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PubMed1985

Evaluation of three serological tests for detection of antibody to Pseudomonas aeruginosa in human sera.

Four hundred and ninety-five sera from 325 patients from whom Pseudomonas aeruginosa had been isolated and 86 control sera were tested for antibody by indirect haemagglutination tests (HAT) and complement fixation tests (CFT) using a polyvalent pseudomonas serotype-specific vaccine antigen, PEV-02. Sera were also tested by countercurrent immunoelectrophoresis (CIE) for precipitins to a species-specific protein antigen. Control sera gave titres of 160 or less by HAT and 20 or less by CFT. 2-Mercaptoethanol resistant antibody titres (immunoglobulin G) were below 40 for all control sera and none of the latter contained precipitins to common antigen. Of 325 patients, 156 (48%) gave titres of 320 or greater by HAT and of these, 114 (73%) showed elevated immunoglobulin G titres. Less patients with positive blood cultures than expected were positive by HAT and more patients with bone infections gave raised immunoglobulin G titres than expected. Cystic fibrosis patients were invariably seropositive by all tests. There was a correlation between positive CIE and CFT tests, especially in patients who were positive by HAT. Approximately half of 83 patients tested gave a serotype-specific antibody response. The tests were of little value in confirming clinically evident acute infections, but in cases of doubtful infection they did provide confirmatory evidence of an antibody response in approximately one-third of patients culture-positive for Pseudomonas aeruginosa.

Antibodies, Bacterial↗
PubMed1983

Comparison of immunodiffusion and crossed-immunoelectrophoresis in the diagnosis of invasive candidiasis.

A retrospective single blind study was conducted to compare the efficacy of crossed-immunoelectrophoresis using concanavalin A intermediate gel with double immunodiffusion tests in the diagnosis of invasive candidiasis. On the basis of cytoplasmic antibody detection, crossed-immunoelectrophoresis with concanavalin A differentiated reliably between Candida albicans fungemia and invasive candida infection, whereas double immunodiffusion did not. With regard to sensitivity, specificity, and predictive value crossed-immunoelectrophoresis with concanavalin A was superior to double immunodiffusion. Analysis of double immunodiffusion precipitin bands revealed fuzzy and sharp precipitin bands which corresponded to mannan (fuzzy) and cytoplasmic (sharp) antibodies in the crossed-immunoelectrophoresis with concanavalin A assay. Therefore, the finding of sharp bands in the double immunodiffusion procedure supports the diagnosis of invasive candidiasis.

Adolescent↗
PubMed2005

The effects of long-term occupational exposure to dust from herbs.

INTRODUCTION: Herbs are a heterogeneous group of many species with several thousand plants, which are used in large quantities in the pharmaceutical and food industries. The aim of the study was to analyse the health effects of long-term occupational exposure to dust from herbs. METHODS: A group of 150 people occupationally exposed to dust from herbs, consisting of farmers and workers from herbs processing industry, was examined. As a reference group, 50 urban dwellers not exposed to any kind of organic dust were examined. Examined people were interviewed with the help of the ATS questionnaire compiled by Ferris and by the questionnaire developed in the Institute of Agricultural Medicine in Lublin, Poland for examination of work-related symptoms caused by organic dust. The lung function examination (vital capacity (VC), forced expiratory volume in the first second (FEV(1)), and FEV(1)/VC (%) of normal ranges) and allergological tests (skin prick test, precipitin test and inhibition of leukocyte migration (MIF) test) with microbial antigens were conducted. RESULTS: 71.3% (95% CI 64.1-78.6%) of the exposed people reported occurrence of work-related symptoms. A post-shift decrease of spirometric values (VC, VC%) was observed in the exposed group (mean decrease 2.6%, P<0.01). A significant relationship was found between the number of work-related symptoms and decrease of FEV(1) values, both before (Spearman correlation coefficient r=-0.21, P<0.05) and after work (r=-0.31, P<0.01). In allergological tests, the frequencies of positive reactions in the exposed group were significantly higher than in the reference group. Precipitins specific to Pantoea agglomerans were found in 30.6% (95% CI 23.2-38.1%) of exposed, compared to 12.0% among unexposed (P=0.01). The frequency of positive results in the migration inhibition test was significantly higher among exposed workers for all antigens tested. CONCLUSION: Long-term exposure to dust from herbs causes work-related symptoms and decrease of lung function parameters, which, finally, may lead to occupational disease.

