Establishment and clinical application of a highly sensitive TAT-2 time-resolved fluorescence immunoassay detection

Abstract Background: A rapid and highly sensitive assay for tumor-associated trypsinogen-2 (TAT-2) based on the time-resolved fluorescence immunoassay (TRFIA) detection technique was developed for the determination of serum TAT-2 levels in cancers. Results: The measurement range of TAT-2-TRFIA was 1.53-300 ng/mL. The within-run and between-run coefficients of variation of TAT-2-TRFIA were 4.38% and 7.82%, respectively. The recovery rate of TAT-2-TRFIA was 103.0%. The cross-reaction rates of trypsin and T-cell immunoglobulin mucin 3 were 0.02% and 0.82%, respectively. The TAT-2-positive rates in lung cancer, liver cancer, nasopharyngeal cancer, cholangiocarcinoma, brain cancer, and pancreatic cancer were 45.9%, 50.0%, 45.0%, 64.3%, 50.0%, and 41.7%, respectively, with the areas under ROC curves of 0.788, 0.734, 0.862, 0.720, 0.887, and 0.585, respectively. In patients with lung cancer, the positive rate of the single indicator CEA was 28.4%, which increased to 60.6% after combined use with TAT-2. In patients with cholangiocarcinoma, the positive rate of CA-199 was 35.7%, which increased to 71.4% after combined use with TAT-2. Conclusions: TAT-2 is expected to be used as an auxiliary diagnostic indicator for the combined use of tumor markers to improve the positive rate and accuracy of detection.

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Assessment of urinary total testosterone production by a highly sensitive time-resolved fluorescence immunoassay

Journal of Clinical Laboratory Analysis ◽

10.1002/jcla.20283 ◽

2008 ◽

Vol 22 (6) ◽

pp. 403-408 ◽

Cited By ~ 5

Author(s):

Shihua Bao ◽

Yifeng Peng ◽

Shile Sheng ◽

Qide Lin

Keyword(s):

Total Testosterone ◽

Fluorescence Immunoassay ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Testosterone Production ◽

Highly Sensitive

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Ultrasensitive Thyrotropin Immunoassay Based on Enzymatically Amplified Time-Resolved Fluorescence with a Terbium Chelate

Clinical Chemistry ◽

10.1093/clinchem/38.4.545 ◽

1992 ◽

Vol 38 (4) ◽

pp. 545-548 ◽

Cited By ~ 24

Author(s):

A Papanastasiou-Diamandi ◽

T K Christopoulos ◽

E P Diamandis

Keyword(s):

Alkaline Phosphatase ◽

Detection Limit ◽

Solid Phase ◽

Thyroid Stimulating Hormone ◽

Fluorescence Immunoassay ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Highly Sensitive ◽

Assay Time ◽

Highly Sensitive Detection

Abstract We describe an ultrasensitive, enzymatically amplified time-resolved fluorescence immunoassay of thyrotropin (thyroid-stimulating hormone) in serum with use of a terbium chelate as the detectable moiety. In this assay, thyrotropin is first simultaneously reacted with a solid-phase (microtiter well) monoclonal antibody and a soluble biotinylated monoclonal detection antibody. After washing, a streptavidin-alkaline phosphatase conjugate is added, followed by another washing. Alkaline phosphatase acts on the substrate 5-fluorosalicyl phosphate (FSAP) to produce 5-fluorosalicylic acid (FSA). FSA, but not FSAP, can then form with Tb3+ and EDTA a highly fluorescent ternary complex of long fluorescence lifetime. This complex is quantified with time-resolved fluorometry. The thyrotropin assay is highly sensitive (detection limit approximately 0.003 milli-int. unit/L when a total assay time of 85 min is used), precise, and accurate. The thyrotropin assay can also be completed in less than 30 min (detection limit 0.013 milli-int. unit/L), thus making this procedure a candidate technology for high-throughput automated analyzers.

