Circulating immune complexes and kidney lesions following 131I administration in mice with thyroglobulin antibodies

Immune complexes containing thyroglobulin have been described in kidneys of some patients with thyroid disease. We investigated the circulating immune complexes (with the Raji cell radioassay) and the kidney histopathology (by immunofluorescence and electron microscopy) in mice that received radioiodine to release thyroglobulin in the circulation, 2 or 4 weeks after immunization with mouse thyroglobulin in Freund's complete adjuvant. Circulating immune complexes and thyroglobulin, antibodies were found in all mice. Granular deposition of IgG, IgM, C3, and thyroglobulin, mainly in the mesangium but also in the capillary walls of the glomeruli, were observed in most of the mice. These experiments suggest that circulating immune complexes composed of thyroglobulin are responsible for the glomerular lesions. Hyperthyroid patients should be tested for thyroglobulin antibodies before treatment with radioiodine to avoid formation of thyroglobulin-containing circulating immune complexes.     

Characterization studies of thyroglobulin antibodies in leprosy: An immunological study of diethylaminoethylcellulose chromatographic fractions

The thyroglobulin antibodies present in the sera of thirteen leprosy patients were shown to be exclusively or predominantly macroglobulin in character when studied by diethylaminoethylcellulose chromatography and preparative ultracentrifugation. This was confirmed by immunoelectrophoresis and analytical ultracentrifugation of the fractions containing thyroglobulin antibodies, as well as by tests with 2-mercaptoethanol. The anti-thyroglobulin specificity of the antibodies showing macroglobulin character was ascertained by absorption experiments with human thyroglobulin; it was ruled out by absorption with FII that the 19S character might result from the formation of complexes with anti-γ-globulin factors. The conditions are discussed which may affect the 7S or 19S character of antibodies, in particular the antibody titre, the amount of antigenic stimulation and the vigour of the subject's immune response.     

The humoral immune response to the influenza virus: the characteristics of the enriched virus-neutralizing fraction of hyperimmune mouse serum

Certain characteristics of immunosorbents prepared on the basis of aminocellulose and oxycellulose were determined. The affinity chromatography of sera from influenza-virus-immunized mice was shown to yield with the former of the immunosorbents a fraction of antibody in which the relative portion of antibodies detectable by neutralization test was higher than that in the original hyperimmune serum. The preparation of pure antibodies obtained with the oxycellulose-based immunosorbent contained antibodies detectable by NT and HI test in a ratio similar to that in the unfractionated hyperimmune serum. It is concluded that aminocellulose and oxycellulose differ in the nature of chemical interaction with influenza virion proteins owing to which differences in the pattern of interaction of immunosorbents prepared on their basis with virus-specific antibodies are observed. The process of influenza virion neutralization by antibodies was also shown to be a multi-hit reaction, the ratio of NT- and HI-detectable antibodies in a preparation affecting the kinetics of the virus-neutralizing effect of the substance. An increase in the preparation of a portion of NT-detectable antibodies reduced the time in which the number of antibody molecules sufficient for virus particle neutralization is adsorbed to it.     

Antigen leakage from immunosorbents Implications for the detection of site-directed auto-anti-idiotypic antibodies

The detection of site-directed anti-idiotypic antibodies is usually based on their ability to inhibit the binding of antigen to idiotype, in either solid- or fluid-phase radioimmunoassays. Passage of serum over antigen-coupled immunosorbents for the purpose of removing the idiotypes from complexes with putative anti-idiotypic antibodies resulted in the release of significant amounts of antigen into the effluents. Normal sera or even isotonic buffers were similarly contaminated with antigen. The amount of antigen released ranged between 200–400 ng/ml, well in excess of the minimal amount required in the inhibition assay. Antigen was detected in effluents passed over a number of antigen coupled-matrices and even in affinity-purified antibody preparations obtained by elution from immunosorbents coupled with dinitrophenyl (DNP)-protein conjugates. Attempts to stabilize the antigen-coupled matrices with glutaraldehyde resulted in a perceptible but insufficient decrease in the amount of antigen released. In the case of anti-hapten antibodies, antigen interference was circumvented by utilizing monovalent haptens such as DNP-lysine coupled to the immunosorbent either directly or through a spacer arm. In the case of protein antigens, the leakage was almost completely prevented by preparing glutaraldehyde-polymerized immunosorbents directly from solution.     

