INFECTION AND IMMUNITY, June 1979, p. 962-964 0019-9567/79/06-0962/03$02.00/0
Vol. 24, No. 3
Antibody to Rickettsia mooseri Erythrocyte-Sensitizing Substance JAMES R. MURPHY, PAUL FISET, LILLIAN B. SNYDER, AND CHARLES L. WISSEMAN, JR.* Department ofMicrobiology, University ofMaryland School ofMedicine, Baltimore, Maryland 21201 Received for publication 29 January 1979
Erythrocyte-sensitizing antigens from Rickettsia mooseri (R. typhi) were found to bind to rabbit erythrocytes. Upon reintroduction into the donor, such antigensensitized cells caused the production of antibodies of restricted serological reactivity.
Rickettsia of the typhus (2, 4) and spotted fever (3) groups possess soluble antigens which are stable in 0.2 N NaOH at 1000C and which spontaneously adsorb to erythrocytes (RBC). These properties allow their isolation from less hardy antigens and provide a reagent which is useful for the serodiagnosis of infections (9, 12). Furthermore, evidence suggests that these erythrocyte-sensitizing antigens (ESS) may be present in tissues and in body fluids early in the course of infection, before specific antibody is detectable (5, 8, 11; Y. A. El Batawi, Ph.D. thesis, University of Maryland, Baltimore, 1964). Therefore, if a suitable anti-ESS antibody were available, this material might be useful for the early diagnosis of rickettsial infection. Furthermore, an antibody specific for ESS could be used to isolate this antigen from NaOH digests of yolk sac-grown rickettsiae, to determine the location of the antigen on the microorganisms, and to determine the significance of anti-ESS antibody to protection from infection. In this report we show that ESS adsorbs to rabbit RBC (RRBC) and that reintroduction of ESS-sensitized RRBC (ESS-RRBC) into the donor raises antibodies which react with a limited spectrum of rickettsial antigens. ESS was prepared by the method of Chang (2, 4) from yolk sac-grown Rickettsia mooseri (R. typhi) Wilmington strain. This ESS preparation adsorbed to both human RBC and RRBC as demonstrated by titration of a positive serum. The highest dilution of ESS which affected complete sensitization of both RBC was 1:50. The diluent was Alsever solution. Based on the demonstration that ESS adsorbed to RRBC, an attempt was made to raise anti-ESS antibody with ESS-RRBC as the immunogen. Each of four New Zealand white rabbits (Bar F Rabbitry, Virginia) was immunized with autologous RBC sensitized with ESS diluted 1:25. The ESS-RRBC were washed twice in Alsever solution, made up to 10%, and thor-
oughly mixed with an equal volume of complete adjuvant (H37Rv, Difco Laboratories, Detroit, Mich.). Each animal received the autologous mixture as follows: 2 ml in the right thigh, 2 ml in the left thigh, and 2 ml intraperitoneally. Each animal was boosted at 7 and 14 days after primary immunization with 3 ml of freshly prepared autologous 10% ESS-RRBC given intravenously without adjuvant. The animals were bled before immunization and 7, 14, and 21 days after primary immunization. The procedures and antigens employed for the complement fixation (1, 7, 10) and microagglutination (6) tests are published elsewhere. The Weil-Felix reactions were carried out with commercially available Proteus antigens OX-19 and OX-2 (Sylvana, Orange, N.J.) at a dilution of 1:10 in the microtiter system. The passive hemagglutination test of Chang (2) was adapted to the microtiter system. The RBC, sensitized with a 1:50 dilution of ESS, were used at a concentration of 2%. Equal volumes (0.025 ml) of serum dilution and sensitized RBC were mixed and allowed to settle overnight at room temperature. Table 1 shows that sera collected after immunization with ESS-RRBC agglutinated autologous ESS-RRBC. Notably, these positive sera did not agglutinate autologous RRBC which had not been sensitized with antigen. Furthermore, sera collected after immunization with ESS-RRBC contained antibodies which reacted in complement fixation with the soluble (group) rickettsial antigen and somewhat more weakly with particulate antigens. There was also a weak Weil-Felix reaction with Proteus OX-19 and OX-2. Antibodies capable of agglutinating purified, ether-treated, particulate rickettsial antigens in the macroagglutination test were not found. Thus, immunization with ESS-RRBC raises antibodies which react strongly with the eliciting antigen and to a lesser degree with some of the other antigens commonly employed for 962
NOTES
VOL. 24, 1979
963
TABLE 1. Antibody response in rabbits after immunization with RBC coated with ESS Titera in indicated test
Damiafteri
PHA (RRBCESS)
Complement fixation"
Weil-Felix OX-19 OX-2
Microagglutination
ET"
SOL'
MT
ET
Vol. 24, No. 3
Antibody to Rickettsia mooseri Erythrocyte-Sensitizing Substance JAMES R. MURPHY, PAUL FISET, LILLIAN B. SNYDER, AND CHARLES L. WISSEMAN, JR.* Department ofMicrobiology, University ofMaryland School ofMedicine, Baltimore, Maryland 21201 Received for publication 29 January 1979
Erythrocyte-sensitizing antigens from Rickettsia mooseri (R. typhi) were found to bind to rabbit erythrocytes. Upon reintroduction into the donor, such antigensensitized cells caused the production of antibodies of restricted serological reactivity.
