Ouchterlony diffusion

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. 

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). 

AM-001, and mannanase B properties are similar to those of P-mannanase M-III. Furthermore, the Ouchterlony double diffusion test showed that these five enzymes gave fused precipitation lines. However, N-terminal amino acid sequences of the five mannanases determined by an automatic amino acid sequencer revealed that the N-terminal amino acid sequence from amino acid 1 (Asn) to 9 (Gin) of the Bacillus sp. AM-001 enzymes coincides with those from amino acid 4 (Asn) to 12 (Asn) of the R coll JMlOl (pMAH3) enzymes as shown in Fig. 4. This may reflect differences in the specificities of the signal peptidases of the two bacteria. 

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.

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. 

In the Ouchterlony method, the double diffusion experiment is carried out with a central well containing the antibody or antibody mixture, surrounded by a circular... 

The most often used procedures for evaluating the specificity of antibody preparations are double diffusion methods developed by Ouchterlony and immunoelectrophoretic methods developed by Grabor and Williams. For a discussion of variations of these techniques as well as other useful procedures the reader is referred elsewhere (7-9). 

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 8-21- 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.

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

In 3 cases of Barta and Tichy (B3) with protein leakage into the gastric juice, serum proteins were identified by the Ouchterlony method. Hollander and Horowitz (H13), using this diffusion technique in canine gastric juice, demonstrated presence of serum albumin in canine gastric juice. 

Ouchterlony, 0., Interpretation of comparative immune precipitation patterns obtained by diffusion-in-gel techniques. In Immunochemical Approaches to Problems in Microbiology (M. Heidelberger and 0. J. Plescia, eds.), pp. 5-19. Rutgers Univ. Press, New Brunswick, New Jersey, 1960. 

Highly purified material gives a single line of identity by Ouchterlony gel diffusion (U2). Unabsorbed h-TSH antibody contains binding sites for gonadotropins and luteinizing hormone (02). Purified h-TSH is separated into several bands by gel electrophoresis (C7), some of which have activity. Human TSH is found just behind albumin when blood is filtered on a Sephadex G-200 column (Ul). Human TSH can also be separated from LATS by gel filtration (M5). 

Ouchterlony, O., Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diffusion. Acta Pathol. Microbiol. Scand. 32, 231 (1953). 

Using Ouchterlony gel double diffusion, no precipitating antibodies against monomeric HSA solution were found in sera of patients with anaphylactoid reactions to HSA (Ring et al. 1979). 

FIGURE 3A and B. Ouchterlony double diffusion assay of a serial dilution of a polysaccharide preparation (A) or soluble starch (B). Central hole antibody. 

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