THE FORMATION AND PROPERTIES OF POLIOVIRUS-NEUTRALIZING ANTIBODY : III. SEQUENTIAL CHANGES IN ELECTROPHORETIC MOBILITY OF 19S AND 7S ANTIBODIES SYNTHESIZED BY RABBITS AFTER A SINGLE VIRUS INJECTION

Rabbits injected intravenously with a single high dose of poliovirus (type 1) produced two major 19S and two 75 type neutralizing antibody populations, with different electrophoretic mobilities. The first 2 to 2½ days after immunization the antibody was of the 19S type and migrated as a single component in the β-region. In contrast, the major 19S antibody synthesized from day 3 on was a γ1-globulin. 7S type antibody, appearing on day 4, was contained only in the γ1-region, while 10 to 14 days following immunization the 7S antibody was associated with both γ1- and γ2-globulins. The electrophoretic distribution of antibody in 2-week sera revealed the presence of at least four antibody populations (19S β, 19S γ1, 7S γ1, and 7S γ2). Serum samples collected 1 or 2 weeks following antigenic stimulation often contained small amounts of antibody which sedimented at a rate (11S to 15S) intermediate to that of 7S and 19S antibodies. The neutralizing activity of the 19S type antibodies and the intermediately sedimenting antibody was destroyed by incubation with 0.1 M (5 days) or 0.2 M (1 day) 2-mercaptoethanol at 3°C. The 7S antibody activity was un-affected by treatment with from 0.1 to 0.8 M mercaptan

THE FORMATION AND PROPERTIES OF POLIOVIRUS-NEUTRALIZING ANTIBODY : IV. NORMAL ANTIBODY AND EARLY IMMUNE ANTIBODY OF RABBIT ORIGIN: A COMPARISON OF BIOLOGICAL AND PHYSICOCHEMICAL PROPERTIES

Rabbit sera were found to possess neutralizing activity (normal antibody) to polioviruses and Coxsackie B viruses. This normal antibody showed high specificity in cross-neutralization and absorption tests. It was associated with heat-stable, mercaptan-sensitive, 19S γ1-β-macroglobulins, which formed weak complexes with the viral antigen. In rare instances, sera with normal macroglobulin antibody, also contained very low activity which was due to 7S γ2-globulins. The neutralization of poliovirus by normal 19S γ1-β-antibody appeared to follow first order kinetics, and the thermodynamic parameters of this reaction were the same as those of serological reactions employing immune antibody. The electrophoretic mobility, sedimentation properties, sensitivity to mercaptan, thermostability, and avidity of normal and early (up to day 3) immune antibodies to poliovirus were similar, but differed in several respects from those of late immune antibodies. Thus, the available evidence suggested, that earlier reported differences between normal and immune antibodies reflected differences between antibodies of diverse physicochemical properties rather than between normal and immune antibodies per se. It is proposed that the normal macroglobulin antibody is associated with an immunological response to repeated stimulation with minute amounts of antigen

FURTHER STUDIES ON THE ULTRASTRUCTURE OF DIMERIC IGA OF HUMAN ORIGIN

Human colostral IgA and myeloma dimer IgA were purified and examined in the electron microscope using a modified technique of negative staining. Both types of preparation contained double Y-shaped structures of the dimensions: Fab region, 35 x 70 A, and the sum of the two Fc regions, 40 x 140–155 A. Colostral IgA as well as myeloma dimer IgA molecules showed a tendency of bending at the point where the Fc regions joined. Secretory component bound to dimer IgA produced no visible alteration of the molecule. Mild reduction and alkylation of colostral IgA yielded single Y-shaped 7S monomers with the dimensions, Fab, 35 x 70 A, and Fc, 40 x 65–70 A

THE FORMATION AND PROPERTIES OF POLIOVIRUS-NEUTRALIZING ANTIBODY : I. 19S AND 7S ANTIBODY FORMATION: DIFFERENCES IN KINETICS AND ANTIGEN DOSE REQUIREMENT FOR INDUCTION

