Extractable Antigens Research Articles

In vitro assembly of 2-D crystals from partially purified EA1 protein secreted by Bacillus anthracis strain Delta Sterne-1

Many bacteria possess surface layers (S-layers) that consist of a two-dimensional protein lattice external to the cell envelope. These S-layer arrays are usually composed of a single species of protein or glycoprotein and are not covalently linked to the underlying cell wall. When removed from the cell, S-layer proteins often reassemble into a lattice identical to that found on the cell, even without supporting cell wall fragments. S-layers exist at the interface between the cell and its environment and probably serve as molecular sieves that exclude destructive macromolecules while allowing passage of small nutrients and secreted proteins. Some S-layers are refractory to ingestion by macrophages and, generally, bacteria are more virulent when S-layers are present.When grown in rich medium under aerobic conditions, B. anthracis strain Delta Sterne-1 secretes large amounts of a proteinaceous extractable antigen 1 (EA1) into the growth medium. Immunocytochemistry with rabbit polyclonal anti-EAl antibody made against the secreted protein and gold-conjugated goat anti-rabbit IgG showed that EAI was localized at the cell surface (fig 1), which suggests its role as an S-layer protein.

Heterophile antigens in serous cystadenocarcinoma of the human ovary.

As the aberrance of carbohydrate moieties, the appearance of heterophile antigens in human ovarian cancer tissue was studied using various heterophile antigens and antibodies together with antisera raised in rabbits by immunization with extracts of human ovarian cancer tissues. In the immunodiffusion test, rabbit antisera reacted with a heat-stable, perchloric-acid extractable antigen that was different from CEA and CA125 and possessed Hanganutziu-Deicher (HD)-type specificity in all 4 specimens of serous cystadenocarcinoma of the ovary. Furthermore, a pathologic serum with strong HD-antibody complex formed a precipitin line with one of them. In the cancer tissue, we also immunochemically demonstrated the presence of rat-erythrocyte antigens. However, other types of heterophile antigens, such as Paul-Bunnell and Forssman, were not detected. These results suggest that HD-type antigens appear in serous cystadenocarcinoma of the human ovary, one in broad distribution and some in specific cases.

Prevalence of non-organ-specific autoantibodies in pregnant and non-pregnant healthy women.

The prevalence of autoantibodies during pregnancy was studied. Sera from 568 women (203 pregnant and 365 non-pregnant) were tested for autoantibodies to double-stranded DNA, cardiolipin and extractable cellular antigens. Nineteen out of 203 pregnant women (9.4%) had at least one autoantibody against double-stranded DNA or cardiolipin, mainly of the IgM class. The non-pregnant control group had significantly higher incidence of autoantibodies (17.8%; chi 2 = 7.39, P < 0.01). Sixty-six out of 568 women had had spontaneous abortions in their past medical history, but there was no correlation between them and the presence of anticardiolipin antibodies. Two of the non-pregnant women had anti-Ro(SSA) antibodies. These findings suggest that (a) the prevalence of autoantibodies decreases during pregnancy; and (b) the presence of anticardiolipin antibodies in healthy pregnant women does not correlate with any pregnancy-related complications. However, our results indicated that autoimmunity is a rather common disorder in healthy women.

Decrease in serum antiphospholipid antibody levels upon development of nephrotic syndrome in patients with systemic lupus erythematosus: Relationship to urinary loss of IgG and other factors

purpose: Having observed a decrease in antiphospholipid antibodies (aPL) upon the development of nephrotic syndrome, as well as a negative association between nephrotic syndrome and secondary antiphospholipid syndrome, in patients with systemic lupus erythematosus (SLE), we sought to determine if this could be due to urinary loss of aPL and/or other factors. subjects and methods: IgG and IgM aPL as well as other autoantibodies were studied by enzyme-linked immunosorbent assay with cardiolipin as antigen in serum and urine from six patients with SLE who had elevated serum aPL levels and developed nephrotic syndrome (cases). For controls, we studied: (1) three SLE patients with nephrotic syndrome but low aPL levels; (2) three patients with non-SLE nephrotic syndrome; (3) three SLE patients with high-titer aPL but no proteinuria; and (4) 10 healthy volunteers. results: We found urinary IgG, but no IgM, aPL in all cases and in one control from Group 2. Serum IgG aPL had gradually decreased after the development of nephrotic syndrome and had become normal. IgM aPL had also decreased in the four patients who had elevated levels, having reached normal levels at the time of the study in two. There was an apparent correlation between serum and urine IgG aPL levels but not between urinary IgG aPL and total proteinuria. By Farr's method, we found no urinary anti-DNA despite high serum titers in three cases. The two cases and one of the controls in Group 1 who had serum antibodies to extractable antigens also had these antibodies in the urine. conclusion: Urinary loss of IgG aPL during nephrotic syndrome does not completely explain the reduction in serum aPL, since IgM also decreases. There could also be decreased synthesis and/or increased catabolism of immunoglobulins.