Immune recognition of serum albumin--XII. Evidence for time-dependent immunochemical cross-reactivity of subdomains 3, 6 and 9 of bovine serum albumin by quantitative immunoadsorbent titration studies

Antisera were raised in rabbits against bovine serum albumin (BSA) or against fragment 377–571 (domain 3) of BSA. Serial bleedings were obtained at different times after immunization. Fragments 115–184, 307–385 and 504–581 were isolated in pure form from BSA. These corresponded essentially to subdomains 3, 6 and 9 respectively of BSA. The capacity of each fragment to bind ¹²⁵I-labelled antibodies of rabbit anti-BSA or anti-domain 3 antisera was determined by a quantitative immunoadsorbent titration technique. With anti-BSA antisera from each rabbit, the maximum (plateau) amount of antibodies bound by each fragment was found to increase with time antisera are obtained after the first immunization. With antisera against domain 3, considerable amounts of antibodies were bound by subdomains 3, 6 and 9 and the fraction of cross-reacting antibodies also increased with time after the initial immunization. Antibodies against each subdomain fragment were isolated on fragment adsorbents and were investigated for their cross-reaction with the other two fragments. Specific antibodies against each subdomain were found to cross-react with the other two subdomains. The degree of cross-reaction increased remarkably with the duration of immunization and reached as high as 100% for the cross-reaction between subdomains 3 and 6. The results are consistent with an unusual functional equivalence of antigenic sites within the same protein molecule. This functional equivalence of the antigenic sites of albumin, which we had previously proposed, improves with time after immunization.     

Purification of human autoantibodies from cross-linked antigen immunosorbents

A method is described whereby autoantibodies to the Sj?gren's syndrome antigen La (SS-B) can be purified from re-usable immunosorbent columns constructed from covalently linked human autoantibodies to which the antigen is cross-linked. Previous attempts to link the antigen directly to CNBr-Sepharose beads resulted in loss of biological activity and thus each purification of antibody required fresh batches of antigen. The present technique is a significant improvement since the cross-linked immunosorbents prepared from a single batch of antigen can be re-used several times over a 6 month period. Furthermore F(ab')2 fragments of anti-La antibodies can be purified from pepsin-digested serum samples. These antibodies react in ELISA, Western blot and immunofluorescence in an identical way to serum and murine monoclonal anti-La antibodies and show no reaction with the Ro antigen. However, being of human origin the affinity-purified anti-La antibodies have the advantages of bearing the same idiotypes and reacting with the same antigenic epitopes as naturally occurring serum autoantibodies.     

Sedimentation Characteristics of Human Thyroid Autoantibodies

Forty-six sera from patients with Hashimoto's thyroiditis were separated into 7S and 19S fractions by zone ultracentrifugation over sucrose density gradients. In each case at least 98 per cent of the anti-thyroglobulin activity was recovered in the 7S fraction. This was confirmed by chromatography on DEAE-cellulose. This also revealed significant antibody activity in a 7S γ-globulin fraction of fast electrophoretic mobility obtained from one serum giving a `clear' line on reaction with thyroglobulin in agar gel. Complement-fixing antibodies reacting with the thyroid microsomal fraction were also of low molecular weight. Thyroglobulin autoantibodies induced experimentally in rats were confined to the 7S fraction of serum. Initially, rabbits respond to the intravenous injection of human thyroglobulin with the production of 19S antibodies but by the 15th day, predominantly 7S antibodies are found.     

Quantitative distribution of human thyroglobulin autoantibodies in different immunoglobulin classes

The antigen-binding capacities of human sera containing thyroglobulin autoantibodies can be determined quantitatively by addition of excess radioactive antigen followed by co-precipitation of the complexes with an anti-immunoglobulin serum. Using a polyvalent anti-Ig serum it was shown that the antigen-binding capacity as measured by the radioactivity in the coprecipitate correlated well with the antibody content determined by precipitin curves. The distribution of antibodies among the major immunoglobulin classes could be estimated by co-precipitation with specific anti-Ig sera and the results obtained accorded with qualitative observations made by radioimmunoelectrophoresis. The specificity of the method was also checked on serum fractions obtained by preparative ultracentrifugation. In twenty-nine Hashimoto sera studied, the thyroglobulin antibodies were predominantly IgG. Sixteen of the sera contained appreciable concentrations of IgA antibodies representing up to 20% of the total antigen binding capacity; these antibodies sedimented with values intermediate between 7S and 19S. In only one serum were significant amounts of IgM antibodies detected by this method but they represented no more than 1% of the total activity. The method was shown to be valuable for following serial antibody levels in patients after operation or during thyroxine therapy.     

