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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.

SUBMITTER: Schoenherr RM

PROVIDER: S-EPMC2839902 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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

Schoenherr Regine M RM Zhao Lei L Whiteaker Jeffrey R JR Feng Li-Chia LC Li Li L Liu Lina L Liu Xiuwen X Paulovich Amanda G AG

Journal of immunological methods 20091201 1-2

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 pote ...[more]

PMID: 19961853

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Project description:Although liquid chromatography/mass spectrometry using selected reaction monitoring (LC/SRM-MS) holds great promise for targeted protein analysis, quantification of therapeutic monoclonal antibody (mAb) in tissues represents a daunting challenge due to the extremely low tissue levels, complexity of tissue matrixes, and the absence of an efficient strategy to develop an optimal LC/SRM-MS method. Here we describe a high-throughput, streamlined strategy for the development of sensitive, selective, and reliable quantitative methods of mAb in tissue matrixes. A sensitive nano-LC/nanospray-MS method was employed to achieve a low lower limit of quantification (LOQ). For selection of signature peptides (SP), the SP candidates were identified by a high-resolution Orbitrap and then optimal SRM conditions for each candidate were obtained using a high-throughput, on-the-fly orthogonal array optimization (OAO) strategy, which is capable of optimizing a large set of SP candidates within a single nano-LC/SRM-MS run. Using the optimized conditions, the candidates were experimentally evaluated for both sensitivity and stability in the target matrixes, and SP selection was based on the results of the evaluation. Two unique SP, respectively from the light and heavy chain, were chosen for quantification of each mAb. The use of two SP improves the quantitative reliability by gauging possible degradation/modification of the mAb. Standard mAb proteins with verified purities were utilized for calibration curves, to prevent the quantitative biases that may otherwise occur when synthesized peptides were used as calibrators. We showed a proof of concept by rapidly developing sensitive nano-LC/SRM-MS methods for quantifying two mAb (8c2 and cT84.66) in multiple preclinical tissues. High sensitivity was achieved for both mAb with LOQ ranged from 0.156 to 0.312 ?g/g across different tissues, and the overall procedure showed a wide dynamic range (?500-fold) and good accuracy [relative error (RE) < 18.8%] and precision [interbatch relative standard deviation (RSD) < 18.1%, intrabatch RSD < 17.2%]. The quantitative method was applied to a comprehensive investigation of the steady-state tissue distribution of 8c2 in wild-type mice versus those deficient in FcRn ?-chain, Fc?IIb, and Fc?RI/Fc?RIII, following a chronic dosing regimen. This work represents the first extensive quantification of mAb in tissues by an LC/MS-based method.

Dataset Information

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

Publications

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

Similar Datasets

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