Ouchterlony plates

Figure 8-18. Intact (a) and exploded (b) views of a disposable micro-Ouchterlony plate. The components include, from bottom to top, (1) a base unit containing a small circular channel into which water is placed to preserve a moist atmosphere during incubation (arrow) (2) agarose through which the antigen and antibody diffuse (3) a plastic center-piece containing the sample reservoirs (4) a moisture seal to prevent the cells from drying out during storage and (5) a cap for the entire cell. (Courtesy of Cordis Laboratories, Miami, Fla.)...

Figure 8-19. Concentration of antigen and antibody at various distances from the wells containing these components. The dashed line describes the concentrations observed when a greater concentration of antibody is used. The lower panel depicts two Ouchterlony plates in cross-section showing the location of the immunoprecipitate at the two antibody concentrations used above. Vertical dotted lines mark the point where the antibody-antigen ratio is 4 1.

Figure 8-21- Precipitin band observed when two concentrations of avidin and avidin-immune serum are permitted to diffuse toward one another. Wells A and B contained 15 and 30 fj,g of avidin, respectively, and well C contained the avidin-immune serum. This photograph was taken 2) days after the components were added to the Ouchterlony plate. Incubation was at 4 C.

Fignre 8-24. Immunoelectrophoresis mode of operation (B) and Ouchterlony plate well pattern (A). 

The following experiments will require (1) one 2.5 to 3.5 kg rabbit, (2) a highly purified preparation of avidin, (3) Freund s complete and incomplete adjuvants, (4) disposable Ouchterlony plates, and (5) C-biotin, which can be obtained from Amersham Searle Corp. 

Double Diffusion of Avidin and Avidin-immune Serum in Ouchterlony Plates... 

Place 0.045 ml avidin-immune serum (step 8-9) in the center well of a disposable micro-Ouchterlony plate such as that shown in Figure 8-18. Make certain that the wells are free of water before the solutions are placed in them and also that no bubbles are trapped at the bottom of the wells. This operation may be successfully performed using Lang-Levy pipettes. 

Antitransferrin sera from rabbits reacted differently with iron-saturated and unsaturated transferrin, as assayed by Ouchterlony plates and immunoelectrophoresis (128, 129). The time course of fight scattering for a reaction between bovine anti-conalbumin antibody and free and complexed conalbumin (130) indicates that the response of the anti-conalbumin antibody is higher towards conalbumin than towards its ferric complex. 

In order to investigate the active sites of these proteins, laccases I and III were subjected to ESR (electron spin resonance) spectroscopic analysis. The ESR spectra shown in Figure 5 indicate clear differences in peaks 2 and 6 which support the concept that the copper atoms in laccases I and III have different conformations in each molecule. Furthermore, immunological similarity between laccases I and III was also investigated. Antibody specific for laccase III was prepared from rabbit serum by conventional methods. When applied to Ouchterlony diffusion plates containing laccase I, no precipitation lines developed (Figure 6). This result showed that there were no conserved epitopes on the surfaces laccases I and III. 

J. G. Feinberg, Int. Arch. Allergy, 11 129 (1957). Identification, Discrimination and Quantification in Ouchterlony Gel Plates. 

W4. Wilson, M. W., and Pringle, B. H., Experimental studies of the agar-plate precipitin test of Ouchterlony. J. Immunol. 73, 232 (1954). 

Fig, 2. Ouchterlony radial immunodiffusion assay of sera and tissues from intact and estrogen-treated female sirtalis. Center well assay A rabbit 337 anti-vitellogenin. Center well assay B vitellogenin, 100 mg/ml. Wells 1 and 4 both assays estrogen-treated female serum. Well 2 both assays untreated female serum. Well 3 both assays dermal tissue from untreated female. Well 5 both assays dermal tissue from untreated female, treated in vitro with estradiol in PBS. Well 6 both assays dermal tissue from the same untreated female as the serum in well 2 and the tissue in well 5, treated as in well 5 but without estradion. Drawn from a plate in Garstka 0-982). 

Gel Diffusion Methods. Many types of gel reactions have been used in the detection of the enterotoxins, the most common ones being some form of either the Ouchterlony gel plate or the microslide. These methods have been used widely in the determination of the enterotoxigenicity of staphylococcal strains. The modification of the Ouchterlony gel plate test that is used in the Food Research Institute and recommended to others is the optimum sensitivity plate (OSP) method (Robbins et al., 1974 Fig. 2). It is easy to use and in conjunction with production of the enterotoxins by the membrane-over-agar or sac culture methods (Robbins et al.,1974) is of adequate sensitivity for detection of most enterotoxigenic... 

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