Utility of immunohistochemistry in the evaluation of necrotic thyroid tumors

We have previously shown that necrotic tumors retain their immunoreactivity for a range of cytokeratin antibodies. Some thyroid tumors undergo extensive necrosis after fine-needle aspiration (FNA) procedures. We evaluated the sensitivity of antibodies on necrotic thyroid tumors by examining a series of thyroid tumors consisting of 10 Hurthle cell neoplasms, 8 carcinomas, and 2 follicular adenomas (12 with post-FNA necrosis). These were stained with antibodies to AE1/3, PANCK, thyroglobulin and S100. Four of the cases of papillary carcinoma were also stained with antibodies to CK19. As a control for the specificity of thyroglobulin immunoreactivity in necrotic tissue, we also stained 11 nonthyroid tumors with extensive necrosis (7 carcinomas, 1 lymphoma, 2 melanomas, 1 sarcoma) for thyroglobulin. Six of 8 thyroid carcinomas were positive for AE1/3 and PANCK; AE1/3 reactivity was retained in necrotic areas of 4 of 6. AE 1/3 was positive in necrotic portions of 5 of 10 Hurthle cell lesions, whereas PANCKwas negative in all but 1. Thyroglobulin reactivity was present in 18 of 20 cases, and was preserved in necrotic portions of 5 of 6 carcinomas, as well as 8 of 10 Hurthle cell neoplasms. S100 cytoplasmic reactivity was present in 4 Hurthle cell neoplasms and 1 papillary carcinoma; this staining was lost in necrotic areas. No staining by thyroglobulin was observed in the viable or necrotic areas of nonthyroid neoplasms. The preservation of cytokeratin reactivity, measured by AE1/3, in thyroid neoplasms is a diagnostically useful feature in spontaneous and post-FNA infarction. PANCK is not a well-preserved marker in necrotic thyroid tissue. This difference may be due to detection of keratin 19 by AE1/3. Thyroglobulin is preserved in some necrotic thyroid carcinomas and in Hurthle cell lesions. Preservation of thyroglobulin reactivity in necrotic tissue is specific in that no staining was observed in nonthyroid neoplasms. These results suggest that thyroglobulin is useful in demonstrating thyroid lineage of both primary and metastatic necrotic tumor masses.     

Major histocompatibility gene complex (MHC)-coded determinants on antigen-specific suppressor factor for delayed-type hypersensitivity and surface phenotypes of cells producing the factor

Antigen-specific suppressor factor for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) was obtained by incubating in vitro spleen cells from CBA mice (H-2k) injected intravenously 3 days previously with 1 x 10(9) SRBC. The suppressor factor was characterized for major histocompatibility gene complex (MHC)-coded antigenic determinants by passing the factor through immunosorbents coupled with appropriate alloantisera. The suppressor factor was absorbed by anti-H-2k, anti-Iak and anti-I-Jk immunosorbents but was not retained by anti-Ias, anti-I-Js, anti-I-Ak, anti-I-E/Ck or anti-H-2Kk immunosorbents. In addition, the factor bound to an immunosorbent coupled with rabbit antibodies against carbohydrate-defined Ia antigens. Furthermore, the suppressive activity that was absorbed was quantitatively recovered in the acid eluates from the immunosorbents. Treatment of the spleen cells with anti-Lyt-1.1 antiserum and complement completely abrogated their ability to elaborate the suppressor factor in vitro. In contrast, treatment with anti-Lyt-2.1 or anti-Iak antiserum and complement had no effect. Thus, it appears that the suppressor factor for DTH to SRBC bears I-J subregion-coded determinants, and its production is dependent on cells which have the Lyt-1+,2- and Ia- phenotype.     

Amino Acid Composition of Proteins Extracted from Endemic Goiter Glands

Proteins were isolated and characterized in thyroid tissue of six patients from Brazil with endemic goiter. Biochemical studies of these thyroidal proteins included gel filtration, electrophoresis, and amino acid analysis. In addition to thyroglobulin, two of the most abundant proteins found in all goiters studied had molecular weights of 68 and 14 kDa. One protein was identified as albumin based on its immunoreactivity with antibodies to serum albumin. The other protein was identified as a hemoglobin subunit using reversephase high-performance liquid chromatography (HPLC). Identification was confirmed by partial N-terminal amino acid sequencing that showed several nonconservative differences in thyroid albumin when compared to human serum albumin (HSA). Biochemical findings were correlated with iodine and hormone contents in serum and thyroglobulin from these patients. Based on these findings, we suggest that hemoglobin and albumin are taken up from the blood by the hyperplastic thyroid tissue. Albumin would be processed by the thyroid follicular cell, becoming iodinated and released into the circulation.     

