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PubMed1987

Lipoarabinomannan and lipid-free arabinomannan antigens of Mycobacterium paratuberculosis.

Lipoarabinomannan (LAM) and lipid-free arabinomannan (AM) were prepared from Mycobacterium paratuberculosis. Purification of LAM was done by ultracentrifugation of the phenol-water-extracted crude polysaccharide, followed by affinity and anion exchange chromatography. AM was purified from the supernatant of the ultracentrifuged polysaccharide or from alkaline-extracted material by gel filtration and anion exchange chromatography. Chemical analysis revealed arabinose and mannose in LAM (1.4:1) and AM (3.5:1) and the presence of palmitic, stearic, and tuberculostearic acids for a total of 7.8% lipid in LAM. Traces of phosphorus were found in the AMs, particularly LAM (0.05%). Nuclear magnetic resonance confirmed the presence of alpha-arabinosyl residues and the acylated nature of LAM. LAM exhibited lipid-dependent aggregation, as indicated by a Triton-induced decrease in molecular weight. By using bovine sera, LAM was found to be active in the complement fixation test, whereas AM was inactive and inhibited this activity. Thus, the presence of AM in crude polysaccharide could explain the variable complement fixation results. Triton-dissociated LAM exhibited a precipitin (Cl) in common with that of AM, confirming shared determinants. LAM in its lipid-dependent aggregated form, however, exhibited a second precipitin (C2), which may be due to the disparity in antigen size or a novel epitope. The lipid content of LAM rendered it 100 times more effective for coating plates in the enzyme immunoassay than lipid-free AM.

Antibodies, Bacterial↗
PubMed1989

Immunochemical and structural characterization of a serotype-specific polysaccharide antigen from Actinobacillus actinomycetemcomitans Y4 (serotype b).

A serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) was extracted from whole cells by autoclaving. The extract was purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300 columns. The purified polysaccharide antigen formed a single precipitin line with anti-type b serum but not with anti-type a serum and anti-type c serum. The antigen was composed of 43.9% L-rhamnose, 49.1% D-fucose, and a trace amount of fatty acid. Methylation analysis, Smith degradation, and optical rotation data showed that the antigen was a polymer consisting of a disaccharide repeating unit, ----3)-alpha-D-fucopyranosyl-(1----2)-beta-L-rhamnopyranosyl-(1----. In quantitative precipitin inhibition tests, D-fucose and L-rhamnose showed very low inhibition, but the partial hydrolysate of the purified antigen was an effective inhibitor, suggesting that the serotype b specific antiserum recognizes the larger oligosaccharide units.

Actinobacillus↗
PubMed1971

Immunochemical comparison of L-asparaginases from Serratia marcescens and Escherichia coli.

Immunochemical properties of l-asparaginases from Serratia marcescens and Escherichia coli were compared by means of quantitative precipitin reactions and quantitative microcomplement-fixation reactions employing homologous and heterologous antisera. The enzyme preparations used in these tests produced one arc of precipitate upon immunoelectrophoresis. According to our results with the precipitin and micro-complement fixation reactions, both enzymes are related immunochemically. On the basis of these tests, distinct structural differences exist between the two enzymes. From results with the micro-complement fixation test, we estimated that the two enzymes could differ by 10 to 15 amino acid residues.

Ammonia↗
PubMed1978

Relationship between phosphate content and immunochemical properties of subfractions of bakers' yeast mannan.

The mannan of bakers' yeast (Saccharomyces cerevisiae) was fractionated on a column of diethylaminoethyl-Sephadex into five subfractions. Phosphate content of these mannan subfractions was proportional to the concentration of NaCl solutions used in the chromatographic separation. Quantitative precipitin reactions showed that the serological reactivities of the subfractions were proportional to the content of phosphate. The result of acetolysis study showed that the amounts of mannotetraose and phosphate-containing oligosaccharide fractions increased proportionally to the acidity, whereas the amount of mannose decreased inversely. The results from quantitative precipitin reaction tests and acetolysis study demonstrated that both phosphate contents and multiplicity of branching moieties of mannan subfractions increased proportionally, i.e., micro-heterogeneity concerning the acidity comprised in the parent bulk mannan is not attributable merely to the coexistence of molecular species containing different amounts of phosphate but also to the presence of more of the branching moieties.

Chemical Phenomena↗
PubMed1968

[Detection by hemagglutination of antibodies to group A and group E streptococci by the use of O-stearoyl derivatives of their cell wall carbohydrate-grouping antigens].

