Double-antibody separation method

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. 

In the double antibody method of separation, a second antibody, produced by injecting the first antibody into another animal, is utilized. This antibody is used to combine with and form an insoluble complex with the first antibody. After... 

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... 

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. 

Use of the Double-Antibody Method to Separate Antibody Bound from Free Ligand in Radioimmunoassay... 

Midgley, A. Hepburn, M.R. Use of double antibody method to separate antibody bound from free ligand in radioimmunoassay. In Methods in Enzymology Immunochemical Techniques Langone, J.J., Ed. Academic Press New York, 1980 Vol. 74, 266-273. 

Traditionally, circulating concentrations of Tg have been measured using double-antibody immunoassays. Commercial RIA kits based on sequential addition techniques have been developed for routine use. These methods involve the preliminary incubation of serum with primary antibody. After a number of hours (2 to 72), the Tg tracer is added and allowed to compete for antibody binding sites. The longer the preincubation the more sensitive the method. Separation of bound from free Tg is then accompHshed by precipitation with a second antibody. 

Heterogenous fluorescence immunoassays can be carried out with the aid of the same separation procedures used in radioimmunoassay. The more expedient homogenous fluorescence immunoassays require quenching, enhancement, polarization, or shifting of the fluorescence of the label upon binding of the labeled analyte to its antibody. Occasionally, a second antibody, directed at the anti analyte antibody, will be used in a double-antibody method to precipitate the bound labeled and unlabeled analyte or to alter the optical properties of the label in such a way as to make the analysis more sensitive. 

Although unlike RIA, enzyme immunoassays can be carried out in homogeneous systems without a separation step (based on the change in enzyme activity during the immune reaction), in practice, heterogeneous (enzyme-linked immunosorbent assay) methods are much more frequently used. The antibody, or in the case of the double-antibody method the second antibody, is immobilized, either covalently or by coating enzyme multiplied immunoassay technique (EMIT). This can be done on the walls of microtiter plates. After the immunogenic reaction, the enzyme activity, which is the equivalent of radioactivity in RIA systems, can be measured by suitable photometric methods on the microtiter plates themselves. 

How is Ab-Ag complex separated from Ag Two methods are mainly followed. Th are the dextran-coated acttvated charcoal method, and the double-antibody method. The first one is given below. 

The possibility of cross-reaction of the second sequence of antibodies with the first sequence may be eliminated by the use of a double immunoenzymatic method in which two specific primary antibodies, produced in two different species, are used in combination with separate species-specific secondary antibodies coupled with... 

Solid-phase procedures are also applied for separation of free antigen (or free antibody). It is also possible to precipitate antibodies by double immunoprecipitation technique in the presence of an excess of anti-IgG. However, this method is not fully reliable since a dissolution of the complex can take place in the presence of an excess of antibodies. 

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