Organ-specific antibodies against ovary in patients with systemic lupus erythematosus

We studied whether patients with systemic lupus erythematosus can have organ-specific antibodies directed against ovaries. With a recently developed enzyme-linked immunosorbent assay system that uses both a soluble and an extractable corpus luteum antigen fraction, we detected both IgG and IgM antibodies in the sera of patients with systemic lupus erythematosus (16 of 19, 84%). The highest levels of IgG antibodies were seen when the extractable organ fraction (ST80) was used as the antigen. (Ann J OBSTET GYNECOL 1989;160:1227-9.)

Identification of Bacillus anthracis by polyclonal antibodies against extracted vegetative cell antigens

The extractable protein antigens EA1 and EA2 of Bacillus anthracis were prepared from electrophoresis transblots of SDS extracts of vegetative bacteria of the Sterne strain. Hyperimmune guinea-pig antiserum against EA2 failed to react with B. anthracis cells in immunofluorescence (IF) tests. Guinea-pig antiserum against EA1 (anti-EA1) reacted strongly in IF tests with non-encapsulated vegetative cell of 10 of 12 strains of B. anthracis and with cells of strains of B. cereus and B. thuringiensis. The unreactive B. anthracis strains were delta-Vollum-1B-1 and Texas. Encapsulated cells of B. anthracis stained poorly except for small bright regions. Absorption of anti-EA1 with cells of B. cereus NCTC 8035 and NCTC 9946 removed activity towards all B. cereus strains tested, but only partly reduced cross-reaction with B. thuringiensis strains. Absorption of anti-EA1 with B. thuringiensis 4041 removed activity towards this strain and B. cereus strains. Evidence is produced that B. thuringiensis cells grown on nutrient agar possess more cross-reacting antigens than cells grown in nutrient broth. The reaction of anti-EA1 with Bacillus spores immobilized in clumps on microscope slides was attributed to contaminating vegetative debris because well-separated individual spores failed to react. A rapid IF test was developed allowing identification of B. anthracis sampled from overnight cultures on blood plates. When sodium dodecyl sulphate extracts of B. anthracis vegetative cells were analysed on immunoblots (Western blots) by reaction with anti-EA1, a number of bands were visualized in addition to the expected 91 kiloDalton EA1 band. Prior absorption of anti-EA1 with B. cereus or B. thuringiensis cells resulted in the disappearance of most or all of the brands in blots of these species, but had less effect on blots of the B. anthracis strains. All six B. anthracis strains that were blotted including delta-Vollum-1B-1 and Texas, could thus be distinguished from B. cereus and B. thuringiensis by their differential reaction with unabsorbed and absorbed anti-EA1.

Non-Goodpasture Anti-GBM Antibodies in Patients with Glomerulonephritis

The sera of 206 consecutive patients with biopsy-proven glomerulonephritis were tested by ELISA for the presence of Goodpasture and non-Goodpasture anti-GBM antibodies. Antigens were solubilised from human GBM with purified bacterial collagenase and with 6 mol/l guanidine-HCl respectively. Only 12 sera reacted when collagenase-resistant GBM proteins were used as antigens in ELISA. Sera from two of these patients also reacted with the Goodpasture antigen, that is the globular domain of collagen IV, purified from collagenase extracts of GBM. These two patients had classical Goodpasture syndrome with linear crescentic nephritis. The other ten sera did not react with the Goodpasture antigen and immunofluorescence microscopy showed granular glomerular immune deposits. Antibodies against antigens present in 6 mol/l guanidine-HCl extracts of human GBM were much more frequent, particularly in lupus nephritis and IgA nephropathy, but relatively common also in patients with glomerulonephritis associated with systemic connective tissue and systemic vasculitic disorders. In contrast, these non-Goodpasture antibodies were only sporadic in primary forms of glomerulonephritis such as minimal-change nephropathy, membranous glomerulopathy, or acute post-infectious glomerulonephritis. The presence of circulating IgG, IgA or IgM antibodies against 6 mol/l guanidine-HCl extractable GBM antigens correlated with granular deposits of corresponding immunoglobulins in both mesangial and capillary loop regions of glomeruli, indicating a possible pathogenic role for non-Goodpasture anti-GBM antibodies in several forms of glomerulonephritis.

