Detection of autoantigens by immunoblotting using a peroxidase-anti-peroxidase complex

An immunoblotting procedure using a peroxidase-anti-peroxidase (PAP) complex was developed for the detection of autoantigens in crude mixtures by human autoimmune sera. Thymus proteins were transferred to a nitrocellulose sheet after electrophoresis in polyacrrylamide gels and probed with a 1:100 dilution of serum. The location and extent of immunoglobulin G (IgG) binding was determined by sequential reaction with: rabbit anti-human IgG, goat anti-rabbit IgG and rabbit peroxidase-anti-peroxidase complex. The peroxidase was allowed to react with chloronaphthol and low levels of autoantigen/autoantibody complex were detectable with virtual absence of background colour. The inclusion of human IgG and its pepsin-generated fragment provided a means of controlling and calibrating the blotting procedure.     

Bullous pemphigoid and cicatricial pemphigoid: immunoblotting detection of involved autoantigens

Bullous pemphigoid (BP) and cicatricial pemphigoid (CP) are subepidermal bullous autoimmune diseases which have distinct clinical features but identical immunological status. In order to determine whether these diseases could be dissociated on the basis of qualitative differences in serum antibodies to basement membrane zone (BMZ) antigens, the reactivity of sera from 7 CP and 29 BP patients with proteins extracted from normal human epidermal sheets (containing most of the lamina lucida components) was analysed using immunoblotting and compared to that of 10 normal sera. 20 out of the 29 BP sera contained antibodies recognizing one or several protein(s) of 240, 200, 180 and 165 kD molecular weight (MW). Antibodies in 4 out 7 CP sera specifically reacted with one or two polypeptides of 240 and 120 kD MW. These data confirm the heterogeneity of BP antigens and show the presence in CP of a novel 120 kD MW polypeptide which is found only in CP but not in BP. Taken together these findings demonstrate that in BP and CP, autoantibodies are directed to both common and specific BMZ antigens, their physiopathological significance need to be understood.     

Synaptophysin identified in metastases of neuroendocrine tumors by immunocytochemistry and immunoblotting

Synaptophysin, an Mr 38,000 integral membrane glycoprotein of neurotransmitter vesicles, has been identified in diverse primary neuroendocrine (NE) tumors of both neural and epithelial origin (Wiedenmann and co-workers, Proc Natl Acad Sci USA 1986; 83: 3500-3504). In the present study, metastases of several types of NE tumors, including medullary thyroid carcinoma, gastrinoma, insulinoma, small (oat) cell carcinoma of the lung, gastrointestinal carcinoid, and neuroblastoma, were examined for the presence of synaptophysin by immunocytochemistry, with the use of tissue sections as well as centrifuged cell suspensions and by immunoblotting of tumor proteins. The results show that expression of synaptophysin can be maintained during formation of metastases. Therefore, the authors propose that synaptophysin antibodies be used for the positive identification of metastatic NE tumors, notably in differential diagnosis. The possible implications of these findings for tumor diagnosis are discussed.     

Antigenically important proteins of Aujeszky's disease (pseudorabies) virus identified by immunoblotting

Summary Immunoblotting was used to identify those Aujeszky's disease virus proteins which elicited major antibody responses in naturally and experimentally infected pigs. Although some proteins comprising purified virus preparations reacted nonspecifically, proteins with mol. wts. of 120K, 90K, 71K and 60K were antigenically important. These corresponded in size to the virus glycoproteins identified by³H-glucosamine labelling. Glycoproteins isolated by affinity chromatography from infected PK15 cells solubilised by Triton X-100 (Triton-soluble glycoproteins) contained antigenic components similar in size to virus glycoproteins and produced minimal non-specific immunoblotting reactions. Antibody responses to the 120K and 71K proteins usually occurred together and were more pronounced than responses to other proteins especially at early times postinfection.     

