Dextran-coated charcoal method

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

T3. Thorpe. S. M., Monoclonal antibody technique for detection of estrogen receptors in human breast cancer Greater sensitivity and more accurate classification of receptor status than the dextran-coated charcoal method. Cancer Res. 47, 6572-6575 (1987). 

Figure 13.22 The dextran coated charcoal method for sqt( ratlr bound radioactivity from unbound radioactive antigen.

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... 

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 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. 

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. 

We have used a method that depends upon (1) the production of high-aflBnity antisera in rabbits injected with Tg-bovine serum conjugate, (2) Merthiolate (final dilution 1 1,000) to inhibit TBG activity, (3) AG AB reaction at 4°C, (4) dextran-coated charcoal to achieve separation of AB-bound and free AG, and (5) the addition of Tg-free serum... 

Nonspecific absorption method (e.g., dextran-coated charcoal, hydroxyapatite)... 

These methods have been widely used. Isotopic dilution methods are based on the dilution of the biotin to be estimated by a known amount of labeled biotin, followed by the determination of the specific activity of the avidin complexed or the free biotin after thorough separation of both species. [ ClBiotin (78,79), [ Hjbiotin (80), and I-labeled derivatives (Fig. 12) of biotin (81) have been used. The separation of free and avidin-complexed biotin has been achieved with bentonite (80), zinc hydroxide (78), dextran-coated charcoal (79), polyethyleneglycol (82), antibodies (83) or by the use of covalently linked avidin on cellulose (84). The sensitivity depends on the specific activity of the labeled biotin and can range from 20 ng with [ Cjbiotin, to 1 ng with [ Hjbiotin, down to 1 pg with I-labeled derivatives. 

Separation for measurement The basis of measurement depends on the ability to separate the unbound or free" Ag from the Ab-Ag complex, which itself relies on differences in properties between the two components while maintaining (that is not disrupting) the Ab-Ag complex (Figure 10.6). There are a number of reagents used for separation including charcoal, hydroxyapatite, ammonium sulfate and polyethylene glycol, but the method that will be considered here relies on the use of dextran-coated activated charcoal (see Figure 10.7). With inherent... 

How is Ab-Ag complex separated from Ag Two methods are mainly followed. Th are the dextran-coated acttvated charcoal method, and the double-antibody method. The first one is given below. 

Dextran-coated activated charcoal method. You must have learnt from your chemistry classes that activated charcoal can adsorb many small molecules. Even large mcdecules like large proteins and even larger complexes like the Ab-Ag complex may bind. However, the binding for the larger molecules is considerably slower. 

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