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Multiple labeling and time-resolvable fluorophores

Clinical Chemistry ◽

10.1093/clinchem/37.9.1486 ◽

1991 ◽

Vol 37 (9) ◽

pp. 1486-1491 ◽

Cited By ~ 34

Author(s):

E P Diamandis

Keyword(s):

Dicarboxylic Acid ◽

Detection Limits ◽

Alpha Fetoprotein ◽

Macromolecular Complex ◽

Fluorescence Immunoassay ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Highly Sensitive ◽

Europium Chelate ◽

New Time

Abstract A new time-resolved fluorescence immunoassay system involving use of the europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid as label is reviewed. This stable chelate by itself is not very fluorescent but, used in multiple labeling strategies, improves the achievable detection limits. By using multiple labeling, streptavidin tailing, and Eu3+ activation, one can create a very stable, easy-to-use reagent that is suitable for devising highly sensitive immunoassays and other biotechnological assays. This reagent, a streptavidin-based macromolecular complex, is able to detect approximately 300,000 molecules (approximately 0.5 amol) of alpha-fetoprotein in a model noncompetitive immunoassay.

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Establishment and Application of a Dual-Labeling Time-Resolved Fluorescence Immunoassay Method for Simultaneous Detection of the Troponin I-C Complex and Full-Size-Troponin I

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2020.596051 ◽

2021 ◽

Vol 7 ◽

Author(s):

Biao Huang ◽

Jian Wu ◽

Hao Chen ◽

Li Zhang ◽

Xiumei Zhou ◽

...

Keyword(s):

Correlation Coefficient ◽

Troponin I ◽

Solid Phase ◽

Simultaneous Detection ◽

Measurement Range ◽

Fluorescence Immunoassay ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Full Size ◽

C Complex

Background: The measurement of cardiac troponin I (cTnI) is widely used in the diagnosis of acute myocardial infarction (AMI). Although existing cTnI detection methods measure total cTnI, the significance of undegraded full-size-cTnI levels is still not well-understood. In this study, we have established a novel dual-labeling time-resolved fluorescence immunoassay (TRFIA) technique that simultaneously detects the cTnI-C complex and full-size-cTnI, allowing us to explore the clinical value of full-size-cTnI determination.Methods: An antibody against the 23–43 amino acid region of cTnI protected by endogenous cTnC is coupled to magnetic beads to provide a solid-phase antibody for capturing all cTnI. An antibody against cTnC in the cTnI-C complex labeled with Eu3+ was used to detect the cTnI-C complex, and an antibody labeled with Sm3+ near the C-terminal 190–203 amino acids of cTnI was used to detect full-size-cTnI. Through dual-labeling TRFIA, cTnI-C complex, full-size-cTnI, and the full-size-cTnI/cTnI-C ratio can be detected simultaneously. The dual-labeling TRFIA technique was used to analyze serum samples collected at different times during treatment and compare their full-size-cTnI/cTnI-C ratios.Results: The sensitivity for the cTnI-C-TRFIA complex was 0.02 ng/mL, the measurement range was 0.02–40 ng/mL, the average intra-batch coefficient of variation (CV) was 4.35%, and the inter-average CV was 6.23%. The correlation coefficient between cTnI-C-TRFIA and commercial cTnI-CLIA methods was R2 = 0.8887. The sensitivity for full-size-cTnI-TRFIA was 0.04 ng/mL, the measurement range was 0.04–40 ng/mL, the average intra-batch CV was 4.95%, and the average inter-batch CV was 7.79%. The correlation coefficient between full-size-cTnI-TRFIA and commercial cTnI-CLIA methods was R2 = 0.7247.Conclusions: Dual-labeling full-size-cTnI/cTnI-C-TRFIA analysis is helpful for determining the length of time of chest pain before admission and the degree of continuous release of cTnI in the myocardium. Thus, it is more for early prognosis than just detecting cTnI.

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