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Time resolved fluoroimmunoassay (TRFIA) is an immunoassay technology developed on the basis of the unique fluorescence properties of rare earth elements. TRFIA combines the advantages of radioimmunoassay, enzyme-linked immunoassay and common fluorescence immunoassay. It has high sensitivity, strong specificity, good stability, wider measurement range, long fluorescence life, simple operation and non-radiation, and shows a good prospect in the field of immunoassay. In this paper, several common TRFIA materials are discussed based on the latest research progress of time-resolved fluorescence in immunoassay. The application of TRFIA in immunodiagnosis, food detection, environmental monitoring and so on is elaborated, and its development direction and application are prospected.
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HSP90 is widely expressed in cells with the main function in assisting the maturation of other proteins that are called clients. Many clients play critical roles in the occurrence and development of cancer. Inhibition of HSP90 can lead to degradation of the oncogenic proteins, and result in potent anti-cancer effects. HSP90-HOP interaction is critical for the chaperone function of HSP90, thereby disruption of the HSP90-HOP interaction is a novel strategy in the inhibition of HSP90. Based on the technology of homogeneous time-resolved fluorescence (HTRF), we developed a new assay for the identification of new inhibitors of HSP90-HOP interaction. This method was evaluated in the study of the HSP90-HOP inhibition activity of the pentapeptide MEEVD from HSP90 C-terminal and its derivatives. This study can provide a basis for the screening and discovery of novel HSP90-HOP disruptors.
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Objective To explore significance of alpha‐fetoprotein isoform L2(AFP‐L2) in the screening of Down syndrome in pregnant women ,so as to provide references for clinical application .Methods A total of 250 healthy pregnant women and 22 preg‐nant women with Down syndrome were enrolled in this study .Serum specimens were collected and AFP‐12 was separated and cap‐tured by using the magnetic bal ,time‐resolved fluorescence immunoassay was used to detect levels of AFP and AFP‐L2 ,and the percentage of AFP‐L2 (AFP‐L2% ) was calculated .Results The serum level of AFP of pregnant women with Down syndrome [(20.2±4.2)ng/mL]was lower than that of healthy pregnant women[(46.7±19.9)ng/mL],and had statistically significant difference(P<0 .05) .Serum AFP‐L2% of pregnant women with Down syndrome was higher than that of healthy pregnant women , and had statistically significant difference(P<0 .05) .Conclusion Detection of AFP level and AFP‐L2% could be an indicator for Dow n syndrome screening .
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Objective To compare the value of time-resolved fluorescence immunoassay (TRFIA) and ELISA for the detection of HBV serological markers .Methods To detect the HBV serological markers in 359 samples with low concentrations of HBsAg by TRFIA and ELISA respectively ,and to compare the results of the two methods .Results The levels of HBV serological markers detected by TRFIA were higher than those detected by ELISA .The positive rates of HBsAg ,HBeAg and HBcAb detected by TR-FIA and ELISA were significantly different(P0 .05) .Conclusion Com-pared with ELISA ,TRFIA has higher sensitivity in the detection of low HBsAg concentration samples ,and it is valuable to be ap-plicated in clinical .
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Objective To make a preliminary study on the results from qualitative and quantitative detection of low concentra-tion hepatitis B surface antigen(HBsAg).Methods 85 HBsAg low concentration serum samples (ELISA method test re-sults were 0.60.05)and 0.60.05).The positive detection rate showed statistically difference between ELISA (70.6%)and CMIA,TRFIA in 0.6TRFIA>ELISA.②The contents of HBsAg showed statistically difference among 0.6TRFIA>ELISA.③There was a positive correlation between the three methods of HBsAg content (t=2.939,2.928,60.915,P<0.05).The correlation between CMIA method and TRFIA method was the best (r=0.989).Con-clusion CMIA was the first choice for testing low concentration HBsAg,TRFIA was the second.For the specimens of the low concentration HBsAg detected by ELISA should be suggested to clinical and retested by CMIA or TRFIA in order to a-void missing detection.And it was not recommended to clinical that different methods of quantitative or half-quantitative re-sults were transverse compared in order to avoid misdiagnosis.
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To establish a method to detect cardiac troponin I by using time-resolved fluoroimmunoassay ( TRFIA) and apply to the clinic.Methods:The assay were measured by TRIFA and double antibody sandwich method .Standard protocols were evaluated with the standard curve , the limit of detection , stability, precision and cross reaction .Healthy reference populations and clinical serum specimens were measured to established the reference interval and evaluated the perspective of the clinical application . Results:The standard curve was Y=7485 .878+1400.924 X with a correlation coefficient of 0.999.The limit of detection was 0.052 ng/ml.The intra-and inter-assay coefficients of variation ( CV) were all 0.05 ).There was significant difference in statistics compared before and after treatment with AMI ( P<0.001 ) .Conclusion: The TRFIA method for detecting cTnI achieves clinical application standards and may be used for the diagnosis and serosurveillance of acute myocardial infarction patients.
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Objective To investigate the similarities and differences of 3 methods for detecting hepatitis B virus S 1(PreS1) ,and select the appropriate method for detecting clinical samples .Methods The PreS1 of the serum samples from chronic hepatitis B pa-tients were tested with enzyme immunoassay analyzer ,time-resolved method and the manual method .To compare the repetition rate ,select PreS1 antigen strongly positive serum and weak positive serum were detected 15 times by three methods ;To compare the explicit rate ,the reaction temperature was raised or lowered by 3 ℃ .Results The positive rate of three methods was 93 .53% , 92 .81% ,92 .81% .Automated ELISA reproducibility CV strong positive CV3 .62% ,weakly positive CV was 13 .42% ,CV of time-resolved method for the 2 kinds of samples were 5 .10% ,7 .92% ,manual methods CV 11 .10% 29 .88% ;changing the reaction incu-bation temperature 3 ℃ ,automatic detection ELISA all specimens S/CO value decreased ,increasing the chance of a false negative . The manual methods and time-resolved detection method for all specimens S/CO values increased ,increasing the chance of a false positive .Conclusion The detection rate and repeatability of automated ELISA were better .The time-resolved method followed and the manual methods were poor .