The streptococcal group A and group E cell wall polysaccharide antigens were extracted with trichloroacetic acid from the cell or cell wall and esterified with stearic acid. The stearoyl derivatives contained 5 to 8% (by weight) of the ester. Sheep or human red blood cells were sensitized with the esterified antigens and were shown to agglutinate in the presence of specific rabbit antisera. Sera from (i) children hospitalized with group A streptococcal respiratory disease and (ii) swine possessing group E streptococcal lymphadenitis were shown to possess antibody titers significantly higher than the controls. The use of the two esterified antigens as controls for each other established the specificity of the reaction in each case. The general shape of the antigen-antibody precipitin curves was not changed when the stearoyl antigens were used; however, the quantitative aspects differed markedly. Oligosaccharides which inhibit the normal antigen-antibody precipitin reaction did not inhibit the hemagglutination reaction. The adsorption of antisera with whole streptococcal cells reduced the hemagglutination titer in relation to the quantity of cells employed. Data are given on the (i) optimal concentration of stearoyl antigen for sensitization, (ii) time of adsorption of antigen to red cells, (iii) use of albumin as diluting fluid, and (iv) condition of red cells. Properties of the esterified antigens and the mechanism of the agglutination reaction are discussed. The results indicate that polysaccharide antigens of other bacteria may be esterified and employed in a similar manner.

Animals↗
PubMed1981

Improved streptococcal grouping antisera containing polyethylene glycol.

Antisera to streptococcal groups A through G containing 4% polyethylene glycol 6000 were prepared and evaluated. Seventy strains of homologous and heterologous beta- and non-beta-hemolytic streptococci were included in the evaluation. Homologous reactions were determined against extracts prepared by four extraction methods: hot hydrochloric acid (Lancefield) extraction, autoclave extraction, hot formamide extraction, and nitrous acid extraction. Enhancement of the precipitin reaction in the presence of polyethylene glycol 6000 permitted a fourfold dilution of all antisera for use in the capillary precipitin test. At this dilution, the potency of the antisera exceeded requirements established for these reagents.

Cross Reactions↗
PubMed1989

Antilipopolysaccharide antibodies and differential diagnosis of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis.

Chronic lung infection in cystic fibrosis is characteristically associated with polyagglutinable, serum-sensitive, mucoid strains of Pseudomonas aeruginosa. Enzyme-linked immunosorbent assay (ELISA) methods for standard-free quantitation of immunoglobulin G (IgG) and IgM antibodies to P. aeruginosa lipopolysaccharides (LPSs) have been developed. We now report the development of assays for quantitation of monomer and dimer total IgA and IgA anti-LPS antibodies. Use of these methods in diagnosis of early chronic P. aeruginosa lung infection was assessed. IgG and IgA anti-LPS levels increased significantly at the onset of chronic infection and continued to increase to very high levels in the later stages of infection. IgM anti-LPS levels also rose at the onset of chronic infection but did not increase further. The function of true- and false-positive rates was illustrated by using various concentrations of IgG, IgA, and IgM anti-LPS for discrimination of patients. Values that gave optimum separations were used for statistical evaluation of the diagnostic sensitivities and specificities of anti-LPS antibody concentrations. The results obtained in these assays were compared with a diagnosis, based on the number of precipitins in crossed immunoelectrophoresis, of serum samples from cystic fibrosis patients. In 64 paired serum samples taken before and immediately after the onset of chronic infection, as defined by crossed immunoelectrophoresis precipitins, the predictive values of a positive ELISA were 86% for IgG and 89% for IgA. The predictive values for a negative ELISA were 98% for IgG and 97% for IgA. Results of the IgM anti-LPS ELISA had a lower predictive value. Immunoblotting and absorption studies showed that IgG anti-LPS antibodies were directed specifically against LPS of P. aeuruginosa. ELISAs were developed to determine the specific IgG sublclasses involved. The increase in IgG anti-LPS involved all four subclasses. Highest anti-LPS titers were seen with IgG1 and IgG4, but the largest relative increases were seen with IgG2 and IgG3.

Antibodies, Bacterial↗
PubMed1977

Precipitating antibodies in mycoplasma infection.

The effectiveness of counterimmunoelectrophoresis (CIEP) for detecting human precipitating antibodies to mcyoplasma antigen was compared with the conventional complement fixation (CF) method in a double-blind experiment. Fifty-one sera from patients suspected of having acute mycoplasma infection were tested by both techniques. Dense precipitin lines to mycoplasma antigen developed in 28 sera with CIEP. Twenty-six of 28 had elevated CF titers to this antigen. No precipitin bands were observed in sera with low antibody titers to mycoplasma. These findings indicate that the CIEP test is a specific method for reliably detecting elevated serum CF antibody levels in patients with acute or recent mycoplasma infection.

Antibodies, Bacterial↗
PubMed1979

Serological study of trichloroacetic acid extracts of Bacteroides fragilis.