Adolescent↗
PubMed1978

Rapid, sensitive detection of myoglobulinemia by improved counterimmunoelectrophoresis in cases of acute myocardial infarction.

A counterimmunoelectrophoresis technique for detection of serum myoglobin (Mb) was improved using non-ionic polymer dextran. Precipitin lines were graded according to their strength, which was ascertained by radioimmunoassay data. By this method, serum Mb in concentrations of 500 ng./ml. before stain and of 200 ng./ml. after stain were detected. Electrophoretic time was 60 minutes. Among 32 cases of acute myocardial infarction (AMI) whose blood samples were collected within 24 hours after disease onset, precipitin lines were detected in 25 cases (78%) before stain and 31 cases (97%) after stain. Considering the early peak concentration time (approximately 10 hours) of serum Mb after AMI onset, diagnosis becomes more rapid and exact with this method, especially in severe cases.

Acute Disease↗
PubMed1976

C1q deficiency associated with urticarial-like lesions and cutaneous vasculitis.

We describe a 46 year old women with a seven year history of urticarial-like symptoms and cutaneous vasculitis with marked deficiency of C1q in the presence of normal levels of C1r and C1s and high titers of low molecular weight (7S) C1q precipitins. Hemolytic C1 activity, which was greatly reduced, was restored upon the addition of purified C1q. The other complement components were present in moderately reduced or normal levels. This patient bears resemblance to several other persons previously described with urticarial-like lesions in association with selective deficiency of C1q. The similarity of the clinical features, pathologic diagnosis, C1q levels disproportionately deficient in relation to other complement components and low molecular weight C1q precipitins support the conclusion that these are causally related in a symptom complex. The underlying basis is yet to be defined.

Complement C1↗
PubMed1983

Rocket and crossed immunoelectrophoresis of proteins solubilized with sodium dodecyl sulfate.

A method for the immunoelectrophoretic analysis of both hydrophilic and hydrophobic proteins from whole-cell extracts solubilized with 2% (w/v) sodium dodecyl sulfate (SDS) is described. For rocket immunoelectrophoresis, Triton X-100 is added to the sample before electrophoresis to sequester non-protein-bound SDS, and polyethylene glycol (PEG) is added to the antibody gel to enhance precipitin formation. With the optimal ratio of Triton X-100 to PEG, the quantitative determination of 5 ng of protein is possible. The SDS-solubilized sample can also be analyzed by crossed immunoelectrophoresis using SDS-polyacrylamide gels in the first dimension and antibody-containing agarose gels in the second. The best results are obtained when intermediate gels without nonionic detergents are used and when ionic detergents are omitted from the cathodal gel. Precipitin peaks of high quality, reproducibility, and without artifacts are obtained using antibody concentrations 5- to 50-fold lower than with other crossed-immunoelectrophoresis procedures.

Chlamydomonas↗
PubMed1981

Human liver prenyltransferase and its characterization.