Immunochemical characterization of the antigen recognized by the murine monoclonal antibody A7 against human colorectal cancer.

The nature of the antigen recognized by the murine monoclonal antibody A7 (Mab A7) against human colorectal carcinoma was investigated using immunochemical and biochemical techniques. Binding activity of 125I-labeled Mab A7 was examined using various human cancer cell lines. Mab A7 gave the highly specific binding to colon cancer cell lines, SW1116 and WiDr, and gave only a very weak or no reactivity to gastric cancer cell lines, pancreas cell lines or lung cancer cell lines. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of the extractable antigen from SW1116 showed a single band at approximately 45,000 dalton formed by 125I-labeled Mab A7. Treatment of SW1116 with sodium periodate, pronase and ficin resulted in the loss of antigenic activity. These data strongly suggest that the antigen recognized by Mab A7 is composed of glycoprotein. Competitive binding analysis to the surface of the colon cancer cell line using polyclonal anti-CEA and Mab A7 as well as immunoblotting analysis using monoclonal anti-CEA and Mab A7 suggested that the antigen recognized by Mab A7 was different from CEA. Moreover, this antigen was also found in surgical specimens of colorectal cancer patients and its molecular property was identical to the antigen extracted from SW1116.

B群溶連菌の型特異的蛋白質抗原

The c protein antigen of group B streptococci has been reported to consist of at least two acid extractable antigens, c alpha and c beta. We previously reported that strain FZ301 contain the c alpha and novel protein antigen, named as S antigen by Denka Seiken, and the c alpha antigen of this strain exhibit a more complicated structure. In this report described the analysis of the diversity of the c alpha antigen by immunoelectrophoresis and immunodiffusion. Hot acid extract from strain A909 produced a single precipitin band against the anti-121424c alpha serum but the 121424 extract produced double precipitin bands by immunoelectrophoresis. These c alpha antigens showed anodic migration, but the 121424c alpha antigens tended to migrate slower than the A909c alpha antigen. Moreover, the anti-A909c alpha serum produced a single precipitin band when tested after electrophoresis of the 121424 extract. When examined by immunodiffusion, the anti-121424c alpha serum produced two distinct precipitin bands with the 121424 and FZ301 extract, and these were reactions of identity. However, the A909 extract produced only one band which joined one of the two bands produced with the 121424 extract. These results shown that the strain 121424 contain c alpha and S antigen as well as strain FZ301. The precipitin band of the c alpha antigen in the A909 extract against the anti-A909 serum formed the spur with the reaction of the c alpha antigen in the 121424 or FZ301 extract on immunodiffusion. Moreover, the c alpha antibody in the anti-A909 serum failed to removed completely by repeated adsorption with strain 121424.(ABSTRACT TRUNCATED AT 250 WORDS)

A microbiological etiology for gastritis and peptic ulceration.

AbstractThe relationship betweenCampylobacter pyloriinfection and gastroduodenal disease was investigated using antral biopsies from 102 patients undergoing endoscopy.C. pyloriwas isolated from the majority of patients with histologically defined gastritis (89%), gastric ulcer (100%), and duodenal ulcer (94%), but only in 6% of patients with normal gastric mucosa. No obvious epidemiological association betweenC. pyloriinfection and recent travel, domestic pets, diet, dental hygiene, drug treatment or symptoms could be established. The susceptibility of 38 of these isolates ofC. pylorito 12 antibiotics was determined.The serum anti‐C.pyloriantibody response was determined by an ELISA technique using an acid extractable antigen on the solid phase, in order to improve the sensitivity and specificity. There was a significant increase in antibody level (p=0.001) of patients with histologically defined gastritis over patients with a normal mucosa. The majority of patients (96%) with a culturableC. pyloriinfection had raised anti‐C.pyloriantibodies. However, some patients with neither C.pyloriinfection nor gastritis had serum antibodies. Possible reasons for this are discussed.The results suggest that a serodiagnostic assay forC. pyloriinfection may be of value in the assessment of gastroduodenal disease.

Immunological analysis of cell-associated antigens of Bacillus anthracis.

