Earl W. Flosdorf

Sonic Extraction of Labile Bacterial Constituents.

Recent investigations have indicated that the killing of certain bacteria by available agents destroys or alters at least some of their antigenic constituents. Thus the investigations of Felix 1 have revealed a labile virulence (Vi) antigen detected only in living, virulent strains of Eb. typhi. Similarly, Mudd, Pettit, Lackman and Czarnetzky 2 have found that the antibody responsible for the phagocytosis of a strain of Streptococcus hemolyticus is elicited in rabbits by the injection of living organisms and that the living organisms will absorb the antibody from immune serum so produced. The corresponding bacterial antigen itself has resisted all ordinary methods of separation or fractionation. Since it has been shown that sonically killed bacteria are disintegrated 3 , 4 , 5 and since some heat labile proteins are not denatured by the sonic treatment, 6 it appeared possible that labile but important antigens such as those referred to above might be extracted by an adaptation of the method of sonic disintegration. The purpose of this paper is to describe the apparatus and technique which we have used; to make brief mention of the antigens obtained thus far; and to point out some implications of these observations. We have used a modified Pierce magnetostriction oscillator as the source of intense vibrations of about 8900 c.p.s. A similar oscillator has been described by Gaines 7 and was used by Chambers and Gaines 3 in their earlier work. The circuit design in our apparatus differs somewhat from that of Gaines but the essential features of the sound source are the same. The electrical circuit we have used is shown in Fig. 1. The arrangement of the vibrating element with relation to polarizing and driving coils is illustrated in Fig. 2. The vibrator itself is a tube of No. 20 Stubbs gauge, seamless, cold-drawn nickel tubing 24.6 cm. in length and 1.875 cm. in outside diameter. The upper end of the tube is closed by a welded nickel cap as in the original Gaines device, but we have found it unnecessary to slit the lower half of the tube. Adequate radiation of heat due to eddy currents is possible, as described below, when no such kerf is present.

Studies with H. Pertussis

Summary Antitoxin for the two known toxins of H. pertussis is not found in human convalescent or hyperimmune human serum. Agglutinins or antibacterial antibodies are found in high titer in the hyperimmune serum of known high clinical potency. Agglutination is recommended as a means of assay of such sera and should be used routinely as a laboratory control test in the production of such sera for clinical use. A procedure for immunization of adult donors is discussed, and the technique for use of agglutination in such assays is presented in detail. Titers of the sera of a group of human donors during the course of immunization are recorded. Most striking is the variation that occurs in different donors in response to immunization and also in any given donor from time to time. This makes it imperative to follow immunization by laboratory assays of the sera and to use the results as a guide in the selection of donors.

Clinical results with the use of agglutinogenfrom phase I hemophilus pertussis as a skin test for susceptibility to whooping cough

Summary The results of a clinical study, using the purified agglutinogen ofphase I Hemophilus pertussis as a skin test for susceptibility to whooping cough, are presented. The test seems to classify immune and susceptible individuals according to their histories, both of disease incidence and vaccination, in a satisfactory way. In addition to its use as a skin test, the agglutinogen in single skin test doses produces a marked increase in the titer of individuals with existing immunity or partial immunity to pertussis. Repeated doses produce a reversal of the test and a titer in those with no initial immunity. Careful study of a small institutional epidemic indicates that susceptibility can be predicted by the use of this skin test.

Studies with hemophilus pertussis

Summary 1. Cross agglutination between B. parapertussis and H. pertussis inphase I has been shown by means of absorptive tests to be due to a common minor antigen. 2. The findings of Miller have been confirmed with respect to a generalwide incidence of parapertussis agglutinins among the general population. By means of agglutinin absorption, it has been shown that these agglutinins for B. parapertussis are specific in large measure, presumably as a result of specific infection by B. parapertussis , and are not the result of the common minor antigen which exists in both B. parapertussis and H. pertussis . Incidence of agglutinins for B. parapertussis in both children and inadults in two institutions, has been found to be lower than in the general population. In the general population, the incidence found has increased with age. 3. As a result of intensive immunization of either man or animals, the agglutinative titer versus the heterologous species is increased through the agency of the common minor antigen, but it is not known whether such agglutinins are effective in clinical cross protection against either disease. Skin testing for susceptibility to whooping cough with purified agglutinogen also stimulates production of these same agglutinins as well as those versus the major phase I antigen. 4. The use of purified agglutinogen as an intradermal reagent forstimulation of immunity, as well as in skin testing for susceptibility to whooping cough, is suggested. 5. The results of the present study suggest that only the relatively serious cases of parapertussis are diagnosed clinically and that these are similar to mild cases of whooping cough; the nature of the typical disease of parapertussis apparently is not recognized clinically in relation to the specific etiologic agent.

The detection of susceptibility to whooping cough

Summary A purified agglutinogen of phase I H. pertussis was used as a skin testreagent in three institutions. An epidemic of whooping cough was studied in detail; all secondary cases occurred in children whose skin tests indicated susceptibility or weak immunity. Routine agglutination tests indicated the value of agglutinogen as a “recall” dose, as shown by the small number of cases following skin testing of the entire population of the institution. Parallel studies were done in an institution in which there was no whoopingcough, in order to rule out the effect of the presence of the disease on the skin test reaction and agglutinative titer of exposed individuals. The effectiveness of the skin test reagent as an antigen was well established by the results obtained in this control institution. In a second institution in which one case of whooping cough appeared, allsecondary cases occurred in individuals who had had negative skin tests.

Studies on H. pertussis. I. Liberation by Sonic Vibration of a Soluble Component That Absorbs Phase I Agglutinins

Summary Filtered extracts of H. pertussis obtained by sonic disintegration of Phase I organisms have been found to absorb to completion the agglutinins from homologous rabbit antiserum of high titer. The soluble preparations now available and known as Endoantigen, Topagen and Detoxified Pertussis antigen were found to be devoid of such activity within the limits of sensitivity of the method used.