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Protein tyrosine kinases (PTKs) are attractive targets in searching for therapeutic agents against many diseases. In this study, a series of dehydroabietylamine derivatives were first determined to show PTK inhibitory activity using a high-throughput screening (HTS) method based on homogeneous time-resolved fluorescence (HTRF) technology. The structure-activity relationships of the dehydroabietylamine derivatives were established, and it was found that the compounds with a nitrogen-containing side chain had better inhibitory activity. Further studies showed that the compounds substituted with halogen in the phenyl ring resulted in higher inhibitory activity on the epidermal growth factor receptor (EGFR), and can be a guide to modify the structure of dehydroabietylamine derivatives. Dehydroabietylamine derivatives might be a new class of multi-targeted and effective PTK inhibitors with structure modifications.
Subject(s)
Humans , Drug Evaluation, Preclinical , Fluorescence , High-Throughput Screening Assays , Kinetics , Molecular Structure , Protein Kinase Inhibitors , Chemistry , Protein-Tyrosine KinasesABSTRACT
Objective: Using homogeneous time-resolved fluorescence (HTRF) technology to screen the Calla Chinensis extracts with anti-epidermal growth factor receptor activity and to analyze the active ingredients in Calla Chinensis by ultra-performance liquid chromatography/quadrupole-time of flight mass-spectrometry (UPLC/Q-TOF-MS). Methods: After percolation with petroleum ether, ethanol extract, ethyl acetate extraction, and boiling with water, four fractions were obtained. HTRF method was applied to detecting the inhibition of ethyl acetate fraction on EGFR, and the inhibitory rate was calculated. The chromatographic separation was performed on Acquity UPLC BEH C18 column with a gradient elufion of 0.1% formic acid water-acetonitrile. The mass spectrometer equipped with electrospay ionization source was used as defector, data were collected under the positive ion modes, and screened at 200-400 nm. Results: The ethyl acetate fraction of Calla Chinensis showed the strong inhibitory activity on EGFR. Fourteen compounds were analyzed and identified, among which tannins were the main active components, the 11 tannin compounds were punicalin, vanillin, di-HHDP-glucose, ellagitannin, myricetin 3-O-rhamnoside, protocatechuic acid, gallotannin-glucose, ellagic acid-hexose, 3, 5-dicaffeoylquinic acid, monogalloyl-glucose, and gallotannin, and the three others were unknown. Conclusion: The ethyl acetate fraction of Calla Chinensis has the significant EGFR inhibitory activity, and the IC50 value is 5.528 μg/mL. Tannins, including Chinese tannin, gallogen, and ellagitannin as main constituents, are identified through the information of positive ion and relative molecular mass determined by Q-TOF-MS. The results indicate that Calla Chinensis contains tannin ingredients to inhibit EGFR, in the hope to provide a theoretical basis of the application in anticancer and to lay the foundation for the further tracking separation of the active ingredients.
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OBJECTIVE: To establish a KDR high throughput screening model with homogeneous time resolved fluorescence(HTRF) detection technology in order to screen small molecular KDR inhibitors from the compound library. METHODS: A 20 μL assay system in 384-well low-volume white microplate was developed with HTRF based fluorescence detection system to determine the enzyme activity. The optimization steps consisted of the following experiments: enzyme concentration and incubation time optimiztion, ATP and substrate Km determination, quality control experiments including signal noise ratio inspection, the inhibitor SU5416 IC50 validation, and Z' value calculation. After a stable assay system was accomplished, a HTS campaign was started with 10560 samples irom the compound library and the IC50 of some compounds were determined. RESULTS: The optimized KDR activity assay conditions were as follows: the kinase concentration was 0.10 ng · μL-1; ATP Km was 0.75μmol · L-1; substrate Km was 94.90 nmol · L-1; biotin/ SA ratio was 2:1; Z' value was 0.85 and the IC50 of SU5416 inhibitor was 1.03 μmol · L-1. Three compounds with the ID of S2-14, S2-16, and S2-38 showed IC50 on KDR of 1.01 × 10-4, 6.04 × 10-5, 7.23 × 10-6 mol · L-1, respectively. CONCLUSION: A KDR high throughput screening model has been successfully established with HTRF methodology and a focused HTS campaign has been accomplished with several leads. This assay system is reliable and the results are stable. The established assay system is suitable for further application in screening natural anti-angiogenesis products.
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Objective: To establish the high-throughput screening system for β-secretase (β-site amyloid precursor protein cleaving enzyme 1, BACE1) inhibitors in vitro. Methods: Using the homogeneous time-resolved fluorescence (HTRF) technique to establish a high-throughput screening system for BACE1 inhibitors according to the extent that test compounds inhibited BACE1 activity by optimizing the incubation time, enzyme concentration and the detector. Results: A high-throughput screening system for BACE1 inhibitors based on HTRF methods was established. The incubation time was 6 h, the BACE1 concentration is 670 U/L. According to the detector of VICTOR3, the signal-to-noise (S/N) ratio reached 649.6, while the signal-to-background (S/B) ratio and Z′ factor were 44.6 and 0.91, respectively. The coefficient of variation (CV) was much lower than 10%. Using this method, the inhibitors with IC50<106mol/L could be screened out. Conclusion: The high-throughput screening system based on HTRF Could assure a high sensitivity, specificity and stability, which could be used to high-through put screening for the BACE1 inhibitors.