γG Antibodies Research Articles

The kinetics of hemolytic plaque formation III. inhibition of plaques by antigen

Equations are presented which can be used to describe the inhibition of plaques by multifunctional antigen which binds γG antibody bivalently. The interaction is treated as a bimolecular reaction which is irreversible within the time of the experiment. It is shown that under these conditions the characteristics of the inhibition curve, and their relationship to kinetic and thermodynamic parameters are strongly dependent upon how antibody interacts with RBCs. When the epitope coating is dense, multivalent attachment of antibody is likely and the interaction is considered irreversible. When the epitope coating is sparse, only rapid, reversible univalent attachment is considered and local equilibrium is assumed to hold. The first case leads to an abrupt inhibition curve whose position is determined by the forward rate constant and RBC density. The second case leads to broad asymmetric curves. For this situation the relation between the extent of inhibition and the affinity of the population is generally complicated and reliable affinity information is difficult to obtain. This is contrasted to results obtained previously for unifunctional inhibitors from which reliable affinity information can, in principle, be obtained. The results emphasize the need for carefully designed experiments if affinity information is to be obtained from inhibition studies.

Antibodies and surface immunoglobulins of immunized congenitally athymic (nu-nu) mice.

SummaryCongenitally athymic mice homozygous (nu/nu) for tile mutation “nude” and control littermates (nu/+) possessing normal thymus function were immunized with E. coli lipopolysaccharide. Nu/nu and nu/+ animals responded well to this thymus‐independent antigen, giving identical titres of serum antibodies. Unexpectedly, nu/nu mice as well as nu/+ animals produced both γM anti γG antibodies by 16 days following a single injection. Surface immunoglobulins of immunized nu/nu and nu/+ mice were isolated by lactoperoxidase‐catalyzed radioiodination, detergent extraction and immunoglobulin coprecipitation. The predominant surface immunoglobulin component on cells of both types of mouse was 7S γM immunoglobulin. The amount of γM surface immunoglobulin recovered from nu/nu mice was generally comparable to that extracted from nu/+ and normal CBA mice.

Local Antibody Response to Experimental Poliovirus Infection in the Central Nervous System of Rhesus Monkeys

By employing the techniques of immunofluorescence and radioimmunodiffusion using (32)P-labeled poliovirus as the antigen, the immunoglobulin response to poliovirus in serum, nasopharynx, spinal fluid, and in different segments of the central nervous system (CNS) was studied after intramuscular, oral, intranasal, and intrathalamic administration of inactivated (Salk), live attenuated (Sabin), or live virulent (Mahoney) type I poliovirus. Spinal fluid gammaG antibody was detected after immunization with Sabin or Mahoney virus and intramuscular administration of Salk vaccine. The response in the CNS was characterized by the appearance of gammaG antibody after oral or intrathalamic administration of Mahoney virus and rarely after intrathalamic inoculation of Sabin vaccine. The antibody activity in CNS was limited to the areas of poliovirus replication. Intrathalamic immunization with Mahoney virus resulted in local gammaG antibody production in the CNS in the absence of any detectable response in serum. Discrete foci of gammaG-containing cells were observed in those areas of CNS which contained poliovirus antibody. No immunoglobulin-containing cells or poliovirus antibody was seen in the CNS of monkeys immunized with intramuscularly or orally administered Sabin or Salk vaccine and in sham-immunized control monkeys. It is suggested that the CNS, when stimulated locally with a potent replicating viral antigen, may manifest a specific local antibody response, which is independent of the response in serum.

