Precipitin Zones Research Articles

Modification of Oudin's method of single immunodiffusion in agar gel for detection of IgA in bronchoalveolar lavage of white mice.

Oudin's principle of single immunodiffusion in agar gel was modified for quantitative determination of IgA in bronchoalveolar lavage (BAL) of normal 20-25 g mice. The reaction took place at 25 degrees C in 0.3% agarose with 16.7% pig serum against mouse IgA, and was evaluated on the basis of a relationship between the progress of the precipitin zone and the square root of time. The linear dependence of the derived constant k on the logarithmic concentration of antibody in the sample permitted to express the results as titre, corresponding to a dilution where k = 0. Examination of seven samples of pooled blood serum of normal mice showed that (1) the IgA level was practically constant, (2) serum IgA possessed under given conditions similar properties as IgA from the bronchoalveolar secretion; it is therefore possible to employ pooled sera as a reliable control of the immunodiffusion system in case of lack of reference standards with defined IgA content. Examination of 82 individual BAL samples of normal mice revealed that the mean IgA concentration in 2.5 mL samples was almost 1000 times lower than in blood serum.

IMMUNODIFFUSION METHOD FOR DETECTION OF CLOSTRIDIUM BOTULINUM TYPES, A, B, E, AND F

Factors affecting the immuno‐gel diffusion method for detecting toxigenic (tox+) C. botulinum type A and nontoxigenic (tax‐) C. sporogenes were studied. This procedure was extended to detect types B, E, and F using homologous and poly A‐F antitoxins for proper tox‐ types. Increasing glucose levels from 0 to 3% in the growth medium caused larger and more intense precipitin zones around colonies of C. botulinum type A. Precipitin zones were detected in TPGYA that contained no glucose, but better zones occurred at 4% and thereafter up to 7% glucose at pH 7.6. The most favorable titers of C. botulinum antitoxins incorporated either in gel‐diffusion agar (GDA) or in growth medium varied with the C. botulinum type. The method differentiates between C. botulinum types A, B, E, F and C. sporogenes.

Immunodiffusion method for detection of type A Clostridium botulinum.

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.

Quantitative immunofixation of proteins following zone electrophoresis in agarose gel: Application to the determination of the stoichiometry of the α1-antitrypsin-elastase interaction

A method for the quantitative determination of proteins by immunofixation-agarose gel electrophoresis is described. The proteins are separated electrophoretically and the resulting circular zones overlaid with specific antibody-impregnated filter paper (10.2 μl/cm 2). Following incubation for 20 h at room temperature the plates were processed, stained and the areas of the precipitin zones determined by planimetry. Intra-plate variation (coefficient of variation) for α 1-antitrypsin (30 samples) and elastase (30 samples) at concentrations of 0.25, 0.5 and 1.0 mg/ml was 2.2–5.8% and inter-plate variation ranged from 2.9 to 5.2%. Overall, the analyses showed that the method is the statistical equivalent of rocket immunoelectrophoresis. The lowest concentration used was 0.25 mg/ml which corresponds to a sensitivity of 1.25 μg (5 μl per sample well). It is conceivable that lower amounts could be successfully determined. Application of the method to the determination of the stoichiometry of the α 1-antitrypsin-elastase interaction permitted the simultaneous quantitative determination of inactivated α 1-antitrypsin (an acidic protein) and excess elastase (a basis protein) in α 1-antitrypsin-elastase reaction mixtures (molar ratio elastase/ α 1-antitrypsin = 1.3) and thus by difference, the amount of elastase and α 1-antitrypsin in the α 1-antitrypsin-elastase complex. The results showed that the molar combining ratio of inhibitor to enzyme was 1.086 : 1 or 1 : 1. Conventional inhibition experiments (residual elastase activity as a function of increasing amounts of α 1-antitrypsin) showed this ratio to be 1.79 : 1 but 1.03 : 1 when the inactivation reaction, as determined by quantitative immunofixation, was taken into account. The quantitative immunofixation method should be applicable to interacting systems which, like elastase and α 1-antitrypsin, result in complexes with electrophoretic mobilities intermediate to the parent proteins.

