Metabolic basis for a drug hypersensitivity: antibodies in sera from patients with halothane hepatitis recognize liver neoantigens that contain the trifluoroacetyl group derived from halothane

Previous studies have demonstrated that antibodies in sera from patients with halothane hepatitis recognize halothane-induced liver microsomal polypeptide neoantigens, and have suggested that these antibodies may play a role in the pathogenesis of the hepatitis. In the present study, the mechanism of neoantigen generation was investigated. Liver microsomes from rats treated in vivo with halothane or deuterated halothane were tested by immunoblotting for reactivity with patients' sera and with an antiserum specific for the covalently bound trifluoroacetyl (TFA) halide metabolite of halothane. Rat liver microsomes incubated aerobically or anaerobically with halothane or deuterated halothane in vitro, +/- NADPH and/or NADH, were also analyzed. The results obtained demonstrate that neoantigen expression involves oxidative halothane metabolism by cytochromes P-450 to TFA halide and covalent binding of the TFA group to the proteins. Incubation of microsomes from halothane-treated rats with 1 M piperidine cleaved the TFA groups from the proteins and abolished antigenicity, confirming this conclusion. Recognition of the neoantigens by the patients' antibodies was inhibited only partially using the hapten derivative N-E-TFA-L-lysine. It appears that the patients' antibodies recognize epitopes consisting of the TFA group plus associated structural features of the protein carriers (100 kDa, 76 kDa, 59 kDa, 57 kDa and 54 kDa), not the TFA hapten alone. To our knowledge, this constitutes the first characterization of drug metabolite-tissue protein neoantigens implicated in a drug hypersensitivity. The approach described may be of general utility for characterization of drug-induced neoantigens associated with other drug hypersensitivities.     

The serology of chronic hepatitis B infection revisited.

The serology of chronic hepatitis B infection has been established through the use of commercial immunoassays to measure the structural antigens of the hepatitis B virus and their respective antibodies in serum. However, the commercial assays have not been designed to detect serum antibodies in the presence of an excess of circulating antigens. A series of serum samples from 200 HBeAg-positive, chronically infected hepatitis B patients with varying degrees of liver disease were analyzed using novel immunoassays designed to detect antibodies in the presence of circulating viral antigens. All patients, regardless of their liver disease, were seronegative for antibodies specific for the envelope antigens or the secreted nucleoprotein antigen (HBeAg) when the commercial assays were used. In contrast, virtually all chronically infected patients with liver disease and approximately 50% of patients without liver disease demonstrated anti-HBe and anti-envelope antibodies when sera were tested in the more sensitive immunoassays. Furthermore, asymptomatic patients could be serologically distinguished from symptomatic patients based on antibody fine specificity, titer, and IgG subclass. This study revealed that the majority of chronically infected hepatitis B patients produce a variety of antibodies for many years, and are not immunologically unresponsive, as suggested by the current assays.     

Frequency and significance of antibody to hepatitis C virus in severe corticosteroid-treated autoimmune chronic active hepatitis.

To determine the frequency and significance of antibody to hepatitis C virus (anti-HCV) in severe autoimmune chronic active hepatitis, we tested sera from 85 cortico-steroid-treated patients by an enzyme immunoassay. Seropositive patients were assessed for specific antibodies to hepatitis C virus-encoded antigens by recombinant immunoblot assay. The findings in patients with and without anti-HCV were contrasted, and the frequency of seropositivity was compared with that in patients who had other types of chronic liver disease and in normal adults. Only 5 of the 85 patients with autoimmune hepatitis (6%) were seropositive for anti-HCV, and only 2 of these patients were reactive by recombinant immunoblot assay. The frequency of seropositivity in autoimmune hepatitis was not significantly different from that in hepatitis B surface antigen-positive (9%) and cryptogenic (18%) disease, but it was significantly less than that in posttransfusion chronic active hepatitis (6% versus 75%; P less than 0.001). Two patients became seronegative after corticosteroid therapy; both had been nonreactive by recombinant immunoblot assay. Four of the seropositive patients entered remission during corticosteroid therapy, including three whose sera were nonreactive to virus-encoded antigens. We conclude that anti-HCV occurs infrequently in corticosteroid-treated severe autoimmune hepatitis and that antibodies detected by enzyme immunoassay may be nonreactive to hepatitis C virus-encoded antigens. Seropositive patients who are nonreactive by immunoblot assay may still respond to corticosteroid therapy and become seronegative during treatment.     

