A solid phase assay with radioactively-labelled antibody for the detection of Brucella abortus.

An assay for the detection of Brucella abortus is described. IgG from rabbit antisera against live brucellae or brucella extracts was chemically linked to cellulose to form a solid phase reagent capable of binding brucella antigens present in buffer solutions or serum. After washing away any unbound material the presence of bound antigen was revealed by incubation with radioactively-labelled anti-brucella antibodies. The assay was capable of detecting less than 100 pg brucella antigen in a 20 microliter sample. Experiments in which IgG of non-related specificity was used in place of anti-brucella IgG showed that the test was specific. Normal human serum had only a slight inhibitory effect but anti-brucella antibodies were strongly inhibitory if present in the test sample. The extent of this effect and its relationship to antibody titre was investigated in 12 sera from brucellosis patients.     

A comparison of immuncapture agglutination and ELISA methods in serological diagnosis of brucellosis

Background: Different serological tests are used in serologic diagnosis of brucellosis. The most widely used of these are Standard Tube Agglutination and Coombs anti-brucella tests. Whereas ELISA Ig M and Ig G tests have been in use for a long time, immuncapture agglutination test has been recently introduced and used in serological diagnosis. The aim of this study was to compare diagnostic values of ELISA Ig M and Ig G and immuncapture agglutination tests with Coombs anti-brucella test.Methods: Sera from 200 patients with presumptive diagnosis of brucellosis were included into the study. Coombs anti-brucella test, ELISA Ig M and Ig G tests and Immuncapture test were investigated in these sera. Then, sensitivity, specificity, negative predictive and positive predictive values were calculated.Results: Sensitivity, specificity, negative predictive and positive predictive values were found to be 90,6 %, 76,3 %, 94,2 %, and 65,9 % respectively for the Immuncapture test, whereas they were found to be 73,7 %, 58,9 %, 84,2 %, and 42,8 % for Ig G and 72,2 %, 67,8 %, 85,2 %, and 48,7 % for Ig M. The Immuncapture test was found to be compatible with ELISA Ig M and Ig G tests but it was statistically incompatible with Coombs anti-brucella test.Conclusions: Immuncapture agglutination test yields similar results to those of Coombs anti-brucella test. This test is a useful test by virtue of the fact that it determines blocking antibodies in the diagnosis and follow-up of brucellosis.     

Determination of brucella immunoglobulin G agglutinating antibody titer with dithiothreitol.

The routine brucella agglutination test measures both immunoglobulin M (IgM) and IgG brucella antibody titers; however, only an elevated IgG titer is significant for differentiating active from inactive disease in patients with symptoms lasting 3 or more weeks. The IgG antibody titer can be determined by treating the serum wih 2-mercaptoethanol to inactivate the IgM brucella antibodies while leaving the IgG brucella antibodies intact. Dithiothreitol, which also inactivates IgM, was compared with 2-mercaptoethanol for the determination of IgG brucella agglutination titers. The dithiothreitol and 2-mercaptoethanol test results agreed within +/- 1 dilution step in 103 of 105 serum specimens tested, for a 98.1% rate of agreement. The results indicate that dithiothreitol can be used in place of 2-mercaptoethanol for determining IgG brucella agglutination titers. Dithiothreitol does not have the offensive odor or the irritant properties of 2-mercaptoethanol.     

Identification and characterization of guinea-pig antibodies that contrasensitize in mice.

Immunological enhancement is a form of active immunoregulation in which humoral antibodies suppress primary sensitization or block reaction in sensitized animals. In mouse serum the antibodies that suppress sensitization in mice (allogeneic enhancement) are predominantly 7Sgamma1 and those that block reaction 7Sgamma2 globulins. But little is known about the identity of xenogeneic enhancing antibodies, e.g. guinea-pig antibodies that can suppress sensitization in mice. We have studied these here as specifically effective against induction of tuberculin-typed delayed hypersensitivity to chicken conalbumin antigen. Guinea-pigs required prolonged and intense immunization with conalbumin to produce readily measurable titres of such antibodies. Their capacity to suppress mouse sensitization was antigenically specific. Of the three major classes of antibodies separated from the immunosuppressive guinea-pig antisera, the IgG2 globulins were the most effective. These antibodies lost immunosuppressiveness if digested to either F(ab.)2 or Fab fragments that retained antigen-binding capacities. Thus, we provide here an example of xenogeneic antibody-mediated contrasensitization, show that only intact antibody molecules are effective, and demonstrate that immunosuppressiveness is concentrated in different immunoglobulin classes for xeno- and allogeneically used antisera.     

