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Related Experiment Videos

Surface immobilized antibody orientation determined using ToF-SIMS and multivariate analysis.

Nicholas G Welch1, Robert M T Madiona1, Thomas B Payten2

  • 1Centre for Materials and Surface Science and Department of Chemistry and Physics, School of Molecular Sciences, La Trobe University, VIC 3086, Australia; CSIRO Manufacturing, VIC 3168, Australia.

Acta Biomaterialia
|April 1, 2017
PubMed
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This study demonstrates a novel method using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and machine learning to confirm antibody orientation on surfaces, improving biosensor performance. The findings show a 50% increase in signal for correctly oriented antibodies on functionalized surfaces.

Area of Science:

  • Biomolecular surface analysis
  • Protein immobilization science
  • Biosensor technology

Background:

  • Antibody orientation is crucial for sensitive biomolecule detection in immunoassays.
  • Surface-sensitive techniques like ToF-SIMS offer detailed analysis but are underutilized for protein orientation.
  • Existing methods often analyze limited mass fragments, leaving significant data unexamined.

Purpose of the Study:

  • To develop and validate a comprehensive methodology for characterizing antibody orientation on solid surfaces.
  • To investigate the role of chromium-functionalized plasma polymers (DGpp+Cr) in promoting preferential antibody orientation.
  • To establish a label-free, direct characterization method for surface-bound proteins.

Main Methods:

  • Utilized Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) to analyze proteolytic antibody fragments.
Keywords:
Antibody fragmentsAntibody orientationArtificial neural networksChromium (III) complexPrincipal component analysisToF-SIMS

Related Experiment Videos

  • Constructed Artificial Neural Network (ANN) and Principal Component Analysis (PCA) models using ToF-SIMS data.
  • Compared mass fragments from F(ab')2 and Fc regions to identify orientation-specific signatures.
  • Quantified antibody orientation by comparing ion intensities of F(ab')2 to Fc fragments.

    • Main Results:

    • ANN and PCA models successfully differentiated correctly and incorrectly oriented antibodies.
    • Identified 20 mass fragments associated with correct F(ab')2 orientation and 23 with incorrect Fc orientation.
    • Demonstrated a 50% increase in the ratio of F(ab')2 to Fc ion intensities on DGpp+Cr compared to DGpp.
    • Confirmed preferential antibody orientation on DGpp+Cr surfaces.

    Conclusions:

    • The developed systematic data analysis methodology provides a robust approach for investigating antibody orientation.
    • This label-free technique is applicable to various substrates and protein types.
    • The findings support the use of DGpp+Cr surfaces for enhanced immunoassay performance due to controlled antibody orientation.