The detection of IgG anti-hepatocyte antibodies by ELISA in sera of patients with chronic active hepatitis: correlation with disease activity

Anti-hepatocyte membrane IgG antibodies were detected in serial dilutions of sera from patients with chronic active hepatitis using an enzyme linked immunosorbent assay with isolated rat hepatocytes as the antigenic substrate. The assay is rapid, reliable and reproducible. Antibodies to hepatocyte membrane antigens were detected in 24 out of 31 (77%) patients with autoimmune chronic active hepatitis. Four of these patients were negative in a 1/10 dilution but became positive on progressive dilution. In 12 patients, liver biopsy was performed at the same time as serum was obtained. In these patients, the titre of antibodies to hepatocyte membrane antigen correlated significantly with the overall biopsy score of disease activity and particularly with the degree of portal tract infiltration. In 2 patients followed serially, titres of anti-hepatocyte membrane antibodies fell progressively in parallel with clinical, biochemical and histological evidence of improvement in the liver disease. The estimation of titres of antibodies to hepatocyte membrane antigens using isolated rat hepatocytes as the antigenic substrate may assist in the diagnosis, assessment of disease activity and follow-up of individual patients with chronic active hepatitis.     

Anti-liver-kidney microsome antibody recognizes a 50,000 molecular weight protein of the endoplasmic reticulum

Children with autoimmune chronic active hepatitis may have high titers of antibodies detected by immunofluorescence staining of hepatocytes and tubular cells in rat liver and kidney sections, respectively. These antibodies are directed against antigens contained in microsomal fractions prepared from these two organs. We have found that sera from these patients recognized a 50,000 mol wt protein present in higher concentration in smooth microsome subfractions compared with rough microsome subfractions. This protein is an integral membrane protein and is not glycosylated. It is exposed on the cytoplasmic face of the endoplasmic reticulum and is rather resistant to proteolysis with proteinase K. Since patients with liver disease of different etiology and similar severity of cell lysis do not give rise to liver-kidney microsome antibody (LKMA), lysis of hepatocytes is apparently not a sufficient condition for their development.     

Serologic analysis of antigen-specific reactivity in patients with systemic candidiasis

Antibody responses to candidal polypeptides and mannans were studied in patients with systemic candidiasis, candiduria, and other fungal and bacterial infections, and in healthy laboratory personnel to determine the diagnostic value of these immunologic responses. When tested by immunoblot analysis, sera from 15 patients with systemic candidiasis frequently contained antibodies to three antigens: 15 of 15 sera from patients with invasive disease reacted to a molecular species having a molecular weight (Mr of 90-200 kd, 13 of 15 reacted with a 45-kd polypeptide, and 12 of 15 reacted with a 17-kd polypeptide. Lesser reactivity was observed in 11 of 15 sera with a 28-kd candidal antigen and in 9 of 15 to a 57-kd candidal antigen. Quantitation of antibody titers against the 45-kd candidal polypeptide demonstrated much higher immunoreactivity in patients with systemic candidiasis than in patients with superficial candidal infections, bacterial infections, other systemic mycoses, and healthy individuals. Antimannan antibody titers were measured by an enzyme-linked immunosorbent assay (ELISA) and these titers were also higher in patients with systemic candidiasis than in patients in the other categories. These differences, however, were less than those observed with the anti-45-kd polypeptide antibody. Therefore, the ability to detect systemic candidiasis is improved by testing sera for immunoreactivity to polypeptide and to mannan antigens from Candida albicans. Detection of polypeptide antibodies improves the serodiagnosis of systemic candidiasis.     

Detection of autoantibodies against M2, LKM-1, and SLA in liver diseases by standardized uniform ELISA-techniques

Antibodies directed against soluble liver antigen (SLA), liver kidney microsomal antigen (LKM-1-AG), and antimitochondrial antigen M2 (M2-AMA) are critical serological markers for the differential diagnosis of autoimmune chronic active hepatitis (AI-CAH) and primary biliary cirrhosis (PBC). The exact diagnosis of autoimmune hepatitis and PBC is of great clinical relevance, as it leads to different therapeutic strategies. In the present work, a simple and reliable ELISA test system is described, which applies the same test principle for the detection of three different species of autoantibodies important for the diagnosis of chronic liver disease. The ELISA assays are based on a competitive inhibition of binding of positive standard antibodies by patients sera containing antibodies of unknown specificity. The purified immunoglobulins of clinically and serologically clearly defined patients with SLA or LKM-1 positive AI-CAH and with M2 positive PBC were used as coating- and detection antibodies in the ELISAs. From homogenized rat liver the fractionated 100,000g supernatant was employed for the SLA ELISA, the microsomal preparation served as antigen for the LKM-1 ELISA and the mitochondrial preparation was used for the M2 ELISA. In 1,500 sera of patients with the differential diagnosis of a hepatobiliary disease, 17 gave a positive signal in the SLA ELISA, 12 in the LKM-1-ELISA, and 72 in the M2-ELISA. The results of the ELISAs were compared with Western blotting and immunofluorescence staining pattern on cryostat sections and Hep2 cells. The antibody profiles of several patients are described in detail. © 1994 Wiley-Liss, Inc.     

