Sandwich assays principles

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.

Very recently, a new noncompetitive assay principle called an open sandwich ELISA has been reported (S7) (Fig. 15). The assay mechanism could be regarded... 

T4. Tanaka, K., Kohno, T, Hashida, S., and Ishikawa, E., Novel and sensitive noncompetitive (two-site) enzyme immunoassay for h tens with amino groups. J. Clin. Lab. Anal. 4,208—212(1990). T5. Towbin, H., Motz, J., Oroszlan, R, and Zingel, O., Sandwich immunoassay for the hapten angiotensin II. A novel assay principle based on antibodies against immune complexes. J. Immunol. Methods 181, 167-176 (1995). 

Principle antibodies used in a sandwich assay do not need to be purified but those to be conjugated need to be as pure as possible. Many different procedures are possible (Tijssen, 1985) only a widely used convenient method of salting-out of IgG and subsequent purification on ion exchangers is presented. 

The principle of a double sandwich assay is also used for the sandwich ELISA, which avoids the use of radioactivity. The second antibody contains an enzyme (e.g., horseradish peroxidase), which serves as catalyst for a color reaction of a suitable substrate. Quantification is performed using UV-spectrophotometry. This type of ELISA may be used for trace impurity analyses. Immunization for such a multiantigen assay requires the representative preparation of all host cell proteins, but this must be completely free from the product protein. 

The immunometric-type assay has also been adapted for use with nonisotopic labels and is typically carried out in a heterogeneous format in which the antibody is immobilized on a solid support, such as a microtiter dish, membrane, or collection of beads. The canonical clinical immunoassay format in toady s laboratories is the enzyme-linked immunosorbent sandwich assay, which employs two antibodies, one to capture the analyte and the other to detect and quantify it. More details of the principles of these and other immunoassay techniques are given elsewhere in this encyclopedia. 

Figure 83. Principle of the patented Capillary-Fill Device. The upper part shows schematically a competitive imniuno-assay the lower part a sandwich assay. The optical waveguide is always the lower baseplate. Here a diode laser beam is used to produced an evanescent field in the sample inside the capillary...

Fig. 21. Principles of ELISA. A In a direct ELISA the unlabeled antigen is attached to the solid phase. Enzyme-conjugated antibody is then added, followed by the enzyme substrate solution and color is allowed to develop. B ELISA with unlabeled antibody attached to the solid support. A variable amount of antigen is then added. A secondary antibody labeled with enzyme, followed by substrate solution, is added to all wells. The amount of color produced is proportional to the amount of antigen present. C Sandwich ELISA assay with the antigen sandwiched between an immobilized, antigen-specific primary antibody and an antigen- or species-specific secondary antibody. An enzyme-labeled tertiary antibody increases the assay specificity and sensitivity...

Noncompetitive ELISA. The usual principle here is the sandwich technique, which requires the antigen to have at least two antibody binding sites (epitopes). Unlabelled antibody is first fixed to microtitre plates a food sample containing antigen (analyte) is then added and allowed to react with the fixed unlabelled antibody (Figure 8.3). Unadsorbed material is washed out and enzyme-labelled antibody then added which reacts with a second site on the bound antigen. Unadsorbed Ab-E is washed off and enzyme activity assayed activity is directly related to the concentration of antigen. 

The principle approach to immunoassay is illustrated in Figure 1, which shows a basic sandwich immunoassay. In this type of assay, an antibody to the analyte to be measured is immobilized onto a solid surface, such as a bead or a plastic (microtiter) plate. The test sample suspected of containing the analyte is mixed with the antibody beads or placed in the plastic plate, resulting in the formation of the antibody—analyte complex. A second antibody which carries an indicator reagent is then added to the mixture. This indicator may be a radioisotope, for RIA an enzyme, for EIA or a fluorophore, for fluorescence immunoassay (FIA). The antibody-indicator binds to the first antibody—analyte complex, free second antibody-indicator is washed away, and the two-antibody—analyte complex is quantified using a method compatible with the indicator reagent, such as quantifying radioactivity or enzyme-mediated color formation (see Automated instrumentation, clinical chemistry). 