Radial immunodiffusion quantitation of proteins by an antibody overlay technique

An economical alternative to the determination of proteins by single radial diffusion in antibody-containing agarose gel is described. The method, which is the equivalent of the above, consists of allowing the sample to diffuse into the gel layer, overlaying of the sample well with antibody-impregnated filter paper (10.2 μl/cm 2), incubation in a moist chamber for 20 hr, determination of precipitin zone size, and plotting concentration vs precipitin zone size. The plot was a straight line equivalent to that obtained by Fahey and McKelvey ( J. Immunol. 94, 84–90, 1965). The average coefficient of variation was 1.6%.

Comparison of procedures for measurement of IgE protein in serum and secretions

Because of conflicting reports in the literature concerning the value of various procedures for measurement of IgE in serum and secretions, we compared four different methods, the radioimmunosorbent test (RIST), the double-antibody radioimmunoassay (RIA), the paper disc immunosorbent test (PRIST), and radial immunodiffusion (RID). The standards used in the assays were tested initially in the double-antibody RIA with the use of the reference from the World Health Organization. The results showed that RID as expected was relatively insensitive and IgE was reliably measured only above approximately 1,000 international units (IU). Moreover, certain sera containing low levels of IgE by the other procedures gave distinct precipitin zones and presumably falsely high levels of IgE protein. Thus RID may yield apparently erroneous results when used as a screening procedure for measurement of IgE levels. Among the other procedures PRIST and the double-antibody RIA showed the best agreement. With serum samples RIST yielded values for IgE in the low level range higher than those given by the PRIST and double-antibody RIA. With breast milk and colostrum, values of IgE between 120 and 690 ng/ml were found by RIST, whereas IgE was not detected by double-antibody RIA and PRIST. No evidence of an inhibitor of IgE was found in breast milk, so that the apparent elevation of IgE in breast milk by the RIST is likely false. These findings confirm prior reports of spurious elevations of IgE with the RIST and indicate the usefulness of the PRIST and double-antibody RIA for the measurement of IgE in sera and secretions.

Relationship between staphylococcal antiserum titer and zone development on immune serum plates.

A workable relationship was established between the standard serum titers of staphylococcal immune antisera and the development of precipitin zones on serum agar around colonies of staphylococcal strains producing homologous antigens (enterotoxins). The standard titer of a serum is defined as the reciprocal of that serum dilution which, with 10 mug of pure enterotoxin per ml, will give a precipitin zone 10 mm in length in single gel-diffusion tubes after 7 days of incubation at 25 C. A numerical scale was set up for determining the intensity of precipitin zones on serum agar. A reading of 3 was considered optimum. This correlated well with a standard serum titer of 25, when 1 ml of such a serum was used per 20 ml of medium per serum plate. From this relationship, the minimum volume of serum required to give optimum precipitin zone development can be calculated.

Relationship Between Staphylococcal Antiserum Titer and Zone Development on Immune Serum Plates 1

A workable relationship was established between the standard serum titers of staphylococcal immune antisera and the development of precipitin zones on serum agar around colonies of staphylococcal strains producing homologous antigens (enterotoxins). The standard titer of a serum is defined as the reciprocal of that serum dilution which, with 10 mug of pure enterotoxin per ml, will give a precipitin zone 10 mm in length in single gel-diffusion tubes after 7 days of incubation at 25 C. A numerical scale was set up for determining the intensity of precipitin zones on serum agar. A reading of 3 was considered optimum. This correlated well with a standard serum titer of 25, when 1 ml of such a serum was used per 20 ml of medium per serum plate. From this relationship, the minimum volume of serum required to give optimum precipitin zone development can be calculated.

A versatile method of radial immunodiffusion assay employing microquantities of antiserum

A method of radial immunodiffusion assay is described. Antiserum is layered on 10 mm diameter discs of agarose gel and enters into the gel by diffusion. Antigen solution is deposited in a central well. Precise measurements of the microquantities of the reagents are obtained with a semiautomatic distributor, which is described. The diameters of the radial precipitin zones formed after 48 hours of diffusion are compared with calibration curves obtained from standard preparations. This method is precise, reproducible, and versatile and consumes only microquantities of antiserum.

Behavior of belladonna mottle virus in serological tests in the presence of organic mercury compounds

The behavior of three isolates of belladonna mottle virus in various serological tests in the presence of Cialit, Merthiolate, and p-Chloromercuribenzoate was studied. All three mercury compounds changed the electrophoretic mobility of the virus in agarose gel. In the Ouchterlony test Merthiolate and Cialit inhibited precipitin line formation. The inhibition by Cialit was reversed at concentrations above 70 μ M. In the presence of crude plant saps or other protein-containing fluids, higher concentrations of mercurials were required for inhibition and reversal of inhibition. In single diffusion tests precipitin zone formation was inhibited only when the virus was the mobile reactant. p-Chloromercuribenzoate did not inhibit in diffusion tests, and none of the three mercurials inhibited precipitate formation in slide precipitin tests.

