analytes or sandwich assays. Furthermore, the gel pads were used for enzyme activity studies. 

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...

Noncompetitive immunoassays This class comprises the following subtypes excess reagent, two-site and sandwich assays. A typical noncompetitive assay has the following major components (i) primary or capture antibody, (ii) sample... 

A sandwich assay for HCG using GOD as label is also described (309). The procedure is based on an amperometric enzyme immunoassay with an electrode-immobilized antibody. The antibody electrode (activated glassy carbon) is used both to separate the assay and to monitor the activity of the bound enzyme label. Two monoclonal antibodies directed against different antigenic sites are used ... 

Fig. 8.1. Immunoassays using antibodies. A. Competitive assay. B. Non-competitive sandwich" assay.

The use of fluorophores and nano-structured metal surfaces or adsorbed colloidal particles led to the formation of metal-enhanced planar immunoassay, such as sandwich assay or DNA hybridization assay as illustrated in Scheme 8.9. 

Figure 12.6 Schematics showing two common types of protein immunoassays. (Left) Direct assay exploiting interactions between target proteins and labeled primary antibodies. (Right) Indirect, sandwich assay relying on interactions, firstly between unlabeled primary antibodies and target proteins, and secondly between target proteins and labeled secondary antibodies.

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. 

Chemiluminescent labels may be employed in sandwich or competitive antigen assays. In sandwich assays, a solid support holds a primary antibody, and incubation with ligand yields a species that is detectable following a second incubation step with a labeled second antibody. Luminol has been tested as an immunoassay label it may be coupled to proteins through its primary amino group. Luminol reacts with hydrogen peroxide and hydroxide in a microperoxidase-catalyzed reaction, which yields light at 430 nm (Eq. 6.8) ... 

Heterogeneous immunoassay in which a piece of single- or double-stranded DNA is used as a label for an antibody in a sandwich assay. Bound DNA label is amplified using the polymerase chain reaction (PCR). The amplified DNA product is separated by gel electrophoresis and quantitated by densitometric scanning of an ethidium stained gel. 

Two-site immunometric or sandwich assays that made use of two or more antibodies directed at different parts of the PRL molecule were next to be developed. As with other two-site IRMA assays, the capture antibody is attached to a solid phase separation system and the second or signal antibody is labeled with a detection molecule (e.g., radio-isotope, enzyme,fluorophor, or chemiluminescence tag ). In some assays, the capture antibody is attached to the wall of test tubes, plastic beads, microtiter plates, ferromagnetic particles, or glass-fiber paper. Other assays have used the strep-avidin approach that couples biotin to the signal antibody with avidin linked to a solid phase. Most of the current immunometric assays for PRL have been adapted to fully automated immunoassay systems. Compared with the older traditional RIA methods, these automated immunometric assays for PRL generally achieve lower detection limits (0.2 to 1.0 ig/L) and improved precision (interlaboratory coefficients of variation of <8% at all concentrations), and have superior specificity (<0.05% crossreactivity with GH). 

The popularity of microtiter plates in diagnostics (e.g., enzyme immunoassays) and the development of systems for washing, measurement of enzyme activity or chemiluminescence, etc., has led to scattered attempts of using these plates for hybridization. Microtiter plates are not suited for immobilization of target nucleic acid but are promising for capture and sandwich assays, particularly for large numbers of samples. 

In the sandwich assay format, two biorecognition elements directed to either the same epitope present multiple times on the analyte surface or the different epitopes on the target analyte are utilized. For immunoassays, this format is useful for large analytes with multiple antigenic sites. Here, one antibody is immobilized onto a porous membrane and serves as the capture antibody, while the other is conjugated to a detectable species. The response is directly proportional to the concentration of analyte in the sample. A band consisting of a secondary antibody may also be present to serve as a control that the assay components worked and the assay was run correctly. This assay format is depicted in Fig. 4. 

Immunoassays can be designed in two formats competitive assays, preferable for quantification of small molecules such as steroid hormones and prostaglandins, and noncompetitive, or sandwich, assays restricted almost exclusively to large molecules... 

This section elucidates some of the practical aspects that should be considered while developing an immunoassay. Although written with an emphasis on the sandwich assay format, and more specifically ELIS As over RIAs and ECL assays, it also applies to the competitive assay format. A systematic approach for immunoassay method development may consist of the steps shown in Fig. 3.3. 

An immunoassay for the detection of PSA in PBS buffer based on a dualchannel SPR instrument with angular modulation (IBIS II) has been reported [24]. This work compared direct and sandwich detection of PSA on planar- and hydrogel-type sensor surfaces. Amplification with colloidal gold and latex microspheres, respectively, was employed in the sandwich assay. Sensor chips with carboxylated matrices of different thicknesses were used. Mouse monoclonal antibodies against PSA were immobilized on the both... 

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