Double-antibody technique

Two methods are commonly employed in RIAs to separate antigen-antibody complexes. The first, the double-antibody technique, precipitates antigen-antibody complexes out of solution by utilizing a second antibody, which binds to the first... 

Double antibody techniques use a second antibody to bind a complex between the antigen and the primary antibody. 

Adsorption with charcoal has been widely used as a separation technique, but a variety of more precise and simpler methods are now available (Table 1). The methods of choice are the double antibody technique, and solid-phase systems. These are also the most easily automated. 

Double Antibody Technique. Although the drug-antibody complex is large it is still soluble. It can be converted into an insoluble form, which can be centrifuged, by the addition of a second antibody (the precipitating antibody) raised against the globulins of the animal in which the first antibody was raised. The free fraction is then simply decanted out of the tube. 

The basic concepts of competitive-binding solid phase EIA have been described elsewhere.The required separation of antibody from the assay mixture can be accomplished in a variety of ways. Double-antibody techniques are quite popular and involve the use of a second antibody, an antibody to the principal antibody, to induce separation. Cross-linking the second antibody serum - with ethyl chloroformate forms an insoluble suspension of small particles, which still maintain a high degree of im-munoreactivity toward the first antibody. Addition of such particles to an EIA assay mixture pulls the first antibody from solution along with enzyme-labeled molecules bound to the antibody. Such a process is depicted in Fig. 1 and employed for all separations in this work. Measurement of enzyme activity in the bound solid phase is desirable because complete purification of the labeled substance is not necessary. 

The method has been termed the double-antibody technique and can be used for a wide variety of analytesJ The double antibody techniques generally require more time because the second antibody reaction can require days to reach equilibrium. The speed of precipitation clearly depends on the concentration of the second antibody. Polyethylene glycol has been used as a cosolvent to increase the precipitation rate. 

Aurell et al. (2004) were able to demonstrate Legionella pneumophila cells in water using a double antibody technique with specific monoclonal antibodies conjugated to FITC. A very simple amplification technique, using a secondary, FITC conjugated, antibody was used to increase the fluorescence intensity. 

The double-antibody technique is also widely used. Here, a second antibody is employed to precipitate the primary antigen-antibody complex. This second antibody is produced by injecting a second animal with the gamma globulin produced in the first animal used to prepare the first antibody. Although the use of a second antibody introduces another source of error, the double-antibody technique has the advantage of being applicable for almost any inununoassay, and the separation is complete. 

The distribution of albumin on the alkylated polymer surface was visualized for a polyether polyurethane. Using a double antibody technique, it was possible to uncover the dense, homogenous nature of the albumin adsorbate on the alkylated surface, and on controls, the patchy coverage with apparent albumin aggregates (Fig. 9) ( 3) Incubation of polyurethane (Biomer) 3 3 alkylated... 

Serum thyroid stimulating hormone (TSH) was measured by a modification of the double antibody technique.The normal range is 5 yoU/ml. 

In the most widely used solid-phase methods no centrifugation step is required. The commercially available kits for steroids contain the antibody (or the second antibody, in the double-antibody technique) immobilized on the walls of a tube, plastic beads, sticks, or membranes. 

Lindstrom, T., Hedman, C.A., and Arnqvist, H.J. (2002) Use of a novel double-antibody technique to describe the pharmacokinetics of rapidacting insulin analogs. Diabetes Care, 25,1049-1054. 

Four types of techniques for separating the bound fraction P Q from the reagent mixture are in common usage, loosely termed double antibody, solid phase, charcoal adsorption and solution precipitation. The first type is used with radioimmunoassay methods specifically, while the other three types can be used with both radioassay and radioimmunoassay methods. 

Tidman N, Janossy G, Bodger M, Granger S, Rung PC, Goldstein G (1981) Delineation of human thymocyte differentiation pathways utilizing double staining techniques with monoclonal antibodies. Clin Exp Immunol 45 457 467... 

Fig. 2. Ouchterlony double-diffusion technique. The antigen is placed in the center well, cut in an agarose gel, and different antisera in a range of dilutions are placed in the sunounding wells. Antigen and antiserum diffuse toward each other and form a white precipitin line where an antibody recognizes the antigen.

Fig. 1. Diagrammatic representation of the double-antibody-labeling technique used in the ELISA.

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. 

Immunofluorescence has become one of the most important tools of contemporary cell biological research, particularly for localising specific antigenic determinants such as newly expressed proteins in the cell. In this context, double immunofluorescence techniques are very powerful use of two antibodies labelled with distinguishable fluorophores allows different cell structural elements to be visualised simultaneously. A similar approach can be used in electron microscopy by labelling different proteins with gold particles of different sizes. 

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