Preparation of rabbit anti-IgE for use in radioimmunoassays of total IgE and specific IgE antibodies

Detailed methodology for preparing and testing isolated rabbit anti-human IgE suitable for radioimmunoassays of total IgE and specific IgE antibodies is presented in a single article for the first time. A method for obtaining a product free of anti-idiotype antibodies is also described.IgE was isolated from E-myeloma serum (PS) by 40 and 50% saturated ammonium sulfate fractional precipitation, followed by DEAE Sephadex ion-exchange chromatography. The purified product was digested with papain, and the Fc fragments were separated from the Fab fragments by G-150 gel filtration and ion-exchange chromatography. Rabbits were immunized with IgE (Fc), and the antisera with the highest precipitin titers were pooled. A globulin fraction was prepared from the pooled antiserum by 50% saturated ammonium sulfate precipitation and the fraction was absorbed using an immunosorbent prepared from whole human serum having a very low IgE level and with immunosorbents prepared from D-myeloma protein and from IgE (Fab). Following absorption, the antibodies were demonstrated by micro-Ouchterlony technique to have no cross-reaction with IgG, IgA, IgM, IgD or any serum protein other than IgE. More than 200 mg of isolated anti-IgE (Fc) was prepared from antiserum globulin by consecutive affinity chromatography on columns of insolubilized IgE. Elution peaks appeared following the application of 0.1 M glycine—HCl, 1.0 M NaCl buffer, pH 2.5, as well as following a subsequent flush with 0.1 M phosphate, 1.0 M NaCl buffer, pH 7.4. The eluates were tested, pooled, radiolabelled with iodine-125 and demonstrated to be effective in RAST analyses. Antibodies to idiotypic determinants of the IgE molecule were eliminated by preparing the affinity chromatography column from a second E-myeloma protein (HL) rather than the E-myeloma protein (PS) used for immunization of the rabbits. Anti-IgE preparations which were free of anti-idiotype antibodies displayed less non-specific binding to IgD and IgG coated discs than preparations containing such antibodies.     

Thyroid Stimulating Antibodies, Thyroglobulin Antibodies and Serum Proteins during Treatment of Graves'' Disease with Radioiodine or Propylthouracil

The relation between serum concentrations of thyroglobulin antibodies (TgAb), thyroid-stimulating antibodies (TSAb) and serum immunoglobulins during treatment of Graves' disease was studied in 36 consecutive patients treated randomly with 131-iodine (n = 16) or propylthiouracil (n = 20). The patients were investigated before treatment was started and on seven occasions within the following year. In the entire patient group 78% were positive for TSAb and 47% for TgAb. There was a significant correlation between TSAb and TgAb in 15 patients concomitantly positive. There were no significant changes in serum immunoglobulins during treatment in either group of patients. In the radioiodine-treated group of patients TgAb was reduced after 1 week, whereas TSAb showed insignificant variations. After 5-10 weeks both antibodies increased, for TgAb with a median peak level 3 time above the initial concentration. Of 16 patients treated with radioiodine five developed myxoedema and four of these were positive for TgAb. There was a relation between the development of myxoedema and the ratio between increases of TSAb and TgAb. Increase in TSAb was not related to serum thyroglobulin (Tg) measured in TgAb-negative patients. Propylthiouracil showed minor effects on the studied variables, but with lower mean values of Tg, TgAb and TSAb at the end of the observation period. The results indicate an immunological relation between TSAb and TgAb, although differences between their course exist in some situations.     

Antigenic markers of V domain of mouse MOPC 21 IgG1 L chain. The conformational basis of VL domain markers and content of MOPC 21 VL-like immunoglobulins in sera of inbred mouse strains

Antisera were raised in rabbits against L chains, isolated from mouse myeloma protein MOPC21 (gamma 1, chi V chi 15 group). Specific antibodies for the V and C domain of MOPC21 L chain were obtained by cross-immunoadsorption of the antisera. The pure anti-V and anti-C antibodies were fixed on diazocellulose and used as immunosorbents. The inhibitory capacity of L chin-monomers and dimers isolated from the L chain preparation was compared to that of intact IgG1 using binding inhibition of 125I-labeled IgG1 on the antibody-containing immunosorbents. It was established that changes of IgG1 quaternary structure influences the conformational state of the L chain V domain only. The inhibitory capacity of the V domain is 1000-fold lower in L monomers, if compared with native IgG1, and only 10-fold lower than in L dimers. The inhibiting capacity of the C domain, however, does not differ in L monomers and intact IgG1. Thus the conformational rigidity of the C domain co-exists with conformational flexibility of the V domain on the same polypeptide chain. We tried to estimate the content of MOPC21 V1-like normal IgG in mouse serum of 6 inbred strains using antibodies against the V1 domain. Data obtained by inhibition of radioimmunoadsorption, indicate that in C57BL/6 mice 0.08% of normal serum Ig carries a V1 region which is idiotypically related to the V1 of MOPC21. In serum Ig of BALB/c mice the percentage is 0.16.     

