Serological properties of γA antibodies to Escherichia coli present in human colostrum

Antibodies against Esch. coli WF 96 and WF 61 present in human colostrum and serum were fractionated by DEAE-cellulose chromatography. Using the haemagglutination test it was found that the antibodies present in colostrum were recovered in the fraction containing the bulk of γA-globulin, whereas the antibodies present in serum were recovered in the fraction containing the bulk of γM-globulin. In the presence of human or guinea-pig complement the antibodies present in colostrum did not lyse red cells coated with bacterial polysaccharides whereas the antibodies present in serum were lytic.

When the properties of γA and γM antibodies were studied using a bacteriolytic system, it was observed that γA-globulin lysed bacteria only in the presence of both complement and lysozyme; in this respect γAbacterial antibodies behaved differently from γM antibodies which were bacteriolytic in the presence of complement alone, without lysozyme.

The effect of treating γA and γM antibodies with 2-mercaptoethanol at neutral pH and of heating at 56° was investigated.

     

Comparative studies of immunoglobulin opsonins in osteomyelitis and other established infections

The opsonic activity of serum and isolated immunoglobulin fractions has been studied in twenty patients with osteomyelitis, using a quantitative assay which measures phagocytosis and killing of bacteria by human polymorphonuclear leukocytes. Opsonic functions as well as agglutinating and complement-fixing antibodies were compared with normal human sera as well as with a control group of eighteen patients with miscellaneous established severe infections. A surprising lack of heat stable γG opsonic activity, particularly for various species of Staphylococcus aureus, was documented among many patients with chronic active osteomyelitis. A similar apparent deficiency or low level of heat stable γG opsonin was also demonstrated among some of the controls with severe soft tissue infections. These weak opsonins showed a marked contrast to the potent heat stable γG opsonin activity previously documented in sub-acute bacterial endocarditis. Heat stable γG opsonin proved to be present in effective concentration in Gram-negative infections of bone and in the control group. Evidence was obtained for competitive blocking of phagocytic mechanisms by relatively weak immune γG opsonins allowed to react with bacteria prior to contact with more potent γG opsonins subsequently added to the test system. High titres of agglutinating anti-bacterial antibodies contrasted with relatively low levels of complement-fixing antibodies in many patients with chronic osteomyelitis. Opsonic activities of both 7S and 19S fractions of heat inactivated sera were studied in sera from osteomyelitis patients and control infections. When phagocytosis-promoting properties were not detectable in such fractions, opsonic capacity could be effectively restored by adding fresh serum devoid of anti-bacterial antibodies but possessing complement activity. Opsonic capacity of serum fractions then depended on the marked facilitative effect of heat labile factors. Activity ratios were derived of opsonic activity to actual amount of immunoglobulin present acting in concert with a constant amount of complement.     

Serological Properties of gammaG and gammaM Antibodies to the Somatic Antigen of Vibrio cholerae During the Course of Immunization of Rabbits

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

The use of immunoadsorbents for the fractionation of rheumatoid factor and rabbit anti-human γG antibodies

Conjugates of diazotized polyaminostyrene and human γ-globulin (PAS-γG) have been employed in adsorption and immunochromatographic† studies of rheumatoid factor serum (RFS) and anti-human γG antiserum. Euglobulin fractions of RFS were chromatographed on PAS-γG columns using stepwise elution with buffers of decreasing pH. At pH 6.3 much of the protein content was eluted but no RF. Between pH 5.25 and pH 4.0 seven fractions of RF agglutinating activity were obtained. In most cases, upon re-chromatography the RF fractions were recovered at the same pH at which they were originally obtained. Treatment of RF fractions with mercaptoethanol resulted in complete loss of agglutinating activity. The capacity of aggregated γG to inhibit agglutinating activity was determined. The earlier fractions obtained from the column required more aggregated γG to inhibit their agglutination in the tanned sheep cell test than did the later fractions.

These results are consistent with previous reports of rheumatoid factor heterogeneity. The variations observed in elutability and inhibitability of the RF subfractions suggest that they differ in there affinity for pooled γG.

The immunochromatographic pattern of an anti-γG antiserum differed from that of the RFS in that no antibody was eluted until pH 4.0 and pH 3.2 where two fractions were obtained. These results reflect the greater affinity of γG antibody as compared to RF, for pooled γG.

