Radial diffusion assay

We will try to test ourselves whether free-ranging (or captive) squirrels discriminate between acorns that have been buried for several weeks and those that have not. In a later laboratory experiment we will analyze tannin levels in the apical and basal poles of acorns, and also compare buried acorns with untreated controls in this regard. We will use the Radial Diffusion Assay for Tannins (Chap. 14). 

Gallinacins, a group of mammalian antifungal peptides. They contain three intramolecular disulfide bonds, are relatively cationic, and are rich in Lys and Arg. It has been reported that gallinadn-l and -la inhibit C. albicans in a radial diffusion assay, whereas gallinacin-2 was not active at up to 400 /xg mL in this assay [M. K. Harwig et al, FEBS Lett. 1994, 342, 281]. 

A separate analysis of the measured response from the reference samples (e.g., if a radial diffusion assay, the ring diameter or if an ELISA assay, the absorbance or if an HPLC assay, the peak height) will indicate the between-assay variation. The analysis of duphcate samples of the reference samples will indicate the within-assay variation. 

Lawrence and Sanderson proposed another micro-method for measuring chymosin and other proteolytic enzymes. Measurement of concentration was based on the rate of radial diffusion of the enzyme through a thin layer of caseinate-agar gel. The limit of diffusion was marked by a zone of precipitated casein (Emstrom and Wong 1974). Holmes et al (1977) developed a microdiffusion assay for residual proteolytic enzymes in curd and whey that is more sensitive than the method of Lawrence and Sanderson or the clotting-time assay of Reyes (1971). 

A number of immunochemical techniques have been used to quantify analytes of clinical interest. They include radial diffusion (RID) and electroimmunoassays, turbidimetric and nephelometric assays, and labeled immunochemical assays. 

Antibiotic diffusion assays are based on the technique of allowing an antibiotic to diffuse throu an agar gel which has been previously seeded with a sensitive test organism. This diffusion may be of two types (a) linear diffusion, i. e., by bringing the antibiotic in contact with a column of seeded agar in a capillary or test tube and (b) radial diffusion around a suitable reservoir on a seeded agar plate. 

Diffusion systems are based upon the ability of the antibiotic to diffuse through agar and cause the inhibition of the sensitive assay strains. Since the substrate to be assayed is applied in a "point source," diffusion occurs radially. A circular zone of inhibition forms and the size of the zone is a function of the concentration. This function is expressed as a linear relationship between the size of the zone of inhibition and the logarithm of the concentration. 

Immunochemical methods are also used for clinical assays because they are rapid and easily automated. Because of the differences in molecular size and corresponding diffusion rates, gel diffusion techniques, such as radial immunodiffusion (RID) require correction for phenotype and are therefore time consuming and inconvenient. Immunoassays in solution, such as nephelometry and turbidimetry, are influenced slightly by size as well, but the differences are relatively insignificant. 

Figure 17. The effects of tissue attenuation on fluorescence measurements, (a) graph showing measured fluorescence versus Photofrin concentration in different tissues (after Panjehpour et al., 1993 [12]), (b-d) correction for attenuation by the ratio of fluorescence to diffuse reflectance at two different radial distances (b) principle, (c) prototype instrument, (d) photosensitizer concentration measured in vivo versus known concentration by assay on tissues ex vivo for three different tissue types (after Weersink et al., 1997 [13]).

Techniques for the detection or assay of various substances based on the reaction of those substances with specific antibodies (or vice versa, i.e. the detection and assay of antibodies using antigens). Such techniques include agglutination reactions, automated immune precipitation, complement fixation tests, crossed electrophoresis, counter electrophoresis, double diffusion, enzyme immunoassay, fluoroimmunoassay, haem-agglutination, immunoelectrophoresis, immunofluoresence, radial immunodiffusion, spin immunoassay, immunofixation, immunoradiometric assay and radioimmunoassay. See separate entries for these subjects. 

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