Scission et réassociation des isoagglutinines traitées par
des agents réducteurs des ponts disulfures.
Préparation d’anticorps mixtes

170

Antibodies to the somatic antigens of Gram-negative bacilli have been demonstrated to occur in three classes of immunoglobulins (1-10). Identification of specific antibody has depended upon the association of immune activity with immunoglobulin-rich fractions prepared from whole serum by various physical and chemical techniques, Immune activity in these fractions has been measured b y examining secondary properties of antigen-antibody interaction such as agglutination of particulate antigens or particulate carriers coated with antigen, preparation of live bacteria for phagocytosis, and complement-dependent bactericidal effect. The results of such experiments have led to the tentative conclusion that all or most of the immune proteins of the serum specific to the somatic antigens of Gram-negative bacilli are of the ~,M-class of immunoglobulins (3,5,(8)(9)(10). I n the studies described below the secondary activities of highly purified rabbit ~M-and ~/G-antibodies to the somatic antigens of Salmonella typhirauriura were examined and were found to differ markedly from each other.Methods and Materials 1. Preparation of the Antigen.--Salmondla typkimurium, strain 7 x (4, 5, 12:z) Salmonella paratypM B 2 (4, 5, 12:b), and Escherichia coli were grown in brain heart infusion broth (Difco Laboratories, Inc., Detroit) with constant agitation for 15 hours at 37°C. The "O" or somatic antigen was prepared as described (12). The heat-killed whole cell antigens used for experiments involving absorption and/or absorption-elution were utilized within 15 hours after preparation. Antigen preparations for assay of agglutination and for immunization were stored at 4°C without preservative. 2.Immunization.--Adult hybrid rabbits were injected with 1 X 101° organisms of S.

Section: Discussionsupporting

confidence: 92%

Section: As Shown Insupporting

confidence: 90%

Section: As Shown Insupporting

confidence: 87%

Section: Analysis Of a Ntibody-containingmentioning

confidence: 83%

See 2 more Smart Citations

The ISOLATION AND BIOLOGICAL ACTIVITIES OF RABBIT ΓM- AND ΓG-Anti-Salmonella TYPHIMURIUM ANTIBODIES

Robbins

Kenny

Suter

1965

170

“…(15) demonstrated that inert indicator erythrocytes of two morphologically distinct species (human and alligator), coated in vitro with differing test antigens, can be readily utilized in mixed agglutination experiments for studies of specificity of rheumatoid serums and for the detection of molecules in such serums capable of reacting with two different antigens (human and rabbit ganuna globulin). Jacot-Guillarmod and Isliker (19) have recently utilized mixed agglutination to demonstrate in vitro formation of antibody of mixed specificity by reduction and reoxidation of a mixture of anti-A and anti-B isoagglutinins of high molecular weight.…”

Section: Discussionmentioning

confidence: 99%

Serologic Demonstration of Dual Specificity of Rabbit Bivalent Hybrid Antibody

Fudenberg

Drews

Nisonoff

1964

Treatment of rabbit 7S antibody with pepsin results in the removal of an inactive fragment, leaving a bivalent residue with an average molecular weight of 106,000 and a sedimentation coefficient of approximately 5S (1, 2). Subsequent reduction of one labile disulfide bond splits this residual molecule into two univalent, non-precipitating fragments which migrate as a single 3.5S peak (3) in the ultracentrifuge. These fragments closely resemble the univalent fractions of a papain digest (4) in a number of their properties.The univalent 3.5S fragments obtained by peptic digestion and reduction of a specifically purified rabbit antibody can be recombined by oxidation, in yields up to 75 per cent, to give 5S antibody which again possesses precipitating activity (5). When a mixture of purified rabbit antibodies of two different specificities is reduced and reoxidized, the recombined product contains a considerable amount of bivalent 5S "hybrid antibody" (6, 7). 1 The recombination of fragments of two different specificities appears to be essentially random, as shown by quantitative analysis of the hapten-binding capacity of hybrid antiovalbumin-antibenzoate antibody before and after treatment with an adsorbent specific for the antiovalbumin (7).Previous reports from these and other laboratories demonstrated that the 5S derivatives of several 7S rabbit antibodies retain most of the original agglutinating activity on a molar basis (8, 9); the 3.5S derivatives lack agglutinating properties but combine with the homologous antigen, as shown by inhibition of hemagglutination (9, 10) or by a modified antiglobulin reaction (8).The present communication extends these hemagglutination studies to 5S antibodies obtained by reoxidation of the 3.5S univalent fragments.

ACTIVITY OF DISSOCIATED AND REASSOCIATED 19S ANTI-γ-GLOBULINS

Schrohenloher

Kunkel

Tomasi

1964

It has been known for some time that 19S maeroglobulins (1) and 19S class antibodies (2) are readily reduced by sulfhydryl compounds into smaller units. In most reported experiments with 19S antibodies this treatment has resulted in the complete loss of biological activity. However, some experiments to the contrary have been reported (3). Previous studies from this laboratory (4, 5) demonstrated that certain 19S anti-~,-globulins regained their activity on oxidative re.association following dissociation with sulfhydryl compounds. Although no activity was demonstrated in the dissociated fragments, these experiments raised such a possibility. Isliker and associates have presented evidence that following treatment with NaBH4-mixed molecules of high molecular weight isoagglutinins could be formed (6) and have recently reported evidence for axztivity in smaller units (7).The present experiments were undertaken to extend the previous observations on the effect of reduction and reassociafion on the biological activity of 19S antibodies. The anti-*t-globulins from human sera were chosen for these experiments because of their ready isolation in workable quantities. The large amount of evidence available that they represent antibodies to T-globulin has been cited previously (8). The results obtained clearly indicate the presence of certain activity in the reduced and blocked dissociated fragments of these 19S proteins and the return of the original type of activity following reassociation. Materials and MethodsThe 19S anti-~-globulins were isolated from the sera of two patients (Do. and Od.) having high titers in the various reactions for the detection of rheumatoid factors. These sara also contained significant amounts of intermediate "~,-globulin complexes (9).