Immunologic Aspects of Hepatitis-Associated Antigen and Antibody in Human Body Fluids

Abstract Employing the techniques of immunoelectrophoresis and indirect immunofluorescence with smears of peripheral blood, we determined the presence of hepatitis-associated antigen (HAA) and anti-HAA antibody in concentrated specimens of serum, nasopharyngeal washings, urine, and fecal extracts from patients with icteric or asymptomatic HAA infection. Highest concentrations of HAA in the serum were demonstrable at the time of initial screening. HAA was detected in the nasopharyngeal washings and urine for a short period in a few subjects. All subjects manifested appreciable quantities of HAA in fecal extracts and the antigen persisted in the feces for as long as 4 months. Anti-HAA antibody response in the serum was characterized by infrequent appearance of small amounts of γG antibody 2 to 3 months after the initial detection of antigen in the serum. No antibody activity was detected after 7 months. The response in the nasopharynx and urine was characterized by transient appearance of small amounts of γA and infrequently of γG class of antibody. The response in the fecal extracts was characterized initially by frequent appearance of HAA-anti-HAA complexes. Subsequently, appreciable levels of free γA antibody to HAA were detected for as long as 8 months. It is suggested that HAA may replicate in various mucosal surfaces, and such a mechanism may explain the nonparenteral transmission of long incubation hepatitis.

Local Antibody Response to Poliovaccine in the Human Female Genital Tract

Abstract Antibody response to poliovirus type I in serum, the nasopharynx, and the secretions of the genital tract was studied in human volunteers after intravaginal, intrauterine, nasopharyngeal, or intramuscular immunization with inactivated poliovaccine. The techniques of radioimmunodiffusion and autoradiography with P32 labeled poliovirus as the antigen were employed to determine antibody activity in the major classes of immunoglobulin in serum and secretions. Intravaginal and intrauterine immunization consistently resulted in the appearance of secretory antibody to poliovirus in the genital tract. The vaginal response was predominantly of γA immunoglobulin, while the response in the uterus was essentially limited to γG immunoglobulin. Intramuscular immunization resulted in a delayed appearance of γG response in the genital tract, which could be correlated with the highest γG antibody titers in the serum. No genital γA response was observed, however, after such immunization. These observations provide evidence for local synthesis of poliovirus antibody in the genital tract, and its implication may be applicable to other genital infections.

Cis- and trans-membrane control of cell surface topography.

AbstractThe topographic distributions of cell surface components can be modified by perturbations from the cell membrane exterior (cis‐membrane effect) or by perturbations from the cell interior that are transferred across the membrane (trans‐membrane effect). Using the human erythrocyte ghost as a model system, cis‐membrane effects on the topography of anionic sites were produced in B+ ghosts with anti‐B sera (but not with anti‐A), R. communis agglutinin and concanavalin A (but not with D. biflorus agglutinin). Cis‐membrane linkage was monitored by the state of aggregation of membrane‐bound colloidal iron hydroxide particles which bind almost exclusively to neuraminidase‐sensitive N‐acetylneuraminic acid residues on the outer surface. Trans‐membrane effects were observed when purified antibodies against an inner surface membrane protein, spectrin, were sequestered inside the ghosts. The sequestered antispectrin bound to spectrin at inner membrane surface and caused aggregation of the anionic sites on the outer membrane surface. The trans‐membrane effects of antispectrin required intact γG antibodies (Fab would not substitute) and was time‐ and concentration‐dependent.

In vitro adherence of soluble immune complexes to macrophages.

The adherence of soluble immune complexes to stimulated alveolar macrophages was studied in vitro using HSA-anti-HSA complexes prepared in antigen excess. Those complexes containing more than two molecules of antibody preferentially adhered to macrophages in the absence of complement. Free gammaG in less than physiological concentrations inhibited the adherence of complexes, and the presence of complement did not significantly alter this inhibition. Complexes prepared with reduced and alkylated antibodies showed a decreased adherence. The strength of binding of soluble complexes to macrophages and their efficiency in fixing complement were greater than seen with small aggregates of homologous gammaG. These differences in biological properties were observed even though the immune complexes and aggregates contained comparable numbers of gammaG molecules. The gammaG receptor on rabbit macrophages exhibited species specificity. Pretreatment of macrophages with proteolytic enzymes led to adherence of larger amounts of soluble complexes. These observations suggest that the strength of binding of soluble immune complexes to macrophages and their efficiency in fixing complement are not determined solely by a random summation of individual binding sites. It is proposed that conformational changes in the gammaG antibodies or a specific molecular arrangement in the lattice work of complexes containing large protein antigens may influence the biological properties of the soluble complexes.

