Saturation Assays

There are a plethora of criteria that should be applied to ensure that the experimentally determined parameters provide a true reflection of the physical interactions that they represent. However, if the data are to be credible they must demonstrate an internal consistency. The equilibrium dissociation constant should, for example, be the same if it has been determined from equilibrium saturation assays or by calculation from the appropriate kinetic constants if it is not, this implies that the physical characteristics of the interaction are outside the criteria for which the equations have been developed, i.e., those rehearsed in Section 2.7. Statistical comparison of data sets must also be carefully assessed here the availability of the powerful computation facilities available on most laboratory desks has taken much of the drudgery out of such analysis. 

The results of this saturation assay were validated by direct comparison to conventionally conducted radioligand binding assays using [ H]NO 711 as marker [80, 100]. Not only due to financial considerations, hot/cold dilutions had to be used in the radioligand binding assays, in contrast to the MS binding assays... 

Two different sequential saturation assays are modified from the ELISA25 to determine association constants of antibody with lysozymes. In both assays, a constant antibody concentration is incubated with varying concentrations of lysozyme until equilibrium is reached. The time necessary must be determined empirically for each antibody for the antibody HyHEL-10, this was determined to be at least 16 hr,26 and all incubations were performed for 16-24 hr. At the end of the incubation, an aliquot of the mixture is treated with an excess of reagents to sample free (unbound) antibody combining sites by incubating with labeled or solid-phase coupled... 

Two main types of assay are usually undertaken, namely, competition and saturation assays. The former is used to measure the binding affinity of an unlabelled compound. A range of concentrations of the compound are mixed with a constant concentration of a carrier-free receptor specific radioligand and... 

PROTOCOL FOR DETERMINATION OF MAXIMUM BINDING CAPACITY (SATURATION ASSAY)... 

Sandwich assays require an Ag to contain at least two distinct antigenic sites and as a result are only applicable to large molecular weight compounds. Optimization of the sandwich assay parameters is also commonly done by a checkerboard approach. This is not a saturation assay, free Ab binding sites must remain after the adsorption of Ag. 

B17. Brown, B. L., Ekins, R. P., ElUs, S. M., and Reith, W. S. A specific saturation assay technique for serum triiodothyronine. In In Vitro Procedures with Radioisotopes in Medicine, pp. 569-583. IAEA, Vienna, 1970. 

Fig. 1. Saturation binding of a cannabinoid receptor ligand to brain membranes. Shown are the results of a typical saturation assay of [ H]SR141716A (a CBi-selective antagonist) in rat cortex membranes. Various concentrations (0.05-5.5 nAf) of radioligand were incubated in assay buffer with 0.1% BSA and rat cortex membrane homogenates (20 jig/tube) for 1 h at 30°C in the absence (total DPM) and presence (nonspecific DPM) of 5 pM unlabeled SR141716A. Specific DPM and nonspecific DPM shown are the mean of three values measured in consecutive assay tubes in the same rack. Specific DPM were calculated by subtracting mean nonspecific DPM from mean total DPM at each concentration of [ H]SR141716A.

Radioimmunoassay represents one type of a somewhat broader classification of assays for which there is no generally accepted name but for which the terms "ligand assays", "saturation assays", "binding assays" or "competitive assays" are sometimes applied. The ccxnmon denominator of these assays may be identified as a reaction involving a ligand and a binder. 

The virtues of this appraoch are that the equipment is relatively inexpensive and widely available, that the procedure can be automated, that results can be obtained on the same day, that the reagents are stable for months, and that the per assay cost of reagents is low (less than 2). Problems include the technical difficulty of synthesizing active enzyme conjugates, less sensitivity than RIA (in part because of steric hindrance of the enzymes), and the statistical problems which affect precision when any saturation assay is performed under non-equilibrium conditions. 

The above methods have opened a new door to tackle quantitative proteomics by element mass spectrometry, although it is still quite difficult to quantify proteins by element mass spectrometry detection after element labeling. In fact, element labeling is not a new concept. For example, a silver saturation assay was applied for measurement of metallothionein in tissue. But this kind of metal-binding assay is based on certain properties of a protein and, being less selective, is applied mainly for a known and isolated protein. Comparatively, bi-functional chelating reagents are established in selective, reproducible reactions and thus are more suitable for protein quantification by element mass spectrometry detection. 

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