Prenyltransferase (dimethylallydiphosphate: isopentenyldiphosphate dimethylallytransferase, EC 2.5.1.1) has been purified to homogeneity from human liver obtained at autopsy. The enzyme is a dimer with a native molecular weight of 74 000 +/- 1 400. The amino acid composition is reported. the enzyme has a broad pH optimum between 7.3 and 8.8 and an absolute requirement for either Mn2+ or Mg2+ for activity; half-maximal activity was observed at 3.7 microM Mn2+ or 89.0 microM Mg2+. Michaelis constants for geranyl pyrophosphate and isopentenyl pyrophosphate were 0.44 and 0.94 microM, respectively; the V value for synthesis of farnesyl pyrophosphate from these substrates was 1.1 mumol . min-1 . mg-1. Isopentenyl pyrophosphate inhibited the reaction rates at concentrations above 2 microM when the concentrations of geranyl pyrophosphate were less than 2 microM. The highest concentration of geranyl pyrophosphate tested, 16 microM, showed no inhibition of reaction rates even when the concentration of isopentenyl pyrophosphate was as low as 0.2 microM. Only one form of human liver prenyltransferase could be observed under conditions which resolved the porcine enzyme into two distinct forms; the human enzyme is akin, physico-chemically, to the B-form of the pig liver enzyme. After dialysis against Tris-HCl buffer, pH 7.8, the enzyme became completely dependent upon dithiols or thiols for its activity. Kinetic experiments with a partially activated enzyme sample showed that the activation by the dithiol greatly enhanced the affinity of the enzyme for geranyl pyrophosphate, but not that for isopentenyl pyrophosphate. The human prenyltransferase is inactivated by phenylglyoxal according to pseudo-first-order kinetics, but is protected against the inactivation by 3,3-dimethylallyl and geranyl pyrophosphate. It is also inactivated by high concentrations (greater than 2 mM) of iodoacetic acid, but is protected against the inactivation by dithiothreitol. Antibodies raised to the B-form of the pig liver enzyme cross-reacted with the human prenyltransferase and were 47% as effective in precipitating the human enzyme as the porcine enzyme. In double immunodiffusion experiments the antiserum was monospecific against the B-form of the porcine enzyme; it also gave a single precipitin line with the A-form, but not identical with that given by the B-form. It gave a precipitin line also with the human enzyme, but not identical with that given by either the A- or B-form of the porcine enzyme.

Animals↗
PubMed1981

Evidence for the presence of cytochrome P-450 functional in lanosterol 14 alpha-demethylation in microsomes of aerobically grown respiring yeast.

Recent studies have shown that a cytochrome P-450 present in microsomes of semi-anaerobically grown cells of Saccharomyces cerevisiae is functional in the 14 alpha-demethylation of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol), but the occurrence of the same cytochrome P-450 in microsomes of aerobically grown yeast cells has not yet been reported. In this study, the microsomal fraction from aerobically grown cells was found to catalyze the lanosterol demethylation in the presence of NADPH and O2 and that this activity was sensitive to CO. In Ouchterlony double diffusion test, antibodies to the yeast cytochrome P-450 formed a single precipitin line with the microsomal fraction as well as with the purified yeast cytochrome P-450 and the two precipitin lines fused with each other. Furthermore, the antibodies inhibited the lanosterol demethylation activity of the microsomal fraction from aerobically grown cells. The quadratic-derivative absorption spectrum of the microsomal fraction measured in the presence of both Na2S2O4 and CO showed an absorption band at 450 nm which is attributable to the reduced CO compound of cytochrome P-450. These facts led to the conclusion that cytochrome P-450 actually exists in aerobically grown yeast and participates in the lanosterol 14 alpha-demethylation which is essential for the ergosterol (5 alpha-ergosta-5,7,22-trien-3 beta-ol) biogenesis by yeast.

Aerobiosis↗
PubMed1975

Leucocyte myosin and its location in the cell.

The intracellular location of the binding site of antibody against purified myosin prepared from equine leucocytes was investigated in neutrophils and lymphocytes by electron microscopy using peroxidase-labelled antibody method. The myosin extracted from equine leucocytes could bind skeletal muscle F-actin and the formed complex showed the biophysical and biochemical properties and electron microscopic appearance of actomyosin. On immunodiffusion, the leucocyte myosin formed a single precipitin line with its antibody prepared in rabbits. The antibody also formed single precipitin lines with myosins from lymphocytes and thrombocytes, fusing with each other. The antibody against the leucocyte myosin did not react with myosins from skeletal or arterial smooth muscle. The specificity of the antibody was further established by determination of K+-EDTA-activated ATPase activity remained in the supernate of antigen-antibody mixture. Under electron microscope, the intracellular immunoreactive products of peroxidase labelled antibody were found in cytoplasm of neutrophils and lymphocytes incubated with antibody against leucocyte myosin, but not in neutrophils or lymphocytes treated with IgG from normal rabbits.

Adenosine Triphosphatases↗
PubMed1988

Immunochemical studies on the N-acetyllactosamine beta-(1----6)-linked trisaccharide specificity of Ricinus communis agglutinin.