Interactions between the mannose receptor and thyroid autoantigens

Thyroid autoantigens require internalization and processing by antigen-presenting cells to induce immune responses. Besides pinocytosis, antigen uptake can be receptor-mediated. The mannose receptor (ManR) has a cysteine rich domain (CR) and eight carbohydrate recognition domains (CRD) that bind glycosylated proteins. The TSH receptor (TSHR), thyroid peroxidase (TPO) and thyroglobulin (Tg) are glycoproteins. To investigate a role for the ManR in thyroid autoimmunity, we tested the interaction between these autoantigens and chimeric ManRs. Plasmids encoding the CR-domain linked to IgG-Fc (CR-Fc) and CDR domains 4-7 linked to IgG-Fc (CDR4-7-Fc) were expressed and purified with Protein A. Enzyme-linked immunosorbent assay (ELISA) plates were coated with human thyroid autoantigens and CR-Fc or CRD4-7-Fc binding detected with peroxidase-conjugated anti-IgG-Fc. CRD4-7-Fc binding was highest for the TSHR, followed by Tg and was minimal for TPO. CR-Fc bound to Tg but not to TSHR or TPO. The interaction between the TSHR and CRD-Fc was calcium-dependent; it was inhibited by mannose (not galactose), and required a glycosylated TSHR A-subunit. Moreover, precomplexing the TSHR A-subunit with CRD-Fc (but not CR-Fc), or adding mannose (but not galactose), decreased in vitro responses of splenocytes from TSHR-immunized mice. Our data indicate that the ManR may participate in autoimmune responses to Tg and the TSHR but not to TPO. Most important, ManR binding of heavily glycosylated TSHR A-subunits suggests a mechanism by which the minute amounts of A-subunit protein shed from the thyroid may be captured by antigen-presenting cells located in the gland or in draining lymph nodes.     

Autoimmune thyroid diseases: genetic susceptibility of thyroid‐specific genes and thyroid autoantigens contributions

Autoimmune thyroid diseases are common polygenic multifactorial disorders with the environment contributing importantly to the emergence of the disease phenotype. Some of the disease manifestations, such as severe thyroid‐associated ophthalmopathy, pretibial myxedema and thyroid antigen/antibody immune complex nephritis are unusual to rare. The spectrum of autoimmune thyroid diseases includes: Graves’ disease (GD), Hashimoto's thyroiditis (HT), atrophic autoimmune thyroiditis, postpartum thyroiditis, painless thyroiditis unrelated to pregnancy and thyroid‐associated ophthalmopathy. This spectrum present contrasts in terms of thyroid function, disease duration and spread to other anatomic location. The genetic basis of autoimmune thyroid disease (AITD) is complex and likely to be due to genes of both large and small effects. In GD the autoimmune process results in the production of thyroid‐stimulating antibodies and lead to hyperthyroidism, whereas in HT the end result is destruction of thyroid cells and hypothyroidism. Recent studies in the field of autoimmune thyroid diseases have largely focused on (i) the genes involved in immune response and/or thyroid physiology with could influence susceptibility to disease, (ii) the delineation of B‐cell autoepitopes recognized by the main autoantigens, thyroglobulin, thyroperoxidase and TSH receptor, to improve our understanding of how these molecules are seen by the immune system and (iii) the regulatory network controlling the synthesis of thyroid hormones and its dysfunction in AITD. The aim of the present review is to summarize the current knowledge regarding the relation existing between some susceptibility genes, autoantigens and dysfunction of thyroid function during AITD.     

Structural features of the autoantigens involved in thyroid autoimmune disease: the thyroid microsomal/microvillar antigen

