anti-HB Antibodies Research Articles

Hemoglobin penetration in the wall of the rabbit basilar artery after subarachnoid hemorrhage and intracisternal hemoglobin injection.

The ability of hemoglobin (Hb) to penetrate the basilar arterial wall in vivo after experimental subarachnoid hemorrhage was examined using immunohistochemistry. The distribution of anti-Hb antibodies in rabbit basilar artery was studied following the injection of autologous blood in the cisterna magna. Vessels removed two or four days after subarachnoid hemorrhage exhibited varying degrees of vasospasm, and exhibited Hb immuno-fluorescence throughout the vessel wall. Hemoglobin immunofluorescence was most conspicuous in the adventitia but was also seen in the smooth muscle and endothelial cell layers in 7 of 10 animals. The degree of vasoconstriction correlated with the total amount of Hb-fluorescence present in the vessel wall. When Hb solution alone was injected into the subarachnoid space, vasoconstriction was evident but penetration into the vascular layers was not as extensive as that observed after injection of autologous blood. These findings demonstrate that Hb is able to penetrate through the arterial wall after subarachnoid hemorrhage. The results provide direct support for the hypothesis that Hb-induced changes in smooth muscle and/or endothelial function can contribute to the pathogenesis of vasospasm.

Antigenic structure of human hemoglobin: Delineation of the antigenic site (site 2) within region 41–65 of the alpha chain by immunochemistry of synthetic peptides

A comprehensive synthetic approach consisting of a series of consecutive, uniform overlapping peptides encompassing the entire protein chain was recently used to determine the full antigenic profile of the α-chain of human hemoglobin (Hb). The peptides synthesized enabled the localization of five major “continuous” antigenic regions within the α chain. The present findings describe the delineation of an antigenic site (site 2) residing within the region 41–65. Ten peptides representing the α-chain regions 41–55, 51–65, 45–54, 45–56, 45–58, 45–60, 48–56, 49–56, 50–56, and 51–56 were synthesized and purified. Quantitative radioimmunoadsorbent titrations were used to determine binding to peptide adsorbents of radioiodinated anti-Hb antibodies that were raised in rabbit, goat, and outbred mouse. In one set of peptides, the N-terminal was fixed while the C-terminal end was increased by increments of two residues from Gln-54 to Lys-60 (i.e., peptides 45–54, 45–45, 45–58, and 45–60). Binding studies revealed that maximum antibody activity resided in peptide 45–45, indicating that Lys-56 marks the C-terminal boundary of the site. In the second set of peptides, the C-terminal was fixed at Lys-56 while the peptides were elongated at their N-terminal by one-residue increments from Gly-51 to Leu-48. Antibody-binding studies with these peptides indicated that Ser-49 defines the N-terminal boundary of the site. Therefore, the antigenic site within region 41–65 of the α chain comprises residues 49–56. The relevance of these findings to the immune recognition of Hb and other proteins is discussed.

Hybridoma autoantibodies to erythrocytes from NZB mice and the induction of hemolytic anemia

We established two clones of monoclonal hybridoma, from a non-immunized NZB mouse, which produce IgM class hemagglutinating autoantibodies reactive with the exposed murine erythrocyte autoantigens. Absorption studies revealed that one monoclonal antibody exhibits cross-reactivity to chick erythrocytes and mouse liver, and the other antibody to rat erythrocytes and mouse brain. Optimal temperatures for the hemagglutination were 22 °C with the former and 4 °C with the latter. The specificity and nature of the autoantibodies are apparently distinct from any of the erythrocyte autoantibodies described to date in NZB mice, anti-X, anti-HB, anti-HOL or anti-I antibodies. Implantation of these hybridoma cells in BALB/c mice induced autoimmune hemolytic anemia associated with a marked splenomegaly. These findings provide evidence that the erythrocyte autoantibodies in NZB mice are more heterogenous than generally assumed and suggest that varieties of erythrocyte autoantibodies may be involved in the development of a naturally occurring hemolytic disease in NZB mice.

Hepatitis B virus and alcoholic liver damage in the Airin district. (Osaka's skid row area).

Healthy adults and non-liver disease patients in the Airin district gave a positivity rate for HBs antigen of approximately 2%, which is not so much different from the average of the entire Japanese population. In this district, the positive rate of HBs antigen 5% (11 out of 244) in chronic liver disease is much lower than that (31%) of control group patients at Osaka City University Hospital (O.C.U.H.). However the positive rate of anti-HBs antibody in this district is 46% which is extremely high compared with that (23.4%) in the rest of Japan. The positive for anti-HB antibodies (including of anti-HBs and anti-HBc antibody) accounted for 67% of non-liver disease patients and 68% of chronic liver disease patients in the Airin district. Corresponding values for patients at O.C.U.H. were 44% and 43% respectively. There were no significant differences in the histological picture between HBs antigen-negative patients with and those without demonstrable anti-HBs antibodies. Changes in the liver exhibited by heavy drinkers inhabitating the Airin district were primarily those of alcoholic liver damage.

