A sensitive solid-phase time-resolved fluorescence immunoassay apparatus

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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Sensitive time-resolved fluorescence immunoassay of somatotropin in serum

Clinical Chemistry ◽

10.1093/clinchem/36.3.503 ◽

1990 ◽

Vol 36 (3) ◽

pp. 503-508 ◽

Cited By ~ 3

Author(s):

I Kahan ◽

A Papanastasiou-Diamandi ◽

G Ellis ◽

S K Makela ◽

J McLaurin ◽

...

Keyword(s):

Monoclonal Antibody ◽

Pediatric Patients ◽

Solid Phase ◽

Nitrogen Laser ◽

Fluorescence Immunoassay ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Sandwich Type ◽

Europium Chelate ◽

Growth Abnormalities

Abstract We describe a new "sandwich"-type non-isotopic immunoassay for human somatotropin (GH, growth hormone) in serum. In the assay, GH is captured by a monoclonal antibody immobilized in a white microtiter well and simultaneously reacted with a second biotinylated monoclonal antibody. The degree of binding of biotinylated antibody, which increases with increasing amount of GH in the sample, is quantified by adding streptavidin labeled with the europium chelate of 4.7 - bis(chlorosulfophenyl) - 1.10 - phenanthroline - 2.9 - dicarboxylic acid. The fluorescent complex on the solid phase is then measured by excitation at 337.1 nm (nitrogen laser) and monitoring the emission at 615 nm in a gated fluorometer/analyzer. The proposed procedure has short incubation times (less than 4 h protocol), uses only 25 microL of serum per microtiter well, and gives precise and accurate results. The method was clinically evaluated with samples obtained from pediatric patients undergoing investigation for growth abnormalities and from a patient with acromegaly.

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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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Evaluation of the time resolved fluorescence immunoassay (TRFIA) for the detection of varicella zoster virus (VZV) antibodies following vaccination of healthcare workers

Journal of Virological Methods ◽

10.1016/j.jviromet.2010.12.021 ◽

2011 ◽

Vol 172 (1-2) ◽

pp. 60-65 ◽

Cited By ~ 13

Author(s):

S.L.R. McDonald ◽

P.A.C. Maple ◽

N. Andrews ◽

K.E. Brown ◽

K.L. Ayres ◽

...

Keyword(s):

Varicella Zoster Virus ◽

Healthcare Workers ◽

Fluorescence Immunoassay ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Varicella Zoster

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Development of a time-resolved fluorescence immunoassay based on immunomagnetic beads for gastrin-17

Journal of Immunological Methods ◽

10.1016/j.jim.2021.113179 ◽

2021 ◽

pp. 113179

Author(s):

Shaoxiong Zheng ◽

Renjing Hu ◽

Xiaomei Yu ◽

Lingli Chen ◽

BinrongWang ◽

...

Keyword(s):

Fluorescence Immunoassay ◽

Time Resolved Fluorescence ◽

Immunomagnetic Beads ◽

Time Resolved

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

10.21203/rs.3.rs-396573/v1 ◽

2021 ◽

Author(s):

Xindong Chen ◽

Jianfeng Hong ◽

Han Zhao ◽

Zhongyi Xiang ◽

Yuan Qin ◽

...

Keyword(s):

Lung Cancer ◽

Nasopharyngeal Cancer ◽

Reaction Rates ◽

Measurement Range ◽

Fluorescence Immunoassay ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Combined Use ◽

Highly Sensitive ◽

Positive Rate

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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