The microsomal/microvillar antigen of the human thyroid gland which provokes thyroid autoimmunity was characterised by immunoprecipitation studies. Sera from patients with Hashimoto's thyroiditis, primary myxoedema or Graves' disease containing autoanti-microsomal antibody specifically precipitated a component, which under reducing conditions migrates with a mol. wt of 105,000 on SDS-polyacrylamide gel electrophoresis. This protein was absent in auto- or xeno-anti-thyroglobulin precipitates, which under reducing conditions display four polypeptides of Mr 260,000, 230,000, 180,000 and 142,000. Under non-reducing conditions, the microsomal/microvillar antigen displayed a small shift in mobility to a mol. wt of 117,000 suggesting the presence of intrachain disulphide bonds. In contrast, under these conditions, anti-thyroglobulin precipitated components displaying polypeptides of approx. mol. wts in the region of 240,000-260,000, 170,000-180,000 and 140,000. Absorption of thyroiditis sera on thyroglobulin-Sepharose followed by immunoprecipitation abolished the anti-thyroglobulin components without affecting the binding of the 105,000-dalton polypeptide, if the sera contained antimicrosomal antibody. No comparable material was identified in microsomal membrane preparations prepared from the stomach which is also commonly involved in organ-specific autoimmunity. The 105,000-dalton component does not bind to a Lens culinaris lectin affinity column. We conclude that the epitopes of the microsomal/microvillar antigen are presented on a poorly glycosylated peptide of mol. wt 105,000, which is probably stabilised by intrachain disulphide bonds and which does not share serological reactivity with membrane-bound thyroglobulin.     

65-70 kD protein identified by immunoblotting as the presumptive gastric microsomal autoantigen in pernicious anaemia.

Sera from 20 of 24 patients with pernicious anaemia reacted by immunoblotting with a 65-70 kD protein in canine and rodent gastric mucosal cells enriched for 80-90% parietal cells, and in microsomal preparations derived from these cells. All 20 reactive sera were positive for parietal cell microsomal antibody demonstrated by immunofluorescence. Eighteen parietal cell microsomal antibody-positive sera from patients with unconfirmed pernicious anaemia also reacted with the same 65-70 kD protein. Serum reactivity with the same 65-70 kD protein was not seen with canine and rodent liver cells or with microsomal preparations derived from these cells. Sera from 10 patients with chronic active hepatitis, 10 with scleroderma and 10 with rheumatoid arthritis and 22 healthy persons did not react with the 65-70 kD protein. These results suggest that the 65-70 kD protein is probably the parietal cell microsomal autoantigen. A second antigen of 85-90 kD mol. wt. present only in canine gastric mucosal preparations also correlates with the presence of parietal cell microsomal antibody. However, the contribution of this second antigen to parietal cell microsomal antibody reactivity remains uncertain.     

Antigens of Brucella abortus S19 immunodominant for bovine lymphocytes as identified by one- and two-dimensional cellular immunoblotting.

Cellular immune responses are influential for protection against intracellular bacteria such as brucellae. Therefore, identification of Brucella abortus antigens that activate primed bovine lymphocytes is fundamental for discerning the breadth of cellular response in bovine brucellosis. Potentially antigenic components of B. abortus S19 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by nitrocellulose blotting. Specific one-dimensional blot segments induced proliferation of peripheral blood lymphocytes from all 25 of the vaccinated cattle tested and were defined as immunodominant. Individual proteins that stimulated lymphocyte proliferation were further characterized by two-dimensional cellular immunoblotting by two different approaches. Individual one-dimensional stimulatory blot segments were eluted, concentrated, and then subjected to two-dimensional cellular immunoblotting. Alternatively, entire two-dimensional gels containing all of the B. abortus components were blotted and nitrocellulose sections containing individual proteins were assayed for lymphocyte activation. Thirty-eight Brucella proteins that induced lymphocyte proliferation were resolved by both procedures. Phenotypic analysis of the proliferating cell population demonstrated the presence of CD4+, CD8+, and immunoglobulin M+ lymphocytes. Two immunogenic proteins, 12 and 31 kDa, identified by two-dimensional cellular immunoblotting, were subjected to partial N-terminal amino acid analysis. The 12-kDa protein was within the area of greatest lymphocyte proliferation, while the 31-kDa protein was chosen for comparison with a 31-kDa protein previously reported by others. A search of the National Biomedical Research Foundation protein data bank showed that the sequences were not homologous with other known proteins. Identification of Brucella proteins immunogenic for bovine lymphocytes provides an important step in distinguishing the various proteins involved in pathogenicity and/or disease resistance.     

