OBJECTIVEThis study aimed to establish chemiluminescent immunoassay (CLIA) for quantitative determination of theophylline levels in human serum.

METHODSTo measure the concentration of theophylline (n=122) and evaluate the assay.

RESULTSThe linear range of the CLIA method was 0.51-40 mg/L (Y=1.02X+0.44, r=0.995). The intra and inter CV (coefficient variance) of CLIA were 3.20% and 3.57%, respectively. The average recovery rate was 102.3%. This method was free from interference by brilirubin (<200 micromol/L), hemoglobin (<10 g/L), and triglycerides (<15 mmol/L).

CONCLUSIONThis method is simple, convenient and precise for clinical pharmacokinetics study of theophylline.

Blood Chemical Analysis ; methods ; Female ; Fluorescence Polarization Immunoassay ; methods ; Humans ; Luminescent Measurements ; methods ; Lung Diseases ; blood ; Male ; Middle Aged ; Reproducibility of Results ; Sensitivity and Specificity ; Theophylline ; blood

OBJECTIVEAn optimal automatic selection method of permissible source region is proposed to reduce the ill-conditioned and ill-posed problems in the reconstruction of the light source in bioluminescence tomography.

METHODSThe 2D images captured by CCD are mapped into surface light irradiance distribution based on the light propagating model. The relation matrix between the source and light distribution is obtained by finite element method. Permissive source region is determined by using the automatic selection method proposed in this paper, and then Tikhonov regularization is applied to reconstruct the light source.

RESULTSThe center point distance between the optimal permissible source region and true source is 1.26 mm, and the center point error of the reconstructed light source and true source is 0.47 mm, the volume error is 9.13 mm3.

CONCLUSIONThe optimal permissive source region selection strategy is effective to locate the permissive source region close to the true source, and reduces the reconstructed error due to subjective orientation of permissible source region. This proposed method is the basis of high precision source reconstruction in bioluminescence tomography.

Algorithms ; Light ; Luminescent Measurements ; Tomography

In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an "accessory protein" whereby a stored substrate is efficiently delivered to the bioluminescent enzyme luciferase. Other types of complexation mediate energy transfer to an "antenna protein" altering the color and quantum yield of a bioluminescence reaction. Spatial structures of the complexes reveal an important role of electrostatic forces in governing the corresponding weak interactions and define the nature of the interaction surfaces. The most reliable structural model is available for the protein-protein complex of the Ca(2+)-regulated photoprotein clytin and green-fluorescent protein (GFP) from the jellyfish Clytia gregaria, solved by means of Xray crystallography, NMR mapping and molecular docking. This provides an example of the potential strategies in studying the transient complexes involved in bioluminescence. It is emphasized that structural studies such as these can provide valuable insight into the detailed mechanism of bioluminescence.
Animals ; Anthozoa ; physiology ; Aquatic Organisms ; physiology ; Bacteria ; metabolism ; Binding Sites ; Calcium ; metabolism ; Crystallography, X-Ray ; Fluorescence Resonance Energy Transfer ; Green Fluorescent Proteins ; metabolism ; Hydrozoa ; physiology ; Imidazoles ; metabolism ; Luciferases ; metabolism ; Luminescent Measurements ; instrumentation ; methods ; Luminescent Proteins ; metabolism ; Models, Molecular ; Protein Binding ; Pteridines ; metabolism ; Pyrazines ; metabolism ; Scyphozoa ; physiology ; Spectrometry, Fluorescence

Animals ; Fluorescence ; Fluorescence Recovery After Photobleaching ; Fluorescence Resonance Energy Transfer ; Fluorometry ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry

OBJECTIVETo develop a new nonisotopic detection method of enzyme-amplified time-resolved fluorescence (EATRF) or enzyme-amplified lanthanide luminescence (EALL) for nucleic acid hybridization assays, which can be applied extensively in clinical diagnosis.

METHODSThe method combines the high affinity of biotin-streptavidin system, amplification of enzyme, and inherent advantage of lanthanide chelate with the background elimination of time-resolved fluorescence detection. The conversion of 5-fluorosalicyl phosphate to 5-fluorosalicylic acid (5-FSA) by alkaline phosphatase. The salicylic acid product forms a luminescent ternary chelate with Tb3+ and EDTA.

