Ouchterlony double diffusion

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.

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

Kolattukudy, 1975b). (2) Ouchterlony double-diffusion analysis showed that the nonspecific esterase from F. solani pisi cross-reacted with rabbit anticu-tinase I, and immunoelectrophoresis showed that this cross-reactivity was not due to contamination of the nonspecific esterase by cutinase (C. L. Soli-day, T. S. Lin, and P. E. Kolattukudy, unpublished). (3) The amino acid composition of the esterase was fairly similar to that of cutinase I (Purdy and Kolattukudy, 1975a). (4) The nonspecific esterase, which has twice the MW of cutinase, contains about 50% carbohydrate, and therefore it appears that the nonspecific esterase might be cutinase with an equal mass of carbohydrate attached to it (Lin and Kolattukudy, unpublished). (5) In both cutinase and the nonspecific esterase, the linkages between the carbohydrates and the protein are similar in both cases the same carbohydrates are attached glyco-... 

Fig. 5. Ouchterlony double diffusion test. Centre well rabbit anti-transketolase serum. Outer wells (1) apotransketolase (0.25 mg ml ), (3) holotransketolase (0.25 mg ml i).

The technique exploits the specificity of reaction between an antigen and antibody and molecular sieving of the gel in which this reaction is taking place for analysis of components of a given sample. The technique was developed by Grabar and Williams and is actually a modification of Ouchterlony double diffusion technique. To understand the technique better a brief discussion of Ouchterlony double diffusion technique is given below. 

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). 

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

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). 

In the agar gel double diffusion test a band with two precipitin lines appeared when normal hemolysate reacted with antiserum (Fig. 1). Two precipitin lines were formed when we used the patients hemolysates in the Ouchterlony test (Fig. 1) Inspite of the deficiency of enzyme activities in the erythrocytes of our patients these precipitin lines showed a complete identity with the lines of the hemolysate from the healthy control person. 

After double diffusion according to Ouchterlony of spinach and maize CF or spinach and maize thylakoids against an antiserum against spinach CF (150) or against maize CF (141), the pattern of only partial identity was seen (spurs). By absorption of the respective antisera with heterologous thylakoids we removed all crossreacting ab and thus produced sera, where only the species-specific non-crossreacting ab had remained (Pucheu,Berzborn, in preparation). 

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