A Receptor for Fc on Mouse B-Lymphocytes

Abstract Antigen-antibody complexes derived from rabbit antibodies bind to mouse spleen lymphocytes. This binding was detected by the inhibition of reverse immune cytoadherence (RICA), a technique detecting surface γ globulin and suggests that these complexes bind to γ globulin carrying B-cells. Complement is not a requirement for binding. Complexes made from 5S bivalent antibodies do not bind nor does 7S antibody or normal γ globulin. Chemically produced microaggregates of ferritin show no binding. Mouse complexes made with mouse γG (7Sγ2a) antibodies also bind to mouse spleen cells. Fc fragments isolated by papain digestion of rabbit antibodies also inhibit RICA and Fc crystals can form around the mouse spleen lymphocytes. Fc from normal rabbit γ globulin is less inhibitory. These results are presented as evidence of the existence of a receptor, provisionally called the Fc receptor, on the surface of mouse spleen lymphocytes which are probably B-cells.

Studies on M and G antibody response of mice to bovine serum albumin. II. Induction of tolerance.

Tolerogenic effect of soluble deaggregated bovine serum albumin (sBSA) on the γM and γG antibody responses was studied. Normal and tolerogen-treated animals were immunized with the intravenous injection of alum-precipitated BSA (net 0.1 mg) and a bacterial endotoxin (0.01 mg), since this immunizing procedure was the most adequate to elicit the strong responses of both types of antibodies. Both γM and γG responses were decreased evenly by a single injection of small dose (0.01 or 0.1 mg) of sBSA. Larger doses (1 mg or 5 mg) caused the marked suppression of γM response, but the γG response was reduced only slightly. The tolerant state was facilitated by weekly injections of 0.1 mg of sBSA, both γM and γG responses being abrogated profoundly by more than three injections. The induction of such a complete tolerance was not ascribed solely to either the total dose or the period of time between the first tolerogen injection and the challenge immunization, but to the retention of antigen more than a certain threshold amount in the body for some duration.

Neutralization of Haptenated Bacteriophage F2 by Anti-Hapten Antibodies: Direct Evidence for a Critical Site

Abstract The neutralization of trinitrophenyl (TNP) derivatives of the small RNA bacteriophage f2 by rabbit anti-DNP antibodies was studied by the plaque assay technique. In agreement with previous work using other hapten-bacteriophage conjugates, we found that whole antibody was much more effective in the neutralization reaction than were corresponding Fab fragments. Measurement of the association constant for whole antibody with heavily substituted phage derivatives (TNP305-f2) gave a result (Ka = 3.2 × 1010) which was 103 greater than the corresponding value for Fab fragments. This difference was, in part, reflected in a 400-fold decrease in the second order rate constant for the phage-antibody association reaction, in going from γG antibody to Fab fragment. Analysis of these results lends further support for a simple steric model of phage neutralization by antibody. The relative effectiveness of antibody in neutralizing TNP-f2 conjugates was in large measure controlled by the degree of phage modification. For example, a lightly substituted f2 derivative (TNP3-f2) remained fully viable in the presence of a large excess of antibody, providing direct evidence for the infectivity of antibody-f2 aggregates. A quantitative study of the infectivity of another lightly substituted TNP-f2 conjugate (TNP30-f2) led directly to a model of f2 neutralization, in which an antibody molecule must directly interact with a single critical site on the surface of f2 in order to inactivate the phage. A consideration of the icosahedral structure of f2 suggests that f2 neutralization occurs by attachment of a single antibody molecule at or in the vicinitv of A-protein.

Studies on M and G antibody response of mice to bovine serum albumin. I. A systematic survey of immunizing conditions.

