Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Messenger RNA isolated from Cocksfoot grass (Dactylis glomerata) anthers has been used to generate a cDNA library in lambda t11. Three cDNA clones (7.8, 8.1, and 8.3) were demonstrated to be recognized by human IgE antibodies in atopic serum and by rabbit polyclonal antiserum raised to a crude aqueous extract of Cocksfoot pollen. The size of the cDNA inserts was determined as approximately 700 bp, and restriction mapping demonstrated them to be identical sequences. Lysogens obtained in Escherichia coli Y1089 allowed expression of a 140 kD beta-galactosidase fusion protein containing 24 kD of cloned allergen protein. Fusion proteins were recognized by IgE antibodies in 75% (6/8) of atopic sera tested, but were not detected by nonatopic sera. On the basis of size and frequency of recognition in the atopic population, the cloned protein may present a major allergen. Monoclonal antibodies specific for the major allergen of Cocksfoot pollen were not reactive with the fusion proteins. Reactivity of human IgE antibodies with the fusion protein could be blocked by crude Cocksfoot pollen extract, but not by the major allergen DG3 purified from the extract by affinity chromatography. Human and rabbit antibodies affinity purified against fusion protein 7.8 did not allow identification of the native protein component in crude extract encoded for by the cDNA clones.
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PMID:Cloning of cDNA coding for an allergen of Cocksfoot grass (Dactylis glomerata) pollen. 280 80

A colored or fluorescent signal is generally evaluated with the naked eye, or by means of different more or less sophisticated and costly instruments. Photodensitometry is an additional technique which is both inexpensive and simple to perform. This technique can satisfactorily quantify a signal without the use of either a spectrophotometer or a fluorometer. In this study we compared readings obtained by spectrophotometry, fluorometry and photodensitometry in 96-well ELISA plates and in Terasaki plates. In ELISA plates, it is possible to detect 1220-300,000 femtograms (fg) of peroxidase by spectrophotometry and 4800-125,000 fg by photodensitometry. In Terasaki plates, we were able to measure between 3.8 and 8000 fg of beta-galactosidase per sample by spectrofluorometry, and from 30 to 8000 fg by photodensitometry. Using a sandwich procedure in Terasaki plates we were able to measure between 100 and 10,000 fg of IgE per sample by spectrofluorometry and from 500 to 10,000 fg by photodensitometry. Photodensitometry is the least expensive technique for the reliable detection of enzyme or enzymatic marker in small sample volumes treated with a fluorogenic substrate.
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PMID:A method for the quantification of a colored or fluorescent signal in enzyme immunoassays by photodensitometry. 311

We have developed a fluorometric enzyme-linked immunosorbent assay for measuring IgE antibody to Dermatophagoides farinae. Polystyrene microplates were coated with proteins extracted from the mites. The IgE antibody which attached to the solid-phase antigen was detected by anti-IgE antibody conjugated with beta-galactosidase. Four-methylumbelliferyl-beta-D-galactoside was used as the enzyme substrate and the fluorescence intensity of the reaction product was measured. The antibody levels determined by this method well correlated with those determined by the radioallergosorbent test (RAST). This method is simpler and less expensive to carry out than the RAST when dealing with a large number of serum specimens for seroepidemiological studies of asthma and nasal allergy.
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PMID:Fluorometric enzyme-linked immunosorbent assay for the measurement of IgE antibody to mite Dermatophagoides farinae. 332 83

The allergen composition of crude extracts of the storage mite Lepidoglyphus destructor were investigated by sodium dodecylsulphate polyacrylamide gel electrophoresis and immunoblotting. The allergen components were detected by sera from allergic farmers with a positive radioallergosorbent test to L. destructor. The allergen-antibody complexes were visualized by rabbit anti-IgE and beta-galactosidase-labelled sheep antirabbit IgG in a chromogenic substrate. Six out of the forty-three sera also had IgE antibodies to the culture medium, which were neutralized by addition of medium antigens before the demonstration of mite allergen components. A total of 11 allergen components were identified. Two of them were found to be major allergen components, identified by more than 50% of the 43 sera used in the study. These 2 IgE-binding components had molecular weights of approximately 18,000 and 16,000 daltons, respectively.
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PMID:Demonstration of allergen components in the storage mite Lepidoglyphus destructor by an immunoblotting technique. 333 56

An enzyme-linked immunosorbent assay (ELISA), employing beta-galactosidase conjugated anti-IgE, was used for the determination of specific IgE antibodies to common inhalant allergens (Dermatophagoides pt. and f., Parietaria and four grass pollens) in serum samples from 82 adult patients with asthma and/or rhinitis. A total of 194 analysis were carried out and the results were compared with those of skin tests and RAST. The correlation coefficient (r) between ELISA and RAST results was high (r = 0.95); the correlation between skin tests and ELISA (r = 0.93) was greater than that between skin tests and RAST (r = 0.90). No significant differences were found among the allergens used. We conclude that the version of ELISA used develops an overall good correlation with skin tests and RAST and seems to provide a satisfactory alternative to RAST for allergy diagnosis.
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PMID:ELISA in diagnosis of respiratory allergy. A comparison with RAST and skin tests. 389 May 99

