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Updated: Jun 6, 2026
High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)
Published on: August 21, 2018
Absolute quantification of a therapeutic domain antibody using ultra-performance liquid chromatography-mass
Matthew E Szapacs1, James J Urbanski, Jonathan R Kehler
1Worldwide Bioanalysis, Drug Metabolism & Pharmacokinetics, GlaxoSmithKline, 709 Swedeland Rd, King of Prussia, PA 19406, USA. matthew.e.szapacs@gsk.com
Background:
Domain antibodies (dAbs; ∼10-15 kDa) are made up of the variable heavy chain or the variable light chain of the antibody structure, and retain binding capability. dAbs have proved difficult to detect in plasma using immunoassay without specific antibodies raised against the dAb.
Results:
A sensitive and selective UPLC-MS/MS method for the absolute quantification of a dAb in monkey plasma was developed (range: 1 to 500 ng/ml) without the need for a specific capture antibody. This method was used to analyze pharmacokinetic studies early on in drug development. Furthermore, an immunoassay was developed and the pharmacokinetic samples were reanalyzed.
Conclusion:
The two assays show good correlation (r(2) = 0.92), giving confidence in using either method for quantification of the dAb.
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