Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A rapid ELISA method has been developed to quantitate myoglobin in serum. An antigen capture enzyme-linked immunoassay with IgG1 mouse monoclonal antibodies produced by cell clones MGB20-4A1.1 and MGB20-3C1.2 were used for myoglobin detection. These monoclonal antibodies are specific for different epitopes of the myoglobin molecule. Monoclonal antibodies from the hybridoma clone MGB20-4A1.1 were adsorbed to microtiter plate wells. The plates were washed with PBS containing 0.05% Tween 20 and then 20 microliter of standard serum or serum of patients and 200 microliter of peroxidase labeled monoclonal antibodies MGB20-3C1.2 were added to each well. Plates were incubated for 90 min at 37 degrees C and enzyme activity was determined using o-phenylenediamine as a substrate. The ELISA assay described is a rapid and sensitive procedure to assess the quantity of myoglobin within the range 2-1000 ng/ml serum. 120 samples can be tested in 3 h.
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PMID:Rapid, simple and sensitive antigen capture ELISA for the quantitation of myoglobin using monoclonal antibodies. 245 49

An enzyme-linked immunosorbent assay (ELISA) was used to measure IgG antibody titers against a synthetic peptide whose sequence was derived from the glycine-alanine repeating region of Epstein-Barr virus nuclear associated antigen 1 (EBNA-1). Antibody titers were determined in sera from 15 normal subjects, sera from 21 normal male siblings of X-linked lymphoproliferative syndrome (XLP) patients, from 20 XLP patients comprising a total of 42 samples, and ten samples before and ten samples after gamma-globulin therapy in ten patients with XLP. Data analysis demonstrated that while there are differences between the ELISA and ACIF, they appear to measure a similar response as demonstrated by their correlation coefficient (0.77) and the GMT to EBNA observed by both methods. No cross-reactivity of cytomegalovirus antibodies to the EBNA-1 peptide was observed by immunoblotting, ELISA or ACIF using adsorption against AD-169 infected MRC-5 cells. However, non-specific binding was observed if samples were not pre-incubated in a 10% goat serum PBS-Tween 20 solution. This pre-treatment removed the non-specific binding that falsely elevated the GMT in approximately 15% of both normal and XLP samples in ELISA. The ELISA system appears to be a sensitive, reproducible and objective test that may be useful for assessing the antibody response of patients to the EBNA-1 protein.
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PMID:Antibody reactivity to a synthetic peptide (P62) of the Epstein-Barr nuclear antigen in sera of patients with X-linked lymphoproliferative syndrome. 303 51

Surfactant protein B (SP-B) is one of the essential constituents of the alveolar surfactant system, presenting mainly in the form of SP-B dimer. The measurement of this molecule in biologic samples has been hampered by its extreme hydrophobicity and intimate association with surfactant lipids. We developed a solid-phase, adsorption-based enzyme-linked immunosorbent assay (ELISA) technique for the quantification of SP-B in aqueous solutions. The ELISA employs the hydrophobicity of SP-B for direct binding of this compound to polystyrol immunosorbent plates. Samples are mixed with propanol (1:1 vol/vol) to achieve a homogeneous dispersion of their lipophilic constituents prior to adsorption to the wells. After fluid removal by evaporation, trifluoroethanol is added to optimize SP-B-polystyrol binding, and is then removed, again by evaporation. Subsequent washing procedures (diisopropyl-ether/butanol; Tween 20 in phosphate buffered saline [PBS]) selectively remove phospholipids. Solid phase-bound SP-B is detected by a monoclonal mouse antibody against porcine SP-B, cross-reacting with the apoprotein of human origin. For amplification, a biotinylated anti-mouse antibody and the avidin/biotin-peroxidase technique are used. Steep calibration curves with an excellent reproducibility are obtained for SP-B dimer (range: 0.3125 to 40 ng/well), either introduced directly into the immunosorbent-plate wells or previously admixed with synthetic phospholipid mixtures. SP-B monomer is detected with approximately 10% efficiency as compared with the dimer (wt/wt). Cross-reactivities with human SP-A and SP-C or albumin are negligible. Experiments with spiking of human bronchoalveolar lavage fluid (BAL) samples with different quantities of SP-B dimer revealed virtually complete apoprotein recovery.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:ELISA technique for quantification of surfactant protein B (SP-B) in bronchoalveolar lavage fluid. 758 90