Identification of HCC-22-5 tumor-associated antigen and antibody response in patients

BACKGROUND: Serological identification of antigens by recombinant expression cloning (SEREX) is a promising method used to analyze tumor-associated antigen (TAA). Nineteen primary HCC-associated antigens have been found from a HCC cDNA library using autogenous serum by the SEREX approach. We searched for HCC-associated antigens and applied them to HCC diagnosis. METHODS: Nine of 19 primaries HCC-associated antigens identified by SEREX method were tested their immune response again with distinct allogeneic sera. One of the screened HCC-associated antigens, HCC-22-5 was recombined and expressed and made the frequency analysis of its seropositivity in various patients using the methods of Western-blot and ELISA. RESULTS: SEREX analysis showed that 9 primary HCC-associated antigens had high-titered IgG antibody in the majority of HCC patients. Western-Blot method confirmed that 3/7 HCC patients had antibodies against HCC-22-5, which demonstrated that expressed HCC-22-5 antigen had the character of antigen. Sera samples from 341 patients and 80 normal individuals have been tested for autoantibodies against HCC-22-5 by ELISA method. The results found that 51/128 of HCC, 11/76 of chronic hepatitis, 11/22 of liver cirrhosis and 8/54 of nasopharynx cancer patients had antibodies against HCC-22-5. No antibody response to HCC-22-5 had been found in the sera of 7 lung cancers, 54 gastric-intestine patients and 80 normal individuals. The groups of HCC and liver cirrhosis had higher antibody positive rate than that of other groups (p<0.05). In the HCC sera with alpha-fetoprotein (AFP) negative, the positive rate of HCC-22-5 was as high as 78.9%. CONCLUSIONS: HCC-22-5 can be used for HCC serologic screening, especially for the patients with AFP negative.     

Toxoplasmosis and cross-reactivity: an analysis of the serological response by immunoblotting

Antibodies which react with toxoplasma antigens (natural antibodies) can be found in patients without other evidence of toxoplasma infection. The natural antibody profile of 37 sera which were Dye Test (DT) negative was investigated by Western blotting with toxoplasma antigens. These 37 sera comprised 11 which were indirect haemagglutination test positive for toxoplasma antibodies, 8 rheumatoid factor positive and 18 from patients with acute viral infections. These three groups of sera could not be distinguished from each other, or from pooled sera from patients not infected with toxoplasma, by characterising the Western blot reactions.     

The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase

Autoantibodies to mitochondrial antigens are characteristic of the autoimmune liver disease primary biliary cirrhosis (PBC), but the precise antigenic determinants recognized by these antibodies have not been defined. Recently, our laboratory identified a 1,370-bp rat liver cDNA clone that coded for a polypeptide recognized specifically by sera from patients with PBC but not by sera from patients with other forms of liver disease. This recombinant protein was identified as the 74-kD M2 mitochondrial inner membrane autoantigen, now known to be dihydrolipoamide acetyltransferase. In the present study, we have identified a 603-bp fragment that codes for a polypeptide containing all of the autoreactivity of the original clone. In addition, based on hydrophobicity/hydrophilicity plots of the amino acid sequence of this polypeptide segment, several peptides were synthesized and tested for reactivity by an inhibition assay using sera from patients with PBC. One peptide, defined by the amino acids AEIETDKATIGFEVQEEGYL, absorbed serum reactivity to the protein product of the original clone. Of particular interest was the finding that this peptide contains the lipoic acid binding site KATIGF of the dihydrolipoamide acetyltransferase found in the inner mitochondrial membrane. Thus, it appears that for this autoantigen, the target of the autoantibodies corresponds to a functional site of the dihydrolipoamide acetyltransferase.     

