Novel sequential immunocapture microflow LC/MS/MS approach to measuring PTH-Fc protein in human serum

Bioanalysis ◽  
2021 ◽  
Author(s):  
Haixing Wang ◽  
Jiang Wu ◽  
Peng Pan ◽  
Thuy Nguyen ◽  
Michael Cwik ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Human Serum ◽  
Clinical Studies ◽  
Binding Assay ◽  
Low Doses ◽  
The Novel ◽  
Ligand Binding Assay ◽  
Significant Challenge ◽  
Sensitivity And Selectivity ◽  
Interday Precision

The quantitation of PTH-Fc in circulation by ligand binding assay presented a significant challenge due to the extremely low doses of administration, interference from the endogenous. A robust LC–MS/MS method to quantify the extremely low concentration of PTH-Fc in human serum utilized sequential immunoaffinity enrichment at PTH and Fc domains in conjunction with microflow LC–MS/MS technology significantly improved the sensitivity and selectivity. The assay displayed a quantitation range of 0.025–5.0 ng/ml and acceptable intraday and interday precision (%CV ≤ 15%) and accuracy (%bias ≤ ±15%) and can be routinely used for pharmacokinetic measurement of the drug. The novel sequential immunocapture workflow described herein can be applied to the quantitation of other recombinant therapeutic proteins to support clinical studies.

scholarly journals A competitive ligand-binding assay for the detection of neutralizing antibodies against dostarlimab (TSR-042)

AAPS Open ◽  
2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Xiaolong Tom Zhang ◽  
Hong Chen ◽  
Weiping Shao ◽  
Zhongping John Lin ◽  
Murad Melhem ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Human Serum ◽  
Neutralizing Antibodies ◽  
Binding Assay ◽  
False Positive Rate ◽  
Ligand Binding Assay ◽  
Drug Interference ◽  
Hook Effect ◽  
System Suitability ◽  
Competitive Ligand

AbstractDostarlimab is a humanized anti–PD-1 monoclonal antibody. Dostarlimab (JEMPERLI; TSR-042) was recently approved in the USA and in the EU. The presence of neutralizing antibodies (NAbs) is a cause for concern because they block the therapeutic function of the antibody and reduce drug efficacy. Therefore, programs developing therapeutic biologics need to develop and validate assays that adequately assess the presence of NAbs in the serum of patients treated with biologic therapies. Presented here is the development and validation of a competitive ligand-binding assay that specifically detects anti-dostarlimab NAbs in human serum. Precision, sensitivity, hook effect, selectivity, assay robustness, stabilities, and system suitability were evaluated. In addition, drug tolerance of the assay with the implementation of a drug removal process was investigated. The cut point factor for the detection of NAbs in human serum at a 1% false-positive rate was determined. The assay’s precision, sensitivity, hook effect, selectivity, robustness, and drug interference were tested and found to be acceptable. With system suitability and stability established, this assay has been used to evaluate NAbs to guide the development of dostarlimab.Trial registration: Clinicaltrials.gov, NCT02715284. Registered 9 March 2016

TBG MEASUREMENTS BY COMPETITIVE LIGAND BINDING ASSAY (CLBA)

1975 ◽  
Vol 80 (1_Suppla) ◽  
pp. S15
Author(s):  
K. H. Rudorff ◽  
H. J. Kröll ◽  
J. Herrmann
Keyword(s):  
Ligand Binding ◽  
Binding Assay ◽  
Ligand Binding Assay ◽  
Competitive Ligand

Development and evaluation of an immunoenzymometric assay for the chicken progesterone receptor

1991 ◽  
Vol 129 (2) ◽  
pp. 189-196 ◽  
Author(s):  
M. K. Bläuer ◽  
P. J. Tuohimaa ◽  
P. J. Vilja
Keyword(s):  
Monoclonal Antibodies ◽  
Small Intestine ◽  
Horseradish Peroxidase ◽  
Progesterone Receptor ◽  
Ligand Binding ◽  
Binding Assay ◽  
Ligand Binding Assay ◽  
Coefficients Of Variation ◽  
First Time ◽  
Determination Range

ABSTRACT A specific and sensitive immunoenzymometric assay (IEMA) was developed for measuring the quantity of chicken progesterone receptor (PR) in tissue cytosol. The assay uses two monoclonal antibodies to the PR. One is used to capture the PR. The second (labelled with biotin) reacts first with the captured receptor and subsequently with avidin-labelled horseradish peroxidase to provide an enzymatic end-point. The method has a determination range from 0·3 to 60 pmol/l. Intra- and interassay coefficients of variation were 3·7% and 9·0% respectively. The assay can be performed with equal results as a rapid (3 h) or an overnight procedure. The IEMA is convenient, especially for signal measurement and the calculation of results. No ultracentrifugation of samples is needed, since the IEMA can be performed on low-speed cytosol samples. Assay results correlated well (r = 0·927) with those obtained by the conventional ligand-binding assay used in our laboratory. Similar results were obtained with the IEMA and the ligand-binding assay after exposure of cytosol samples to increased temperatures: at 20 °C the PR remained stable for the 4-h period examined, whereas at 37 °C almost complete degradation of the PR was observed in 30 min. Being more than 100 times as sensitive as the ligand-binding assay, the IEMA enabled the quantification of PR for the first time in such tissues as the bursa and small intestine even of immature animals. Journal of Endocrinology (1991) 129, 189–196

Matrix effect in ligand-binding assay: the importance of evaluating emerging technologies

Bioanalysis ◽  
2014 ◽  
Vol 6 (8) ◽  
pp. 1033-1036 ◽  
Author(s):  
Rebecca M Crisino ◽  
Linlin Luo ◽  
Brian Geist ◽  
Jad Zoghbi ◽  
Franklin Spriggs
Keyword(s):  
Ligand Binding ◽  
Matrix Effect ◽  
Emerging Technologies ◽  
Binding Assay ◽  
Ligand Binding Assay

Performance evaluation of three platforms with ultrasensitive ligand-binding assay potential

Bioanalysis ◽  
2017 ◽  
Vol 9 (12) ◽  
pp. 937-946 ◽  
Author(s):  
Kyra Juliana Cowan ◽  
Albert Geiger ◽  
Hans Hornauer ◽  
Robyn Rourick ◽  
Patricia Yanira Siguenza
Keyword(s):  
Performance Evaluation ◽  
Ligand Binding ◽  
Binding Assay ◽  
Ligand Binding Assay
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