Dextran coated charcoal

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). 

Quickly add 0.5 ml of cold dextran-coated charcoal suspension to tubes 3 through 18 and to all patient sample tubes so as to get rid of free cortisol. Do not add to tubes 1 and 2,... 

The ice-cold dextran-coated-charcoal suspension (0.1 ml) is added to all the above tubes, followed by immediate mixing and incubation for 10 minutes at 4°C,... 

Adsorption of free fraction Dextran-coated charcoal... 

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

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. 

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. 

Serum also contains steroid hormones and, in experiments where these are the object of study, they can be removed by treatment of the serum with dextran-coated charcoal, as follows -... 

Figure 10.7 Illustration of dextran-coated charcoal method for separating bound and unbound antigen.

In a radioimmunoassay, dextran-coated charcoal is primarily used to ... 

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.

To develop charcoal dose-response curves, we suggest routinely using dextran-coated charcoal because it is stickier than uncoated charcoal, thus forming a distinct and firm pellet on centrifugation. Which size dex-tran is used to coat is unimportant, but once selected, continue to use Se-phadex of that size. The charcoal is placed in buffer (10 1, v/w) (10 xnM... 

In hquid phase adsorption, the free antigen is adsorbed onto particles of activated charcoal or dextran-coated charcoal that are added directly to the reaction mixture. The particles of charcoal and the adsorbed antigen are then removed by allowing them to settle or by centrifugation. The disadvantage of this separation technique is that the time of contact between the adsorbent and the incubation mixture is critical, especially for the more active adsorbents, such as activated charcoal. 

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. 

PEG, polyethylene glycol AS, ammonium sulfate SP, solid phase DC, dextran-coated charcoal. 

N6. Nugent, C. A., and Mayes, D. M. (with technical assistance of S. Morrett) Plasma corticosteroids determined by use of corticosteroid-binding globulin and dextran-coated charcoal. J. Clin. Endocrinol. Metab. 26, 1116-1122 (1966). 

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. 

Al. Anderson, K. M., Bonomi, P., Marogil, M., Hendrickson, G., and Economou, S., Comparison of dextran-coated charcoal and sucrose density gradient analyses of estrogen and progesterone receptors in human breast cancer. Cancer Res. 40, 4127-4132 (1980). 

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). 

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