A method for detecting specific anti-C100 protein antibodies of IgM isotype in hepatitis C virus infection

Summary A method for the determination of specific IgM antibodies to C100 protein, a hepatitis C virus-associated antigen, was developed which employed fraction of serum proteins by gel chromatography and an enzyme-linked immunosorbent assay. Detection of IgM anti-C100 proved to be specific and reproducible in purified IgM fractions. Separation of IgM from IgG was necessary before IgM could be measured, because of the detrimental effect of the simultaneous presence of IgG antibodies with anti-C100 rectivity on IgM determination. IgM anti-C100 was not found in sera containing rheumatoid factors or IgM antibodies to other hepatotropic viruses. IgM anti-C100 was detected in 19 (44%) of 43 patients with hepatitis C virus-related chronic liver disease. When compared with the histological picture of liver disease, IgM anti-C100 was absent in patients with minimal changes and was most common (66.7%) in patients with progressive disease. It is suggested that IgM anti-C100 could reflect an active state of hepatitis C virus infection.     

A method for detecting specific anti-C100 protein antibodies of IgM isotype in hepatitis C virus infection.

A method for the determination of specific IgM antibodies to C100 protein, a hepatitis C virus-associated antigen, was developed which employed fractionation of serum proteins by gel chromatography and an enzyme-linked immunosorbent assay. Detection of IgM anti-C100 proved to be specific and reproducible in purified IgM fractions. Separation of IgM from IgG was necessary before IgM could be measured, because of the detrimental effect of the simultaneous presence of IgG antibodies with anti-C100 rectivity on IgM determination. IgM anti-C100 was not found in sera containing rheumatoid factors or IgM antibodies to other hepatotropic viruses. IgM anti-C100 was detected in 19 (44%) of 43 patients with hepatitis C virus-related chronic liver disease. When compared with the histological picture of liver disease, IgM anti-C100 was absent in patients with minimal changes and was most common (66.7%) in patients with progressive disease. It is suggested that IgM anti-C100 could reflect an active state of hepatitis C virus infection.     

Species cross-reactivity of anti-LSP antibodies in acute viral hepatitis

Previous reports have suggested that circulating antibodies reacting with species cross-reactive determinants in the liver-specific membrane lipoprotein (LSP) complex are rare in acute viral hepatitis (AVH) and that their high frequency in chronic active hepatitis (CAH) indicates a fundamental change in antigen specificity of anti-LSP autoantibodies associated with progression to chronic liver disease. In the present study, sera from 20 patients with uncomplicated AVH (10 with virus A and 10 with virus B infection) were examined by radioimmunoassay for antibodies reacting with human, rabbit and rat LSP. All 20 patients were found to be seropositive for antibodies against all 3 LSP preparations. Titres of anti-human and anti-rat LSP were significantly higher (p less than 0.001) than those of anti-rabbit LSP but a strong linear correlation (p less than 0.005) was found between titres of antibodies against the 3 LSP preparations. Cross-inhibition experiments with sera from 10 patients (5 with AVH-A and 5 with AVH-B) failed to reveal any major differences in antigen specificity between anti-rabbit and anti-rat LSP in 8 but results with the remaining 2 patients suggested that their anti-LSP antibodies were not reacting with the same antigens in the 2 LSP preparations. The results indicate that antibodies reacting with species cross-reactive LSP determinants are a normal occurrence in AVH and suggest that, if anti-LSP antibodies are involved in progression to (and/or development of) CAH, this is more likely to be related to a defect in the control of autoreactivity than to inherent differences in target antigen specificities of these autoantibodies in acute and chronic liver diseases, although the latter possibility cannot be totally excluded.     

Anti-liver endoplasmic reticulum autoantibodies are directed against human cytochrome P-450IA2. A specific marker of dihydralazine-induced hepatitis.

Sera from patients with dihydralazine-induced hepatitis were shown to contain anti-liver microsomal autoantibodies (anti-LM) by indirect immunofluorescence. These anti-LM antibodies were different from anti-liver/kidney microsomes (anti-LKM) 1 or 2 autoantibodies which have been previously described. Sera recognized a single 53,000 = Mr polypeptide in human liver microsomes as judged by immunoblotting, and the target antigen was identified as cytochrome P-450IA2 (P-450IA2) by (a) comparison of immunoblotting patterns with anti-human P-450IA2 and anti-rat P-450IA2 and with five anti-LM sera, and (b) specific immunoinhibition of microsomal ethoxyresorufin and phenacetin O-deethylation activities (both P-450IA2 supported reactions) by anti-LM antibodies. Finally, purified human P-450IA2 was recognized by these anti-LM sera. The anti-LM antibodies are specific for the disease because none of the other antisera tested behaved in the same manner as anti-LM, even those from patients treated with dihydralazine and without hepatic disease. A possible role of P-450IA2 in the metabolism of dihydralazine was suggested by competitive inhibition of ethoxyresorufin-O-deethylase observed in microsomal incubations. Thus, a new example is presented in which a cytochrome P-450 may be a target for autoantibodies in drug-induced hepatitis.     

Frequency and significance of antibody to hepatitis C virus in severe corticosteroid-treated cryptogenic chronic active hepatitis

To determine the frequency and significance of antibody to hepatitis C virus (anti-HCV) in severe cryptogenic chronic active hepatitis (CAH), we tested sera from 17 corticosteroid-treated patients by an enzyme immunoassay. Specificity of the antibodies to HCV-encoded antigens was assessed by recombinant immunoblot assay. The findings in patients with and without anti-HCV were contrasted, and the frequency of seropositivity was compared with that in patients who had other types of chronic liver disease and in normal adults. Only three patients (18%) with severe cryptogenic CAH had anti-HCV. Sera from two of these patients were reactive by recombinant immunoblot assay; the other sample produced an indeterminate reaction. The frequency of seropositivity in patients with cryptogenic disease was not statistically different from that in patients with autoimmune CAH (6%), hepatitis B surface antigen-positive CAH (9%), or alcoholic liver disease (0%), but it was significantly less than in those with posttransfusion CAH (18% versus 75%; P less than 0.01). Seropositive patients tended to have lower serum aspartate aminotransferase, gamma-globulin, and bilirubin levels than seronegative counterparts, and they did not have histologic features of confluent necrosis at initial assessment. Two of the three seropositive patients, both of whom had been reactive by recombinant immunoblot assay, entered remission during therapy, and one, with an indeterminate reaction, died of liver failure. We conclude that anti-HCV occurs infrequently in severe corticosteroid-treated cryptogenic CAH. Seropositive patients may have less severe inflammatory activity than seronegative counterparts. Cryptogenic disease may improve during corticosteroid treatment, a result suggesting an underlying immunologic disorder in some patients.     

Performance of an enzyme-linked immunosorbent assay system for antibodies to hepatitis C virus with two new antigens (c11/c7).

We developed an enzyme-linked immunosorbent assay (ELISA) system for antibodies to the hepatitis C virus (HCV), using two new recombinant antigens (c11 and c7) derived from the HCV genome. The performance of this ELISA system (Imucheck HCV Ab) was examined. The CV values for both intra-assay precision and reproducibility of identifying HCV antibody in the panel sera ranged from 3.5% to 6.4%. The blood elements in serum and anticoagulants did not interfere in this ELISA system. The specificity of Imucheck HCV Ab to samples from patients with non-A, non-B (NANB)-type chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma was 93.7%, 93.5%, and 81.4%, respectively. These results are more sensitive than those obtained by the first-generation anti-HCV ELISA system. In the samples from patients with NANB-type acute hepatitis, Imucheck HCV Ab enabled detection of HCV antibodies at an early stage. This system increased the sensitivity for blood donor screening and for monitoring patients with acute hepatitis.     

Anti-GOR in hepatitis D: specific association with hepatitis C virus superinfection

Summary A group of 113 patients with chronic hepatitis D was investigated for the presence of anti-GOR and liver kidney microsomal antibodies. Eight patients were anti-GOR positive and also positive for hepatitis C virus-infection. In sera from 16 patients liver-kidney microsomal antibodies were detectable by immunofluorescence. They were classified as LKM-3 due to their fluorescence pattern. Two of the LKM-3-positive sera were also anti-hepatitis C virus and anti-human immunodeficiency virus positive. None of these patients were positive for anti-GOR. Fourteen sera from LKM-3-positive patients reacted in Western blot with a microsomal protein at 55 kDa that differs from the 50-kDa LKM-1 (cytochrome P450IID6) antigen. Our studies demonstrate that hepatitis D virus itself does not induce an autoimmune reaction against the GOR antigen and that autoimmunity to the LKM-3 antigen induced by hepatitis D virus infection does not correlated with anti-GOR. These studies support the specificity of the anti-GOR response for hepatitis C virus infection.     

Lymphocytotoxins in sera from highly sensitized multiparous dialysis patients: antibody class, relationship with the HLA and with paternal antigens.