The 45 kilodalton molecule of Mycobacterium tuberculosis identified by immunoblotting and monoclonal antibodies as antigenic in patients with tuberculosis

The object of this study was to discover new M. tuberculosis antigens which are recognized by patients with tuberculosis, because effective serodiagnostic tests are likely to require combinations of different antigens. In our early experiments using immunoblotting, the findings suggested that human sera from smear-negative tuberculosis patients bound to an antigen in the 45 kDa region. Subsequently, estimates of molecular weight in the immunoblots confirmed that the murine monoclonal antibody (MAB) HGT-6 and sera from patients both recognized the same 45 kDa molecule. An antibody-antibody competition assay between MAB HGT-6 and sera from smear-positive tuberculosis patients yielded a positive result in 23 out of 43 sera from patients, but in only four out of 23 from controls. This is further evidence that the 45 kDa antigen is recognized by tuberculous patients. We analysed whether a combination of the 45 kDa antigen results and those of known antigens might better discriminate between minimal smear-negative disease and healthy controls than could test with single antigens. There is no clinically useful laboratory test for smear-negative tuberculosis. In immunoblotting, combining the results with the 65, 45, 38 and 10 kDa antigens gave the best discrimination. This suggests that future serodiagnostic tests for minimal disease, such as the antibody-antibody competition assay, should contain a MAB against the 45 kDa antigen and possibly also against the 10 kDa antigen.     

Microfilariae identified in FNA of a thyroid nodule

A patient presented with hyperthyroidism and a thyroid swelling, clinically thought to be malignant. Fine-needle aspiration of the thyroid nodule revealed microfilariae of Wuchereria bancrofti. After therapy the thyroid swelling subsided and the patient became euthyroid. The possible etiologic role of microfilariae in the genesis of the thyroid tumor and hyperthyroidism is discussed. Diagn. Cytopathol. 16:149–150, 1997. © 1997 Wiley-Liss, Inc.     

The 45 kilodalton molecule of Mycobacterium tuberculosis identified by immunoblotting and monoclonal antibodies as antigenic in patients with tuberculosis.

The object of this study was to discover new M. tuberculosis antigens which are recognized by patients with tuberculosis, because effective serodiagnostic tests are likely to require combinations of different antigens. In our early experiments using immunoblotting, the findings suggested that human sera from smear-negative tuberculosis patients bound to an antigen in the 45 kDa region. Subsequently, estimates of molecular weight in the immunoblots confirmed that the murine monoclonal antibody (MAB) HGT-6 and sera from patients both recognized the same 45 kDa molecule. An antibody-antibody competition assay between MAB HGT-6 and sera from smear-positive tuberculosis patients yielded a positive result in 23 out of 43 sera from patients, but in only four out of 23 from controls. This is further evidence that the 45 kDa antigen is recognized by tuberculous patients. We analysed whether a combination of the 45 kDa antigen results and those of known antigens might better discriminate between minimal smear-negative disease and healthy controls than could test with single antigens. There is no clinically useful laboratory test for smear-negative tuberculosis. In immunoblotting, combining the results with the 65, 45, 38 and 10 kDa antigens gave the best discrimination. This suggests that future serodiagnostic tests for minimal disease, such as the antibody-antibody competition assay, should contain a MAB against the 45 kDa antigen and possibly also against the 10 kDa antigen.     

Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens

AKR/N mice injected with fibroblasts expressing MHC class II (RT4.15HP cells) and the TSH receptor (TSHR) develop antibodies similar to those in Graves' disease. We were unable to analyse the subclass of these antibodies because of unexpectedly high non-specific binding by ELISA or flow cytometry. The non-specific binding reflected generalized immune activation which occurred even when the fibroblasts did not express the TSHR. However, the IgG subclasses were determined for thyroid peroxidase (TPO) antibodies induced using TPO-expressing RT4.14HP cells and found to be IgG2a > IgG1. This Thl pattern is consistent with spontaneous secretion of interferon-gamma (but not IL-4 or IL-10) by splenocytes from injected mice. The Th1 bias was related to fibroblast injection because conventional immunization of the same mouse strain with purified TPO and adjuvant induced a Th2 response (IgG1 > IgG2a). Further, untransfected fibroblasts themselves induced powerful, non-specific proliferative responses when used as antigen-presenting cells (APC) in vitro. Flow cytometry revealed that the RT4.15HP fibroblasts (and TSHR- and TPO-transfected derivatives) expressed B7-1. Unexpected constitutive expression of this key molecule may bypass the requirement for up-regulation of other costimulatory molecules involved in T cell stimulation. Our data support the concept that RT4.15HP fibroblasts present the TSHR (or TPO), at least for initiating the immune response. However, the accompanying generalized immune stimulation creates difficulties for analysis of TSHR-specific T and B lymphocytes. On the other hand, extension of the model to TPO, an easier antigen to study, will facilitate analysis of murine T cell responses likely to resemble those in human thyroid autoimmunity.     

