Protein-binding assays

Practical Concepts of Competitive Protein Binding Assays... 

Radioimmunoassay is a competitive protein binding assay which utilizes an antibody as the binding protein. This assay also employs a highly purified antigen which has been radio-labeled (tagged). 

Radioassay is a competitive protein binding assay which employs a natural binding protein instead of an antibody. For example, transcortin is the binding protein for cortisol in nature and also in the radioassay procedure for cortisol. 

The basic theory of competitive protein binding assays employing a radioactive label is as follows ... 

This equation illustrates the components of a competitive protein binding assay system. That is, the reaction system contains both radioactive and non-radioactive free ligand (P and P) and both radioactive and non-radioactive protein bound ligand (P Q and PQ). This type of assay assumes that binding protein will have the same affinity for the labeled or non-labeled material that is being measured. Although this assumption is not always completely valid, it usually causes no problems of consequence with most radioassays or radioimmunoassays. 

If only one counter can be purchased, then a gamma counter is the instrument of choice since most assays are now performed with gamma emitting isotopes. Table I lists the isotopes in common usage for competitive protein binding assays. 

Besides counting equipment, automatic pipettes and dilutors are also needed in doing competitive protein binding assays. 

Assays are usually set up according to the instructions provided with the reagents. These directions are usually very detailed and should be referred to in establishing an assay. However, the following illustrates the most common type of assay and may be helpful in explaining some of the various nomenclature used in the field of competitive protein binding assays. 

While much care has to be used in performing competitive protein binding assays, most well-equipped and staffed clinical laboratories should have no serious problem in undertaking such assays. The biggest problem that may be encountered is the selection of a dependable and reliable manufacturer for reagents. Problems that may arise are non-purity of standards and label non-specificity of antibodies or the inability to maintain any of these characteristics from lot to lot. It therefore is a good practice to evaulate a few manufacturers before selecting one for routine use. 

Odiell, W. D. and Daughaday, W. Principles of Competitive Protein-Binding Assays. J. B. Lippincott (1971). 

The assays that utilize protein instead of antibody are normally termed as competitive protein binding assays. As an antibody is also a protein, therefore, a radioimmunoassay may be looked upon as a type of competitive protein binding assay. 

The first applications in calibration problems appear to have been in the field of competitive protein binding assays. Marschner (19) have introduced the concept of smoothing... 

Continuous-flow Multi-protein Binding Assays Using Electrospray MS... 

We will synthesize a chemical library of small molecules with maximal structural diversity and a format compatible with protein binding assays. 

Y Ishihama, T Miwa, N Asakawa. Drug-plasma protein binding assay by electrokinetic chromatography-frontal analysis. Electrophoresis 23 951-955... 

Polybrominated Diphenyl Ethers. Results of in vitro estrogen receptor and thyroid hormone transport protein binding assays and in vivo studies of thyroid hormone homeostasis indicate that PBDEs have the potential to disrupt normal endocrine function. 

The Coomassie dye-based protein-binding assays have the advantage of being the fastest and the easiest to perform (Fig. Bl.1.7). In addition, the assay is performed at room temperature and no special equipment, other than a spectrophotometer, is required. Briefly, the sample is added to the ready-to-use reagent and, following a short incubation, the resultant blue color is measured at 595 nm versus deionized water. 

When determining naturally occurring vitamin D in animal products for nutritional evaluation purposes, 25-hydroxyvitamin D3 should be included, because this metabolite contributes significantly to the total biological activity, particularly in milk. 25-Hydroxy vitamin D3 is present in dairy products, eggs, and meat tissues in sufficient concentration to permit its determination by HPLC using an absorbance detector. In bovine milk the concentration of this metabolite is less than 1 ng/ml (63) hence it is usually determined by a competitive protein-binding assay after fractionation of the extracted sample by HPLC (64). 

Table 23 HPLC Methods for Quantitating Biotin in Foods (C18 Columns Detection by Fluorophore-Linked Protein Binding Assay)... 

In a protein-binding assay with fluorescence detection a microarray of biotin, HPYPP-peptide and WSHHPQFEK-peptide was screened against streptavidin-Cy3 and avidin-Cy5. By following the same principle an anti-human insulin monoclonal antibody was also screened against a set of different peptides. 

A protein-binding assay (BA) coupled with hplc provided a highly sensitive post-column reaction detection system for the biologically important molecule biotin and its derivative biocytin, biotin ethylenediamine, 6-(biotinoylamino) caproic acid, and 6-(biotinoylamino)caproic acid hydrazide (71). This detection system is selective for the biotin moiety and responds only to the class of compounds that contain biotin in their molecules. In this assay a conjugate of streptavidin with fluorescamine isothiocyanate (streptavidin—FITC) was employed. Upon binding of the analyte (biotin or biotin derivative) to streptavidin—FITC, an enhancement in fluorescence intensity results. This enhancement in fluorescence intensity can be directly related to the concentration of the analyte and thus serves as the analytical signal. The hplc/BA system is more sensitive and selective than either the BA or hplc alone. With the described system, the detection limits for biotin and biocytin were found to be 97 and 149 pg, respectively. 

Jordan KH, Bruner J, Doan M et al. (2000) Automated Plasma Protein Binding Assay Implementation on a Tecan Genesis 150 and and Zymark RapidPlate. Poster presented at the LRIG SouthEast October 25 2000 Judd RL and Pesce AJ (1982) Free drug concentrations are constant in serial fractions of plasma ultrafiltrates. Clin Chem 28 1726... 

Morris DL, Ledden DJ, Boguslaski RC (1987) Dry-phase technology for immunoassays. J Clin Lab Anal 1 243-249 Moss AJ, Dalrymple GV, Boyd CM (1976) Practical Radioimmunoassay. The C.V. Mosby Company, St. Louis Odell WD, Franchimont P (1983) Principles of Competitive Protein-Binding Assays. John Wiley and Sons, New York Read B (2001) Long-distance runners of the fluorophore family. Amer Biotechnol Lab 44... 

Butterfly Wings as SERS Substrates for Protein-Binding Assaying... 

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