     

Immunoglobulin production in the European pond tortoise, Emys orbicularis, immunized with serum protein antigens

The immunological responses of the European pond tortoise, Emys orbicularis, to BGG and sheep serum proteins indicate that in this tortoise there is no serum component corresponding with mammalian albumin and that both γG (7S) and γM (19S) immunoglobulins are involved when antibodies are produced. A prolonged period of γM antibody production occurs after primary immunization. The separation of the γM and γG immunoglobulins was performed using starch block electrophoresis and Sephadex G-200 gel filtration. The separated immunoglobulins were characterized by immunodiffusion and by starch gel, agar gel and immunoelectrophoresis.     

Autoimmune haemolytic anaemias. III. Preparation and examination of specific antisera against complement components and products, and their use in serological studies

The preparation and examination of agglutinating antisera, specific for β1E, β1A, α2D and the /β1C/ determinant of the β1C molecule are described. With these reagents it could be established that anticomplement serum does not contain antibodies against the B determinant of the β1C molecule, while antiserum prepared with β1C globulin does. Further, the complement factors present on red cells, the complement coating of which was effected in various ways, were studied.

Whereas red cells incubated in vitro with haemolytic iso- or auto-antibodies are agglutinated by anti-β1E, anti-β1A and anti-α2D sera, the cells of patients with autoimmune haemolytic anaemia of any of the serologically defined groups of this disease, only reacted with anti-α2D. The possible significance of these findings is discussed.

     

Antibodies against degradation products of polysaccharide antigens

Specific antibodies against periodate oxidized capsular polysaccharides isolated from different Klebsiella types, have been demonstrated in rabbit immune sera against five out of eleven Klebsiella types investigated. It is assumed that an oxidation similar to the periodate oxidation must occur in vivo. Antibodies specifically directed against the oxidized polysaccharide may appear early in the immunization period, and decrease or disappear again later.

Reduction and alkylation of the antibodies against oxidized polysaccharide in one serum indicated that they probably belonged to the γM-immunoglobulins, whereas the antibodies against the unoxidized polysaccharide appear to be mainly γG.

     

The circulating immunoglobulins involved in protective immunity to the intestinal stage of Nippostrongylus brasiliensis in the rat

Antisera taken from rats after one, two or three larval infections with Nippostrongylus brasiliensis or after one infection of adult worms were separated by chromatography and the fractions were tested for passive protective activity.

Irrespective of the number or type of infections given, protection against the parasite was most frequently obtained with fractions of antisera containing predominantly 7Sγ₁-globulin. γM- and γA-globulins sometimes appeared to contribute to protective activity, but γM-globulin was not the major source of protective antibodies in antisera taken after a primary infection either of adult worms or of larvae. 7Sγ₂-protective antibodies were found only in a pool of antiserum taken after three infections.

Rats were passively protected with antiserum or fractions of antiserum, which were free of reagins and of other identifiable anaphylactic antibodies. It appears that rats can be passively immunized with antisera which do not have anaphylactic activity.

     

Immune responses in congenitally thymus-less mice. II. Quantitative studies of serum immunoglobulins, the antibody response to sheep erythrocytes, and the effect of thymus allografting

The concentrations of immunoglobulins in the sera of congenitally thymus-less `nude' (nu/nu) mice of various ages were studied by single radial diffusion and compared with those in phenotypically normal nu/+ controls. γM was present in normal concentrations. γA and γG2a concentrations were greatly reduced. γG1 was normal at the time of weaning (28 days of age), but fell progressively thereafter until it was indetectable in most animals more than 60 days old. The primary response to 4 × 10⁷ sheep erythrocytes was studied by haemolytic plaque and serum haemagglutination techniques. Nu/nu mice made subnormal quantities of 19S and little or no 7S antibody. Subcutaneous grafting of neonatal CBA/H thymus at 10–25 days of age was followed by increased survival of nu/nu mice and by the appearance of normal concentrations of γG2a and γG1 globulins and near-normal concentrations of γA. Intraperitoneal injection of 10⁸ CBA/H thymus cells resulted in still higher concentrations of these three proteins. It is concluded that the establishment of normal serum concentrations of γA, γG2a and, especially, γG1 depends upon the presence of funtional T-lymphocytes. γM shows no such T-dependence.     