An enzyme-linked immunosorbent assay (ELISA) for the quantitation of 2,4-dinitrophenyl (DNP)-specific murine immunoglobulin (Ig) E is described. The assay uses beta-galactosidase, which is conjugated to goat anti-rabbit gamma-globulin (GARG) via a method using a mild heterobifunctional cross-linking reagent, m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). The assay has a sensitivity for detection of about 200 pg/ml of anti-DNP IgE. Analyses of murine serum samples using this ELISA correlate well with those obtained using the passive cutaneous anaphylaxis (PCA) reaction in rats. With the use of automated 96-well reader, data acquisition is rapid and, therefore, this ELISA is ideal for analyses of large numbers of samples. The assay can be easily modified for the measurement of other Ig classes and of IgE of other antigen specificities.
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PMID:A sensitive enzyme-linked immunosorbent assay for the quantitation of antigen-specific murine immunoglobulin E. 616 64

Spleen cells from Balb C mice immunized with purified Yu human myeloma IgE were fused with NS-1 mouse myeloma cells. After initial EIA screening for antibody-secreting cells, 20 hybrids were further characterized for cell growth, ascites production, antibody titer, specificity and affinity. Immunoglobulins purified from ascites fluid obtained from selected clones were labelled with beta-galactosidase. Combinations were made using either antibodies as capture and as conjugate against calibrated human IgE plasma samples. The combination of monoclonal anti IgE X b 10-22 as a capture antibody and X b 6-16 as a conjugate gave the best sensitivity and slope in EIA. It was successfully used in a sensitive two-step-enzyme-immunoassay for total IgE. The X b 6-16 conjugate was also assayed for the detection of allergen specific IgE antibodies. The results presented and discussed indicate that monoclonal antibodies could favourably substitute for polyclonal anti IgE antibodies in such assays.
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PMID:Monoclonal antibodies to human IgE: utilization for total IgE quantification and estimation of allergen specific IgE antibodies. 639 32

Enzyme immunoassay techniques are widely use to quantify various antigens and antibodies. The final step of these techniques (i.e. enzyme reaction) may be carried out in several ways (e.g. chromogenic, fluorogenic, or radioactive substrate and thermometric measurement). This paper compares the effectiveness of the chromogenic and the fluorogenic substrates in the beta-galactosidase immunoassay. Using microtitration plates (150 microliter samples) coated with anti-human IgE, and anti-human IgE labeled with E. coli beta-galactosidase, the lowest concentrations of IgE that one could detect employing either the chromogenic (o-nitrophenyl-beta-D-galactopyranoside) or the fluorogenic (4-methyl-umbelliferyl-beta-D-galactopyranoside) substrate were determined. It was found that both substrates were almost equally effective in measuring the lowest concentration of IgE (0.075-0.13 IU/ml) under the optimal conditions. But, using fluorogenic substrate and suitable apparatuses the enzyme immunoassay can be miniaturized. Thus by using decreasing volumes of reagents, progressively smaller amounts of antigen were quantified: as the sample volumes were reduced from 150 to 10 microliter and finally to 0.3 microliter a progressive decrease from 7 x 107 molecules of IgE to 2.9 x 107 molecules and to 1.5 x 106 molecules was observed. The corresponding lowest detection limits were 0.075 IU/ml, 0.46 IU/ml and 0.8 IU/ml.
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PMID:Miniaturization of beta-galactosidase immunoassays using chromogenic and fluorogenic substrates. 679 80

A cDNA clone encoding a new allergen from the Dermatophagoides farinae cDNA lambda gt11 library was isolated and sequenced. There was no amino acid sequence homology with other known allergens. The gene product, beta-galactosidase fusion protein, of the truncated cDNA on blot reacted with IgE in 13 of 43 sera from patients allergic to mites. The affinity-purified fusion protein had a potent ability to release histamine from washed blood cells of the mite-allergic patients. Human specific IgE eluted from the fusion protein band on blots recognized a 39-kD component on blots of a mite body extract.
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PMID:Cloning and characterization of cDNA coding for a new allergen from the house dust mite, Dermatophagoides farinae. 751 May 58

Indoor allergens comprise a group of allergenic proteins that are commonly derived from house dust mite and cat and dog dander. In addition to the two major dog allergens (molecular weights: 19 and 23 kd), dog albumin represents an important allergen for up to 35% of patients who are allergic to dogs. In IgE immunoblot inhibition studies and histamine release tests it has been demonstrated that patients who react to dog albumin exhibit IgE reactivity with purified albumins from cat, mouse, chicken, and rat. The proportion of dog-specific IgE directed against dog albumin was determined for patients allergic to dog albumin, and it ranges from 70% to 90%. By IgE immunoscreening of a lambda gt11 expression library from a dog salivary gland, we identified a number of reactive complementary DNA clones. All patients with IgE reactivity against natural dog albumin displayed IgE reactivity to the beta-galactosidase fusion protein encoded by clone 54c, which was therefore assumed to contain major IgE epitopes of dog albumin. The deduced amino acid sequence of clone 54c was compared with the Swiss-Prot library, and significant sequence homologies were found with albumins from different species (human: 82.6%, pig: 81.8%, cattle: 77.3%, sheep: 78.8%, mouse: 75.8%, and rat: 76.2%). Several other IgE-positive clones hybridized with oligonucleotides that were prepared according to this sequence. Partial complementary DNA coding for dog albumin fragments may be considered a useful tool for further characterization of major IgE epitopes of dog albumin.
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PMID:Molecular characterization of dog albumin as a cross-reactive allergen. 751 2


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