The sandwich principle achieved its breakthrough when monoclonal antibodies became available because of the need of higher amounts of antibody compared to competitive assays. 

The RIA-gnost Ferritin kit (no more commercially available) from formerly Behringwerke AG, Radiochemical Laboratory described below uses the principle of an immunoradiometric assay (IRMA). It is a two-site solid phase assay of the sandwich type, based on a plastic bead as solid phase to which the antiferritin antibody adheres. The antibody-solid phase is incubated with standards or serum samples containing ferritin and in this process the ferritin in the solution is bound quantitatively to the solid phase via the antibody. The amount of ferritin bound to the solid phase is then determined by a reaction with 125I-labeled anti-ferritin antibody. An antibody-ferritin-125I-antibody complex is thus formed. 

Kinase or phosphatase assays based on the AlphaScreen principle are similar to TR-FRET assays in that they usually require a biotinylated substrate peptide and an anti-phosphoserine or tyrosine antibody. These two reagents are sandwiched between biotin and protein A-functionalized acceptor and donor beads. A kinase assay would show an enzyme-dependent increase in antibody binding (and thus signal) over time and a phosphatase assay would show an enzyme-dependent decrease in antibody binding over time. In some cases, the phosphorylation of an epitope will block the antibody binding and thus a phosphatase assay in principle can be constructed as a signal increase assay (Von Leoprichting and Kumpf, 2004 Warner et al., 2004). 

The most popular form of this technique is solid-phase heterogeneous ELISA, and the inherent use of passive adsorption facilitates flexibility in assay design. ELISA is generally classified as direct, indirect, sandwich (double-antibody) or competitive. Principles of each of these formats are briefly discussed below. 

The binding assays that are performed in this flow injection system may be either competitive or sandwich type. An assay for the serum protein transferrin using a competitive ELISA (129) illustrates the principle (Fig. 5). An immunoafiinity purified polyclonal antiserum raised in rabbits against human transferrin is the binder. The immobilized antibody is packed in a small column (100-200 pL), which is placed in a continuous flow of buffer. The experimental setup is shown in Fig. 6. 

On the basis of an enzyme thermistor, Mattiasson et al. (1977) developed one of the first immunosensors. Immobilized antibodies against albumin are placed in a column and set into an ET. After injection of an albumin-sample and a known amount of enzyme-labeled albumin, both are separated from the sample matrix by antibody-antigen-interaction. After injection of a substrate, the change in heat is a measure of analyte concentration. The less heat produced means that more albumin has been bound. An elution step regenerates the ELISA. Due to its thermal detection principle, the procedure is called TELISA (thermometric enzyme-linked immunosorbent assay). Figure 3 shows the principle of the TELISA procedure in its sandwich configuration. 

Sandwich hybridization, using affinity-based hybrid collection, is based on two nonoverlapping nucleic acid probes (one is labeled, the other can be collected by the affinity matrix) (Syvanen et al., 1986 Jalava et al., 1990). The principles are shown in Fig. 8.3. Target nucleic acid thus mediates binding of labeled probe to the matrix. The detectability is about 10 molecules with a linear range to at least 10 molecules with radioisotopes as labels. In contrast to capture hybridization assays, the immobilization of the complex is at 22-37°C (leaching is then usually less important). 

Principles and characteristics of lateral flow strip assays are reviewed. Recent technology developments permit the use of inexpensive electronic readers for interrogating lateral flow strip test results, thus avoiding the inevitable variation and subjectivity of visual inspection to assess the capture of reporter-labeled analyte on test lines of the strip. Protocols for developing lateral flow assays are described, including two specific case studies for assaying cotinine (a small-molecule metabolite of nicotine) in a competitive format, and assaying HIV antibodies in a sandwich-type assay format. 

The number of components for the indirect sandwich ELISAs is increased and consequently, the number of reagent combinations. The reader should by now be familiar with the descriptions in diagrammatic form so that the next series of assays exploiting the indirect sandwich ELISAs can be examined more briefly, with the principles involved being highlighted. 

Fig. 9 Three test modes for immune assays based on the Ru(bpy)3 /TPrA system adopted by commercial instrument, (a) competitive principle, (b) sandwich principle (c) bridging principle.

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