Antibody for analyte

The most obvious way to use immobilized antibodies for analytical affinity chromatography is to simply use it in a traditional single-column method to determine an antigen s concentration and/or purity. However, there are a number of ways this technique can be advanced to more sophisticated analyses. For example, instead of immobilizing an antibody, the antigen may be immobilized to quantify the antibody as has been done with the Lewis Y antigen [3]. However, the analysis is still a single-column method. 

An alternative procedure is to isolate the B-cells, fuse them with a tumor line (myeloma), and separate these hybrid cells into individual clones, to produce antibody producing hybrids called hybridomas [10-12]. These separate cell lines can be screened to select a clone that produces an antibody with a single structure, called a monoclonal antibody. For analytical purposes, one advantage of a monoclonal antibody is that the cell line can be preserved so that a consistent source of antibody can be obtained. Again, usually researchers prepare their own monoclonals or obtain cell lines from other investigators who have published reports about the use of their monoclonals. Many researchers deposit their cultures with the American Type Culture Collection, Rockville, MD. 

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). 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Organic fluorescent dyes with the appropriate spectral properties also can be paired with lanthanide chelates in FRET systems. For instance, many rhodamine dyes and the cyanine dye Cy5 have ideal excitation wavelengths for receiving energy from a nearby europium chelate. The LeadSeeker assay system from GE Healthcare incorporates various Cy5-labeled antibodies for developing specific analyte assays. In addition, if using a terbium chelate as the donor, then a Cy3 fluorescent dye can be used in assays as the acceptor. 

The development of techniques for the production of monoclonal antibodies by Kohler and Milstein has enormously expanded the potential of antibodies as analytical and therapeutic agents. A monoclonal antibody is one that is produced by a clone of cells all derived from a single lymphocyte. Any lymphocyte can probably produce only a single immunoglobulin and hence the antibody produced by a clone of identical cells is very restricted in the antigens to which it will bind, making it a very specific reagent. 

A third area of development in carbohydrate l.c. analyses is in the combined techniques (see Section IV,3) and other methods that provide qualitative, as well as quantitative, information about sample constituents, such as high-performance liquid affinity chromatography. The use of specific lectin- and monoclonal antibody-based, stationary phases for analytical and preparative applications is now being considered. The basic concepts of these techniques have been reviewed - and their applications to carbohydrates have been discussed. 

A. Lucking, M. Horn, H. Eickhoff, K. Bussow, H. Lehrach, and G. Walter. 1999. Protein Microarrays for Gene Expression and Antibody Screening. Analytical Biochemistry 270 103-111. 

For these reasons, microbial sensors are less suitable for the determination of individual analytes. However, some practical apphcations for biosensors based on enzymes or antibodies for the specific determination of environmentally relevant compounds can be expected soon [11]. Furthermore, in some cases defined specific metabolic pathways in microorganisms are used, leading to microbial sensors for more selective analysis for those environmental pollutants which cannot be measured by the use of simple enzyme reactions, e.g., aromatic compounds and heavy metals. In this context it is also important to mention the aspect of bio availability, a parameter which is included by the measuring procedure of microbial sensors as an integral effect. 

This biosensor consists of two surface-tethered components. The biorecognition element can be an antibody (Section 19-5), DNA, RNA, or carbohydrate with specific affinity for analyte. A structural analog of the analyte is bound to a flexible arm adjacent to the recognition element. In the absence of analyte, the tethered analog binds to the recognition element. 

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. 

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