Bacteriostatic effects of horse sera and serum fractions on Clostridium welchii type A, and the abolition of bacteriostasis by iron salts

Under a variety of conditions of concentration, Eh, and pH, horse anti-Clostridium welchii serum and normal horse serum exerted similar bacteriostatic effects against Cl. welchii Type A. Ferric iron abolished the bacteriostatic effect when added during the first 2 hours of incubation at Eh+60 mV. Ferrous iron abolished the bacteriostatic effect when added after 3 hours. Ferric iron abolished the bacteriostatic effect at—140 mV. A mixture consisting of horse β₂- and γ-globulins together with human transferrin exerted a bacteriostatic effect similar to that of whole serum. This system responded in the same way as whole serum to the addition of iron. A mixture of horse β₂- and γ-globulins exerted an immediate bactericidal effect which could not be prevented by ferric iron.     

γ1-Antibodies in guinea-pigs infected with the cattle lungworm

The parameters of a system for testing both actively acquired and passive immunity to the cattle lungworm in the guinea-pig are described. It was found that the passive transfer of guinea-pig immunoglobulins conferred a strong immunity. The globulins responsible for immunity were present in a serum fraction containing electrophoretically fast moving globulins and anaphylactic activity. Guinea-pigs were not protected by artificial immunization with adult worm homogenate even though precipitating and anaphylactic antibodies were produced. Artificial immunization may have failed because γ₁- and γ₂-antibodies were produced against irrelevant antigens or because blocking γ₂-antibody was produced in excess.     

Characterization of the antibody response against Plasmodium falciparum erythrocyte membrane protein 1 in human volunteers

The immune response against the Plasmodium falciparum variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a key component of clinical immunity against falciparum malaria. In this study, we used sera from human volunteers who had been infected with the P. falciparum 3D7 strain to investigate the development, specificity, and dynamics of anti-PfEMP1 antibodies measured against six different strain 3D7 Duffy binding-like domain 1α (DBL1α) fusion proteins. We observed that a parasitemia of 20 to 200 infected erythrocytes per μl was required to trigger an antibody response to DBL1α and that antibodies against one DBL1α variant cross-react with other DBL1α variants. Both serum and purified immunoglobulin Gs (IgGs) were able to agglutinate infected erythrocytes, and purified anti-DBL1α IgGs bound to the live infected red blood cell surface in a punctate surface pattern, confirming that the IgGs recognize native PfEMP1. Analysis of sera from tourists naturally infected with P. falciparum suggests that the anti-PfEMP1 antibodies often persisted for more than 100 days after a single infection. These results help to further our understanding of the development of acquired immunity to P. falciparum infections.     

Biological and physiocochemical properties of purified anti-DNP guinea-pig non-precipitating antibodies

Methods for isolation and purification of precipitating and non-precipitating guinea-pig antibodies are described.

The physicochemical properties of γ₁ and γ₂ non-precipitating antibodies are similar to γ₁ and γ₂ precipitating ones. Biological properties are also similar excepting the reverse Arthus reaction, which is positive with the precipitating and negative with the non-precipitating antibodies.

Bivalence of these antibodies was experimentally demonstrated. Precipitating antibodies K₀ do not differ greatly from those obtained with the corresponding non-precipitating ones. The incapacity to precipitates with the antigen may be a consequence of a steric impediment.

     

Effect of Antibody on Heterologous and Homologous Cells in Tissue Culture

In order to compare antibody against heterologous and homologous tissue in the same serum, rabbits were immunized with rat kidney and complete adjuvant. The resulting sera showed antibody against both rat and rabbit kidney. Cultures of rat kidney cells were killed by exposure to these sera. A concentration of 0.3 per cent γ globulin (ammonium sulphate fraction) was adequate to kill cultures in the absence of complement, but smaller concentrations were effective when guinea-pig complement was added. The cell surfaces were shown to have taken up antibody by the fluorescent antibody technique. Cytoplasmic staining could only be shown in cells which had previously been injured by freezing and thawing or by fixation. Rabbit kidney cells in culture were unaffected when exposed to whole rabbit anti-rat serum, but were killed and their cell membranes stained on exposure to γ globulin derived by (NH₄)₂SO₄-fractionation from such serum and having the same complement-fixation titre as the parent serum. At least 0.6 per cent γ globulin had to be added to kill rabbit cell cultures. It was found that normal rabbit serum had a partial protective effect against this antibody. Fractionation of sera by gradient centrifugation or chromatography on DEAE-cellulose showed that while antibody against heterologous tissue was found both in the 7S γ globulin and macroglobulin fractions, antibody against homologous tissue was confined to the latter. It is considered that the findings do not support a concept of an in vivo pathogenic role for circulating antibody.     