The use of siliceous immunosorbents in hemosorption

Experiments were conducted on rabbits and monkeys for approbation of immunosorbents by covalent immobilization of human serum albumin or antibodies to human apoprotein B on the surface of silo-chromium and subsequent incubation with 1% albumin solution. Extracorporeal immunosorption in immunized rabbits led to reduction of the level of specific antibodies by 60% and the number of blood platelets by 10-30%. The initial antibody level was restored in 5-10 days. Study of immunosorbents on monkeys showed a satisfactory compatibility of the sorbent with blood, its ability for regeneration, and the possibility of its repeated use. The specific capacity was 66% of the capacity according to human apoprotein B.     

Concentrations of Free Thyroxine,Total Thyroxine,and Total Triiodothyronine in Rabbits Immunized with Human Thyroglobulin

Levels of serum free thyroxine, total triiodothyronine and total thyroxine were measured in two rabbits (TG-1,TG-2) which had been immunized with human thyroglobulin and produced both anti-thyroglobulin and anti-thyroid hormone antibodies. Despite the presence of both anti-triiodothyronine and -thyroxine antibodies, levels of serum free thyroxine in serial sera obtained from a rabbit (TG-1) stayed within normal range up to 249 days after the first immunization, whereas in the other rabbit (TG-2),serum free thyroxine concentrations of serial sera declined after three immunizations and stayed at lower levels than normal range up to 249 days. Extraction of serial sera from both rabbits disclosed the presence of larger amounts of triiodothyronine and thyroxine in immune sera than preimmune sera. From these results, it is concluded that the presence of anti-thyroid hormone antibodies per se can reduce serum level of free thyroxine in rabbits.     

Effect of Antibodies to Nerve Growth Factor and Serum Albumin on the Development and Behavior of Mice

We studied physical development, behavioral characteristics, and learning capacity in the offspring of mice immunized with nerve growth factor and bovine serum albumin. High titer of antibodies to these factors in the blood of pregnant females determines high levels of these antibodies in the blood of their pups. These changes modulate physical development, behavior, and learning capacity of rat pups. The effects of these antibodies differed in the strength and directionality. Antibodies to nerve growth factor more markedly retarded physical development, reduced learning capacity, and considerably increased pain thresholds in animals.     

Concentrations of Free Thyroxine, Total Thyroxine, and Total Triiodothyronine in Rabbits Immunized with Human Thyroglobulin

Levels of serum free thyroxine, total triiodothyronine and total thyroxine were measured in two rabbits (TG-1,TG-2) which had been immunized with human thyroglobulin and produced both anti-thyroglobulin and anti-thyroid hormone antibodies. Despite the presence of both anti-triiodothyronine and -thyroxine antibodies, levels of serum free thyroxine in serial sera obtained from a rabbit (TG-1) stayed within normal range up to 249 days after the first immunization, whereas in the other rabbit (TG-2),serum free thyroxine concentrations of serial sera declined after three immunizations and stayed at lower levels than normal range up to 249 days. Extraction of serial sera from both rabbits disclosed the presence of larger amounts of triiodothyronine and thyroxine in immune sera than preimmune sera. From these results, it is concluded that the presence of anti-thyroid hormone antibodies per se can reduce serum level of free thyroxine in rabbits.     

Whole organisms and purified cell walls compared as immunosorbents for the detection of IgE antibodies to Staphylococcus aureus

We have developed an immunoradiometric assay for IgE antibodies to Staphylococcus aureus (Staph IgE-Ab) which uses purified cell walls (PCW) from the Wood 46 strain of S. aureus as an immunosorbent. We compared Wood 46 PCW and whole organisms (WO) as immunosorbents for Staph IgE-Ab by performing tests on sera from patients with atopic dermatitis (AD) or the hyperimmunoglobulin E syndrome (hyper IgE syndrome). Sera with Staph IgE-Ab demonstrated dose-dependent binding to PCW and WO, but the ratio of specific to non-specific binding was much greater with PCW. Mean non-specific binding to WO was greater than to PCW, 5% versus 2%; and non-specific binding to WO varied directly with the serum concentration of IgE. Results of tests on patients' sera indicated that PCW are required in screening assays for Staph IgE-Ab to avoid false positive results caused by high levels of non-specific binding to WO.