The combining site of Ricinus communis agglutinin (RCA1) was studied by quantitative precipitin and precipitin inhibition assays. Of 31 complex carbohydrates tested, all except active and inactive antifreeze glycoproteins, Streptococcus group C polysaccharide, and native rat salivary glycoprotein, reacted strongly, and 22 completely precipitated the lectin, indicating that RCA1 has both a broad range of affinity and a low solubility of its carbohydrate-bound complex. Of the monosaccharides and glycosides tested for inhibition of precipitation, p-nitrophenyl beta-D-galactopyranoside was the best. It was about 6.4 times better than methyl beta-D-galactopyranoside. The beta anomer of glycosides of D-galactose was much more potent than the corresponding alpha anomer. Among the oligosaccharides tested, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-D-Gal was the best inhibitor, which was approximately 2/3 as active as p-nitrophenyl beta-D-galactopyranoside. It was approximately 1.4 times as active as beta-D-Gal-(1----4)-D-GlcNAc (N-acetyllactosamine), twice as active as beta-D-Gal-(1----3)-D-GlcNAc, and 4.5 times more active than lacto-N-tetraose. From the results, it can be concluded that; (a) hydrophobic interaction is important for binding; (b) the combining site of this lectin is at least as large as a trisaccharide; and (c) of the compounds studied, the trisaccharide beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-D-Gal was the most complementary to the human blood group I Ma determinant beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-D-Gal.

Amino Sugars↗
PubMed1977

Properties of amylase-linked immunoglobulins.

In this report, the properties of eight cases of amylase-linked immunoglobulins were gel filtration using Sephadex G-200 superfine. The class of heavy chain of amylase-linked immunoglobulins was proved to be gamma in four cases and alpha in three cases by immunoelectrophoresis followed by amylase activity staining. In one of the cases, the precipitin line against light chain lambda could be visualized only after treatment with 0.1 M 2-mercaptoethanol. Then, the type of light chain was determined to be exclusively lambda irrespective of heavy chain classes. In two cases, the immunoglobulin complexes were partially dissociated into normal amylase and immunoglobulin G at pH 8.6, and completely dissociated at pH 9.0. In three cases, the complexes were completely dissociated at pH 8.6. The precipitin line of papain treated amylase-linked immunoglobulin G against anti-Fc was not. This fact suggests that the binding site of amylase-linked immunoglobulins G was located in the Fab portion of immunoglobulin molecule and that the complexes are specific antigen-antibody complexes. Treatment with Con-A Sepharose caused the dissociation of the amylase-immunoglobulin complexes. It is suggested that the changes in the conformation of amylase-linked immunoglobulin causes the dissociation of this immuno-complex. Thus, it is elucidated that the complexes of amylase and immunoglobulins are specific antigen-antibody complexes, and that these complexes must be recognized as one of the circulating autoantibodies in plasma, and must be clearly distinguished from the other unknown macromolecular amylase complexes.

Amylases↗
PubMed1985

Development of a radial immunodiffusion technique employing monoclonal antibodies for apolipoprotein B determination in human plasma.

Twenty monoclonal antibodies obtained from two different fusions of SP2/0-Ag 14 cell line (non-secretor hybridoma) with human plasma low density lipoproteins have been selected. We found that a mixture formed by the 20 monoclonal antibodies was able to form a single precipitin line with human plasma low density lipoproteins by a double gel diffusion technique. Further studies revealed that only two monoclonal antibodies were needed to precipitate low density lipoproteins in gel. However, a minimum of four particular monoclonal antibodies was required to obtain an optimal precipitin ring and a linear standard curve within 24 h using a radial immunodiffusion technique. We have then compared the radial immunodiffusion performed with a monoclonal antibody mixture to those employing conventional goat and rabbit antibodies in terms of plasma apolipoprotein B determinations. The apolipoprotein B values determined by monoclonal antibodies significantly correlate with the values obtained by the technique using goat (r = 0.95; p less than 0.001) and rabbit (r = 0.95; p less than 0.001) antibodies. Our data indicate that a mixture of monoclonal antibodies can mimic conventional antibodies in terms of immunoprecipitation and apolipoprotein B determination.

