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PubMed1975

Isolation and antigenic characteristics of axial filaments from the Reiter Treponeme.

Axial filaments were isolated and purified from Reiter treponemes after detergent solubilization of the cells" outer envelope. The axial filaments were separated from the spirochetal cells by shearing, purified by density gradient centrifugation, and fragmented by ultrasonication. Acrylamide gel electrophoresis of dissociated filaments revealed two major protein bands. Gel diffusion precipitin tests and immunoelectrophoresis between a purified axial filament suspension and anti-Reiter treponeme serum gave a single precipitin line. Checkerboard complement fixation tests also gave results consistent with a single antigen-antibody system. Tests with immune sera to other cultivable spirochetes were positive with some and negative with others. In addition, strongly positive reactions were obtained in complement fixation and precipitin tests with sera from rabbits and humans with syphilis and other treponematoses. However, both serological tests gave reactions of partial identity between the antigen(s) of Reiter treponeme axial filaments and those of the pathogenic treponemes. It was concluded from these studies that the axial filaments were probably the cellular locus of the so-called "Reiter protein" antigen of syphilis serology.

Animals↗
PubMed1971

Biological characteristics of Clostridium perfringens type A enterotoxin.

An enterotoxin with the ability to induce fluid accumulation in rabbit ileal loops, erythema in the skin of guinea pigs, and lethality in mice appears in cell extracts (CE) and culture filtrates (CF) of sporulating cells of some Clostridium perfringens type A strains. All activities in CE and CF were eluted simultaneously from a Sephadex G-200 column. Different elution patterns were obtained for these activities present in CE and CF. Rabbit immune serum against CF and the active CE fractions eliminated the three biological activities in CE and CF. These activities present in CF and CE were not eliminated by any of the known antitoxins present in diagnostic serum against C. perfringens types A, B, C, D, and E. Immunodiffusion studies with immune serum against active CE fractions and CF indicated a precipitin line of identity between CF and CE of NCTC 8798 and other enterotoxin-positive strains but not enterotoxin-negative strains. Disc electrophoresis of active G-200 fractions on 7.0% polyacrylamide gels revealed a single area containing erythemal activity and mouse lethality. Immunodiffusion with acrylamide gels, containing crude fractionated enterotoxin, and immune serum against partially purified enterotoxin revealed a single precipitin band in the same area as the biological activities. Immunoelectrophoresis of CE of enterotoxin-positive and enterotoxin-negative strains also showed one precipitin band which occurred only with enterotoxin-positive strains. These findings suggest that one component is responsible for the biological activities attributed to the enterotoxin.

Animals↗
PubMed1983

Immunogenicity of Pseudomonas aeruginosa outer membrane antigens examined by crossed immunoelectrophoresis.

By crossed immunoelectrophoresis 36 different anode-migrating antigens were demonstrated in sonicated antigen preparations of Pseudomonas aeruginosa. We numbered these antigens to establish a reference precipitin pattern. Antigen no. 31 was identified as the lipopolysaccharide (LPS) antigen, because it was found to be responsible for the O-group specificity and because it reacted with anti-LPS monoclonal antibodies and with Limulus amoebocyte lysate. Purified outer membrane proteins F (porin), H2, and I used as antigens formed precipitins with the reference antibodies, thus establishing their antigenicity. LPS that copurified with protein F and slightly contaminated protein H2 was detectable as an extra precipitin (antigen no. 31). The use of monoclonal antibodies specific for smooth LPS and rough LPS revealed different antigenic determinants in the LPS molecule and suggested that antigen no. 5 could be the core region of the LPS which is equivalent to the rough LPS. Antibodies against these outer membrane antigens were detected in patients with chronic P. aeruginosa pneumonia and in patients with acute P. aeruginosa bacteremia. Antibodies with the same specificity were also found in rats chronically infected with P. aeruginosa 7 days postinfection. This demonstrates the surface accessibility and antigenic reactivity of outer membrane antigens.

Animals↗
PubMed1984

Naturally occurring pasteurellosis in laboratory rabbits: chemical and serological studies of whole cells and lipopolysaccharides of Pasteurella multocida.

Whole cells and lipopolysaccharides (LPS) of 10 isolates of Pasteurella multocida from laboratory rabbits were subjected to chemical and serological analysis. LPS of most of these isolates possessed pyrogenic potency comparable to LPS from Salmonella minnesota 9700, although their average ketodeoxyoctonate content was only 18% of that of salmonella. A gel diffusion precipitin test for somatic antigens extracted in a formal-saline solution demonstrated several isolates with three to four somatic antigens, with some variation in the major somatic type from one test to another. Conversely, the use of LPS as antigen in the gel diffusion precipitin test (i) eliminated cross-reactivity with reference antisera and (ii) often resulted in the organism being typed as serotype 12 even when the type 12 antigen was a minor antigen in the formal-saline extracts. Antisera from specific pathogen-free rabbits immunized with either whole cells or LPS of two isolates were tested against whole cells of LPS of the 10 isolates by enzyme immunoassay and indirect hemagglutination. Both whole cells and LPS of one of the isolates (isolate 2) were serologically specific, whereas those of the other isolate (isolate 1) were moderately to strongly cross-reactive with other isolates. The data indicate that although LPS is the major antigen responsible for typing based on the gel diffusion precipitin test, substances other than LPS (probably capsular polysaccharide) are responsible for the type specificity that forms the basis for the A, B, D, or E classification of this organism.

Animal Diseases↗
PubMed1984

Identification and characterization of a major surface antigen of Giardia lamblia.

The surface antigens of Giardia lamblia trophozoites were characterized by crossed immunoelectrophoresis, radioiodination, and immunoprecipitation. Crossed immunoelectrophoretic analysis of trophozoites with hyperimmune rabbit anti-trophozoite antiserum revealed a prominent precipitin peak that disappeared upon adsorption of the antiserum with live or formaldehyde-fixed trophozoites. This peak was intensely labeled when the antigen was derived from surface-radioiodinated trophozoites. An antiserum monospecific for the antigen contained in this precipitin peak was prepared. The precipitin peak was shown to contain an antigen with an apparent molecular weight of 82,000 by Western blotting. The antiserum also detected this 82,000-molecular-weight antigen on nitrocellulose blots of trophozoites analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On radioiodination of live trophozoites, an iodinated molecule of 82,000 apparent molecular weight was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was immunoprecipitated by the monospecific antiserum. Preliminary characterization of this antigen with the monospecific antiserum in crossed immunoelectrophoresis revealed that the surface antigen is hydrophobic and thus may be anchored in the plasma membrane, and that it is heat sensitive, but only partially sensitive to pronase or periodate. This antigen was shared by the four G. lamblia strains examined.

Antibody Specificity↗
PubMed1980

Immunochemical studies of the mannans of Saccharomyces cerevisiae X2180-1A-5 and Saccharomyces cerevisiae 4484-24D-1 mutant strains, with special reference to their phosphate content.

The mannans from Saccharomyces cerevisiae mutant strains X2180-1A-5 and 4484-24D-1, both of which were shown to contain small amounts of phosphate (less than 0.2%), were fractionated on a column of diethylaminoethyl-Sephadex into five subfractions designated as fractions I to V. These subfractions contain different amounts of phosphate, ranging from 0.03 to 0.09 (strain X2180-1A-5) and from 0.01 to 0.17% (strain 4484-24D-1). Fractions I to IV from strain X2180-1A-5 showed nearly identical precipitin activities against the homologous anti-whole cell serum, whereas fraction V, containing the largest amount of phosphate and protein among this mannan subfraction series, showed unexpectedly weaker precipitin activity than those of the other fractions. A synthetic mannan consisting or consecutive alpha-1 leads to 6-linked D-mannopyranosyl residues was found to be cross-reactive with all the mannan subfractions of strain X2180-1A-5 against anti-X2180-1A-5 serum. On the other hand, antibody-precipitating activities of the mannan subfractions of the latter strain were proportional to their phosphate content, although the increments of precipitated antibody nitrogen among the subfractions were quite small. However, fraction V of this mannan subfraction series, containing the largest amounts of phosphate and protein, showed lower precipitin activity than did the other four fractions. These findings indicate that mannans containing no phosphate or relatively small amounts of phosphate, such as those investigated in the present study, are less heterogeneous in the densities of the branching moieties than are highly phosphorylated mannans. These findings suggest that the transfer step of mannosyl-1-phosphate into the precursor(s) of the wild-type strain mannans during the biosynthetic process corresponds to the key reaction responsible for the anionic heterogeneity due to the density heterogeneity of the antigenic determinants.