Phenothiazine-induced increase in thyroid autoantigens and costimulatory molecules on thyroid cells: a pathophysiological mechanism for drug-induced autoimmunity?

We have previously demonstrated (J Immunol 1995; 154 :3593) that MHC class II antigens can be induced on thyroid epithelial cells (TEC) by alimemazine, a member of the phenothiazine group. Although this expression of MHC class II antigens on TEC confers the theoretical ability to behave as antigen-presenting cells (APC), the simultaneous expression of self antigens and co-receptor(s) must also occur for efficient presentation of self antigens. Therefore, we investigated whether alimemazine applied at pharmacologic doses would modify the expression of thyroid antigens, and simultaneously, the expression of intercellular adhesion molecule-1 (ICAM-1), B7, and LFA-1 co-receptors in human TEC in culture. Using polymerase chain reaction (PCR) amplification and Northern blot analysis, we showed that alimemazine induces increases in thyroglobulin (Tg) and thyroid-stimulating hormone receptor (TSH-R) cDNA, within the first 2 h following its addition. This phenomenon is followed 48 h later by an increase of Tg and TSH-R protein expression on the surface of TEC. Furthermore, increases in the expression of ICAM-1 and B7 co-receptors were concomitantly observed. These results suggest that alimemazine, a drug currently used in paediatrics, could play a role in the induction and perpetuation of thyroid autoimmune disorders by transforming TEC into functional APC.     

Proteins ofNippostrongylus brasiliensis analyzed by immunoblotting

Antigenic proteins were characterized by the immunoblotting technique with sera from rats and mice after infection as well as hyperimmune sera. The immune response of infected animals was mainly directed toward five proteins of adult worms (190, 118, 110, 98, and 52 kDa) and four proteins of the third larval stage (L3; 92, 78, 58, and 24 kDa). The immunoblots indicated that stage-specific proteins of the homogeneates were recognized. Three stage-specific proteins of L3 larvae (150, 135, and 125 kDa) and three proteins typical to the adult worm (100, 82, and 67 kDa) were identified. The majority of the worm proteins elicited an IgG response. IgE synthesis was induced by living and dead parasites and was directed towards four proteins (190, 150, 125, and 98 kDa). Three proteins additionally induced an IgG or IgM antibody response. The immune response as shown by the immunoblotting technique seems to be directed towards (1) antigens that are present for the duration of an infection and (2) stage-specific antigens that are expressed for only a short time during the life cycle of the parasite.Abbreviations EDTA ethylenediaminetetraacetate - IMS sera from infected mice - IRS sera from infected rats - kDa kilodalton - L3 larvae, third-stage larvac - MS imm. L3 sera from mice immunized with dead L3 larvae - MS imm. W sera from mice immunized with dead adult worms - PBS phosphate-buffered saline - PCMB p-chloromercuribenzoate - PMSF phenylmethylsulfonylfuoride - RMS sera from reinfected mice - RRS sera from reinfected rats - TBS TRIS-buffered saline - TLCK p-tosyl-l-lysine-chloromethylketone - TPCK l-1-tosylamido-2-phenylethyl-chloromethylketone - TRIS tris(hydroxymethyl)-aminomethane - SDS sodium dodecyl sulfate Supported by Deutsche Forschungsgemeinschaft Bo 740/1-2; the work by U. Dorzok comprises the partial fulfillment of a PhD thesis