John Laycock (Presenter)
SPEware Corporation
Authorship: John Laycock (1), Karsten Liegmann(1), Qi Huang (1), Phil Dimson (1), Bonnie Baker (2), Carmen Fernandez-Metzler (2), and Rick King (2)
(1) SPEware Corporation, Baldwin Park, CA (2) PharmaCadence Analytical Services, LLC, Hatfield, PA
| Short Abstract We present a novel automated workflow utilizing 96 well plates with control flow properties for immunoaffinity capture and tryptic digestion. The novel plate airgap design restricts the flow of liquid during conditions applicable to many assay incubation types and steps. The flow is restricted completely until positive pressure is applied, providing a simple and cost-effective alternative to magnetic bead based cleanup protocols. In this presentation we discuss application of controlled-flow plates (CFP) to immunoaffinity capture of a recombinant human antibody for isolation from rat plasma, followed by tryptic digestion and LCMSMS. |
Long Abstract
Introduction
LCMSMS measurement of signature peptides derived from digestion of whole monoclonal antibodies has emerged as an alternative to traditional ligand binding assay measurement techniques [1]. Current methodologies for immunoaffinity capture followed by tryptic digestion require numerous transfer steps that can result in errors and loss of peptide analytes due to non-specific binding to tip surfaces. Magnetic bead workflows and platforms are available but require a high cost platform and loss of beads during multiple wash steps can be problematic [2].
We present a novel automated workflow utilizing 96 well plates with control flow properties for immunoaffinity capture and tryptic digest. The novel plate airgap design restricts the flow of liquid during conditions applicable to many assay incubation types and steps. The flow is restricted completely until positive pressure is applied providing a simple and cost-effective alternative to magnetic bead based cleanup protocols.
Methods
Preliminary evaluation for the controlled flow plate was performed with an aqueous dye solution to demonstrate complete lack of liquid breakthrough. The conditions tested included incubation at temperatures up to 100C, continuous vigorous vortexing for up to 4 hours, and sonication for up to 45 minutes.
SILu™Lite SigmaMAb Universal Antibody Standard human was spike into rat plasma in the concentration range from 2-400 µg/mL. Immunoaffinity capture procedure was perfomed in in a controlled flow 96 well plate as follows: Capture and immobilize target antibody from diluted rat plasma. Incubate diluted biological matrix with antibody and Protein A/G agarose for 60 minutes at RT. Purification of the captured antibody is achieved by application of positive pressure to remove liquid and three repeated washes agarose digestion buffer.
Purification is followed by direct on-bead digestion of the whole antibody using a standard tryptic digestion procedure: Denature target protein by heating in the presence of DTT at 60℃ for 30 minutes. Alkylate target protein by reaction with iodoacetamide at RT for 30 minutes in the dark. Digestion is performed by reaction with trypsin at 37℃ for 18 hours. Digested peptides are passed directly into a collection plate for LCMSMS analysis on a Sciex QTRAP® 5500 LC/MS/MS system operating with a standard reversed-phase LC gradient. The peptide MRMs we monitored in scheduled mode for the surrogate peptides GPSVFPLAPSSK, STSGGTAALGCLVK, TPEVTCVVVDVSHEDPEVK, and FNWYVDGVEVHNAK.
Results
Based on measured peak are response uncorrected with internal standard, the results for the 4 peptides monitored were linear in the assay range and reproducible. Linearity in the range from 2-400 µg/mL was achieved with %CV of less than 20% at the LLOQ and less than 15% throughout the calibration range. Recovery of all peptides exceeded 80%.
Conclusions
An immunocapture workflow with a novel controlled flow plate has been demonstrated to provide a simple and cost-effective assay platform for measurement of surrogate peptides resulting from digestion of whole antibodies. Acceptable data was achieved even without the presence of an internal standard. The workflow eliminates the need for attention to pipetting detail when handling magnetic beads manually and does not require a high cost dedicated magnetic bead platform.
References & Acknowledgements:
[1] Anal. Chem., 2012, 84 (3), pp 1267–1273
[2] Bioanalysis (2015) 7(3)
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | SPEware |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |