Determination of a limit of detection (LoD) for surface bound antibodies is crucial for the development and deployment of sensitive bioassays. The measurement of very low concentrations of surface bound antibodies is also important in the manufacturing of pharmaceutical products such as antibody-conjugated pharmaceuticals. Low concentrations are required to avoid an immune response from the target host. Enzyme-linked immunosorbent assay (ELISA), x-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to determine the LoD for the surface bound antibody (antiepidermal growth factor receptor antibody) on silicon substrates. Antibody solution concentrations between 10 μg/ml and 1 ng/ml and a control (antibody-free buffer solution) were employed, and the detection performance of each technique was compared. For this system, the ELISA LoD was 100 ng/ml and the XPS LoD was 1 μg/ml, corresponding to an estimated surface concentration of 49 7 ng/cm2 using a 1 μg/ml solution. Due to the multivariate complexity of ToF-SIMS data, analysis was carried out using three different methods, peak ratio calculations, principal component analysis, and artificial neural network analysis. The use of multivariate analysis with this dataset offers an unbiased analytical approach based on the peaks selected from ToF-SIMS data. The results estimate a ToF-SIMS LoD between applied antibody concentrations of 10 and 100 ng/mL. For surface bound antibodies on a silicon substrate, the LoD is below an estimated surface concentration of 49 ng/cm2. The authors have determined the LoD for this system using ELISA, XPS, and ToF-SIMS with multivariate analyses, with ToF-SIMS offering an order of magnitude better detection over ELISA and 2 orders of magnitude better detection over XPS.
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August 2017
Research Article|
August 11 2017
Determining the limit of detection of surface bound antibody
Robert M. T. Madiona;
Robert M. T. Madiona
Centre for Materials and Surface Science and Department of Chemistry and Physics, School of Molecular Sciences, La Trobe University
, Melbourne, VIC 3086, Australia
and CSIRO Manufacturing
, Clayton, VIC 3168, Australia
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Nicholas G. Welch;
Nicholas G. Welch
CSIRO Manufacturing
, Clayton, VIC 3168, Australia
and Centre for Materials and Surface Science and Department of Chemistry and Physics, School of Molecular Sciences, La Trobe University
, Melbourne, VIC 3086, Australia
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Judith A. Scoble;
Judith A. Scoble
CSIRO Manufacturing
, Clayton, VIC 3168, Australia
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Benjamin W. Muir;
Benjamin W. Muir
CSIRO Manufacturing
, Clayton, VIC 3168, Australia
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Paul J. Pigram
Paul J. Pigram
a)
Centre for Materials and Surface Science and Department of Chemistry and Physics, School of Molecular Sciences, La Trobe University
, Melbourne, VIC 3086, Australia
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a)
Electronic mail: p.pigram@latrobe.edu.au
Biointerphases 12, 031007 (2017)
Article history
Received:
June 05 2017
Accepted:
July 21 2017
Citation
Robert M. T. Madiona, Nicholas G. Welch, Judith A. Scoble, Benjamin W. Muir, Paul J. Pigram; Determining the limit of detection of surface bound antibody. Biointerphases 1 August 2017; 12 (3): 031007. https://doi.org/10.1116/1.4986377
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