Quantitative determination of soluble cellular proteins by radial diffusion in agar gels containing antibodies

Radial diffusion of protein antigens through gels containing antibodies has been used to quantitate proteins derived from cellular origins. For quantitation, the diameters of radial precipitin zones formed by tissue extracts were compared with zones formed by known concentrations of the standard proteins. The method employs immunological features of specific proteins to measure concentrations within the range of sensitivity usually obtained only with enzymic analyses.

Immunodiffusion Techniques in Clinical Medicine

Progress in protein chemistry made during the ten years has resulted in the preparation of well-defined human plasma proteins and, consequently, of highly specific antiserums to the proteins. The most important contribution during recent years to quantitation of serum protein components has been development of the single diffusion type of precipitin reaction. The term "radial immunodiffusion" is applied to a system in which the antibody is incorporated in the agar and the gel spread out on a surface with the antigen diffusing radially, starting from a circular reservoir. The diameter of the precipitin zone formed is directly proportional to the amount of antigen present in the test serum, and inversely proportional to the concentration of antibody incorporated in the agar gel. Mancini contributed handsomely to this technique with kinetic studies of the system. By standardizing the technical conditions of the test, one can utilize this principle for immunochemical quantitation of

Immunotaxonomic Investigations of the Coleoptera

Immunological comparisons of beetles involving 66 species from 24 families were made by photodensitometrically measuring the precipitin zones that developed in agar-gels. Comparisons at the intrageneric and intergeneric levels were most suitable for study using the photodensitometric technique, and results obtained at these levels agreed with predicted results based on morphological studies. Unusually high immunological correspondences of Melantotuis (Family Elateridae) and Oryzaephillus (Family Cucujidae) to Tenebrio molitor suggested previously unsuspected relationships. The high degree of reciprocal correspondence between members of the families Cerambycidae and Chrysomelidae indicated that relegation of these families to subfamilial status would be reasonable. An interfamilial cluster analysis revealed systematically useful data.

Electrophoretic and immunologic studies on the chicken serum and egg volk.

Electrophoretic and immunologic studies on the partitions of the sera of laying hens and of cocks and egg yolk obtained by ultracentrifugation showed the presence of 2 specialized components, (a low density phospholipo-P-free protein and a phosphoprotein or a phosphoprotein complex) either in the serum of laying hens or in egg yolk, which were not found in the serum of cocks. Each component was capable of producing the specific antibody or antibodies. The specific antibody or antibodies to one specialized component could not be exhausted by absorption with the other component of the serum. Low density phospholipo-P-free protein, found in the top layer of the serum of laying hens, obtained by ultracentrifugation is almost identical with that found in the supernatant solution of egg yolk obtained by ultracentrifugation. This protein is a single antigenic substance as shown by the appearance of only one precipitin zone in the agar-antiserum gel. Phosphoprotein or phosphoprotein complex, found in the sediment of the serum of laying hens, is not identical with that found in the sediment of egg yolk, since the former is soluble in isotonic saline, while the latter is quite insoluble in isotonic saline. The pattern of precipitin reaction zone to the material in sediment of serum in agar-antiserum gel, which showed several zones, differed from that to the material in sediment of yolk.

Serum Imbalances in Rats 24 to 96 Hours after Exposure to X-Irradiation

An agar column diffusion method together with direct photometric quantitation was used to detect statistically significant changes in the diffusion of precipitin zones in rat sera. Zones of precipitation asscciated with albumin, globulin(s), and mucoproteins were measured. In addition to a control group, serum samples were taken from each animal before exposure. The animals were exposed to 500 r and 400 r of whole-body X irradiation and sacrificed at 24-hour irtervals. Disproportional changes in the serum components were found, beginning with the first post-irradiation bleeding (24 hours) and increased up to 72 hours. Imbalances were slightly less at 96 hours postirradiation. The quantitative application of the agar column immunochemical technique to irradiation studies of rats is believed to indicate a new horizon. Some advantages of the method over others generally used are discuesed. Considerations for finding valid changes in diffusion studies are also mentioned. (auth)