Response of CBA mice with γM and γG antibodies to bovine serum albumin (BSA) was studied in relation to a variety of conditions of antigen administration. The variables in the conditions were doses and physical forms of antigen, and injection routes. It was realized that γG antibody response to soluble BSA and both γM and γG antibody responses to particulate forms of BSA were augmented as the dose was increased. The γM response to soluble BSA was not elevated by an increase in the amount of antigen up to 1 mg. The soluble form was not so immunogenic as the particulate forms, in which alum-precipitated BSA was capable of inducing both γM and γG antibodies to high titers, and heat-denatured BSA elicited preferably antibody. Alum-precipitated BSA and the emulsified BSA were strong inducers for γG antibody response when injected subcutaneously. In any antigen form, γM response was markedly influenced by changing the injection route, the order of decreasing efficiency for antibody being intravenous, intraperitoneal and subcutaneous. The γG antibody response was hardly affected by the injection route. The effect of a single intravenous injection of 0.01 mg of endotoxin, given 1 to 2 hr after antigen injection, on γM and γG antibody production differed according to the antigen administration procedures. Generally speaking, this agent had an enhancing effect when the antigen was given in the particulate forms, and it depressed the response when the antigen was given in the soluble form.

The influence of polyvalency on the binding properties of antibodies

We present a general formalism for predicting the relationship between the binding affinities in several kinds of antigen-antibody interactions. The reference binding constant is that of monovalent antibody for monovalent antigen. The equations then allow one to predict the ratio of this constant to that for attaching divalent antibody to divalent antigen. We further develop the theory to consider the binding of multivalent antibodies to a multideterminant antigen particle, in the restricted case of small fractional occupancy of the determinant sites by antibodies. There is good agreement between the calculated results and certain data from the literature.In situations where multisite adherence to a single particle and cross-linking of discrete particles are both possible, the former is predicted to predominate strongly. That the opposite appears sometimes to be experimentally the case means that special features favoring agglutination reactions must be present in these instances.Finally, those factors that potentially make polyvalent γM antibodies more effective agglutinators than bivalent γG antibodies are delineated briefly.

The gammaG subclass of antinuclear and antinucleic acid antibodies.

AbstractSelected sera from patients with systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS) were studied to determine the γG subclass in which the antibody activities were present. These sera were known to contain γG antibodies to nuclear antigens, cytoplasmic antigens or nucleic acids. Antibodies to DNA and those giving a peripheral pattern in the fluorescent antinuclear antibody test (ANF) primarily were of the γG1 and γG3 types; antibodies giving a speckled pattern primarily were of the γG3 type; and antibodies giving a diffuse pattern in the ANA test primarily were of the γG3 type, and less so of the γG1 type. These findings indicate: that antibodies to some nuclear antigens and nucleic acids may be restricted to certain γG subclasses; and that anti‐DNA antibodies, which have been most strongly associated with low serum complement levels and active lupus nephritis, are found primarily in those γG subclasses which fix complement most efficiently—namely, γG1 and γG3.

86.Studies on the mechanisms of γM and γG antibody response in mice.I.Difference in responses with antigen administrations

86.Studies on the mechanisms of γM and γG antibody response in mice.I.Difference in responses with antigen administrations

Induction and properties of rabbit secretory γA antibody directed to group a streptococcal carbohydrate

