Radioimmunoassay bound/free separations

In a radioimmunoassay procedure, 1 pmol of labelled analyte was added to the sample. Antiserum equivalent to 75 pmol of analyte was then added, and after incubation the bound and free fractions separated by precipitation with ammonium sulphate solution. The activity ratio, bound free, was measured to be 2.88. Calculate the number of moles of analyte in the original sample. 

Radioimmunoassay requires a separation of the antibody-bound from the free labelled hormone. Various methods have been proposed to accomplish this separation. 

Immunoassays based on fluorescence polarization have become very popular because, in contrast to radioimmunoassays, they require no steps to separate free and bound tracer. In a fluoroimmunoassay, the fluorescently labeled antigen is... 

The principal components in radioimmunoassay (RIA) are a drug labelled with a suitable radioisotope, an antibody specific to that drug, and the assay sample. The drug-antibody complex (bound drug) must be separated from the free drug in the later stages of the assay. 

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

Charcoal is widely used to separate antibody-bound ligand (bound) from non-antibody-bound ligand (free) in competitive protein binding assays. Its use was originally described by Miller for vitamin Bi assay and later by Herbert et al. for B12. In 1965, Herbert suggested its use to separate bound and free in the radioimmunoassay of insulin. Since then it has been used for a large number of radioimmunoassays and radioreceptor assays. 

Separation of free antigen from antigen-antibody complexes is a universal procedure in radioimmunoassay and related techniques and is necessary to determine the distribution of the antigen between the bound and the free forms. It requires that the free fraction be physically separated from the bound fraction, and a variety of techniques have been developed for this purpose, all of which depend on physicochemical differences between the two forms. For example, both ammonium sulfate and polyethylene glycoF can be used at concentrations that will precipitate the antibody molecules and thus the antigen-antibody complex, but will not precipitate the free antigen. 

The efficiency of a separation procedure in radioimmunoassay can be defined as the completeness with which the free and bound fractions are separated. This concept is illustrated diagrammatically in Fig. . ... 

Precipitation of proteins with organic solvents or salts was one of the earliest methods for the separation of free and bound antigen in radioimmunoassays and related systems. The use of water-soluble nonionic polymers in protein fractionation has been reviewed by Fried and Chun. ... 

A protocol for the use of either ammonium sulfate or polyethylene glycol in the separation of free and bound antigen in a radioimmunoassay is given in Table II. This protocol can be considered as virtually universal for all those antigens for which the method has been shown to be appropriate, i.e., if it satisfies the criteria of the type of experiment shown in Fig. 7. 

Any radioimmunoassay analysis relies upon the efficiency of the separation of the unbound (free) antigen or hapten from that bound to antibody. When using immunoassay to estimate low molecular weight compounds such as steroids, the problem is perhaps somewhat simpler than in protein immunoassay. Because of the large difference in molecular weight between steroid haptens and their appropriate antibodies, relatively simple methods of separation, such as dialysis, can be used. Although nonspecific precipitation of the antibody-bound steroid by ammonium sulfate and polyethylene glycoH are widely used, the utilization of second-anti-body immunoprecipitation of the steroid-antibody complex is not com-... 

Determination of the distribution of Ag between the antibody-bound and free fractions, in radioimmunoassay procedures, requires a separation step which isolates Ag from Ag -Ab. Once separated, measurement of the radioactivity of the label in one or both the fractions provides a signal that is approximately exponentially related to the concentration of unlabeled antigen. The graphical relationship can have either a positive or a negative slope depending on whether the antibody-bound or free fraction is measured. The exact shape of the calibration curve however, is dependent on the antibody-binding equilibrium constant. ... 

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. 

Thorell JI. Internal sample attenuator counting (ISAC) A new technique for separating and measuring bound and free activity in radioimmunoassays. Clin Chem 1981 27 1969-73. 

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. 

A fluorescence-quenching immunoassay for cortisol, based upon the use of a tracer comprising fluorescein linked to the steroid via a 21-amino-group, claims a good correlation with radioimmunoassay results. Separation of free and bound fractions is unnecessary with this system.141... 

Many different systems have been used successfully for the separation of free and antibody-bound hormones in radioimmunoassays. These depend on (1) differences in physicochemical properties of the glycoprotein hormones and the glycoprotein-y-globulin complex (2) immuno-... 

The most prominent of these assays are radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). In the case of RIA, the analyte sample is either extracted or used directly, and mixed with a constant amount of antibody and radiolabeled analyte [tracer mostly fi-emit-ters (e.g., H, " C) or y-emitters (e.g., I)]. After equilibration and separation of the free and bound antigen, radioactivity is measured for quantification of the analyte. In the case of sandwich-RIA, two antibody preparations are used the first serves as the binding partner of the analyte, whilst the second - which is radiolabeled - is directed either against the analyte or the first... 

Antigen-antibody reactions occur in aqueous solution. Therefore, after separating "free" and "bound" radioactivity in a radioimmunoassay, one must frequently measure the radioactivity of an aliquot of an aqueous solution. Here we are faced with the problem of dispersing water, a polar solvent, in a non-polar toluene or xylene based scintillator system. To minimize quenching problems, especially serious when counting weak beta particles such as those emitted by tritium, it is best if a homogenous mixture of the sample and liquid scintil-lant results. That is, two-phase systems should be avoided if possible. 

In RIA it is necessary to separate the free from the protein bound activity before counting. A novel approach to separation is to partition unbound radioactivity directly into a non-polar scintillation cocktail (Castanier et al, 1970). In using this technique it is necessary that the scintillator be non-polar and hence immiscible with the aqueous phase. In addition, the free form of the radioactivity must be soluble in non-polar solvents. This separation method has been used successfully by Jowett ot al (1973) for the radioimmunoassay of... 

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