Competitive protein-binding assay

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... 

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). 

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... 

Charcoal is widely used to separate antibody-bound ligand (bound) from non-antibody-bound ligand (free) in competitive protein binding assays. Its use was originally described by Miller for vitamin Bi assay and later by Herbert et al. for B12. In 1965, Herbert suggested its use to separate bound and free in the radioimmunoassay of insulin. Since then it has been used for a large number of radioimmunoassays and radioreceptor assays. 

Odell, W.D. Daughaday, W.H. Principles of Competitive Protein Binding Assays Lippincott Philadelphia 1971. Christian, G.D. Clinical chemistry. Analytical Chemistry John Wiley and Sons Inc. New York, 1994 611-628. Ullman, E.E., Langen, J., Clapp, J.J., Eds. Liquid Assay Analysis of International Development on Isotopic and Non-Isotopic Immunoassay Masson New York, 1981 113. Sharma, A. Schulman, S.G. Fluorescence analytical methods and their applications. Introduction to Fluorescence Spectroscopy, WHey-lntexsdeace.l ew York, 1999 123-158. 

Campfield, L.A. Mathematical analysis of competitive protein binding assays. In Principles of Competitive Protein Binding Assays, 2nd Ed. Odell, W., Franchimont, P., Eds. John Wiley Sons New York, 1983 125-148. 

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). 

Competitive Protein-Binding Assays with Vitamin D-Binding Protein. Before the development of immunoassays, 25(OH)D was measured primarily with CPBA with DBP as the specific binder, and with tritiated 25(OH)D3 (>100 Ci/mmol) as tracer. Rat serum, diluted approximately... 

Horst RL, Reinhardt TA, Beitz DC, Littledike ET. A sensitive competitive protein binding assay for vitamin D m plasma. Steroids 1981 37 581-91. 

Jones G, Byrnes B, Palma F, Segev D, Mazur Y. Displacement potency of vitamin Dj analogs in competitive protein-binding assays for 25-hydroxyvitamin Ds, 24,25-dihydroxyvitamin D3 and 1,25-dihydroxyvita-min D3. J Clin Endocrinol Metab 1980 50 773-5. 

In laboratories which use an NSILA bioassay, however, somatomedin activity has been expressed in units of insulinlike activity, generally p,U/ml, a convention which has to some extent continued as radioligand assays were introduced. Expressed in these units, normal human samples have a mean value of 350 66 pU/ml by competitive protein-binding assay (Z4). In the same laboratory, a mean normal value ( SD) of 148 45 ng/ml was determined by IGF-I RIA (Z5). The close correspondence between reference values obtained by RIA in different laboratories, when expressed as ng/ml of SM-C or IGF-I, suggests that mass units, if uniformly adopted, would allow better comparison of results. With the increasing availability of highly pu-... 

Schalch, D. S., Heinridt, U. E., Koch, J. C., Johnson, C. J., and Schlueter, R. J., Non-suppressible insulin-like activity of human serum. 1. Development of a new sensitive competitive protein-binding assay. /. Clin. Endocrinol. Meted). 46, 664-671 (1978). 

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). 

Shaw, W., Slade, B. A., Harrison, J. W., and Nino, V., Assay of serum folate Difference in serum folate values obtained by L. casei bioassay and competitive protein binding assay. Clin. Biochem. (CHtawa) 7, 165-178 (1973). 

GE Abraham, PK Grover. Covalent linkage of hormonal haptens to protein carriers for use in radioimmunoassay. In WD Odell, WH Daughaday, eds. Principle of Competitive Protein-Binding Assays. Philadelphia Lippincott, 1974, p 134. 

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