Rapid formation of poliovirus-neutralizing antibody was observed in the rabbit. 19S type antibody was detectable 8 to 12 hours following a single intravenous virus injection and the induction period was of the order of 4 to 5 hours or less. The production of 7S antibody had a longer lag phase (1½ to 2 days) and it was formed at slower rate. The observed rate of early 19S and 7S antibody formation as well as the peak titers of the two antibodies were antigen dose dependent. Normal rabbit sera showed low neutralizing activity to several viral antigens in a sensitive assay system. Following intravenous inoculation of poliovirus either transitory (⩽ 1 month) or enduring (¾ to 1½ year) antibody formation resulted depending upon the dose of antigen employed. In transitory responses, which could be induced by a single small antigen dose, only 19S antibody was demonstrable and there was an abrupt cessation of antibody synthesis on day 4 or 5. In enduring responses, both 19S and 7S antibody were formed and the minimum antigen dose required for initiation of such a response was equal to the dose needed for induction of 7S antibody formation. Thus, enduring antibody formation was an all-or-none phenomenon depending upon whether or not 7S antibody formation was induced. The antigen dose requirement for induction of 7S antibody was much higher (by 50-fold or more) than that for 19S antibody. This allowed a determination of antigen dose regions, within which predictably transitory (19S) or enduring (19S + 7S) antibody formation was obtained. These pronounced differences in antigen dose requirement for induction and kinetics of formation of 19S and 7S antibody suggest that the same cells do not participate in the formation of the two antibodies

THE FORMATION AND PROPERTIES OF POLIOVIRUS-NEUTRALIZING ANTIBODY : II. 19S AND 7S ANTIBODY FORMATION: DIFFERENCES IN ANTIGEN DOSE REQUIREMENT FOR SUSTAINED SYNTHESIS, ANAMNESIS, AND SENSITIVITY TO X-IRRADIATION

Transient 19S antibody formation was induced in rabbits by single or repeated stimuli with a small dose of poliovirus. Available evidence indicated that cessation of 19S synthesis was due to lack of continuous antigenic stimulation and not to loss of cells participating in antibody formation. "Immunological memory" in 19S antibody formation was demonstrable only within 2 to 3 days following discontinuation of synthesis but not thereafter. Following stimulation with a high dose of polio-virus both 19S and 7S antibodies were formed. The kinetics of their formation differed in several respects: (a) 19S antibody preceded 7S antibody by ⩾1½ days; (b) 19S antibody rose to peak titers at a rapid exponential rate within 1 week, while 7S antibody increased at a slow decelerating rate for ⩽3 weeks; (c) 19S antibody formation was short-lasting while 7S antibody synthesis endured. A renewed formation of both antibodies occurred following restimulation with a high antigen dose. The secondary 19S and 7S antibody responses were similar to the respective primary responses, and the preexistence of 7S antibody synthesis did not detectably alter the secondary 19S response. Both 19S and 7S antibodies were formed and the kinetics of their formation was similar (a) for infectious and non-infectious (UV-) poliovirus antigen; (b) for the serologically unrelated poliovirus and Coxsackie B-4 virus; (c) when poliovirus was administered by different routes; (d) when 1-day-old or adult rabbits were immunized; (e) in antibody responses to poliovirus in rabbit, guinea pig, and man. Whole body x-irradiation 20 hours prior to antigenic stimulus (high dose) resulted in delayed but markedly prolonged 19S antibody formation and inhibition of 7S antibody synthesis. Thus, the formation of 19S and 7S antibody differed in (a) antigen dose requirements for induction and maintained synthesis; (b) kinetics; (c) retention of memory; and (d) sensitivity to prior x-irradiation. These differences are best explained on the assumption that the two antibodies are produced by different cells

ULTRASTRUCTURE OF PAPAIN AND PEPSIN DIGESTION FRAGMENTS OF HUMAN IgM GLOBULINS

The ultrastructure of papain and pepsin-digested products of human IgM globulins has been analyzed. Papain digestion was performed both in the presence and absence of cysteine. The Fcµ fragment was found to represent the central ring structure in the intact IgM molecule, plus a minor part of the appendages extending from the ring. The Fcµ ring structure was occasionally seen to be composed of dimers of short rods, probably identical with the endpieces of two µ-chains. Such dimeric structures, released from the intact Fcµ rings, had a tendency to aggregate sidewise, producing complexes of varying size. The dimensions of the Fcµ fragments were: outer diameter approximately 85 A, inner diameter about 40 A. The length of the protrusions varied from 20–30 A. The Fabµ preparations contained long strands of sidewise aggregated, short rod-shaped fragments. No aggregates were seen in the F(ab'')2µ preparations. The two Fab''µ units in the dimeric F(ab'')2µ fragments were usually parallel to each other. The dimensions of the Fabµ and F(ab'')2µ fragments were 50–80 A x 30 A and 75–80 A x 55 A, respectively. These findings provide morphological evidence that the C-terminal ends of the µ-chains (the Fcµ fragment) make up the central ring structure in the IgM molecule. They further indicate that the F(ab'')2µ fragments constitute about ¾ of the appendages extending from this ring structure

The production and properties of poliovirus neutralizing antibody of rabbit origin, Virology

