Electrochemiluminescence immunoassays

Figure 11. Electrochemiluminescent PDMS-graphite biochip formats (a) nucleic acid-based biochip (b) immunochip (competitive immunoassay).

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

Deiss F, LaFratta CN, Symer M, Blicharz TM, Sojic N, Walt DR (2009) Multiplexed sandwich immunoassays using electrochemiluminescence imaging resolved at the single bead level. J Am Chem Soc 131 6088-6089... 

Some electrochemically active substances that can generate photons on an electrode surface are suitable labels for homogeneous immunoassays. A labelled antigen exhibits an electrochemical reactivity and produces luminescence, but when it is immunochemically complexed, the labelled antigen loses its electrochemiluminescent properties. One optical immunosensor for homogeneous immunoassays was assembled by spattering platinum on the end surface of an optical fibre. Spattered platinum maintains optical transparency and functions as an electrode. An optical electrode efficiently... 

On the other hand, the main types of immunoassays that can be performed by using labelled antibodies or antigens are direct sandwich, competitive and indirect assays. The labels can be enzymes (alkaline phosphatase, peroxidise or glucose oxidase) metal NPs (gold) fluorescent or electrochemiluminescent probes. 

Fig. 9. Enzyme electrochemiluminescence (ECL) immunoassay protocol, (a) Trinitrotoluene (TNT) and dini-trophenyl of haptenylated dextran compete for antibody-binding sites, (b) Antibodies attached to dextran bound to streptavidin-coated paramagnetic beads, (c) ECL detection of enzyme-labelled antibodies magnetically concentrated on electrode. Reprinted from Wilson et al. [68], Copyright (2003), with permission...

Immunogenicity assays for investigating the frequency and consequences of antibody development against a protein therapeutic agent are typically based on an immunoassay technique (mostly ELIS As of various types). However, other assay formats are available such as radioimmunoprecipitation assay, surface plasmon resonance, and electrochemiluminescence [3]. Assays for measuring antibody response should be established in the early preclinical stage of development to estimate the value of the applied animal models (see Chapters 16 and 20). 

An example for an anti-HBe test with a direct label is the electrochemiluminescence immunoassay ECLIA from Roche Diagnostics (automated on Elecsys immunoassay analyser), whereas the Im-mulite anti-HBe test from Diagnostics Products Corp. represents an enzyme-amplified chemiluminescence immunoassay with sustained signal (automated on the Immulite chemiluminescent system). 

Chemiluminescence immunoassay These assays rely on the use of chemiluminescent labels, which may be labelled antigens or antibodies. For example, the chemiluminescent label isoluminol is oxidized in the presence of hydrogen peroxide and a catalyst, producing long-lived light emission that is measured, thus allowing determination of the unknown antigen or antibody concentration in a sample. Another useful variant is electrochemiluminescence immunoassay that is commonly used in biomolecular detection, and in particular the measurement of native and recombinant peptides and proteins. 

Obenauer-Kutner, L.J. Jacobs, S.J. Kolz, K. Tobias, L.M. Bordens, R.W.A. A highly sensitive electrochemiluminescence immunoassay for interferon alfa-2b in human serum. J. Immunol. Meth. 1997, 206 (1-2), 25-33. Rabbany, S.Y. Donner, B.L. Ligler, F.S. Optical immuno-sensors. Crit. Rev. Biomed. Eng. 1994, 22 (5-6), 307-346. Wink, T. van Zuilen, S.J. Bull, A. van Bennekom, W.P. Liposome-mediated enhancement of the sensitivity in immunoassays of proteins and peptides in surface plasmon resonance spectrometry. Anal. Chem. 1998, 70 (5), 827-832. Gaudin, V. Pavy, M.-L. Determination of sulfamethazine in milk by biosensor immunoassay. J. AOAC Int. 1999, 82 (6), 1316-1320. 

Blackburn GF, Shah HP, Kenten JH, et al. Electrochemiluminescence development of immunoassays and DNA probe assays for clinical diagnostics. Clin Chem 1991 37 1534-9. 

Ruthenium (II) tris(bipyridyl) (Figure 9-17, B) undergoes an electrochemiluminescent reaction (620 nm) with tripropylamine at an electrode surface, and this chelate is now used as a label in competitive and sandwich electrochemiluminescence immunoassays. Using this label, various assays have been developed in a flow cell using magnetic beads as the solid phase. Beads are captured at the electrode surface, and an unbound label is washed out of the cell by a wash buffer. Label bound to the bead undergoes an electrochemiluminescent reaction, and the light emission is measured by an adjacent photomultiplier tube. ... 

Figure 9-17 Luminescent labels. A, Chemiluminescent acrid in turn ester label. (From Law S-j, Miller T, Piran U, et al. Novel polysubstkuted aryl acridlnium esters and their use In immunoassay, j Eioium Cftem/7um / 989 4 88-98. Reprinted by permission of John Wiley Sons, Ltd.). B, Electrochemiluminescent ruthenium (11) tris bipyridyi) NHS ester label.

Automated chemistry analyzers have traditionally relied on photometers and spectrophotometers for measurement of absorbance. Alternative approaches now being incorporated into analyzers include reflectance photometry and fluorom-etry. Immunoassay systems have used fluorescence (IMX), chemiluminescence (Centaur and Immulite), and electrochemiluminescence (ELECSYS) to enhance sensitivity. Ion-selective electrodes and other electrochemical teclmiques are also widely used. Principles of these measurement techniques have been discussed previously (see Chapter 4). This section reviews the special features and application of the various approaches to automated analysis. 

As of 2004, the College of American Pathologists Interlaboratory Survey listed five manufacturers as providing first generation noncompetitive immunoassays for intact PTH. Most of these commercially available methods are on fully automated immunoassay analyzers using chemiluminescence detection including ALP with 1,2-dioxetane phosphate, acridinium ester, or electrochemiluminescence (ruthenium chelate). Signal antibody is less commonly radiolabeled with L... 

For each type of assay, the label or tag involved is listed along with typical substrates, type of absorbance, and type of instrument required. RIA radioimmunoassay IRMA immunoradiometric assay CPM counts per minute ELISA enzyme linked immunosorbent assay EIA enzyme immunoassay TMB 5,5 tetramethylbenzidine HPA p hydroxyphenylacetic acid AMPPD 4 methoxy 4 (3 phosphatephenyl) spiro(l,2 dioxetane) 3,2 adamantane p NPP p nitrophenyl phosphate 4 MUP 4 methylumbelliferyl phosphate ONPG o nitrophenyl P galactopyranoside MUG 4 methylumbelliferyl P D galactopyrano side AMPGD 3 (4 methoxyspiro( 1,2 dioxetane 3,2 tricyclo(3.3.1.1(3,7))decan) 4 yl)phenylgalacto pyranoside ECL electrochemiluminescence. 

Horninger, D., Eirikis, E., Pendley, C., Giles Komar, J., Davis, H.M., and Miller, B.E. (2005) A one step, competitive electrochemiluminescence based immunoassay method for the quantification of a fully human anti TNFa antibody in human serum. Journal of Pharmaceutical and Biomedical Analysis, 38, 703 708. 

Grimshaw, C., Gleason, C., Chojnicki, E., and Young, J. (1997) Development of an equilibrium immunoassay using electrochemiluminescent detection for a novel recom binant protein product and its application to pre clinical product development. Journal of Pharmaceutical and Biomedical Analysis, 16, 605 612. 

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