Sera from Hartley guinea pigs vaccinated with a veterinary live spore anthrax vaccine were compared with sera from guinea pigs vaccinated with the human anthrax vaccine, which consists of aluminum hydroxide-adsorbed culture proteins of Bacillus anthracis V770-NP-1R. Sera from animals vaccinated with the spore vaccine recognized two major B. anthracis vegetative cell-associated proteins that were either not recognized or poorly recognized by sera from animals that received the human vaccine. These proteins, termed extractable antigens 1 (EA1) and 2 (EA2), have molecular masses of 91 and 62 kilodaltons, respectively. The EA1 protein appeared to be coded by chromosomal DNA, whereas the EA2 protein was only detected in strains that possessed the pXO1 toxin plasmid. Both of the extractable antigen proteins were serologically distinct from the components of anthrax edema toxin and lethal toxin. Following vaccination with the live spore vaccine, the EA1 protein was the predominant antigen recognized, as determined by electrophoretic immunotransblots. Vaccine trials with partially purified EA1 demonstrated that it neither elicits protective antibody against anthrax nor delays time to death in guinea pigs challenged intramuscularly with virulent Ames strain spores. In addition, animals vaccinated with sterile gamma-irradiated cell walls had significant antibody titers to the N-acetylglucosamine-galactose polysaccharide of B. anthracis but were neither protected nor had a delay in time to death following challenge.

Antibodies to extractable cellular antigens in Spanish systemic lupus erythematosus (SLE) patients.

We studied the frequency of antibodies to extractable cellular antigens in 90 Spanish SLE patients. Antibodies to Sm, nRNP, Ro and La were found, respectively, in 6 (6.6%), 18 (20%), 30 (33.3%) and 6 (6.6%) patients. In spite of HLA differences, these results are similar to those found by other authors in Caucasian SLE patients. Our only relevant association was the finding of severe renal disease and high frequency of anti-DNA and hypocomplementemia in patients with antibodies to Ro. Based on these and earlier findings, we discuss the possibility that the immune response to Ro in Spanish patients might be located in a unique extended HLA-complement haplotype.

Antibodies to Saline Extractable Tissue Antigen in Sera of Patients with Renal Diseases

Multiple serum samples originating from 110 renal allograft recipients were examined against saline extract of normal human kidney by means of double diffusion gel precipitation. Eleven recipients were found to be positive; 99 of 106 sera from these patients were positive. Pretransplantation sera were available from 7 of these recipients and 6 patients were found "positive." The precipitation reaction was composed of one line. Identity reactions were formed between the lines produced by sera from all patients except 1. Sera of patients from end-stage renal disease produced similar reaction; however, only 3 of 234 sera from patients with nonrenal diseases precipitated the kidney extract. None of 154 normal sera were positive. Several positive sera also were positive in complement fixation tests with human kidney extract. Evidence was presented that the antibodies under study combined with a nonorgan-specific but species-restricted tissue antigen. The hypothesis was advanced that these antibodies are autoantibodies formed in response to a sequestered antigen released as a result of tissue damage. Apparently, the antigen is released frequently in immunogenic form from injury to kidney but infrequently from injury to other organs.

Immunological detection of moulds in food: relation between antigen production and growth

In a study of Notermans and Heuvelman ( Int. J. Food Microbiol. (1985) 2, 247–258) an enzyme linked immunosorbent assay (ELISA) for detection of moulds was described. The ELISA used was based on detection of a heat stable, water extractable and genus specific antigen(s) produced by moulds. The results indicated the possibility of testing food products, whether or not heated, for mould contamination. In this study antigen(s) production by moulds was tested under different growth conditions. It was observed that production of antigen(s) was correlated with mycelium weight. Type of medium, including different fruit juices, did not affect the production of antigen(s). Also no differences between surface culturing and submerged culturing were observed. Both incubation temperature and water activity showed no significant effect on production of antigen(s). Within the genus Penicillium 43 out of 45 species tested produced identical antigen(s) in comparable quantities. The quantity of antigen(s), expressed as minimal detectable quantity of mould mycelium produced, varied from 6–108 ng/ml with an average of 32 ng/ml.

Enzyme-linked immunosorbent assay for detection of antibodies to extractable muscle antigens in myasthenia gravis.

A purified citric acid soluble extract from human skeletal muscle (AEMA) and a phosphate-buffered saline extract from rabbit muscle acetone powder (EMA) were used to coat polystyrene beads in an enzyme-linked immunosorbent assay (ELISA). From 54 patients with myasthenia gravis, positive results were observed in 14. Five of 6 patients with thymoma had high levels of antibodies. With the diagnostic difficulties in detecting small and medium-sized thymoma, a sensitive assay for detection of antibodies to muscle antigen may be an important supplementary tool to detect tumors at early stages.

