Immunoassays sandwich

In this method, we used Z1O2 NP as a recognition agent to capture the OP adduct on AChE. To complete the immunoassay sandwich, QD-labeled primary anti-AChE antibodies were used. Here, AChE-OP was prepared by incubating AChE and paraoxon, with a subsequent purification step to remove free paraoxon. 

Fig. 1. A principal approach to immunoassay, the sandwich immunoassay, where the thick line represents the soHd matrix.

Most immunoassay kits and many commercial immunoassay analyzers are based on heterogenous EIA or FIA. These include an immunoassay system that uses FIA linked to radial partition chromatography of the antibody—antigen complex (39) a system that uses antibody-coated tubes for enzyme immunoassay of a variety of hormones and dmgs (40) and a system that uses either a sandwich or competitive FIA format to measure a variety of analytes (41). 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

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)...

A sandwich electrochemical enzyme immunoassay has been described for IgG Alkaline phosphatase was again used as the enzyme label with the conversion of phenyl phosphate to phenol being determined electrochemically by LCEC. A detection limit of 10 pg/mL was reported. 

The microplate ELISA testis conducted in standard 96-well microplates. A microplate consists of a 12 X 8 grid of wells for test solutions. The three most widely used ELISA formats are immobilized antigen competitive immunoassay, immobilized antibody competitive immunoassay and sandwich immunoassay. " ... 

Sandwich ELISAs (Eigure 4) are the most common type of immunoassay used for the detection of proteins. A capture antibody is immobilized on the wells of a microplate. The solution containing the analyte is introduced and antibody-analyte... 

Figure 4 Sandwich immunoassay. A capture antibody (Y) is passively adsorbed on a solid phase. The target protein contained in the sample and the enzyme-labeled reporter antibody (Y-E) are added. Both the capture antibody and enzyme-labeled reporter antibody bind to the target protein at different sites, sandwiching it between the antibodies. Following a wash step, the substrate (<>) is added and colored product ( ) formed. The amount of colored product is directly proportional to the amount of target protein captured...

As an alternative, extremely sensitive detection can be achieved with reporter antibody probes tagged with intensely SERS-active compounds or with enzymes that react with substrates to yield SERS-active products. These methods often involve sandwich immunoassay techniques, which increase the number of required steps but offer the advantages of excellent sensitivity and the potential for label multiplexing. For example, Nie and coworkers recently reported the simultaneous detection of two types of antigens in a... 

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

Fujiwara, K., Matsumoto, N., Yagisawa, S., Tanimori, H., Kitagawa, T., Hirota, M., Hiratani, K., Fukushima, K., Tomonaga, A., Hara, K., and Yamamoto, K. (1988) Sandwich enzyme immunoassay of tumor-associated antigen sialosylated Lewisx using b-D-galactosidase coupled to a monoclonal antibody of IgM isotype./. Immunol. Meth. 112, 77-83. 

Hashida, S., and Ishikawa, E. (1985) Use of normal IgG and its fragments to lower the nonspecific binding of Fab -enzyme conjugates in sandwich enzyme immunoassay. Anal. Lett. 18(B9), 1143-1155. 

S.A. Fernando and G.S. Wilson, Studies of the hook effect in the one-step sandwich immunoassay. J. 

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],... 

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