Gel diffusion techniques

Demonstration of circulating antibodies to Plasmodium falciparum by gel diffusion techniques. Nature London) 210, 1384-1386 (1966). 

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. 

F6. Finger, I., Use of simple gel diffusion techniques to assign antigenic markers to native proteins. Nature 208, 1035-1039 (1964). 

Immunological methods Methods such as gel-agglutination tests were introduced many years ago (according to Outcherlony) however they are not sufficiently sensitive. Reversed passive latex agglutination kits (SET-RPLA and TST-RPLA) are presently applied for enterotoxin detection and are more sensitive than gel diffusion (Wieneke, 1988). The ELISA technique is also very popular - toxins are detected via VIDAS STAPH ENTEROTOXIN SET and indirect double sandwich ELISA (Meyrand et al., 1998). 

Fig. 2. Ouchterlony double-diffusion technique. The antigen is placed in the center well, cut in an agarose gel, and different antisera in a range of dilutions are placed in the sunounding wells. Antigen and antiserum diffuse toward each other and form a white precipitin line where an antibody recognizes the antigen.

This can be done by the Ouchterlony diffusion technique (see Fig. 2 and ref. 6). by enzyme-linked immunosorbent assay (see Chapter 15), or by Western blot, either using the purified protein or a more complex mixture of proteins containing the antigen of interest separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (see Chapter 20). 

By utilizing the Ouchterlony technique of two-dimensional, agar-gel diffusion, Slodki and his colleagues investigated the interaction of con A with O-phosphonomannans.363 They suggested a possible correlation between the extent of precipitate formation with con A and the content of a-D-(l— 2)-mannosidic linkages. 

These additives can also be placed into microcapsules with a thin polymer skin. For example, they are filled with natural aromas and applied to the textile from a water dispersion in combination with a polyurethane or silicone binder. The ratio of micro bubbles to binder determines the efficiency and permanence of the finish. Good results after 4-12 washing cycles are reported. The encapsulated materials are released during wearing as the micro bubbles burst from rubbing caused by body movement or by diffusion through the thin layer of the capsules. A market available encapsulation in micro bubbles, built from chitosan, is described by Hampe. The incorporation and controlled release of fragrance compounds is also provided by the sol-gel nano-technique described in Section 18.4. 

Fig. 3. A-H are examples in double diffusion in two dimensions of shape and position of a various number of reservoirs in a gel plate. Other examples may be found in Oudin, L analyse immunochimique par la methode des gels in Techniques de laboratoire (Loiseleur, secretaire de redaction), p. 1415. Masson, Paris, 1963, and in the references given therein.

Changes in temperature, which cause, in diffusion techniques, changes in the number of collisions between molecules of antigen and antibody and, therefore, in the density of the precipitate in a very thin gel layer. These artifacts differ from the mobile precipitation zones not only in their appearance (thinness) but also in their necessary immobility. For the latter reason, they do not occur when the precipitation zone itself is immobile but mainly when its displacement is fairly rapid, as is frequently the case in simple diffusion (see below). 

Leakage of a reagent between the gel and the glass wall can greatly disturb the reaction. A technical precaution to prevent such an accident with agar gels is always used in the one dimension diffusion techniques and has been described above. 

Five to seven days after the last injection, check the antibody production using the double diffusion technique in 1 % agar gel as described below. 

Tg antibodies are directed against the Tg protein, a major constituent of thyroid colloid. Several different techniques have been used to detect and quantify TgAb in peripheral blood. These include passive hemagglutination, the agar gel diffusion precipitin technique, immunofluorescence of tissue sections, enzyme-linked immunosorbent assay (ELISA), radioassay techniques, and chemiluminescence-based immunometric assays. 

Arnaud and Littledyke (A5) and Hargis et al. (HI) overcame the problem of antigenicity by conjugating relatively pure porcine thyro-calcitonin to rabbit albumin before injection, and claim that the antisera obtained were specific to thyrocalcitonin, judged by gel diffusion and immunoelectrophoresis results. The former workers have briefly described the use of this antiserum in a radioimmunoassay procedure similar to that devised by Berson et al. for parathormone (B4). They claim that the technique will detect 0.5 ng of thyrocalcitonin/ml of serum. These workers found no difference in the immunological behavior of human and porcine hormone, and quote a normal range of 30-85 ng/ml in human plasma. In animal studies, elevated values were found in older animals and in response to hypercalcemia. 

A requirement for making YAG crystals is a homogeneous, high-purity starting material for use in the Czochralski technique. This means that the application of mechanical mixing and solid-state diffusion techniques are limited. Accordingly, a variety of synthesis techniques have been developed, most of which are sol-gel based. 

Highly purified h-TSH and b-TSH showed no cross reactivity by gel diffusion methods and radioimmunological precipitation techniques (U2). However, in several bioassay procedures limited cross reactivity has been shown between anti-b-TSH antibodies (L4, M9, U2) and human TSH. A radioimmune assay described by Lemarchand-Beraud (L2) is based on the use of anti-b-TSH antibodies developed in rabbits. Recently cross reactivity has been reported between h-TSH and antibodies to porcine TSH (F9). Radioimmunoassay procedures were used in these studies. It would appear that there may be two groups of antigenic sites on human TSH, one specific and one shared in common with b-TSH and p-TSH. 

Macroporous gels Another technique to obtain fast-responsive hydrogels is to create voids (pores) inside the hydrogel matrix, so that the response rate becomes a function of the microstructure rather than the size or the shape of the gel samples (Okay 2000). For a polymer network having an interconnected pore structure, absorption or desorption of water occurs through the pores by convection, which is much faster than the diffusion process that dominates the non-porous hydrogels. 

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