Immunotherapy in Chronic Brucellosis. Effect of Levamisole and Interferon; Mechanisms of Action and Clinical Value

Thirty two anergic patients with chronic brucellosis treated with a)interferon-alpha2b(group 1), b)levamisole (group 2) and c)conventional therapy(group 3) were studied. the effect of treatment on T lymphocyte blast formation in the presence of PHA, specific cell mediated immunity against brucella antigens, titers of brucella antibodies and clinical symptoms were evaluated T lymphocyte blast formation was shown to range in normal levels in all patients before treatment compared to 10 normal controls suggesting against a generalized impairment of cell mediated immunity. Titers of brucella antibodies were significantly decreased in group 1, almost significantly in group 2 and were significantly increased in group 3 at the end of treatment. A significant improvement of symptoms as well as production of leukocyte migration inhibition against brucella antigens were noted in both groups 1 and 2, in contrast to group 3. This response to treatment was however greater in group 1. These findings demonstrate that immunotherapy resulted in both clinical and immunological improvement and that interferon seems to be a more promising therapeutic approach of chronic brucellosis.     

Anti-Gamma Globulins and Chronic Infection: Comparative Studies of the Immune Response to Various Bacteria and Gamma Globulin Preparations

A study of the relationship of clinical states associated with prolonged infection (bacterial endocarditis and osteomyelitis) and generation of serum anti-gamma globulins was made with particular reference to quantitative amounts of staphylococcal protein A in various infecting strains. No correlation between individual strain amounts of protein A and presence of anti-gamma globulins was detected. Thirty-eight rabbits were immunized intravenously with various strains of bacteria (Staphylococcus aureus, enterococci, Streptomyces viridans, pneumococci, pseudomonas, and Escherichia coli) for periods of 6 weeks, and antibacterial as well as anti-gamma globulin antibodies were assayed. No single group or strain of bacteria stood out as being more prone to produce anti-gamma globulins than others tested. Most rabbits developed anti-gamma globulins reacting with human gamma globulins, whereas the specificity for rabbit gamma globulin appeared more restricted. In 16 rabbits immunized with eight different strains of S. aureus, quantitative elevation of serum gamma globulin above 2.5 g per 100 ml often seemed to be correlated with presence of detectable serum anti-gamma globulins. By contrast 15 rabbits immunized with autologous or isologous rabbit gamma globulins in many instances developed extremely high titers of anti-gamma globulins showing primary specificity for human rather than rabbit gamma globulin. These studies further amplify the remarkably heterogeneous anti-gamma globulin reactivity associated with various types of immune response.     

Effect of intravenous human immunoglobulins on bacterial clearance and mortality in experimental Escherichia coli K 1 septicemia in chicken

The antibacterial effect of intravenous immune globulins (IVIG) was tested in 4 to 6-week-old chickens experimentally infected with a K1 E. coli strain from a neonate with meningitis. Following inoculation of 10(7) E. coli K1 per animal, the chickens were randomized to receive a placebo, 0.25 g/kg, 0,50 g/kg or 1 g/kg of intravenous immune globulins. Injections were performed 4 hours after the bacterial inoculation. Numbers of animals in each group were 30, 19, 30 and 10 for 6-week-old chickens and 9, 10, 9 and 0 for 4-week-old chickens. Bacterial diffusion was evaluated using quantitative cultures of blood from breast capillaries. In the 6-week-old chickens given 0.25 g/kg or 0.50 g/kg of intravenous immune globulins, bacteremia 48 hours after inoculation and mortality were significantly lower than in the placebo group. Administration of intravenous immune globuline in a dose of 1.0 g/kg, however, was followed by increased blood bacteria counts 48 and 72 hours after inoculation. In the 4-week-old chickens, no reduction in bacterial counts or improvement in survival was seen after administration of intravenous immune globulins. These data should provide a basis for a more rational use of intravenous immune globulins in neonates and children.     