RESULTSThe dynamic range of the standard curve of EATRFA for nucleic acid hybridization assay was very wide, the range was more than third order of magnitude. The detection sensitivity was about 10 pg of target sequence. When the known target sequence was 20, 10 and 2 ng, the ratio of measured amount to known amount was 110%, 90% and 115% respectively. The main experimental conditions, for example, the irradiating time of ultraviolet rays, the concentrations of biotinylated probe, AP-SA, 5-FSAP and Tb-EDTA and the methods of washing in the related steps, have been optimized. A new stable technology of fluorescence has been developted.

CONCLUSIONSEATRF detection for nucleic acid hybridization assays is a new sensitive simple method, which has a great prospect.

Alkaline Phosphatase ; analysis ; Blotting, Southern ; DNA ; genetics ; Fluoroimmunoassay ; methods ; Luminescent Measurements ; Metals, Rare Earth ; Nucleic Acid Hybridization ; methods ; Spectrometry, Fluorescence ; Substrate Specificity

AIMTo develop a simple, fast and inexpensive approach as well as an instrument for detection of gene mutation.

METHODSPyrosequencing based on bioluminometry assay was employed to detect gene mutation. Pyrosequencing is a method of sequencing by synthesis step-by-step using four enzymes, DNA-polymerase, ATP sulfurylase, luciferase and apyrase. The signal was produced by detecting pyrophosphate released during a dNTP incorporation. For mutation detection, a DNA fragment was amplified by PCR at first, followed by a single-stranded DNA preparation. In the second step, a short primer was annealed to the position just before the mutation point. Finally, specific dNTPs were added in terms of the template sequence. The mutation species can be readily determined by the sequence.

RESULTSA new instrument was developed for gene mutation detection by pyrosequencing. To iteratively inject small amount of each dNTP for the sequencing reaction, capillaries were used to connect dNTP reservoirs and the reaction chamber. Each dNTP was delivered by adding a gas pressure on the top of a corresponding dNTP reservoir, by which 0.2 microL of dNTP can be exactly added each time. It was theoretically proved that undesired liquid seep through the capillary did not affect the sequencing reactions in pyrosequencing. In addition, the three possible variants (wildtype, mutant and heterozygote) of a mutant point Cys275Ser in P53 gene exon 8 were determined by pyrosequencing using the instrument. A simple method was also described for rapidly distinguishing the type of a variant.

CONCLUSIONThe developed method is very simple, and the corresponding instrument is inexpensive and easy to operate, which can be used to detect many types of mutation.

Exons ; genetics ; Genes, p53 ; genetics ; Humans ; Luminescent Measurements ; Point Mutation

BACKGROUND: Multiple allergosorbent test chemiluminescent assay (MAST CLA) is a simple method for measuring total and allergen-specific IgE in human serum. Total IgE level, however, was much frequently high, even if no allergen-specific IgE could be detected in serum. The aim of this study was to evaluate the total IgE class of the MAST CLA system. METHODS: We studied 649 patients in whom MAST CLA (MAST Immunosystems, Inc., Mountain View, CA, USA) was tested and compared the results with those of total IgE, Phadiatop FEIA & RAST FEIA using Pharmacia CAP system (Pharmacia AB, Uppsala, Sweden). RESULTS: MAST CLA specific IgE was positive in 139 (21.4%) among 649 patients. Total IgE was increased over class 2 in 379 (74.3%) among 510 MAST CLA allergen-specific IgE -negative patients. Total IgE assayed by Pharmacia CAP system was increased over 100 kU/L in 33 (54.1%) among 61 MAST CLA allergen-specific IgE -negative patients. Especially, total IgE assayed by Pharmacia CAP system was increased over 100 kU/L in 25 (69.4%) of 36 patients when MAST CLA total IgE class is over 2. Phadiatop FEIA which is a screening test for inhalant allergy was positive in 11 (50.0%) of 22 MAST CLA allergen-specific IgE -negative patients, and especially, positive in 8 (66.7%) of 12 MAST CLA allergen-specific IgE -negative patients who had MAST CLA total IgE class over 2. Nine of 11 patients with positive Phadiatop FEIA were also found to be positive in d1 and/or d2 by RAST FEIA. CONCLUSIONS: It is concluded that another allergen screening tests should be used for detecting allergen-specific IgE, when MAST CLA total IgE is increased over class 2 with no detectable MAST CLA specific IgE because the sensitivity of MAST CLA allergen-specific IgE could be low.
Humans ; Hypersensitivity ; Immunoglobulin E* ; Luminescent Measurements ; Mass Screening