Stool samples were examined by capture ELISA for the presence of T. saginata antigens. The specimens were from 303 patients infected with T. saginata, 43 samples from individuals suffering from bacterial, fungal, protozoan and nematode infections and 36 ones deriving from uninfected persons. The samples were diluted 1:10 (w:v) in PBS + 0.35% Tween 20, centrifuged twice at 5000 g for 20 min at room temperature and resulting supernatant at 12,000 g for 30 min at 0 degree C. The capturing and detecting polyclonal antibodies were prepared in rabbits immunizing them with T. saginata surface antigens. The stool samples freshly obtained, stored at 4 degrees C not longer than 72 h, or kept at at -20 to -30 degrees C even for 24 months showed reliable results in ELISA. The samples kept for a long time at 4 degrees C, dried and covered with moult were negative in the test. All samples taken from patients infected with other than T. saginata parasites or uninfeced were negative.
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PMID:Detection of Taenia saginata antigens in faeces by ELISA. 868 59

The conditions which optimize detection of T. saginata antigens in faeces were subject of examination. The most appropriate appeared diluent of PBS + 0.3% Tween 20. The highest effectiveness was achieved using capturing and detecting antibodies to surface antigens in comparison with somatic and metabolic ones. Two step centrifugation and final dilution of samples 1:10 (w:v) were recommended. The stability of freezed faecal supernatants after the first centrifugation was limited to 2 months. The sensitivity of capture ELISA established in our examinations allow to signalize 1 ng/ml antigen in high absorbance values. The antigens recognized by the test had MW bigger than 100,000.
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PMID:Factors conditioning detection of Taenia saginata antigens in faeces. 868 66

The application of peptide nucleic acid (PNA) probes for detection of Epstein-Barr Virus (EBV) snRNA in fixed cells is described. Fluorescein labelled PNA probes were used to detect EBER1 and EBER2 snRNA in Raji, Daudi and HS-Sultan cells. The fixation and permeabilization of cells were optimized. The optimal fixation was found to be 5% acetic acid plus 4% paraformaldehyde in PBS and the optimal permeabilization 0.5% Tween 20 in PBS whereas no proteolytic digestion was needed. The hybridization time needed with the PNA probes was only 1 h. When running mixed samples of Ramos (EBV neg.) Raji, Daudi and HS-Sultan (EBV pos.) cells in flow cytometry a strong fluorescence signal was seen in Raji, Daudi and HS-Sultan cells whereas no fluorescence signal was seen in the Ramos cells. In total 0.5% EBER positive Raji cells could easily be identified in a mixture of Raji and Ramos cells. The results were verified by fluorescence microscopy. It is concluded that PNA probes can be used for in situ hybridization in solution and the analysis can be done using flow cytometry or fluorescence microscopy. PNA probes therefore may facilitate and enhance the potential use of the in situ hybridization/flow cytometry combination.
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PMID:Flow cytometric detection of EBV (EBER snRNA) using peptide nucleic acid probes. 976 87

Ochratoxin A (OA) contamination of black pepper, coriander seeds, powdered ginger and turmeric powder was estimated using indirect competitive ELISA. Samples (1 g) were extracted with 0.5% potassium chloride (KCl) in 70% methanol (5 ml) and diluted subsequently to give two-fold to ten-fold step-wise dilutions in phosphate-buffered saline containing 0.05% Tween 20 and 0.2% bovine serum albumin (PBS-T BSA). For extracts from the spices analysed, ELISA estimates of OA concentrations were compared with those made by HPLC. All estimates were within 1-2 standard deviation of the ELISA values. More than 90% of OA added to spice samples was recovered from samples containing between 5 and 100 microg/kg OA. Extracts of OA-free spice samples contained substances that interfered with ELISA, presumably because of non-specific reactions. This effect was avoided by preparing all the test solutions in extracts of OA-free spice samples. In 126 samples obtained from retail shops, OA was found to exceed 10 microg/kg in 14 (in the range of 15-69 microg/kg) of 26 black pepper samples, 20 (in the range of 10-51 microg/kg) of 50 coriander samples, two (23 microg/kg and 80 microg/kg) of 25 ginger samples and nine (in the range of 11-102 microg/kg) of 25 turmeric samples. This is the first record in India of the occurrence of OA in what are some of the most widely used spices in Indian cooking.
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PMID:Occurrence of ochratoxin A in black pepper, coriander, ginger and turmeric in India. 1155 50