Immunodiffusion techniques were used on trichloroacetic acid extracts from 10 strains of Bacteroides fragilis in detecting precipitating antibodies against this species in immune rabbit sera. Species and even strain specificities were observed in these precipitin reactions. Multiple antigens were detected in the extracts from some strains, whereas only one precipitin band per extract developed during agar-gel diffusion tests of others. The antigen extracts were found to be both heat stable and resistant to hydrolysis by alpha-chymotrypsin. Four serological patterns were demonstrated in homologous and heterologous reactions with the B. fragilis. antigen-antibody systems used. The results showed that some strains were serologically distinct from others, indicating that the strains tested are of more than one serotype.

Animals↗
PubMed1979

Modified coagglutination procedure for the serological grouping of streptococci.

Cowan I staphylococci coated with antisera to streptococcal groups A, B, C, D, F, and G were used as coagglutination reagents in a modified coagglutination procedure (MCAP). Streptococcal group antigens were extracted with a Streptomyces albus-lysozyme enzyme mixture for 30 min at 55 degrees C and centrifuged, and the supernatant was tested by slide coagglutination. Positive coagglutination reactions occurred within 30 s. The cell pellets from overnight broth cultures and colonies taken directly from sheep blood agar plates were tested and compared with the results of the Lancefield capillary precipitin method. Of the 102 strains of broth-grown cells tested, 100 were grouped by the MCAP and the Lancefield capillary precipitin method. The remaining two isolates were serologically identified only by the MCAP. Of the original 102 strains, 97 were tested by MCAP after extraction of five well-isolated colonies from a sheep blood agar plate. When this latter method was used, 95.9% of the strains were correctly identified. Nonspecific reactions were observed only while testing the MCAP with the direct plate assay. These cross-reactions were remedied promptly by either absorption or dilution of the antisera involved. The MCAP was found to be a rapid and reliable technique for the serological grouping of streptococci.

Agglutination Tests↗
PubMed1972

Immunoelectrophoretic analysis of avian ribonucleic acid tumor virus group-specific antigens.

Tumors induced in pigeons by inoculation with the Schmidt-Ruppin strain of Rous sarcoma virus regressed after about 6 weeks. Sera from these pigeons, taken 8 weeks after inoculation, had complement-fixing group-specific antibody titers of 1:2 to 1:256. In immunoelectrophoresis with the pigeon serum, disrupted BAI strain A (myeloblastosis) avian tumor virus showed at least five precipitin arcs. The pattern of precipitin lines was dependent in part on the means used for virus disruption, and ethyl ether and nonionic detergents appeared to be both effective and relatively mild reagents. Immunoelectrophoretic comparison of pigeon serum with serum from a tumor-bearing hamster and that from virus-inoculated rabbits yielded similar, though not identical, results.

Animals↗
PubMed1978

Budgerigar-fancier's lung: the commonest variety of allergic alveolitis in Britain.

A questionnaire survey of 1005 consecutive attenders at four outpatient clinics yielded 117 (12%) budgerigar fanciers (exposed to budgerigars- known in North America as parakeets-for at least three months) and 296 (29%) former fanciers. Twnety had precipitins to budgerigar serum or droppings or both, and 10 of these together with 39 precipitin-negative patients reported undue breathlessness on exertion during exposure to buderigars. These 59 patients were investigated further, seven completing a series of inhalation provocation tests with budgerigar antigens designed to confirm or exclude budgerigar-fancier's lung (BrFL). Typical positive responses were obtained from four current and one former fancier. The prevalence of confirmed BrFL among the 11n current budgerigar fanciers was 3.4% (four cases). This was biased, however, by the inclusion of one patient whose attendance at the surveyed clinic was attributable to the disease. With the exclusion of this patient, confidence limits suggested that the true prevalence of BrFL among current budgerigar fanciers in the general population lies between 0.5% and 7.5%, which is similar to the prevalence of farmer's lung in farm workers. In view of the enormous population at risk, however, this implies that BrFL rather than farmer's lung is by far the commonest type of allergic alveolitis in Britain.

Adolescent↗
PubMed1972

A fluorescent antibody test in the diagnosis of farmer's lung.

A test for farmer's lung is described employing the indirect fluorescent antibody technique. The test compared very favourably with standard precipitin tests, detecting 88% of a series of positive cases, as opposed to 38% detected by a precipitin test. The fluorescent antibody test, in addition, introduced some measure of quantitation of the serological response in the disease.

Bacteriological Techniques↗
PubMed1975

Use of a serologically distinct strain of Thermoactinomyces vulgaris in the diagnosis of farmer's lung disease.

