Automated screening of monoclonal antibodies for SISCAPA assays using a magnetic bead processor and liquid chromatography-selected reaction monitoring-mass spectrometry

Abstract

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) utilizes antibodies to enrich peptides from complex matrices for quantitation by stable isotope dilution mass spectrometry. SISCAPA improves sensitivity and limits the sample handling required for plasma-based analysis. Thus far, SISCAPA assays have been performed using polyclonal antibodies, yet monoclonal antibodies are an attractive alternative since they provide exquisite specificity, a renewable resource, and the potential for isolation of clones with very high affinities (10(-9) M or better). The selection of a good monoclonal antibody out of hundreds-to-thousands of clones presents a challenge, since the screening assay should ideally be in the format of the final SISCAPA assay, but performing the assays manually is labor- and time-intensive. In this manuscript, we demonstrate that monoclonal antibodies can be used in SISCAPA assays, and we describe an automated high-throughput SISCAPA method that makes screening of large numbers of hybridomas feasible while conserving time and resources.

Figure 1

KingFisher plate set-up and processing steps for monoclonal antibody screening SISCAPA assay. The magnetic beads are picked up by the magnetic PCR head (not shown) and transferred sequentially from one plate to the next, starting at Plate 1. Peptides are eluted from the antibodies in Plate 7 (with subsequent return of the beads to Plate 1), and the eluates are transferred manually to an autosampler 96-well plate for LC-SRM-MS analysis.

Figure 2

Workflow of the recovery efficiency SISCAPA assay. Antibody performance is assessed by determining an antibody's peptide recovery (the percentage of a peptide amount captured by the antibody and detected by LC-SRM-MS upon elution of the peptide from the antibody). A capture sample is prepared by adding a known amount of light peptide to an aliquot of diluted, digested human plasma, and subsequently adding antibody (previously coupled to magnetic beads). After capture, light peptide bound to the antibody is eluted and a known amount of heavy peptide (equivalent to the light peptide amount initially used) is added to the eluate. A pre-capture sample is prepared using equal amounts of light and heavy peptide (equivalent to the amounts used for the capture sample). Both samples are analyzed by LC-SRM-MS and the light-to-heavy peptide peak area ratios are compared to arrive at the recovery efficiency (the peak area ratio of the capture sample is divided by the ratio of the pre-capture sample).

Figure 3

Response curve for the mouse ADAM17(VDN)-83-17 monoclonal antibody SISCAPA assay. Varying amounts of heavy peptide and a constant amount of light peptide were spiked into 10 μL of human plasma digest, the sample was diluted to 100 μL, and the peptides were captured, eluted, and detected using SISCAPA LC-SRM-MS. Linear regression analysis was performed on log-transformed peak area ratios of the heavy-to-light peptide signals versus the theoretical protein concentrations (back-calculated to undiluted plasma). Calculated protein concentrations were obtained with the resulting regression coefficients.

Figure 4

Pipeline for hybridoma screening using peptide-glutaraldehyde-BSA ELISA and KingFisher SISCAPA LC-SRM-MS assays. An initial first-pass screen using pep-glu-BSA ELISA will eliminate a majority of primary clone hybridomas that would not be effective in SISCAPA assays. This will reduce the number of available hybridomas from thousands to tens to hundreds. Some hybridomas are expected to pass the ELISA screen that will subsequently be negative in SISCAPA (false ELISA positives). These false positives will be identified by KingFisher SISCAPA screening using fixed supernatant volumes and will hence be eliminated from further consideration. Quantitative mouse IgG ELISA assays will be performed with SISCAPA positive supernatants, and KingFisher SISCAPA experiments using adjusted supernatant volumes can then be performed to enable rank-ordering of the primary clones. Up to five best-performing primary clones can be subcloned, and the KingFisher SISCAPA assay strategy of ‘fixed supernatant volume KingFisher SISCAPA—quantitative ELISA—adjusted supernatant volume KingFisher SISCAPA’ can be repeated to select the best subclone for production and purification.