Although various functions of CD99 have been reported, such as apoptosis and homotypic aggregation of thymocyte and transendothelial migration of immune cells, biochemical/molecular natures of CD99 are still elusive. Using mouse CD99 gene, we show that CD99 forms homodimer through its extracellular domain. Expression of mouse CD99 is up-regulated on T cells after CD3-mediated activation, like the case for human CD99. The potential of CD99 to form homodimer was tested with a recently developed bimoleular fluorescence complementation analysis (BiFC). In BiFC analysis, the dimerization-induced fluorescence was strong near the perinuclear region and was faded at the cell membrane. However, surface expression of CD99 was still detected by flow cytometry, suggesting that CD99 either in monomer form or in association with other molecules exists on the cell surface. In BiFC analysis using CD99 mutants with its extracellular, transmembrane, or cytosolic domains changed to corresponding human CD4 domains, the mutant replaced with human CD4-extracellular domain did not produce fluorescence. Purified soluble CD99-Fc fusion proteins bound to CD99-Fc immobilized onto the gold sensor chip in surface plasmon resonance analysis, confirming that the extracellular domain was responsible for dimer formation. Intracytoplasmic staining for CD99 expression in the thymocytes and mature T cells showed that most of the cells, even the cells with low surface level of CD99, contained the molecule inside the cell. Our results suggest that majority of CD99 homodimers may exit in the cell and be exported to the cell surface, dissociating from each other, after a certain regulatory signal is delivered.
Animals ; Antigens, CD/chemistry/*isolation & purification ; Cell Adhesion Molecules/chemistry/*isolation & purification ; Flow Cytometry ; *Fluorescence ; Luminescent Measurements/*methods ; Mice ; Molecular Biology/*methods ; T-Lymphocytes/immunology

No abstract available.
Cyclosporine* ; Fluorescence Polarization Immunoassay* ; Fluorescence Polarization* ; Fluorescence*

BACKGROUND: The chemiluminescent assay(CLA) is a new in vitro non-radioactive modification of the radioallergosorbent test(RAST). The CLA permits quick, siultaneous detection of total and specific IgE in human serum up to 35 different allergens, as well as their semiquantitative concentrations into classes from 0(negative) to 4(very high). The CLA has shown sensitivity, specificity, and a good correlation with the RAST and also with skin pick testing. OBJECTIVE: The purpose of this study was to find the causatix llergens and to evaluate the clinical significance of CLA comparing the results of tbe CLA wi! h hose of the prick test in patients with chronic urticaria. METHODS: We performed the CLA with 35 allergens(MAST immunosunosystems, Inc., California, America) and the prick test with 51 allergens(Bencard Ltd., Bricantord, England) in 70 patients with chronic urticaria. The CLA detects the allergen-IgE reactic say the use of an enzyme-labeled anti-IgE and a coupled photoreagent mixture. lhe amount of the luminescent energy is measured by exposing a polarcid film and then semiquantitation is a sessed by using a densitometer. RESULTS: Except for those over the age of 60, all age-groups ealed a similar positive reaction to the CLA and prick test, but the positive reaction rate o the prick test(91.4%) was higher than that of the CLA(38.6%). The positive reaction rate oringle allergen and compound allergens was 10.0% and 28.6% in the CLA, but 10.0% and 81.4% in the prick test, respectively. In the CLA, the rate of positive reaction was 17(24.3%) in Dermatophagoides(D.) farinae, 15(21.4% ) in D. pteronyssinus, 5(7.1% ) in house dust, an 13(4.3%) in shrimp in that order. In the prick test, the rate of positive reaction was 39(55.7%) in house dust, 35(50.0%) in D. farinae, 29(41.4%) in D. pteronyssinus, and 21(30.0%) in haydrst(30.0%) in that order. The results of the CLA to 3 comrnon allergens showed a concordance rare of 67.6%, sensitivity of 34.3%, and specificity of 97.3% compared with those of the prick case. The levels of total IgE and eosinophils were elevated in 59(n=70, 843%) and in 5(n=56, 84.3%), respectively. CONCLUSION: The results of this study suggest that the CLA may be used as an alternative method for the diagnosis of chronic urticaria because it is and coiomic, safe, simple procedure with very high specificity and trelative low sensitivity, and is sirnilt. neously capable of testing to multiple allergens.
Allergens ; California ; Diagnosis ; Dust ; Eosinophils ; Humans ; Immunoglobulin E ; Luminescent Measurements* ; Sensitivity and Specificity ; Skin ; Urticaria*