Antigens, Fungal↗
PubMed1982

Formation of molecular complexes between a structurally defined M protein and acylated or deacylated lipoteichoic acid of Streptococcus pyogenes.

The orientation of lipoteichoic acid (LTA) molecules on the surface of bacterial cells undoubtedly is determined by the ability of the LTA, during its transit through the cell wall, to bind via its polyglycerophosphate backbone or its glycolipid moieties to other constituents of the cytoplasmic membrane and the cell wall. We have investigated the possibility that LTA may become anchored to the cell surface by binding through its polyanionic backbone to positively charged regions of cell wall proteins. LTA was found to prevent the precipitation of partially purified HCl extracts of several strains of streptococci as well as a structurally defined streptococcal M protein molecule (pep M24) in 83% solutions of ethanol. The formation of complexes between LTA and M protein was demonstrated further by immunoelectrophoresis of pep M24 protein with increasing concentrations of radiolabeled LTA and by using antiserum against pep M24 to develop precipitin arcs. Pep M24 electrophoresed alone produced a single precipitin arc close to the origin. In contrast, when electrophoresed as a mixture with LTA or deacylated LTA, the M protein produced a second precipitin arc toward the anode coinciding with the area of migration of the radioactive LTA. Increasing concentrations of LTA or deacylated LTA shifted increasing amounts of the pep M24 antigen to the region of the second arc. Maleylation of M protein to block the positively charged free amino groups before mixing it with LTA prevented the formation of complexes. The complexes formed by the M protein with LTA, but not with deacylated LTA, showed the capacity to bind bovine serum albumin; LTA had been shown previously to bind to the fatty acid binding sites on bovine serum albumin. These results indicate that the LTA molecule is able to bind via its polyanionic backbone to positively charged residues of surface proteins of cells of S. pyogenes. The implications of such interaction as to the orientation of LTA molecules on the surface of cells of S. pyogenes are discussed.

Acylation↗
PubMed1964

IMMUNE DIFFUSION ANALYSIS OF THE EXTRACELLULAR SOLUBLE ANTIGENS OF TWO STRAINS OF RHIZOBIUM MELILOTI.

Dudman, W. F. (Commonwealth Scientific and Industrial Research Organization, Canberra, Australia). Immune diffusion analysis of the extracellular soluble antigens of two strains of Rhizobium meliloti. J. Bacteriol. 88:782-794. 1964.-Immune diffusion techniques applied to cultures of two strains of Rhizobium meliloti grown in liquid defined medium showed the presence of multiple antigens. Improved resolution of precipitin patterns was obtained with concentrated antigens separated from the cultures as the extracellular soluble fraction or as suspensions of washed cells. The extracellular fraction contained the same diffusible antigens as the washed cells, but additional antigens were found in the cells after ultrasonic disruption. The extracellular soluble antigens were shown by analysis to contain polysaccharide and protein components. In immune diffusion systems, they gave rise to three groups of precipitin bands, two of which were characterized as polysaccharides by their susceptibility to periodate oxidation, and the third as protein by its lability to heat. All the extracellular antigens of both strains were shared except a fast-diffusing polysaccharide, which was specific for each strain. Despite the sharing of all but one of their antigens, cells of these strains showed only a low degree of cross-agglutination, suggesting that their surfaces are dominated by the specific polysaccharide. No differences could be found in the composition of the polysaccharides in the unfractionated extracellular antigens of the two strains, the main components of which were glucose (66%) and galactose (12%) in the presence of several other unidentified sugars in smaller amounts. The pattern of precipitin bands produced in immune diffusion systems by the extracellular soluble fraction could be changed by altering the cultural conditions.

Agglutination↗
PubMed1975

Rapid identification of group B streptococci by counterimmunoelectrophoresis.

