Labels equilibrium label-free technique

Most problems with this procedure have involved tracer impurities and the separation of bound and free labeled fractions. Several separation techniques have been used, including equilibrium dialysis, membrane ultrafiltration, and steady-state gel filtration. Their deficiencies include a requirement for a large sample volume, the need for complicated correction of sample volume changes that occur during the separation, and difficulties of collecting and measuring radioactivity in numerous fractions of each sample. Equilibrium dialysis has been used most often in the past, but serious errors often arise from the sample dilution required by this method. Symmetrical dialysis of undiluted samples is reported to be less susceptible to tracer contamination and dilution effects. Ultrafiltration also appears to overcome these problems and to obviate errors caused by dilution. 

Equilibrium dialysis is an analytical technique which is usually performed with commercial apparatus. In this technique, the two dialysis chambers, separated by a membrane, are filled respectively with macromolecule solution and with ligand, which is usually radioactively labelled. After equilibrium has been reached, samples are removed from the two chambers the concentration of free ligand is determined from one sample, and free plus bound ligand from the other. Parameters characterising the binding equilibrium can be determined by appropriate analysis of the data. 

Labeled-Analogue Methods, In a typical one-step RIA for FT4, endogenous free hormone and a I-labeledT4 analogue compete for solid-phase antibody binding sites in the presence of serum protein the amount of labeled analogue bound by the specific antibody is inversely related to the amount of FT, in the specimen. Test results are expressed relative to secondary serum calibrators that have been independently calibrated using equilibrium dialysis or ultrafiltration techniques. A number of one-step RIAs have been developed and are commercially available. 

In practice, the concentration of the free drug is obtained by ultrafiltration, equilibrium dialysis or by measurement of the ultraviolet absorption spectrum which is often displaced by the binding. The use of radioactively labelled drugs increases the precision of some of these techniques. 

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