Immunoglobulin a concentrations in commercial immune globulins

Commercial immune globulins contain varying concentrations of IgA as a minor constituent. For patients who are both IgA deficient and IgA hypersensitive, the administration of most immune globulins is contraindicated due to potentially lethal anaphylactic reactions. We have measured the IgA levels in a broad range of commercial immune globulin preparations by means of a competitive-binding solid-phase radioimmunoassay. Of the products tested, all immune globulins intended for intramuscular administration and most immune globulins for intravenous use had IgA levels of over 150 µg/ml. Only two of the intravenous products had IgA concentrations in the range of 10 µg/ml or less.     

Seroepidemiological survey of the prevalence of antibodies to a strain of human calicivirus

Batches of pooled immune globulins and sera were tested by immune electron microscopy (IEM) for the presence of antibodies to a strain of human calicivirus (HCV, UK1). The results show that this strain of HCV is prevalent throughout many parts of the world and that the majority of the population experience infection by the age of 12 years. The survey carried out in the United Kingdom indicates that the presence of maternal antibody correlates with some degree of protection during the first few weeks of life; the peak incidence of cases and acquisition of antibody occur between 3 months and 6 years. Tests on sera from Japan show a similar pattern of acquisition of antibodies and demonstrate that infection with more than one strain of HCV commonly occurs during childhood.     

Cyclic beta-1,2-glucan is a Brucella virulence factor required for intracellular survival

Pathogenic brucella bacteria have developed strategies to persist for prolonged periods of time in host cells, avoiding innate immune responses. Here we show that the cyclic beta-1,2-glucans (CbetaG) synthesized by brucella is important for circumventing host cell defenses. CbetaG acted in lipid rafts found on host cell membranes. CbetaG-deficient mutants failed to prevent phagosome-lysosome fusion and could not replicate. However, when treated with purified CbetaG or synthetic methyl-beta-cyclodextrin, the mutants were able to control vacuole maturation by avoiding lysosome fusion, and this allowed intracellular brucella to survive and reach the endoplasmic reticulum. Fusion between the endoplasmic reticulum and the brucella-containing vacuole depended on the brucella virulence type IV secretion system but not on CbetaG. Brucella CbetaG is thus a virulence factor that interacts with lipid rafts and contributes to pathogen survival.     

Monoclonal Anti Thy 1.2 Antibodies from Hybridoma Ho13-4 do not React with Mouse Natural Killer Cells

The susceptibility of NK cells and immune cytotoxic T-cells to treatment with (a) monoclonal anti thy 1.2 antibodies from hybridoma HO13-4, (b) rabbit anti-mouse T-cell antiserum and (c) gamma globulins prepared from AKR/J anti C3H/HeJ antiserum was studied in the presence of rabbit complement. Monoclonal anti thy 1.2 antibody treatment completely abolished the cytotoxic activity of immune T-cells derived from C57BL/6J mice (H-2^b) immunized with (C57BL/6J x DBA/2)F₁spleen cells (H-2^bd) against P815 (H-2^b) target cells. The same treatment had no significant effect on the NK activity of spleen cells from unimmunized mice against YAC target cells. Rabbit anti-mouse T-cell and mouse anti theta antisera also abrogated completely the immune T cell activity of spleen cells. This treatment however also resulted in a partial loss of NK activity. These results indicate that conventional anti theta antisera contain antibodies which recognize antigenic specificities on T-cells as well as on a population of NK cells. The cross reactivity is not a result of thy 1.2 antigen expression on NK cells and T-cells as recognized by the monoclonal antibodies. The specificity recognized by the monoclonal antibody (HO13-4) is only expressed on T-cells.     

Detection and quantitation of chicken antibodies to streptococcal carbohydrate by high-salt reverse radial immunodiffusion

A comparative study was made of the reverse radial immunodiffusion technique and quantitative precipitin analysis for determining the amount of precipitating chicken anti-streptococcal carbohydrate antibodies in ‘high-salt’ buffers. For most immune sera the two techniques gave similar values but with some the reverse radial immunodiffusion technique underestimated the antibody content. In most cases this was correlated with the presence of considerable polymeric (IgM and IgA) immunoglobulin antibody. As little as 45 μg/ml of anti-carbohydrate antibody could be detected using this method. This ‘high-salt’ radial immunodiffusion technique is also useful for detecting chicken antibodies to bovine serum albumin, dinitrophenylated proteins, blood group substances and gamma globulins.     