Beta-hemolytic streptococcal isolates have been examined by counterimmunoelectrophoresis (CIE) with group B antiserum to determine whether this techinque is of value in the rapid identification of group B strains. Ninety stock cultures and 100 clinical isolates of beta-hemolytic streptococci including representatives of groups A, D, C, G, and B were inoculated into Todd-Hewitt broth; after incubation at 37 C for 1, 2, 3, and 4 h, aliquots of the whole broth cultures were removed and tested by CIE. Antigen was not regularly detected in the 1-, 2-, and 3-h samples, but after 4 h all 126 group B streptococcal strains identified by the capillary precipitin reaction gave CIE precipitin bands with group B antiserum. None of the 58 non-group B strains gave precipitin reactions with this antiserum. Cerebrospinal fluid from an infant with group B streptococcal meningitis and peritoneal fluid from a patient with group B streptococcal peritonitis had free group B antigen detected by the CIE technique. CIE of broth cultures and direct body fluids appears to be a rapid and sensitive method for the identification of group B streptococcal strains.

Adult↗
PubMed1979

Microimmunodiffusion test for nocardiosis.

Sera from 71 patients with culturally proven nocardiosis were tested for precipitins against a pool of Nocardia asteroides and N. brasiliensis culture filtrates and against antigens from the supernatant of homogenized N. asteroides cells. A human nocardiosis case serum was used as a reference. Sera from 56 of the 71 cases were reactive with either the culture filtrate antigen, the homogenate antigen, or both antigens, resulting in an overall sensitivity of 79%. Sera from 35 of the patients (49%) were positive with the homogenate antigens, and 28 (39%) showed bands of identity with the reference serum. Sera from 50 nocardiosis cases (70%) were positive with the pooled culture filtrate antigens, and 29 (41%) produced bands of identity with the reference serum. Of 89 sera from patients with various systemic mycotic diseases, tuberculosis, or actinomycosis, 24 (27%) were positive with the nocardial homogenate antigens and 4 (4.5%) showed precipitin bands of identity. Thirty-five of the 89 sera (42%) were positive with the nocardial culture filtrate antigens, and 6 (6.7%) showed bands of identity. The majority of sera demonstrating false-positive reactions were from tuberculosis and actinomycosis cases. One of seven sera from well individuals produced a precipitin band with the culture filtrate antigen, but this was not a band of identity with reference serum. These antigens did not distinguish antibodies from patients with N. asteroides, N. brasiliensis, or N. cavia infections.

Actinomycosis↗
PubMed1975

Application of counterimmunoelectrophoresis in the identification of Streptococcus pneumoniae in clinical isolates.

The use of counterimmunoelectrophoresis (CIE) as a diagnostic tool in infectious diseases is becoming more widespread. This study was undertaken to determine the possible use of CIE in the more rapid identification of Streptococcus pneumoniae in clinical isolates. Typing sera were obtained from the Statens Seruminstitut, Denmark. Sixty-seven out of 68 pneumococcal isolates that were optochin sensitive and bile soluble were typed by CIE. One isolate was nontypable even after mouse passage. An additional three isolates that were optochin resistant were considered to be pneumococci on the basis of the bile solubility test and their typability by CIE. Seventy-seven alpha-streptococci were tested for the presence of cross-reacting capsular antigens. Fifty-three alpha-streptococci showed no cross-reactions using the omniserum. Precipitin bands were obtained with the omniserum with 10 of the isolates, but these did not react with type-specific antisera. However, 14 isolates did react with the type-specific pneumococcal antisera. The sensitivity of the test was increased by sonicating whole-cell suspensions before electrophoresis was carried out. Mueller-Hinton broths were inoculated with presumptive pneumococcal colonies that formed the predominant or only colony type on primary blood agar plates. These cultures required a 4-h incubation period with an initial inoculum of 10(6) viable organisms/ml before a precipitin band could be detected. Precipitin bands were observed in 54 out of 56 (97%) broth cultures of pneumococci that had been incubated for 4 h at 37 C. These data suggest that CIE could be a useful tool in the identification of S. pneumoniae isolated from clinical specimens.

Antigens, Bacterial↗
PubMed1986

Antinuclear antibodies in the relatives and spouses of patients with systemic sclerosis.