A previous communication (1) in this series reported that transient, very rapid macroglobulin antibody responses could be induced in rabbits by small doses of poliovirus (PV) antigen. 19S antibody appeared 8 to 16 hours following immunization in such responses. It was also observed that almost all normal rabbit sera (NRS) contained low background neutralizing activity to several viral antigens, among them PV (2). In the present study the physicochemical nature, cross-reactivity, and avidity of the serum factor responsible for this background neutralization of PV and its relationship to the rapid 19S antibody response, were explored. Normal antibody has been reported to differ from immune antibody in specificity (3) and thermostability (3-6). While in those studies normal antibody was compared with immune antibody collected at a fairly late stage in the course of immunization, in the present study, normal antibody was compared with the earliest detectable induced antibody. Under these circumstances it was found that the physicochemical properties, cross-reactivity, and avidity of normal an

FURTHER STUDIES ON THE ULTRASTRUCTURE OF DIMERIC IGA OF HUMAN ORIGIN*

We have previously reported on the architecture of secretory IgA (s-IgA) 1 protein molecules (1) using electron microscopy and the conventional technique for negative staining described by Brenner and Home (2). A modification of this technique, the so-called mica technique, was introduced by Valentine (3). The mica technique has been found particularly useful in studies of certain macromolecules such as immunoglobulins (4, 5) which are difficult to distribute evenly on a hydrophobic carbon film by the conventional method. The aim of the present investigation was to procure further ultrastructural information on dimeric human IgA and to compare the merits of the mica technique with the conventional method for negative staining. Materials and Methods Purification of IgA.--Secretory IgA from human colostrum, collected within 24 hr after delivery, was purified by a combination of ultracentrifugation and gel filtration chromatography on a tall (2.5)< 400 cm) Bio Gel A 15 m column (Bio-Rad Laboratories, Richmond, Calif.) as earlier described (1). Five different human colostral IgA preparations were prepared and examined. Serum from a patient with IgA myeloma ~ was fractionated by a two-step procedure. 10 ml of serum was gel filtered on the same tall Bio Gel column as was used for purification of the s-IgA material. Tubes containing high polymer IgA, based on immunodiffusion analyses and position on the elution curve, were concentrated by vacuum dialysis. This material also contained some ~-lipoprotein, a2M globulin, and haptoglobln. These contaminating components were removed on an immunoadsorbent column by polymerizing a protein solution (50 mg/ml) containing specific antibodies against these contaminants. The protein solution was dialyzed for 24 hr agMnst 0.15 M NaC1 buffered with 0.01 ~ phosphate, pH 7.2 (PBS), and polymerization was performed by adding glutaraldehyde (TAAB, Reading, England) at a final concentratio

The formation and properties of poliovirus neutralizing antibody. IL 19S and 7S antibody formation: differences in antigen dose requirement for sustained synthesis, anamnesis, and sensitivity to x-irradiation

The use of plaque-forming techniques in animal virology has markedly increased flae sensitivity and precision of measuring neutralizing antibody, and this together with other developments such as the ready availability of large quantities of relatively pure viral antigens and new techniques for characterizing antibody have prompted a reexamination of several aspects of the formation and properties of viral neutralizing antibody. In the work being reported a poliovirus-rabbit antibody system has been extensively studied with particular emphasis on experiments concerned with the length of the induction period, changes in properties of antibody produced at different times after an antigenic stimulus, differences in!9S and 7S antibody formation and on the ability of animals to display "immunological memory. " The combination of high sensitivity and high accuracy in measuring antibody to poliovirus (PV) makes an excellent tool permitting for example the study of the very early appearance of circulating antibody. In this work, to be reported in a series of papers, we have sought to (a) analyz

ULTRASTRUCTURAL STUDIES OF HUMAN AND RABBIT

biological function has remained obscure for a long time. In 1962 Haverback et al. (2) described an otrgiobulin ill human plasma which combined with trypsin or chymotrypsin. This combination did not render the enzymes inactive but made them inaccessible to certain inhibiting proteins. Mehl et al. (3) reported that the trypsin-binding protein was the macro-constituent of the a2region. Recent studies have indicated that human a2-macroglobulin plays a significant role in a number of enzymological reactions. Different authors have described its binding to plasmin (4), thrombin (5), insulin (6, 7), growth hormone (8, 9), and elastase (10). Ganrot (11) has demonstrated a correlation between the a~M-concentration and the esterolytic activity of the trypsin-a~YI complex in human sera. Elevated a2-macroglobulln levels, normally seen in pregnant women and in children, can also be found in patients with nephrosis. The orielmolecule is considered to be rather unstable, and its degradation products migrate faster than the complete molecule in starch electrophoresis (12). The viscosity data of SchSnenberger et al. (13) suggested that the a~Ylmolecul