Bound antibodies

Fig. 5. Scheme of the last steps for the sandwich ELISA. Where Ah 1 represents the the surface-bound antibody, Ag, the antigen, and Ah 2, the... 

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). 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Elution of the bound antibody-enzyme conjugate occurs by only a slight shift in pH to acidic conditions or through the inclusion of a metal-chelating agent like EDTA or imidazole in the binding buffer. Either method of elution is mild compared to most immunoaffinity separation techniques (discussed in the previous section). Thus, purification of the antibody-enzyme complex can be done without damage to the activity of either component. 

M. Aizawa, A. Morioka, S. Suzuki, and Y. Nagamura, Enzyme immunosenser III. Amperometric determination of human cherienic gonadotropin by membrane-bound antibody. Anal. Biochem. 94,22—28 (1979). 

I immediate Soluble Clonal expansion B cells Cyto-philic antibody (IgE) generated binds to mast cells Antigen binds to cell bound antibody crosslinks receptors, causing release of mediators Anaphylactic response to bee sting immediate response in allergic asthma... 

Fig. 1. Diagram of an EM immunogold assay localizing a protein on plastic sections. The primary antibody binds to an exposed surface epitope of the embedded cells. The antibody is then visualized by binding a second antibody coupled to a colloidal gold particle. The electron-dense gold particle visibly marks the position of the bound antibodies when visualized with the electron microscope. 

Major progress in the analytical use of antibodies occurred with the development of several fundamental techniques the ability to detect cell-bound antibody (Coombs Test, 1945) immunoprecipitation in gels (Ouchterlony, 1953) radioimmunoassay (Yalow and Berson, 1960) and monoclonal antibodies (Kohler and Milstein, 1975). These, together with a con-... 

An IgG-antibody against an individual ribosomal protein binds specifically only to this protein in a ribosomal subunit. Since the antibody is divalent it can form a bridge between the identical proteins in two subunits, leading to a dimer that can be examined under Ae electron microscope. The location of the bound antibody on the subunit surface can be determined, defining the position of the antigenic determinant of a particular protein. The method relies on the fact that IgG-antibodies are able to react with specific proteins within the intact ribosomal subunits and that both subunits have discernible shapes with recognizable morphological landmarks. 

Wash cultured cells attached to 35-mm plastic tissue-culture dishes in Dulbecco s PBS, then incubate in a blocking buffer consisting of BSA-PBS for 5 min, and cool to 4°C (see protocol flow chart in Fig. 1). Cooling prevents subsequent endocytosis of any added antibody reagents, as well as minimizing lateral mobility of bound antibody in the plane of the plasma membrane (see Notes 5 and 6). 

Diluted human serum (1 pL) was incubated with the peptide microarray and bound antibodies were detected using a rhodamine-labeled anti-human IgG. Signal was detected using a slide scanner (Affymetrix model 418) with data collection in the Cy3 channel. A reported eightfold gain in sensitivify at 100% specificity over standard ELISA was achieved using the peptide microarray. 

It has often been realized that the separation efficiency in the competitive CEIA mode is favorable compared to the noncompetitve mode, especially if free and bound antibodies are utilized. Ou and co-workers presented a relatively simple approach to solve this problem (15,16). They used nondenaturing SDS capillary gel electrophoresis (CGE) to analyze anti-bovine serum albumin (BSA) antibodies with UV detection. 

Non-labelled immunosensors rely on various principles (Fig. 3.27.A). Either the antibody or the antigen is immobilized on the solid matrix to form a sensing device. The solid matrix should be sensitive enough at the surface to detect immunocomplex formation. Electrode, membrane, piezoelectric and optically active surfaces may in principle be used to construct non-labelled immunosensors. The antigen or antibody to be determined is dissolved in a solution and reacted with the complementary matrix-bound antibody or antigen to form an immunocomplex that alters the physical e.g. the electrode potential or intrinsic piezofrequency) or optical properties of the... 

Immunochemical detection is possible in one step, if the detecting antibody carries the signal-forming principle. But also cascades of steps are used to identify the first-bound antibody and by it the antigen immobilized by blotting. Antibodies form these cascades, i.e., if the first bound antibody is from species A, e.g., rabbit, a second antibody from other species, e.g., goat, directed against the primary, is used. 

The EIA described in this protocol is a so-called indirect EIA, because the antigen is immobilized onto the surface of the microtiter plate and a second species-specific antibody enzyme conjugate detects the bound antibody of the antiserum. As an example. 

Fig. 11. Principle of antibody directed enzyme prodrug therapy. Hatched circles represent cells that have not bound antibody-enzyme conjugate. Internalized drug is abbreviated with the letter d . (From [172] with permission)...

Proteins can be detected in tissue sections or cell cultures using similar immune detection systems. Use of an antibody to detect specific proteins in tissues is called immunohistochemistry, whereas detection of proteins in cell suspensions is called immunocyto-chemistry. Tissues can be prepared by fixation and embedding in paraffin wax, or by rapid freezing in a compound that inhibits ice formation in the tissue, so as to preserve cell morphology. Some antibodies do not work well with paraffin-embedded tissues, probably because the antibody cannot access the antigen properly (133). The most common labeling system used for detection of the bound antibody is an enzyme-coupled secondary antibody that produces a color reaction... 

Furthermore, certain cells such as cytotoxic T lymphocytes and macrophages have Fc receptors, which bind to the Fc portion of the bound antibodies (e.g., IgG). As a result, the cytotoxic T cell or macrophage is activated, and lyses the cell to which the antibody is bound. 

Figure 19-13 illustrates the principle of an enzyme-linked immunosorbent assay, abbreviated ELISA in biochemical literature. Antibody 1, which is specific for the analyte of interest (the antigen), is bound to a polymeric support. In steps 1 and 2, analyte is incubated with the polymer-bound antibody to form a complex. The fraction of antibody sites that bind analyte is proportional to the concentration of analyte in the unknown. The surface is then... 

A column is prepared with covalently bound antibodies to TNT. Fluorescence-labeled TNT is passed through the column to saturate the antibodies with labeled TNT. The column is washed with excess solvent until no fluorescence is detected at the outlet. 

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