During investigations into farmer's lung disease it was noticed that Micropolyspora faeni (MF) was isolated and identified with ease, but difficulty was encountered in the identification of Thermoactinomyces vulgaris (TV), although an organism resembling TV was frequently isolated. Extracts prepared from the isolates resembling TV (called Thermoactinomyces vulgaris variant, TVV) when tested against the standard TV antiserum by double diffusion, did not produce any precipitin lines. When TVV extracts were tested against the serum of a patient from whom TVV had been isolated from the sputum, +++ precipitin lines were observed. With the TVV extracts the authors have demonstrated the existence of a serologically distinct strain of TV, and also that it would be a useful addition to the routine testing of antibodies to farmer's lung disease.

Dust↗
PubMed1977

Avian erythrocyte agglutination tests with the sera of bird fanciers.

The agglutination of avian red cells, particularly pigeon and budgerigar cells, by sera from patients who had been exposed to birds and had respiratory symptoms, as well as from healthy bird fanciers, has been used to detect the presence of avian specific antibody. Agglutination tests performed by standard techniques using bird erythrocytes can be read within 45 minutes. One hundred and sixty-five sera were tested, and the 44 which were precipitin positive gave a positive agglutination test with one exception. Of 61 sera which gave positive agglutination tests, 43 gave positive precipitin tests with unconcentrated serum and seven others after fourfold concentration of the serum. Avian antibodies were demonstrated in the remaining 11 sera by radioimmunoelectrophoretic tests. Absorption of the sera with pigeon IgG and IgM and the use of animal antisera directed against pigeon IgG showed that the agglutination reactions were mediated by antibodies directed against pigeon IgG and IgM, and against light chains derived from pigeon IgG.

Alveolitis, Extrinsic Allergic↗
PubMed1977

Observations on the use of the double diffusion test in the diagnosis of vaginal candidiasis.

Precipitins to mannan and cytoplasmic antigens of three Candida species were determined in an unselected series of 289 non-pregnant women. Precipitins were present in 20% of sera of women with vaginal candidiasis, in 23% of women harbouring yeasts in the vagina without clinical signs of infection, and in 21% of women harbouring yeasts in sites other than the vagina. Of the 47 patients who reacted with Candida albicans mannan or cytoplasmic antigens, 98% reacted with mannan antigen but only 13% with cytoplasmic antigen. The inclusion of mannan and cytoplasmic antigens of C. guilliermondii and C. parapsilosis did not increase the specificity or sensitivity of the test in the diagnosis of vaginal candidiasis. It is suggested that the double diffusion test is of doubtful value as an adjunct to the diagnosis of vaginal candidiasis.

Adolescent↗
PubMed1981

Sensitisation against environmental antigens and respiratory symptoms in swine workers.

Adverse effects caused by airborne material to the respiratory tract are due either to non-specific irritation or to hypersensitivity. In this study 20 people employed in swine barns and 18 controls were tested for sensitisation against dusts present in the barn. Immunoprecipitation and enzyme-linked immunoassay (ELISA) were used to test for IgG antibodies; IgE antibodies against swine epithelium were tested using solid phase radioimmunoassay. Precipitins against swine antigens were found in two swine workers; while ELISA found six to be sensitised. Sensitisation against swine antigens correlated with exposure but not with the presence of symptoms. No IgE antibodies were found. Precipitins against feed antigens were detected in 12 workers; in nine of the 12 with symptoms, and in three of the eight asymptomatic workers. No single antigen was of special importance as an inducer of sensitisation. Sensitisation against feed dusts in barns, as indicated by the presence of circulating antibodies, suggests an immunological background for persistent symptoms. A large antigen penal should be used in testing for sensitisation because of the many immunogenic dusts present in the air in swine barns.

Adult↗
PubMed1979

Spiroplasmas: serological grouping of strains associated with plants and insects.

Spiroplasma strains from plant and arthropod hosts, and from surfaces of flowers, were classified into three serological groups (designated I, II, and III) based on results from growth-inhibition tests. No significant cross reactions were observed among groups. The groupings were confirmed by ring-interface precipitin and microprecipitin tests, using membrane preparations as test antigens, and by organism-deformation tests. Serogroup I contained three subgroups: subgroup A (Spiroplasma citri strains Maroc R8A2 and C189), subgroup B (strain AS 576 and closely related strains from honeybee or flowers), and subgroup C (corn stunt spiroplasma strains). Serogroup II contained strains 23-6 and 27-31 isolated from flowers of the tulip tree (Liriodendron tulipifera L.) growing in Maryland. Serogroup III contained strains SR 3 and SR 9 isolated from flowers of the tulip growing in Connecticut. The subgroups of serogroup I were based on organism deformation, microprecipitin, and ring-interface precipitin tests. The data are consistent with the hypothesis that the three serogroups represent no less than three distinct spiroplasma species.

Animals↗