Rickettsia of the typhus (2, 4) and spotted fever (3) groups possess soluble antigens which are stable in 0.2 N NaOH at 1000C and which spontaneously adsorb to erythrocytes (RBC). These properties allow their isolation from less hardy antigens and provide a reagent which is useful for the serodiagnosis of infections (9, 12). Furthermore, evidence suggests that these erythrocyte-sensitizing antigens (ESS) may be present in tissues and in body fluids early in the course of infection, before specific antibody is detectable (5, 8, 11; Y. A. El Batawi, Ph.D. thesis, University of Maryland, Baltimore, 1964). Therefore, if a suitable anti-ESS antibody were available, this material might be useful for the early diagnosis of rickettsial infection. Furthermore, an antibody specific for ESS could be used to isolate this antigen from NaOH digests of yolk sac-grown rickettsiae, to determine the location of the antigen on the microorganisms, and to determine the significance of anti-ESS antibody to protection from infection. In this report we show that ESS adsorbs to rabbit RBC (RRBC) and that reintroduction of ESS-sensitized RRBC (ESS-RRBC) into the donor raises antibodies which react with a limited spectrum of rickettsial antigens. ESS was prepared by the method of Chang (2, 4) from yolk sac-grown Rickettsia mooseri (R. typhi) Wilmington strain. This ESS preparation adsorbed to both human RBC and RRBC as demonstrated by titration of a positive serum. The highest dilution of ESS which affected complete sensitization of both RBC was 1:50. The diluent was Alsever solution. Based on the demonstration that ESS adsorbed to RRBC, an attempt was made to raise anti-ESS antibody with ESS-RRBC as the immunogen. Each of four New Zealand white rabbits (Bar F Rabbitry, Virginia) was immunized with autologous RBC sensitized with ESS diluted 1:25. The ESS-RRBC were washed twice in Alsever solution, made up to 10%, and thor-
oughly mixed with an equal volume of complete adjuvant (H37Rv, Difco Laboratories, Detroit, Mich.). Each animal received the autologous mixture as follows: 2 ml in the right thigh, 2 ml in the left thigh, and 2 ml intraperitoneally. Each animal was boosted at 7 and 14 days after primary immunization with 3 ml of freshly prepared autologous 10% ESS-RRBC given intravenously without adjuvant. The animals were bled before immunization and 7, 14, and 21 days after primary immunization. The procedures and antigens employed for the complement fixation (1, 7, 10) and microagglutination (6) tests are published elsewhere. The Weil-Felix reactions were carried out with commercially available Proteus antigens OX-19 and OX-2 (Sylvana, Orange, N.J.) at a dilution of 1:10 in the microtiter system. The passive hemagglutination test of Chang (2) was adapted to the microtiter system. The RBC, sensitized with a 1:50 dilution of ESS, were used at a concentration of 2%. Equal volumes (0.025 ml) of serum dilution and sensitized RBC were mixed and allowed to settle overnight at room temperature. Table 1 shows that sera collected after immunization with ESS-RRBC agglutinated autologous ESS-RRBC. Notably, these positive sera did not agglutinate autologous RRBC which had not been sensitized with antigen. Furthermore, sera collected after immunization with ESS-RRBC contained antibodies which reacted in complement fixation with the soluble (group) rickettsial antigen and somewhat more weakly with particulate antigens. There was also a weak Weil-Felix reaction with Proteus OX-19 and OX-2. Antibodies capable of agglutinating purified, ether-treated, particulate rickettsial antigens in the macroagglutination test were not found. Thus, immunization with ESS-RRBC raises antibodies which react strongly with the eliciting antigen and to a lesser degree with some of the other antigens commonly employed for 962
NOTES
VOL. 24, 1979
963
TABLE 1. Antibody response in rabbits after immunization with RBC coated with ESS Titera in indicated test
Damiafteri
PHA (RRBCESS)
Complement fixation"
Weil-Felix OX-19 OX-2
Microagglutination
ET"
SOL'
MT
ET
