A.H. Sehon

Demonstration of antibodies in the sera of grass-sensitive persons by the bis-diazotized-benzidine hemagglutination technique

Abstract The BDB-hemagglutination technique has been adapted for the demonstration of antibodies to grass pollen constituents in the sera of immunized rabbits and grass-sensitive hay fever patients. The grasses used were timothy, June grass, orchard grass, redtop, sweet vernal, and Bermuda grass. The method was shown to be highly sensitive. Titers of the order of 10 4 and 10 2 were obtained with sera of immunized rabbits and of allergic human subjects, respectively. The specificity of the technique was demonstrated by inhibition of the hemagglutination with free soluble pollen constituents. The inhibition of hemagglutination obtained with rabbit antisera was used for establishing antigenic relationships among the various grasses. Timothy, June grass, orchard grass, redtop, and sweet vernal were found to contain common antigens, which were different from those of Bermuda grass and ragweed. The results of Prausnitz-Kustner tests using the cross-neutralization technique with desensitizing doses of each of the five grasses (timothy, June grass, orchard grass, redtop, and Bermuda grass) suggest that redtop grass pollen contains all the allergens common to the other grasses and that timothy, June grass, and orchard grass pollens have a complex and in many respects similar allergenic composition and contain additional allergens to those found in Bermuda grass pollen. Absorption of an allergic serum with adsorbents prepared by coupling grass pollen constituents to polystyrene or erythrocytes resulted in the removal of the hemagglutinating factor(s), which was paralleled by the removal of skin-sensitizing antibodies.

Electrophoretic separation of skin-sensitizing antibody from the sera of ragweed-sensitive patients

Abstract 1.1. The separation of ten sera from ragweed-sensitive patients into their electrophoretically separable protein components, using starch electrophoresis, is described. The advantages of the method are discussed and several modifications are presented. 2.2. The resulting sera fractions were tested by passive transfer techniques to determine the localization of skin-sensitizing antibody present in the whole sera. 3.3. It was found that the albumin and alpha globulin fractions have no skin-sensitizing activity, while both beta and gamma globulin fractions demonstrate significant skin-sensitizing activity. These findings and the results of previous work of this nature have been discussed.

Physico-chemical and immunologic studies on macroglobulins.

Normal sera are resolved in the ultracentrifuge into two major peaks with sedimentation constants of the order of 4.5 and 6.5 S 2 and a minor peak of heavy materials, amounting to less than 3 per cent of the total proteins, with a sedimentation constant of about 20 S. In 1944, Waldenstrdm (1) reported the presence of a large amount of fast sedimenting moieties (19 to 20 S) in sera of some patients with marked hyperglobulinemia. This finding prompted him to assume that these serum components were of high molecular weight and he named them macroglobulins. He also reported that these serum components could be precipitated out on dilution of the serum with 16 volumes of distilled water. In recent years a number of workers have reported the presence of components in pathological sera with properties similar to those assigned by Waldenstrdm to macroglobulins (2-10). However, in a number of cases the macroglobulins detected by the precipitation test with distilled water had sedimentation constants as low as 12 S and as high as 30 S. On the basis of electrophoretic mobilities, macroglobulins have been reported to occur either in the 8 or y-globulin regions (2, 3, 5, 6, 8-11). Vogler, Oberhinsli, and Kofler (12) found that the diffusion coefficient for some macroglobulins was 1.2 x 10-7 cm2. per sec. in contrast to the accepted value for normal y-globulins of about 3.8 X 10-7 cm2. per sec. This finding was considered

Studies on ragweed pollen. I. Preliminary fractionation of ragweed pollen: Characterization and allergenicity of the isolated fractions☆

Abstract 1.1. Defatted ragweed pollen was extracted with a number of solvents and the various fractions were analyzed by free and paper electrophoresis and ultracentrifugation. All fractions were compared for their allergenic reactivity by skin tests. 2.2. Extraction of defatted ragweed pollen with different solvents yielded fractions of different composition. 3.3. All the extracts soluble in the different solvents displayed allergenic reactivity, while the water-insoluble ragweed was the only one of the insoluble residues to display reactivity consistently. 4.4. The results of this study suggest that zone electrophoresis is suitable for the further fractionation of the various ragweed pollen fractions.

Studies on the allergens of ragweed pollen: A review

Abstract The results reported by numerous investigators who have attempted over the last forty years to isolate and establish the composition of the allergen(s) in the pollens of short and giant ragweed are conflicting. None of the investigations reviewed has provided a satisfactory proof that the allergen(s) have been separated in a pure form. It would seem that the following two studies represent the most advanced stages in the characterization of allergens in ragweed pollens: The work of Goldfarb and associates 58 has led to the isolation from giant ragweed pollen, of a fraction (Trifidin A) which was shown to be chemically, electrophoretically, and immunologically a single entity. On standing in the cold in distilled water, however, it was degraded into at least three components: a ninhydrin-stainable material, arabinose, and another reducing substance. We 117 have recently isolated, by use of paper electrophoresis, a highly allergenically active fraction from short ragweed pollen (AA 1 -D), which was shown to contain a pigment and peptide(s) composed of eight amino acids: arginine, lysine, glutamic acid, glycine, alanine, hydroxyproline, valine, and norleucine. The pigment could be removed from this fraction without loss of allergenic activity. On the other hand, hydrolysis of the peptide(s) resulted in a complete loss of allergenic activity; therefore, it was concluded that the peptide(s) appeared to be the allergen(s) in short ragweed pollen. Furthermore, it was shown that 10 −7 mg. of fraction AA 1 -D was allergenically as potent as 10 −4 mg. of the whole water-soluble extract of ragweed pollen. In view of the extremely high biologic potency of fraction AA 1 -D, and in view of the fact that allergenically active fractions isolated in previous studies all contained some nitrogen material, it may be suggested that the allergenicity of ragweed pollen is due to peptide(s), which may be complexed with other carriers, such as proteins, carbohydrates, or pigments.

Localization of blocking antibody in sera of ragweed-sensitive individuals by starch electrophoresis

Abstract Using starch electrophoresis, blocking activity of four sera of treated ragweed-sensitive patients was demonstrated to reside mainly in the slow-moving gamma globulins. No blocking antibodies were detected in other serum fractions. The method of detection and titration of blocking antibodies is described in detail.

Characterization of the dialyzable constituents of the aqueous extract of ragweed pollen

Abstract Four fractions of the dialyzable components of the aqueous extract of ragweed pollen were prepared and their physicochemical and immunologic properties were examined. All these fractions were skin active with respect to sera from both treated and nontreated individuals. All the antigenic and allergenic determinant groups of the dialysate fractions were found to be present in the nondialyzable components of the whole aqueous extract. Contrary to previous conclusions, it was demonstrated by cross-neutralization studies that new reagin(s) directed exclusively against components of the dialysate is not produced by hyposensitization treatment with the whole aqueous extract. Rather, any increase in reagin concentration caused by hyposensitization is considered to be the result of a rise in the concentration of reagin (s) already being produced by the allergic individual.