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

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 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 establish an in vitro homogeneous time-resolved fluorescence immunoassay method for high throughput screening of protein tyrosine kinase (PTK) inhibitors.

METHODSSpecific fluorescence signals at 670 and 612 nm were measured by multifunctional microplate reader when the fluorescence was emitted through a resonance energy transfer between fluorescent materials (EuK and XL-665). The inhibitory activity of Sunitinib, a standard PTK inhibitor, on vascular endothelia growth factor receptor 2 (VEGFR-2) kinase activity was investigated.

RESULTSA homogeneous time-resolved fluorescence immunoassay was established for high throughput screening of PTK inhibitor. In this system, the concentrations of VEGFR-2, adenosine triphosphate (ATP) and poly-peptide substrate were 5 ng/microl, 100 micromol/L and 1 micromol/L, respectively. Sunitinib inhibited VEGFR-2 kinase activity with an IC50 value of 86.7 nmol/L, which was close to the values tested using other methods.

CONCLUSIONThe homogeneous time-resolved fluorescence immunoassay we established can be easily used for high throughput screening of PTK inhibitors.

Fluoroimmunoassay ; methods ; High-Throughput Screening Assays ; methods ; Indoles ; pharmacology ; Peptides ; metabolism ; Phosphorylation ; drug effects ; Protein Kinase Inhibitors ; pharmacology ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; metabolism ; Pyrroles ; pharmacology ; Time Factors ; Vascular Endothelial Growth Factor Receptor-2 ; antagonists & inhibitors ; metabolism

OBJECTIVETo prepare a time-resolved fluoroimmunoassay (TRFIA) kit for clinical detection of IgM antibodies to hepatitis B core antigen (HBc).

METHODSImmunocapture method was used to develop the TRFIA kit for detection of the anti-HBc IgM antibodies, and the precision, cross-reactivity and sensitivity of the kit were tested with the clinical serum samples.

RESULTSThe intra- and inter-assay coefficients of variation of the TRFIA kit were 4.8%-7.2% and 7.5%-8.6%, respectively, and no cross-reactivity with anti-HBs, anti-HBc-IgG or anti-HBe was found. Comparison of the results of the TRFIA kit and enzyme-linked immunosorbent assay (ELISA) demonstrated greater sensitivity of the kit than ELISA in detecting the anti-HBc IgM antibodies in 584 serum samples. According to the detection results in 300 serum samples from healthy donors, the cutoff value of the TRFIA kit was 4.5 times of the fluorescence value of the negative control.

CONCLUSIONThis TRFIA kit for detecting anti-HBc IgM antibodies meets the demand for clinical application and can replace the ELISA kits.

Fluoroimmunoassay ; methods ; Hepatitis B ; immunology ; virology ; Hepatitis B Antibodies ; blood ; Hepatitis B Core Antigens ; immunology ; Humans ; Immunoglobulin M ; blood ; Reagent Kits, Diagnostic ; Sensitivity and Specificity

OBJECTIVE: To explore the expression of tryptase and chymase in human lung tissue of anaphylactic shock and its value for forensic medicine. METHODS: With ten carbon monoxide poisoning cases as control group, the levels of tryptase and chymase were observed by immunofluorescence and analyzed using the Image Analyze and the Image-pro plus 5.0.2. The positive mast cells were counted and the levels of the tryptase and chymase were calculated respectively. RESULTS: There was a statistically significant difference (P < 0.05) for the tryptase and chymase concentrations in the lung tissue between the anaphylactic shock group and the control group. CONCLUSION The levels of the tryptase and the chymase expression are greatly increased in human lung tissue of anaphylactic shock, which may provide the morphological evidence and reference for the diagnosis of anaphylactic shock in forensic practice.
Adolescent ; Adult ; Anaphylaxis/pathology* ; Cadaver ; Carbon Monoxide Poisoning/pathology* ; Child ; Child, Preschool ; Chymases/metabolism* ; Female ; Fluoroimmunoassay/methods* ; Forensic Pathology ; Humans ; Infant ; Lung/pathology* ; Male ; Mast Cells/enzymology* ; Middle Aged ; Staining and Labeling ; Tryptases/metabolism* ; Young Adult

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 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

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