The families of 65 patients with systemic sclerosis were examined clinically and serum samples from each subject were tested for antinuclear antibodies (ANA) by immunofluorescence on HEp2 cells and for precipitating antibodies to soluble cellular antigens including Scl-70. Of 217 blood relatives, 58 (27%) had ANA (42 speckled, 13 nucleolar, one centromere, two homogeneous); 22 (10%) had precipitins, one anti-Scl-70, one anti-PM-Scl, one anti-nRNP, two anti-Ro(SSA), the remainder unidentified). Family members tended to share ANA patterns. Of 38 spouses, nine (24%) had ANA (all speckled) and two showed unidentified precipitins. This compares with an incidence of ANA and precipitins in a control population of 8% and 1% respectively. Antibodies were more common in female than male relatives (particularly in mothers and sisters of probands). Twenty one of the 58 family members with ANA had clinical features of connective tissue disease; the remainder were asymptomatic. The presence of genetic factors influencing autoimmunity is suggested by the incidence of autoantibodies in first degree relatives. Similar observations in spouses, however, indicate that environmental factors may also have a role in these immune abnormalities.

Antibodies, Antinuclear↗
PubMed1980

Reversible airway obstruction in cystic fibrosis.

Fourteen (29%) of 48 children with cystic fibrosis had a greater than 15% improvement in forced expiratory volume in one second, or in forced vital capacity after inhalation of salbutamol. All these children were atopic (one or more positive prick tests) and had a significantly higher mean serum IgE than either non-atopic subjects or those atopic subjects without airways reversibility (p less than 0.02). Half of those with airways reversibility had or subsequently developed the clinical picture of allergic bronchopulmonary aspergillosis. Of the whole group 81% were atopic, of whom 77% had a positive reaction to A fumigatus, 64% to housedust, and 56% each to grass pollen and cat hair. Children who were not atopic had significantly better spirometry (p greater than 0.05) than those who were. Children with skin hypersensitivity to A fumigatus had identical spirometry to those who were atopic without reactivity to A fumigatus. Serum precipitins to A fumigatus were present in 48%. Serum precipitins to pancreatin were present in 71%, but the presence of these precipitins did not correlate with atopy, airways reversibility, or serum IgE.

Adolescent↗
PubMed1994

Rise in total IgE as an indicator of allergic bronchopulmonary aspergillosis in cystic fibrosis.

BACKGROUND: Allergic bronchopulmonary aspergillosis is a serious complication of cystic fibrosis and may be difficult to diagnose. The aim of this study was to define the usefulness of measuring total IgE compared with other major criteria in the diagnosis of allergic bronchopulmonary aspergillosis in children with cystic fibrosis. METHODS: A retrospective analysis was carried out of the case records of 160 children attending a tertiary referral paediatric cystic fibrosis clinic. RESULTS: Sixteen children had a total IgE level above 500 IU/ml. Eleven children had six or more other major criteria and were considered to have allergic bronchopulmonary aspergillosis. These 11 children had a fourfold rise in IgE in association with clinical deterioration. A further child had a fourfold rise in IgE to 341 IU/l, and was also thought to have allergic bronchopulmonary aspergillosis. Eleven had a fall in IgE with successful treatment; one patient died with uncontrolled disease. Only one of these 12 children had negative precipitins to Aspergillus fumigatus. The five children with a raised IgE not thought to have bronchopulmonary aspergillosis had four or fewer major criteria and were not treated; none had positive precipitins. CONCLUSIONS: A fourfold rise in total IgE, particularly to above 500 IU/ml, is strongly suggestive of the diagnosis of allergic bronchopulmonary aspergillosis in children with cystic fibrosis. The measurement of total IgE has the merit of being simple to perform and objective. Positive aspergillus precipitins provide useful confirmatory evidence. These two criteria, taken in conjunction with clinical deterioration and new radiological shadowing, allow simplification of the diagnosis of allergic bronchopulmonary aspergillosis in cystic fibrosis.

Adolescent↗
PubMed1984

Studies on precursor proteins PIVKA-II, -IX, and -X in the plasma of patients with 'hemorrhagic disease of the newborn'.