1. Sera from 11 highly sensitized multiparous dialysis patients were studied in order to define the target antigens, antibody class and relationship with paternal HLA class I antigens of the underlying lymphocytotoxic antibodies. All sera contained lymphocytotoxic antibodies to over 70% of a panel of lymphocytes from 24 donors (panel reactivity greater than 70%). 2. Inhibition of cytotoxic activity against paternal lymphocytes by monoclonal antibodies to HLA framework determinants indicated that all 11 sera contained lymphocytotoxic antibodies to paternal class I antigens. In addition, five sera contained lymphocytotoxic antibodies to paternal class II antigens. 3. In order to determine the extent to which lymphocytotoxic antibodies were directed to paternal antigens, the panel reactivity of sera was compared before and after absorption with paternal peripheral blood lymphocytes. Over 50% of panel reactivity was absorbed from eight out of 11 sera, and in three of these 11 over 80% was absorbed. In the majority of patients this change in panel reactivity could be ascribed to binding of lymphocytotoxic antibodies to specific paternal class I antigens. 4. Digestion of sera with dithiothreitol had no significant effect on panel reactivity, indicating that the lymphocytotoxic antibodies were of immunoglobulin G class. 5. No sera reacted with either autologous lymphocytes or K562 cells, indicating an absence of autoantibodies. 6. These studies imply that panel-reactive lymphocytotoxic antibodies in the sera of highly sensitized multiparous patients are those which mediate hyperacute renal allograft rejection. Their development may be related to secondary humoral responses to antigens in blood transfusions from donors who share paternal class I specificities.     

Methodology and significance of the detection of liver-kidney-microsomal (lkm) autoantibodies in autoimmune-type chronic active hepatitis

Liver-kidney-microsomal (LKM) autoantibodies are diagnostic markers for a subgroup of HBsAg-negative chronic active hepatitis, presumably owing to autoimmunity. They were originally detected by indirect immunofluorescence and can now be evaluated by radioimmunoassay, enzyme-linked immunosorbent assay, and immunoblotting. In immunoblotting LKM-positive sera react strongly with a 50-kilodalton (KD) polypeptide band of microsomes. In immunoelectron microscopy, LKM-positive sera show a binding with membranes of the endoplasmic reticulum. The LKM antigen was further identified on various isoenzymes of cytochrome P-450. Immunofluorescence is still the method of choice for screening sera routinely. However, cytplasmic antigen-antibody systems often can hardly be distinguished by this method. A specific radioimmunoassay and an enzyme-linked immunosorbent assay can differentiate the various autoantibodies against cytoplasmic antigens. Immunoblotting and immunoelectron microscopy are specific tools for the characterization of the target antigens on an ultrastructural or molecular level. So far they have no use in routine testing of sera. However, since LKM antigen was localized on isozymes of cytochrome P-450, this subgroup of CAH might turn out to be a drug-induced autoimmune liver disease. Clinically, these patients are characterized by chronic active hepatitis or cirrhosis in liver histology, a slight predominance of females, low IgA immunoglobulin levels, and an often rapidly progressing disease. Patients can be treated with immunosuppressive drugs. However, controlled therapeutical trials are missing. Furthermore, an immunogenetic background still has to be proven for this autoimmune liver disease.     

Species-specific sera against surface antigens of Bacillus anthracis strains

The species-related specificity of sera against 94-KD proteins isolated from culture filtrates of B. anthracis strains with different levels of virulence plasmids was studied to determine whether they might be used to identify the pathogen of anthrax. Sera against fractions 1 of culture filtrates of B. anthracis strains CTI (pXO1+ pXO2-), 81/1TR (pXO1- pXO2-), Davies (pXO1- pXO) separated by gel chromatography on Sephacryl S-300 were examined. In the gel immunodiffusion test with growing cultures, the sera exhibited non-identical antigens and differed in the presence of antibodies to antigens of related bacilli. The sera against fractions 1 of culture filtrates of toxin-producing and plasmidless strains displayed antigens produced only by B. anthracis strains into nutrient agar. Electroimmunotransblotting revealed that they contained antibodies mainly to 94-kD proteins and failed to react with B. cereus proteins with a molecular weight of 94 kD and with B. thuringiensis proteins with a molecular weight of 97 kD, which were extracted from autonomous cells. In the immunofluorescence test, immunoglobulins of sera against fractions 1 of culture filtrates of three strains stained autonomous cells and spores of 23 B. anthracis strains with different levels of virulence plasmids. In working dilutions, they did not react with antigens of 18 strains of related bacilli, which presents a possibility of using them for species identification of B. anthracis.