The Extraction of Hemagglutinin from Strains of Hemophilus Aegyptius

A serologically active substance was extracted from cells of Hemophilus aegyptius with dilute NAOH. This substance directly hemagglutinated human erythrocytes. The activity of this material was demonstrated at a near neutral pH and was precipitated at acid pH. Preliminary biochemical determination showed this serologically active material to be predominately polysaccharide. Davis, Pittman, and Griffiths (1950) reported that strains of Hemophilus aegyptius (Koch-Weeks Bacillus) possessed the capacity to hemagglutinate human red blood cells. This property was relatively stable to aging, not lost by frequent transfer of the culture, exposure to low temperatures, or upon treatment with formalin or ether. Erythrocytes treated with the receptor-destroying enzyme of Vibrio cholerae were still hemagglutinated by strains of this organism, thereby differing from the influenza and similar viruses. However, the hemagglutinating capacity of this organism was inhibited by antiserum to H. aegyptius, prepared in rabbits. It was also determined that this property could not be completely removed from the organism by successive washings with normal saline, and that these washings did not contain appreciable quantities of the hemagglutinin. Supernates or culture nitrates of the organism also possessed this activity, but in quantities less than that observed with intact cells. The study to be described was initiated to further characterize this property. MATERIALS AND METHODS Hemophilus aegyptius strains 18, 46, 128, 145, 180a, 181a, and 763 were generously provided by Dr. Margaret E. Pittman, USDHEW. Cultures were routinely passed and maintained on Brain Heart Infusion medium fortified with five per cent digest of sheep blood (which supplies the necessary X and V factors) after the method of Fildes (1920). Large quantities of H. aegyptius cells were obtained by cultivating the various strains in 32-oz prescription bottles containing the fortified BHI agar. Organisms were harvested after 18 to 24 hr incubation by washing the cells from the surface with unbuffered 0.85 per cent saline. The resulting suspensions were thrice washed in normal saline and tested for the capacity to directly hemagglutinate human erythrocytes. The extraction of hemagglutinin was accomplished by resuspending the harvested bacterial cells in physiological saline and adjusting the pH to 10 or 11 by the addition of dilute NaOH. This alkalinized suspension was incubated in a 37 C water bath for 30 min and centrifuged lightly for 5 min in a Servall Superspeed Angle Centrifuge. The supernate was recovered and filtered through a millipore filter to insure a bacteria-free solution. The remaining extracted cells were saved for additional serological studies. A portion of the bacteria-free alkaline extract was adjusted to a pH of 6.5 by the addition of dilute HC1. At this pH, a precipitate formed which was recovered by centrifugation, washed three times in saline, resuspended to the original volume and tested for hemagglutinability. The unadjusted alkaline extract was also tested for this capacity. Manuscript received May 30, 1964. THE OHIO JOURNAL OF SCIENCE 65(3): 149, May, 1965. 150 M. S. RHEINS, JERRY M. MANN AND JAMES F. CLEMENTS Vol. 65 Bacterial cells which had been subjected to alkaline extraction for the removal of hemagglutinin retained to some extent the hemagglutinating property, and therefore were repeatedly re-extracted at a pH range of 10 to 11 in an attempt to remove the remaining serologically active material and also to obtain hemagglutinin-free cells. The resulting cell suspensions were employed for bacterial agglutination studies, hemagglutination tests, and antiserum-adsorption procedures. All preparations of whole cells, hemagglutinin-free (denuded) cells, and extracts were tested for hemagglutinability. Serial doubling saline dilutions of the test materials were prepared in 0.5-ml quantities. To each tube in the titration series was added 0.5 ml of a 0.5 per cent suspension of washed normal human red cells. The specific type or group of the red cells employed apparently was of no consequence. All tests were incubated in a 37 C water bath for 30 min and then the suspended red cells permitted to settle completely at room temperature. The end point of the titration was considered to be the last tube exhibiting a definite pattern of hemagglutination. Bacterial hemagglutination-inhibition and extract hemagglutination-inhibition studies were performed using standard doubling saline dilutions of antiserum in 0.25-ml volumes against four units of either the hemagglutinating intact cell or of the active extract contained in 0.25 ml saline. A suspension of 0.5 per cent washed normal human red cells in 0.5-ml quantities was employed as the indicator system. After incubation at 37 C for 30 min, the tubes were allowed to settle at room temperature and read for pattern. The last dilution tube of the series in which there was no evidence of hemagglutination represented the titration end-point. A suspension of approximately 1X10 whole cells per ml served as antigen in bacterial agglutination studies. Tests were incubated at 37 C for 1 hr, refrigerated over-night, and read after light centrifugation. The last dilution presenting evidence of macroscopic agglutination, as compared with a cell-saline control, was considered the end point of the titration. Antisera against the various strains of H. aegyptius were prepared by injecting rabbits with vaccines prepared from washed bacterial cells that had been heatinactivated for 30 min at 56 C and standardized to a turbidity of 10 billion cells per ml by the addition of saline containing 1:10,000 merthiolate. These vaccines were administered intravenously in 1.0-ml quantities at three day intervals for a nine day period. On the fifteenth day following the first injection, each animal was bled by cardiac puncture, the serum recovered and stored at — 4 C until needed. In an attempt to obtain antibodies specifically directed against the hemagglutinin without interference from extraneous material from the medium or the bacterial cell, normal human red cells were sensitized with the extract and injected as antigens. These antigens were prepared by incubating one volume of washed human erythrocytes with two volumes of the active extract material (pH 6.5) for 30 min in a 37 C water bath. These modified red cells were washed three times in saline to remove unadsorbed material, reconstituted with saline to a volume twice that of the original packed red cells and 1.0 ml injected. After several injections the animals were bled and the recovered antisera were heat-inactivated and repeatedly adsorbed with washed packed human erythrocytes to remove antibodies directed to the red cell carrier. These antisera then were tested for inhibition of both bacterial and extract hemagglutination and for bacterial agglutination. In the course of this investigation, precipitin ring tests were employed using the alkaline extract (pH 8.5 to 9.0) as the antigen to determine the reactivity of the various antisera. This method was of value for screening purposes, but due to the capacity of this antigen to precipitate at a near neutral pH, the tests were not considered to be as valid as desired. A micro-gel diffusion technique after the method of Yakulis and Heller (1959) was utilized for the detection of antibody. No. 3 HEMOPHILUS AEGYPTIUS HEMAGGLUTININ 151 The adjusted alkaline extract was diffused against antisera to red cells modified with the alkaline extract, and to antisera against intact bacterial cells. These preparations were examined for lines of precipitation after 24 hr. The capacity of the alkaline H. aegyptius extracts to sensitize red cells was investigated. Highly alkaline extracts (pH 10 to 11) as described above were adjusted to pH 8.0 with dilute HC1 and the preparation rendered isotonic by the addition of dry NaCl. One tenth ml of washed packed normal erythrocytes and 1.0 ml of the adjusted isotonic extract were mixed and incubated for 30 min in a 37 C water bath after which the excess extract was removed by washing with physiological saline and finally the red cells resuspended to 0.5 per cent by volume. This red cell suspension then was tested for sensitizing capacity of the extract by the addition of antiserum prepared against the whole bacterial cell.