Radioimmunoassay for anticollagen — antibodies using 14C-labelled collagen

A sensitive radioimmunoassay for anticollagen antibodies is described. ¹⁴C-labelled human acid-soluble collagen of high specific activity (5 × 10⁶ dpm/mg) is used as antigen either in native or denatured state. Experimentally induced anticollagen antibodies or RA synovial fluids containing antibodies to collagen are reacted with the labelled antigen. The immune complexes formed are precipitated with goat antiserum to rabbit globulins (‘second antibody’). A systematic investigation of the labelled collagen in regard to cleavage by enzymes, fibril formation and specificity showed that no gross alteration had been caused by the labelling procedure. The assay furnishes information on the avidity, specificity and immunoglobulin class of experimental or pathological anticollagen antibodies. It can also be used as sensitive assay for collagen in biological fluids.     

Conditioned reflex regulation of the serum globulin level

Summary Experiments were conducted on oxen to study the conditioned-reflex mechanism responsible for maintaining the serum globulins at a high level. The results confirmed the theory that the immunological reactions of the organims are regulated by a conditioned-reflex mechanism and shed light on the physiological mechanisms of the cortical control of antibody synthesis. Apparently conditioned-reflex stimuli modify the intensity of the general nonspecific function of the immunogenetic apparatus, namely the synthesis of immune globulins, and thereby regulate the synthesis and production of specific antibodies.(Presented by Active Member AMN SSSR N. N. Zhukov-Verezhnikov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 55, No. 6, pp. 80–83, June, 1963     

in vitro : Interference of Non-specific Gamma Globulins with Passive • Anaphylactic Sensitization of Isolated Guinea-Pig Intestine

Non-specific γ globulins, when added to the incubating medium containing rabbit anti-egg albumin antibody, have been found to impede passive in vitro sensitization of the isolated guinea-pig ileum. Although this interference has been observed with the various γ globulins studied, there is a consistent difference in the intensity depending upon their zoological origin. When rabbit antibody is used for sensitization, the impeding effect of the γ globulins decreases in the following order: rabbit>man>dog>guinea pig>rat>horse>cattle>pig>chicken>goat.

A certain degree of interference has been observed even with horse, cattle, chicken, and goat γ globulins, although it is impossible to passively sensitize the guinea pig with the antibodies originating from those animal species in vivo.

The results reported here are difficult to reconcile with the hypothesis that the non-specific γ globulins interfere with the sensitization process by `competing' with the antibody for the same specific receptors. They rather suggest that the impeding effect of the γ globulins is due to their interaction with the antibody by some physico-chemical process, producing thus an alteration of its specific properties.

     

Biophysical and immunological studies on bovine immune globulins with evidence for selective transport within the mammary gland from maternal plasma to colostrum

Three immune globulins in maternal serum and colostrum and newly born calf serum, have been characterized and compared. An examination was made to determine first, which of the maternal serum immune globulins accumulate in the circulation of the calf and secondly, the selectivity of the mammary gland for these proteins compared with the intestinal mucosa of the newly born calf.

By difference in their electrophoretic mobilities three antigenically related immune globulins were isolated from bovine serum.

The immune lactoglobulins in bovine colostrum were qualitatively similar to those in serum. However, marked differences were observed between the relative concentrations in serum and colostrum of the three immune globulins.

An electrophoretically fast immune globulin (C1), present in colostrum at high concentration, was shown to be antigenically similar to an immune globulin (S1) present in the maternal serum at low concentration.

These findings indicate that the mammary gland showed a highly selective preference for, and hence ability to concentrate in, colostrum, the electrophoretically fastest serum immune globulin.

The slowest serum immune globulin and the component with intermediate electrophoretic mobility (S3 and S2 respectively) were both present at high concentration in bovine maternal serum, but were transmitted at different rates into the colostrum, so that the slowest serum immune globulin (S3) was present in the colostrum as a comparatively minor component (C3).