Agglutinating and non-agglutinating antibodies in rabbits inoculated with a particulate antigen (Salmonella typhimurium)

Agglutinating and non-agglutinating anti-Salmonella typhimurium antibodies were specifically purified from the sera of immunized rabbits. Both types of antibody had the same electrophoretic mobility and were localized in the IgG fraction. It was not possible to find antigenic differences between agglutinating and non-agglutinating antibodies by immunodiffusion.

Agglutinating antibody activated the complement system, while non-agglutinating antibody lacked this capacity. Only the former increased clearance of antigen from the blood. When serum samples with different antibody titres determined by agglutination (agglutinating antibody) and Coombs test (non-agglutinating antibody) were injected in mice, clearance of antigen from the blood showed changes. These results were similar to those previously observed by us when different precipitating: co-precipitating antibody ratios were used, and indicated that competition of both antibodies for the antigen depends on their respective amounts.

When mice protection tests were set up by injection of agglutinating and non-agglutinating antibody before the inoculation of 10 LD₅₀ S. typhimurium, non-agglutinating antibody was found to be less effective than agglutinating antibody.

Non-agglutinating antibody was detectable during the whole course of immunization. Its serum concentration was higher than that of the agglutinating antibody.

Non-agglutinating antibody behaves in a similar way to co-precipitating antibody. The initially proposed hypothesis that such antibodies could interfere with immunity to certain chronic infections was extended to include the non-agglutinating antibodies demonstrated here.

     

Expression of human–Torpedo hybrid acetylcholine receptor (AChR) for analysing the subunit specificity of antibodies in sera from patients with myasthenia gravis (MG)

The nicotinic AChR, a pentamer composed of α₂βγ(or ε)δ subunits, is the autoantigen in the human autoimmune disease MG. Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore determination of their specificities requires the use of intact AChR. Indirect antibody competition studies have suggested that most MG antibodies are inhibited from binding to AChR by MoAb to the main immunogenic region (MIR) on the α-subunits. More recently, based on the knowledge that MG antibodies show little detectable cross-reaction with Torpedo AChR, we have shown, using mouse–Torpedo hybrid AChR, that most MG antibodies that detectably cross-react with the mouse AChR bind to the α-subunit. To analyse the whole anti-AChR antibody repertoire in MG sera, we expressed on stably transfected fibroblasts a novel human α+Torpedoβγδ AChR and compared the antibody titres against human, Torpedo, and the hybrid AChR. Direct information was provided for the subunit specificity of several MoAbs and sera from 50 MG patients. On average, at least 48% of the anti-AChR antibodies in the sera were directed against the α-subunit. Interestingly, the anti-α-subunit antibodies predominated in low titre (0.6–7.4 nm) but not in high titre (10–386 nm) sera, where they comprised on average 68% versus 23% of the antibodies, respectively. Finally, the directly determined anti-α-subunit antibodies and the anti-MIR antibodies defined by antibody competition were significantly correlated, thus suggesting that at least a significant fraction of the anti-MIR antibodies in MG sera bind to the α-subunit.     

Immunological Identification of the Pathological Cold Auto-Antibodies of Acquired Haemolytic Anaemia as β2M-Globulin

Cold antibodies were separated from the sera of six patients suffering from the cold-haemagglutinin syndrome and from one patient with acquired haemolytic anaemia secondary to lymphosarcoma by dissociation of the specific antigen-antibody complexes. The eluted antibodies were studied (a) by immuno-electrophoresis along with the parent sera against horse anti-human serum and (b) by double diffusion in agar gel along with electrophoretically separated `γ<sub>1</sub> globulin'† against anti-19S `γ-globulin' rabbit sera.

The protein forming the cold antibody was localized in the β₂-M position on immuno-electrophoresis in each instance. It was found by double diffusion in agar gel to be immunologically identical with the protein forming the abnormal `γ₁-globulin' electrophoretic peak in the parent serum.

The results of these experiments indicate that the cold antibodies derived from patients with the cold-antibody type of acquired haemolytic anaemia are macro-molecular globulins.

