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

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 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

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

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

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

OBJECTIVETo screen and diagnose Down's syndrome during mid-term pregnancy to reduce the number of babies with Down's syndrome.

METHODSWith the multi-level of stratified cluster sampling, twenty thousand and eight hundred and three women at 15-20 weeks gestation were screened by maternal serum AFP and beta-hCG using the time resolved fluoroimmunoassay (TRFIA). Then the screened high-risk women were diagnosed by amniocentesis, cell culture and chromosome analyses. The born children were diagnosed by follow-up and peripheral blood chromosome analyses.

RESULTSSix fetuses were diagnosed by serum screening and amniotic fluid chromosome analyses, and 3 born children were diagnosed by follow-up and peripheral blood chromosome analyses. Nine cases of Down's syndrome were detected in total, with the positive prenatal screen rate being 67% (6/9).

CONCLUSIONThe prenatal screening and diagnosis can reduce the birth of Down's syndrome patients and improve the population quality. However, the diagnosis accuracy still needs to be improved to further reduce the false negative rate and prevent misdiagnosis.

Adult ; Chorionic Gonadotropin, beta Subunit, Human ; blood ; Chromosome Aberrations ; Down Syndrome ; blood ; diagnosis ; genetics ; metabolism ; Female ; Fluoroimmunoassay ; Humans ; Pregnancy ; Prenatal Diagnosis ; methods ; Young Adult ; alpha-Fetoproteins ; metabolism

OBJECTIVETo prepare a rapid and sensitive diagnostic kit for detection of CA50 based on time-resolved fluoroimmunoassay.

METHODSA sandwich-TRFIA diagmostic kit was developed using anti-CA50 monoclonal antibody and all parameters of the kit were evaluated.

RESULTSThe linear measurement range of the kit was (5 - 300) U/ml. The sensitivity was 0.2 U/ml. The intra- and inter-assay coefficients of variation were 4.3% - 8.2% and 7.7% - 11.2%, respectively. There was no cross-reaction with CEA, CA12-5, CA15-3 and AFP. The cross reactivity with CA19-9 was 0.7 U/ml. The correlation coefficient of detection results of 107 blood samples between this newly developed kit and commercially available CA50 RIA kit was 0.901.

CONCLUSIONThis newly developed CA50-TRFIA kit is a valuable test tool for clinical application with even better sensitivity, specificity and accuracy.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, Tumor-Associated, Carbohydrate ; blood ; Female ; Fluoroimmunoassay ; instrumentation ; methods ; Humans ; Male ; Middle Aged ; Radioimmunoassay ; Reagent Kits, Diagnostic ; standards ; Reference Standards ; Reproducibility of Results

Digoxin plays a part in healing of congestive heart failure in clinic. Its therapeutic dose is very approximate to toxic dose and even they overlap each other sometimes. There are many influencing factors on blood drug level of digoxin. Pharmacodynamics and pharmacokinetics varies with different individuality. It is indispensable to detecting blood drug level in order to treat disease and prevent intoxication. Integrating with the detecting-methods of blood drug level of digoxin home and broad, characteristic of many methods are summarized from sensitivity, linearity range, cross-reaction and precision. These methods include radio immunoassay, enzyme immunoassay, chemiluminescence immunoassay, fluorescence immunoassay and HPLC-MS-MS. These methods are popular for their specialized ascendancy. The cost of radio immunoassay is low. Enzyme immunoassay has good specificity. Sensitivity and stability of chemiluminescence immunoassay is very excellent. Fluorescence polarization immunoassay is sensitive and convenient. HPLC-MS-MS has high resolution and good specificity. One of the development tendencies is to combine two or more methods in detecting the blood drug level of digoxin which contribute to these methods integrated use.
Chemistry Techniques, Analytical ; methods ; Chromatography, High Pressure Liquid ; Digoxin ; blood ; Enzyme-Linked Immunosorbent Assay ; Fluorescence Polarization Immunoassay ; Fluoroimmunoassay ; Humans ; Radioimmunoassay ; Reproducibility of Results ; Tandem Mass Spectrometry