Animals↗
PubMed1986

Absence of argininosuccinate lyase protein in the liver of two patients with argininosuccinic aciduria.

The enzyme defects in two cases of argininosuccinic aciduria were examined at the molecular level by enzymatic and immunological methods. No argininosuccinate lyase activity was detected in the liver or erythrocytes of either patient nor in the kidney or brain of one of the patients even in the presence of high concentrations of the substrate. The titration curve of antiserum to human argininosuccinate lyase with the liver extract from a control subject was not affected by the addition of the liver extracts from one of the patients. Double immunodiffusion analysis revealed a single precipitin line between the purified antiserum and the liver extract from a control, but no precipitin lines between the antisera and the liver extracts from the two patients. These results indicate a complete or almost complete defect of an immunologically cross-reactive material in the liver of the patients.

Amino Acid Metabolism, Inborn Errors↗
PubMed1995

Native and/or asialo-Tamm-Horsfall glycoproteins Sd(a+) are important receptors for Triticum vulgaris (wheat germ) agglutinin and for three toxic lectins (abrin-a, ricin and mistletoe toxic lectin-I).

The binding properties of human Tamm-Horsfall Sd(a+) urinary glycoprotein (THGP) and asialo-THGP with Triticum vulgaris agglutinin(WGA) and three toxic lectins (abrin-a, ricin, and Mistletoe toxic lectin-I) were investigated by quantitative precipitin and precipitin inhibition assays. Both glycoproteins reacted strongly with abrin-a, precipitating over 80% of the lectin nitrogen tested. THGP also bound well to mistletoe toxic lectin-I and precipitated 86% of this lectin added, while the precipitability of its asialo product decreased by 28%. The native glycoprotein completely precipitated the WGA added, but its reactivity was reduced dramatically after desialylation. On the contrary, the poor reactivity of THGP with ricin increased substantially after removal of sialic acid and completely precipitated the lectin added. The glycoprotein-lectin interactions were inhibited by one or several of the following haptens, p-NO2-phenyl alpha GalNAc, p-NO2-phenyl beta GalNAc, Gal beta 1-->4GlcNAc, Gal beta 1-->4Glc, GlcNac beta 1-->4GlcNAc and/or GlcNAc. From the above results, it is concluded that native and/or Tamm-Horsfall glycoproteins serve as important receptors for these three toxic lectins and for WGA.

Abrin↗
PubMed1996

Interaction of a human blood group Sd(a-) Tamm-Horsfall glycoprotein with applied lectins.

Unlike the human blood group Sd(a+) Tamm-Horsfall glycoprotein (THGP), the Sd(a-) one lacks terminal GalNAcbeta1--> residues at the nonreducing ends. The binding properties of this glycoprotein and its asialo product with lectins were characterized by quantitative precipitin (QPA) and precipitin inhibition assays. Among 20 lectins tested by QPA, both native and asialo Sd(a-) THGP reacted best with Abrus precatorius and Ricinus communis and completely precipitated the lectin added. They also precipitated well Wistaria floribunda (WFA), Glycine max (SBA), Bauhinia purpurea alba, abrin-a and ricin, all of which recognize the Galbeta1--> 4GlcNAcbeta1--> sequence, although at different strength. The lectin-glycan interactions were inhibited by Galbeta1--> 4GlcNAc and Galbeta1--> 4Glc. When the precipitability of Sd(a-) THGP was compared with that of the Sd(a+) phenotype, the native Sd(a-) THGP exhibited a 40% lesser affinity for WFA, SBA, WGA and mistletoe lectin-I (ML-I). Mapping the precipitation and inhibition profiles of the present study and the results of THGP Sd(a+), it is concluded that Sd(a-) THGP showed a strongly diminished affinity for GalNAcbeta1--> active lectins (SBA and WFA) than the Sd(a+) phenotype.

Adjuvants, Immunologic↗
PubMed1977

Immunoadsorbents for the isolation of high-affinity anti-hapten antibodies in high yield.