     

Analysis of Human Serum Immunoglobulin G against O-Acetyl-Positive and O-Acetyl-Negative Serogroup W135 Meningococcal Capsular Polysaccharide

The capsular polysaccharide of Neisseria meningitidis serogroup W135 is expressed in both O-acetyl-positive (OA⁺) and O-acetyl-negative (OA⁻) forms. This study investigates the impact of OA status (OA⁺ versus OA⁻) on serological measurements of anti-W135 immunoglobulin G (IgG) antibodies in immunized adults. W135-specific serum antibody assignments were made for 28 postimmunization sera from adults by enzyme-linked immunosorbent assay using the meningococcal standard reference serum CDC1992. The established IgG concentration in micrograms per milliliter ([IgG]μg/ml) for CDC1992 against OA⁺ antigen (16.2 μg/ml) was used as a reference to assign a concentration of 10.13 μg/ml IgG against OA⁻ antigen by cross-standardization. Overall, the IgG assignments for these sera were higher against OA⁺ antigen (geometric mean concentration [GMC] = 7.16 μg/ml) than against OA⁻ antigen (GMC = 2.84 μg/ml). However, seven sera showed higher specific [IgG]μg/ml values against the OA⁺ antigen than against the OA⁻ antigen. These sera were also distinguished by the inability of fluid-phase OA⁻ antigen to compete for antibody binding to OA⁺ solid-phase antigen. Although there was no overall difference in functional activity measured by complement-mediated serum bactericidal assay (SBA) against OA⁺ and OA⁻ target bacteria (geometric mean titers of 9,642 and 9,045, respectively), three serum specimens showed a large difference in SBA antibody titers against OA⁺ versus OA⁻ W135 target bacteria, which may reflect different epitope specificities for these sera. Our data indicate that, for some sera, the agreement in anti-OA⁺ versus anti-OA⁻ W135 IgG assignments is serum specific and does not reflect the functional (killing) activity in vitro.     

Peyer's patches are required for intestinal immunoglobulin A responses to Salmonella spp

Previous studies have shown that Peyer's patches (PP) are not required for intestinal immunoglobulin A (IgA) responses to orally administered soluble protein. However, the roles of PP in regulation of mucosal immune responses against bacterial antigen remain to be clarified. In the present study, we generated several gut-associated lymphoreticular tissue-null mice by treatment with anti-interleukin-7 receptor antibody, the fusion protein of lymphotoxin β receptor and IgG Fc, and/or tumor necrosis factor receptor p55 and IgG Fc. These mice were then immunized with recombinant Salmonella expressing the C fragment of the tetanus toxin (rSalmonella-Tox C). Orally immunized PP-null mice as well as isolated lymphoid follicle (ILF)-null, PP/ILF-null, and PP/ILF/mesenteric lymph node-null mice induced identical levels of tetanus toxoid (TT)-specific systemic IgG responses to those of control mice. However, PP-null mice, but not ILF-null mice, failed to induce TT-specific intestinal IgA antibodies. Analysis of TT-specific CD4⁺ T-cell responses showed a reduction of gamma interferon (IFN-γ) synthesis in the intestinal lamina propriae of PP-null mice given oral rSalmonella-Tox C. In contrast, TT-specific IFN-γ responses in the spleen and delayed-type hypersensitivity responses were intact in those immunized mice. Interestingly, Salmonella lipopolysaccharide (LPS)-specific fecal IgA responses were not elicited in PP-null mice, while serum IgG anti-LPS antibodies were identical to those of control mice. These results suggest that while none of the gut-associated lymphoreticular tissues are required for the induction of systemic immune responses, PP are an essential lymphoid tissue for induction and regulation of intestinal IgA immunity against orally administered rSalmonella.     

Immunological studies of phytohaemagglutinin: I. Reaction between phytohaemagglutinin and normal sera

Studies of the precipitation of normal human serum by the kidney bean phytohaemagglutinin, Phaseolus vulgaris (PHA), showed that the main serum components involved were α₂-macroglobulin, β-lipoproteins and γM immunoglobulins. Preparations of α₁-glycoprotein, orosomucoid and some γA immunoglobulins were also precipitated by PHA. Antisera prepared against the PHA—NHS precipitate recognized α₂-macroglobulin, β-lipoproteins and γM immunoglobulins, γG immunoglobulins and at least four other unidentified antigens in the β and α regions. PHA did not react with B or A, O blood group substances.

Concanavalin A, a jack bean agglutinin, precipitated the same proteins which were precipitated by Phaseolus vulgaris PHA but a Dolichos biflorus extract did not react with human serum. Incomplete chemical studies of this interaction suggested that even though the same serum proteins were precipitated by Concanavalin A and PHA, the sugar specificity, if involved, is different for both lectins.

Incomplete studies of the component or components of PHA involved in the PHA—NHS interaction suggested that three cathodally migrating proteins were recognized by both the antisera to PHA and to the precipitate formed by PHA and normal human serum.