Secretory γA antibodies were induced in the colostrum of New Zealand Red rabbits by inejection of Group A streptococcal vaccine into the mammary glands. Colostral antibodies were purified by elution from Group A cells used as solid immunoadsorbent. Antibodies of both secretory γA and γG class were obtained upon elution with 5 per cent N-acateyl glucosamine (NAG) or pH 2·5 buffer. The eluates were filtered through Sepharose 6B to separate the 11 S γA from the 7 S γG antibodies. From 4·2 to 13·1 μg of γA per ml of original colostrum was obtained by elution with 5 per cent NAG and from 20·4 to 208·8 μg of γA per ml original colostrum was obtained by elution with pH 2·5 buffer. The amount of γG antibody obtained in these eluates varied greatly, ranging from 90 to 10 per cent of the total antibody recovered. Purified γA anti-NAG antibodies isolated from a single rabbit exhibited the following properties: (1) They agglutinated Group A cell was. (2) They bound to Group A cell was as demonstrated by indirect fluorescent antibody straining using monospecific fluorescin-labeled goat anti-rabbit γA. (3) They failed to precipiate with Group A carbohydrate under conditions in which the γG anti-NAG antibody was found to precipitate. (4) Secretory γA antibodies, however, inhibited the precipitation of the γG antibodies with Group A carbohydrate. (5) They contained secretory component as detected by gel diffusion tests. (6) The secretory γA antibodies bound triated p-nitrophenyl-β-N-acetyl glucosaminide in equilibrium dialysis experiments. A plot of r vs c for the binding of the hapten by γA antibodies shows that r increased with increasing hapten concentration and that there was a plateau as r approached 4, indicating that secretory γA may have a valence of 4. An association constant of the order of 10 5 l/m could be estimated from the data; however, because of the low association constant there was considerable sctter of points. The association constant for γG anti-NAG, isolated from the same colostrum was about one log lower when tested with the same hapten. Neither γA or γG antibodies bound detectable amounts of triated N-acetyl glucosamine, suggesting that the β-linkage makes an important contribution to binding. Many of properties of described for secretory γA antibody can be explained by a preference of this antibody to bind most of its combining sites to a single molecule of possessing multiple identical antigenic determinants as opposed to cross-linking two or more such molecules. This property, termed monogamous polyvalency, could significantly enhance certain biologic functions of secretory γA.

Passive protection of mice against intraperitoneally injected Vibrio cholerae by G and M antibody.

Fractions of rabbit anti-Vibrio cholerae serum containing gammaG antibodies were compared with fractions containing gammaM antibodies for their ability to protect mice against lethal infection resulting from the intraperitoneal injection of organisms suspended in mucin. About twice as much gammaG as gammaM (estimated by quantitative precipitation) was required to protect against approximately 1,000 50% lethal doses when the antibody was given intraperitoneally 4 hr before challenge. When protective serum fractions were given subcutaneously, however, the amount of gammaM required to protect was increased about 40-fold, whereas gammaG was about equally effective subcutaneously and intraperitoneally.

Studies of the Mechanism of Binding of Chemically Modified Cytophilic Antibodies to Macrophages

Abstract Chemical modification of lysine or tryptophan groups of rabbit γG antibody led to marked reduction in its cytophilic binding ability. The procedures used, carbamylation, amidination and benzylation, were highly selective in their effect on the various properties of antibody. Thus, although they led to loss of complement-fixing ability and diminution in the rate of immune aggregation, they had no significant effect on the primary union of antibody with antigen or on the ability of antibody to mediate anaphylaxis in the guinea pig. This selectivity suggested that the modifications led to specific alteration of Fc binding sites rather than nonspecific alteration of the molecule as a whole. The fact that these chemical alterations affect both cytophilic binding and complement fixation does not imply a functional relationship between the two. Cytophilic binding did occur in the absence of complement. Moreover, the various treated antibodies had dissociation of these activities; concentrations of conjugated antibody sufficient for cytophilic rosette formation were not capable of fixing enough complement for detectable erythrocyte lysis. In this regard, differences in the ability of untreated antibody to mediate rosette formation and complement fixation in the direct and indirect procedures for cytophilic binding suggest that the mechanisms of complement-antibody and macrophage-antibody interaction are distinct, even though both reactions can be a property of the same antibody molecule.

