No abstract available.
Fluoroimmunoassay* ; Immunoenzyme Techniques*

This study was firstly conducted to detect antinuclear antibody(ANA) titer by using number influorescence density analysis assay instead of serum diluted assay. The best camera explore time was selected. Then 4,140 ANA positive sera were detected to determine the relationship between number influorescence density (detected by number camera system Spot 32 and computer analysis software ipwin32) and serum diluted titer. The consistent rates in different ANA patterns used by the two methods were compared. 4 seconds was found to be the best explore time and the relationship between number influorescence density and serum diluted titer was 29-50 vs 1:100, 51-85 vs 1:320, 86-175 vs 1:1000, 176-215 vs 1:3200, 216-237 vs 1:10,000. According to this standard we detected 3140 ANA positive sera by use of the two methods and observed a total consistency rate of 89.4%. The consistency rates of three ANA patterns including speckled, homogenous, mixture of speckled and homogenous were as high as 98.9%, 99.5%, 99.8% respectively. The lower consistency rate patterns included nucleolar (5.3%), centromere (1.8%), ribosome(12.6%) and other special patterns(0%). For practical purpose, number influorescence density analysis assay can be used in detecting the three main ANA patterns (speckled, homogeneous, mixture of speckled and homogenous) titer instead of serum diluted assay. The number influorescence density analysis assay is more objective, economical and simple than the serum diluted assay.
Antibodies, Antinuclear ; analysis ; Fluorescence ; Fluoroimmunoassay ; methods ; Software

BACKGROUND: Eosinophils play an important role in asthmatic airway inflammation collaborateing with other inflammatory cells. Nitric oxide (NO) may amplify and perpetuate allergic inflammation. OBJECTIVE: The present study was aimed to determine whether NO metabolites and eosinophil activation markers in sputum reflect the severity of asthma. METHODS: Sputum was obtained in 27 asthmatic patients. We processed freshly expectorated sputum separated from saliva and by a treatment with equal volume of dithiothreitol 0.1%, cytospins for cell count and special stain, we collected the supernatant for biochemical assay. We used immunocytochemical staining to detect EG2+ eosinophils, and fluoroimmunoassay to detect eosinophil cationic protein (ECP). NO metabolites were assayed by using modified Griess reaction. RESULTS: Moderate and severe asthmatic patients had a significantly higher proportion of eosinophils (37.9+/-7.8% vs 58.4+/-7.9% vs 8.9+/-2.0%, p<0.01) and lower proportion of macrophages (46.5+/-5.5% vs 18.1+/-4.6% vs 75.6+/-4.0%, p<0.01) compared to mild asthmatics. The proportion of EG2+ eosinophils, and ECP levels in the sputum were significantly higher in moderate and severe asthmatic patients than in mild asthmatic patients (p<0.01, respectively). The level of NO metabolites in the sputum was significantly increased in severe asthmatic patients compared to that in mild asthmatic patients (1372.0+/-168.5micromole/L vs 658.3+/-186.4micromole/L, p<0.05). The proportion of eosinophils in sputum was inversely correlated with FEV1 and FEV1/FVC. EG2+ eosinophils and ECP also had an inverse relationship with FEV1 and FEV1/FVC. CONCLUSION: These findings demonstrate that the proportion of eosinophils, ECP, EG2+ eosinophils, and NO metabolites in the sputum of patients with asthma may be correlated with asthma severity.
Asthma* ; Cell Count ; Dithiothreitol ; Eosinophil Cationic Protein* ; Eosinophils* ; Fluoroimmunoassay ; Humans ; Inflammation ; Macrophages ; Nitric Oxide* ; Saliva ; Sputum*

Biomarkers ; blood ; Enzyme-Linked Immunosorbent Assay ; Fluoroimmunoassay ; Hepatitis B ; blood ; Hepatitis B virus ; Humans ; Luminescent Measurements

OBJECTIVETo prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA).

METHODSThe CFP10 gene was amplified by PCR from Mycobacterium tuberculosis strain H37Rv and cloned into pET24b, pET24b-streptavidin (SA) or pET21a-SA expression vectors. The recombinant proteins CFP10, CFP10-SA and SA-CFP10 were expressed in Rosetta cells, purified via nickel affinity chromatography and refolded by dialysis. The sensitivity and stability of the resultant proteins as reference samples were evaluated by double-antibody sandwich TRFIA.

RESULTSCFP10-SA and SA-CFP10 fusion proteins were expressed as inclusion bodies, whereas CFP10 was expressed in a soluble form. The resultant purity of the 3 recombinant proteins all exceeded 95%. TRFIA results showed that CFP-SA fusion protein possessed the best sensitivity (0.02 µg/L) and stability.

CONCLUSIONThe reference samples of CFP10 for TRFIA detection have been successfully prepared and can be used in the development of a diagnostic kit for Mycobacterium tuberculosis.

