Coated Charcoal Assay

The dextran-coated charcoal (DCC) assay measures the hormone-binding capacity of the cytosol fraction of the tumor tissue (Lee and Chan, 1994). Fresh tissue (more than 100 mg) is immediately frozen, homogenized, and the cytosol fraction is extracted. Aliquots of the cytosol are incubated with radiolabeled [3H] estradiol with and without 100-fold molar excess of nonradiolabeled competitor (diethylstilbestrol) to displace radiolabeled steroid from the low-capacity specific binding sites (ER), but not from the high-capacity nonspecific binding sites in the cytosol. The unbound steroid is removed by selective adsorption on DCC. 

The receptor binding capacity, reported in femtomoles of radiolabeled steroid bound per milligram of cytosol protein, is calculated by subtracting the level of nonspecific cytosol-bound radioligand (radiolabeled steroid with excess of competitor) from the total cytosol-bound radioligand (radiolabeled steroid without competitor). 

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In situ hybridization overcomes the problem of cellular localization, but it is difficult to relate the expression of a particular mRNA to the expression of the functional protein. Moreover, this method is difficult to carry out. Immunohistochemistry, on the other hand, is a relatively simple technique that overcomes these problems by identifying the precise cellular localization of the functional protein. This technique, using paraffin sections, provides information on the ER status of tumors very simply and rapidly. In addition, this approach is superior to frozen section immunohistochemistry, the dextran-coated charcoal assay (DCC) (see page 276), or the enzyme-linked immunosorbent assay (ELISA) for predicting the response to endocrine therapy. 

Menendez-Botet, C. J., Stankievic, R., Schwartz, D., and Schwartz, M. K., Comparison of the quantitative measurement of human estrogen and progesterone receptor in tissue cytosol by the dextran-coated charcoal assay and the Abbott monoclonal enzyme immunoassay. Clin. Chem. (Winston-Salem, N.C.) 34(6), 1225 (1988). 

Since both of these compounds are very large they cannot enter the pores, thus the pores are still free to hold small molecules. When the coated charcoal is added to the assay reagent mixture, the free (unbound) material is adsorbed while the larger bound fraction is not. Final separation is accomplished by centrifugation and decantation of the supernatant. 

Adrenal Tumours The assay-method is entirely based on the Schwartz-Mann Kit. According to this method, cortisol is first extracted from the plasma using CH2C12 (methylene chloride). In the actual radioimmunoassay the cortisol present in the extract competes with Cortisol-H3 i.e., the radioactive tracer) for the common binding sites on transcortin, which is incidently not an antibody but a cortisol-binding protein. Now, the free cortisol is quantitatively removed by adsorption on dextran-coated charcoal from the one bound to the transcortin. Finally, the bound radioactivity (due to Cortisol-H3) is measured which is then employed to calculate exactly the amount of cortisol present in the sample by the help of a Standard Curve (or Calibration Curve). 

Biochemical assays such as the dextran-coated charcoal (DCC) assay, certain signal amplification techniques, and other cytosol-based methods have been mostly replaced... 

Fig. 1. Dose-response curve for amount of dextran-coated charcoal added versus percentage free and bound, adsorbed. These are theoretical curves drawn from generalizations derived from several assays. —, Curve showing binding of free ligand —, curve showing binding of antibody bound ligand. The exact amounts of charcoal giving a particular bound value vary with each assay (e.g., growth hormone assay differs from insulin assay). For significance of arrows A and B, see the section on selection of charcoal dose.

The classic quantitative biochemical method for assaying steroid receptors in tumor tissue specimens is the multiple-point dextran-coated charcoal (DCC) titration assay. However, in comparison with the classic DCC assays, enzyme immunoassays are preferred as they cost less and are simpler, require less time, and can be performed using less tissue than DCC titration assays. 

Waxman, Schreiber, and Herbert first described a radioisotopic assay for the measurement of serum folate in 1971 (W14) which they claimed gave almost identical results to those obtained by with the L. casei microbiological assay and could separate low, borderline, and normal folate levels. Serum was mixed with PHjmethyltetrahydrofolic acid in a phosphate buffer and a folate binder was added which, in this case, was Carnation brand instant powdered milk. Following incubation, free vitamin was separated from that which was bound using hemoglobin-coated charcoal. The radioactivity of the supernatant was determined, and from this the folate concentration in the serum sample could be calculated using appropriate standards and blanks. 

Several investigators have utilized fine needle aspirates for hormone receptor evaluation using an immunocytochemical method (ERICA Abbott Laboratories, North Chicago, IL) and compared the data with the quantitative values obtained by chemical methods [dextran-coated charcoal (DCC)] for both ERP and PRP. These data are shown in Table 1. There was a variation of 65 to 100% in concordance in the separate studies. The ERP immunocytochemical assay in fine needle aspirates was also compared with the immunochemical assay in biopsies with a concordance of 87% (Rl). The role of the immunocytochemical assay of receptors is primarily in patients in whom surgical biopsies cannot be obtained or fi om whom the surgical specimen is too small. 

Raven, J.L., Robson, M.B., Walker, P.L., and Barkhan, P., 1968. The elfect of cyanide, serum and other factors on the assay of vitamin B12 by a radioisotope method using CoB12, intrinsic factor and coated charcoal. Guy s Hospital Reports. 117 89-109. 

Properties.—Massive, sometimes fibrous and radiated like stibnite also finely granular. Before the blowpipe, on charcoal, a yellow coating of Bi O, near the assay, and, beyond, white Sb Oj. 

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