Delineation of Spontaneous Erythrocyte Autoantibody Responses of NZB and Other Strains of Mice

Four anti-erythrocyte autoantibody responses (anti-X, anti-HB, anti-HOL, and anti-I) that occur spontaneously in mice have been characterized with regard to antigenic specificities, predominant immunoglobulin class, and pathogenetic importance. Each autoantibody response exhibits specificity for an independent erythrocyte membrane autoantigen (X, HB, HOL, or I) or a soluble analogue (SEA-X or SEA-HB) present in the plasma. The anti-X response, unique to NZB mice, is directed to a normally exposed murine erythrocyte autoantigen, whereas the anti-HB response is directed to a cryptic erythrocyte autoantigen exposed by limited enzymatic cleavage of the membrane. The anti-I response also is directed to a cryptic but distinct autoantigen, and anti-HOL autoantibodies react with an erythrocyte autoantigen located at the cytoplasmic surface of the membrane. Analysis of the predominant immunoglobulin class of each of the autoantibodies has demonstrated that anti-HB and anti-I antibodies are predominantly of IgM class, whereas anti-X and anti-HOL antibodies are IgG immunoblobulins. Only anti-X and anti-HB autoantibodies are recovered from Coombs' positive erythrocytes from NZB mice and erythrocytes with surface C3 are detected only in NZB mice greater than 9 months of age. These data suggest that only the anti-X and anti-HB responses are pathogenetically implicated in the autoimmune hemolytic anemia of NZB mice.

Hepatitis B circulating immune complexes. Characterization by radioimmunoprecipitation — PEG assay (RIPEGA)

Incidence of circulating immune complexes (IC) was investigated in carriers of hepatitis B antigen (HBAg) and/or anti-HB antibodies (anti-HBAb). Three methods were used: radiolabelled C1q binding test (C1qBT), complement fixation test (CFT), and optical density (OD) measurement after dissolution of 3% polyethylene glycol (PEG) precipitate of serum. A highly significant correlation was obtained between these three techniques. The level of IC was higher in carriers of HBAg without anti-HBAb, than in others. The characterization of HBAg and anti-HBAb in IC was carried out by a new procedure, the radioimmunoprecipitation-PEG assay (RIPEGA). This sensitive and reproducible test was performed by incubation of 125I-HBAg or 125I-HBAg with 3% precipitate of the carriers' sera. Separation of free from complexed 125I-HBAg or 125I-HBAb was achieved by PEG precipitation. A highly significant correlation was found between the levels of circulating IC evaluated by the C1q-BT and the quantities of HBAg or anti HBAb measured by RIPEGA. RIPEGA was used to quantify HBAg and anti-HBAb present in serum from HBAg and/ or anti-HBAb carriers, confirmed by a radioimmunoassay. In preliminary results, RIPEGA was shown to be more sensitive than classical radioimmunoassay.

Isolation of hepatitis B surface antigen (HB sAg) by affinity chromatography on antibody-coated immunoadsorbents

Hepatitis B surface antigen (HB sAg) was isolated from human serum by two steps of affinity chromatography on antibody-coated gels. HB sAg-positive serum was passed through a column packed with guinea pig anti-HB sAg antibodies covalently bound to CNBr-activated beaded agarose gel. The majority of non-specifically bound protein was removed by washing the gel with increased concentrations (0.5 M) of NaCl in Tris buffer. Elution of the specifically bound HB sAg was carried out with 3 M NaSCN. Residual normal human serum proteins present in the eluate were removed by passing the partially purified HB sAg through an immunoadsorbent coated with rabbit antibodies directed against human serum proteins. After this treatment normal human serum proteins could no longer be demonstrated by passive haemagglutination in the isolated HB sAg. Cross-reactions between HB sAg and normal human serum proteins could not be demonstrated. Both antibody-coated immunoadsorbents could be used over ten times without significant loss of their binding capacity.

Fulminant hepatitis-virus B in a three months old infant: therapeutic trial with specific anti-HB globulins in antibody excess.

A newborn of normal birthweight received fresh blood transfusions and procoagulant fractions during his first days for a haemorrhagic disease. Three months later the infant had acute liver failure. Liver biopsy revealed a massive hepatic necrosis and an Australia antigen was detected. 1ml/kg of anti-HB gamma globulin was administered after each of two exchange transfusions (200 ml/kg). Anti-HB antibodies were detected after gamma globulin therapy. Once the passively administered antibodies had disappeared, the HB titer increased, and the child seemed physically and mentally normal seven months after the infection. These observations are difficult to interpret but immunity of the child seems possible.