Anti-hapten antibodies with affinities close to 10(8)/M can be isolated in high yield by elution under mild conditions from immunoadsorbents prepared by coupling haptens directly to an aminoethylamino (AEA) derivative of beaded agarose. In experiments with rabbit anti-2,4-dinitrophenyl (DNP) antibody with an average association constant (Ko) of 1.2 X 10(9)/M, elution of antibody bound to a Sepharose-AEA-DNP column produced a yield of 57%. With rabbit antibody to the 5-dimethylaminonaphthalene-1-sulfonyl (DANS) hapten with Ko=2.7 X 10(8)/M and a Sips heterogeneity index a=0.52, a 20% yield of the antibody found in the starting material by quantitative precipitin analysis was obtained by specific precipitation and dissolution of the precipitate with 0.18 M Na DANSulfonate; this antibody had Ko=6.6 X 10(8)/M and a=0.66. Purification of another aliquot of the same antibody preparation by immunoadsorption on DANS-AEA-Sepharose and elution with the same hapten concentration produced a yield of 75% of antibody with Ko=5.7 X 10(7)/M and a=0.29. In 3 production runs with the new method, apparent yields of 181, 101 and 94% (based on quantitative precipitin analysis of the starting material) of anti-DANS antibody were obtained. In 2 of these runs, the immunoadsorbent was further eluted with 0.1 M acetic acid and then with 0.1 M acetic acid in 10% dioxane; additonal yields of 9 and 4% were obtained with acetic acid and further yields of 15 and 11% with acetic acid/dioxane; the antibody eluted with acetic acid was damaged as shown by its failure to mediate passive immune lysis of DANS-coupled sheep erythrocytes, but the antibody eluted with acetic acid/dioxane was recovered intact.

Animals↗
PubMed1981

Cascade immunoelectrophoresis: combined electrophoretic and solid-phase processing of immunoreactive protein by zonal immobilization.

A new approach to immunochemical analysis of complex mixtures of proteins has been devised through development of a system for (1) sequentially absorbing, desorbing, and cascading antibodies in a profile indicative of both the quantity and electrophoretic characteristics of the antigen, and (2) for carrying out multiple tests of antigenic determinants at sub-picomole levels regardless of the solubility or precipitin forming characteristics of the immunoreactive protein. The procedure employs zonal immobilization in which a novel, aldehyde-rich gel (glyoxyl agarose) is used interchangeably as an inert support for electrophoresis and as an immobilizing matrix to fix the protein for analysis by solid-phase techniques. Proteins may be separated on the gel as with ordinary agarose, and then driven to combine covalently with the gel upon exposure to NaCNBH3. After removing NaCNBH3, the distribution of specified antigens is then established by exposing the gel to antibody an profiling the pattern of antibody uptake by a cross-electrophoretic technique in which the absorbed antibody is (1) desorbed with dodecyl sulfate, then (2) transferred through a gel containing potassium ion to halt migration of the detergent, and (3) displayed by immunoprecipitation with anti-IgG antibodies. To cascade the process further, the IgG may be immobilized and used as a surrogate antigen to bind additional antibody protein. In addition to enhancing sensitivity, the cross-electrophoretic measurement of uptake of antibody by immobilized antigen eliminates dependence on direct immunoprecipitation of the antigen, and may accordingly be of particular advantage with non-precipitin forming antigens and monoclonal antibodies.

Aldehydes↗
PubMed1983

Radioimmunoelectrophoresis, a sensitive method for detecting cleavage of the fifth component of human complement (C5).

A method has been developed for detecting cleavage of human C5 in serum and whole blood as a consequence of complement activation. Standard, single-dimension immunoelectrophoresis was performed using as antibody a radioiodinated IgG fraction prepared from a commercially available antiserum to human C5. Autoradiographs developed after radioimmunoelectrophoresis of either normal human serum or functionally pure human C5 revealed only one precipitin band. In contrast, when either zymosan-treated serum or trypsin-treated human C5 were examined with this technique, two additional precipitin bands were detected. One migrated more anodally than native C5 while the other remained at the origin (cathode). Radioimmunoelectrophoresis was significantly more sensitive as an indicator of complement activation in human serum than either measurements of total hemolytic complement or a standard assay for complement (C5)-derived chemotactic activity.

Complement Activation↗