In contrast to the mammary gland, the intestine of the newly born calf (permeable to undegraded protein during the first 24 hours of life) showed no selectivity. Immune globulins showing the three electrophoretic mobilities were absorbed equally readily.

Thus, while the bovine mammary gland showed a highly selective preference for certain electrophoretically different serum proteins, no comparable selectivity was shown by the intestinal mucosa of the newly born calf.

The results emphasize the heterogeneity of bovine immune globulins and show that the calf receives into its circulation from ingested colostrum selected maternal serum immune globulins. This selection of proteins from maternal plasma, for admission to the calf's circulation, occurs within the mammary gland during the formation of colostrum but not during absorption across the calf's intestinal mucosa.

     

Modulation of the immune response by passive antibodies. IV. Effects of IgG1 and IgG2 anti-hapten antibodies

The effects of IgG1 and IgG2 anti-hapten antibodies waere studied on celluar and humoral reactions induced by immunization with a hapten-carrier complex. IgG2 was shown to depress delayed hypersensitivity reactions to the carrier and to enhance anaphylactic reactions to both the hapten and the carrier whereas IgG1 appeared to have no activity except a little enhancing effect on antibody synthesis to the carrier. The regulatory role of IgG2 antibodies, which were cytophilic for macrophages, in the immune response to a hapten-carrier conjugate is discussed.     

Fate of skin allografts in normal mice injected with antigraft strain globulin in relation to the IgG1 and IgG2 class of the antibodies

A recent study of the effects on skin allografts of alloantibody-containing globulins of the mouse indicated that such globulins can have a variety of effects on the fate of the grafts, ranging from accelerated rejection to prolonged retention. We have also shown evidence of the presence in such globulins of anti-graft strain antibodies of IgG2 class by complement-dependent reactions of such globulin, and of antibodies of IgG1 class by the finding that the titres of these reactions were elevated on treatment of the globulin with anti-mouse IgG1 serum. Further, it was shown that the increasing titre of these antibodies on treatment with increasing amounts of the anti-IgG1 serum reached a plateau level which is the actual titre of an IgG2 anti-graft strain antibody, such as the suppressive antibody, in these preparations. The difference between the plateau level and the original level of suppressive titre was therefore considered to provide a measure of the competing, IgG1-class, antibody.The present study has examined the relation, in anti-graft strain ascitic fluid globulin preparations, between this measure of relative content of anti-graft strain antibody of IgG1 class and the effects of these globulins on the fate of skin allografts. Of globulin preparations which had caused various effects on the skin grafts, portions were treated by adding increasing amounts of anti-mouse IgG1, removing the resulting precipitates, and testing the supernatant fluids for suppressive titre, to determine the plateau level of titre attained. All pools which caused accelerated rejection of BALB/c skin grafts showed increases from original to plateau titres in the lowest range, log₂ 0.1–1.2; all those causing prolonged retention showed such differences in the highest range observed, log₂ 2.1–3.1. Globulins giving partial effects of accelerated rejection, or negative results, showed corresponding intermediate of ranges the increases in titre produced by the anti-IgG1 serum.For a test of this relation, use was made of an earlier observation of a failure to produce accelerated rejection by later-day globulins of mice which had, at 11–13 days, produced rejecting globulins. The failure to cause accelerated rejection by such later-day globulins was confirmed, and these later globulins also showed consistently greater increases of titre from original to plateau level. Similarly, of mice whose 11–13-day globulin pools had caused no significant difference from normal rejection time, later globulins showed, again, greater differences between original and plateau titres than the 11–13-day globulins, and again a difference in effect on skin graft rejection, in this case a significant prolongation of graft retention, in comparison with the negative results given by the 11–13-day pools.     

Humoral Immune Response to the Antigen Administered as an Immune Complex

Antigen (HSA) bound in immune complexes at equivalence with syngeneic anti-HSA antibodies elicit much stronger humoral immune response then soluble HSA. On the other hand, administration of immune complexes formed with xenogeneic (rabbit) anti-HSA antibodies suppressed humoral immune response against HSA, but not against rabbit IgG in mice. We suggest that immunization with antigen bound in immune complex might represent a powerful tool in enhancing humoral immune responses.