Double antibody precipitation

II.R.l Competitive RIA of Apidra (Insulinanalogue) with Double Antibody Precipitation 647... 

The assay described below in more detail uses the principle of a competitive immunoassay with a double antibody precipitation (Chard 1990). The antibody (from guinea pig) bound tracer is separated from the free tracer by the second antibody present in the precipitating reagent (goat anti guinea pig antibody). After... 

The traditional radioimmunoassay (RIA) for TSH was based on competition between endogenous and radiolabeled hormone for bmding sites on a fimited amount of antibody. Separation of antibody-bound and free radioligands was conveniently performed by double-antibody precipitation (enhanced by the addition of polyethylene glycol) or by using a solid-phase, second-antibody procedure. The amount of labeled TSH bound to the antibody was inversely related to the amount of unlabeled TSH present in the serum specimen. 

Immunoglobulins G from antisera specific for human chorionic gonadotrophin, carcinoembryonic antigen, a-foetoprotein, and casein have been isolated by affinity chromatography on columns of immobilized staphylococcal protein A, before being themselves immobilized on supports.The resulting coupled antibodies provide a greater sensitivity and convenience in radioimmunoassay studies than the conventional double antibody-precipitation methods. 

Fig. 3. Comparison of the antigenicity of calf ADA and its mPEG derivative. Graph shows the activity of ADA remaining in the supernatant after double antibody precipitation using calf ADA antiserum Unmodified calf ADA (-0—0-), mPEG modified calf ADA (- - ), CS is...

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. 

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 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 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. 

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. 

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. 

---