Fascioliasis in cattle is one of the most common and very serious trematode diseases in Korea. In the present study, the enzyme linked immunosorbent assay (ELISA) was applied in the diagnosis of fascioliasis using antigen of Fasciola hepatica, peroxidase of conjugate anti-cattle IgG and orthophenylenediamine as a substrate by micro-method technique of Voller et al. (1976b) and MacLaren (1978) with a slight modification. Results obtained from the present study are as follows: In assay for optimal dilution of stock antigen, the antigen (protein contents; 0.8 mg/ml) was diluted from 1/50 to 1/600 with carbonate buffer (pH 9.6), and then absorbance values were measured with 1/100 diluted sera. The regression equations between the OD values of ELISA and dilution of antigen were log Y=-0.181-0.00127X in infected sera, and log Y= -0.578- 0.000879X in normal sera. The significantly higher (p<0.05) OD value was observed in the former. In assay for optimal dilution of sera, the sera were diluted from 1/25 to 1/400 with in PBS/ Tween 20(pH 7.4), and absorbance values were measured with 1/200 diluted antigen. The regression equation between the OD values of ELISA and dilution of sera were log Y=-0.1540-0.0007238X in infected sera and log Y=-0.4834-0.00116X in normal sera. The former was higher than the latter (p<0.05). In the 27 cases of negative intradermal test, OD values of the ELISA are 0.447 +/- 0.144, the 95 percent confidence interval (Mean + 2 x SD) of the values was 0.735, and there was no case over the values. Therefore, the sensitivity of the antigen to diagnose fascioliasis was 100 percent in the negative case. The OD value 0.7 which is designed as a criterion (detection level of positive one) is useful for the performance of the ELISA in fascioliasis. According to the OD value of criterion in the regression equations, the optimal dilutions of stock antigen and serum were 1/250 and 1/100, respectively. In the 58 cases of fascioliasis from which the adult could be found in the bile ducts, the OD value was 0.846 +/- 0.224). The 75 percent (44 cattle) among them had higher value with compared to the criterion, and the 60 percent (20 cattle) of the cases of proliferative cholangitis of 33 cattle which had been infected previousely with Fasciola sp. is higher than the criterion. Prevalence of fascioliasis was 43.4 percent in the application of the ELISA to 272 cattle which were reared in Jeonbug district.
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PMID:[Application of micro-ELISA in serodiagnosis of fascioliasis in cattle] 1288 91

Aptamer-based microarrays for the quantitation of multiple protein analytes have been developed. A multiplex aptamer microarray was generated by printing two RNA aptamers (anti-lysozyme and anti-ricin) and two DNA aptamers (anti-IgE and anti-thrombin) on to either streptavidin (SA) or neutravidin (NA)-coated glass slides. However, substantial optimization was required in order to ensure the simultaneous function of the aptamer:analyte pairs. The effects of protein labeling, assay buffer, surface coating, and immobilization chemistry and orientation were investigated. A single buffer (PBS buffer containing 5 mM MgCl2 and 0.1% Tween 20) was found to work well with all the aptamers, even though this was not the buffer originally used in their selection, while neutravidin-coated slides yielded a lower detection limit, wider detection range, and more uniform background than streptavidin-coated slides. Incubation with Cy3-labeled proteins yielded sensitive, target-specific, and dose-dependent responses to each protein. Target protein concentrations as low as 72 pg/mL (5 pM, lysozyme), 15 ng/mL (0.5 nM, ricin), 1.9 ng/mL (0.01 nM, IgE), and 170 ng/mL (5 nM, thrombin) could be detected. These results show that aptamer arrays can potentially be used with numerous proteins in parallel, furthering the notion that aptamer arrays may be useful in proteomics.
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PMID:Optimization of aptamer microarray technology for multiple protein targets. 1772 65

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification and quantification of growing importance. However, saturation labeling of thiols with fluorescent dyes results in poor protein recuperation and therefore requires the use of large quantities of starting material. This is especially important in sequential dye-labeling steps when applied for an identification of cysteine modifications. First, we studied the effects of different detergents during labeling procedure, i.e. Tween 20, Triton X-100 and CHAPS, on protein yield and composition. Tween 20 and Triton X-100 resulted in yields of around 50% labeled proteins compared to only 10% with PBS alone and a most diversified 2-DE protein pattern. Secondly, Tween 20 was used for serial protein labeling with maleimid fluorophores, first to conjugate to accessible thiols and after a reduction to label with another fluorophore previously masked di-sulphide and/or oxidized proteins in frontal cortex autopsy tissue of a subject with mild Alzheimer's disease. Two-DE DIGE revealed a complex protein pattern of readily labeled thiols and di-sulphide and/or oxidized proteins. Seventeen proteins were identified by MALDI-TOF and by peptide fingerprints. Several proteins were oxidized and involved in Alzheimer's disease. However methionine oxidation was prevalent. Infrared DIGE may provide an additional tool for an identification of oxidation susceptible proteins.
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PMID:Serial protein labeling with infrared maleimide dyes to identify cysteine modifications. 1855 56


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