Polyagglutinability due to loss of O-antigenic determinants in Pseudomonas aeruginosa strains isolated from cystic fibrosis patients.

O-polyagglutinable P. aeruginosa are prevalent among chronically infected cystic fibrosis patients. In the present work, one O-group 3, one O-group 9 and one polyagglutinable 0-3/9 strain were analysed by use of both corresponding group specific antisera against the O-antigen and polyspecific antisera against all extractable antigens of the 3 strains. Quantitative immunoelectrophoresis and classical tube-agglutination methods were employed in the analysis. The results showed that the polyagglutinable strain contained 0.1% and the 0-9 strain 1%, respectively, of the O-antigen-containing LPS present in the 0-3 strain. It is discussed whether these differences represent a gradual antigenic adjustment to the defence mechanisms of the chronically infected patients.

Carbohydrate antigen defined by a monoclonal antibody raised against a gastric cancer xenograft.

A monoclonal antibody, ST-4-39, was obtained by using a human gastric cancer xenograft, St-4, as an immunogen. Immunization was achieved by transferring immunocompetent mouse spleen cells into a nude mouse bearing St-4. Hybridomas were produced with the spleen cells of the mouse after rejection of the tumor and screened for immunohistochemical reactivity with cancers and normal tissues on formalin-fixed paraffin sections. ST-4-39 immunohistochemically reacted with various cancers including gastric, colorectal and pancreatic cancers as well as some normal tissues. ST-4-39 and NS 19-9 differed in immunohistochemical reactivity, although they reacted with some cancers and a few normal tissues in common. PBS extracts of normal and cancer tissues were examined for antigen reactive with ST-4-39 by sandwich enzyme immunoassay. Extractable antigen was detected in adenocarcinomas of colon, stomach and lung, while it was detected only in salivary gland and trachea among normal tissues examined. Gel filtration analysis of the antigen indicated a molecular weight of greater than or equal to 1 X 10(6), and the antigenic determinant was suggested to be a carbohydrate chain with terminal sialic acid by studies using periodic acid, neuraminidase and pronase treatments. Furthermore, the ST-4-39 antigen affinity-purified from two gastric cancer strains was shown to contain multiple carbohydrate determinants including sialyl-Lewisa and sialyl-Lewisx, suggesting the antigen to be a mucin.

Application of electroblotting technique to studies of the intestinal antibody response to extractable fecal antigens.

Water-soluble and sodium dodecyl sulfate (SDS)-extractable fractions were prepared from feces of four patients with inflammatory and six with non-inflammatory bowel disease. Both types of fractions were run on polyacrylamide gel electrophoresis, followed by electroblotting on nitrocellulose sheets of the separated components. The antibody reactivities in serum and in the intestinal wall against the extracted and separated fecal components were investigated. A striking lack of reactivity was observed when serum was used as antibody source against the fecal extracts both in patients with inflamed and in those with non-inflamed intestinal mucosa. The locally produced gut-associated IgG reacted more intensely with SDS-extractable fecal components than with water-soluble components. A strong reaction between intestinal-wall IgG extracts and a water-soluble antigen of 46-48 kD in the feces of two colitis patients was, however, observed. No clear correlation between the single bands and the occurrence of inflammatory bowel disease could be established because of the small number of patients.

Antibodies to extractable cellular antigens specific of chronic liver disease (CLD)

Antibodies to extractable cellular antigens specific of chronic liver disease (CLD)

Investigation of a Campylobacter jejuni outbreak by serotyping and chromosomal restriction endonuclease analysis.

Fifty Campylobacter jejuni isolates, including 29 from humans associated with an outbreak of enteritis, 20 from cattle, and 1 from a milk source, were serotyped on the basis of extractable thermostable antigens and examined by bacterial chromosomal restriction endonuclease digest analysis. Serotyping showed specific differences between the human isolates and the milk isolates, but each of these generally, although not consistently, reacted with 4 of the 42 C. jejuni typing antisera. Restriction patterns of all of the human isolates and some of the cattle isolates were indistinguishable, confirming the suspected link between the cattle and the human outbreak. The single milk isolate had a restriction pattern unlike those of the human isolates, and therefore its involvement in the transmission of infection from the cattle to the humans could not be confirmed.