Automated screening of monoclonal antibodies for SISCAPA assays using a magnetic bead processor and liquid chromatography-selected reaction monitoring-mass spectrometry.
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.
Schoenherr
Regine M
RM
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., P.O. Box 19024, Seattle, WA 98109-1024, USA.
Zhao
Lei
L
Whiteaker
Jeffrey R
JR
Feng
Li-Chia
LC
Li
Li
L
Liu
Lina
L
Liu
Xiuwen
X
Paulovich
Amanda G
AG
D051722:ADAM Proteins / Q000097:blood / Q000276:immunology; D000072198:ADAM17 Protein; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D057205:Automation, Laboratory; D015415:Biomarkers / Q000097:blood; D002097:C-Reactive Protein / Q000032:analysis / Q000276:immunology; D002853:Chromatography, Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas; D018189:Immunomagnetic Separation; D007201:Indicator Dilution Techniques; D013058:Mass Spectrometry; D051379:Mice; D010446:Peptide Fragments / Q000097:blood / Q000276:immunology
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D010446:Peptide Fragments; D002097:C-Reactive Protein; D051722:ADAM Proteins; D000072198:ADAM17 Protein