Agglutinating and precipitating capacity of rabbit anti-Salmonella typhosa yG and yM antibodies during prolonged immunization. J. Bacteriol. 92:880-886. 1966.-Antibody produced in rabbits immunized with acetone-dried typhoid bacilli was followed over a period of 445 days by agglutination and by quantitative precipitation. Repeated injections of vaccine resulted in suppression of antibody titers. Both-yG and yM antibodies were rapidly increased by booster injections after rest periods during which titers had decreased to low levels. The 0 agglutinin titers and the amount of antibody protein, as determined by precipitation with endotoxin, generally were parallel, except in serum specimens in which unusually large proportions of the agglutinating activity were found in the yG fraction. These exceptions were explained by the greater agglutinating capacity of the-yM. Endotoxin precipitated about 10 times as much antibody from-yG preparations as it did from yM fractions of equivalent agglutinating strength. A much higher proportion of the serological

The major portion of the agglutinating and bactericidal activity of the sera of rabbits immunized with live Vibrio cholerae or with cholera vaccine was found in the yM fractions during the early stages of immunization. After 5 weeks or more, yG fractions accounted for more than half of the agglutinating activity. When late

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...

Directand passive-agglutinating, complement-fixing, and bactericidal properties of-yG and yM antibodies produced in rabbits inoculated with live Vibrio cholerae were determined at intervals over a period of 345 days. Although yM antibody titers increased more rapidly than yG during the initial stages of antibody production, the titers of-yG and-yM declined proportionally during a 3-month rest period and increased proportionally after a booster injection. The relative titers of yM as determined in the four serological procedures remained fairly constant throughout the period of observation. In contrast, early yG was less effective than late yG in vibriocidal, complement-fixing, and passive-hemagglutinating activity. At no stage of immunization was the agglutinating ability of yG affected by 2-mercaptoethanol, but its complement-dependent activity was markedly reduced, more so in early serum than in late. The heat lability of early 'yG approached that of yM, but yG became more resistant to heat in later stages of immunization.

A solution of 2-mercaptoethanol began to lose its reducing capacity after storage for a few days.The loss of serological activity on treatment of a preparation with 2-mercaptoethanol (ME) is often taken as an indication of the presence of