Poliovirus antibody response in patients withacute leukemia

Poliovirus antibody response in γG, γA, and γM classes of immunoglobulins in serum was determined after immunization with inactivated (Salk) polio vaccine in children with acute lymphocytic leukemia, in healthy children (control), and in children with solid tumors: Ewing's, Wilms', and osteogenic sarcoma. Serum response in healthy children and in children with solid tumors was characterized by the regular appearance of high levels of γM and γG and occasionally of γA poliovirus antibody. Primary immunization in children with acute leukemia elicited low or undetectable γG and no γA poliovirus antibody response. Most significantly, however, γM poliovirus antibody response was conspicuously absent. Reimmunization with inactivated vaccine in patients with acute leukemia manifested a 2- to 3-fold rise in the pre-existing γG antibody levels. Although reimmunization resulted consistently in the reappearance of γM poliovirus antibody in previously immunized control subjects, patients with acute leukemia failed to elicit any γM antibody response.

IMMUNE RESPONSES IN VITRO

We have demonstrated for the first time that mouse spleen cells stimulated in vitro with heterologous erythrocytes developed immunoglobulin class-specific gammaM, gamma(1), gamma(2a+2b), and gammaA plaque-forming cell (PFC) responses. A modification of the hemolytic plaque technique, the addition of goat anti-mouse micro-chain antibody to the assay preparation, specifically prevented development of all gammaM PFC and enabled accurate and reproducible enumeration of immunoglobulin class-specific PFC after treatment with appropriate monospecific anti-globulins and complement. Culture conditions, with regard to medium, atmosphere, agitation, and spleen cell densities, were similar to those previously shown to support only gammaM PFC responses. Evaluation of the kinetics of appearance of PFC showed that gammaM PFC reached maximum numbers on days 4-5; the magnitude of this response was 3-10 times greater than gamma(1) gamma(2a+2b), or gammaA PFC which reached maximum numbers on days 5-6. Optimal erythrocyte antigen dose for gammaM PFC responses was 10(7)/culture, whereas a dose of 10(6) erythrocytes/culture consistently stimulated optimal gamma(1) gamma(2a+2b), or gammaA PFC responses. Investigations of the effects of anti-erythrocyte antibody on gammaM and gammaG PFC responses indicated that antibody suppressed these responses by neutralizing the effective antigenic stimulus at the macrophage-dependent phase of the response. At the same antibody concentration, gammaG PFC responses were more effectively suppressed than gammaM PFC responses. Further, gammaG responses could be almost completely suppressed by antibody as long as 48 hr after initiation of cultures, whereas gammaM PFC responses could only be completely suppressed during the first 24 hr. These results were discusssed in terms of the role of antigen in the stimulation gammaM and gammaG antibody.

Regulation of Homocytotropic Antibody Formation in the Rat

Abstract The effects of four representative immunosuppressants, i.e., 5-bromouridine deoxyriboside (BUDR), actinomycin D, cyclophosphamide and cortisone, on the formation of homocytotropic antibody (HTA) were observed in rats immunized with dinitrophenylated ascaris extracts (DNP-As) and killed Bordetella pertussis, with the following results. 1) A single injection of 35 mg/kg of BUDR given simultaneously with or shortly after the start of immunization caused enhancement and prolongation, after some delay, of HTA formation, as measured by the mean of reciprocal PCA titers. 2) Pretreatment with 0.3 mg/kg of actinomycin D caused a delayed response of HTA, but the subsequent mean peak titer was about the same as that of the control untreated rats. Such treatment after immunization was started caused an absolute stimulation of HTA, as judged by high peak titers and prolonged production of the antibody. 3) Pretreatment with a single injection of 150 mg/kg of cyclophosphamide caused delayed but enhanced formation of HTA which lastedo lnger than the normal period of time, whereas the same treatment started 2 days after the antigen was given caused suppression. 4) Pretreatment with 200 mg/kg of cortisone caused temporary enhancement, whereas treatment during immunization was moderately suppressive. The enhancement or suppression of HTA formation did not correlate with γM and γG antibodies in the serum, as measured by passive hemagglutination titers before and after 2-mercaptoethanol treatment.