Protein, estimation competition

Cost estimates of producing ethanol from com have many uncertainties (11). Most estimates fall into the range of 0.26 to 0.40 per Hter ( 1 to 1.50/gal), after taking credits for protein by-products, although some estimates are lower. These estimates do not make ethanol competitive with oil until... 

Alternatively, competitive ELISA can be used to estimate the hapten density if an antibody that specitically recognizes the hapten is available. At first observation this approach seems circular because the immunoassay developed is used to determine hapten density on proteins used for immunization. However, if a small molecule mimic of the protein conjugate is used as a standard, the method can be accurate. For example, a hapten containing a carboxylic acid can be coupled to phenethylamine or tyramine, its structure confirmed and the material used to generate a calibratron curve to estimate hapten density. 

Figure 19.7. Competition curves for two compounds versus a known radioligand. (Top) These data represent the competition of two compounds with a known radioligand (in this case a radioligand that labels the dopamine D receptor, a member of the G protein-coupled superfamily). It is important to note not only the left-right difference between Compound A and Compound B, but also the difference in the shape of their competition curves. (Bottom) A Hill plot [based on Eq. (19.20)] of the competition curves shown in the top figure provides two pieces of data. First, the slopes of the lines are different (Compound A = -1.0 Compound B = -0.6), which has important mechanistic meaning that is discussed in Section 19.3.4b. Hill plots also allow more precise estimation of IC50s. By definition, at 50% inhibition, the Hill coefficient is 0. As shown, one can estimate the IC50 for each compound from this plot.

Antibodies can have distinct advantages over the natural receptor molecules that are used in some competitive binding assays. They are stable, soluble proteins with known chemical and physical properties. - Procedures for their purification are available, " but usually diluted antisera is used in fluid phase radioimmunoassays. It has been estimated that an individual animal has the potential to produce antibodies specific for approximately 10 diverse immunodominant moieties. These molecules may possess binding constants for individual antigens on the order of... 

Artefactual increases of as much as 50% in total thyroxine, estimated by a competitive protein-binding assay, and of as much as 30% in triiodothyronine resin uptake are probably due to rapid and continuing lipolytic hydrolysis of triglycerides after blood has been drawn (126). Thyroid function tests should therefore always be performed on blood samples taken before (or a sufficient time after) heparin treatment (127). An increase in serum-free thyroxine concentrations has also been reported after low molecular weight heparin, by up to 171% in specimens taken 2-6 hours after injection. When specimens were obtained 10 hours after injection, the effects were smaller, but with concentrations still up to 40% above normal the results can still cause errors of interpretation (128). 

Only a small fraction (1% to 2%) of imconjugated testosterone exists fireely (non-protein bound) in serum or plasma. None of the conventional assay methods, including RIA, is sufficiently sensitive to quantify the free steroid directly in a protein-free ultrafiltrate of plasma. Instead, the free steroid is estimated in plasma by adding a known amount of radiolabeled compound to the sample and allowing labeled and unlabeled compounds to reach equilibrium in their competition for the same binding sites on the proteins. Bound and free radiolabeled fractions are then separated and the ratio of free labeled to total labeled compound is determined. At equilibrium, this ratio is taken as a measure of the free testosterone fraction. An estimate of serum free testosterone can then be calculated by multiplying the free testosterone fraction by the total testosterone concentration. 

A3. Archibald, E. L., Mincey, E. K., and Morrison, R. T., Estimation of serum folate levels by competitive protein binding assay. Clin. Biochem. (Ottawa) 5, 232-241 (1972). 

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