Chromatographic Analysis and Sulfhydryl Sensitivity of Antileptospira Agglutinins in Rabbit and Human Sera.

Abstract

Antibody activity for various antigens has been found chiefly in the yG (7s) and the yM (19s) fractions of globulin. The molecular type of antibody which predominates appears to depend to some extent on the chemical nature of the antigen involved. Protein antigens such as serum albumin and bacterial flagella induce the early formation of yM antibody followed by yG which soon comprises the major portion of the antibody( 1 ) . Other antibodies, notably those produced in response to the somatic antigens of Gramnegative bacilli (1,2,3) and the infectious mononucleosis antibody that reacts with the beef thermostable antigen (4), are predominantly or exclusively in the yM globulin.Since only limited information is available regarding the antigenic composition of leptospirae and the relation of antigenic components to various serologic reactions involving these organisms ( 5 ) , it seemed of interest to determine the types of globulin associated with the agglutinins in antileptospira sera.Materials and methods. Rabbits were immunized by 4 intravenous injections, each consisting of 1 ml of live culture of leptospirae, given during a period of 6 days. Three of the sera from patients with leptospirosis had been preserved in the frozen state for 6 to 7 years without apparent reduction in agglutinin titer. Serum from one patient, 812, was examined after 6 weeks refrigeration without being frozen. Antibodies were determined by the microscopic agglutination test employing live cultures in Korthof's medium( 6). Chromatographic separation of fractions from 5 ml samples of serum was accomplished as previously described (3,4) or, when the amount of serum was limited, 1 ml of serum was placed on a column containing 1 g of DEAE cellulose and from 8 to 20 4-ml fractions were eluted with each *This work was supported by a grant from National Science Foundation.of the 4 buffers. Individual fractions were screened for agglutinins and the positive fractions eluted witheach buffer were pooled and concentrated with the aid of carbowax(3). Sensitivity of antibody to disulfide bond reduction was determined by mixing equal volumes of diluted serum and 0.2 M 2-mercaptoethanol (ME), allowing the mixture to stand at room temperature for 16 to 18 hours, and testing for agglutinins without further treatment. A control consisting of equal parts of serum dilution and saline was included in each instance.Results. Human sera. The data obtained on analysis of serum specimens from 4 patients with leptospirosis are shown in Table I. Each serum reacted with at least 4 serotypes. If one assumes that the type showing the highest agglutinin titer was the type responsible for the infection, it appears that patients 328 and 812 were Leptospira canicola infections, that patient 336 was infected with L . pomona, and that L. grippotyphosa was the infecting type in patient 406. Although the differences between titers for L. sejroe and the presumed infecting type was only one tube, it is unlikely that all 4 patients had multiple infections, especially since neithe...

Genus-specific leptospiral antigen and its possible use in laboratory diagnosis

“…No history as to the stage of illness when these samples were collected was available. In human leptospirosis the 19S agglutinins have been known to appear earlier than the 7S agglutinins (Hartmann, Filitti-Wurmser, Jacquot-Armand, Mailloux, Hurez, and Fauvert, 1964;Lataste-Dorolle, Eyquem, and Buri, 1964;Pike, McBrayer, Schulze, and Chandler, 1965;Van Dalen, Seijen, and Gruber, 1967). In the present investigation an early appearance and disappearance of HA titres as compared with the microscopic agglutination titres was noted in the case of rabbits injected with the ethanol-precipitated antigen (Fig.…”

The Antibodies Involved in the Human Immune Response to Leptospiral Infection

“…Earlier workers (Pike et al, 1965;Tong et al, 1971;Sulzer et al, 1975) either failed to isolate and identify the infecting serovar, or examined sera only in the early stages of illness, or gave insufEcient information. += Protection; -= no protection.…”

“…The titres did not necessarily correlate, but the times of appearance and disappearance of the two types of antibody were similar, although F4 antibodies sometimes appeared earlier than agglutinins. Unlike agglutinins, F4 antibodies were confined to the IgM class, a finding characteristic of antibodies to polysaccharide-type antigens (Pike, 1967).…”

“…Pike et al (1965) found that in four patients in whom serological tests had indicated infection with serovars canicola, grippotyphosa or pomona, the predominant homologous agglutinins were IgM. Sera at different stages of illness were not examined, but in one patient with serovar pomona infection, IgM agglutinins still predominated in serum taken 2 months after the onset of illness.…”

Evaluation of an immunoenzimatic test (ELISA) for the diagnosis of leptospirosis in Italy

“…One false positive was found in the controls. Adler et al (2) employed homologous whole cell sonicated strains for ELISA to detect leptospiral IgM and IgG in sera from patients suffering from leptospirosis, IgM remained the predominant class of antibodies in human leptospirosis (1,16,20,25). These authors employed for antigen preparations one or two serovars dominant in a particular region and tested human sera which originated from patients infected by homologous serovar.…”