Bacterial Proteins ; genetics ; standards ; Fluoroimmunoassay ; methods ; Gene Amplification ; Mycobacterium tuberculosis ; isolation & purification ; Reference Standards

The aim of this study was to apply a new type piezo-electric immunosensor in immunology classification for acute leukemia. Based on the plasma-polymerized film and the nanogold particles self-assembly technology, a new type piezo-electric immunosensor was firstly developed to absorb and fix the monoclonal antibodies for detecting leukemia cell antigens in patient blood samples. The results showed that the positive rate of detection with this method was quite close to that with the fluoroimmunoassay which are commonly used in clinic work. There was no significant difference between the positive rates (chi(2) = 3.4, P > 0.05). It is concluded that this piezo-electric immunosensor detection has many advantages, such as convenient operation, real time operation and control, qualitative detection, and less cost. This method can be used for acute leukemia immunology classification.
Acute Disease ; Antigens, Neoplasm ; analysis ; Biosensing Techniques ; methods ; Fluoroimmunoassay ; Humans ; Leukemia ; classification ; immunology

A convenient, highly sensitive and highly specific method using time-resolved fluoro immunoassay (TRFIA) for quantitative detection of hepatitis B virus is described. Using EU-DTTA as the tracer molecule, the assay showed a detection range of the standard curves of 0.2-300 ng/ml for HbsAg with a sensitivity of 0.05 ng/ml. The within-run coefficient of variations for standard samples were less than 10%, and the correlation coefficient between radioimmunoassay and TRFIA assay results reached 95% for the same sample, demonstrating the advantages of TRFIA for its wide detection range, high sensitivity and simple operation and its great potentials for clinical use.
Fluoroimmunoassay ; methods ; Hepatitis B ; blood ; Hepatitis B Surface Antigens ; blood ; Humans ; Sensitivity and Specificity

AIM AND METHODSBased on the reaction that 2',7'-dichlorodihydrofluorescein (DCFH) can be oxidized by reactive oxygen species (ROS) to yield the highly fluorescent 2',7'-dichlorofluorescin (DCF), ROS production in mitochondria can be observed dynamically as well as quantified directly by spectrofluorometer.

RESULTS AND CONCLUSIONDCF fluorescence showed linear increase in State 4 mitochondria, which suggest ROS produced at constant rate. The slopes of the linear increase in fluorescence with time were computed performing a linear regression that took into account all relevant data points in selected time windows. The slopes were proportional to ROS production in mitochondria. Addition of sodium azide and malonic acid increased and decreased the rate of ROS production respectively during measurement. DCF fluorescence varied linearly with increasing concentration of mitochondria, which showed quantitative relations in definite range. Repeated measures showed low coefficients of variation. This method is reliable and efficient for determining ROS in mitochondria.

Animals ; Fluoresceins ; Fluoroimmunoassay ; methods ; Male ; Mice ; Mice, Inbred Strains ; Mitochondria ; metabolism ; Reactive Oxygen Species ; analysis

OBJECTIVETo develop a time-resolved fluoroimmunoassay (TRFIA) for detection of pertussis toxin (PT) S1 subunit for quality control of human PT vaccine.

METHODSA double antibody sandwich one-step method was used to establish the TRFIA for detecting PT S1 subunit in the vaccine.

RESULTSThe sensitivity of c peptide analysis reached 2.5 ng/ml without cross-reactions with other antigens. This assay could be used in detecting S1 subunit in the vaccine.

CONCLUSIONThe TRFIA for detecting PT S1 subunit is simple, sensitive and rapid for quality control of the PT vaccine.

Cross Reactions ; Fluoroimmunoassay ; methods ; Pertussis Toxin ; analysis ; Pertussis Vaccine ; chemistry ; standards ; Quality Control ; Sensitivity and Specificity

BACKGROUND: Eosinophilic leukocytes are prominent cellular participants in the pathogenesis of allergic disease and asthma. Chemotaxis is still a very useful method in evaluating the response of human eosinophil to novel modulators. Degranulated mast cells and activated T lymphocytes are responsible for the pathophysiology of asthma and tryptase is one of most important proteases released after activation of mast cells. The purpose of this study was to investigate the actions of trypsin and chymotrypsin on eosinophils in terms of chemotaxis and activation. METHOD: Eosinophils were isolated by negative immunoselection from the peripheral blood of atopic donors. Chemotaxis was studied by using micro-Boyden chambers and ECP release was assayed by fluoroimmunoassay. RESULTS: Eosinophil showed a chemotactic response to trypsin. Maximal chemotactic response was with 1000microg/ml trypsin (56.52 +/- 14.50/HPF) which was comparable to PAF. But chymotrypsin showed no significant chemotactic response to eosinophils. Trypsin at the concentration of 10, 100,1000microg/ml induced secretion of ECP, which at the concentration of 10microg/ml represented about 2.7 times of the spontaneous rate of release. Soybean protease inhibitor reduced trypsin induced ECP release. CONCLUSION: Trypsin can induce chemotactic response to eosinophils and activation of eosinophils that can induce secretion of ECP. On the contrary, chymotrypsin showed no direct effect on eosinophils. We propose a role of trypsin on the chemotaxis and activation of eosinophils.
Asthma ; Chemotaxis* ; Chymotrypsin ; Eosinophils* ; Fluoroimmunoassay ; Humans ; Leukocytes ; Mast Cells ; Peptide Hydrolases ; Protease Inhibitors ; Soybeans ; T-Lymphocytes ; Tissue Donors ; Trypsin* ; Tryptases