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Protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches.

ABSTRACT: Developing antibodies with high specificity against post-translationally modified epitopes remains a challenge. Yeast biopanning is well suited in screening for high-specificity binders. Here, we present a protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches. We describe steps for screening a yeast surface display library for antibodies and other binders. We then detail procedures for validating the antibodies found by analyzing their specificity through whole-well image analysis in 96-well plates. For complete details on the use and execution of this protocol, please refer to Arbaciauskaite et al.¹.

SUBMITTER: Arbaciauskaite M

PROVIDER: S-EPMC11345596 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches.

Arbaciauskaite Monika M Pirhanov Azady A Paoloni Justin J Lei Yu Y Cho Yong Ku YK

STAR protocols 20240801 3

Developing antibodies with high specificity against post-translationally modified epitopes remains a challenge. Yeast biopanning is well suited in screening for high-specificity binders. Here, we present a protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches. We describe steps for screening a yeast surface display library for antibodies and other binders. We then detail procedures for validating the antibodies found ...[more]

PMID: 39093705

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Project description:Research in biosensing approaches as alternative techniques for food diagnostics for the detection of chemical contaminants and foodborne pathogens has increased over the last twenty years. The key component of such tests is the biorecognition element whereby polyclonal or monoclonal antibodies still dominate the market. Traditionally the screening of sera or cell culture media for the selection of polyclonal or monoclonal candidate antibodies respectively has been performed by enzyme immunoassays. For niche toxin compounds, enzyme immunoassays can be expensive and/or prohibitive methodologies for antibody production due to limitations in toxin supply for conjugate production. Automated, self-regenerating, chip-based biosensors proven in food diagnostics may be utilised as rapid screening tools for antibody candidate selection. This work describes the use of both single channel and multi-channel surface plasmon resonance (SPR) biosensors for the selection and characterisation of antibodies, and their evaluation in shellfish tissue as standard techniques for the detection of domoic acid, as a model toxin compound. The key advantages in the use of these biosensor techniques for screening hybridomas in monoclonal antibody production were the real time observation of molecular interaction and rapid turnaround time in analysis compared to enzyme immunoassays. The multichannel prototype instrument was superior with 96 analyses completed in 2h compared to 12h for the single channel and over 24h for the ELISA immunoassay. Antibodies of high sensitivity, IC50's ranging from 4.8 to 6.9ng/mL for monoclonal and 2.3-6.0ng/mL for polyclonal, for the detection of domoic acid in a 1min analysis time were selected. Although there is a progression for biosensor technology towards low cost, multiplexed portable diagnostics for the food industry, there remains a place for laboratory-based SPR instrumentation for antibody development for food diagnostics as shown herein.

Dataset Information

Protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches.

Publications

Protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches.

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