Radioimmunoassays double-antibody

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. 

Fig. 6. Comparison of immunoassays using clonotype antibodies. The EDso values (thyroxine concentration corresponding to 50% of bound form at zero concentration of thyroxine) in thyroxine immunoassays using clonotype antithyroxine antibody prepared by fast protein liquid chromatography (FPLC). RIA, double-antibody radioimmunoassay FLA, fluorescence polarization immunoassay EIA, enzyme immunoassay. [Cited from Miyai et al. (M5).]...

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. 

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. 

Insulin-, somatostatin-, ACTH-, and relaxin-related materials were detected in the partially purified extracts using specific double antibody radioimmunoassays (12,8,16,7). 

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 availability of radioimmunoassay (RIA) procedures for environmental agents holds some promise in minimizing the need for the more sophisticated and expensive instrumental methods of analysis by eliminating negative samples and for routine monitoring of exposure in environments known to be contaminated by certain classes of compounds. There are a number of fundamental problems involved in development of such RIA procedures and in their use (Table 3). Double-antibody RIA s have been developed (29) for quantitating a number of chlorinated aromatic hydrocarbons of current concern from environmental samples including animal tissues. These chlorinated hydrocarbons include members of the dibenzo-p -dioxin, dibenzofuran, and biphenyl classes of compounds. The use of RIA procedures for trace residue analysis is discussed further in another paper in this conference. 

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. 

The development of antisera to prostaglandins pg 2a and their analysis using solid-phase and double antibody radioimmunoassay methods. Br J Pharmac (55) 503-514, 1975. 

A solid-state radioimmunoassay has been developed for the detection and measurement of porcine pancreatic a-amylase. A double-antibody radioimmunoassay that is able to determine and detect small amounts of pancreatic amylase, even in the presence of amylase isoenzymes, has been reported. The assay was used to ascertain the effects of pancreatectomy on the levels of amylase in porcine tissues, and it was concluded that the pancreas contributes significantly to the levels of circulating amylase. 

Sodium warfarin added in vitro to the blood of healthy individuals does not affect the double antibody radioimmunoassay of insulin or influence insulin secretion from samples of rat pancreas incubated in vitro for 40 min. (S ). This observation might be important, in view of the fact that heparin, unlike coumarin, is thought to be a potent and direct inhibitor of insulin secretion (9 ). 

In two sensitised workers exposed to diphenylmethane diisocyanate (MDI), used for coating pipes with a polyurethane foam, tests with an MDI-human serum albumin conjugate showed the presence of specific IgE antibodies as measured by polystyrene tube radioimmunoassay (PTRIA) procedures (Zeiss et al. 1979). High levels of specific IgG antibody were found, with some cross-reactivity with a TDI-human serum albumin conjugate. The serum of one of the workers gave a positive precipitin reaction in the double diffusion test in gel. The authors suggest that these antibodies may be related to the respiratory reactions to the inhaled chemical. 

Techniques for the detection or assay of various substances based on the reaction of those substances with specific antibodies (or vice versa, i.e. the detection and assay of antibodies using antigens). Such techniques include agglutination reactions, automated immune precipitation, complement fixation tests, crossed electrophoresis, counter electrophoresis, double diffusion, enzyme immunoassay, fluoroimmunoassay, haem-agglutination, immunoelectrophoresis, immunofluoresence, radial immunodiffusion, spin immunoassay, immunofixation, immunoradiometric assay and radioimmunoassay. See separate entries for these subjects. 

In this kind of work, monospecificity was operationally defined by the experimental demonstration that only one molecule of the antibody population can bind to one molecule of the antigen. Under these conditions, the complex formed is soluble and a radioimmunoassay or a double precipitation must be performed to quantitate the determination. 

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