Factors II, IX, and X in the plasmas of 10 patients with 'hemorrhagic disease of the newborn' were investigated by means of electroimmunoassay and crossed immunoelectrophoresis (CIE). The biological activity was within 4.2-30 U/dl for factor II, 5.1-20 U/dl for factor IX, and 5.2-24 U/dl for factor X, whereas the immunological antigen was within 33-58, 25-50, and 35-60 U/dl, respectively. Thus, for all factors, 1.7 times more antigen than activity was present. The CIE pattern of factors II and IX in the presence of Ca++ ions clearly showed a biphasic precipitin arc, and in the absence of Ca++ ions, only one precipitin arc was observed. These results implied the presence of precursor proteins PIVKA-II and -IX in the plasma. However, even in the presence of Ca++ ions the CIE pattern of factor X antigen in the patient plasmas only showed a single precipitin arc with a slightly faster than normal electrophoretic mobility. PIVKA-II, -IX, and the abnormal factor X antigen (PIVKA-X) disappeared within 24 h after the patients were treated with vitamin K.

Adult↗
PubMed1985

Identification of crustacea allergens by crossed radioimmunoelectrophoresis.

Crossed immunoelectrophoresis (CIE) detected 18 precipitating antigens in extracts of shrimp. Of these antigens, crossed-line immunoelectrophoresis (CLIE) of shrimp extract demonstrated that 5 cross-reacted with crayfish, 3 with lobster and 1 with crab extract. Allergens present in the shrimp CIE plates were identified by crossed radioimmunoelectrophoresis (CRIE) using sera from 6 study subjects who were skin-test and RAST positive to shrimp extract. Of the 7 allergens detected, 3 (precipitins 1, 3 and 6) reacted with most of the 6 sera tested from shrimp-sensitive subjects. Precipitins 1 and 6 appear to be common crustacea allergens (present in shrimp, crayfish, lobster and crab) whereas precipitin 3 may be a specific allergen since it is present only in shrimp.

Allergens↗
PubMed1996

Early sensitization to farming-related antigens among young farmers: analysis of risk factors.

It was the aim of this study to investigate the prevalence of and risk factors for IgE- and IgG-mediated allergy in young farmers. We investigated 147 young men attending a farming college in the Austrian Tyrol and 57 age-matched pupils at a grammar school. All individuals completed a questionnaire and had spirometry, skin prick tests, total and specific IgE analysis, and testing for precipitins against thermophilic actinomycetes and true fungi. In the farming group, a family history of atopy or asthma was reported less frequently than in the control group (p < 0.001) but there were no differences in the frequencies of actual respiratory symptoms. Forced expiratory volume in the first second (FEV1) was slightly lower in the farmers than in the pupils (p < 0.05). There was no difference in the prevalence of IgE-mediated allergy. Nine of the farmers but none of the pupils had precipitating antibodies against Faeni rectivirgula (p < 0.05). Overall reactivity (as defined by at least one positive finding with the skin prick test, specific IgE, or precipitins) was associated with larger farming estates, lack of a haydrying device, use of mouldy hay, and positive family history of asthma (p < 0.05). We conclude that in farmers, precipitin formation may occur at an early age and that even in younger age groups there is a clear relationship between allergic sensitization and occupational allergen exposure.

Adolescent↗
PubMed1975

Further studies on the antigenic properties and distribution of the putative bovine leukemia virus.

The C-type viruses found in long-term cultures. New Bolton Center (NBC) cell lines, of peripheral lymphocytes from leukemic cattle and in short-term cultures of bovine buffy coat(BC) cells share an immunofluorescent(IF)antigen detected in the cytoplasm of infected cells as well as an antigen demonstrable in gel diffusion experiments. Therefore the viruses from these cultures most likely represent different isolates of the putative bovine leukemia virus (BLV). The BLV precipitin antigen is analogous to the group specific (gs) antigens of the leukemia viruses of other species in that it is soluble, ether resistant, and apparently located within the virion. These observations, together with results showing that the specificity of the BLV precipitin antigen differs from that of the gs antigen of other mammalian leukemia viruses, indicate that the former antigen represents the intraspecies (gs-1) determinant of BLV. Antibodies to the precipitin viral antigen were found in 82% of cattle with leukemia and in 40% of clinically normal adult cattle in multiple-case herds. These groups of animals also had fluorescent antibodies to the virus, but with significantly higher frequencies (100% and 76%, respectively). On the other hand, in leukemia-free herds, precipitating antibodies were not found and the incidence of fluorescent antibodies was only 3%.

Animals↗